β2 adrenergic receptors orchestrate neutrophil demargination and recruitment to the ischemic heart following myocardial infarction

Domain: neuroscience
Published: 2026-04-11
Editorial outcome: major_revision

Abstract
Neutrophils rapidly accumulate in the heart after myocardial infarction (MI), yet bone marrow granulopoiesis peaks later, suggesting an alternative early source. Here we show in male mice that the first wave of neutrophils recruited to the ischemic heart arises from the vascular marginated pool rather than newly generated cells in the bone marrow or spleen. Using hematopoietic stem cell ablation, flow cytometry and proteomic analyses, we find that early MI-induced neutrophilia displays hallmarks of stress-induced demargination, a process in which adherent neutrophils are rapidly released into circulation. Pharmacological or genetic inhibition of norepinephrine synthesis or β-adrenergic receptor signaling in male mice suppresses neutrophil demargination and limits cardiac infiltration. Although sustained β-adrenergic blockade does not improve long-term outcomes, transient inhibition enhances cardiac remodeling and function. These findings identify stress-induced demargination as the dominant source of early post-MI neutrophils and suggest that temporally targeted β2-adrenergic modulation may help restrain acute inflammatory injury.

Introduction
Acute myocardial infarction (AMI) induces a rapid and robust inflammatory response characterized by infiltration of different immune cell types to the infarcted heart. The recruitment of immune cells is a dynamic process and consists of sequential infiltration of neutrophils, pro- and anti-inflammatory monocytes, and lymphocytes to the injured myocardium1-6. While the excessive inflammatory response after MI is responsible for cardiomyocyte death, infarction expansion and adverse remodeling, it also has an essential role in orchestrating the reparative response for optimal infarct healing7. The inflammatory response following MI can be mapped into three distinct but overlapping phases8-11. The first phase, also known as the acute inflammatory phase, occurs at the onset of MI and is characterized by a significant increase in innate immune cell (neutrophils as well as monocytes/ macrophages) infiltration to the myocardium mainly to clear debris and dead cells. In the second phase, reparative monocytes, macrophages, dendritic cells, and lymphocytes are mobilized to resolve the initial inflammatory response and support the formation of vascularized granulation tissue (from 3-4 days to 2 weeks after infarction). The third phase takes place from 2 weeks onward where the vascularized granulation leads to a stable myocardial scar that is almost free of immune cells. It is therefore important that a timely and well-balanced inflammatory response is established to successfully dampen the inflammatory response and initiate infarct healing in patients surviving MI. However, there are no clear strategies in clinical practice to regulate the inflammatory response optimal enough to favorably modulate the remodeling processes.

Studies from our lab and elsewhere have shown that infiltration of neutrophils to the infarcted myocardium have the ability to influence the nature of the ensuing inflammatory response. This is because neutrophils are the first responders to ischemic injury and have the ability to amplify the initial inflammatory response by deploying key alarmins such as S100A8 and S100A9. These alarmins interact with Toll-Like-Receptor (TLR4) on naïve neutrophils, prime the NLRP3 inflammasome and facilitate their migration to the bone marrow (BM). The reverse-migrated neutrophils are preferentially retained within the BM sinusoids by certain adhesion receptors, a process that is essential for the activation of Gasdermin D- dependent membrane pore formation and secretion of IL-1β12,13. All these events culminate in amplified granulopoiesis and ensure a steady supply of neutrophils to facilitate the clearance of debris and dead cells and, to initiate wound healing. Interestingly, the process of granulopoieses in the BM does not reach the climax until 24 hours post-MI, yet neutrophils begin to appear in the heart much faster than their production rate in the BM. These findings suggest that sources other than granulopoiesis may help replenish neutrophils for immediate deployment to the ischemic heart. However, the exact source of these neutrophils is unclear.

Under steady-state conditions, mature neutrophils are present in major reservoirs across the body (spleen, liver, BM, lungs, and vasculature) and are recruited following changes in homeostasis14. The unique feature of neutrophils in the vasculature is their distribution into two distinct groups, the marginated and the demarginated. Firmly adherent to the vasculature walls, the marginated pool of neutrophils represents ~ 51% of neutrophils in circulation and are believed to serve as early responders. The demargination process in neutrophils is attributed to loss of adhesion molecules, and rearrangement of actin cytoskeleton within the cells15,16. Under certain stress and inflammatory conditions, adhesion of neutrophils is altered, and the marginated cells detach and mobilize into the bloodstream, expanding the percentage of demarginated cells in the blood17. We reasoned that the marginated pool of neutrophils may be mobilized to meet the sudden and increased demand of the ischemic heart. Thus, deciphering the signaling pathways that stimulate the demargination of neutrophils and their recruitment to the ischemic heart may provide a viable approach to regulate neutrophil trafficking to the heart post-MI.

Results
Neutrophil recruitment to the ischemic heart precedes the peak of BM granulopoiesis.
We previously have shown that large-scale infiltration of neutrophils to the ischemic heart post-MI12 is facilitated by a robust activation of HSPCs in the BM (~18-24 hours). Furthermore, the first wave of recruited neutrophils releases alarmins (e.g., S100A8 and S100A9) via NETosis, a crucial step in sustaining granulopoiesis in the BM13. However, the pathways that regulate granulopoiesis in the BM are activated between 12- and 24-hours post-MI and thus, the mechanisms that trigger the recruitment of the first wave of neutrophils are unknown. We hypothesized that the circulating neutrophils including the marginated pool are sourced to meet the immediate demand of the ischemic heart. To test this hypothesis, we first performed a temporal study to examine the kinetics of neutrophil recruitment to the heart and their production from sites in the BM and spleen. We employed a mouse model of permanent ligation of the left anterior descending (LAD) artery as described previously12,13.

Following MI, an increase in neutrophil recruitment to the heart occurred swiftly and reached a submaximal threshold at around 6 hours (Figure 1A). To independently validate that neutrophil infiltration occurs as early as 6 hours post-MI, we re-analyzed a publicly available bulk RNA-seq dataset (GSE153485) of infarcted mouse myocardium18. A curated neutrophil gene signature comprising S100a8, S100a9, Mmp8, Mmp9, Fpr1, and Fpr2 was strongly upregulated at 6 hours compared with sham controls and became even more pronounced by 24 hours (Supplementary Figure 1A). Consistent with this, neutrophil-specific biological processes, such as neutrophil degranulation, chemotaxis, and activation, were significantly enriched at 6 hours and exhibited further amplification at 24 hours post-MI, indicating progressive engagement of neutrophil effector programs (Supplementary Figure 1B). Direct visualization of the infarct zone by confocal microscopy corroborated these transcriptomic findings, revealing Ly6G+ neutrophils with classical multilobulated nuclear morphology in the myocardium at 6 hours post-MI (Supplementary Figure 1C). It is important to note that these heart-infiltrating neutrophils were likely sourced from circulation since blood also showed a significant increase in neutrophil number at 6 hours after MI (Figure 1B).

The neutrophils are matured and not “newly produced” because the EdU (a nucleoside analog of thymidine incorporated into DNA during active DNA synthesis) label did not increase significantly in neutrophils in both the heart (Figure 1C) and blood (Figure 1D) up until 24 hours post-MI. We also confirmed that the increase in neutrophil number in the heart and blood during the first 6 hours was not due to mobilization of matured neutrophils from the BM (Figure 1E) since the neutrophil stocks in BM remain undepleted. Interestingly, the number of neutrophils in the spleen increased (Figure 1F). However, as expected, the EdU label did not increase in either BM (Figure 1G) or splenic neutrophils (Figure 1H) suggesting a secondary role for granulopoiesis during the initial hours after MI. This was also reflected in BM progenitor cells (i.e. CMPs, GMPs) that did not show any sign of enhanced granulopoiesis up until 24 hours post-MI (Figures 1I-K). This led us to conclude that the initial wave of neutrophils recruited to the heart (< 6 hours post-MI) was not sourced from the BM/ splenic reservoirs or granulopoiesis (Summarized in Figure 1L).

Neutrophil demargination from the vasculature is the primary source of neutrophils in the ischemic heart during the early hours following MI.
Since we found no major role for either BM or spleen in supplying neutrophils to the ischemic heart immediately after-MI, we hypothesized that neutrophils within the vasculature are the predominant source. To verify this notion, we performed a series of experiments utilizing the total body irradiation (TBI) strategy along with the administration of classical demarginating agents such as dexamethasone (DEX) and nor-epinephrine (NE). We reasoned that TBI of mice would lead to complete ablation of proliferating HSPCs and, thus granulopoiesis in the BM/ spleen would be severely suppressed. If our hypothesis is true, administration of DEX or induction of MI post-TBI would still increase the number of demarginated neutrophils in the absence of routine granulopoiesis.

First, to confirm that DEX and NE can be used as experimental agents, we performed a temporal study. We injected healthy WT mice with a single dose of either NE or DEX and quantified the number of neutrophils demarginated over a period of 24 hours. Using flow cytometry, we quantified the neutrophil numbers in blood and found a rapid increase starting at 2 hours and reaching the peak at 4 hours following NE administration, while the neutrophil population in DEX-treated mice reached a maximum at around 8 hours (Supplementary Figure 2A). These data suggest that both NE and DEX induce demargination but may follow different trajectories perhaps due to different mechanisms of demargination. Next, to confirm that TBI does prevent granulopoiesis without adversely impacting matured blood neutrophils, healthy WT mice were lethally irradiated (TBI) following which the number of neutrophils in the blood and their progenitor cells in the BM and spleen were analyzed over a period of 24 hours. TBI of mice led to a significant decrease in the number of progenitor cells in the BM (Supplementary Figure 2B) and spleen (Supplementary Figure 2C) by 6 hours while the number of matured neutrophils in the blood (Supplementary Figure 2D), BM (Supplementary Figure 2E) and spleen (Supplementary Figure 2F) began to decline after 6 hours and remained constantly lower thereafter. The decline in the number of blood neutrophils can be attributed to the lack of normal hematopoiesis and replenishment due to radiation damage caused to HSPCs. After initial characterization of neutrophil response separately to TBI and demarginating agents, we selected a time point (~18 hours) wherein the blood neutrophil count is slightly decreased, and the BM is unable to mount a granulopoietic response (Summarized in Figure 2A). We choose this time point as a window of opportunity to study the impact of DEX or MI on neutrophil response. As expected, administration of DEX to irradiated mice (Study outline, Figure 2B) significantly increased the number of neutrophils in the blood (Figure 2C) despite a dramatic depletion of progenitor cells in the BM and spleen (Supplementary Figures 2B, C). Similarly, induction of MI in TBI mice (Study outline, Figure 2D) also increased the number of neutrophils in the infarcted heart (Figure 2E) given that a significantly elevated number of demarginated neutrophils was available in the blood (Figure 2F) for rapid recruitment to the heart. This increase in the number of circulating neutrophils indicated that a population of neutrophils is actively present and remains marginated in the vasculature until certain stress signals induce their demargination.

To confirm that demarginated neutrophils are the predominant source of heart infiltrating neutrophils during the early hours after-MI, we tested an alternate hypothesis wherein we first induced MI in two groups of healthy WT mice, followed by injection of DEX (Study outline, Supplementary Figure 2G). We reasoned that administration of DEX to MI mice would not further increase the number of neutrophils in the blood or heart. This is because a prior MI would have exhausted all the marginated neutrophils, and there are no more neutrophils left for DEX to demarginate them. As expected, injection of DEX to MI mice did not increase the number of neutrophils in the blood (Supplementary Figure 2H) or the heart (Supplementary Figure 2I) confirming that there were no more marginated neutrophils left in the vasculature.

Following MI, matured neutrophils can be sourced from the BM or spleen by emptying, a process termed as forced mobilization. To assess their contribution to the demarginated pool and their potential recruitment to the heart, we induced MI in a separate group of mice and immediately injected granulocyte colony-stimulating factor (G-CSF) (Study outline, Supplementary Figure 2J). As expected, the administration of G-CSF induced a rapid efflux of neutrophils (perhaps premature) from the BM with a proportional decrease in BM, a response that is typical to G-CSF administration (Supplementary Figure 2K). These data suggest that MI did not mobilize/ empty the BM of neutrophils during the first 6 hours. Interestingly, the increase in the number of circulating neutrophils (Supplementary Figure 2L) in response to G-CSF did not translate into greater cardiac neutrophil burden (Supplementary Figure 2M). These data not only highlight the importance of the source of neutrophils (i.e., demargination vs BM emptying) but also confirm that matured neutrophils from vasculature are preferred over immature cells from the BM for recruitment to the infarct.

Following MI, neutrophils display phenotypic hallmarks characteristic of demarginated neutrophils.
Constitutively expressed adhesion molecules such as P- and L-selectin (CD62L) are important for neutrophils to remain anchored to the vascular endothelium. During neutrophil migration (i.e., demarginated) and trafficking into injured tissues, CD62L is rapidly shed. It is long known that anti-inflammatory glucocorticoids (e.g., DEX) and catecholamines (e.g., NE) induce neutrophil demargination and, thus neutrophilia, by suppressing CD62L gene expression and/ or shedding of surface CD62L by Adam17, a sheddase16,19. Recent studies have shown that, in addition to the shedding of CD62L, a purely mechanical phenomenon caused by cell softening also plays a major role in neutrophil demargination20. To validate this mechanism, we compared established markers of neutrophil demargination in MI with those observed following DEX or NE treatment. Flow analysis of neutrophils from MI mice displayed phenotypes strikingly similar to those from DEX- or NE-treated animals, including reduced surface CD62L expression (Figures 2G-I) and elevated levels of soluble CD62L in the plasma (Figures 2J-L), consistent with CD62L shedding as a key driver of demargination. In parallel, F-actin content was significantly reduced in neutrophils from both MI and DEX/ NE groups (Figures 2M-O), supporting the engagement of a shared cytoskeletal remodeling program. Confocal imaging revealed that control neutrophils exhibited a bright, continuous cortical actin ring with prominent localized actin structures. In contrast, neutrophils isolated 6 hours after MI or NE treatment showed a dimmer yet continuous cortical actin ring, whereas DEX-treated neutrophils displayed the greatest heterogeneity, characterized by fragmented and discontinuous cortical actin (Figures 2P, Q). Collectively, these data show that MI triggers neutrophil demargination through CD62L shedding and cortical actin remodeling. We next assessed how these demarginated neutrophils are recruited to the injured myocardium.

Proteomic analysis of demarginated neutrophils after MI or NE administration reveals activation of pathways involved in cellular movement and cytoskeletal reorganization.
Neutrophils undergo transcriptional and translational changes while in the circulation and during their recruitment to injured tissues13. Having confirmed that the demarginated neutrophils contribute to increased neutrophil burden in the heart post-MI, we next focused on identifying the signaling pathways that promote demargination of neutrophils. To achieve this, we induced MI in healthy WT mice and sorted blood neutrophils at 6 hours post MI and subjected the samples to proteomics (Study outline, Figure 3A) using a high-resolution SLC-MS/MS (label-free spectral counting approach). Purified neutrophils harvested from the blood at 6 hours after sham surgery served as controls (Figure 3B).

