Kenneth C. Bedi

BACKGROUND: No therapies exist to reverse right ventricular failure (RVF), and the molecular mechanisms that drive RVF remain understudied. We recently reported that the developmentally restricted noncanonical WNT receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) is upregulated in human RVF in proportion to severity of disease. Here we test the mechanistic role of ROR2 in RVF pathogenesis. METHODS: ROR2 was overexpressed or knocked down in neonatal rat ventricular myocytes and then characterized using confocal microscopy, RNA sequencing, proteomics, proteostatic functional assays, and pacing to assess contractile properties. The impact of cardiac ROR2 expression was evaluated in mice by AAV9-mediated overexpression and by AAV9-mediated delivery of short hairpin RNA to knockdown ROR2 in a pulmonary artery banded pressure overload model of RVF. ROR2 -modified mice were evaluated by echocardiography, histology, and right ventricle protein synthesis and proteasome capacity. RESULTS: In neonatal rat ventricular myocytes, we find that ROR2 profoundly dysregulates the coordination between protein translation and folding. This imbalance leads to excess protein clearance by the ubiquitin proteasome system with dramatic impacts on sarcomere and cytoskeletal structure and function. Inhibiting the ubiquitin proteasome system or restoring chaperone expression is sufficient to partially rescue ROR2-induced structural and contractile deficits in cardiomyocytes. In mice, forced cardiac ROR2 expression is sufficient to disrupt proteostasis and drive RVF, whereas conversely Ror2 knockdown partially rescues proteostasis and right ventricle structure and function in a pressure overload model of RVF. CONCLUSIONS: In sum, ROR2 is a key driver of RVF pathogenesis through proteostatic disruption and thus provides a promising target to treat RVF.

Right ventricular failure (RVF) independently predicts mortality in heart failure, yet its pathophysiology is poorly understood. Here we present a multi-modal atlas of human right ventricular pressure loading and failure, including bulk ( n = 142), single-nucleus ( n = 11) and Xenium spatial ( n = 9) transcriptomics in adults, plus pediatric ( n = 14) and mouse pulmonary artery banding cohorts. Subclustering resolved 34 cell subtypes across 12 lineages along a two-phase trajectory. Phase 1 (non-failing to pressure-loaded) accounts for most of the transcriptional change detectable by bulk and single-nucleus RNA sequencing and is marked by loss of resident macrophage identity, fibroblast activation, endothelial expansion and erosion of tissue-protective programs. Phase 2 (pressure-loaded to RVF) produces little change in bulk RNA sequencing but is resolved by spatial transcriptomics, engaging multi-lineage fibrotic, endothelial-activation and cardiomyocyte-reactivation programs, with a gain of extracellular matrix (collagen, laminin, thrombospondin) signaling over a stable cell contact adhesion baseline. Mitochondrial respirometry revealed respiratory dysfunction in adult and mouse RVF but not pediatric RVF. Multi-lineage remodeling of the cardiac microenvironment emerges as the central molecular program of RVF, meriting further study as possible disease-modifying targets.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce mortality in heart failure, but their pharmacological target remains unclear. In this study, we showed that SGLT2i directly activate pantothenate kinase 1 (PANK1), the rate-limiting enzyme in coenzyme A (CoA) synthesis. Using stable isotope infusions, we established that SGLT2i activate CoA synthesis and broadly stimulate fuel use in human cardiac tissue. We also demonstrated that SGLT2i bind PANK1 at physiological concentrations, directly inducing conformational changes and increasing enzymatic activity. In silico modeling identified the site of SGLT2i binding on PANK1, which was confirmed by amino acid mutagenesis. Finally, we showed that SGLT2i-mediated PANK activation is necessary and sufficient to increase contractility of human cardiomyocytes. In summary, we demonstrate off-target activation of PANK1 and promotion of CoA synthesis by SGLT2i, which may explain their marked clinical benefits.

