tbuckley/cabot-search huggingface dataset:
Abstract Mo069: Phospholamban Acetylation Enhances Cardiomyocyte Calcium Cycling Under Conditions of High-Fat Feeding
Authors: Bellina Mushala, Ellen Cho, Michael Stoner, Greg Gibson, John Sembrat, Paramesha Bugga, Iain Scott, Seth Robia, Janet Manning
Journal: Circulation Research (2024)

Background: Diet-induced obesity (DIO) is an increasing driver of cardiovascular disease and cardiac dysfunction. The breakdown of excessive fat and sugar intake increases the presence of intracellular acetyl-CoA, which promotes the acetylation of available lysines, altering the stability and activity of these proteins. While numerous proteins involved in cardiac health have been identified as targets of acetylation, the impact of acetylation on calcium handling sarcoplasmic reticulum (SR) proteins, particularly the SERCA2a regulator phospholamban, remains poorly understood. Hypothesis: Phospholamban acetylation is upregulated by exposure to high fat conditions, in turn driving SERCA2a activity and promoting faster calcium SR reuptake. Approach: Left ventricle (LV) tissue from DIO mice and controls, and human LV tissue from control and diabetic patients, was collected and analyzed using immunoblotting and acetylation was quantified by immunoprecipitation with acetyl-lysine antibodies.Isolated working hearts from DIO and control mice were assessed before and after treatment with acute HDAC activator ITSA-1 and SIRT1 activator SRT1720 to promote deacetylation. Adult ventricular cardiomyocytes were isolated from DIO and control mice, and DIO cells were incubated in media containing excess lipid substrates (1% Lipid Mix 1, Millipore Sigma) and ITSA-1 orSRT1720. Changes in stimulated calcium dynamics were assessed using Fura-2AM and Fluo-4AM imaging. Acetylmimetic/acetylation incompetent PLN mutants were transfected into cultured cells to evaluate SERCA2a binding, oligomerization, and FRET efficiency. Results: Phospholamban acetylation significantly increased in LV tissue from both DIO mice and human diabetic patients. Acute treatment of isolated hearts and isolated cardiomyocytes from DIO mice with HDAC and SIRT1 activators significantly reduced contractility, relaxation, and SR calcium reuptake rates. Mutations in PLN mimicking or preventing lysine acetylation altered oligomerization and SERCA2a binding. Conclusions: Obesity increases the acetylation of phospholamban and enhances the reuptake of calcium into the SR, while deacetylation reverses these effects. These data directly tie nutritive status to a novel phospholamban post-translational modification that may directly impact myocardial calcium handling and contractile function.
Abstract We082: Contribution of local hematopoiesis to endocardial cushion remodeling during development
Authors: Norika Liu, Atsushi Nakano
Journal: Circulation Research (2024)

Introduction: The cells that comprise the circulatory system not only share their origins but also mutually promote each other’s differentiation during the formation of functional circulatory system. We previously reported a subset of endocardial cells is hemogenic during early embryogenesis. They are enriched in the cushion region, the primordia of the cardiac valves and septa, where active remodeling via endothelial-mesenchymal transition takes place. Hemogenic endocardial cells undergo endocardial-hematopoietic transition (EHT) via Nkx2-5-dependent manner, suggesting that Drosophila tinman-dependent cardio-hematopoietic program is conserved in mammals. Methods: In this study, we analyzed the regulatory network of Nkx2-5-dependent endocardial hematopoiesis using a scRNA-seq data from wildtype and Nkx2-5-null embryonic hearts. In vivo genetic analysis were performed to confirm the candidate signal pathways driven under Nkx2-5 transcription. Results and Conclusion: Nkx2-5-null hearts were devoid of clusters for hemogenic endocardium and cushion endocardium. Interestingly, scRNA-seq analysis further revealed that genes related to Notch signaling pathway are significantly downregulated in Nkx2-5-null endocardium. A further gene network analysis identified that Dhrs3, an enzyme that attenuates retinoic acid (RA) signal by catalyzing the reduction of all-trans-retinaldehyde to all-trans-retinol, is a signature gene of the hemogenic endocardial cells downstream of Nkx2-5. Although RA signal is known to induce the formation of multipotent progenitor, our ex vivo hematopoietic colony forming assay revealed that macrophage formation is strongly inhibited by RA signal. Notch inhibition also suppressed the formation of macrophages. Consistently, in vivo forced activation of NICD drastically increased the number of CD41 + hemogenic endocardial cells as well as macrophages in the cardiac cushion. Strikingly, impaired hematopoiesis and cushion defects in the Nkx2-5-null heart were both rescued by overexpression of NICD, suggesting that Notch signaling promotes endocardial hematopoiesis downstream of Nkx2-5. Taken together, our study demonstrated that the Nkx2-5/RA/Notch signaling axis plays a pivotal role in EHT during early embryogenesis, thereby facilitating local tissue remodeling by inducing macrophage differentiation. The talk will include the latest findings on the specific role of endocardially-derived tissue macrophages.
Abstract We045: Development of SA Node Organoids: A Multicellular Approach to Biological Pacemaking
Authors: Jihee Won, Young Hoon Son, Hee Cheol Cho, Sung Park
Journal: Circulation Research (2024)