MI induced a dramatic shift in neutrophil proteome as demonstrated by PCA plot depicting a clear spatial discrimination of the proteomes between the two groups (Supplementary Figure 3A). A total of 3517 protein entries were identified (Figure 3C). Among them, 587 proteins were upregulated, and 188 proteins were downregulated (with a 1.5-fold difference) in response to MI compared to sham group. A heat map view of the impacted proteins highlights the distinctive quantitative features between the 2 groups as well as the homogeneity of the biological replicates within each group (Figure 3D). Many of the proteins in demarginated neutrophils are unique to MI with dormant proteins flaring up compared to resting neutrophils in sham mice. Hierarchical pathway cluster analysis of the differentially abundant proteins revealed enrichment of various cellular processes and pathways unique to MI group (Supplementary Figures 3B-G). Among the pathways most impacted by MI (Figure 3E) are those that are associated with cellular movement and immune cell trafficking (24.7%). An equal or slightly lower number of upregulated proteins are involved in cellular functions such as immune cell responses (15.8%), immunological/ inflammatory disease, (11.6%), immune cell function, and maintenance (10.5%). Delving deeper into different categories, pathways were then attributed to activation scores based on the level of combined altered functions of its constituent proteins and their effect in modifying neutrophil functions in MI (Figure 3F). We found a large number of proteins related to enhanced immune cell movement, migration, and organization of the cytoskeleton. Interestingly, the initial stages of MI also promoted cell survival, immune functions (phagocytosis) as well as a general increase in the alert state of an inflammatory response, while shutting down most pathways that are involved in the resolution.

Signaling pathways activated in neutrophils during an event such as MI are governed by a diverse set of extracellular ligands. Among the major canonical pathways engaged during MI, our proteomic analyses revealed substantial modifications in proteins involved in G-protein–coupled receptor (GPCR) signaling. GPCR signaling from multiple ligand classes converges downstream on actin polymerization and cytoskeletal reorganization, largely through calcium-dependent pathways21. These include activation of Rac, Pak, and Rho family GTPases, as well as proteins regulating polarization and depolarization such as ROCK, COFILIN (CFN), PROFILIN, and LIM kinase (LIMK) 22 (Supplementary Figure 2H). For example, downstream Gα12/13-mediated signaling upregulates RhoGEFs, leading to increased ROCK1 and ROCK2 activity. ROCK activation inhibits myosin light-chain phosphatase (MLCP), thereby enhancing phosphorylation of myosin light-chain proteins and promoting reorganization of phosphorylated myosin complexes that favor cytoskeletal remodeling23. ROCK proteins additionally phosphorylate LIMK, inhibiting the active forms of CFN that stabilize actin filaments24. In parallel, GPCR-driven activation of GBD- and FGD1/3-containing proteins enhances serine/threonine kinase PAK and WASP signaling, both of which regulate cytoskeletal architecture, cell motility, actin polymerization, and actin filament turnover25,26. FGD1/3 also activates the plasma-membrane–associated small GTPase CDC4227,28, which, in its active state, interacts with multiple effector proteins to regulate cellular attachment and actin polymerization29,30. We also observed a slight upregulation of the glucocorticoid receptor NR3C1; however, predicted downstream activation was minimal. Taken together, these findings indicate that the major impact of MI on neutrophil signaling converges on GPCR-dependent pathways controlling actin polymerization, cytoskeletal reorganization, and cellular movement, which align mechanistically with the demargination phenotype and mirror the NE-induced proteomic program.

Activation of β-adrenergic receptors by NE induces cellular movement and cytoskeletal reorganization, mirroring the effects observed following MI.
The sympathetic stress response mediated through β-adrenergic receptors (β-ARs) represents a major GPCR signaling pathway in neutrophils31. To test the hypothesis that catecholamine release during MI is a primary driver of neutrophil demargination and migration, we performed an additional proteomic study in which healthy WT mice were acutely treated with NE, with or without propranolol (Figure 3G). NE administration induced an immune and cytoskeletal remodeling program that closely resembled the MI-induced proteomic state. Although we did not formally quantify protein-level overlap, both MI and NE activated immune-related pathways especially those governing cell movement and trafficking to a similar extent (MI: 24.7; NE: 22.6), highlighting a comparable downstream biological response (Figure 3H). Among these, pathways related to cellular movement, cytoplasmic organization, and actin cytoskeleton dynamics exhibited the strongest activation (Figure 3I). Notably, propranolol markedly suppressed the majority of NE-induced pathways, indicating that NE-driven demargination signals occur predominantly through β-ARs (Figure 3J).

To further define the specificity of NE-β-AR interactions, we employed pharmacological inhibitors targeting catecholamine synthesis or individual β-AR subtypes. Inhibition of tyrosine hydroxylase with AMPT (Figure 4A) significantly reduced neutrophil demargination (Figure 4B) and subsequent recruitment to the heart (Figure 4C). Broad β-AR blockade with propranolol (Figure 4D) similarly suppressed both demargination (Figure 4E) and cardiac recruitment (Figure 4F). In contrast, selective β1-AR blockade (Figure 4G) had no effect on either process (Figures 4H, I), whereas inhibition of β2-ARs with butoxamine (Figure 4J) significantly diminished both demargination (Figure 4K) and recruitment to the heart (Figure 4L). These findings indicate that NE signaling through β2-ARs is the critical trigger mobilizing neutrophils from the vascular wall to the infarcted myocardium. Consistent with this, we also found that β₂-ARs are the most abundantly expressed ARs on neutrophils (Supplementary Figure 4A). Publicly available RNA-seq datasets from purified mouse32 and human neutrophils33 similarly demonstrate that Adrb2 is the predominantly expressed AR, whereas Adrb1 and Adra1 isoforms are minimally expressed or undetectable. Only low-level expression of Adra2 isoforms is observed, and at levels far below Adrb2 (Supplementary Figures 4B, C). Together, these transcriptomic datasets support the conclusion that β₂-AR is the principal AR expressed on neutrophils.

To determine whether glucocorticoid stress contributes to MI-induced neutrophil demargination, we tested whether blocking glucocorticoid receptor signaling altered neutrophil responses to MI. Although DEX is a classical pharmacologic inducer of demargination, prior treatment with mifepristone, a potent glucocorticoid receptor antagonist (Study outline, Supplementary Figure 4D), had no effect on neutrophil demargination (Supplementary Figure 4E) or on their recruitment to the heart (Supplementary Figure 4F) following MI. These findings indicate that catecholamine stress, not glucocorticoid signaling, is the predominant driver of early MI-induced neutrophil demargination.

Hematopoietic cell specific deletion of -ARs suppresses neutrophil demargination in response to MI.
Given the ability of NE in stimulating neutrophil demargination by involving one or more -ARs, we hypothesized that myeloid cell-specific deletion of 2 or both 1and 2 (DKO) should significantly dampen demargination of neutrophils and their potential recruitment to the infarct. To test this hypothesis, we performed bone marrow transplant (BMT) studies. Briefly, we transplanted BM from WT, 2 or 12 AR knockout mice into age and sex matched WT mice and subjected them to MI (Study outline, Figure 5A). Following a 6-week BM reconstitution period, MI was induced, and a group of mice was euthanized within 6 hours to examine the markers of granulopoiesis, neutrophil demargination and recruitment to the heart. The remaining mice were monitored for cardiac dysfunction and survival over a period of 30 days. As expected, induction of MI significantly increased the number of neutrophils at 6 hours post-MI in the blood (Figure 5B) and hearts (Figure 5C) of recipient mice transplanted with BM from WT mice. However, deletion of 2 or both 1 and 2, significantly decreased the number of neutrophils in the blood and heart. Among the chimeric mice, mice lacking 2 ARs showed the most robust decline in neutrophil recruitment (63.5% vs 37.2%) to the heart suggesting that 2 ARs are vital for neutrophil demargination and recruitment during early hours post-MI. Interestingly, the number of neutrophils remained lower in the blood (Supplementary Figure 5A) but increased in spleens (Supplementary Figure 5B) of both chimeric mice at 30 days post-MI suggesting a sympathetic hypo-responsiveness to the loss of  AR on leukocytes34. However, as expected, the number of HSPCs including CMPs and GMPs in the BM did not differ either at 6 hours or 30 days post MI (Supplementary Figures 5C, D) among the different groups. These data reiterates that early neutrophil response during MI is not driven by emergency granulopoiesis in the BM.

Despite a significant decrease in both neutrophil demargination and recruitment at 6 hours post-MI in 2 and DKO mice, all chimeric mice demonstrated similar survival rates (Figure 5D). Furthermore, all cardiac functional paraments tested including left ventricular ejection fraction (Figure 5E), cardiac output (Figure 5F), End Diastolic (Figure 5G) and End Systolic Volumes (Figure 5H) were similar among all 3 groups. The hearts from all groups showed a similar degree of hypokinesis (Figure 5I) although the size of scar (Figure 5J) and fibrosis (Figure 5K) was significantly increased in both 2 and DKO mice compared to WT mice. Thus, while blockade of  ARs reduces neutrophil recruitment to the heart, the lack of functional  ARs over a prolonged period post-MI does not translate into better survival rates or cardiac functional outcomes.

Short-term, but not long-term, inhibition of β2-ARs ameliorates cardiac remodeling and improves functional parameters following MI.
Previous studies have reported that depletion of neutrophils beyond the inflammatory stage (> 3 days) during MI have a negative impact on cardiac remodeling and heart failure35. Similarly, deletion of  ARs, particularly the 2 ARs in hematopoietic cells showed increased mortality due to impaired recruitment of leukocytes over a prolonged time following MI34. To examine this notion within a framework of neutrophil demargination induced by activation of  ARs during MI, we performed an in vivo study comparing the short-term (ST) vs long-term (LT) impact of  AR blockade using propranolol. For short-term  AR inhibition, mice were administered with one dose of propranolol 8 hours prior to and another dose immediately after surgery. Long-term (LT) inhibition involved 2 doses of propranolol every day (12 hours apart) continuously for 6 days. The control group was treated with a vehicle for 6 days (Study outline, Figure 6A). All mice were observed for 30 days while assessing cardiac function by echocardiography at day 0, day 3, day 7, day 14, and day 30 (Study outline, Supplementary Figure 6A).

Contrary to the survival data in DKO mice from BMT studies, the survival rate of mice in ST group was significantly higher than those receiving LT propranolol treatment (90% vs 40%) or vehicle (90% vs.30%) (Figure 6B). This improved survival is likely due to a transient but substantial reduction in neutrophil infiltration into the heart (Supplementary Figures 6B and C), which corresponded with decreased cardiac injury, as indicated by lower levels of cardiac troponin I (Supplementary Figure 6D). The mice that survived MI in ST group demonstrated a significant improvement in cardiac functional parameters including preserved ejection fraction (Figure 6C), cardiac output (Figure 6D) and lower end diastolic (Figure 6E) and systolic volumes (Figure 6F) measured at 30 days post-MI. The vector diagrams illustrating the three-dimensional regional wall displacement also supported an overall improvement in contraction and relaxation parameters (Figure 6G). The size of scar (Figures 6H, J) as well as percentage of fibrosis (Figures 6I and K) in ST group was also much lower than LT and vehicle groups. Additional markers including α-SMA (Supplementary Figures 6E, F), ERK (Supplementary Figures 6E, G), Col1A1 (Supplementary Figure 6I), Col3A1 (Supplementary Figure 6J), and Fibronectin 1 (Supplementary Figure 6K) further support the conclusion that short-term inhibition of β1/β2-ARs significantly improves cardiac remodeling and functional recovery.

The beneficial effects of ST inhibition of  ARs with propranolol on cardiac phenotype is mainly due to the blockade of 2 ARs rather than 1 ARs. This is because, short-term inhibition of 1 AR with metoprolol (Study outline, Supplementary Figure 7A) did not improve any of the cardiac functional parameters including hypokinesia (Supplementary Figure 7B), ejection fraction (Supplementary Figure 7C), cardiac output (Supplementary Figure 7D), end diastolic (Supplementary Figure 7E) or systolic volumes (Supplementary Figure 7F) while 2 AR blockade with butoxamine significantly improved both the cardiac functional parameters (Supplementary Figures 7B-F), scar size (Supplementary Figures 7G, I) and fibrosis (Supplementary Figures 7H, J). Together, these data suggest that blockade of NE binding to 2 ARs on neutrophils during the peak inflammatory stage (< 1 day) suppresses neutrophil mobilization and recruitment to the heart thus, facilitating a favorable cardiac remodeling process and better functional outcomes.

Short-term inhibition of β1/β2-ARs using propranolol also decreases neutrophil infiltration and enhances cardiac function following ischemia-reperfusion (IR) injury.
While the permanent ligation model is widely used to study ischemic injury, it does not necessarily reflect the clinical scenario of AMI, which typically involves timely reperfusion. However, since many patients still face delays in revascularization and because both permanent ligation and reperfusion injury trigger neutrophil recruitment, we aimed to determine whether ST inhibition of β1/β2 ARs in a reperfusion injury (IR) model would yield outcomes similar to those observed in the permanent ligation model. In brief, ischemia was induced by ligating the LAD artery for 50 minutes, followed by reperfusion (experimental design, Supplementary Figure 8A). One group of mice was euthanized at 6 hours post-IR to evaluate the effect of short-term propranolol (ST) treatment on cardiac neutrophil infiltration, while the remaining mice were monitored for an additional 30 days to assess long-term cardiac outcomes. As anticipated, IR injury led to a marked increase in the number of neutrophils in the heart as assessed by flow cytometry (Supplementary Figures 8B, C) and IHC (Supplementary Figures 8D, E). This was associated with elevated levels of cardiac troponin I in the plasma (Supplementary Figures 8F, G), an indicator of cardiac tissue damage. Short-term propranolol treatment significantly reduced neutrophil infiltration into the heart as well as cardiac damage as shown by reduced cTnI levels. This resulted in a significant improvement in cardiac function including all the saliant features of hypokinesia (Supplementary Figure 8H), ejection fraction (Supplementary Figure 8I), cardiac output (Supplementary Figure 8J), end systolic and diastolic volumes (Supplementary Figures 8K, L). While the hearts of vehicle treated group showed increased scar size (Supplementary Figure 8M, N) and fibrosis (Supplementary Figures 8M, O), short term blockade of 1/2 ARs with propranolol not only prevented this but also preserved the cardiac function. These findings suggest that both ischemic and reperfusion injuries promote neutrophil mobilization to the injured heart and blocking 1/2 ARs transiently with propranolol may be effective in preventing adverse cardiac remodeling and preserving cardiac function.

Methods
Mice and Treatments: Male mice (10-12 weeks old) on a C57BL/6 background were used, including wild-type (WT), β2-adrenergic receptor–deficient (β2AR−/−), and β1/β2-adrenergic receptor double-knockout (DKO) mice. WT mice were obtained from The Jackson Laboratory. β2AR−/− and β1/β2AR−/− mice were provided by Dr. Doug Tilley (Temple University). Mice were housed under specific pathogen–free conditions at The Ohio State University (OSU) and the University of Oklahoma Health Sciences Center (OUHSC). All animal experiments were approved by the Institutional Animal Care and Use Committees (IACUC) at each institution (Ohio State University IACUC, protocol #2019A0056); University of Oklahoma Health Sciences Center IACUC, protocol #23-067-SFCHX) and were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Animals were monitored daily by trained personnel for general health, signs of distress, and post-procedural recovery. Humane endpoints were applied in accordance with institutional guidelines. Mice were euthanized using carbon dioxide (CO₂) inhalation followed by cervical dislocation to ensure death, in accordance with institutional guidelines. Mice were maintained on standard chow and randomly assigned to experimental groups. All experiments were performed in male mice.

