bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract Tu059: Single-Nucleus Transcriptomics Demonstrates Endothelial Cell Expansion in Failing Human Right Ventricles
Authors: Kuznetsov Ivan , Li Kristina , Simonson Bridget , Chaffin Mark , Guedira Yasmine , Bedi Kenneth , Margulies Kenneth , Ellinor Patrick , Arany Zoltan , Edwards Jonathan
Published: October 2024

Background:
Right ventricular failure (RVF) is most commonly observed as a sequela of left ventricular failure (LVF)—for which it more than doubles the risk of mortality. Moreover, isolated RVF is the most common cause of death in pulmonary hypertension and is a leading cause of major adverse events in systemic right ventricle congenital heart diseases. Therapies which effectively treat LVF show poor efficacy for RVF. For example, beta blockade, a cornerstone of LVF therapy, has no efficacy in RVF. Therefore, there is a need for broader exploratory research to identify pathways which are dysregulated in RVF and which could serve as targets for future therapies.

Methods/Results:
To this end, we performed single-nucleus RNA sequencing (N=11) on RV myocardium from nonfailing or dilated cardiomyopathy (DCM) hearts subgrouped by preserved systolic and diastolic RV function (pRV) or RVF. After cell-type assignment and differential expression analysis, we find a progressive upregulation of pro-angiogenic signaling in vascular endothelial cells (ECs) from nonfailing to pRV and RVF. There is a concomitant expansion of the EC pool from nonfailing to pRV and RVF (EC percentage 5%, 9%,15% for NF, pRV, and RVF; p < 0.05 for one-way ANOVA). We find increased HIF-mediated and VEGF-mediated signaling in these ECs, which may be mediating this expansion. Surprisingly, there is also increased expression of genes regulated by type 1 interferon signaling, which is thought to be anti-angiogenic.

Discussion:
In summary, we have conducted a single-nuclei transcriptomic study of human RVF and found evidence for increased angiogenic signaling and EC expansion. These findings are significant, as the balance of pro-angiogenic and anti-angiogenic factors in RVF is still unclear, with studies in animal models showing conflicting results in terms of changes in macro/microvasculature in RVF. Here, we provide evidence that in human RVF the balance tends to lead toward a pro-angiogenic phenotype, even in dysfunctional RVs. Orthogonal studies to validate the causal nature of these observed changes in pathogenesis of RVF are currently ongoing.
Abstract Tu062: FLNC Deficiency Triggers Unfolded Protein Response and Leads to Dilated Cardiomyopathy
Authors: Xuan He , Li Jiacheng , Lian Hong , Nie Yu
Published: October 2024

Background:
The
FLNC
gene encodes filamin C, which is highly expressed in cardiomyocytes. Dysfunction of filamin C, due to different types of
FLNC
mutations, has been reported to be associated with various types of cardiomyopathy. Truncating mutations in
FLNC
often result in insufficient expression of filamin C, resulting in dilated cardiomyopathy (DCM). However, the specific processes via which the deficiency of filamin C causes DCM remain incompletely comprehended.

Aims:
To investigate the pathogenic mechanisms of DCM caused by the deficiency of filamin C in cardiomyocytes using a mouse model with cardiac-specific knockout of
Flnc
and to explore potential therapeutic approaches.

Methods and Results:
We used tamoxifen to induce cardiac-specific
Flnc
gene knockout in cardiomyocytes and found that mice with cardiac-specific
Flnc
knockout exhibited left ventricular dilation, heart failure, increased heart weight, cardiomyocyte enlargement and fibrosis, suggesting that
Flnc
deficiency in cardiomyocytes causes DCM. Transcriptional profiling of primary cardiomyocytes isolated on the seventh day after tamoxifen injections showed the activation of ER stress and the unfolded protein response (UPR) upon
Flnc
deletion. Then we measured the expression levels of proteins in UPR pathway in cardiac tissue and found that PDI and pIRE1a proteins were significantly upregulated, while there were no changes in the other two branches of UPR, including the PERK and ATF6 branches. Intraperitoneal injection of a PDI inhibitor E64FC26 significantly improved heart function and reduced fibrosis in cardiac-specific
Flnc
deletion mice.

Conclusion:
Cardiac-specific knockout of
Flnc
leads to DCM in mice, during which the UPR pathway is activated. Treatment with the PDI inhibitor E64FC26 improves the heart function and the fibrosis in
Flnc
-deficient DCM mice, offering novel perspectives on the pathological mechanisms of DCM and providing new insights into therapeutic strategies in treating DCM caused by FLNC dysfunction.
Abstract Tu064: Abnormal Calcium Regulation Leads to Pathological Cardiac Hypertrophy During Pregnancy in the GSNOR-Deficient Mouse Model of Preeclampsia
Authors: Dulce Raul , Balkan Wayne , Hare Joshua , Kulandavelu Shathiyah
Published: October 2024

Introduction:
Preeclampsia (PE), a leading cause of maternal mortality, is linked to cardiac hypertrophy and impaired relaxation, predisposing the mother to an increased risk of cardiovascular (CV) injury during pregnancy. Mice lacking S-nitrosoglutathione reductase (GSNOR
–/–
mice) exhibit all the clinical features of PE. Calcium regulation plays an important role in cardiac relaxation and hypertrophy, but whether alterations in calcium handling contribute to the impairment in these PE mice is unknown.

Hypothesis:
Cardiac hypertrophy and impaired relaxation in pregnant GSNOR
–/–
mice may be due in part to altered Ca
2+
handling mechanisms.

Methods:
Pregnant control (WT [C57B6]) and GSNOR
–/–
mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) were isolated (N=3-4 mice/group; n=8-24 CMs/heart) and assessed for intracellular Ca
2+
and sarcomere shortening using IonOptix. Ca
2+
decay and sarcomere relaxation rates were recorded. Ventricular tissue homogenates were assessed for Ca
2+
handling and sarcomere-associated protein expression and post-translational modifications by Western Blot.

