Human Heart Failure Research Articles

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Myocardial DNA damage combined with genotype predicts heart failure outcome in various underlying diseases

Abstract Background Reliable predictors of treatment efficacy in heart failure have been long awaited. DNA damage has been implicated as a cause of heart failure. Purpose To investigate the association of DNA damage in myocardial tissue with treatment response and prognosis of heart failure from various etiologies; and to elucidate the relationships between genotypes and DNA damage, as well as their additive effect in prognosis prediction. Methods We performed immunostaining of DNA damage markers, poly(ADP-ribose) (PAR) and γ-H2A.X on biopsy specimens from 175 patients with heart failure with reduced ejection fraction (HFrEF) of various underlying etiologies. We calculated the percentage of nuclei positive for each DNA damage marker (%PAR and %γ-H2A.X). The primary outcome was left ventricular reverse remodeling (LVRR) at ∼1 year. The secondary outcome was a composite of cardiovascular death, heart transplantation, and ventricular assist device implantation. Whole-exome data from consenting patients were further analyzed to classify pathogenic/likely pathogenic (P/LP) variants in cardiomyopathy-related genes. We compared the degree of DNA damage among different genotypes, and examined the additive effect of combining DNA damage markers and genotypes in predicting treatment response. Results The participants had a mean age of 56 years, of whom 72% were men. Among them, 55% achieved LVRR at ∼1 year. Patients who did not achieve LVRR after the optimization of medical therapies presented with significantly higher %PAR and %γ-H2A.X when compared with those who did [%PAR, 11 (6–19) % vs. 2 (1–4) %, P < 0.001; %γ-H2A.X, 22 (11–40) % vs. 5 (2–9) %, P < 0.001] (Figure, left bottom). The receiver-operating characteristic (ROC) analysis demonstrated good performance of both %PAR and %γ-H2A.X for predicting LVRR (area under the curve 0.87 and 0.85, respectively). Patients with lower %PAR or %γ-H2A.X had reduced long-term clinical events [PAR, hazard ratio 1.63 (95%CI 1.31- 2.01), P < 0.001; γ-H2A.X, 1.48 (1.27-1.72), P < 0.001] (Figure, left bottom). In addition, there was a strong negative correlation between the mean proportion of DNA damage marker positive nuclei and the probability of LVRR, across different underlying diseases (Figure, middle). Genotypes were identified in 105 patients (Figure, right top). Non-sarcomeric mutation (LMNA, etc) carriers presented with more pronounced DNA damage than sarcomeric mutation carriers (TTN, MYBPC3) (Figure, right bottom). Combination of genotypes and %γ-H2A.X augmented the area under the ROC for predicting 1-year LVRR to 0.90, from that of %γ-H2A.X alone at 0.85. Conclusions The degree of DNA damage accumulation in myocardial tissue differs by underlying diseases and genotypes, and determines the consequences of human heart failure. Assessment of DNA damage, especially when combined with genotype, is useful to predict treatment efficacy and prognosis of HFrEF patients with various underlying etiologies.graphical abstract

Abstract 13305: Energetic Basis of Recovered Ejection Fraction in Human Heart Failure

Introduction: Left ventricular ejection fraction (LVEF) improvement in HFrEF patients is classified as heart failure with improved ejection fraction (HFimpEF) and is associated with better long-term outcomes than persistent HFrEF. HFimpEF is difficult to predict and underlying mechanisms remain poorly understood. Hypothesis: Better myocardial high-energy phosphate metabolism at baseline predicts future LVEF recovery and outcomes in a general HFrEF population Methods: We measured cardiac energetics, the ratio of the two main high-energy phosphates, phosphocreatine and ATP (PCr/ATP), and the rate of ATP synthesis through the primary cardiac energy reserve reaction creatine kinase (“CK flux”), in 37 nonischemic HFrEF patients (LVEF ≤40%) with 31 P cardiac magnetic resonance spectroscopy (MRS) at 3T. Baseline transthoracic echocardiogram (TTE) was obtained prior to 31 P MRS and follow-up consecutive TTEs with the two highest LVEFs meeting HFimpEF criteria (LVEF >40% and ≥10% increase over baseline LVEF) over a median follow-up of 6.9 years were averaged and used to classify HFimpEF. Results: There were no significant differences in demographics, HF etiology, baseline TTE metrics, or medications between persistent HFrEF (n=22) and eventual HFimpEF (n=15). Cardiac CK flux was significantly higher at baseline in patients who later recovered to HFimpEF than in those who remained HFrEF (p=0.031). Better baseline cardiac CK flux was associated with increases in LVEF (p=0.025) and decreases in LV end-diastolic diameter (p=0.016) over time. By both dichotomous and continuous metrics, better preserved CK flux was associated with future HFimpEF. Fourteen subjects (38%), all persistent HFrEF, experienced cardiac death, transplant, or LVAD and had significantly lower baseline CK flux than outcome-free patients (p=0.017). Cardiac PCr/ATP was not associated with eventual HFimpEF, outcomes, or changes in TTE indices. Conclusion: Better preserved cardiac CK flux is associated with future LVEF recovery and LV reverse remodeling while lower CK flux is associated with worse cardiac outcomes in HFrEF patients. Interventions restoring CK flux, and possibly other energy-generating metabolic reactions, may elicit myocardial recovery in HFrEF patients.