Drugs and Administration: Drug solutions were prepared under sterile conditions and administered according to IACUC-approved protocols. The following agents were used: propranolol (30 mg/kg, i.p.), butoxamine (12.5 mg/kg, i.p.), metoprolol (12.5 mg/kg, i.p.), α-methyl-DL-tyrosine (AMPT; 40 mg/kg, s.c.), dexamethasone (5 mg/kg, i.v.), granulocyte colony-stimulating factor (G-CSF; 100 mg/kg, i.p.), norepinephrine (2 mg/kg, i.p.), mifepristone (30 mg/kg, s.c.), and 5-ethynyl-2′-deoxyuridine (EdU; 16 mg/kg, i.p.). Key reagents and resources are listed in Supplementary Table 1.

Method Details
Mouse Model of Myocardial Infarction: Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending artery. Twenty-four to 48 hours before surgery, the fur on ventral surface in and around left chest area was cleared off using depilatory cream. Briefly, the mice were anesthetized with 2% isoflurane anaesthesia using a continuous isoflurane delivery system and with an endotracheal tube respiration was controlled with use of a rodent ventilator. A small longitudinal incision (≤5 mm) was made in the center of the throat to locate the trachea for intubation. After confirming the intubation, 4-0 vicryl sutures were used to close the skin in the throat. The line between left pectoralis and major muscles was identified and then an oblique incision (~7mm) was made to expose the heart and left coronary artery. An 8-0 suture (silk or nylon) was placed around the coronary artery and ligated to create the ischemia. Animals were monitored for the depth of anaesthesia at least every 10 to 15 minutes throughout the surgery. After controlling the exhaust pressure to restore the lungs to normal, 5-0 vicryl sutures were used to close the muscle layer while 4-0 vicryl sutures were then to close the skin on the chest. The animals were gradually weaned off the ventilator during recovery from anaesthesia and monitored further to confirm that they have resumed spontaneous respiration in a normal rhythm without assistance during the postoperative period. The sham group underwent the same surgical procedure, except that the ligature was passed under the LAD but not tied. Immediately after surgery, all mice were injected with a long-acting analgesic, buprenorphine. No-MI mice did not undergo either LAD or sham surgery.

Mouse Model of Myocardial Ischemia-Reperfusion Injury (IRI): Myocardial ischemia-reperfusion injury was induced in adult male C57BL/6J mice (10–12 weeks old) as previously described, with minor modifications. Briefly, mice were anesthetized with isoflurane (1–2% in oxygen) and placed on a heating pad to maintain body temperature. A left thoracotomy was performed to expose the heart, and a 7-0 silk suture was placed around the left anterior descending (LAD) coronary artery approximately 1–2 mm below the tip of the left atrium. To induce ischemia, the suture was tightened and secured with a small piece of PE-10 tubing to occlude the LAD for 50 minutes. Ischemia was confirmed by pallor of the left ventricle and ECG changes. After 50 minutes, the ligature was released to allow reperfusion, verified by return of myocardial color. Mice were monitored during the reperfusion period and euthanized at indicated time points for tissue and plasma collection. The sham group underwent the same surgical procedure, except that the ligature was passed under the LAD but not tied. Immediately after surgery, all mice were injected with a long-acting analgesic, buprenorphine. No-MI mice did not undergo either ligation or sham surgery.

Transthoracic Echocardiography: Echocardiography was performed using the Vevo 3100 high-resolution ultrasound imaging system with a MX400 transducer (Visual Sonics Inc., Toronto CA). Mice were sedated with 2% isoflurane and placed on temperature-maintained platform in a supine position. Hair was removed from the chest using depilatory cream, warmed ultrasound transmission gel placed on the chest and left parasternal long axis view and basal short axis views acquired. Ejection fraction, stroke volume and cardiac output were evaluated on the left parasternal long axis view while fractional shortening was calculated from the short axis view using the Vevo 3100 software. Core body temperature was maintained at 37°C using a heated platform and infrared heat lamp throughout the procedure. Mice with heart rates below 400 bpm (beats per minute) were excluded from the analysis.

Bone Marrow Transplants (BMT): WT mice were lethally irradiated (with 13 Gy from a Cesium gamma source in 2 doses separated by 4 hours) and transplanted with BM (5x106 cells) from donor mice. Chimerism was confirmed using either flow cytometry or qRT-PCR analysis of respective proteins/ genes (mRNA) in peripheral blood cells at ~6 weeks after transplantation. Once the chimerism was confirmed, MI was induced as described above.

White Blood Cell (WBC) Counts: Total WBC in freshly collected mouse blood was measured using hematology analyzer (Element HT-5, Heska, Inc.,) or a K2 Fluorescent Viability Cell Counter (Nexcelom Biosciences, MA).

Flow Cytometry Analysis.
Blood and Bone Marrow Leukocytes: Briefly, fresh blood from mice was collected via tail bleeding directly into microcentrifuge tubes containing EDTA (5 mM) using heparin coated capillary tubes and immediately placed on ice. A small amount of blood was set aside for separation of plasma and for analysis of complete blood cell count (CBC) while the remaining blood was transferred to 15 ml centrifuge tubes and red blood cells were lysed using BD Pharm Lyse (BD Biosciences, San Jose, CA), washed, rinsed and resuspended in PBS or FACS buffer (Hanks balanced salt solution, HBSS + 0.1% BSA w/v, 5mM EDTA) for staining.

For isolation of BM, femurs and tibias from each mouse were isolated, cleaned and their ends were cut open with a sharp scissor to expose lumen. Bone marrow was flushed with ice cold PBS using a 23-gauge, 10 ml syringe directly through a 40 mM nylon mesh strainer on a 50 ml centrifuge tube. The BM samples were next spun at 300g for 10 min at 4ºC. Following aspiration of supernatant, red blood cells were lysed using RBC lysis buffer, washed twice with PBS, and immediately stained with a fixable Live/Dead marker (1 ml per million cells in 1 ml for 30 minutes, Molecular Probes, Eugene OR). The cells were then washed and resuspended in 100 ml of FACS buffer containing a cocktail of antibodies directed against different types of leukocytes. In experiments that required intracellular staining, the surface labelled cells were fixed and permeabilized with fix/ perm buffer (30 minutes) and then stained with intracellular antibodies (e.g., S100A9, IL1b) in perm/ wash buffer. The stained cells were analyzed on a LSRII flow cytometer using FACS DiVa software or Cytek Northern Lights flow cytometer (Cytek). Once the debris, doublets (by FSC-H vs. FSC-A) and dead cells, leukocytes were identified as described previously12,13. Representative flow cytometry gating strategies used for the identification of leukocyte populations in blood and BM are shown in Supplementary Figure S9.

Heart Leukocytes: For flow cytometric analysis of leukocytes in the heart, a single cell suspension was first prepared. Briefly, hearts were perfused with ice cold PBS to remove blood cells, harvested, cleared of adhering tissues and atria, minced into 2-3 large pieces, transferred to GentleMACS C tubes (Miltenyi Biotec) containing either collagenase type II (1mg/ml) alone or a cocktail of collagenase I (450U/ml), collagenase XI (125U/ml), hyaluronidase type I-s (60U/ml) and DNase (60U/ml) and processed into a fine suspension using a tissue dissociator (GentleMACS Octo Dissociator, Miltenyi Biotec). Digestion was enabled by alternating between dissociation and incubation in a thermomixer (37ºC, 15 minutes each time) with gentle agitation. The single cell suspension was then filtered through a 100 μm strainer into a 50 ml tube, rinsed with FACS buffer, centrifuged at 300g for 10 min at 4ºC. The resultant pellet was washed, resuspended in 1 ml FACS buffer to determine cell count (K2 Cell Counter, Nexcelom Biosciences) followed by staining for Live/Dead marker, surface and intracellular proteins as described above. Data were acquired on Cytek Northern lights or LSR Fortessa flow cytometer (BD Biosciences, San Jose CA) using FACS Diva software, and analysis was performed with FlowJo software (Ashland, OR). Once the doublets (by FSC-H vs. FSC-A) and dead cells (Live vs. Dead) were excluded, neutrophils were identified as CD45+, Ly6Ghi cells and monocytes as CD45+, Ly6G-, F4/80-, CD11b+, CD115+ and further classified as Ly6-Chi and Ly6-Clo cells. Representative flow cytometry gating strategies used for the identification of leukocyte populations in the heart and spleen are shown in Supplementary Figure S10.

Hematopoietic Stem Cells in the BM: Hematopoietic stem and progenitor cells from the BM were prepared as described above. After cell count and staining for Live/Dead markers, the cells were resuspended in a cocktail of antibodies first against lineage-committed cells (B220, CD19, CD11b, CD3e, TER-119, CD2, CD8, CD4, Ly6-C/G) followed by markers of stem cells, proliferation (e.g., Ki67) and EdU. Hematopoietic stem and progenitor cells were first identified as Lin-, Sca1+ and ckit+ (LSK) while the hematopoietic progenitor subsets were separated by using antibodies to CD16/CD32 (FcgRII/III) and CD34. Common Myeloid Progenitors (CMP) were identified as Lin-, Sca1-, ckit+, CD34int, FcgRII/IIIint, Granulocyte Macrophage Progenitors (GMP) as Lin-, Sca1-, ckit+, CD34int, FcgRII/IIIhi. Common lymphoid progenitors (CLP) were identified as Lin-, Sca1lo, ckitlo and IL7R+ cells. Hematopoietic stem cells (HSCs) were identified as Lin-, Sca1+, ckit+, CD48- and CD150+. Cellular proliferation in hematopoietic stem and progenitor cells was assessed by measuring the incorporation of EdU and /or Ki67 according to the manufacturer’s protocol (Click-iT™ Plus EdU, Molecular Probes, Eugene OR). Flow cytometry was performed using an LSR Fortessa or Cytek Northern Lights (Cytek) using FACS DiVa or SpectroFlo software. All flow cytometry data were analyzed using FlowJo software. Representative flow cytometry gating strategies used for the identification of HSCs and HSPCs in the BM are shown in Supplementary Figure S11.

Confocal Microscopy: Neutrophils from whole blood were sorted using Neutrophil Isolation kit (130-097-658 Miltenyi Biotec), neutrophils were then permeabilized with BD Cytoperm/Cytofix (BD554722), Fc receptors blocked with Fc Block (TruStain FcX PLUS anti-mouse CD16/CD32, 156603, Biolegend) and stained with Phalloidin, Fluorescein isothiocyanate labelled (P5282, Millipore Sigma) for 60mn and mounted using ProLong Gold antifade reagent with DAPI (P36935, Invitrogen). Neutrophil purification efficiency was assessed by flow cytometry (CD45, CD11b, Ly6G). Confocal images were obtained using Olympus FV3000 Multi confocal.

Histology of Heart Sections: The hearts were harvested in diastole with saturated KCl and CdCl (100 mM) injected through the apex into the left ventricular (LV) cavity. The LV apex was then cannulated, and the heart perfused with PBS, followed by 10% buffered formalin at 75 mmHg, with the severed inferior vena cava serving as the outlet. The hearts were then cut into 2-mm cross-sectional slices and processed for paraffin embedding. The slices were cut into 4 μM sections for histologic examination and picrosirius staining. Images were acquired digitally, and the areas measured using NIH Image J software.

Assessment of scar size and fibrosis
Quantification of Infarct Size: Scar size was evaluated using Masson’s trichrome staining. Deparaffinized and rehydrated sections were incubated in Bouin’s solution, followed by sequential staining with Weigert’s iron hematoxylin (nuclei), Biebrich scarlet–acid fuchsin (cytoplasm and muscle), and aniline blue (collagen). Infarcted myocardium was identified by collagen-rich blue/whitish staining with loss of red-stained viable myocardium. Whole-section images were acquired at low magnification. Infarct area was quantified using ImageJ (NIH) by an investigator blinded to genotype and treatment. The entire myocardial cross-section was manually outlined to obtain the whole-heart (WH) area, and the infarct region was traced based on the presence of collagen-rich tissue. Infarct size was calculated using the equation, Infarct Size (%) = (Infarct area/ Whole heart area) X 100.

Fibrosis Assessment: Interstitial and replacement fibrosis were assessed using Picrosirius Red staining. Rehydrated paraffin sections were stained with 0.1% Sirius Red in saturated picric acid and rinsed in acidified water. Sections were imaged under both brightfield and polarized light, the latter allowing visualization of birefringent collagen fibers. Fibrosis was quantified using ImageJ by applying color thresholding to identify Sirius Red–positive collagen. Fibrotic area was expressed as the percentage of total tissue area. At least 2–3 sections per heart and multiple fields per region were analyzed, with all quantification performed blinded to experimental group.

Proteomics
Cell Lysis: Cell pellets were washed with PBS three times before resuspend in 100 µL of 5% SDS buffer in 50 mM TEAB (Triethylammonium bicarbonate) solution. Samples then were vortexed briefly before proceeding to sonication by using the Bioruptor® Pico (Diagenode, Denville, NJ) following manufacturer’s suggested protocol. Briefly, sonication temperature was set at 4C; sonication cycle was set at 30 sec on and 30 sec off. A total of 10 cycles were done for the cell pellets. (Sometimes, DNAs are not sheared well in SDS buffer so a probe homogenizer can be used to shear DNAs in that case). After sonication, samples were centrifuged at 14,000 rpm for 15 min at 4°C to remove any remaining insoluble material. Concentration of the proteins were measured using Qubit fluorometer (ThermoFisher Scientific).

Protein Digestion via Strap: 50 µg of the samples were taken for trypsin digestion. 5 µL of 50 mM ABC containing 5 µg/µL DTT was added, and the sample was incubated at 650C for 15 min followed by addition of 5 µL of 50 mM ABC containing 15 µg/µL iodoacetamide (incubated at RT for 15 min in the dark). Samples were then acidified by adding 12% phosphoric acid (1:10 v/v acid to sample). For every 25 µL of samples, 165 µL of TEAB (Triethylammonium bicarbonate, 1M)/MeOH (10:90 v/v) was added and then loaded to S-trap for further washes. Samples were centrifuged at 4000g for 3 min (4C) to remove supernatant. 150 µL of TEAB (Triethylamonium bicarbonate, 1M)/MeOH (10:90 v/v) was added to the trap as wash solution and the trap was washed 3-6 times depends on the initial loading volume. After the final wash, sequencing grade trypsin dissolved in 50 mM TEAB was added and digested O/N at 370C. The following day, peptides were eluted from the trap by adding 40 µL of 50mM TEAB, 0.1% FA and 0.1% FA in Acetonitrile (50:50), sequentially. The sample was pooled together and dried in a vacufuge and resuspended in 20 µL of 50 mM acetic acid. Peptide concentration was determined by nanodrop (A280 nM).