Results:
At late pregnancy, the CMs of GSNOR
–/–
mothers were significantly wider and systolic and diastolic Ca
2+
levels were elevated as compared to WT. At 17.5 dpc, expression of Ca
2+
handling proteins, RyR2, L-TCC and NCX1 were increased, and phosphorylation of RyR2 and L-TCC were increased in the GSNOR
–/–
mothers as compared to WT. The Ca
2+
decay rate improved in CMs from pregnant versus NP WT CMs but not in GSNOR
–/–
CMs. PLB phosphorylation at Thr-17 was augmented in pregnant WT hearts, while phosphorylation at Ser-16 was reduced in pregnant GSNOR
–/–
. Sarcomere relaxation parameters and sarcomere resting length were improved in CMs from pregnant WT mice but were impaired in pregnant versus NP GSNOR
–/–
mice in association with increased expression of MyBPC in pregnant GSNOR
–/–
.

Conclusion:
The PE-related cardiac hypertrophy and impaired relaxation at late pregnancy in GSNOR
–/–
mice were associated with elevated cytosolic Ca
2+
levels, altered expression and post-translational modifications of Ca
2+
handling and sarcomeric proteins, which may predispose these mice to higher CV injury during pregnancy.
Abstract Tu067: ROCK2 Specific Inhibition Prevents Isoproterenol Induced Takotsubo Cardiomyopathy in Mice
Authors: Fehrenbach Daniel , Wassenaar Jean , Smart Charles , Zhong Lin , Miller Francis , Madhur Meena
Published: October 2024

Takotsubo Cardiomyopathy (TCM) is an acute, life-threatening condition characterized by a drop in ejection fraction (EF) and apical ballooning of the left ventricle (LV). TCM is most frequently observed in women and is associated with an emotional or physical stressor. Recent work has demonstrated that the Rho kinase ROCK2 contributes to heart failure phenotypes in mice in other models of heart failure. The role of ROCK2 in TCM is unknown. We tested the hypothesis that ROCK2 inhibition attenuates isoproterenol (ISO)-induced TCM in mice. To induce TCM, male and female mice were given a single dose of 200mg/kg ISO, i.p. and cardiac function was assessed by conscious echocardiography. The effect of ISO overdose on acute heart failure over a 7-day time course was determined followed by an intervention study in which mice were treated with the ROCK2 specific inhibitor KD025/belumosudil (50mg/kg, i.p.) concurrent with heart failure induction. Though ISO induced a drop in EF in both sexes after 24 hours, females exhibited lower EF (46.64±3.11%) than males (68.36±2.62, p<0.0001) mirroring the increased prevalence of clinically defined TCM in females in humans. Female mice were used for the remaining experiments. Invasive hemodynamics revealed reduced contraction and relaxation (dp/dt max and dp/dt min) as well as an increased relaxation time constant Tau 24 hours after ISO injection. A time course study demonstrated that EF begins to normalize by Day 3 and is restored to baseline levels by Day 7. Importantly, ROCK2-specific inhibition prevented ISO-induced drop in EF, thinning of the LV wall, and LV volume expansion. Here, we demonstrate that female mice are more susceptible to ISO-induced cardiac dysfunction. Furthermore, we show that ROCK2 activation is necessary for the development of TCM in mice. These results suggest that FDA-approved KD025/belumosudil could potentially be repurposed for treatment in TCM.
Abstract Tu070: Impact of Hypertrophic Cardiomyopathy in Hospitalizations with Aortic Stenosis : A National Inpatient Sample survey
Authors: Sekandlapuram Rohit , Nanavaty Dhairya , Voruganti Hima Varsha , Vachhani Bhavyakumar , Jayakumar Jayalekshmi , Devarakonda Pradeep Kumar , Singh Sohrab , Patel Nishant , Sanka Sujana , Sinha Rishav , Ayala-Rodriguez Cesar , Hotwani Priya , Nanavaty Sukrut
Published: October 2024

Background:
The management of patients with combined aortic stenosis (AS) and hypertrophic cardiomyopathy (HCM) is tricky. HCM causes a dynamic obstruction, while AS causes a fixed obstruction, both causing a hindrance to left ventricular output. We aim to study the outcomes of hospitalizations with co-prevalent HCM and AS.

Methods:
We queried the National Inpatient Sample 2018-2020 for adult hospitalizations with AS. This sample was stratified based on the prevalence of HCM. Mortality, arrhythmia prevalence, and acute cardiac complications were compared in aortic stenosis hospitalizations with and without

AS. A multivariate regression analysis was performed to study the impact of HCM on mortality, arrhythmias, and other complications in patients with AS.

Results:
In hospitalizations with aortic stenosis (n=2483854), a total of 11820 had underlying HCM (0.47%). The mean age was 76 years and 64% were females. Hospitalizations with AS and HCM as compared to those without HCM had a higher prevalence of arrhythmias including first degree AV block, atrial flutter, atrial fibrillation, ventricular tachycardia and ventricular fibrillation (Table 1). The occurence of cardiogenic shock was also higher in this subgroup. The difference in mortality, acute coronary syndrome, acute heart failure and cardiac arrest was not statistically significant in AS hospitalizations with and without HCM (Table 1). Multivariate regression analysis revealed a higher odds of arrhythmias (OR-1.44; C.I: 1.36-1.62; P<0.001) and cardiogenic shock (OR-1.58; C.I: 1.22-2.06; P<0.001) with HCM in AS hospitalizations.

Conclusion:
Prior studies have demonstrated worse outcomes in AS patients with pre-existing HCM, especially post TAVI. Our study concludes that HCM was associated with a higher odds of arrhythmias and cardiogenic shock in AS hospitalizations.
Abstract Tu073: Mst1 induces cardiac dysfunction through the activation of PERK
Authors: Mukai Risa , Usui Soichiro , Zhai Peiyong , Nakada Yasuki , Sadoshima Junichi
Published: October 2024

Introduction:
Mammalian sterile 20-like kinase 1 (Mst1), a major component of the Hippo pathway, promotes cell death and differentiation, but inhibits cell growth, thereby contributing to the development of heart failure. We discovered that Mst1 activates protein kinase RNA-like endoplasmic reticulum kinase (PERK), a kinase responsible for the integrated stress response (ISR).

