Mesenchymal stromal cells for improvement of cardiac function following acute myocardial infarction: a matter of timing.

Acute myocardial infarction (AMI) is the leading cause of cardiovascular death and remains the most common cause of heart failure. Reopening of the occluded artery, i.e., reperfusion, is the only way to save the myocardium. However, the expected benefits of reducing infarct size are disappointing due to the reperfusion paradox, which also induces specific cell death. These ischemia-reperfusion (I/R) lesions can account for up to 50% of final infarct size, a major determinant for both mortality and the risk of heart failure (morbidity). In this review, we provide a detailed description of the cell death and inflammation mechanisms as features of I/R injury and cardioprotective strategies such as ischemic postconditioning as well as their underlying mechanisms. Due to their biological properties, the use of mesenchymal stromal/stem cells (MSCs) has been considered a potential therapeutic approach in AMI. Despite promising results and evidence of safety in preclinical studies using MSCs, the effects reported in clinical trials are not conclusive and even inconsistent. These discrepancies were attributed to many parameters such as donor age, in vitro culture, and storage time as well as injection time window after AMI, which alter MSC therapeutic properties. In the context of AMI, future directions will be to generate MSCs with enhanced properties to limit cell death in myocardial tissue and thereby reduce infarct size and improve the healing phase to increase postinfarct myocardial performance.

Selective loss of resident macrophage-derived insulin-like growth factor-1 abolishes adaptive cardiac growth to stress.

Hypertension affects one-third of the world's population, leading to cardiac dysfunction that is modulated by resident and recruited immune cells. Cardiomyocyte growth and increased cardiac mass are essential to withstand hypertensive stress; however, whether immune cells are involved in this compensatory cardioprotective process is unclear. In normotensive animals, single-cell transcriptomics of fate-mapped self-renewing cardiac resident macrophages (RMs) revealed transcriptionally diverse cell states with a core repertoire of reparative gene programs, including high expression of insulin-like growth factor-1 (Igf1). Hypertension drove selective in situ proliferation and transcriptional activation of some cardiac RM states, directly correlating with increased cardiomyocyte growth. During hypertension, inducible ablation of RMs or selective deletion of RM-derived Igf1 prevented adaptive cardiomyocyte growth, and cardiac mass failed to increase, which led to cardiac dysfunction. Single-cell transcriptomics identified a conserved IGF1-expressing macrophage subpopulation in human cardiomyopathy. Here we defined the absolute requirement of RM-produced IGF-1 in cardiac adaptation to hypertension.

BET bromodomains mediate transcriptional pause release in heart failure.

Heart failure (HF) is driven by the interplay between regulatory transcription factors and dynamic alterations in chromatin structure. Pathologic gene transactivation in HF is associated with recruitment of histone acetyl-transferases and local chromatin hyperacetylation. We therefore assessed the role of acetyl-lysine reader proteins, or bromodomains, in HF. Using a chemical genetic approach, we establish a central role for BET family bromodomain proteins in gene control during HF pathogenesis. BET inhibition potently suppresses cardiomyocyte hypertrophy in vitro and pathologic cardiac remodeling in vivo. Integrative transcriptional and epigenomic analyses reveal that BET proteins function mechanistically as pause-release factors critical to expression of genes that are central to HF pathogenesis and relevant to the pathobiology of failing human hearts. This study implicates epigenetic readers as essential effectors of transcriptional pause release during HF pathogenesis and identifies BET coactivator proteins as therapeutic targets in the heart.

Role of the Epigenome in Heart Failure.

Gene expression is needed for the maintenance of heart function under normal conditions and in response to stress. Each cell type of the heart has a specific program controlling transcription. Different types of stress induce modifications of these programs and, if prolonged, can lead to altered cardiac phenotype and, eventually, to heart failure. The transcriptional status of a gene is regulated by the epigenome, a complex network of DNA and histone modifications. Until a few years ago, our understanding of the role of the epigenome in heart disease was limited to that played by histone deacetylation. But over the last decade, the consequences for the maintenance of homeostasis in the heart and for the development of cardiac hypertrophy of a number of other modifications, including DNA methylation and hydroxymethylation, histone methylation and acetylation, and changes in chromatin architecture, have become better understood. Indeed, it is now clear that many levels of regulation contribute to defining the epigenetic landscape required for correct cardiomyocyte function, and that their perturbation is responsible for cardiac hypertrophy and fibrosis. Here, we review these aspects and draw a picture of what epigenetic modification may imply at the therapeutic level for heart failure.