Uterine smooth muscle (myometrium) is a highly energetic tissue that depends on mitochondria to sustain powerful contractions during labor. Despite their central role in generating energy for contractions, human myometrial mitochondria have unknown bioenergetic properties. We developed methods to isolate myometrial mitochondria and dissect the electron transport system in live and frozen pregnant human myometrium. Compared to unlabored myometrial mitochondria, those from laboring women display bioenergetic rewiring characterized by depressed Complex I (CI)-dependent respiratory capacity and a blunted respiratory response, consistent with impaired adaptation to high energetic stress (simulating active labor). These alterations occur independent of changes in uncoupled electron transport capacity, mitochondrial content, or apoptosis, suggesting a defect in coupled, pyruvate dehydrogenase-driven respiration. In vivo contraction strength correlates positively with paired CI-dependent respiration. Addition of succinate to labored mitochondria rescues the labor-induced CI-dependent respiratory bottleneck, unveiling new avenues for mitochondria-targeted therapies to reduce labor arrest.

Heart failure with preserved ejection fraction (HFpEF) has become the dominant heart failure phenotype, with many patients impacted by marked obesity. Lipid dyshomeostasis is thought to contribute, but human myocardial lipidomic data remain lacking. We performed untargeted lipidomics (>1,000 species) of ventricular samples for non-failing (NF) controls (n=40), HFpEF (n=29), and HFrEF (n=13) patients, integrating results with myocardial transcriptomics, ultrastructure, and plasma lipidomics, across a broad obesity spectrum. Multiple lipid classes increased in HFpEF myocardium including phospholipids, sphingolipids, neutral lipids, and free fatty acids, but acylcarnitines declined. HFrEF had far fewer lipid changes, and cardiolipin and phosphatidylethanolamine depletion. Lipids were minimally altered in NF hearts from obese individuals. Plasma lipidomics identified few inter-group disparities. Heart lipidomics from major animal HFpEF models (high fat diet+L-NAME, ZSF-1 rat, obese-DOCA-salt pig) all differed from human HFpEF. Thus, human HFpEF myocardium has distinctive cardiac lipid accumulation not found with obesity alone or HFrEF, nor mirrored by common preclinical HFpEF models.

Heart failure with preserved ejection fraction (HFpEF) causes substantial morbidity and mortality and has few effective therapies. Its phenotype has changed over time, with morbid obesity and metabolic defects supplanting hypertension and cardiac hypertrophy. We reveal that cardiomyocytes from patients with severe obesity and HFpEF have very depressed contractile reserve, including reduced calcium- and length-stimulated tension, power, and myosin activation compared with less-obese HFpEF and nonfailing (NF) controls with or without obesity but similar to those with advanced HF and reduced ejection fraction. Myocyte defects correlate with body mass index and exercise hemodynamics in patients with HFpEF but not NF and appear reversible upon weight loss. Increased troponin I phosphorylation at threonine 181 occurs only in heart failure with obesity, contributing to sarcomere dysfunction. Weight reduction and sarcomere enhancers may offer benefits in HFpEF with obesity.