Aim:
We investigated whether Mst1-induced PERK activation is involved in the development of heart failure.

Methods and results:
Mst1 induces phosphorylation of the cytoplasmic domain of PERK, thereby activating it. We generated knock-in mice expressing a PERK mutant, which is not phosphorylated by Mst1, (PERK-KI). Although PERK was activated by tunicamycin, an ER stress inducer, it was not activated by transverse aortic constriction (TAC) in PERK-KI mice
in vivo
. To investigate the effect of Mst1-induced PERK phosphorylation on cardiac function, cardiac-specific Mst1 transgenic mice (Tg-Mst1) mice were crossed with heterozygous (h) PERK-KI mice (Tg-Mst1-hPERK-KI). The level of phosphorylation of eIF2α, a PERK substrate, was decreased in Tg-Mst1-hPERK-KI mice compared to in Tg-Mst1 mice (0.45-fold, p<0.05). Left ventricular ejection fraction (LVEF) was maintained in Tg-Mst1-hPERK-KI mice compared to in Tg-Mst1 mice (46% vs Tg-Mst1 34%, p<0.05), which was accompanied by reduced fibrosis (3.5% vs Tg-Mst1-hPERK-KI 2.2%, p<0.05) in the heart. We investigated the role of the Mst1-PERK pathway during TAC. Mst1 in wild-type (WT) mice was activated by TAC for 4 weeks (2.4-fold). LVEF was preserved in hPERK-KI mice compared to in WT mice (67% vs WT 46%, p<0.05) after 8 weeks of TAC. Increases in lung weight/tibia length were attenuated in hPERK-KI mice than in WT mice (9.6 vs WT 11.5, p<0.05).

Conclusion:
Mst1 triggers ISR through phosphorylation of PERK, which in turn plays a significant role in mediating heart failure during pressure overload.
Abstract Tu075: Cardiac macrophages expressing CD206 and IL-4Ra are required for adverse LV remodeling in HF
Authors: Wang Qiongxin , Ismahil Ameen , Rokosh Gregg , Hamid Tariq , Zhou Guihua , Zhu Yujie , Prabhu Sumanth
Published: October 2024

Cardiac macrophages significantly expand in chronic heart failure (HF). We tested the hypothesis that CD206
+
macrophages expressing interleukin (IL)-4Ra in the failing heart are key drivers of left ventricular (LV) remodeling and ischemic cardiomyopathy. Cardiac macrophages were profiled using flow cytometry in adult male C57BL/6 (WT) mice following non-reperfused myocardial infarction (MI) to induce HF or sham operation. CD206
+
macrophages progressively expanded in post-MI hearts during the progression of LV remodeling and comprised ~85% of macrophages at 8 w. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2)
–
and major histocompatibility complex (MHC)II
hi
in failing hearts, with the majority (92%) of CD206
+
CCR2
–
macrophages expressing LYVE-1, an embryonically-derived resident macrophage marker. Nearly half of cardiac CD206
+
macrophages expressed IL-4Ra; the abundance of CD206
+
IL-4Ra
+
directly correlated with LV dysfunction and fibrosis. Intramyocardial adoptive transfer of IL-4-polarized bone marrow derived CD206
+
macrophages induced progressive LV dilation, dysfunction and fibrosis over 4 w in naïve recipient mice. To evaluate the role of CD206
+
IL-4Ra
+
macrophages in LV remodeling, we used male and female LysM
CreERT2
;IL-4Ra
F/F
mice that allow for inducible myeloid-specific IL-4Ra gene deletion. Administration of tamoxifen from at 4 to 10 w post-MI (in established HF), effectively deleted IL-4Ra in cardiac macrophages and significantly reduced CD206
+
macrophage abundance and proliferation in the remodeling heart. Myeloid-specific IL-4Ra deletion further prevented LV remodeling progression, improved fibrosis and neovascularization, and alleviated LV systolic dysfunction, while suppressing expansion of cardiac immune cells and blood Ly6C
high
monocytosis.
We conclude
that an expanded and proliferative population of CD206
+
IL-4Ra
+
macrophages primarily derived from resident macrophages are key mediators of pathological LV remodeling and fibrosis. Inhibition of CD206
+
macrophage IL-4Ra signaling may be a fruitful therapeutic approach to alleviate disease progression in HF.
Abstract Tu085: The β
IV
-spectrin/STAT3 Complex regulates the orientation of cardiac hypertrophic growth
Authors: Nassal Drew , Mohta Shivangi , Shaheen Rebecca , Buck Benjamin , Lococo Simon , Leahy Nicholas , Winkle Alex , xu Xianyao , Hund Thomas
Published: October 2024

Background:
Cardiac hypertrophy is a major risk factor for adverse cardiovascular events including heart failure and arrhythmia. However, the precise orientation of cell and organ growth varies considerably depending on stress type and duration with important implications for cardiac function. Despite this, little is known regarding the mechanisms that regulate hypertrophic orientation. Here we evaluate the role of the cytoskeletal protein β
IV
-spectrin and signal transducer and activator of transcription (STAT3) in directing the orientation of pressure overload-induced hypertrophy.

Methods:
Transgenic mouse models with altered STAT3 signaling through modified interaction with its scaffolding partner β
IV
-spectrin, or phospho-regulation of STAT3 directly, were evaluated at baseline and transaortic constriction (TAC) for its role in promoting concentric versus eccentric morphologies and resulting impact on systolic function. Unbiased screening of gene expression from these structurally divergent states were evaluated for pathways responsible for directing myocyte length/width. These pathways were tested
in vitro
using primary mouse myocytes and
in vivo
to tune growth patterns for therapeutic intervention.