The rising prevalence of sinoatrial (SA) node dysfunction, attributed to the aging population, is emerging as a critical health issue. The SA node, a natural organoid within the heart, comprises the pacemaker cells embedded within a connective tissue matrix, insulated electrically from the surrounding atrial muscle cells. This isolated architecture, along with a heterogeneous cellular composition, is crucial for the SA node's robust pacemaking and electrical conduction. Developing methods to reconstruct this crucial heterogeneous structure could offer a long-term solution for individuals with SA node dysfunction. However, the use of current biological pacemakers, which typically involve a single cell type, has often led to unstable heart rhythms during one-month in vivo integration, challenging their long-term clinical application as biological pacemakers. To address existing SA node model limitations, we developed a SA node organoid that reproduces the human SA node's multicellular tissue structure. Initially, we developed a protocol for generating cardiac SA node organoids by employing a lentivirus to deliver dox-inducible genes for key pacemaker transcription factors, specifically TBX-18 or Shox-2. We mixed wild-type human-induced pluripotent stem cells (hiPSCs) with those modified to express TBX-18 or Shox-2. These cell mixtures were aggregated, embedded in a diluted Matrigel, and then exposed to a cardiac differentiation protocol, activating TBX-18 or Shox-2 at specific times (D1 or D7) via Dox administration. By D9, these organoids began to exhibit contraction, which lasted beyond 60 days, with notable upregulation of two transcription factors and pacemaker-specific genes following Dox treatment. Subsequently, we integrated the SA node organoid into 2D hiPSC-derived atrial muscle tissue constructs, maintaining geometric isolation within the cardiac tissue to enhance source-sink mismatch. This isolation allowed the SA node organoid to successfully trigger activity in adjacent dormant cardiomyocytes, preserving the rhythm for over 60 days. These findings could redefine the role of tissue architecture and cell diversity in the SA node's pacemaking function, potentially leading to a high-fidelity, tissue-level biological pacemaker as a new treatment strategy for SA node dysfunctions.
Abstract We071: Identifying Preventive and Therapeutic Effects of SGLT2 Inhibitor on Atrial Fibrillation
Authors: Hyewon Park, Bokyeong Park, Hyelim Park, Junbeom Park
Journal: Circulation Research (2024)

Background: The frequency and prevalence of atrial fibrillation (AF) and heart failure are expected to increase over time, which are expected to result in enormous medical expenses and socioeconomic losses. Previous studies have confirmed that SGLT2 inhibitor (SGLT2i) reduces cardiovascular mortality and hospitalization rates in heart failure patients. Also, follow-up studies have been reported that the use of SGLT2i reduces atrial fibrillation, and its administration is recommended in clinical practice guidelines. In spite of beneficial effects of SGLT2i on atrial fibrillation, the exact mechanism by which SGLT2i reduces atrial fibrillation has not been revealed. Methods: We performed experiments by creating an AF cell model using Ang II and an AF animal model using Acetylcholin-Calcium chloride. Fibrosis markers and calcium regulatory proteins were confirmed by Western blot, and the occurrence and burden of AF were evaluated by using a 1F catheter in animal models. Results: In our cell studies and animal experiments, the inhibitory effect of SGLT2i administartion on atrial fibrillation was confirmed. First, fibrosis markers tend to reduce in atrial fibrillation models treated with SGLT2i, compared to non-treated atrial fibrillation models. In addition, the phosphorylation of the calcium handling proteins, such as CaMKII, RyR2, and PLB, increased in the AF group, whereas the degree of phosphorylation in SGLT2i treated group decreased to the phosphorylation level in control group. Conclusion: In AF-induced cell and animal models, the effect of SGLT2i was shown to be highly effective against atrial fibrillation. In particular, the results indicate that administration of SGLT2i before inducing atrial fibrillation could bring out the preventive and therapeutic effects through amelioration of fibrosis and reduction of calcium handling proteins phosphorylation.
Abstract We066: Macropinocytosis is essential for macrophage activation in acute ischemia.
Authors: Jinhua Wen, mariasanta Mangione, Xiang Luo, Dian Cao
Journal: Circulation Research (2024)