Orbitrap Fusion: Capillary-liquid chromatography-nanospray tandem mass spectrometry (Capillary-LC/MS/MS) of protein identification was performed on a Thermo-Scientific orbitrap Fusion mass spectrometer equipped with an EASY-Spray™ Sources operated in positive ion mode. Samples (6.4µL) were separated on a easy spray nano column (PepmapTM RSLC, C18 3µ 100A, 75µm X150mm Thermo Scientific) using a 2D RSLC HPLC system from Thermo Scientific. Each sample was injected into the µ-Precolumn Cartridge (Thermo Scientific) and desalted with 0.1% Formic Acid in water for 5 minutes. The injector port was then switched to inject, and the peptides were eluted off the trap onto the column. Mobile phase A was 0.1% Formic Acid in water and acetonitrile (with 0.1% formic acid) was used as mobile phase B. Flow rate was set at 300 nL/min. Mobile phase B was increased from 2% to 20% in 105 min and then increased from 20-32% in 10 min and again from 32-95% in 1 min and then kept at 95% for another 4 min before being brought back quickly to 2% in 1 min. The column was equilibrated at 2% of mobile phase B (or 98% A) for 15 min before the next sample injection.

MS/MS data was acquired with a spray voltage of 1.95 KV and a capillary temperature of 305 °C is used. The scan sequence of the mass spectrometer was based on the preview mode data dependent TopSpeed™ method: the analysis was programmed for a full scan recorded between m/z 375-1500 and a MS/MS scan to generate product ion spectra to determine amino acid sequence in consecutive scans starting from the most abundant peaks in the spectrum in the next 3 seconds. To achieve high mass accuracy MS determination, the full scan was performed at FT mode, and the resolution was set at 120,000 with internal mass calibration. Three compensation voltage (cv=-50, -65 and -80v) were used for samples acquisition. The AGC Target ion number for FT full scan was set at 4 x 105 ions, maximum ion injection time was set at 50 ms and micro scan number was set at 1. MSn was performed using HCD in ion trap mode to ensure the highest signal intensity of MSn spectra. The HCD collision energy was set at 32%. The AGC Target ion number for ion trap MSn scan was set at 3.0E4 ions, maximum ion injection time was set at 35 ms and micro scan number was set at 1. Dynamic exclusion is enabled with a repeat count of 1 within 60s and a low mass width and high mass width of 10ppm.

The raw files were searched using Mascot Daemon by Matrix Science version 2.5.1 (Matrix Science, Boston, MA) via ProteomeDiscoverer (Thermo) against the database provided by the user. The mass accuracy of the precursor ion was set to 10 ppm, accidental pick of 113C peaks was also included into the search. The fragment mass tolerance was set to 0.5 Da. Carbamidomethylation (Cys) is used as a fixed modification and considered variable modifications were oxidation (Met) and deamidation (N and Q). Four missed cleavages for the enzyme were permitted. A decoy database was also searched to determine the false discovery rate (FDR) and peptides were filtered according at 1% FDR. Proteins identified with at least two unique peptides were considered as reliable identification. Any modified peptides are manually checked for validation.

Quantitation: Label free quantitation was performed using the spectral count approach, in which the relative protein quantitation is measured by comparing the number of MS/MS spectra identified from the same protein in each of the multiple LC/MSMS datasets. Scaffold (Proteome Software, Portland, OR) was used for data analysis. Student-t test was performed by scaffold to evaluate if the folder change for certain proteins is significant (p<0.05).

Protein inference and bioinformatic analysis: The resulted MS raw data were processed by Proteome Discoverer V2.4 (Thermo Fisher Scientific), using the UniProtKB/Swiss-Prot Mus musculus reference protein database for protein identification. Search parameters included: enzyme: trypsin; maximum miss cleavages: 2; fixed modification: cysteine carbamidomethylation; variable modifications: oxidation of methionine and deamidation of asparagine and glutamine. The criteria for protein identification also contained the detection of at least 2 unique peptide per protein and the protein false discovery rate (FDR) target to be below 0.05. Label-free relative protein quantitation on the precursor level was performed using the same software platform, which aligns chromatograms, extracts ion peaks and integrates them to compare peptide/protein spectral abundance along the different datasets. We enabled 90% of replicate features and the ANOVA hypothesis test to determine the statistical significance associated with the protein ratio calculation. Normalization was done using the total peptide amount on controls average.

The differentially abundant proteins (DAPs) were identified with an abundance ratio ≥ 1 and an adjusted P-value ≤ 0.05, based on the Benjamini-Hochberg algorithm, was set as the cut-off criterion.

Measurement of Cardiac Troponin I (cTnI) Levels
Plasma samples were collected from mice at designated time points following myocardial infarction (MI) or IR, with or without propranolol treatment. Cardiac troponin I levels were quantified using the Mouse Cardiac Troponin I (TNNI3) ELISA Kit (Thermo Fisher Scientific, Cat# EEL112), following the manufacturer’s protocol. Concentrations were determined using a standard curve generated from known calibrators provided with the kit.

Measurement of soluble CD62L Levels
The levels of soluble L-selectin (sCD62L) were measured in the plasma of the indicated groups at various time points using a commercially available ELISA kit (The RayBio Mouse L-Selectin, Cat# ELM-L-Selectin). Plasma samples were diluted 200-fold before incubation. The plasma levels of the unknown samples were determined based on the standard curve generated using known mouse L-selectin protein standards.

Western Blot Analysis in heart tissue
Mouse left ventricular area was dissected and used for the protein extraction using RIPA lysis buffer supplemented with complete protease and phosphatase inhibitor cocktail. The lysate was mixed with NuPAGE lds sample buffer and heat denatured at 75Soc for 10-15 min. The proteins were separated using 10 or 12% NuPAGE Bis-Tris gels and transferred onto a PVDF membrane. Further, the membrane was blocked with 3% BSA for 30-60 min at room temperature. The membrane was probed for the indicated targets ( -SMA (ThermoFisher, #14-9760-82, 1:1000), p-ERK (CST, #4370S, 1:1000), T-ERK (CSIT, #4695S, 1:1000 and GAPDH (CST, #5174S, 1:1000) and developed using SuperSignal™ West Femto Maximum Sensitivity Substrate and captured/visualized using LI-COR odyssey XF system.

Gene expression analysis
Quantitative real-time polymerase chain reaction (qRT-PCR) experiments in LV of hearts were performed using TaqMan assays for target genes (Col1a1 assay ID: Mm00801666_g1; Col3a1 assay ID: Mm00802296_g1; FN1 assay ID: Mm01256744_m1) using TaqMan Fast Advanced Master Mix (ThermoFisher). Changes in the expression levels of target genes were determined using Comparative CT (ΔΔCT) analysis, with 18S (ThermoFisher #4319413E) acting as an endogenous control.