Results:
Loss of β
IV
-spectrin or direct STAT3 activation promoted a preferential increase in myocyte length over width, resulting in dilation of the left ventricular (LV) chamber (eccentric hypertrophy) and decreased systolic function. Conversely, preservation of β
IV
-spectrin favored an increase in myocyte width without LV dilation (concentric hypertrophy) and preserved systolic function in response to TAC. Differential expression of genes associated with microtubule dynamics were identified in concentric vs. eccentric hypertrophic states. In vitro assays revealed a relationship between β
IV
-spectrin/STAT3 signaling and microtubule stability which impacted spatial distribution of mRNA for the sarcomeric gene
actc1
. Finally, intervention with pharmacologic STAT3 inhibition following chronic 6-week TAC successfully recovered concentric growth with improved systolic function.

Conclusions:
These data identify a novel and pivotal role for β
IV
-spectrin/STAT3 to modify microtubule dynamics and sarcomeric transcript distribution to direct myocyte geometry and therapeutically serve in the prevention or recovery from HF. These mechanisms further illustrate the unique separation of hypertrophic drivers from growth orientation as distinct pathways in cardiac remodeling.
Abstract Tu087: Knockout of TIGAR Rescues Cardiac Dysfunction in SIRT3 Knockout Mice
Authors: Besanson Jessie , Cantrell Aubrey , Williams Quinesha , Zeng Heng , Chen Jian-Xiong
Published: October 2024

Introduction:
Heart failure (HF) is the leading cause of death in the United States, despite advances in treatment, it maintains the highest disease mortality rate in the country. Previously it has been established that Sirtuin 3 (SIRT3), a mitochondrial NAD+- dependent deacetylase, and the Tp53-induced glycolysis and apoptosis regulator (TIGAR) are associated with the development of HF. SIRT3 plays a key role in the maintenance of cardiac function. Decreases in SIRT3 are associated with human aging and diabetes, whereas knockout results in cardiac dysfunction in mice. We have shown: (1) overexpression of SIRT3 downregulates TIGAR and attenuates diabetic cardiomyopathy; (2) knockout of TIGAR enhances myocardial glycolysis and reduces pathological remodeling while maintaining cardiac function in pressure-overload-induced heart failure. To date, the direct interactions between TIGAR and SIRT3 in the development of HF remain unexplored.

Aim:
By crossing TIGAR knockout mice (TIGARKO) with SIRT3 knockout (SIRT3KO) mice, this study tested if genetic targeting of TIGAR could rescue cardiac dysfunction in SIRT3KO mice.

Methods and Results:
Echocardiography was performed on SIRT3KO, TIGARKO, TIGARKO/SIRT3KO, and SIRT3 Control mice. Analysis of cardiac function showed that SIRT3KO mice had significant decreases in coronary flow reserve (CFR), ejection fraction (EF) SIRT3KO (41.52 ± 4.50) vs SIRT3 Control (60.91 ± 4.91, p<0.001) and fractional shortening (FS) SIRT3KO (20.11 ± 2.48) vs SIRT3 Control (32.07 ± 3.37, p<0.001). The diastolic E’/A’ ratio was decreased; IVRT and MPI were significantly increased. Knockout of TIGAR in mice maintained systolic function by increasing EF (62.25 ± 9.84) vs (41.52 ± 4.50, p<0.001), FS (33.33 ± 7.12 vs 20.11 ± 2.48, p<0.05), and CFR, as well as improved diastolic function by decreasing IVRT, MPI, and increasing E’/A’ ratio in SIRT3KO mice. SIRT3KO and TIGARKO mice had a significant increase in weight/tibia length ratio (HW/TL). Interestingly, the knockout of TIGAR significantly reduced LV mass and HW/TL in SIRT3KO mice.

Conclusions:
The knockout of TIGAR can rescue the HF phenotype of SIRT3KO mice, but the underlying mechanisms require further investigation to develop potential treatments for HF.
Abstract Tu091: Mechanism of beta1-adrenergic receptor signaling by IgG3 subclass of autoantibodies
Authors: Mohan Maradumane , Liu Chia-Feng , Ashok Anushruti , Khuraijam Dhanachandra Singh , Karnik Sadashiva , Tang Wai Hong , Naga Prasad Sathyamangla
Published: October 2024

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop (ECL) 2 of β1-adrenergic receptor (β1AR) is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. We have previously shown that IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blocker compared to non-IgG3(+) β1AR AAbs. Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, it is critical to determine the mechanistic basis of the unique beneficial signaling pathway that may have therapeutic potential. HEK 293 cells stably expressing human β1AR (HEK-β1AR) was generated and treated with β1AR AAbs. Minimal changes in phosphorylation of β1AR was observed following IgG3(+) β1AR AAbs, while significant phosphorylation of β1ARs was observed with non-IgG3(+) β1AR AAbs. To dissect the diverse signaling responses mediated by IgG3(+) and non-IgG(+) β1AR AAbs, HEK-β1AR cells were treated with IgG3(+) or non-IgG3(+) β1AR AAbs. β1ARs were immunoprecipitated and subjected to mass-spectrometry analysis to identify unique co-immunoprecipitating proteins that could complex with receptor in presence of IgG3(+) or non-IgG3(+) β1AR AAbs. One of the proteins that was differentially recruited to the β1AR complex was Insulin Receptor Substrate 4 (IRS4). Traditionally, IRS4 is a signaling hub downstream of insulin/insulin growth factor receptors mediating glucose uptake. The differential interaction of IRS4 with β1AR in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs reflects that IRS4 could be a key signaling regulator of uniquely biased signal transduction pathway in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs. Mechanisms underlying this unique IRS4 mediated signaling modulation will be discussed.
Abstract Tu092: Role of Z-disc as a Driver of Hypertrophy and Sarcomere Assembly in Hypertrophic Cardiomyopathy
Authors: GIRI PRERNA , Zaoralova Magda , Fajardo Giovanni , Heinrich Paul , Wu Sean , Dahlberg Peter , Dunn Alexander , Bernstein Daniel
Published: October 2024

