Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

Spatially clustered type I interferon responses at injury borderzones.

Sterile inflammation after myocardial infarction is classically credited to myeloid cells interacting with dead cell debris in the infarct zone1,2. Here we show that cardiomyocytes are the dominant initiators of a previously undescribed type I interferon response in the infarct borderzone. Using spatial transcriptomics analysis in mice and humans, we find that myocardial infarction induces colonies of interferon-induced cells (IFNICs) expressing interferon-stimulated genes decorating the borderzone, where cardiomyocytes experience mechanical stress, nuclear rupture and escape of chromosomal DNA. Cardiomyocyte-selective deletion of Irf3 abrogated IFNIC colonies, whereas mice lacking Irf3 in fibroblasts, macrophages, neutrophils or endothelial cells, Ccr2-deficient mice or plasmacytoid-dendritic-cell-depleted mice did not. Interferons blunted the protective matricellular programs and contractile function of borderzone fibroblasts, and increased vulnerability to pathological remodelling. In mice that died after myocardial infarction, IFNIC colonies were immediately adjacent to sites of ventricular rupture, while mice lacking IFNICs were protected from rupture and exhibited improved survival3. Together, these results reveal a pathological borderzone niche characterized by a cardiomyocyte-initiated innate immune response. We suggest that selective inhibition of IRF3 activation in non-immune cells could limit ischaemic cardiomyopathy while avoiding broad immunosuppression.

Adult c-kit(pos) cardiac stem cells are necessary and sufficient for functional cardiac regeneration and repair.

The epidemic of heart failure has stimulated interest in understanding cardiac regeneration. Evidence has been reported supporting regeneration via transplantation of multiple cell types, as well as replication of postmitotic cardiomyocytes. In addition, the adult myocardium harbors endogenous c-kit(pos) cardiac stem cells (eCSCs), whose relevance for regeneration is controversial. Here, using different rodent models of diffuse myocardial damage causing acute heart failure, we show that eCSCs restore cardiac function by regenerating lost cardiomyocytes. Ablation of the eCSC abolishes regeneration and functional recovery. The regenerative process is completely restored by replacing the ablated eCSCs with the progeny of one eCSC. eCSCs recovered from the host and recloned retain their regenerative potential in vivo and in vitro. After regeneration, selective suicide of these exogenous CSCs and their progeny abolishes regeneration, severely impairing ventricular performance. These data show that c-kit(pos) eCSCs are necessary and sufficient for the regeneration and repair of myocardial damage.

Phospholipase Cε hydrolyzes perinuclear phosphatidylinositol 4-phosphate to regulate cardiac hypertrophy.

Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.

Spatial multi-omic map of human myocardial infarction.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

Incomplete-penetrant hypertrophic cardiomyopathy MYH7 G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the ß-myosin heavy chain (MYH7) can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant MYH7 G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions. We developed a collaborative pipeline to characterize myosin function across protein, myofibril, cell, and tissue levels to determine the multiscale effects on structure-function of the contractile apparatus and its implications for gene regulation and metabolic state. The G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 33%, resulting in more myosin heads available for contraction. Myofibrils from gene-edited MYH7WT/G256E human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exhibited greater and faster tension development. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. We demonstrated consistent hypercontractile myosin function as a primary consequence of the MYH7 G256E mutation across scales, highlighting the pathogenicity of this gene variant. Single-cell transcriptomic and metabolic profiling demonstrated upregulated mitochondrial genes and increased mitochondrial respiration, indicating early bioenergetic alterations. This work highlights the benefit of our multiscale platform to systematically evaluate the pathogenicity of gene variants at the protein and contractile organelle level and their early consequences on cellular and tissue function. We believe this platform can help elucidate the genotype-phenotype relationships underlying other genetic cardiovascular diseases.

SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

BACKGROUND: Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. METHODS: Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. RESULTS: The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. CONCLUSIONS: Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

Ubiquitin Ligase RBX2/SAG Regulates Mitochondrial Ubiquitination and Mitophagy.

BACKGROUND: Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. Apart from Parkin, little is known about additional Ub (ubiquitin) ligases that mediate mitochondrial ubiquitination and turnover, particularly in highly metabolically active organs such as the heart. METHODS: In this study, we have combined in silico analysis and biochemical assay to identify CRL (cullin-RING ligase) 5 as a mitochondrial Ub ligase. We generated cardiomyocytes and mice lacking RBX2 (RING-box protein 2; also known as SAG [sensitive to apoptosis gene]), a catalytic subunit of CRL5, to understand the effects of RBX2 depletion on mitochondrial ubiquitination, mitophagy, and cardiac function. We also performed proteomics analysis and RNA-sequencing analysis to define the impact of loss of RBX2 on the proteome and transcriptome. RESULTS: RBX2 and CUL (cullin) 5, 2 core components of CRL5, localize to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, increased cardiomyocyte cell death, and has a global impact on the mitochondrial proteome. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to the rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. The action of RBX2 in mitochondria is not dependent on Parkin, and Parkin gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 (PTEN-induced kinase 1) in mitochondria. CONCLUSIONS: These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that regulates mitophagy and cardiac homeostasis in a Parkin-independent, PINK1-dependent manner.

Cardiac Fibroblastic Niches in Homeostasis and Inflammation.