Abstract 18222: FAM210A Regulates Mitochondrial Translation and Maintains Cardiac Mitochondrial Homeostasis

Introduction: Mitochondria play a vital role in cellular metabolism and energetics and support cardiac function. FAM210A (family with sequence similarity 210 member A) is a novel hub gene in mouse cardiac remodeling. Human FAM210A mutations are associated with sarcopenia. However, the physiological and molecular function of FAM210A remain elusive in the heart. Hypothesis: We aim to determine the biological and molecular mechanism of FAM210A in regulating mitochondrial function and cardiac health. Methods and Results: Tamoxifen-induced conditional knockout of Fam210a in mouse cardiomyocytes (CMs) induced progressive dilated cardiomyopathy and heart failure (HF), ultimately causing mortality. Fam210a deficient CMs exhibit severe mitochondrial morphological disruption and functional decline accompanied by myofilament disarray at the late stage of cardiomyopathy. Moreover, we observed increased mitochondrial reactive oxygen species production, disturbed mitochondrial membrane potential, and reduced respiratory activity in CMs at the early stage before contractile dysfunction and HF. Multi-omics analyses indicate that FAM210A deficiency persistently activates integrated stress response (ISR), resulting in transcriptomic, translatomic, proteomic, and metabolomic reprogramming, ultimately leading to pathogenic progression of HF. Mechanistically, mitochondrial polysome profiling analysis shows that FAM210A loss of function compromises mitochondrial mRNA translation and leads to reduced mitochondrial encoded proteins, followed by disrupted proteostasis. We observed decreased FAM210A protein expression in human ischemic heart failure and mouse myocardial infarction tissue samples. To further corroborate FAM210A function in the heart, AAV9-mediated overexpression of FAM210A promotes mitochondrial-encoded protein expression, improves cardiac mitochondrial function, and partially rescues murine hearts from cardiac remodeling and damage in ischemia-induced HF. Conclusions: These results suggest that FAM210A is a mitochondrial translation regulator to maintain mitochondrial homeostasis and normal CM contractile function. This study also offers a new therapeutic target for treating ischemic heart disease.