BACKGROUND: Enhanced late Na ⁺ current (I _Na,L ) in heart failure (HF) contributes to cardiomyocyte proarrhythmia. In addition to CaMKII (Ca ²⁺ /calmodulin-dependent protein kinase II), FGF (fibroblast growth factor) homologous factors 1–4 (FHF1–4) also modulate I _Na,L , and targeting these pathways may provide benefits in HF. METHODS: Reverse-transcriptase quantitative polymerase chain reaction analysis of cardiac Na ⁺ channel and FHF splice isoforms was performed in human arrhythmogenic HF with reduced ejection fraction (HFrEF) and in translational animal models of HFrEF and HF with preserved ejection fraction. We tested the effects of an FixR (FHF inhibiting x region) on cardiomyocyte I _Na,L and electrophysiology in rabbit HFrEF and murine models of HFrEF and HF with preserved ejection fraction. We also tested the in vivo electrophysiological effects of FixR via adenoviral delivery in transgenic mice with cardiomyocyte-specific overexpression of CaMKIIδ _C . RESULTS: Expression of key FHF long isoforms (FHF1 _A in humans, FHF2 _S in rabbits, and FHF2 _VY in mice) that have inhibitory effects on I _Na,L was reduced in human, rabbit, and murine failing hearts. I _Na,L was markedly enhanced in both HFrEF and HF with preserved ejection fraction, and the FixR cell-penetrating peptide significantly reduced pathological I _Na,L in both forms of HF. FixR had no effect on transient peak Na ⁺ current, L-type Ca ²⁺ current, or major K ⁺ currents in rabbit ventricular myocytes. FixR markedly attenuated proarrhythmogenic action potential changes (increased action potential duration, short-term variability, and alternans susceptibility) and delayed afterdepolarizations in both rabbit and murine failing cardiomyocytes. These cellular antiarrhythmic effects were mimicked by the selective I _Na,L inhibitor GS967 and the CaMKII inhibitor AIP (autocamtide-2 inhibitory peptide). FixR also attenuated QT prolongation and in vivo arrhythmia susceptibility in transgenic mice with cardiomyocyte-specific overexpression of CaMKIIδ _C . CONCLUSIONS: In HF, I _Na,L is increased, and FHF splice isoform expression is altered, allowing a novel mechanism to therapeutically target I _Na,L . FixR, an FHF1 _A -derived peptide, is a potent and selective I _Na,L inhibitor and has antiarrhythmic properties in both HFrEF and HF with preserved ejection fraction cardiomyocytes.

BACKGROUND: Heart failure with preserved ejection fraction (HFpEF) is a poorly understood, multisystem disease with high morbidity and mortality. To improve understanding of its pathobiology, we analyzed single-nucleus RNA sequencing in human HFpEF myocardium versus controls. METHODS: Septal myocardial biopsies from 19 HFpEF and 24 nonfailing controls were analyzed using the 10× Genomics Chromium platform, with nuclei isolated from combined samples (6 patients/pool). Genotype-based demultiplexing was performed with souporcell, and gene expression was quantified with CellRanger and CellBender. After quality control, nuclei were annotated by cell types, and differential expression was performed between HFpEF versus controls using limma-voom. Functional analysis was performed using Gene Set Enrichment Analysis. Data were compared with prior single-nucleus RNA sequencing in dilated cardiomyopathy versus controls. RESULTS: We successfully demultiplexed pooled myocardial biopsies, assigning >70% of nuclei to individuals. After quality control, we recovered 48 886 nuclei and identified 14 cell types. Many differentially expressed genes across cell types were detected in HFpEF versus controls (fibroblasts, 5905; cardiomyocytes, 5159; endothelial cells, 2143; pericytes, 1812; and macrophages, 1405). Enriched pathways common to multiple cell types included immune activation, transcription/translation, metabolism, and protein quality control. They were particularly shared between cardiomyocytes and fibroblasts. Vascular smooth muscle cells had a more synthetic, proliferative phenotype. Immune cell analyses suggested enhanced T-cell activation and reduced macrophage clearance programs. Comparative analysis between HFpEF and dilated cardiomyopathy identified transcriptional differences primarily in cardiomyocytes. Two of 3 cardiomyocyte differential expression genes unique to HFpEF were validated to have concordant protein expression changes in HFpEF ( MAP2K6 and PLPP3 ). CONCLUSIONS: Our findings reveal a distinct, cell-type–specific transcriptomic landscape in the human HFpEF myocardium. While HFpEF and dilated cardiomyopathy share significant molecular pathways across most cell types, the profound divergence within cardiomyocytes suggests a unique pathological driver for HFpEF. These signatures may provide a high-resolution roadmap for identifying precision therapeutic targets in HFpEF.