Macrophages (Mφ) play central roles in inducing the robust inflammatory response to acute ischemia and promoting anti-inflammatory actions necessary for myocardial repair. The broad range of functional repertoire stems from Mφ plasticity dictated by their interaction with the dynamic ischemic milieu. The limited understanding of such interactions contributes to the lack of means to mitigate inflammatory injury in MI patients. We hypothesize that the dynamic Mφ plasma membrane-mediated process macropinocytosis (MP) is essential for Mφ’s activation and function evolvement in ischemia. By MP, Mφs engulf large amounts of soluble contents from the ischemic environment, and the macropinosomes formed from MP become signaling hubs of DAMP (Damage-Associated Molecular Pattern)-PRR (Pattern Recognition Receptor). We aim to determine if ischemia activates MP in Mφs, the signaling pathways that MP promotes, and the role of this process in post-ischemia repair. The rationale is based on our data demonstrating only soluble DAMP (sDAMP), not tissue debris, activates Mφs . We tested our hypothesis using the murine MI model and bone marrow-derived Mφs (BMDM) and used unpaired t-tests for statistical analyses. We uncover that ischemia markedly increases MP in Mφs; blocking MP by the inhibitors or siRNA-mediated knock-down of the Na+H+ exchanger abolished the production of potent cytokines such as IL1β from Mφs in vitro and in vivo . Furthermore, MP activates NF-κB and MAP kinase triggered by sDAMP and ischemia, critical pathways to produce inflammatory cytokine and chemokine. Intriguingly, mTORC1 activation in BMDM and Mφs in the ischemic myocardium depends on MP. These data suggest MP organizes the activation signal and the pathway required for metabolic rewiring in inflammatory Mφs. Suppressing MP at the earlier stage of ischemia (72h) when sDAMP is abundant in the ischemic tissue significantly reduces mortality and improves cardiac function. In conclusion, we identify MP functionalizes the danger signals in bulk and integrates activation signals with metabolic rewiring to promote and sustain the inflammatory function. Targeting MP is better equipped to reduce inflammatory injury as it modifies the dynamic interaction of Mφs with the ischemic environment.
Abstract Mo001: Inhibition of TAOK1-mediated Cardiomyocyte Death Attenuates Doxorubicin-Induced Cardiotoxicity
Authors: Masaya Kogure, Tomoya Kitani, TAKAOMI SUGA, Takehiro Ogata, Koji Ikeda, Satoaki Matoba
Journal: Circulation Research (2024)

Background and Objective: Doxorubicin (Dox)-induced cardiotoxicity (DIC) is one of the most serious side effects of cancer treatments. However, no effective treatment is currently available. In this study, we aimed to identify novel therapeutic targets using CRISPR/Cas9-based screening of human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Methods and Results: We performed a kinase-focused CRISPR gene knockout screen in hPSC-CMs and identified thousand and one amino acid protein kinase 1 (TAOK1 ) as a potential regulator of DOX-induced cardiomyocyte death. We generated TAOK1-knockout (KO) hPSC-CMs using CRISPR/Cas9 and found that TAOK1-KO hPSC-CMs showed higher cell viability than control KO cells after 1μM DOX treatment (49.3% vs. 30.5%, P = 0.03). TAOK1-KO decreased DOX-induced ROS production (-58.8%, P = 0.01) and DNA damage (-28.8%, P &lt; 0.01) in hPSC-CMs. Transcriptome analysis using RNA-seq of TAOK1-KO hPSC-CMs identified 483 differentially expressed genes, and Hallmark pathway analysis revealed upregulation of the oxidative phosphorylation pathway (FDR &lt; 0.01) in TAOK1-KO hPSC-CMs compared to the control. Extracellular flux analysis demonstrated higher basal and maximal respiratory capacity in TAOK1-KO hPSC-CMs compared to control after DOX treatment (1.2 vs. 0.6, and 3.8 vs. 2.0 pmol/min/μg, respectively, P &lt; 0.01 for all). Next, we overexpressed TAOK1 in hPSC-CMs using adenovirus vector and found that TAOK1 overexpression (OE) augmented DOX-induced cell viability reduction in hPSC-CMs compared to control after 0.5μM DOX treatment (-63.4 % vs. -19.2%, P &lt; 0.01). Western blot analysis showed that DOX-induced p38 phosphorylation was suppressed by TAOK1-KO (P &lt; 0.01) and enhanced (P = 0.01) by TAOK1-OE in hPSC-CMs. Augmented DOX-induced cardiomyocyte death in TAOK1-OE hPSC-CMs was partially rescued by inhibitors of p38 MAPK or apoptosis. Lastly, we demonstrated that TAOK1 suppression using AAV-mediated gene silencing mitigated the decline in the left ventricular ejection fraction in a mouse model of DIC (-30.0% vs. -17.7%, P &lt; 0.01). Histological analysis of heart tissue revealed that TAOK1 suppression reduced myocardial fibrosis and apoptosis of cardiac cells in a mouse model of DIC (6.6% vs. 4.3%, P = 0.02, and 0.31% vs. 0.18%, P&lt; 0.01, respectively). Conclusion: Our results suggest that TAOK1 regulates DOX-induced cardiomyocyte death; thus, its inhibition could be a potential therapeutic approach for DIC.
Abstract Or113: Identification of Newly Synthesized Protein in Cardiomyocytes after Myocardial Infarction
Authors: Feng Zhang, Jie Liang, Jenna Cusick, Fangfei Wang, Ainyu Li, Shijie Liu
Journal: Circulation Research (2024)