The Z-disc is an electron-dense structure demarcating the lateral boundaries of the sarcomere and postulated to play a key role in both cell signaling and sarcomere assembly. We performed EM on both HCM patient septal myectomies and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with 3 different HCM MYH7 mutations, finding marked Z-disc widening along with a hallmark feature of HCM, myofibrillar disarray. Mutations in MYH7 that cause HCM do so by increasing force at the sarcomere level, leading to the proposal that the Z-disc may act as a mechanosensor that converts myosin-generated force into the intracellular signaling that drives hypertrophy. We focused on understanding the mechanisms of Z-disc mechanotransduction of both hypertrophy and sarcomere disarray. RNA-seq from HCM myectomies demonstrated 52 Z-disc differentially expressed genes (DEGs). scRNA-seq from HCM hiPSC-CMs identified 24 Z-disc DEGs. 14 Z-disc DEGs were shared across both datasets, most notably 5 upstream regulators of calcineurin signalling. To correlate changes in gene expression with altered force we transfected hiPSC-CMs with a FRET-based tension sensor in the Z-disc protein vinculin allowing the direct measurement of force experienced by the Z-disc. Live-cell confocal imaging of hiPSC-CMs expressing EGFP-α-actinin-2 showed that indistinct α-actinin-2 foci coalesced into sarcomeres over the course of 24-48 h after replating. Imaging at nm scale using cryo-electron tomography revealed marked disorder in
individual thick filaments
in HCM hiPSC-CMs,
distinct from the classical disarray at the level of whole myofibrils
and which would not be visible on conventional EM. Our data suggest that early alterations in sarcomeric micro-structure and signaling induced by altered myosin force may precede hypertrophy and could occur decades before clinical signs (hypertrophy on ECG or echo) are present, with potential implications for early preventive intervention.
Abstract Tu094: p53 Regulates the Extent of Fibroblast Proliferation and Fibrosis in Left Ventricle Pressure Overload
Authors: Liu Xiaoyi , Burke Ryan , Lighthouse Janet , Baker Cameron , Dirkx Ronald , Kang Brian , Chakraborty Yashoswini , mickelsen deanne , twardowski jennifer , mello Stephano , ashton John , Small Eric
Published: October 2024

Background:
Cardiomyopathy is characterized by the pathological accumulation of resident cardiac fibroblasts that deposit ECM (extracellular matrix) and generate a fibrotic scar. However, the mechanisms that control the timing and extent of cardiac fibroblast proliferation and ECM production are not known, hampering the development of antifibrotic strategies to prevent heart failure.

Methods:
We used the Tcf21 (transcription factor 21) MerCreMer mouse line for fibroblast-specific lineage tracing and p53 (tumor protein p53) gene deletion. We characterized cardiac physiology and used single-cell RNA-sequencing and in vitro studies to investigate the p53-dependent mechanisms regulating cardiac fibroblast cell cycle and fibrosis in left ventricular pressure overload induced by transaortic constriction.

Results:
Cardiac fibroblast proliferation occurs primarily between days 7 and 14 following transaortic constriction in mice, correlating with alterations in p53-dependent gene expression. p53 deletion in fibroblasts led to a striking accumulation of Tcf21- lineage cardiac fibroblasts within the normal proliferative window and precipitated a robust fibrotic response to left ventricular pressure overload. However, excessive interstitial and perivascular fibrosis does not develop until after cardiac fibroblasts exit the cell cycle. Single-cell RNA sequencing revealed p53 null fibroblasts unexpectedly express lower levels of genes encoding important ECM proteins while they exhibit an inappropriately proliferative phenotype. in vitro studies establish a role for p53 in suppressing the proliferative fibroblast phenotype, which facilitates the expression and secretion of ECM proteins. Importantly, Cdkn2a (cyclin-dependent kinase inhibitor 2a) expression and the p16Ink4a-retinoblastoma cell cycle control pathway is induced in p53 null cardiac fibroblasts, which may eventually contribute to cell cycle exit and fulminant scar formation.

Conclusions:
This study reveals a mechanism regulating cardiac fibroblast accumulation and ECM secretion, orchestrated in part by p53-dependent cell cycle control that governs the timing and extent of fibrosis in left ventricular pressure overload.
Abstract Tu096: The Aryl Hydrocarbon Receptor Agonist, L-Kynurenine, Modulates Cardiac Fibroblast Activation and Antigen Presentation
Authors: Theall Brandon , Smolgovsky Sasha , Bayer Abraham , Sanders Erin , Zambrano Maria , Robbe Zachary , aronovitz mark , kaur Kuljeet , Alcaide Pilar
Published: October 2024

Background:
The Aryl Hydrocarbon Receptor (AHR) is a ubiquitously expressed transcription factor that regulates the cellular response to external stimuli through the binding of ligands including tryptophan metabolites such as L-Kynurenine (L-Kyn). Alterations in tryptophan metabolic pathways and downregulation of myocardial AHR are associated with cardiac inflammation and fibrosis in experimental heart failure (HF) induced by transverse aortic constriction (TAC). We hypothesized cardiac fibroblast (CFB) AHR and its agonism by L-Kyn prevent the pro-fibrotic and pro-inflammatory activity of CFBs in experimental HF.

Methods and Results:
We performed TAC or sham surgery on
Ahr -/-
mice and WT littermates for 4 weeks and found that
Ahr-/-
mice showed significantly decreased contractile function measured by echocardiography, and increased collagen deposition in left ventricular histological sections after TAC compared to WT mice. We performed bulk RNA sequencing in sorted CFB (CD31-CD45-MEFSK4+) from
Ahr
-/- and WT mice. Gene set enrichment analysis showed
Ahr -/-
CFB had increased expression in pathways for fibroblast activation, chemokine production, and immune cell recruitment.