Statistical Analysis: Normality was assessed using the Shapiro–Wilk test. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test for normally distributed data or a Mann–Whitney test for non-normally distributed data. Comparisons among three or more groups were analyzed using one- or two-way analysis of variance (ANOVA) followed by Holm–Šidák or Tukey’s multiple comparisons tests, as appropriate. Non-parametric multi-group comparisons were performed using the Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Survival analyses were conducted using the log-rank (Mantel–Cox) test. Longitudinal data (e.g., echocardiography measurements) were analyzed using two-way ANOVA with a mixed-effects model to account for missing values due to post-MI mortality, followed by Bonferroni’s multiple comparisons test. All statistical tests were two-sided unless otherwise specified. A two-sided P value ≤ 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism (version 8). Extended methodological details are provided in the Supplementary Information.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Dahdah et al. presents a comprehensive study on the early recruitment of neutrophils to the ischemic heart following myocardial infarction (MI), focusing on the role of vascular demargination rather than granulopoiesis in the bone marrow (BM) or spleen. Neutrophil demargination is a process by which neutrophils are mobilized from the vascular wall into the bloodstream. The authors employ a variety of advanced methodologies, including sophisticated in vivo experiments, flow cytometry, confocal microscopy, and proteomics to elucidate the signalling pathways that regulate neutrophil demargination and subsequent recruitment to the infarct. Specifically, they explored the role of β-adrenergic receptor (β-AR) signalling in mediating this process.\n\nThe research is novel, and the findings are of significant interest, as they provide insights into the temporal dynamics of neutrophil recruitment and highlight the potential of targeted pharmacological intervention in early MI management.\nHowever, I would recommend a revision to further strengthen the mechanistic understanding of neutrophil demargination and the potential for therapeutic intervention.\n\nThe use of dexamethasone (DEX) and norepinephrine (NE) as demarginating agents in mice needs careful consideration. While these agents can induce demargination, they may also have additional systemic effects, including influencing the behavior of other immune cells, eg:\nMacrophages: Dexamethasone can inhibit the activation and pro-inflammatory cytokine production of macrophages, promoting a more anti-inflammatory phenotype.\nT cells: Dexamethasone can suppress T cell proliferation and cytokine production, leading to reduced inflammation.\nDendritic cells: Dexamethasone can impair dendritic cell maturation and antigen presentation, thereby reducing T cell activation.\nFurthermore, TBI has a big impact on the other immune cell populations and it can not be ruled out, that the observed effects are due to altered interactions between neutrophils and macrophages, DCs and T cells.\nTherefore, when using TBI , BM chimeric approaches and dexamethasone as a tool to study neutrophil demargination, it is crucial to consider its potential effects on other immune cell populations.\nTo isolate the specific effects on neutrophils, comparing the effects of dexamethasone on neutrophil demargination in TBI wild-type mice to those after ablation of specific immune cell types (using e.g., macrophage-depleted mice or CD11c-cell depleted mice) could help to elucidate the direct and indirect effects of dexamethasone.\nTo control for these effects, it is also recommended to measure systemic inflammatory markers (e.g., cytokines) after DEX and NE administration.\nPlease also consider to discuss whether the observed demargination is a localized response (specific to ischemic tissue) or part of a systemic phenomenon. To gain a more comprehensive understanding of the mechanisms underlying neutrophil demargination in MI, it would be beneficial to explore the interplay between β-AR signalling and other relevant pathways, such as those mediated by chemokines and adhesion molecules. Additionally, considering the long-term consequences of excessive neutrophil recruitment, such as impaired tissue repair and adverse cardiac remodelling, would provide valuable insights into the clinical implications of the authors findings.\nTo directly measure β2-AR activation in neutrophils, phospho-specific antibodies (to measure phosphorylated proteins downstream of the receptor, e.g., p-ERK1/2, p-Akt, p-p38 MAPK) should be employed in isolated neutrophils stimulated with a β2-adrenergic agonist like isoproterenol or inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, and CXCL8) to mimic the inflammatory response. This could provide further evidence for the role of β2-ARs in mediating neutrophil demargination.\nTo complement the proteomics analysis of demarginated neutrophils post- MI or NE administration, which revealed the activation of processes involved in cellular movement and cytoskeletal reorganization, visualizing changes in cellular morphology and cytoskeletal reorganization would provide additional insights. Immunofluorescence microscopy should be used to show changes in cell shape, formation of pseudopodia, and cytoskeletal dynamics in response to β-AR activation.\n\nMinor:\nFigure 5 j,k and Figure 6 h,i: Please check your scale bars! From my point of view, magnifications are not the same, so the scale bars cannot be the same size!", "reviewer_2": "In this paper the authors propose that neutrophil demargination induced by NE via beta-2 adrenergic receptors, rather than neutrophil production and mobilization from the BM or spleen, is responsible for the first wave of neutrophil infiltration in the heart after myocardial infarction in mice. Figure 1 examines kinetics of granulopoiesis and granulocyte dynamics in various compartments i.e. BM blood spleen heart, showing that peak heart infiltration occurs before peak granulopoiesis suggesting non-BM/spleen source of neutrophils for early recruitment. Figure 2 uses a model of total body irradiation to disrupt granulopoiesis, and treat mice with demargination inducing agents (DEX, NE), providing data supporting the notion that demarginating neutrophils are recruited to the heart early after MI. In Figure 3 the authors performed proteomics analysis of blood neutrophil 6 hours after MI or NE and NE+propranolol treatment. Figure 4/5: the authors use various treatments to prevent NE synthesis of block different beta adrenergic receptors (Fig 4) or use mice with myeloid specific deficiency in beta2 or beta1+beta2 AR (Fig 5), proposing a role of beta2-AR in neutrophil demargination and recruitment to the heart after MI. Figure 6 shows that short term, but not long term, propranolol treatment improves cardiac repair after permanent MI in mice.\n\nThe paper is clearly written, the proposed conclusions are an important and a new contribution to the field. There are however several technical and conceptual issues, including insufficiently powered experiments and unexpected results in the LAD permanent ligation model. I would furthermore suggest toning down the conclusions as most evidence is indirect, and discuss the weaknesses and limitation of the different experimental approaches in more details.\n\n-One weakness of the paper is that it is technically not possible to clearly distinguish neutrophil origin and most of the provided evidence is indirect. Thus, most of the conclusion are overstated e.g. line 155 “Since we found no role for either BM or spleen”. While experiments such as those presented in Figure 1 are informative, they only provide indirect evidence as the different compartments observed are not merely ‘communicating vases’, and the number of neutrophils at a given time point in the BM represents a complex balance between production, retention, mobilization and reverse-migration. The EdU experiments show neutrophils originating from HSPCs that have proliferated during the labeling period, but mature neutrophils originating from BM or spleen reservoirs and mobilized early after MI cannot be distinguished from demarginating neutrophils. However, the cumulative amount of evidence provided by the authors indeed suggests that demargination is a substantial source of early neutrophil recruitment to the heart.\n-Figure 1A-B: please show pre-gating strategy. There is possibly an error in percentages showed as they are exactly the same between heart and blood\n-The authors conclude that neutrophil numbers are not affected in the BM at 6 hours but the data shown in Figure 1E is not very robust, the experiment being insufficiently powered with clear outlier observations.\n-Line 182: “wherein the blood neutrophil count is slightly decreased”. According to Figure S1D the neutrophil count might be close to 0 at 18h, but there is actually no measurement performed at this time point\n-Neutrophil counts in the bone marrow and spleen should be provided for all interventions.\n-Many experiments are quite underpowered with sometimes as few as n=3 for functional readouts in a model known for its quite substantial variability. Interexperiment variability also seems quite high with e.g. a very wide range of reported blood neutrophil counts in the control groups across experiments.\n-Figure 6B shows a 90% death rate in the control MI group, this is rather unusual in this model. The effect of short-term propranolol treatment with almost no scar formation after LAD ligation (Figure 6H-K) also does not seem very realistic, this would represent a spectacular cardioprotective effect in this permanent ischemia model. Whether and how blinding and randomization were performed is not indicated in the methods.\n-The methods do not mention the use of analgesics prior to or after MI surgery.\n-Previous work showing splenic retention of immune cells in mice with myeloid beta2-AR deficiency early (24 hours) after MI suggests that some of the effects observed here might also be caused by splenic or medullar retention of neutrophils.\n-Assessment of neutrophil purity after magnetic sorting for proteomics studies is missing\n-Full list of differentially abundant proteins to provide as supplementary table, here only unannotated heatmap + pathway analyses are shown. It would be a good addition to provide some cross-validation of proteomics and flow cytometry data e.g. do the authors see downregulation of CD62L in the proteomics data, and can some up/down proteins identified by proteomics be validated by flow?\n-Lines 307-308: ‘Administration of NE resulted in the activation of an immune response that was very similar to that induced by MI’ : how was this formally quantified? Overlap of up/down proteins induced by MI or NE?", "reviewer_3": "In this report Dahdah and colleagues set out to investigate the mechanisms whereby neutrophils are supplied to the heart in the early hours after myocardial infarction as part of the complex innate immune response to ischemic injury. Because myocardial infiltration of neutrophils is thought to occur much quicker than granulopoiesis in the bone marrow, the authors hypothesized that the early wave of neutrophils recruited to the heart is exclusively sourced from the vasculature (the marginating pool) and not from increased production in the bone marrow or release from the spleen. Using a combination of flow cytometry, BM ablation of hematopoietic stem cells, confocal microscopy and multiple proteomics analysis, they propose that the MI-evoked neutrophilia is mediated by a demargination process. Finally, using genetic and pharmacological strategies (they report that suppression of norepinephrine synthesis or disruption of β-adrenergic signaling reduced both neutrophil demargination and recruitment to the heart, while only short-term inhibition of β-ARsignaling improved cardiac remodeling and function in mice subjected to permanent LAD ligation.\n\nGeneral comments\nOverall, the central research question in this study evolves on the innate immune response after ischemic myocardial damage and the specific role of one of the early responders in this process, the neutrophils. The inflammatory response after MI is required for the induction of cardiac repair processes to which many cell types contribute and has received ample attention over the past 2 decades in both preclinical and a myriad of clinical studies. Disappointingly, all interventional studies targeting the immune response in the acute setting of MI have failed highlighting the need for a better mechanistic understanding of key molecular and cellular pathways driving either myocardial damage or repair. The authors state that deciphering the signaling pathways that stimulate the demargination of neutrophils and their recruitment to the ischemic heart may provide a viable approach to regulate neutrophil trafficking to the heart post-MI and conclude that their “findings support a pharmacological strategy to contain the initial onslaught of neutrophils on the ischemic heart using β2-AR blockers to regulate the otherwise runaway inflammatory response”.\n\nWhile in general the topic addresses a timely problem and while the demargination hypothesis of neutrophil trafficking post MI is supported by experimental data, the study suffers from many methodological limitations and flaws. At face value the additional knowledge brought to the field by the data as presented is at best incremental as the proper experimental design to identify the role of beta2-AR blockers in the response to acute myocardial infarction and their impact on neutrophil trafficking to the heart remains elusive.\n\nSpecific comments.\n1. A major limitation of the study is the use of the permanent ligation of the LAD to induce myocardial infarction in mice, a species devoid of collateral coronary circulation (which in contrast is abundantly present in the canine model). Full LAD ligation will prevent influx of inflammatory cells (neutrophils and monocytes) in the infarcted area in the first few hours after ligation, despite the statements to the contrary by the authors. They do not provide any histological data at 6h post MI that would corroborate the presumed influx of neutrophils in the infarct territory. The FACS data, listed in figure 1 to underpin their claim, do not provide the required scientific evidence to substantiate this critical statement, as the data are derived from whole LV homogenates. The authors need to provide histological sections prepared at 6h post MI and stain for the presence of infiltrating neutrophils. They should next (semi)quantitate the degree of trafficking in their subsequent studies with different pharmacological and genetic interventions.\n2. The cited references #32, 44, and 50 are not well poised to back up the statement that “ischemic damage caused by both permanent ligation and reperfusion injury attracts neutrophils to the infarct”. None of these studies provides histological data on neutrophil cell infiltration in the myocardium. Moreover, the 37-year-old study by Chatelain et al (ref 50) was performed using a canine model of both ligation and temporary (3h) occlusion and showed significantly less radiolabeled neutrophils in the infarct region following full ligation (even in a species with abundant coronary collaterals). Therefore, the limitation reported by the authors that more temporal studies focusing on earlier time points than 6hrs are required, is not convincing. To the contrary, additional studies using an ischemia reperfusion model are indispensable, as well as a larger number of animals per group to increase the power of the study (at least 10 animals per group rather than 4-6 as is).\n3. The histological data presented in figure 5 are very limited and do not allow proper evaluation of the extent of fibrotic remodeling of the infarcted LV, especially at 30 days post ligation. One would expect to see full transmural infarction over a large part of the LV free wall in WT (as suggested by the echo image Fig 5, panel I and as explicitly highlighted in reference 44).\n4. Also, mortality rates are extremely high with only 1 or 2 mice surviving at the 30 day follow up time point. Such high fatality rates do not instill confidence in the experimental approach when studying novel strategies to regulate what the authors call “runaway inflammatory responses”.\n5. The finding that DEX and NE contribute to neutrophil demargination via CD62L shedding and cell softening is not novel and the finding that MI is capable of inducing a similar phenomenon is of interest and points toward potential pharmacological interventions to interfere with this early demargination. Unfortunately, the methodological limitations and lack of outcome data using beta2-AR blockers do not allow to address our knowledge gaps.\n6. The authors report that bone marrow transfer experiments with myeloid cell-specific ablation of beta 1 and beta1 and beta2 receptors impact on the trafficking to the infarcted myocardium at 6hrs post ligation (again no convincing data given the methodological limitations and absence of neutrophil-specific immunohistochemistry) but fail to impact on survival (staggering mortality rates – see above #4.) or on functional echo parameters. Unfortunately, more robust MRI imaging data allowing comprehensive function structure analysis are not available but have become the contemporary standard.\n7. The major translational value from this study is the potential role of short term inhibition of beta receptor blockade (combined beta1-2 versus selective beta2) on neutrophil trafficking and subsequent infarct functional and structural remodeling. As presented in figure 6, it is unclear to this reviewer exactly how many animals were incorporated in the respective study groups. Again survival data in the control group do not instill great confidence in the model and major state of the art diagnostic criteria to evaluate early ischemic burden are lacking. The authors should definitely integrate troponin injury markers in their study to provide more convincing evidence of cardioprotection and rescue of myocardial viable tissue despite full ligation of nutrient blood supply. In the absence of these corroborating data and confirmation in a reperfusion model, the data reported here are too preliminary.\n8. The manuscript is excessively long and, in many instances, repetitious. The discussion reiterates many sections already discussed in the results. Overall, it can be easily reduced by 30-40%."}, "author_response": "Response to reviewer’s comments\nReviewer #1\nComment-1: The manuscript by Dahdah et al. presents a comprehensive study on the early\nrecruitment of neutrophils to the ischemic heart following myocardial infarction (MI), focusing on\nthe role of vascular demargination rather than granulopoiesis in the bone marrow (BM) or\nspleen. Neutrophil demargination is a process by which neutrophils are mobilized from the\nvascular wall into the bloodstream. The authors employ a variety of advanced methodologies,\nincluding sophisticated in vivo experiments, flow cytometry, confocal microscopy, and\nproteomics to elucidate the signaling pathways that regulate neutrophil demargination and\nsubsequent recruitment to the infarct. Specifically, they explored the role of β-adrenergic\nreceptor (β-AR) signaling in mediating this process.\nThe research is novel, and the findings are of significant interest, as they provide insights into\nthe temporal dynamics of neutrophil recruitment and highlight the potential of targeted\npharmacological intervention in early MI management. However, I would recommend a revision\nto further strengthen the mechanistic understanding of neutrophil demargination and the\npotential for therapeutic intervention.\nResponse-1: We sincerely appreciate the reviewer’s constructive feedback. In response, we\nhave incorporated additional data to further clarify the mechanistic basis of neutrophil\ndemargination. Below, we provide detailed responses to each specific comment.