Compared to mRNA, proteins perform ultimate functions in the heart and can be rapidly upregulated or downregulated in response to environmental inputs. Identifying how the proteome is altered in cardiomyocytes (CMs) after myocardial infarction (MI) will improve our understanding of the stress response signaling after MI and contribute to developing new therapeutic strategies. Here, we developed a method for determining which proteins in CMs are newly synthesized after MI by using mice expressing a mutant methionyl-tRNA synthetase that incorporates the non-canonical amino acid azidonorleucine into the newly synthesized peptides of CMs. We found that the proteins synthesized at the highest levels in homeostasis are involved in sarcomere structure and metabolism, which aligns with immunofluorescence staining results that showed enrichment of newly synthesized proteins localized at the sarcomere and intercalated disc. We showed that α-actin, a critical component of the sarcomere, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an enzyme for glucose metabolism, are highly dynamic and require constant protein synthesis for maintenance. After MI, protein synthesis in CMs within the infarct area is halted, and the border zone CMs showed reduced protein synthesis. Most notably, proteins associated with mitochondrial oxidative phosphorylation are reduced compared with sham. By integrating with spatial transcriptomics, we also discovered that the MI-induced genes are enriched in CMs within the infarcted areas. These CMs exhibit decreased general protein synthesis, indicating a decoupling of transcriptional and translational regulation following MI. Collectively, our findings revealed that CMs rapidly respond to injuries by controlling protein synthesis, which is independent of the expression of mRNAs.
Abstract Mo058: Cellular and Subcellular Ventricular Localization of the Fast Transient Outward Potassium Channels in the Murine Heart
Authors: Renee Gorman, Peter Backx
Journal: Circulation Research (2024)

Introduction: Cardiac fast transient outward potassium currents (I to,f ) are formed by KCND2 (Kv4.2) and KCND3 (Kv4.3) channels in mice and primarily KCND3 in humans. Rapid I to,f activation induces early phase 1 repolarization of action potentials (APs), which has been linked strongly to the modulation of excitation-contraction coupling (ECC). Consequently, variations in I to,f densities underlie regional differences in ventricular APs and myocardial contractile properties. To investigate further the role of I to,f in cardiac function, we created mice with fluorescently labelled KCND2 (KCND2-GFP) and KCND3 (KCND3-RFP). Methods and Results: The expression patterns of I to,f in the ventricles were examined using frozen histological sections. Remarkably, KCND2 and KCND3 were preferentially localized to the intercalated discs (ICDs) by &gt;50%, with expression also in lateral membranes and within t-tubules (i.e. a sarcomeric pattern), yet KCND3 showed a greater t-tubule localization. Additionally, KCND2 expression was, as expected, greater ( P &lt;0.048, two-way ANOVA; n=3) in the epi- versus endo-myocardium of both ventricles, whereas KCND3 lacked ( P &gt;0.172; n=3) a distinct gradient. Moreover, KCND2 expression inversely correlated with the temporal sequence of ventricular activation, whereby the posterobasal regions that depolarize last show elevated Kv4.2 ( P =0.009, unpaired t-test; n=3) compared to the anterolateral endocardium activated first. In contrast, Kv4.3 demonstrates a relatively uniform expression. Conclusions: The regional expression patterns of I to,f in ventricles and preferential I to,f expression in t-tubules support previous studies suggesting that I to,f controls the regional variations in the timing and amplitude of ECC. We are currently exploring the possible differential actions of KCND2 versus KCND3 in ECC and the potential consequence of ICD localization of I to,f on ephaptic coupling.
Abstract We061: Cardiomyocytes HIPK2 Regulates Myocardial Inflammation And Heart Function Through Purinergic Signaling
Authors: Tousif Sultan, Prachi Umbarkar, Rohan Sethi, Angelica Toro Cora, Yunxi Chen, Jin He, Praveen Dubey, qinkun zhang, Min Xie, Prasanna Krishnamurthy, Hind Lal
Journal: Circulation Research (2024)