SARS-CoV-2 spike-induced syncytia are senescent and contribute to exacerbated heart failure.

SARS-CoV-2 spike protein (SARS-2-S) induced cell-cell fusion in uninfected cells may occur in long COVID-19 syndrome, as circulating SARS-2-S or extracellular vesicles containing SARS-2-S (S-EVs) were found to be prevalent in post-acute sequelae of COVID-19 (PASC) for up to 12 months after diagnosis. Although isolated recombinant SARS-2-S protein has been shown to increase the SASP in senescent ACE2-expressing cells, the direct linkage of SARS-2-S syncytia with senescence in the absence of virus infection and the degree to which SARS-2-S syncytia affect pathology in the setting of cardiac dysfunction are unknown. Here, we found that the senescent outcome of SARS-2-S induced syncytia exacerbated heart failure progression. We first demonstrated that syncytium formation in cells expressing SARS-2-S delivered by DNA plasmid or LNP-mRNA exhibits a senescence-like phenotype. Extracellular vesicles containing SARS-2-S (S-EVs) also confer a potent ability to form senescent syncytia without de novo synthesis of SARS-2-S. However, it is important to note that currently approved COVID-19 mRNA vaccines do not induce syncytium formation or cellular senescence. Mechanistically, SARS-2-S syncytia provoke the formation of functional MAVS aggregates, which regulate the senescence fate of SARS-2-S syncytia by TNFα. We further demonstrate that senescent SARS-2-S syncytia exhibit shrinked morphology, leading to the activation of WNK1 and impaired cardiac metabolism. In pre-existing heart failure mice, the WNK1 inhibitor WNK463, anti-syncytial drug niclosamide, and senolytic dasatinib protect the heart from exacerbated heart failure triggered by SARS-2-S. Our findings thus suggest a potential mechanism for COVID-19-mediated cardiac pathology and recommend the application of WNK1 inhibitor for therapy especially in individuals with post-acute sequelae of COVID-19.

Indole-3-Propionic Acid Protects Against Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a common but poorly understood form of heart failure, characterized by impaired diastolic function. It is highly heterogeneous with multiple comorbidities, including obesity and diabetes, making human studies difficult. METHODS: Metabolomic analyses in a mouse model of HFpEF showed that levels of indole-3-propionic acid (IPA), a metabolite produced by gut bacteria from tryptophan, were reduced in the plasma and heart tissue of HFpEF mice as compared with controls. We then examined the role of IPA in mouse models of HFpEF as well as 2 human HFpEF cohorts. RESULTS: The protective role and therapeutic effects of IPA were confirmed in mouse models of HFpEF using IPA dietary supplementation. IPA attenuated diastolic dysfunction, metabolic remodeling, oxidative stress, inflammation, gut microbiota dysbiosis, and intestinal epithelial barrier damage. In the heart, IPA suppressed the expression of NNMT (nicotinamide N-methyl transferase), restored nicotinamide, NAD+/NADH, and SIRT3 (sirtuin 3) levels. IPA mediates the protective effects on diastolic dysfunction, at least in part, by promoting the expression of SIRT3. SIRT3 regulation was mediated by IPA binding to the aryl hydrocarbon receptor, as Sirt3 knockdown diminished the effects of IPA on diastolic dysfunction in vivo. The role of the nicotinamide adenine dinucleotide circuit in HFpEF was further confirmed by nicotinamide supplementation, Nnmt knockdown, and Nnmt overexpression in vivo. IPA levels were significantly reduced in patients with HFpEF in 2 independent human cohorts, consistent with a protective function in humans, as well as mice. CONCLUSIONS: Our findings reveal that IPA protects against diastolic dysfunction in HFpEF by enhancing the nicotinamide adenine dinucleotide salvage pathway, suggesting the possibility of therapeutic management by either altering the gut microbiome composition or supplementing the diet with IPA.