BACKGROUND: CPT1 (carnitine palmitoyltransferase 1) is a rate-limiting enzyme for long-chain fatty acid oxidation. In adult hearts, CPT1b predominates, while CPT1a is coexpressed at lower levels. Pathological stress on the heart induces CPT1a expression, coinciding with a reduction in fatty acid oxidation, yet the role of CPT1a in pathological remodeling is unknown. METHODS: CPT1 isoform expression was assayed in the myocardium of patients with heart failure with nonischemic cardiomyopathy and a preclinical mouse model of heart failure. Mice were subjected to afterload stress via transverse aortic constriction (TAC) or sham surgery (sham) with cardiac-specific CPT1a knockdown or cardiac-specific, adeno-associated virus serotype 9 (AAV9)–mediated CPT1a overexpression (AAV9.cTnT [cardiac troponin T].Cpt1a) versus empty virus or PBS infusions as controls. MicroRNA 370, known to suppress hepatic CPT1a, was assayed and overexpressed to determine if microRNA 370 regulates cardiac CPT1a expression. RESULTS: CPT1a protein was elevated and microRNA 370 reduced in the myocardium of male and female patients with nonischemic cardiomyopathy, as well as in failing mouse hearts. AAV9–mediated microRNA 370 overexpression in mouse hearts suppressed CPT1a expression and attenuated the response of CPT1a to TAC. Preventing CPT1a upregulation in response to TAC in cardiac-specific CPT1a knockout mice exacerbated adverse remodeling, severe dysfunction, and increased mortality. In contrast, CPT1a overexpression (2.8-fold) attenuated impaired ejection fraction (by 54%) versus control TAC hearts ( P <0.05). Delivery of AAV9.cTnT.Cpt1a 4 weeks after TAC surgery led to significant rescue of ejection fraction and mitigated the exacerbated dysfunction of cardiac-specific CPT1a knockout mice TAC hearts. RNA-seq revealed a novel function of CPT1a in suppressing hypertrophic, profibrotic, and cell death gene programs in both sham and TAC hearts, irrespective of changes in fatty acid oxidation, with reduced histone acetylation. CONCLUSIONS: The effects of CPT1a in the heart extend beyond fatty acid oxidation including noncanonical regulation of gene programs. CPT1a upregulation occurs in nonischemic cardiomyopathy and is a critical cardioprotective adaptation to pathological stress.

Background: Truncating variants in desmoplakin ( DSP tv), are a leading cause of arrhythmogenic cardiomyopathy (ACM), often presenting with early fibrosis and arrhythmias disproportionate to systolic dysfunction. DSP is critical for cardiac mechanical integrity, linking desmosomes to the cytoskeleton to withstand contractile forces. While loss-of-function is implicated, direct evidence, both for DSP haploinsufficiency in human hearts and for the impact of mechanical stress on cardiomyocyte adhesion, has been limited, leaving the pathogenic mechanism unclear. Methods: We analyzed explanted human heart tissue from patients with DSP tv (N=3), titin truncating variants ( TTN tv, N=5), and controls (N=5) using RNA-sequencing and mass spectrometry. We generated human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) harboring patient-derived or CRISPR-Cas9 engineered DSP tv to model a range of DSP expression levels. Using a 2D cardiac muscle bundle (CMB) platform enabling live visualization of cell junctions, we developed an assay to assess cell-cell adhesion upon heightened contractile stress in response to the contractile agonist endothelin-1. CRISPR-interference (CRISPRi) was used to confirm the role of DSP loss, and CRISPR-activation (CRISPRa) was tested for therapeutic rescue. Results: Compared to both control and TTN tv hearts, DSP tv human hearts exhibited reduced DSP at both the mRNA and protein level, as well as broadly disrupted desmosomal stoichiometry. Transcriptomic and proteomic analyses implicated cell adhesion, extracellular matrix, and inflammatory pathways. iPSC-CM models recapitulated DSP haploinsufficiency and desmosomal disruption. DSP tv CMBs showed normal baseline contractile function. However, they displayed marked cell-cell adhesion failure with contractile stress (75% failure vs. 8% in controls, pDSP tv CMBs (9% failure post-CRISPRa, pDSP transcript 1 loss of function. Conclusions: DSP haploinsufficiency is the major cause of DSP cardiomyopathy with a primary consequence of conferring vulnerability to cardiomyocyte cell-cell adhesion failure under heightened contractile stress. Transcriptional activation of DSP reverses this defect in preclinical models, establishing proof-of-concept for a potential therapeutic strategy in DSP cardiomyopathy.