Abstract 15535: SGK1 is a Key Mediator of Pathological Cardiac Fibrosis

Background: Cardiac fibroblasts initially respond to injury adaptively, proliferating and differentiating into myofibroblasts to maintain cardiac structure and function. However, prolonged cardiac stress leads to pathological cardiac fibrosis, ultimately resulting in heart failure (HF). Thus, there is a major interest in developing therapeutic strategies to constrain fibroblast differentiation and sustained activation. We recently identified the cytosolic protein kinase, serum/glucocorticoid regulated kinase 1 (SGK1), as being largely involved with pathological remodeling in the cardiac myocyte. While there have been reports that SGK1 may also play a role in fibrosis, the mechanistic role for SGK1 in cardiac fibroblasts in response to injury remains unknown. Hypothesis: SGK1 promotes pathological cardiac fibrosis and exacerbates progression to HF. Methods: Transforming growth factor beta, (TGFβ) was used in primary cardiac fibroblasts (CFBs), in vitro , and transverse aortic constriction (TAC) was used as a HF model in mice, in vivo . CFBs were transfected with siRNA to knockdown SGK1, in vitro , and SGK1 floxed mice were injected with a novel AAV9 utilizing a myofibroblast-specific promoter to knockout SGK1 specifically in myofibroblasts (SGK1 mfbKO), in vivo . Results: SGK1 signaling was elevated in cardiac explants obtained from ischemic human HF patients which led us to further evaluate SGK1 activity in CFBs. SGK1 knockdown in CFBs attenuated TGFβ-mediated differentiation and fibrotic signaling. SGK1 mfbKO mice subjected to TAC showed preserved systolic and diastolic cardiac function, measured by echocardiography. At the biochemical level, pathological remodeling and gene markers of fibrosis were attenuated in SGK1 mfbKO mice as determined by qRT-PCR of cardiac extracts. Finally, treatment of CFBs with a small molecule SGK1 inhibitor attenuated differentiation and fibrotic signaling in response to TGFβ treatment. Conclusion: SGK1 knockdown is protective against cardiac fibrosis, attenuating differentiation and fibrotic signaling in CFBs. Further, SGK1 mfbKO protected against pathological cardiac remodeling and functional decline in a mouse model of HF, highlighting the specific pathophysiological contributions of CFBs.

Myocardial DNA Damage Predicts Heart Failure Outcome in Various Underlying Diseases

BackgroundReliable predictors of treatment efficacy in heart failure have been long awaited. DNA damage has been implicated as a cause of heart failure. ObjectivesThe purpose of this study was to investigate the association of DNA damage in myocardial tissue with treatment response and prognosis of heart failure. MethodsThe authors performed immunostaining of DNA damage markers poly(ADP-ribose) (PAR) and γ-H2A.X in endomyocardial biopsy specimens from 175 patients with heart failure with reduced ejection fraction (HFrEF) of various underlying etiologies. They calculated the percentage of nuclei positive for each DNA damage marker (%PAR and %γ-H2A.X). The primary outcome was left ventricular reverse remodeling (LVRR) at 1 year, and the secondary outcome was a composite of cardiovascular death, heart transplantation, and ventricular assist device implantation. ResultsPatients who did not achieve LVRR after the optimization of medical therapies presented with significantly higher %PAR and %γ-H2A.X. The ROC analysis demonstrated good performance of both %PAR and %γ-H2A.X for predicting LVRR (AUCs: 0.867 and 0.855, respectively). There was a negative correlation between the mean proportion of DNA damage marker–positive nuclei and the probability of LVRR across different underlying diseases. In addition, patients with higher %PAR or %γ-H2A.X had more long-term clinical events (PAR HR: 1.63 [95% CI: 1.31-2.01]; P < 0.001; γ-H2A.X HR: 1.48 [95% CI: 1.27-1.72]; P < 0.001). ConclusionsDNA damage determines the consequences of human heart failure. Assessment of DNA damage is useful to predict treatment efficacy and prognosis of heart failure patients with various underlying etiologies.

The effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats treated with the ACE2 inhibitor MLN-4760

BackgroundAngiotensin converting enzyme 2 (ACE2) plays a crucial role in the infection cycle of SARS-CoV-2 responsible for formation of COVID-19 pandemic. In the cardiovascular system, the virus enters the cells by binding to the transmembrane form of ACE2 causing detrimental effects especially in individuals with developed hypertension or heart disease. Zofenopril, a H2S-releasing angiotensin-converting enzyme inhibitor (ACEI), has been shown to be effective in the treatment of patients with essential hypertension; however, in conditions of ACE2 inhibition its potential beneficial effect has not been investigated yet. Therefore, the aim of the study was to determine the effect of zofenopril on the cardiovascular system of spontaneously hypertensive rats, an animal model of human essential hypertension and heart failure, under conditions of ACE2 inhibition induced by the administration of the specific inhibitor MLN-4760 (MLN).ResultsZofenopril reduced MLN-increased visceral fat to body weight ratio although no changes in systolic blood pressure were recorded. Zofenopril administration resulted in a favorable increase in left ventricle ejection fraction and improvement of diastolic function regardless of ACE2 inhibition, which was associated with increased H2S levels in plasma and heart tissue. Similarly, the acute hypotensive responses induced by acetylcholine, L-NAME (NOsynthase inhibitor) and captopril (ACEI) were comparable after zofenopril administration independently from ACE2 inhibition. Although simultaneous treatment with zofenopril and MLN led to increased thoracic aorta vasorelaxation, zofenopril increased the NO component equally regardless of MLN treatment, which was associated with increased NO-synthase activity in aorta and left ventricle. Moreover, unlike in control rats, the endogenous H2S participated in maintaining of aortic endothelial function in MLN-treated rats and the treatment with zofenopril had no impact on this effect.ConclusionsZofenopril treatment reduced MLN-induced adiposity and improved cardiac function regardless of ACE2 inhibition. Although the concomitant MLN and zofenopril treatment increased thoracic aorta vasorelaxation capacity, zofenopril increased the participation of H2S and NO in the maintenance of endothelial function independently from ACE2 inhibition. Our results confirmed that the beneficial effects of zofenopril were not affected by ACE2 inhibition, moreover, we assume that ACE2 inhibition itself can lead to the activation of cardiovascular compensatory mechanisms associated with Mas receptor, nitrous and sulfide signaling.