Mitochondrial Structure and Function in Human Heart Failure.

Despite clinical and scientific advancements, heart failure is the major cause of morbidity and mortality worldwide. Both mitochondrial dysfunction and inflammation contribute to the development and progression of heart failure. Although inflammation is crucial to reparative healing following acute cardiomyocyte injury, chronic inflammation damages the heart, impairs function, and decreases cardiac output. Mitochondria, which comprise one third of cardiomyocyte volume, may prove a potential therapeutic target for heart failure. Known primarily for energy production, mitochondria are also involved in other processes including calcium homeostasis and the regulation of cellular apoptosis. Mitochondrial function is closely related to morphology, which alters through mitochondrial dynamics, thus ensuring that the energy needs of the cell are met. However, in heart failure, changes in substrate use lead to mitochondrial dysfunction and impaired myocyte function. This review discusses mitochondrial and cristae dynamics, including the role of the mitochondria contact site and cristae organizing system complex in mitochondrial ultrastructure changes. Additionally, this review covers the role of mitochondria-endoplasmic reticulum contact sites, mitochondrial communication via nanotunnels, and altered metabolite production during heart failure. We highlight these often-neglected factors and promising clinical mitochondrial targets for heart failure.

Blunted Cardiac Mitophagy in Response to Metabolic Stress Contributes to HFpEF.

BACKGROUND: Metabolic remodeling and mitochondrial dysfunction are hallmarks of heart failure with reduced ejection fraction. However, their role in the pathogenesis of HF with preserved ejection fraction (HFpEF) is poorly understood. METHODS: In a mouse model of HFpEF, induced by high-fat diet and Nω-nitrol-arginine methyl ester, cardiac energetics was measured by 31P nuclear magnetic resonance (NMR) spectroscopy and substrate oxidation profile was assessed by 13C-isotopmer analysis. Mitochondrial functions were assessed in the heart tissue and human induced pluripotent stem cell-derived cardiomyocytes. RESULTS: HFpEF hearts presented a lower phosphocreatine content and a reduced phosphocreatine/ATP ratio, similar to that in heart failure with reduced ejection fraction. Decreased respiratory function and increased reactive oxygen species production were observed in mitochondria isolated from HFpEF hearts suggesting mitochondrial dysfunction. Cardiac substrate oxidation profile showed a high dependency on fatty acid oxidation in HFpEF hearts, which is the opposite of heart failure with reduced ejection fraction but similar to that in high-fat diet hearts. However, phosphocreatine/ATP ratio and mitochondrial function were sustained in the high-fat diet hearts. We found that mitophagy was activated in the high-fat diet heart but not in HFpEF hearts despite similar extent of obesity suggesting that mitochondrial quality control response was impaired in HFpEF hearts. Using a human induced pluripotent stem cell-derived cardiomyocyte mitophagy reporter, we found that fatty acid loading stimulated mitophagy, which was obliterated by inhibiting fatty acid oxidation. Enhancing fatty acid oxidation by deleting ACC2 (acetyl-CoA carboxylase 2) in the heart stimulated mitophagy and improved HFpEF phenotypes. CONCLUSIONS: Maladaptation to metabolic stress in HFpEF hearts impairs mitochondrial quality control and contributed to the pathogenesis, which can be improved by stimulating fatty acid oxidation.

TRIM35 Monoubiquitinates H2B in Cardiac Cells, Implications for Heart Failure.