To investigate the role of L-Kyn agonism of CFB AHR in transformation and contractile function, murine CFB were treated with Transforming Growth Factor β (TGFβ) (100ng/ml) or seeded in collagen discs, in the presence or absence of L-Kyn(100ug/ml). L-Kyn attenuated TGFβ mediated induction of αSMA and collagen 1 protein expression by immunofluorescence and western blot, as well as decreased disc contraction 72h after mechanical activation and release.

The role of AHR agonism by L-Kyn on the pro-inflammatory function of CFB was determined in WT CFB treated with IFNγ (100U/ml) in the presence or absence of L-Kyn. IFNγ induction of chemokines
Cxcl9
and
Cxcl10,
as well as MHC-II surface expression were attenuated by L-Kyn, so was T cell adhesion to CFB in co-culture assays. In contrast, L-Kyn had no effect on the ability of CFB to take up and process antigens, as determined by DQ-Ovalbumin internalization quantified by flow cytometry.

Conclusions:
Our results demonstrate that AHR regulates activation and pro-inflammatory signatures in CFB at baseline, and that stimulation of CFB transformation and pro-inflammatory activity is dampened by agonism with L-Kyn. Current studies are aimed at investigating AHR agonism in the context of pressure overload induced HF in CFB reporter mice.
Abstract Tu101: Measuring Fibrosis Progression in Duchenne Cardiomyopathy Using Cardiac Magnetic Resonance in mice
Authors: Hawkins India , Mitchell Josh , Mauro Adolfo , Carter Nigeste , Corwin Frank , Salloum Fadi , Raucci Frank
Published: October 2024

Introduction:
Transthoracic echocardiogram (TTE) is currently the gold standard for measuring cardiac function in mouse models of cardiomyopathy, however, it has its limitations. Challenges include poor acoustic windows, lower reproducibility, larger margin of error, and inability to accurately detect fibrosis. The detection of fibrosis is critical for assessing the severity of cardiac involvement and monitoring disease progression in Duchenne muscular dystrophy (DMD). Cardiac magnetic resonance imaging (CMR) can be used to more accurately measure cardiac function, chamber volumes, and tissue characterization to monitor the progression of fibrosis, including with parametric T1 mapping and late gadolinium enhancement (LGE). In this study, we have used a dystrophic mouse model (D2.
mdx
) on the DBA/2J genetic background as a preclinical model of DMD.

Hypothesis:
We hypothesize that CMR can detect worsening cardiac function and signs of fibrosis in the left ventricle of D2.
mdx
mice compared to wildtype (DBA) controls.

Methods:
In a study group of 12 age-matched male mice (8 mdx, 4 DBA), CMR was performed with contrast to determine cardiac function and fibrotic developments in the heart.

Results:
D2.
mdx
mice displayed a higher left ventricular end-diastolic volume compared to DBA mice (32.8 ± 3.6 µL vs 27.4 ± 1.5 µL,
p
< 0.05). Global longitudinal strain was decreased in D2.
mdx
compared to DBA mice (-11.0 ± 3.5% vs -18.2 ± 2.2%,
p
< 0.05). Native T1 mapping (Fig 1A) showed significantly higher mean T1 times in D2.
mdx
mice compared to DBA controls (Fig 1B). LGE in the myocardium of the left ventricle was noted in 6 of 8 of the D2.
mdx
mice (Fig 1C), but not in DBA mice. Logistic regression modeling showed strong predictive value detecting LGE with increasing native T1 times (Fig 1D).

Conclusion:
CMR can detect tissue changes in D2.
mdx
myocardium representing fibrotic changes. Native T1 may be useful in longitudinal studies of antifibrotic therapies in these models.
Abstract Tu102: The Role of the β
IV
-spectrin/STAT3 Complex in Regulating Ischemic Cardiac Remodeling
Authors: Shaheen Rebecca , Nassal Drew , Rivera Riece , Lococo Simon , xu Xianyao , Hund Thomas
Published: October 2024

Adverse fibrotic remodeling contributes to high morbidity and mortality in patients following myocardial infarction (MI). The healing process after MI requires necrotic tissue to be cleared and replaced with fibrosis, which is driven by the coordinated effort of several distinct cardiac cell populations, including macrophages and cardiac fibroblasts (CFs). While previous studies have focused on identifying the stimuli that drives fibrotic remodeling in the infarcted myocardium, the contribution of specific cell populations to the signaling cascade and the manner of how these communication networks are tuned in response to chronic stress remains unclear. Prior work has identified a role for the cytoskeletal protein, β
IV
-spectrin, in coordinating the activity of the transcription factor, STAT3, with implications for CF activation and profibrotic signaling. Specifically, it was found that mice expressing truncated β
IV
-spectrin (

qv
3J

) are resistant to cardiac fibrosis in the setting of chronic pressure overload, with maintained β
IV
-spectrin expression and STAT3 localization/activity in CFs. Further, β
IV
-spectrin deficiency causes increased secretion of profibrotic and proinflammatory factors, including matrix metallopeptidases (MMPs). Therefore, we hypothesized that stress-induced loss of β
IV
-spectrin and subsequent dysregulation of STAT3 signaling in CFs proximal to the infarct promotes fibroblast activation, an important first step in repairing the infarct region. In this study, we subjected WT and

qv
3J

mice to permanent ligation of the left anterior descending artery and assessed cardiac function and survival through 28 days. A subset of mice were sacrificed at 7 days for histology, qPCR, and flow cytometry. Compared to WT,

qv
3J

mice showed a significant decrease in 28-day survival with increased mortality in the first week post-MI. Autopsy identified increased incidence of cardiac rupture in

qv
3J

animals. Immunohistochemistry revealed impaired scar formation at 7 days post-MI in

qv
3J

compared to WT. Quantitative PCR showed altered expression of matrix metallopeptidases (MMPs 3, 8, 9, and 14) in the

qv
3J

hearts at day 7. MMP activity was assessed in whole heart lysates using gelatin-zymography. Collagen uptake was assessed in CFs and bone marrow-derived macrophages isolated from WT and

qv
3J

mice. Our work identifies a novel role of the spectrin-based pathway in facilitating fibrotic remodeling in response to ischemic stress.
Abstract Tu116: Neurofibromin 2 regulates metabolic gene expression and cardiac responses to pressure overload stress
Authors: Zhang Yu , Mareedu Satvik , Mizushima Wataru , Francisco Jamie , Guan Jin , Yang Zhi , Oka Shinichi , Zhai Peiyong , Abdellatif Maha , Sadoshima Junichi , Del Re Dominic
Published: October 2024