\nComment-2. The use of dexamethasone (DEX) and norepinephrine (NE) as demarginating\nagents in mice needs careful consideration. While these agents can induce demargination, they\nmay also have additional systemic effects, including influencing the behavior of other immune\ncells, e.g.:\nMacrophages: Dexamethasone can inhibit the activation and pro-inflammatory cytokine\nproduction of macrophages, promoting a more anti-inflammatory phenotype.\nT cells: Dexamethasone can suppress T cell proliferation and cytokine production, leading to\nreduced inflammation.\nDendritic cells: Dexamethasone can impair dendritic cell maturation and antigen presentation,\nthereby reducing T cell activation.\nFurthermore, TBI has a big impact on the other immune cell populations, and it cannot be ruled\nout, that the observed effects are due to altered interactions between neutrophils and\nmacrophages, DCs and T cells. Therefore, when using TBI , BM chimeric approaches and\ndexamethasone as a tool to study neutrophil demargination, it is crucial to consider its potential\neffects on other immune cell populations.\nTo isolate the specific effects on neutrophils, comparing the effects of dexamethasone on\nneutrophil demargination in TBI wild-type mice to those after ablation of specific immune cell\ntypes (using e.g., macrophage-depleted mice or CD11c-cell depleted mice) could help to\nelucidate the direct and indirect effects of dexamethasone.\nTo control for these effects, it is also recommended to measure systemic inflammatory markers\n(e.g., cytokines) after DEX and NE administration.\nResponse-2: We fully agree with the reviewer that dexamethasone exhibits potent anti-\ninflammatory properties and is widely used as an anti-inflammatory agent in various clinical\nsettings. However, it is important to emphasize that these effects generally manifest after\nprolonged corticosteroid treatment. In our studies, dexamethasone was used solely as a\nneutrophil demarginating agent in conjunction with NE as an experimental tool. Our data is\nconsistent with data from a recent study in humans that showed successful application of both\nDEX and NE as demarginating agents (PMID: 26858400) to elucidate the mechanisms of\nneutrophil demargination.\npoint that interactions between neutrophils and macrophages, DCs, and T cells cannot be ruled\nout. However, TBI is primarily used in our models to deplete stem cells to completely halt\ngranulopoiesis. Despite the absence of routine granulopoiesis, both MI induction and DEX\nadministration were still able to increase circulating neutrophil numbers, suggesting that\nmarginated (matured cells that were not killed by TBI) neutrophils serve as the primary source\nof neutrophils in the heart immediately after MI. While it remains challenging to determine\nwhether this increase results from interactions between neutrophils and other immune cells, our\nstudy primarily aimed to identify the sources of neutrophils post-MI. That said, we cannot\ncompletely exclude the involvement of other cytokines in neutrophil demargination during DEX\nand NE treatment. As suggested by the reviewer, we conducted a cytokine array to evaluate\nsystemic inflammatory markers following DEX/ NE treatment. The resulting data are presented\nin Table 1. As shown in the Table 1, DEX/NE treatment for 6 hours has no significant impact on\nany of the major cytokines tested. We also present serum cytokine data (Table -2) in sham and\nMI mice at different time points post-MI to show how MI increase certain inflammatory cytokines\nas early as 6 hours but not DEX or NE.\nComment-3. Please also consider discussing whether the observed demargination is a\nlocalized response (specific to ischemic tissue) or part of a systemic phenomenon. To gain a\nmore comprehensive understanding of the mechanisms underlying neutrophil demargination in\nMI, it would be beneficial to explore the interplay between β-AR signaling and other relevant\npathways, such as those mediated by chemokines and adhesion molecules. Additionally,\nconsidering the long-term consequences of excessive neutrophil recruitment, such as impaired\ntissue repair and adverse cardiac remodeling, would provide valuable insights into the clinical\nimplications of the authors findings.\nResponse-3: This is an important point. As suggested, we have included this in our discussion\nunder the limitations (Page 20). We believe that neutrophil demargination represents a systemic\nresponse driven by sympathetic nervous system (SNS) activation. However, it is likely triggered\nby ischemic damage (i.e., MI) to the heart, as sham surgeries—while likely activating the SNS\nto some extent—did not cause a similar neutrophil demargination response.\nThe interplay between β-AR signaling and those mediated by chemokines and adhesion\nmolecules is indeed complex, and defining this interplay in detail is beyond the scope of this\nstudy. Indeed, it is well established that chemokines and cytokines activate the endothelium,\npromoting the adhesion of leukocytes to the vessel wall. In contrast, β-adrenergic receptor (β-\nAR) stimulation on neutrophils promotes their migration away from the endothelium by\nsuppressing the expression of adhesion molecules. This facilitates neutrophil movement into\nboth the circulation and the affected tissues. The delicate balance between adhesion and\nmigration is critical for regulating neutrophil trafficking during inflammatory responses and\nensuring proper immune responses without excessive tissue damage.\nRegarding the long-term consequences of neutrophil recruitment, previous studies, including\nour own, have shown that excessive neutrophil infiltration into the heart can exacerbate infarct\nexpansion and impair cardiac remodeling (PMID:31941367). On the other hand, indiscriminate\ndepletion of neutrophils beyond the inflammatory phase post-MI (after day 3) may negatively\naffect wound healing. The findings presented in this manuscript are particularly significant, as\nthey identify β2 ARs as a promising therapeutic target to modulate excessive neutrophil\nrecruitment to the heart, particularly in the critical early hours following MI.\nComment-4. To directly measure β2-AR activation in neutrophils, phospho-specific antibodies\n(to measure phosphorylated proteins downstream of the receptor, e.g., p-ERK1/2, p-Akt, p-p38\nMAPK) should be employed in isolated neutrophils stimulated with a β2-adrenergic agonist like\n2\nisoproterenol or inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6, and CXCL8) to\nmimic the inflammatory response. This could provide further evidence for the role of β2-ARs in\nmediating neutrophil demargination.\nResponse: This is an important consideration, as neutrophils express a range of cytokine\nreceptors that could influence their behavior during demargination. To explore this, we\nconducted immunoblotting studies using representative ligands: isoproterenol (a β-adrenergic\nreceptor agonist), TNF-α (a pro-inflammatory cytokine), and CXCL1 (a chemokine that signals\nthrough CXCR1 and CXCR2). Notably, mice do not express CXCL8; instead, they produce\nGCP-2/LIX, which also signals via CXCR1/2. Therefore, we used CXCL1 to mimic relevant\nchemokine signaling. As shown in Figure-R1, stimulation of bone marrow neutrophils with these\nligands for 6 hours did not induce activation of ERK, AKT, or p38 MAPK pathways. In fact,\nphosphorylation of some signaling proteins (e.g., P-AKT) was reduced following treatment with\nTNF-α, CXCL1, or isoproterenol. These results suggest that MAPK pathways are unlikely to\nmediate neutrophil demargination in response to cytokine or β2-adrenergic receptor activation.\nThis conclusion is further supported by our proteomics data (Supplemental Figure 2), which\nshowed no enrichment of MAPK pathway activation despite general GPCR engagement.\nInstead, we observed significant changes in proteins involved in actin polymerization and\ncytoskeletal remodeling, particularly through calcium-regulated pathways. These included Rac,\nPak, Rho, as well as key regulators of cell polarity and motility such as ROCK, Cofilin (CFN),\nProfilin, and LIM kinase (LIMK). Collectively, these findings support a model in which β2-AR-\nand cytokine-driven neutrophil demargination is primarily mediated through cytoskeletal and\ncalcium-dependent signaling rather than MAPK activation.\nComment-5. To complement the proteomics analysis of demarginated neutrophils post- MI or\nNE administration, which revealed the activation of processes involved in cellular movement\nand cytoskeletal reorganization, visualizing changes in cellular morphology and cytoskeletal\nreorganization would provide additional insights. Immunofluorescence microscopy should be\nused to show changes in cell shape, formation of pseudopodia, and cytoskeletal dynamics in\nresponse to β-AR activation.\nResponse-5: Previous studies (PMID: 26858400) have demonstrated that cytoskeletal\nreorganization—specifically changes in F-actin intensity and its localization—can reliably\nindicate neutrophil motility in response to DEX or NE stimulation. While we acknowledge the\nreviewer’s suggestion to include additional markers, we believe that incorporating them would\nnot significantly change the primary conclusions of our study. Nonetheless, to address this\nfeedback, we performed additional immunofluorescence staining on mouse blood cells treated\nex vivo with either PBS or NE for 6 hours. As shown in Figure-R2, NE stimulation led to a\npronounced spreading of F-actin and the formation of pseudopodia, consistent with cytoskeletal\nremodeling. These findings support the conclusion that β-adrenergic receptor (β-AR) activation\nby NE promotes cytoskeletal reorganization, a hallmark of neutrophil motility and migration.\nComment-6: Figure 5 j,k and Figure 6 h,i: Please check your scale bars! From my point of view,\nmagnifications are not the same, so the scale bars cannot be the same size!\nResponse: Thank you for bringing this to our attention. We have now corrected the error and\nincluded the appropriate scale bar.\nComment-1: In this paper the authors propose that neutrophil demargination induced by NE via\nbeta-2 adrenergic receptors, rather than neutrophil production and mobilization from the BM or\nspleen, is responsible for the first wave of neutrophil infiltration in the heart after myocardial\ninfarction in mice. Figure 1 examines kinetics of granulopoiesis and granulocyte dynamics in\nvarious compartments i.e. BM blood spleen heart, showing that peak heart infiltration occurs\nbefore peak granulopoiesis suggesting non-BM/spleen source of neutrophils for early\nrecruitment. Figure 2 uses a model of total body irradiation to disrupt granulopoiesis, and treat\nmice with demargination inducing agents (DEX, NE), providing data supporting the notion that\ndemarginating neutrophils are recruited to the heart early after MI. In Figure 3 the authors\nperformed proteomics analysis of blood neutrophil 6 hours after MI or NE and NE+propranolol\ntreatment. Figure 4/5: the authors use various treatments to prevent NE synthesis of block\ndifferent beta adrenergic receptors (Fig 4) or use mice with myeloid specific deficiency in beta2\nor beta1+beta2 AR (Fig 5), proposing a role of beta2-AR in neutrophil demargination and\nrecruitment to the heart after MI. Figure 6 shows that short term, but not long term, propranolol\ntreatment improves cardiac repair after permanent MI in mice.\nThe paper is clearly written, the proposed conclusions are an important and a new contribution\nto the field. There are however several technical and conceptual issues, including insufficiently\npowered experiments and unexpected results in the LAD permanent ligation model. I would\nfurthermore suggest toning down the conclusions as most evidence is indirect and discuss the\nweaknesses and limitation of the different experimental approaches in more details.\nResponse-1: We appreciate the constructive feedback and thank you for your suggestion. We\nhave repeated most of the previously underpowered experiments and have included new\nfigures. Additionally, we have addressed the limitations of the LAD model and have refined our\nconclusions accordingly.\nComment-2: One weakness of the paper is that it is technically not possible to clearly\ndistinguish neutrophil origin and most of the provided evidence is indirect. Thus, most of the\nconclusion are overstated e.g. line 155 “Since we found no role for either BM or spleen”. While\nexperiments such as those presented in Figure 1 are informative, they only provide indirect\nevidence as the different compartments observed are not merely ‘communicating vases’, and\nthe number of neutrophils at a given time point in the BM represents a complex balance\nbetween production, retention, mobilization and reverse-migration. The EdU experiments show\nneutrophils originating from HSPCs that have proliferated during the labeling period, but mature\nneutrophils originating from BM or spleen reservoirs and mobilized early after MI cannot be\ndistinguished from demarginating neutrophils. However, the cumulative amount of evidence\nprovided by the authors indeed suggests that demargination is a substantial source of early\nneutrophil recruitment to the heart.\nResponse-2: We completely agree with the reviewer. Delineating the various mechanisms by\nwhich neutrophils are sourced from different compartments and recruited to the heart post-MI is\nindeed challenging. As the reviewer correctly pointed out, the number of neutrophils in\ncirculation at any given time reflects a delicate balance between their production, mobilization,\nretention, and elimination.\nNotably, our total body irradiation (TBI) studies provide crucial evidence that mature neutrophils\n(protected from radiation) are mobilized from the vasculature during bone marrow ablation. We\nappreciate the reviewer’s recognition that our findings support the notion that demargination is a\nsignificant source of early neutrophil recruitment to the heart.\nComment-3: Figure 1A-B: please show pre-gating strategy. There is possibly an error in\npercentages showed as they are exactly the same between heart and blood\nblood and heart have now been included. Additionally, we have provided the gating strategy for\nthe data in figures1A and B (Figure. R3).\nComment-4: -The authors conclude that neutrophil numbers are not affected in the BM at 6\nhours, but the data shown in Figure 1E is not very robust, the experiment being insufficiently\npowered with clear outlier observations.\nResponse-4: A slight reduction in neutrophil numbers was observed at 6 hours post-myocardial\ninfarction (MI) compared to the Sham group; however, this difference was not statistically\nsignificant. We repeated the experiment, and similar results were obtained (Figure 1E).\nImportantly, inclusion of these additional data points did not alter the overall statistical outcome.\nComment-5: Line 182: “wherein the blood neutrophil count is slightly decreased”. According to\nFigure S1D the neutrophil count might be close to 0 at 18h, but there is actually no\nmeasurement performed at this time point\nResponse: That is correct. Both neutrophil and total WBC counts drastically decrease following\nbone marrow ablation. Please note that since we were able to completely deplete neutrophils by\n12 hours, we did not measure them again to avoid causing unnecessary stress to the irradiated\nanimals. Additionally, the 18-hour time point was selected carefully after several preliminary\nstudies to identify a time when neutrophils are completely depleted without resulting in mortality\nin the mice.\nComment-6: Neutrophil counts in the bone marrow and spleen should be provided for all\ninterventions.\nResponse-6: Thank you for the suggestion. We do have data on neutrophil counts in both the\nbone marrow and spleen for all treatments. These results have now been included in Figure-R4\nfor completeness.\nComment-7: Many experiments are quite underpowered with sometimes as few as n=3 for\nfunctional readouts in a model known for its quite substantial variability. Interexperiment\nvariability also seems quite high with e.g. a very wide range of reported blood neutrophil counts\nin the control groups across experiments.\nResponse-7: Yes, some of our initial histological analyses were underpowered due to\nunanticipated loss of animals during surgery, which prevented us from reaching the originally\nplanned group sizes. To address this, we have repeated the experiments and included\nadditional animals in the previously underpowered groups to strengthen the robustness and\nreliability of the results. Please see figures 1, 2, 4, 5L-M, 6J-K and supplementary figures S1H,\nS1I, S5I and S5J with new statistical analysis.\nComment-8: Figure 6B shows a 90% death rate in the control MI group, this is rather unusual\nin this model. The effect of short-term propranolol treatment with almost no scar formation after\nLAD ligation (Figure 6H-K) also does not seem very realistic, this would represent a spectacular\ncardioprotective effect in this permanent ischemia model. Whether and how blinding and\nrandomization were performed is not indicated in the methods.\nResponse-8: Please note that Figure 6B shows 70% mortality (not 90%) in the control MI\ngroup. This is quite typical, as the untreated mice experience extensive ventricular damage,\nsuch as thinning and rupture. Additionally, our MI surgeries resulted in a large infarct size.\nRegarding the short-term propranolol treatment, these data are not entirely surprising, as\nneutrophils are the first cells to arrive at the infarct site, and their presence influences the\nsubsequent inflammatory response. This leads to the secretion of various proteases, which in\nturn contribute to cardiac tissue damage. We believe that by preventing excessive neutrophil\n5\ninfiltration, we could reduce the extent of damage to the heart. Please note that these mice were\nadministered a dose of propranolol 8 hours prior to MI induction as part of a preemptive\nstrategy. Regarding blinding and randomization, the IHC data were performed by a technician\n(Nitin), while surgeries were conducted by Dr. Park, and drugs were injected by Dr. Dahdah.\nAlthough Dr. Dahdah was aware of the group assignments and drugs, the technician was\nblinded to the group allocations.\nComment-9: The methods do not mention the use of analgesics prior to or after MI surgery.\nResponse-9: We apologize for any oversight. We used a long-acting analgesic\n(Buprenorphine) for all our MI surgeries, and this information has now been included in the\nmethods section. Please see supplemental file page 3.\nComment-10: Previous work showing splenic retention of immune cells in mice with myeloid\nbeta2-AR deficiency early (24 hours) after MI suggests that some of the effects observed here\nmight also be caused by splenic or medullar retention of neutrophils.\nResponse-10: While we acknowledge previous studies reporting splenic retention of immune\ncells in mice with myeloid β2-AR deficiency early after MI, it is unclear whether this mechanism\nis relevant to our model. We quantified neutrophil numbers in both the BM and spleen of β2⁻/⁻\nand DKO mice at 6 hours and 30 days post-MI. As shown in Figure R5, there were no\nstatistically significant differences at either time point. These findings suggest that neutrophil\nretention in these reservoirs does not account for the reduced neutrophil numbers observed in\nthe heart or blood post-MI in β2 BMT mice. This interpretation is further supported by prior data\nshowing that NE injections do not mobilize neutrophils from the BM or spleen under steady-\nstate conditions, indicating that the absence of β2-ARs does not result in abnormal retention in\nthese tissues, at least in the short term.\nComment-11: Assessment of neutrophil purity after magnetic sorting for proteomics studies is\nmissing\nResponse: Before subjecting neutrophils to any downstream applications, including\nproteomics, we always conduct a quick flow cytometry analysis to confirm their purity. This data\nhas now been included in the proteomics figure 3B.\nComment-12: Full list of differentially abundant proteins to provide as supplementary table,\nhere only unannotated heatmap + pathway analyses are shown. It would be a good addition to\nprovide some cross-validation of proteomics and flow cytometry data e.g. do the authors see\ndownregulation of CD62L in the proteomics data and can some up/down proteins identified by\nproteomics be validated by flow?