BACKGROUND: The tumor suppressor and proapoptotic transcription factor P53 is induced (and activated) in several forms of heart failure, including cardiotoxicity and dilated cardiomyopathy; however, the precise mechanism that coordinates its induction with accessibility to its transcriptional promoter sites remains unresolved, especially in the setting of mature terminally differentiated (nonreplicative) cardiomyocytes. METHODS: Male and female control or TRIM35 (tripartite motif containing 35) overexpression adolescent (aged 1-3 months) and adult (aged 4-6 months) transgenic mice were used for all in vivo experiments. Primary adolescent or adult mouse cardiomyocytes were isolated from control or TRIM35 overexpression transgenic mice for all in vitro experiments. Adenovirus or small-interfering RNA was used for all molecular experiments to overexpress or knockdown, respectively, target genes in primary mouse cardiomyocytes. Patient dilated cardiomyopathy or nonfailing left ventricle samples were used for translational and mechanistic insight. Chromatin immunoprecipitation and DNA sequencing or quantitative real-time polymerase chain reaction (qPCR) was used to assess P53 binding to its transcriptional promoter targets, and RNA sequencing was used to identify disease-specific signaling pathways. RESULTS: Here, we show that E3-ubiquitin ligase TRIM35 can directly monoubiquitinate lysine-120 (K120) on histone 2B in postnatal mature cardiomyocytes. This epigenetic modification was sufficient to promote chromatin remodeling, accessibility of P53 to its transcriptional promoter targets, and elongation of its transcribed mRNA. We found that increased P53 transcriptional activity (in cardiomyocyte-specific Trim35 overexpression transgenic mice) was sufficient to initiate heart failure and these molecular findings were recapitulated in nonischemic human LV dilated cardiomyopathy samples. CONCLUSIONS: These findings suggest that TRIM35 and the K120Ub-histone 2B epigenetic modification are molecular features of cardiomyocytes that can collectively predict dilated cardiomyopathy pathogenesis.

Cardiomyocyte PANX1 Controls Glycolysis and Neutrophil Recruitment in Hypertrophy.

BACKGROUND: PANX1 (pannexin 1), a ubiquitously expressed ATP release membrane channel, has been shown to play a role in inflammation, blood pressure regulation, and myocardial infarction. However, the possible role of PANX1 in cardiomyocytes in the progression of heart failure has not yet been investigated. METHOD: We generated a novel mouse line with constitutive deletion of PANX1 in cardiomyocytes (Panx1MyHC6). RESULTS: PANX1 deletion in cardiomyocytes had no effect on unstressed heart function but increased the glycolytic metabolism and resulting glycolytic ATP production, with a concurrent decrease in oxidative phosphorylation, both in vivo and in vitro. In vitro, treatment of H9c2 (H9c2 rat myoblast cell line) cardiomyocytes with isoproterenol led to PANX1-dependent release of ATP and Yo-Pro-1 uptake, as assessed by pharmacological blockade with spironolactone and siRNA-mediated knockdown of PANX1. To investigate nonischemic heart failure and the preceding cardiac hypertrophy, we administered isoproterenol, and we demonstrated that Panx1MyHC6 mice were protected from systolic and diastolic left ventricle volume increases as a result of cardiomyocyte hypertrophy. Moreover, we found that Panx1MyHC6 mice showed decreased isoproterenol-induced recruitment of immune cells (CD45+), particularly neutrophils (CD11b+ [integrin subunit alpha M], Ly6g+ [lymphocyte antigen 6 family member G]), to the myocardium. CONCLUSIONS: Together, these data demonstrate that PANX1 deficiency in cardiomyocytes increases glycolytic metabolism and protects against cardiac hypertrophy in nonischemic heart failure at least in part by reducing immune cell recruitment. Our study implies PANX1 channel inhibition as a therapeutic approach to ameliorate cardiac dysfunction in patients with heart failure.

Targeting Cyclophilin A in the Cardiac Microenvironment Preserves Heart Function and Structure in Failing Hearts.