Background:
Neurofibromin 2 (NF2) is a tumor suppressor that can engage multiple signaling pathways to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and cardiac injury caused by acute myocardial infarction.

Research Aim:
The role of NF2 in heart failure remains uncharacterized. This study sought to determine whether NF2 modulates heart failure due to chronic stress.

Approach:
We generated cardiomyocyte-specific NF2 knockout (cKO) mice, and used transverse aortic constriction (TAC) to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. Complementary cell-based experiments were performed in neonatal rat ventricular myocytes (NRVMs). We analyzed cardiac function by echocardiographic and hemodynamic analysis. We used RNAseq, ChIP, Seahorse metabolic profiling, and biochemical analyses to investigate underlying mechanisms.

Results:
We found that NF2 is transiently upregulated in wild-type mouse myocardium in response to early phase of PO, but is downregulated during heart failure. Following TAC, NF2 cKO hearts unexpectedly showed significantly worsened cardiac function, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways and impaired ERR activity in NF2 cKO hearts. qPCR confirmed significantly reduced ERRβ and ERRγ transcripts and decreased metabolic gene expression. Experiments employing NRMVs confirmed that NF2 promoted expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Analysis of NRVM bioenergetics demonstrated that NF2 depletion impaired mitochondrial respiration, which was reversed by concomitant expression of ERRγ. Moreover, AAV9-mediated restoration of ERRγ in NF2 cKO mice normalized cardiac function in response to PO. Because NF2 does not directly bind DNA, ongoing studies are leveraging a proteomics-based approach to identify the mediator linking NF2 to ERR isoform expression.

Conclusion:
Based on these findings, we conclude that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.
Abstract Tu117: Unraveling the Role of Mitochondrial Ribosomal Protein L7/L12 (MRPL12) in Diabetic Cardiomyopathy
Authors: Rai Amit , Sanghvi Shridhar , Natrajseenivasan Suriya Muthukumaran , Chandrasekera Dhananjie , Kadam Ashlesha , Kyriazis Ioannis , Gopalkrishna Sreejit , Tomar Dhanendra , Ponnalagu Devasena , Khan Mahmood , Singh Harpreet , Nagareddy Prabhakara , Goukassian David , Katare Rajesh , Garikipati Venkata
Published: October 2024

Diabetic cardiomyopathy (DMCM) is a major cause of death in diabetic patients. Emerging evidence shows that mitochondrial dysfunction contributes to heart failure in diabetes. However, the molecular mechanisms of mitochondrial dysfunction mediating heart failure in diabetes are still poorly understood. The current study aimed to investigate the role of mitochondrial ribosomal protein L7/L12 (MRPL12) in human and mouse models of type II diabetes (db/db mice, and high-fat diet fed (HFD) mice) with or without induction of myocardial infarction (MI). Mitochondrial respiration was measured by using sea horse and mitochondrial membrane potential was measured by using confocal microscopy. Data was analyzed by using the mean of the groups was compared using a student t-test (for 2 groups) and ANOVA. We found increased MRPL12 levels in diabetic patients with ischemic heart disease compared to non-diabetic patients. Further we found elevated MRPL12 levels in the LV tissue of HFD fed mice with MI compared to low-fat diet-fed mice with MI. With the overexpression of MRPL12 under hyperglycemic conditions, the level of oxidative phosphorylation (OXPHOS) was found downregulated, but cellular ATP and human cardiomyocyte cell death remained unchanged, However, there was notable impairment in mitochondrial membrane potential (MMP) in hyperglycemia condition, along with changes in basal respiration oxygen consumption rate (OCR) and maximal respiratory capacity OCR. Overall, our results suggest MRPL12 may have a compensatory role in the diabetic hearts with ischemic heart disease. Therefore, our finding proposes new insights into the role of MRPL12 in the pathophysiology of MI in diabetes.
Abstract Tu120: Sarm1 promotes diabetic cardiomyopathy by regulating HIF signaling, lipotoxicity and mitochondrial dysfunction
Authors: Minor Keaton , Nizami Hina Lateef , Gorecki Ignacy , Light Christine , Gu Haiwei , Lee Chi Fung
Published: October 2024