Downregulation of apoptotic repressor AVEN exacerbates cardiac injury after myocardial infarction

Myocardial infarction (MI) is a leading cause of heart failure (HF), associated with morbidity and mortality worldwide. As an essential part of gene expression regulation, the role of alternative polyadenylation (APA) in post-MI HF remains elusive. Here, we revealed a global, APA-mediated, 3' untranslated region (3' UTR)-lengthening pattern in both human and murine post-MI HF samples. Furthermore, the 3' UTR of apoptotic repressor gene, AVEN, is lengthened after MI, contributing to its downregulation. AVEN knockdown increased cardiomyocyte apoptosis, whereas restoration of AVEN expression substantially improved cardiac function. Mechanistically, AVEN 3' UTR lengthening provides additional binding sites for miR-30b-5p and miR-30c-5p, thus reducing AVEN expression. Additionally, PABPN1 (poly(A)-binding protein 1) was identified as a potential regulator of AVEN 3' UTR lengthening after MI. Altogether, our findings revealed APA as a unique mechanism regulating cardiac injury in response to MI and also indicated that the APA-regulated gene, AVEN, holds great potential as a critical therapeutic target for treating post-MI HF.

Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A.

The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

FRI118 3D Reconstruction Of Mitochondria From Human Heart Failure Structures Is Associated With Alterations In CHCHD6 And CHCHD3 Expression

Abstract Disclosure: A.G. Marshall: None. Z. Vue, PhD: None. E. Garza-Lopez: None. H. Beasley, PhD: None. V. Exil: None. K. Actkins: None. T. Miller-Fleming: None. A.O. Hinton: None. Heart failure (HF) is accompanied by symptoms and signs caused by cardiac dysfunction, resulting in reduced longevity. Critically, HF has been associated with mitochondria dysfunction, causing pathophysiology that might contribute to age-dependent decrease in cardiac function. Thus, we hypothesize that reduced cardiac function in response to HF, affects mitochondrial function and morphology. To test this, we investigated mitochondrial morphological changes using 3D reconstruction from 4 control human hearts and 4 human heart failure patients. Mitochondrial samples were 3D rendered and quantified using serial block facing-scanning electron microscopy and the Amira software. From our HF reconstructions, there are many phenotypes, including fragmentation, nanotunnels, and mitochondrial clustering, compared to controls, which may be associated to mitochondrial dysfunction. Surprisingly, we saw altered expression of CHCHD6 and CHCHD3 in human heart failure samples, based on immunofluorescence. Thus, we knocked out CHCHD6 and CHCHD3 in mouse fibroblasts, myotubes and human-induced pluripotent stem cell-derived cardiomyocytes and saw fragmentation in all three models. Presently, HF represents an unmet need with no approved clinical therapies for damaged myocardium replacement. Here, we suggest that CHCHD3 and CHCHD6 may be a molecular target that functions in the development of HF and possibly develop novel therapeutics for the treatment of HF. Presentation: Friday, June 16, 2023

Measuring Cardiac Dyssynchrony with DENSE (Displacement Encoding with Stimulated Echoes)-A Systematic Review.