BACKGROUND: Cardiac hypertrophy is characterized by remodeling of the myocardium, which involves alterations in the ECM (extracellular matrix) and cardiomyocyte structure. These alterations critically contribute to impaired contractility and relaxation, ultimately leading to heart failure. Emerging evidence implicates that extracellular signaling molecules are critically involved in the pathogenesis of cardiac hypertrophy and remodeling. The immunophilin CyPA (cyclophilin A) has been identified as a potential culprit. In this study, we aimed to unravel the interplay between eCyPA (extracellular CyPA) and myocardial dysfunction and evaluate the therapeutic potential of inhibiting its extracellular accumulation to improve heart function. METHODS: Employing a multidisciplinary approach encompassing in silico, in vitro, in vivo, and ex vivo experiments we studied a mouse model of cardiac hypertrophy and human heart specimen to decipher the interaction of CyPA and the cardiac microenvironment in highly relevant pre-/clinical settings. Myocardial expression of CyPA (immunohistology) and the inflammatory transcriptome (NanoString) was analyzed in human cardiac tissue derived from patients with nonischemic, noninflammatory congestive heart failure (n=187). These analyses were paralleled by a mouse model of Ang (angiotensin) II-induced heart failure, which was assessed by functional (echocardiography), structural (immunohistology, atomic force microscopy), and biomolecular (Raman spectroscopy) analyses. The effect of inhibiting eCyPA in the cardiac microenvironment was evaluated using a newly developed neutralizing anti-eCyPA monoclonal antibody. RESULTS: We observed a significant accumulation of eCyPA in both human and murine-failing hearts. Importantly, higher eCyPA expression was associated with poor clinical outcomes in patients (P=0.043) and contractile dysfunction in mice (Pearson correlation coefficient, -0.73). Further, myocardial expression of eCyPA was critically associated with an increase in myocardial hypertrophy, inflammation, fibrosis, stiffness, and cardiac dysfunction in vivo. Antibody-based inhibition of eCyPA prevented (Ang II)-induced myocardial remodeling and dysfunction in mice. CONCLUSIONS: Our study provides strong evidence of the pathogenic role of eCyPA in remodeling, myocardial stiffening, and dysfunction in heart failure. The findings suggest that antibody-based inhibition of eCyPA may offer a novel therapeutic strategy for nonischemic heart failure. Further research is needed to evaluate the translational potential of these interventions in human patients with cardiac hypertrophy.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy.

BACKGROUND: Calcium (Ca2+) uptake by mitochondria occurs via the mitochondrial Ca2+ uniporter. Mitochondrial Ca2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca2+]m) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca2+-sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: MICU3 knock out hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca2+]m than wild-type hearts following acute isoproterenol infusion. In contrast, heart with overexpression of MICU3 exhibited an enhanced increase in [Ca2+]m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, mice with overexpression of MICU3 exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in hearts with overexpression of MICU3 compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca2+]m in the heart. We conclude that MICU3 plays an important role in regulating [Ca2+]m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

Glucagon Receptor Antagonist for Heart Failure With Preserved Ejection Fraction.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is an emerging major unmet need and one of the most significant clinic challenges in cardiology. The pathogenesis of HFpEF is associated with multiple risk factors. Hypertension and metabolic disorders associated with obesity are the 2 most prominent comorbidities observed in patients with HFpEF. Although hypertension-induced mechanical overload has long been recognized as a potent contributor to heart failure with reduced ejection fraction, the synergistic interaction between mechanical overload and metabolic disorders in the pathogenesis of HFpEF remains poorly characterized. METHOD: We investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction in lean C57Bl/6J or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice, followed by single-nuclei RNA-seq and targeted manipulation of a top-ranked signaling pathway differentially affected in the 2 experimental cohorts. RESULTS: In contrast to the post-transverse aortic constriction C57Bl/6J lean mice, which developed pathological features of heart failure with reduced ejection fraction over time, the post-transverse aortic constriction ob/ob mice showed no significant changes in ejection fraction but developed characteristic pathological features of HFpEF, including diastolic dysfunction, worsened cardiac hypertrophy, and pathological remodeling, along with further deterioration of exercise intolerance. Single-nuclei RNA-seq analysis revealed significant transcriptome reprogramming in the cardiomyocytes stressed by both pressure overload and obesity/diabetes, markedly distinct from the cardiomyocytes singularly stressed by pressure overload or obesity/diabetes. Furthermore, glucagon signaling was identified as the top-ranked signaling pathway affected in the cardiomyocytes associated with HFpEF. Treatment with a glucagon receptor antagonist significantly ameliorated the progression of HFpEF-related pathological features in 2 independent preclinical models. Importantly, cardiomyocyte-specific genetic deletion of the glucagon receptor also significantly improved cardiac function in response to pressure overload and metabolic stress. CONCLUSIONS: These findings identify glucagon receptor signaling in cardiomyocytes as a critical determinant of HFpEF progression and provide proof-of-concept support for glucagon receptor antagonism as a potential therapy for the disease.

Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

A Carrier-Based Quantitative Proteomics Method Applied to Biomarker Discovery in Pericardial Fluid.

Data-dependent liquid chromatography tandem mass spectrometry is challenged by the large concentration range of proteins in plasma and related fluids. We adapted the SCoPE method from single-cell proteomics to pericardial fluid, where a myocardial tissue carrier was used to aid protein quantification. The carrier proteome and patient samples were labeled with distinct isobaric labels, which allowed separate quantification. Undepleted pericardial fluid from patients with type 2 diabetes mellitus and/or heart failure undergoing heart surgery was analyzed with either a traditional liquid chromatography tandem mass spectrometry method or with the carrier proteome. In total, 1398 proteins were quantified with a carrier, compared to 265 without, and a higher proportion of these proteins were of myocardial origin. The number of differentially expressed proteins also increased nearly four-fold. For patients with both heart failure and type 2 diabetes mellitus, pathway analysis of upregulated proteins demonstrated the enrichment of immune activation, blood coagulation, and stress pathways. Overall, our work demonstrates the applicability of a carrier for enhanced protein quantification in challenging biological matrices such as pericardial fluid, with potential applications for biomarker discovery. Mass spectrometry data are available via ProteomeXchange with identifier PXD053450.

Proximal telomeric decompaction due to telomere shortening drives FOXC1-dependent myocardial senescence.

Telomeres, TTAGGGn DNA repeat sequences located at the ends of eukaryotic chromosomes, play a pivotal role in aging and are targets of DNA damage response. Although we and others have demonstrated presence of short telomeres in genetic cardiomyopathic and heart failure cardiomyocytes, little is known about the role of telomere lengths in cardiomyocyte. Here, we demonstrate that in heart failure patient cardiomyocytes, telomeres are shortened compared to healthy controls. We generated isogenic human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with short telomeres (sTL-CMs) and normal telomeres (nTL-CMs) as model. Compared to nTL-CMs, short telomeres result in cardiac dysfunction and expression of senescent markers. Using Hi-C and RNASeq, we observe that short telomeres induced TAD insulation decrease near telomeric ends and this correlated with a transcription upregulation in sTL-CMs. FOXC1, a key transcription factor involved in early cardiogenesis, was upregulated in sTL-CMs and its protein levels were negatively correlated with telomere lengths in heart failure patients. Overexpression of FOXC1 induced hiPSC-CM aging, mitochondrial and contractile dysfunction; knockdown of FOXC1 rescued these phenotypes. Overall, the work presented demonstrate that increased chromatin accessibility due to telomere shortening resulted in the induction of FOXC1-dependent expression network responsible for contractile dysfunction and myocardial senescence.

TRF2 rescues telomere attrition and prolongs cell survival in Duchenne muscular dystrophy cardiomyocytes derived from human iPSCs.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease caused by the lack of dystrophin. Heart failure, driven by cardiomyocyte death, fibrosis, and the development of dilated cardiomyopathy, is the leading cause of death in DMD patients. Current treatments decrease the mechanical load on the heart but do not address the root cause of dilated cardiomyopathy: cardiomyocyte death. Previously, we showed that telomere shortening is a hallmark of DMD cardiomyocytes. Here, we test whether prevention of telomere attrition is possible in cardiomyocytes differentiated from patient-derived induced pluripotent stem cells (iPSC-CMs) and if preventing telomere shortening impacts cardiomyocyte function. We observe reduced cell size, nuclear size, and sarcomere density in DMD iPSC-CMs compared with healthy isogenic controls. We find that expression of just one telomere-binding protein, telomeric repeat-binding factor 2 (TRF2), a core component of the shelterin complex, prevents telomere attrition and rescues deficiencies in cell size as well as sarcomere density. We employ a bioengineered platform to micropattern cardiomyocytes for calcium imaging and perform Southern blots of telomere restriction fragments, the gold standard for telomere length assessments. Importantly, preservation of telomere lengths in DMD cardiomyocytes improves their viability. These data provide evidence that preventing telomere attrition ameliorates deficits in cell morphology, activation of the DNA damage response, and premature cell death, suggesting that TRF2 is a key player in DMD-associated cardiac failure.