Background: Homeodomain-interacting protein kinase 2 (HIPK2) is a serine/threonine kinase that belongs to an evolutionary conserved HIPK family of kinases. HIPK2-mediated regulation of the inflammation axis of signaling is a comparatively new concept. Purinergic signaling is critical to myocardial inflammation and function. However, there is no literature on HIPK2 regulating purinergic signaling. Methods and Results: Herein, we identified HIPK2, as a novel regulator of purinergic signaling. Cardiomyocyte-specific HIPK2 knockout (CM-HIPK2-KO) hearts exhibited reduced expression of multiple key players of canonical purinergic signaling, including ectonucleases CD39 and CD73, and led to excessive inflammation and cardiac dysfunction. Multiple in vitro experiments with gain-of-function (adenovirus expressing HIPK2, Ad-HIPK2), and loss-of-function (Ad-sh-RNA-HIPK2) approaches were performed to establish that HIPK2-mediated regulation of purinergic signaling is a conserved mechanism in many cell types of diverse backgrounds. Mechanistically, we identified that the signaling circuit of HIPK2-ERK-CREB exerts its effects on purinergic signaling through transcriptional control on CD39 and CD73. Aberrant activation of purinergic signaling and inflammation was critical to cardiac pathologies because interventions with purinergic signaling inhibitor apyrase or NLRP3 inflammasome inhibitor CY09 largely rescued the detrimental cardiac phenotype of CM-HIPK2 KOs. Conclusion: Herein, we identified HIPK2 as a novel regulator of purinergic signaling. Its deletion leads to excessive inflammation and cardiac dysfunction. Therefore, strategies to maintain HIPK2 are critical to sustaining cardiac health.
Abstract We040: Molecular Characterization of Human Highly Proliferative Cells Isolated from Coronary Artery Bypass Graft Surgery Patients Identifies IGFBP3, SOST, and ISLR as Downstream Targets of BMP9/ALK1 Signaling
Authors: Michayla Moore, Sergey Ryzhov, Douglas Sawyer, Calvin Vary
Journal: Circulation Research (2024)

Stem/progenitor cells are being investigated as a potential cell source to reverse cardiac remodeling and restore cardiac function, but the molecular mechanisms required for improved repair are not well understood. Circulating BMP9 is negatively associated with heart disease risk factors. BMP9/ALK1 signaling is a critical pathway involved in several mechanisms of vascular repair such as angiogenesis and inflammation. We recently identified ALK1 expression in human highly proliferative cells (hHiPCs) with progenitor capabilities isolated from patients undergoing coronary artery bypass graft (CABG) surgery as measured by flow cytometry. We have determined that circulating BMP9 in patients is negatively associated with number of hHiPC (p&lt;0.001) and positively associated with endothelial cell (EC) number (p=0.008) suggesting that BMP9 may play a role in hHiPC differentiation into EC. Further, we found upregulation of novel BMP9 regulated proteins, IGFBP3, SOST, and ISLR, in hHiPC upon BMP9 treatment using LC-MS/MS analysis of the secretome (p&lt;0.01, Fold-change (FC)&gt;2.0) and RT-qPCR (p&lt;0.001, FC&gt;2.0). Conditioned media collected from BMP9 treated hHiPCs or ECs improved tube formation in the tube formation assay. IGFBP3, SOST, and ISLR upregulation by BMP9 is inhibited upon knockdown and pharmacological inhibition of ALK1 as measured by proteomics (p&lt;0.01) and RT-qPCR analysis (p&lt;0.05). ISLR is a novel marker of mesenchymal stem cells found to have an important role in maintaining “stemness” and colony formation. hHiPCs treated with novel BMP9 target, ISLR, were analyzed using LC-MS/MS and STRING. We found ISLR treatment enriched for differential regulation of cell adhesion, cell migration, and angiogenesis proteins such as CXCL12, EMILIN1, and ANXA5 (p&lt;0.05). Together, these data lay the foundation for investigating BMP9/ALK1 autocrine signaling in potential regulation of progenitor cell maintenance and angiogenesis in heart disease.