In this review, we introduce the displacement encoding with stimulated echoes (DENSE) method for measuring myocardial dyssynchrony using cardiovascular magnetic resonance (CMR) imaging. We provide an overview of research findings related to DENSE from the past two decades and discuss other techniques used for dyssynchrony evaluation. Additionally, the review discusses the potential uses of DENSE in clinical practice. A search was conducted to identify relevant articles published from January 2000 through January 2023 using the Scopus, Web of Science, PubMed and Cochrane databases. The following search term was used: (DENSE OR 'displacement encoding with stimulated echoes' OR CURE) AND (dyssynchrony* OR asynchron* OR synchron*) AND (MRI OR 'magnetic resonance' OR CMR). After removing duplicates, researchers screened a total of 174 papers. Papers that were not related to the topic, reviews, general overview articles and case reports were excluded, leaving 35 articles for further analysis. Of these, 14 studies focused on cardiac dyssynchrony estimation with DENSE, while the remaining 21 studies served as background material. The studies used various methods for presenting synchronicity, such as circumferential uniformity ratio estimate (CURE), CURE-singular value decomposition (SVD), radial uniformity ratio estimate (RURE), longitudinal uniformity ratio estimate (LURE), time to onset of shortening (TOS) and dyssynchrony index (DI). Most of the dyssynchrony studies concentrated on human heart failure, but congenital heart diseases and obesity were also evaluated. The researchers found that DENSE demonstrated high reproducibility and was found useful for detecting cardiac resynchronisation therapy (CRT) responders, optimising CRT device settings and assessing right ventricle synchronicity. In addition, studies showed a correlation between cardiac fibrosis and mechanical dyssynchrony in humans, as well as a decrease in the synchrony of contraction in the left ventricle in obese mice. DENSE shows promise as a tool for quantifying myocardial function and dyssynchrony, with advantages over other cardiac dyssynchrony evaluation methods. However, there remain challenges related to DENSE due to the relatively time-consuming imaging and analysis process. Improvements in imaging and analysing technology, as well as possible artificial intelligence solutions, may help overcome these challenges and lead to more widespread clinical use of DENSE.

Fibulin-3 is necessary to prevent cardiac rupture following myocardial infarction

Despite the high prevalence of heart failure in the western world, there are few effective treatments. Fibulin-3 is a protein involved in extracellular matrix (ECM) structural integrity, however its role in the heart is unknown. We have demonstrated, using single cell RNA-seq, that fibulin-3 was highly expressed in quiescent murine cardiac fibroblasts, with expression highest prior to injury and late post-infarct (from ~ day-28 to week-8). In humans, fibulin-3 was upregulated in left ventricular tissue and plasma of heart failure patients. Fibulin-3 knockout (Efemp1−/−) and wildtype mice were subjected to experimental myocardial infarction. Fibulin-3 deletion resulted in significantly higher rate of cardiac rupture days 3–6 post-infarct, indicating a weak and poorly formed scar, with severe ventricular remodelling in surviving mice at day-28 post-infarct. Fibulin-3 knockout mice demonstrated less collagen deposition at day-3 post-infarct, with abnormal collagen fibre-alignment. RNA-seq on day-3 infarct tissue revealed upregulation of ECM degradation and inflammatory genes, but downregulation of ECM assembly/structure/organisation genes in fibulin-3 knockout mice. GSEA pathway analysis showed enrichment of inflammatory pathways and a depletion of ECM organisation pathways. Fibulin-3 originates from cardiac fibroblasts, is upregulated in human heart failure, and is necessary for correct ECM organisation/structural integrity of fibrotic tissue to prevent cardiac rupture post-infarct.

Multi-omic analyses and network biology in cardiovascular disease.

Heart disease remains a leading cause of death in North America and worldwide. Despite advances in therapies, the chronic nature of cardiovascular diseases ultimately results in frequent hospitalizations and steady rates of mortality. Systems biology approaches have provided a new frontier toward unraveling the underlying mechanisms of cell, tissue, and organ dysfunction in disease. Mapping the complex networks of molecular functions across the genome, transcriptome, proteome, and metabolome has enormous potential to advance our understanding of cardiovascular disease, discover new disease biomarkers, and develop novel therapies. Computational workflows to interpret these data-intensive analyses as well as integration between different levels of interrogation remain important challenges in the advancement and application of systems biology-based analyses in cardiovascular research. This review will focus on summarizing the recent developments in network biology-level profiling in the heart, with particular emphasis on modeling of human heart failure. We will provide new perspectives on integration between different levels of large "omics" datasets, including integration of gene regulatory networks, protein-protein interactions, signaling networks, and metabolic networks in the heart.