Proteomics and phosphoproteomics of failing human left ventricle identifies dilated cardiomyopathy-associated phosphorylation of CTNNA3.

The prognosis and treatment outcomes of heart failure (HF) patients rely heavily on disease etiology, yet the majority of underlying signaling mechanisms are complex and not fully elucidated. Phosphorylation is a major point of protein regulation with rapid and profound effects on the function and activity of protein networks. Currently, there is a lack of comprehensive proteomic and phosphoproteomic studies examining cardiac tissue from HF patients with either dilated dilated cardiomyopathy (DCM) or ischemic cardiomyopathy (ICM). Here, we used a combined proteomic and phosphoproteomic approach to identify and quantify more than 5,000 total proteins with greater than 13,000 corresponding phosphorylation sites across explanted left ventricle (LV) tissue samples, including HF patients with DCM vs. nonfailing controls (NFC), and left ventricular infarct vs. noninfarct, and periinfarct vs. noninfarct regions of HF patients with ICM. Each pair-wise comparison revealed unique global proteomic and phosphoproteomic profiles with both shared and etiology-specific perturbations. With this approach, we identified a DCM-associated hyperphosphorylation cluster in the cardiomyocyte intercalated disc (ICD) protein, αT-catenin (CTNNA3). We demonstrate using both ex vivo isolated cardiomyocytes and in vivo using an AAV9-mediated overexpression mouse model, that CTNNA3 phosphorylation at these residues plays a key role in maintaining protein localization at the cardiomyocyte ICD to regulate conductance and cell-cell adhesion. Collectively, this integrative proteomic/phosphoproteomic approach identifies region- and etiology-associated signaling pathways in human HF and describes a role for CTNNA3 phosphorylation in the pathophysiology of DCM.

Phosphorylation-Regulated Dynamic Phase Separation of HIP-55 Protects Against Heart Failure.

BACKGROUND: Heart failure (HF), which is the terminal stage of many cardiovascular diseases, is associated with low survival rates and a severe financial burden. The mechanisms, especially the molecular mechanism combined with new theories, underlying the pathogenesis of HF remain elusive. We demonstrate that phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 (hematopoietic progenitor kinase 1-interacting protein of 55 kDa) protects against HF. METHODS: Fluorescence recovery after photobleaching assay, differential interference contrast analysis, pull-down assay, immunofluorescence, and immunohistochemical analysis were used to investigate the liquid-liquid phase separation capacity of HIP-55 and its dynamic regulation in vivo and in vitro. Mice with genetic deletion of HIP-55 and mice with cardiac-specific overexpression of HIP-55 were used to examine the role of HIP-55 on ß-adrenergic receptor hyperactivation-induced HF. Mutation analysis and mice with specific phospho-resistant site mutagenesis were used to identify the role of phosphorylation-regulated dynamic liquid-liquid phase separation of HIP-55 in HF. RESULTS: Genetic deletion of HIP-55 aggravated HF, whereas cardiac-specific overexpression of HIP-55 significantly alleviated HF in vivo. HIP-55 possesses a strong capacity for phase separation. Phase separation of HIP-55 is dynamically regulated by AKT-mediated phosphorylation at S269 and T291 sites, failure of which leads to impairment of HIP-55 dynamic phase separation by formation of abnormal aggregation. Prolonged sympathetic hyperactivation stress induced decreased phosphorylation of HIP-55 S269 and T291, dysregulated phase separation, and subsequent aggregate formation of HIP55. Moreover, we demonstrated the important role of dynamic phase separation of HIP-55 in inhibiting hyperactivation of the ß-adrenergic receptor-mediated P38/MAPK (mitogen-activated protein kinase) signaling pathway. A phosphorylation-deficient HIP-55 mutation, which undergoes massive phase separation and forms insoluble aggregates, loses the protective activity against HF. CONCLUSIONS: Our work reveals that the phosphorylation-regulated dynamic phase separation of HIP-55 protects against sympathetic/adrenergic system-mediated heart failure.