\nResponse-12: We have included the complete list of proteins identified in our proteomics\nanalysis in the supplementary table; please refer to Sheet 1 labeled “Proteins” for relative\nabundance data. CD62L was not detected, likely due to its well-documented shedding upon\nneutrophil activation following MI. As a surface protein, CD62L is rapidly cleaved and released\ninto the extracellular space, and since our proteomics analysis was performed on cell pellets—\nwhich exclude shed or secreted proteins—it was likely not captured. Similarly, several other\nsurface proteins were also absent from our dataset for this reason.\nComment-13: -Lines 307-308: ‘Administration of NE resulted in the activation of an immune\nresponse that was very similar to that induced by MI’ : how was this formally quantified?\nOverlap of up/down proteins induced by MI or NE?\nResponse-13: This conclusion was based on the observation that both MI and NE similarly\ninfluenced pathways related to immune cell movement and trafficking, with comparable pathway\nactivation scores (MI: 22.6; NE: 24.7). However, we acknowledge that the original wording may\n6\nhave overstated the similarity. We have revised the sentence to more accurately reflect the data\nand clarify that while there is a comparable impact on specific pathways, we did not formally\nquantify the overlap of individual upregulated or downregulated proteins between the MI and NE\ngroups.\nReviewer #3\nComment -1: A major limitation of the study is the use of the permanent ligation of the LAD to\ninduce myocardial infarction in mice, a species devoid of collateral coronary circulation (which in\ncontrast is abundantly present in the canine model). Full LAD ligation will prevent influx of\ninflammatory cells (neutrophils and monocytes) in the infarcted area in the first few hours after\nligation, despite the statements to the contrary by the authors. They do not provide any\nhistological data at 6h post MI that would corroborate the presumed influx of neutrophils in the\ninfarct territory. The FACS data, listed in figure 1 to underpin their claim, do not provide the\nrequired scientific evidence to substantiate this critical statement, as the data are derived from\nwhole LV homogenates. The authors need to provide histological sections prepared at 6h post\nMI and stain for the presence of infiltrating neutrophils. They should next (semi)quantitate the\ndegree of trafficking in their subsequent studies with different pharmacological and genetic\ninterventions.\nResponse-1: We recognize that the LAD ligation model does not fully replicate the clinical\npresentation of human acute coronary syndrome (ACS). However, our study design was\ninformed by recently published guidelines for in vivo murine models of myocardial infarction\n(PMID: 34623181; PMID: 34114891), which provide standardized recommendations for both\nreperfused and non-reperfused MI models. These guidelines outline key benchmarks for\nevaluating left ventricular (LV) remodeling and function post-MI and support the use of the non-\nreperfused LAD ligation model as the preferred approach for mechanistic studies of post-MI\ninflammation.\nTo further address this limitation, we performed additional studies using an ischemia-\nreperfusion injury (IRI) model to assess early neutrophil infiltration into the heart at 6 hours post-\ninjury. We also investigated the effects of short-term β-adrenergic blockade with propranolol on\nneutrophil recruitment, and evaluated cardiac structure, function, and injury using cardiac\ntroponin I (cTnI) as a biomarker. These additional findings are now presented in Supplemental\nFigure S7.\nRegarding neutrophil influx during coronary ligation, it is important to note that neutrophils retain\nthe ability to access injured cardiac tissue even in the absence of reperfusion (i.e., during\nocclusion). These cells can traverse the vascular endothelium in a luminal-to-abluminal direction\nto reach the site of injury and contribute to systemic inflammation. This migratory behavior has\nbeen elegantly demonstrated using 4D imaging by the Nourshargh group (PMID: 21706006)\nand in vivo imaging by Paul Kubes (PMID: 28983053).\nTo confirm neutrophil presence in infarcted tissue in our model, we performed\nimmunohistochemical staining of ischemic WT hearts at 6 hours post-MI and observed robust\nneutrophil infiltration, indicated by the presence of multilobulated nuclei (Figure R6). These\nfindings support the suitability of the LAD ligation model for studying early neutrophil responses\nfollowing myocardial injury.\ntissue, it is important to emphasize that neutrophils are not cardiac-resident cells like\nmacrophages; they are recruited only in response to injury (PMID: 31941367). Therefore, the\nnumber of neutrophils remains consistent whether quantified using only infarcted tissue or the\nentire ventricle. Flow cytometry, as the most widely accepted method for neutrophil\nquantification in the heart, supports this approach (PMID: 28158426, 26358260, 36034743).\nComment -2. The cited references #32, 44, and 50 are not well poised to back up the\nstatement that “ischemic damage caused by both permanent ligation and reperfusion injury\nattracts neutrophils to the infarct”. None of these studies provides histological data on neutrophil\ncell infiltration in the myocardium. Moreover, the 37-year-old study by Chatelain et al (ref 50) was performed using a canine model of both ligation and temporary (3h) occlusion and showed\nsignificantly less radiolabeled neutrophils in the infarct region following full ligation (even in a\nspecies with abundant coronary collaterals). Therefore, the limitation reported by the authors\nthat more temporal studies focusing on earlier time points than 6hrs are required, is not\nconvincing. To the contrary, additional studies using an ischemia reperfusion model are\nindispensable, as well as a larger number of animals per group to increase the power of the\nstudy (at least 10 animals per group rather than 4-6 as is).\nResponse-2: We acknowledge that some of our initial experiments were underpowered. To\naddress this limitation, we repeated several key studies, particularly those with fewer than six\nmice per group, to strengthen the statistical rigor of our findings. Please see new figures 5L, 5M,\n6K, 6J, S1H, S1I, S5H and S5J. Additionally, we now include new immunohistochemical (IHC)\ndata demonstrating neutrophil infiltration in both reperfused and non-reperfused models at 6\nhours post-ischemia (Figure R6). Interestingly, we observed greater neutrophil accumulation in\nthe infarcted tissue during ischemic-reperfusion injury (50-minute ligation followed by 6-hour\nreperfusion) compared to that of permanently ligated MI model, highlighting differences in the\nearly inflammatory response between these two injury types (Figure R7 and Figure S7).\nComment -3. The histological data presented in figure 5 are very limited and do not allow\nproper evaluation of the extent of fibrotic remodeling of the infarcted LV, especially at 30 days\npost ligation. One would expect to see full transmural infarction over a large part of the LV free\nwall in WT (as suggested by the echo image Fig 5, panel I and as explicitly highlighted in\nreference 44).\nResponse-3: We would like to clarify that we are referring to the extent of transmural infarction.\nAs shown in Figure 5J (Masson’s Trichrome), the infarct is extensive, encompassing the entire\nventricular wall downstream of the ligation in WT+ MI mice. This observation is further\ncorroborated by the echocardiographic data, as recommended by the reviewer.\nComment -4. Also, mortality rates are extremely high with only 1 or 2 mice surviving at the 30\nday follow up time point. Such high fatality rates do not instill confidence in the experimental\napproach when studying novel strategies to regulate what the authors call “runaway\ninflammatory responses”.\nResponse-4: This is partly due to the larger-than-usual infarct size (>30%) induced during\nligation. While we initially started with more than 10 mice per group, the combined effects of\nBMT and MI led to significant mortality rates, ranging from 70% in WT mice to 30% in β2 AR KO\nmice. High mortality rates are commonly observed in permanent ligation models, and our\nfindings are consistent with these expected outcomes.\nshedding and cell softening is not novel and the finding that MI is capable of inducing a similar\nphenomenon is of interest and points toward potential pharmacological interventions to interfere\nwith this early demargination. Unfortunately, the methodological limitations and lack of outcome\ndata using beta2-AR blockers do not allow to address our knowledge gaps.\nResponse: We agree with the reviewer and do not claim that the mechanisms of DEX/NE-\ninduced demargination (via CD62L shedding or cell softening) are novel. Our experiments were\nprimarily aimed at identifying a potential target for regulating initial neutrophil recruitment to\nprevent the amplification of granulopoiesis, as we recently reported (PMID: 31941367,\n34788059). We used butaxamine, a specific β2 blocker, to investigate its effects on neutrophil\ndemargination and recruitment in mouse models. Additionally, we included propranolol to\nassess the translational relevance of our findings to humans, as propranolol and carvedilol are\ncommonly used in clinical settings post-MI.\nComment-6. The authors report that bone marrow transfer experiments with myeloid cell-\nspecific ablation of beta 1 and beta1 and beta2 receptors impact on the trafficking to the\ninfarcted myocardium at 6hrs post ligation (again no convincing data given the methodological\nlimitations and absence of neutrophil-specific immunohistochemistry) but fail to impact on\nsurvival (staggering mortality rates – see above #4.) or on functional echo parameters.\nUnfortunately, more robust MRI imaging data allowing comprehensive function structure\nanalysis are not available but have become the contemporary standard.\nResponse-6: We would have delved deeper into characterizing the histological and functional\ndata if we had identified greater relevance for these studies. The primary objective of these\nexperiments was to emphasize that long-term depletion of neutrophils, achieved by deleting β1,\nβ2, or both, is detrimental to cardiac health. These findings align with those observed in studies\ninvolving long-term neutrophil depletion using antibodies (PMID: 28158426) or blocking\nneutrophil-derived proteins, such as S100A8/A9 (PMID: 31292614).\nComment-7. The major translational value from this study is the potential role of short-term\ninhibition of beta receptor blockade (combined beta1-2 versus selective beta2) on neutrophil\ntrafficking and subsequent infarct functional and structural remodeling. As presented in figure 6,\nit is unclear to this reviewer exactly how many animals were incorporated in the respective\nstudy groups. Again, survival data in the control group do not instill great confidence in the\nmodel and major state of the art diagnostic criteria to evaluate early ischemic burden are\nlacking.\nResponse-7: We initially started with 10 mice per group, but some groups experienced\nsubstantial mortality, with rates as high as 70% in the vehicle-treated group. It is important to\nnote that these are non-reperfused models, where a mortality rate of 70% in untreated mice is\nconsidered typical. However, short-term treatment with propranolol significantly improved\nsurvival, increasing it to 90%.\nComment-8. The authors should definitely integrate troponin injury markers in their study to\nprovide more convincing evidence of cardioprotection and rescue of myocardial viable tissue\ndespite full ligation of nutrient blood supply. In the absence of these corroborating data and\nconfirmation in a reperfusion model, the data reported here are too preliminary.\nResponse: Thank you for this suggestion. In response, we conducted additional studies using\nboth myocardial infarction (MI) and ischemia-reperfusion injury (IRI) models to evaluate cardiac\ntroponin I levels at various time points following myocardial injury. These data provide a\nquantitative assessment of myocardial injury and allow us to further examine the impact of\nshort-term (ST) propranolol treatment on cardiac injury, structure, and function. The results\nhave now been incorporated into the revised manuscript and are presented in Figure S6.\ndiscussion reiterates many sections already discussed in the results. Overall, it can be easily\nreduced by 30-40%.\nResponse: We have substantially shortened the manuscript, particularly the Discussion section\n(highlighted), reducing the word count from 2,066 to 1,400 words.\nReviewer 3\nComment #1: Reviewer 3 arguments that insufficient data have been presented of neutrophil\ninfiltration in the early phase (6h) post MI. Reviewer 3 wants to see more specific\nimmunohistological data on this aspect. As requested, the authors show in their\nimmunohistological assessments, that even in the permanently ligated coronary artery MI model\n(LAD), myocardial neutrophil infiltration can be observed, which supports the current hypothesis.\nIn Figure-R7 the authors show that Ly6G-positive infiltrating cells are visible (B) which, however,\nis not reflected in the H&E stainings (nucleoli are not really visible in infiltrating cells). Basically,\nthis is a critical question of Reviewer 3 which needs to be answered in more details and in depth\nand not just to provide three histological sections for this critical aspect. Most of the initial\nhypothesis relies on the assumption, that early infiltration of neutrophils occurs post-MI and is of\ncrucial pathophysiological consequence for function and survival thereafter. Thus, the Reviewer\npoints to a critical issue, but the Response is positive but not totally convincing. Further, these\nimportant data need to be shown and validated in the text and figures of the main manuscript\n(not just comments to the Reviewers).\nResponse: Thank you for emphasizing the importance of rigorously validating early neutrophil\ninfiltration after MI and for recommending that these data be incorporated into the main\nmanuscript. While flow cytometry provides a robust and quantitative assessment of cardiac\nneutrophils, we complemented these analyses with histology and IHC in response to the\nreviewer’s initial concerns. Our Ly6G immunostaining clearly identifies neutrophils within the\nmyocardium at 6 hours post-MI; although multilobed nuclei are not easily visualized on H&E\nsections—likely due to technical limitations in staining or resolution—this does not contradict the\nfindings obtained by flow cytometry or Ly6G IHC. We would also like to highlight that we\npreviously provided confocal images (Reviewer Fig. R6) showing Ly6G neutrophils with distinct\nmultilobed nuclear morphology, further confirming their early presence in the infarcted\nmyocardium. We have now included this in the manuscript as Suppleme⁺ntal Figure 1C.\nTo strengthen this conclusion further, we additionally analyzed an independent, publicly\navailable bulk RNA-seq dataset profiling healthy mouse myocardium (C57BL6) at 6 h and 24 h\nafter permanent LAD ligation (PMID: 33972017). At 6 h post-MI, we observed significant up-\nregulation of canonical neutrophil-associated chemokines, receptors, and effector genes—\nincluding Cxcl1, Cxcl2, Cxcr2, S100a8, S100a9, Mmp8, Mmp9, Trem1, Sell, Fpr1, and Fpr2—\nwith log fold-changes of ~2–3 (FDR < 0.05). These transcriptional signatures strongly indicate\nearly neutrophil mobilization and activation and align closely with our flow cytometric and IHC\nfindings₂. As expected, these pathways were further amplified at 24 h, consistent with the well-\nestablished progression of neutrophil-driven inflammation following MI. We have now\nincorporated these transcriptomic analyses (Supplemental Figures 1A and 1B) and expanded\nconfocal microscopy data (Supplemental Figure 1C) directly into the revised main manuscript.\nTogether, these orthogonal approaches—flow cytometry, IHC, confocal microscopy, and RNA-\nseq provide strong and convergent evidence that neutrophil-driven inflammatory programs are\ninitiated within the first few hours of myocardial ischemic injury.\nComment #2: The Reviewer criticizes low numbers of animals per group and wants to see a\ncomparison to I/Rmyocardium. The authors responded with new experiments including I/R\nmodels and claim that neutrophil infiltration occurs in both MI and I/R. The authors claim from\ndata shown in Figure-R7, that neutrophil infiltration is higher in the I/R group compared with MI\ngroup. However, this is not confirmed in Figure-R7, part B (missing statistical significance).\nResponse: We appreciate the reviewer’s careful assessment and apologize for the confusion\ncaused by our earlier interpretation. Our previous statement regarding increased neutrophil\naccumulation in the I/R model relative to permanent LAD ligation was based on H&E staining,\nwhich quantifies total nuclei rather than neutrophils specifically, and therefore is not an\nappropriate method for comparing neutrophil abundance between groups. As the reviewer\ncorrectly notes, the corresponding Ly6G immunostaining which directly identifies neutrophils\nshowed no significant differences in neutrophil numbers between MI and I/R hearts.\nTo avoid further confusion, we have removed this reviewer-only figure from the revised\nmanuscript. Importantly, our goal was not to highlight differences between MI and I/R models,\nbut rather to demonstrate that cardiac injury, irrespective of model, results in rapid neutrophil\nrecruitment to the myocardium. Our updated analyses and revised presentation now reflect this\nmore accurately.\nComment #3: The Reviewer is right to question the histological assessment of fibrosis of the\ninfarcted LV- myocardium. It is not clear how the scar formation was assessed and calculated. A\nmore convincing approach seems to be missing still. Usually, gross pathological assessment\nand stainings showing cross sections of the whole LV are more adequate.\nResponse: We appreciate the reviewer’s request for clearer information regarding how scar\nformation and fibrosis were assessed. To clarify, Masson’s trichrome staining was used to\nevaluate scar formation, with infarcted regions identified by collagen-rich blue/whitish tissue and\nthe corresponding loss of viable myocardium. Scar size was quantified as a percentage of the\nwhole-heart (WH) area using standardized image-analysis workflows. To assess interstitial\nfibrosis, we used Picrosirius Red staining, which provides enhanced sensitivity and specificity\nfor collagen, particularly when viewed under polarized light. These complementary approaches,\nMasson’s trichrome for macroscopic scar architecture and Picrosirius Red for detailed collagen-\nbased fibrosis were applied to whole heart cross-sections, as now described in detail in the\nrevised Methods section (Please see supplemental document, page 8) . As recommended by\nthe reviewer, we have also incorporated whole heart/ LV cross-sectional images into the revised\nfigures to provide a more comprehensive visualization of scar extent and fibrosis. Please see\nfigures 5, 6 and supplemental figure 7.\nComment #4: The Reviewers argues the low number of MI mice that survived 30-days after\nligation. This is a critical aspect that is not met by the authors response. Thus, the data shown in\nfigure 7B is not convincing (due to low numbers of survivors. It is also not clear how much\nindividual mice could be analyzed by means of echocardiographic assessments.\nResponse: We presume that the reviewer is referring to the Kaplan–Meier survival data shown\nin Figure 6B, as there was no Figure 7 in the submitted manuscript. We agree that long-term\nsurvival is an important consideration when evaluating post-MI remodeling. In our model, 30-day\nsurvival ranged from 30% to 90%, depending on the treatment group. Each experimental cohort\nwas initiated with 16-20 mice, and therefore even under the most conservative condition of\n~70% mortality (seen in both the vehicle and LT propranolol groups), approximately 6 mice per\ngroup remained available for echocardiographic assessment at 30 days. This sample size is\nwell within the norms of murine MI literature and is generally considered sufficient to provide\nstatistically and scientifically robust echocardiographic data, particularly in permanent LAD\nligation models, where inter-animal variability is typically lower than in I/R models.