Heart failure occurs at twice the rate in diabetic patients as compared to normal patients, and reduced NAD levels are associated with diabetic cardiomyopathy. Sterile Alpha and Tir Motif Containing 1 (SARM1) is an intracellular NAD hydrolase. We hypothesized that SARM1 promotes NAD decline and diabetic cardiomyopathy. We subjected wild type (WT) and global SARM1 knockout mice to chronic diabetic stress induced by streptozotocin injections. We showed that 16-week diabetic stress promoted progressive decline in systolic and diastolic function, and in NAD levels, which were ameliorated by SARM1 deficiency despite similar plasma glucose and lipid levels. Gene Module Network Analysis of cardiac transcriptomics identified upregulation of genes in fatty acid and glucose metabolic processes, and suppression of genes in mitochondrial processes in diabetic WT hearts. SARM1 deficiency reversed upregulated fatty acid metabolic genes but had no impact on glucose metabolic and mitochondrial gene expressions in diabetic hearts. By transmission electron microscopy and Oil Red O staining, we observed increases in lipid droplet sizes and lipid accumulation in diabetic WT hearts, which were suppressed by SARM1 deficiency. SARM1 deficiency did not reverse mitochondrial gene expression, but it mitigated the reduction in ADP-driven respiration, mitochondrial fragmentation, and abnormal cristae structure in diabetic WT hearts. Diabetic WT hearts had increased phospho-PDH and PDK4 levels as well as activated HIF signaling, exhibiting an increase in expression of HIF-1a target genes (55 genes) , many of which were related to lipid storage. SARM1 deficiency suppressed expression of HIF1-a target genes (53 genes) , and reduced phospho-PDH and PDK4 levels. These data suggest that SARM1 regulates pseudohypoxic HIF-dependent signaling in diabetic hearts. In summary, our current data support the hypothesis that SARM1 regulates HIF-dependent transcription events, particularly those influencing lipid metabolism of diabetic hearts. However, SARM1 promotes mitochondrial dysfunction in diabetic hearts independent of transcription of mitochondrial genes. Further studies will continue to elucidate the role of SARM1 in diabetic cardiomyopathy, and its potential as a novel therapeutic target.
Abstract Tu121: Investigating Mechanisms Of MCUB Inhibition Of Mitochondrial Calcium Uptake
Authors: Rai Neeraj , Chaudhuri Dipayan
Published: October 2024

The mitochondrial calcium uniporter mediates Ca
2+
entry into the matrix, and matches energetic supply to demand during cardiac activity. However, many cardiac diseases are characterized by mitochondrial Ca
2+
overload, such as ischemia/reperfusion (I/R) injury. The uniporter channel is typically formed by a tetramer of MCU subunits. These subunits have two transmembrane domains and create a pore lined with negative charges to allow rapid and selective Ca
2+
permeation. Intriguingly, many studies have shown that a close homolog, MCUB can potentially oligomerize with MCU subunits to alter the pore. When the tetramer is made of MCU Ca
2+
transmission is facilitated, but the presence of MCUB inhibits Ca
2+
intake through the channel, suggesting that it is a dominant negative subunit. Moreover, recent studies in animals subject to ischemic cardiovascular injury has revealed endogenous upregulation of the MCUB subunit, proposed as an endogenous mechanism to prevent Ca
2+
overload. Deletion of MCUB enhances activation and uptake when exposed to high Ca
2+
, making MCUB
−/−
cells prone to mitochondrial Ca
2+
overload and pore opening. Here, we investigate the mechanisms by which MCUB regulates the behavior of the channel, focusing on how MCUB may influence complex composition and regulate its targeting. For this study, we performed structure-function studies of reconstituted MCUB in MCU
-/-
MCUB
-/-
double knockout HEK 293T cells. Using modified MCUB constructs and a range of MCU to MCUB transfection ratios, we queried how many MCUB subunits can successfully incorporate into uniporter complex. We subsequently created a range of tetrameric concatemers featuring different ratios of MCU to MCUB, allowing us to observe that increasing amounts of MCUB incorporation lead to greater inhibition of mitochondrial Ca
2+
uptake. Finally, we examined whether replacing portions of MCUB with the corresponding MCU sequence was able to rescue Ca
2+
uptake, finding that these substitutions failed to significantly increase mitochondrial Ca
2+
uptake. Our findings clarify how MCUB incorporation into the uniporter complex leads to dominant-negative inhibition of mitochondrial Ca
2+
uptake.
Abstract Tu125: Loss of mitochondrial magnesium leads to hepatic ketogenic insufficiency and accelerates the progression of cardiac hypertrophy
Authors: Ponnusamy Thiruvelselvan , Santhanam Shanmughapriya
Published: October 2024

Magnesium (Mg
2+
) is an abundant intracellular divalent cation and is an important co-factor. Like calcium, Mg
2+
is also compartmentalized to mitochondria. Mrs2 is the only known molecular machinery associated with mitochondrial Mg
2+
influx. Although we know that Mg
2+
plays a crucial role in cellular homeostasis, our understanding of how mMg
2+
homeostasis alters the metabolic state of the cell remains incomplete. To begin elucidating the importance of mMg
2+
, we made a liver-specific Mrs2 knockout mouse model (Mrs2
Dhep
) using the Cre-loxP system. Since liver is the central hub of lipid metabolism, we hypothesized that loss of mitochondrial Mg
2+
could alter hepatic lipid homeostasis. Enigmatically we observed increased hepatic lipid accumulation, in spite of increased PPARa activation and Fatty acid oxidation (FAO). In our previous work we showed in Mrs2
Dhep
the FAO-derived acetyl CoA to be channeled to
de novo lipogenesis (DNL)
via cataplerosis, instead of ketogenesis. Further, our results show markedly blunted ketonemia in Mrs2
Dhep
revealing a state of ketogenic insufficiency. In the presence of increased ketone bodies (KB), normal hearts preferentially use both KB and FAO while glucose metabolism tend to be reduced. During the progression of heart failure (HF), the failing heart depends on alternate ATP generating substrates like KB as there is a metabolic deficit in both FAO and glucose metabolism. Because we saw a decrease in the KB, we hypothesize that hepatic ketogenic insufficiency will lead to cardiac hypertrophy and heart failure in pressure overload model due to energy insufficiency. Interestingly we found the heart weight and the heart to body weight ratio, left ventricular (LV) wall thickness, expression of genes linked to cardiac hypertrophy to be increased in Mrs2
Dhep
compared to control. We also observed an adverse structural remodeling of LV and LV dysfunction. Masson’s trichrome staining showed increased collagen deposition and fibrosis in the Mrs2
Dhep
mice when compared to the control. Our results show altered hepatic mitochondrial Mg
2+
homeostasis to have profound direct effect on hepatic lipid metabolism and indirect inter-organ effect on the heart following pressure overload.