Fibroblasts are essential for building and maintaining the structural integrity of all organs. Moreover, fibroblasts can acquire an inflammatory phenotype to accommodate immune cells in specific niches and to provide migration, differentiation, and growth factors. In the heart, balancing of fibroblast activity is critical for cardiac homeostasis and optimal organ function during inflammation. Fibroblasts sustain cardiac homeostasis by generating local niche environments that support housekeeping functions and by actively engaging in intercellular cross talk. During inflammatory perturbations, cardiac fibroblasts rapidly switch to an inflammatory state and actively communicate with infiltrating immune cells to orchestrate immune cell migration and activity. Here, we summarize the current knowledge on the molecular landscape of cardiac fibroblasts, focusing on their dual role in promoting tissue homeostasis and modulating immune cell-cardiomyocyte interaction. In addition, we discuss potential future avenues for manipulating cardiac fibroblast activity during myocardial inflammation.

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.

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.

Dysferlin Enables Tubular Membrane Proliferation in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy compensates for increased biomechanical stress of the heart induced by prevalent cardiovascular pathologies but can result in heart failure if left untreated. Here, we hypothesized that the membrane fusion and repair protein dysferlin is critical for the integrity of the transverse-axial tubule (TAT) network inside cardiomyocytes and contributes to the proliferation of TAT endomembranes during pressure overload-induced cardiac hypertrophy. METHODS: Stimulated emission depletion and electron microscopy were used to localize dysferlin in mouse and human cardiomyocytes. Data-independent acquisition mass spectrometry revealed the cardiac dysferlin interactome and proteomic changes of the heart in dysferlin-knockout mice. After transverse aortic constriction, we compared the hypertrophic response of wild-type versus dysferlin-knockout hearts and studied TAT network remodeling mechanisms inside cardiomyocytes by live-cell membrane imaging. RESULTS: We localized dysferlin in a vesicular compartment in nanometric proximity to contact sites of the TAT network with the sarcoplasmic reticulum, a.k.a. junctional complexes for Ca2+-induced Ca2+ release. Interactome analyses demonstrated a novel protein interaction of dysferlin with the membrane-tethering sarcoplasmic reticulum protein juncophilin-2, a putative interactor of L-type Ca2+ channels and ryanodine receptor Ca2+ release channels in junctional complexes. Although the dysferlin-knockout caused a mild progressive phenotype of dilated cardiomyopathy, global proteome analysis revealed changes preceding systolic failure. Following transverse aortic constriction, dysferlin protein expression was significantly increased in hypertrophied wild-type myocardium, while dysferlin-knockout animals presented markedly reduced left-ventricular hypertrophy. Live-cell membrane imaging showed a profound reorganization of the TAT network in wild-type left-ventricular myocytes after transverse aortic constriction with robust proliferation of axial tubules, which critically depended on the increased expression of dysferlin within newly emerging tubule components. CONCLUSIONS: Dysferlin represents a new molecular target in cardiac disease that protects the integrity of tubule-sarcoplasmic reticulum junctional complexes for regulated excitation-contraction coupling and controls TAT network reorganization and tubular membrane proliferation in cardiomyocyte hypertrophy induced by pressure overload.

Therapeutic Inhibition of LincRNA-p21 Protects Against Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. METHODS: We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. RESULTS: lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 (nuclear factor of activated T cells/myocyte enhancer factor-2) pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1 (KRAB-associated protein-1). lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. CONCLUSIONS: These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

3D Imaging Reveals Complex Microvascular Remodeling in the Right Ventricle in Pulmonary Hypertension.

BACKGROUND: Pathogenic concepts of right ventricular (RV) failure in pulmonary arterial hypertension focus on a critical loss of microvasculature. However, the methods underpinning prior studies did not take into account the 3-dimensional (3D) aspects of cardiac tissue, making accurate quantification difficult. We applied deep-tissue imaging to the pressure-overloaded RV to uncover the 3D properties of the microvascular network and determine whether deficient microvascular adaptation contributes to RV failure. METHODS: Heart sections measuring 250-µm-thick were obtained from mice after pulmonary artery banding (PAB) or debanding PAB surgery and properties of the RV microvascular network were assessed using 3D imaging and quantification. Human heart tissues harvested at the time of transplantation from pulmonary arterial hypertension cases were compared with tissues from control cases with normal RV function. RESULTS: Longitudinal 3D assessment of PAB mouse hearts uncovered complex microvascular remodeling characterized by tortuous, shorter, thicker, highly branched vessels, and overall preserved microvascular density. This remodeling process was reversible in debanding PAB mice in which the RV function recovers over time. The remodeled microvasculature tightly wrapped around the hypertrophied cardiomyocytes to maintain a stable contact surface to cardiomyocytes as an adaptation to RV pressure overload, even in end-stage RV failure. However, microvasculature-cardiomyocyte contact was impaired in areas with interstitial fibrosis where cardiomyocytes displayed signs of hypoxia. Similar to PAB animals, microvascular density in the RV was preserved in patients with end-stage pulmonary arterial hypertension, and microvascular architectural changes appeared to vary by etiology, with patients with pulmonary veno-occlusive disease displaying a lack of microvascular complexity with uniformly short segments. CONCLUSIONS: 3D deep tissue imaging of the failing RV in PAB mice, pulmonary hypertension rats, and patients with pulmonary arterial hypertension reveals complex microvascular changes to preserve the microvascular density and maintain a stable microvascular-cardiomyocyte contact. Our studies provide a novel framework to understand microvascular adaptation in the pressure-overloaded RV that focuses on cell-cell interaction and goes beyond the concept of capillary rarefaction.

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.