\nTo ensure complete transparency, we now explicitly report the exact n-values at each time point\nin the Kaplan–Meier curve in the revised manuscript, making the progression of sample sizes\nfully traceable.\nReviewer 4\nComment #1. Vascular demargination. This is a critical aspect for the whole hypothesis of the\nmanuscript and needs to be substantiated. The authors show that dexamethasone elevates\ncirculating blood neutrophils of cells with low CD62L surface expression. The argument that\n“shedding” of CD62L results in demargination seems logical. It might be of interest to directly\nmeasure soluble sCD62L in plasma samples. Perhaps the authors can reanalyze the blood\nsamples.\nResponse: Shedding of L-selectin (CD62L) from activated neutrophils is a well-established\nprocess in human leukocytes and leads to the appearance of soluble CD62L (sCD62L) in the\ncirculation. In patients with acute myocardial infarction or myocardial ischemia, dynamic\nchanges in plasma sCD62L levels have been reported and are thought to reflect leukocyte\nactivation and recruitment to the injured myocardium (PMID: 1044240). Although sCD62L has\nnot been systematically examined in murine MI models, we followed the reviewer’s suggestion\nand directly measured plasma sCD62L in mice subjected to MI or dexamethasone treatment.\nIn both settings, sCD62L levels were significantly increased and closely paralleled the\nexpansion of circulating CD62Llow neutrophils. Notably, both MI and dexamethasone induced a\nsustained elevation of sCD62L, with peak levels observed at approximately 6 hours post-\ntreatment, consistent with prolonged neutrophil activation and demargination (please see figure\n2, J-L) . These changes were mirrored by corresponding reductions in neutrophil surface CD62L\nexpression. Together, these findings extend the established biology of CD62L shedding to acute\nMI in mice and provide strong mechanistic support for the concept that β-adrenergic activation\npromotes neutrophil demargination via CD62L shedding, in agreement with the reviewer’s\nrecommendation.\nComment #2. It would be of interest to see the effect of norepinephrine on circulating\nneutrophils.\nResponse: Yes, we performed this analysis. We found that norepinephrine (NE) administration\ninduces rapid neutrophil demargination, accompanied by reduced surface CD62L expression\nand a significant, transient increase in plasma soluble CD62L (sCD62L). The NE-induced rise in\nsCD62L occurred earlier than that observed after MI or dexamethasone treatment, peaking at\n~3 hours and returning toward baseline by 6 hours, in parallel with recovery of neutrophil\nsurface CD62L expression (Please see Fig 2, H and K). In contrast, MI, which is associated with\nsustained sympathetic activation produced a more prolonged elevation of sCD62L. These\nfindings further support our conclusion that β-adrenergic signaling drives neutrophil mobilization\nvia CD62L shedding.\nComment #3. Myocardial infiltration of neutrophils. A better description and validation of\nneutrophil infiltration in MI by immunohistochemistry in addition to the flow cytometric data will\nstrengthen the conclusion. Topographical assessment of myocardial infiltration in respect to\nischemic areas etc.\nResponse: Please see our response to comment 1, Reviewer 3.\nComment #4. Do the authors have data how neutrophils in the early stage of MI alter tissue\ninflammation? Do the authors know whether metalloproteinases and extracellular\nrearrangement occurs in areas of neutrophil accumulation? What about S100A8/A9 which was\nshown by the group to be of significant importance.\nResponse: We thank the reviewer for raising this important point. It is well established that\nexcessive or prolonged neutrophil accumulation in the myocardium can induce collateral tissue\ninjury through the release of proteolytic enzymes, including matrix metalloproteinases (MMPs)\nand other granule-associated proteases. These enzymes contribute to extracellular matrix\ndegradation, structural destabilization, and amplification of local inflammation during the early\nphase of MI (comprehensively reviewed in PMIDs: 3566486, 35410418, 35276496 and others)\nAlthough the primary focus of our study was not to delineate the downstream injury pathways,\nthese mechanisms have been extensively characterized in prior work. At the same time,\nmounting evidence indicates that complete or sustained neutrophil depletion is detrimental, as\nneutrophils also play essential roles in the resolution and repair phases of post-MI healing\n(PMID: 28158426). A key example is S100A8/A9, which our group and others have shown to\nexhibit dual, time-dependent functions:\n early after MI, S100A8/A9 amplifies inflammation and promotes leukocyte recruitment\n(PMIDs: 31941367, 34788059 and 32878477)\n whereas later, it supports resolution and macrophage-mediated reparative processes\n(PMID: 32434457, 31262614).\nAccordingly, the concept proposed in this manuscript—short-term modulation of neutrophil\nactivity by targeting β-adrenergic receptor–mediated demargination is mechanistically aligned\nwith this emerging body of work. Similar to the short-term therapeutic window described for\nS100A8/A9 blockade by Marinković et al. (PMID: 32434457, 31262614), our data support the\nrationale that transient, rather than sustained, neutrophil-directed interventions may attenuate\nearly injury while preserving reparative functions.\nComment #5. The proteome data is interesting. However, it is not totally clear as shown in\nFigure 3, why the proteome data points specifically to the impact of ß-ARs in this context. The\nauthors might think about to provide some more specific data on the effect of NE and NE plus\npropranolol on proteome profile. Was the effect of NE similar than that observe in MI mice?\nResponse: We thank the reviewer for this insightful comment. In our original submission, we\nincluded a detailed explanation of how the proteomic signatures pointed toward GPCR- and β-\nadrenergic–mediated cytoskeletal activation in neutrophils during MI. During revision, some of\nthis mechanistic context was streamlined for brevity, and we appreciate the opportunity to clarify\nthe direct link between the proteomic data and β-AR signaling.\nAs presented in the Figures 3 and Figure S3, we performed parallel proteomic profiling of\nneutrophils isolated following acute MI, NE administration, and NE plus propranolol treatment.\nThese data show that NE alone elicits a proteomic program that strongly parallels the MI-\ninduced state, including activation of pathways governing cytoskeletal remodeling, actin\npolymerization, mechanotransduction, immune cell trafficking, and inflammatory signaling.\nImportantly, co-treatment with propranolol (a pan–β-AR antagonist) markedly suppressed the\nmajority of NE-induced pathway activations, demonstrating that these effects are mediated\npredominantly through β-adrenergic receptors. The substantial overlap between the MI-induced\nand NE-induced proteomic signatures and, the ability of propranolol to abrogate NE-driven\nchanges provides direct mechanistic evidence that the proteomic alterations observed after MI\nreflect β-AR–dependent signaling. Together, these findings support the conclusion that MI\nactivates a β-AR–mediated cytoskeletal program in circulating neutrophils, and that NE\nadministration faithfully recapitulates the major MI-associated proteomic features, while\npropranolol effectively blocks them. Please see the underlined text in pages 12 and 13 for\nupdated version of the text.\nComment #6. Figure 5 shows that bone-marrow transplanted wild type mice with ß2- or ß1/ß2-\ndeficient bone marrow indicate that blood and myocardial neutrophils are decreased following\nMI. No effect on cardiac function or survival was found. However, fibrosis was significantly\nenhanced in KO mice compared to wild type. This observation is not easy to understand. What\nis the interpretation and explanation of this finding?\nResponse: We appreciate the reviewer’s thoughtful question. Our prior work demonstrated that\nthe early wave of neutrophils recruited to the infarcted heart serves as an essential trigger for\nthe amplification of bone-marrow granulopoiesis, which peaks around 24 hours post-MI. At that\ntime, the upstream mechanism driving this early recruitment had not been defined, but we\nhypothesized that limiting this initial influx could help tone down the subsequent expansion of\nneutrophils and potentially provide a therapeutic window. However, it is now well-recognized\nthat prolonged or excessive suppression of neutrophils beyond the early inflammatory window\n(>3 days post-MI) can be detrimental, as neutrophils play key roles in inflammation resolution,\ncollagen remodeling, and coordination of reparative macrophage responses. Sustained\nneutrophil deficiency is therefore associated with impaired healing and adverse remodeling,\nincluding increased fibrosis.\nIn the β2- or β1/β2-deficient bone-marrow chimeras, loss of β-AR signaling reduced early\nneutrophil demargination and myocardial recruitment, as expected. However, because these\nmice lack β-AR signaling throughout both the inflammatory and reparative phases, they exhibit a\nsustained reduction in neutrophil influx into the heart. This prolonged deficit likely disrupts the\nnormal transition to reparative inflammation, impairs appropriate collagen remodeling, and\nultimately leads to the increased fibrosis observed at later time points. In other words, reducing\nneutrophil recruitment for longer than the early inflammatory window does not confer additional\nbenefit and may in fact be detrimental. These findings support the emerging consensus that\nshort-term modulation of neutrophil activity can be protective, whereas long-term suppression\ninterferes with healing and promotes adverse structural remodeling.\nComment #7. Short-term administration of propranolol improves survival, function and reduces\nfibrosis formation associated with reduced neutrophil infiltration. In contrast, long-term\nadministration has virtually no effect. Do the authors have data whether LT-propranolol leads to\nonly an initial neutrophil demargination?\nResponse: We thank the reviewer for this important question. We performed both short-term\n(ST) and long-term (LT) propranolol studies to assess the effects of β1/β2-AR blockade on\nneutrophil demargination, myocardial recruitment, and downstream cardiac remodeling. As\nshown in Figure 4E, short-term propranolol markedly reduces early neutrophil infiltration into the\nheart, which in turn improves survival, cardiac function, and limits fibrosis. In contrast, long-term\npropranolol continues to suppress neutrophil recruitment well beyond the early inflammatory\nwindow. While this further reduces total neutrophil numbers, it also diminishes the population of\nneutrophils required for transition to reparative inflammation, including coordination with\nmacrophages during resolution and matrix remodeling. Consequently, LT treatment fails to\nconfer the benefits observed with short-term inhibition.\nWith respect to the reviewer’s specific question: demargination is an acute event that occurs\nwithin minutes to hours post-MI, and thus both ST and LT propranolol block the initial\ndemargination response similarly. What differs is the duration of neutrophil suppression: only\nshort-term, time-limited blockade preserves the subsequent reparative neutrophil functions\nnecessary for optimal healing.\nComments on the rebuttal to comments of Reviewer 3.\nComment #1 of Reviewer 3\nReviewer 3 arguments that insufficient data have been presented of neutrophil infiltration in the early\nphase (6h) post MI. Reviewer 3 wants to see more specific immunohistological data on this aspect. As\nrequested the authors show in their immunohistological assessments, that even in the permanently\nligated coronary artery MI model (LAD), myocardial neutrophil infiltration can be observed, which\nsupports the current hypothesis. In Figure-R7 the authors show that Ly6G-positive infiltrating cells are\nvisible (B) which, however, is not reflected in the H&E stainings (nucleoli are not really visible in\ninfiltrating cells). Basically, this is a critical question of Reviewer 3 which needs to be answered in more\ndetails and in depth and not just to provide three histological sections for this critical aspect. Most of\nthe initial hypothesis relies on the assumption, that early infiltration of neutrophils occurs post-MI and\nis of crucial pathophysiological consequence for function and survival thereafter.\nThus, the Reviewer points to a critical issue, but the Response is positive but not totally convincing.\nFurther, these important data need to be shown and validated in the text and figures of the main\nmanuscript (not just comments to the Reviewers).\nComment #2 of Reviewer 3\nThe Reviewer criticizes low numbers of animals per group and wants to see a comparison to I/R\nmyocardium. The authors responded with new experiments including I/R models and claim that\nneutrophil infiltration occurs in both MI and I/R. The authors claim from data shown in Figure-R7,\nthat neutrophil infiltration is higher in the I/R group compared with MI group. However, this is not\nconfirmed in Figure-R7, part B (missing statistical significance).\nComment #3 of Reviewer 3\nThe Reviewer is right to question the histological assessment of fibrosis of the infarcted LV-\nmyocardium. It is not clear how the scare formation was assessed and calculated. A more convincing\napproach seems to be missing still. Usually gross pathological assessment and stainings showing cross\nsections of the whole LV are more adequate.\nComment #4 of Reviewer 3\nMortality rates. The Reviewers argues the low number of MI mice that survived 30-days after\nligation. This is a critical aspect that is not met by the authors response. Thus, the data shown in\nfigure 7B is not convincing (due to low numbers of survivors. It is also not clear how much\nindividual mice could be analyzed by means of echocardiographical assessments.\nComment #5 of Reviewer 3\nComment is justified by not convincingly responded.\nComment #6 of Reviewer 3\nComments are justified. MRI imaging would be a more timely assessment of LV-function and scare\nformation. However, it is understandable that MRI imaging is not routinely available everywhere and\nlogistically complex, especially in mouse models with high mortality rates.\nComment #7 of Reviewer 3\nComment is justified and not convincingly answered.\nComment #8 of Reviewer 3\nDetermination of troponin levels have been adequately performed and shown.\nComment #9 of Reviewer 3\nAs suggested by Reviewer 3 the manuscript has been revised, shortened and the discussion has been\nfocused.\nIn summary, the comments and criticism of Reviewer 3 are well justified, however, only partially and\nconvincingly responded and answered by the authors. The Reviewer (and also me) are not totally\nconvinced."}, {"round": 2, "reviewer_comments": {"reviewer_1": "All comments are sufficiently addressed now.", "reviewer_2": "Although I do not necessarily agree with all the arguments presented in their rebuttal, the authors have reasonnably addressed my comments, and especially increased the statistical power of key experiments. The authors could consider including the data provided for reviewers only as supplemental figures.", "reviewer_4": "The manuscript authored by Dahdah and coworkers elaborates the role of neutrophils in the early phase of myocardial ischemia in mice. The general hypothesis is that rapid demargination and myocardial infiltration of neutrophils occurs in the first ours of cardiac ischemia. The authors claim that myocardial infarction promotes norepinephrine-dependent stress-response that in turn liberates “vascular-resident” neutrophils via activation of the ß-adrenergic receptors (ß-AR) on neutrophils. The authors show that short-term but not long-term administration attenuates myocardial neutrophil infiltration, preserves cardiac function and has positive effects on post-MI survival in mice.\n\nGeneral comments:\nThe hypothesis is clear and of potential clinical relevance. The significance of elevation of sympathetic tone in myocardial infarction has been well described which is a major justification of ß-blocker administration following MI. The finding that ß-ARs regulate neutrophil demargination and infiltration in ischemic myocardium is novel. Understanding the pathophysiological role of neutrophils demargination is an experimental challenge and not easily validated. The results shown in the present manuscript rely often on indirect methods. For example, dexamethasone and norepinephrine have been shown to liberate marginated immunocompetent cells within the microvasculature. However, this effect does not seem to be specific for neutrophils. Further, the systemic immunomodulatory effects of both dexamethasone and norepinephrine may not be underestimated although the authors do not find significant changes in a chemokine/cytokine plasma profiling. Although flow cytometry is a well-established method for characterizing circulating and tissue neutrophils, the findings derived from these data often need substantiation through additional strategies to identify subtypes. I admit that this is not an easy task, however, some of the present data needs to be further substantiated and the speculation on the role of ß-ARs and their inhibition in the setting of MI needs to be very critically assessed and discussed, since the data will have an impact in the role of ß-blockers in acute MI, which is currently under intense debate.\n\nSpecific comments\n1. Vascular demargination. This is a critical aspect for the whole hypothesis of the manuscript and needs to be substantiated. The authors show that dexamethasone elevates circulating blood neutrophils of cells with low CD62L surface expression. The argument that “shedding” of CD62L results in demargination seems logical. It might be of interest to directly measure soluble sCD62L in plasma samples. Perhaps the authors can reanalyze the blood samples.\n2. It would be of interest to see the effect of norepinephrine on circulating neutrophils.\n3. Myocardial infiltration of neutrophils. A better description and validation of neutrophil infiltration in MI by immunohistochemistry in addition to the flow cytometric data will strengthen the conclusion. Topographical assessment of myocardial infiltration in respect to ischemic areas etc.\n4. Do the authors have data how neutrophils in the early stage of MI alter tissue inflammation? Do the authors know whether metalloproteinases and extracellular rearrangement occurs in areas of neutrophil accumulation? What about S100A8/A9 which was shown by the group to be of significant importance.\n5. The proteome data is interesting. However, it is not totally clear as shown in Figure 3, why the proteome data points specifically to the impact of ß-ARs in this context. The authors might think about to provide some more specific data on the effect of NE and NE plus propranolol on proteome profile. Was the effect of NE similar than that observe in MI mice?\n6. Figure 5 shows that bone-marrow transplanted wild type mice with ß2- or ß1/ß2-deficient bone marrow indicate that blood and myocardial neutrophils are decreased following MI. No effect on cardiac function or survival was found. However, fibrosis was significantly enhanced in KO mice compared to wild type. This observation is not easy to understand. What is the interpretation and explanation of this finding?\n7. Short-term administration of propranolol improves survival, function and reduces fibrosis formation associated with reduced neutrophil infiltration. In contrast, long-term administration has virtually no effect. Do the authors have data whether LT-propranolol leads to only an initial neutrophil demargination?"}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_4": "The authors adequately addressed all my comments. I don’t have any further comments"}, "author_response": ""}]

Decision letter