Abstract 075: Ketone Bodies Rescue Mitochondrial Dysfunction And Improve Maladaptive Epigenetic Remodeling In Heart Failure

CATEGORY: Genetics, Epigenetics and Epidemiology Ketone Bodies Rescue Mitochondrial Dysfunction and Improve Maladaptive Epigenetic Remodeling in Heart Failure Fahimeh Varzideh 1 , Stanislovas S. Jankauskas 1 , Urna Kansakar 1 , Simone Sidoli 1 , Jessica Gambardella 1 , Gaetano Santulli 1 1 : Albert Einstein College of Medicine, 10461, New York, NY, USA Background: The exact molecular processes leading to the progression of post-ischemic heart failure (HF) are not fully understood. Methods: we carried out a comprehensive and coordinated set of in vivo and in vitro experiments using human cardiac specimens from patients with post-ischemic HF and healthy controls, a mouse model of HF, and mechanistic studies in vitro . Results: We identified, for the first time to our knowledge, a specific pattern of maladaptive chromatin remodeling, namely a double methylation of histone 3 at lysine 27 and one methylation of lysine 36 (H3_K27me2K36me1) consistently induced by ischemic injury in all these settings: human HF, murine HF, and in vitro models. To translate our findings in vivo , we used an established murine model of HF induced by myocardial infarction, obtained by permanent ligation of the left anterior descending coronary artery. After surgery, the mice were fed standard diet or diet supplemented with β-hydroxybutyrate (BHB), the most abundant ketone body in humans, for one month. Mechanistically, we demonstrate that the histone modification H3_K27me2K36me1 mediates the ischemia-induced transcriptional repression of PPARG Coactivator 1α (PGC1α), a master regulator of mitochondrial function and biogenesis. Strikingly, both the augmented H3_K27me2K36me1 and the PGC1α-mediated mitochondrial dysfunction were significantly attenuated by the treatment with BHB, revealing an unprecedented molecular pathway coupling metabolism to gene expression. The increased circulating levels of ketone bodies observed in HF patients treated with SGLT2 inhibitors further supports our preclinical results. Conclusion: Collectively, our findings establish maladaptive chromatin remodeling as a key mechanism in post-ischemic heart injury, functionally modulated by ketone bodies.

High expression of Circ-PALLD in heart failure is transcriptionally regulated by the transcription factor GATA4

To determine the changes in the expression of circular RNA Circ-PALLD in heart failure and explore the biogenesis of Circ-PALLD. We analyzed second-generation sequencing results of human and murine heart failure samples to identify the candidate CircRNAs. Sanger generation sequencing was performed after PCR amplification, and the sequencing results were compared to determine the reverse splicing pattern of the corresponding CircRNAs. We further examined the expressions of CircRNAs and linear RNAs in 8 patients with heart failure admitted in our hospital, and RT-qPCR was performed to detect the expression levels of Circ-PALLD and PALLD in the failing myocardium. Bioinformatic analysis was performed to predict the transcription factors that may regulate PALLD. Small interfering RNAs (siRNAs) against GATA4 were used to determine the regulatory effect of the transcription factor GATA4 on PALLD. Sanger sequencing and sequence alignment verified the reverse splicing of Circ-VWA8, Circ-VMP1, Circ-PRDM5, Circ-PLCL2, Circ-PALLD, Circ-NFATC3, Circ-MLIP, Circ-FAM208A, Circ-ANKIB1, and Circ-AGTPBP1, demonstrated their loop-forming nature and determined the exon arrangement of reverse splicing. Semi-quantitative PCR results showed that the expression levels of CircPALLD, Circ-NFATC3 and Circ-AGTPBP1 were significantly increased while the expression level of linear PALLD was significantly decreased in the myocardial tissues of heart failure patients. Bioinformatic analysis suggested that the transcription of PALLD was regulated possibly by the transcription factor GATA4. RT-qPCR showed that the expression level of Circ-PALLD was significantly increased, while PALLD expression was significantly decreased in the failing myocardium, which was consistent with the results of semi-quantitative PCR. In primary mammary rat cardiomyocytes, GATA4 knockdown resulted in lowered expressions of both Circ-PALLD and PALLD. Circ-PALLD is highly expressed in heart failure and can be used as a novel molecular marker for chronic heart failure, and GATA4 may play important role in regulating its transcription. Circ-PALLD points a new direction for investigating the molecular mechanism of heart failure and may also serve as a potential therapeutic target for heart failure.

Galectin-3 is able to differentiate dogs with myxomatous mitral valve disease from healthy control dogs.

Galectin-3 is a cardiac biomarker for heart failure in humans. However, it has not been investigated in dogs with naturally occurring heart disease. This study aimed to compare plasma galectin-3 concentration in healthy dogs and those with myxomatous mitral valve disease (MMVD) and explore the potential association of galectin-3 with other cardiac biomarkers, inflammatory cytokines, echocardiographic estimates, and dog characteristics. 10 healthy dogs and 30 dogs with MMVD were prospectively recruited. In this case-control study, plasma galectin-3, inflammatory cytokines, echocardiographic estimates, and other cardiac biomarkers were measured, and dog characteristics were recorded. Plasma galectin-3 concentration was significantly higher in dogs with MMVD (2.94 [interquartile range, 1.61 to 5.20] ng/mL) than in healthy controls (1.56 [0.69 to 1.84] ng/mL, P = .009). Logistic regression analysis revealed that galectin-3 concentration and age predicted the presence of MMVD (predictive accuracy = 90.0%, P < .05). A cut-off value ≥ 1.9 ng/mL for galectin-3 differentiated healthy dogs from dogs with MMVD (70% sensitivity; 90% specificity AUC, 0.77; P = .01). Plasma galectin-3 concentration was higher in dogs with MMVD than in healthy dogs, indicating that it is a novel cardiac biomarker in dogs with MMVD although there was no significant difference between MMVD stages.

SERCA2a Protein Levels Are Unaltered in Human Heart Failure.

SERCA2a Protein Levels Are Unaltered in Human Heart Failure.

Abstract P1151: Exploring The Role Of Bh3-like Motif Containing Cell Death Inducer In Cardiomyocyte Death: Insights From Human Failing Hearts And Ipsc-derived Cardiomyocytes

Background: Cardiac cell death plays a critical role in the development and progression of heart failure. Identifying new molecular targets to limit cardiac cell death could improve patient lives. BLID (BH3-Like Motif-Containing Cell Death Inducer) is a member of the BCL family of proapoptotic genes containing a BH3-like motif that induces cell death. Previous studies have shown that in many human cancers, downregulation of BLID is associated with tumor progression, while overexpression promotes cell death through a caspase-dependent mechanism. However, its role in cardiomyocyte death under various stress conditions is unknown. Methods and Results: Our RNAseq data from human failing and non-failing hearts revealed that mRNA expression of the BLID gene was significantly upregulated in human heart failure. We further validated our RNAseq findings by measuring the expression of BLID in RNA and protein isolated from the same set of human failing and non-failing hearts. As expected, the expression of BLID was significantly upregulated at both the mRNA and protein levels in human failing hearts compared to non-failing hearts. Furthermore, in cultured human cardiomyocytes (AC16 cells) and iPSC-derived cardiomyocytes exposed to stressors (such as hypoxia or H 2 O 2 ) for 2 hours, we observed a significant upregulation of BLID expression at both the mRNA and protein levels. We also overexpressed human BLID protein in human cardiomyocytes, leading to cardiomyocyte death. To determine their localization, we fractionated the overexpressed BLID proteins into cytoplasmic, nuclear, and mitochondrial fractions. We found that BLID was localized in the cytoplasm, nucleus, and mitochondria, with higher levels in the nucleus and mitochondria. These findings suggest that increased expression of BLID in failing human hearts may lead to cardiomyocyte death. Conclusion: Our data suggest that increased expression of BLID in human heart failure may be a key molecule that drives cardiomyocyte death. Inhibiting its expression during heart failure could mitigate cardiomyocyte death and improve cardiomyocyte function and survival.

Abstract P1183: Phosphorylation Signatures Of Human Heart Failure: Characterizing Dilated Cardiomyopathy-associated Signaling Pathways At The Cardiomyocyte Intercalated Disc

Heart failure remains a highly prevalent condition with diverse etiology, yet the underlying signaling mechanisms are not fully understood. Despite the profound effects of post-translational protein modifications on downstream signaling, limited studies have investigated the cardiac phosphoproteome in human heart failure. We hypothesized that a combined proteomic and phosphoproteomic analysis of human dilated (DCM) and ischemic (ICM) cardiomyopathy would reveal novel etiology-associated disease pathways. Integrative analyses of left ventricular explants from DCM patients ( n =4) vs. non-failing controls ( n =4), and left ventricular infarct vs. non-infarct, and peri-infarct vs. non-infarct regions of ICM patients ( n =4) identified 5,570 unique proteins with 13,624 corresponding phosphorylation sites. Each pair-wise comparison revealed shared and etiology-specific signatures, with a unique DCM-associated enrichment of cell-cell adhesion pathways. We focused our attention on αT-catenin (CTNNA3) as a cardiomyocyte intercalated disc candidate phosphoprotein with a unique cluster of 4 hyperphosphorylated sites in DCM hearts ( P &lt;0.0001). Overexpression of non-phosphorylatable hCTNNA3 in ex vivo isolated adult mouse cardiomyocytes showed internalized protein expression and weaker cell-cell adhesion vs. wildtype (WT) and phospho-mimetic forms. We established an in vivo mouse model using recombinant adeno-associated virus 9 (rAAV9) harboring hCTNNA3-WT, hCTNNA3-phospho-null, or empty rAAV9 control. Phospho-null CTNNA3 mice developed left ventricular dilation and contractile dysfunction (% EF; 51.25±1.17 phospho-null vs. 62.07±1.20 WT vs. 66.76±1.42 empty; n ≥10) with impaired left ventricular conduction velocity (cm/s; 36.83±1.10 phospho-null vs. 47.74±2.04 WT; n =6), by echocardiography and ex vivo optical mapping. Loss of CTNNA3 phosphorylation led to intercalated disc remodeling with internalization and dissociation of CTNNA3, connexin 43, N-cadherin, β-catenin, and plakophilin 2 from the adherens junction, using high-resolution confocal imaging. Together these findings reveal a compensatory role for αT-catenin phosphorylation in maintaining cardiomyocyte intercalated disc organization in human DCM.

Abstract P3079: Integrating Existing Cardiac Single-cell RNA Sequencing Datasets To Uncover The Role Of B-cells In Heart Disease

Introduction: The role of B-cells in human heart failure remains unclear. To address this knowledge gap, we analyzed publicly available datasets of scRNAseq and snRNAseq from patients with various forms of cardiomyopathy. Methods and Results: We used two public datasets including single cells or single nuclei RNAseq data collected from the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), dilated cardiomyopathy (DCM), idiopathic cardiac diseases (ICD), or healthy controls. The first dataset comprised 30 healthy controls and 22 non-ischemic DCM; the second contained 18 controls and 61 with DCM, ARVC, or ICD. Data were normalized and scaled, a UMAP plot was generated, and cells were classified using ScType. B-cells were merged into a single matrix, and the final dataset contained 1100 cells from 32 healthy controls, 59 DCM, and 8 ARVC patients. Four different B-cell clusters were identified. The top 100 biomarkers from each cluster were used to identify B-cell subtypes. Cluster 1 was identified as transitional splenic B-cells; Cluster 2 as plasma cells; Cluster 3 as follicular splenic B-cells; Cluster 4, present only in DCM, as immature B cells. Ingenuity Pathway Analysis (IPA) of the genes enriched in each cluster highlighted that different B cell clusters displayed distinct biological activity, ranging from phagosome formation to B cell receptor signaling and cytokine production. Differentially expressed gene analysis comparing healthy controls to DCM and ARVC showed both similar and disease-specific gene expression patterns in the various B cell clusters. For instance, cluster 2 showed upregulation of oxidative phosphorylation in both DCM and ARVC. On the other hand, DCM cluster 2 showed downregulation of CD40 signaling, while in ARVC the same cluster showed downregulation of IL-6 signaling (Z score &gt;2, p&lt;0.05). Discussion: Our findings suggest that human heart failure is associated with perturbations in B cell gene expression, with both shared and disease-specific features. This data supports the notion that B cells likely play a role in human cardiomyopathy. Further, this data suggests that B-cells play distinct roles in disparate forms of cardiac disease.