A therapeutic leap: how myosin inhibitors moved from cardiac interventions to skeletal muscle myopathy solutions.

The myosin inhibitor mavacamten has transformed the management of obstructive hypertrophic cardiomyopathy (HCM) by targeting myosin ATPase activity to mitigate cardiac hypercontractility. This therapeutic mechanism has proven effective for patients with HCM independent of having a primary gene mutation in myosin. In this issue of the JCI, Buvoli et al. report that muscle hypercontractility is a mechanism of pathogenesis underlying muscle dysfunction in Laing distal myopathy, a disorder characterized by mutations altering the rod domain of ß myosin heavy chain. The authors performed detailed physiological, molecular, and biomechanical analyses and demonstrated that myosin ATPase inhibition can correct a large extent of muscle abnormalities. The findings offer a therapeutic avenue for Laing distal myopathy and potentially other myopathies. This Commentary underscores the importance of reevaluating myosin activity's role across myopathies in general for the potential development of targeted myosin inhibitors to treat skeletal muscle disorders.

Myosin folding boosts solubility in cardiac muscle sarcomeres.

The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.

A novel gene-trap line reveals the dynamic patterns and essential roles of cysteine and glycine-rich protein 3 in zebrafish heart development and regeneration.

Mutations in cysteine and glycine-rich protein 3 (CSRP3)/muscle LIM protein (MLP), a key regulator of striated muscle function, have been linked to hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, the roles of CSRP3 in heart development and regeneration are not completely understood. In this study, we characterized a novel zebrafish gene-trap line, gSAIzGFFM218A, which harbors an insertion in the csrp3 genomic locus, heterozygous fish served as a csrp3 expression reporter line and homozygous fish served as a csrp3 mutant line. We discovered that csrp3 is specifically expressed in larval ventricular cardiomyocytes (CMs) and that csrp3 deficiency leads to excessive trabeculation, a common feature of CSRP3-related HCM and DCM. We further revealed that csrp3 expression increased in response to different cardiac injuries and was regulated by several signaling pathways vital for heart regeneration. Csrp3 deficiency impeded zebrafish heart regeneration by impairing CM dedifferentiation, hindering sarcomere reassembly, and reducing CM proliferation while aggravating apoptosis. Csrp3 overexpression promoted CM proliferation after injury and ameliorated the impairment of ventricle regeneration caused by pharmacological inhibition of multiple signaling pathways. Our study highlights the critical role of Csrp3 in both zebrafish heart development and regeneration, and provides a valuable animal model for further functional exploration that will shed light on the molecular pathogenesis of CSRP3-related human cardiac diseases.

Deficiency of Transcription Factor Sp1 Contributes to Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is the most prevalent monogenic heart disorder. However, the pathogenesis of HCM, especially its nongenetic mechanisms, remains largely unclear. Transcription factors are known to be involved in various biological processes including cell growth. We hypothesized that SP1 (specificity protein 1), the first purified TF in mammals, plays a role in the cardiomyocyte growth and cardiac hypertrophy of HCM. METHODS: Cardiac-specific conditional knockout of Sp1 mice were constructed to investigate the role of SP1 in the heart. The echocardiography, histochemical experiment, and transmission electron microscope were performed to analyze the cardiac phenotypes of cardiac-specific conditional knockout of Sp1 mice. RNA sequencing, chromatin immunoprecipitation sequencing, and adeno-associated virus experiments in vivo were performed to explore the downstream molecules of SP1. To examine the therapeutic effect of SP1 on HCM, an SP1 overexpression vector was constructed and injected into the mutant allele of Myh6 R404Q/+ (Myh6 c. 1211C>T) HCM mice. The human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a patient with HCM were used to detect the potential therapeutic effects of SP1 in human HCM. RESULTS: The cardiac-specific conditional knockout of Sp1 mice developed a typical HCM phenotype, displaying overt myocardial hypertrophy, interstitial fibrosis, and disordered myofilament. In addition, Sp1 knockdown dramatically increased the cell area of hiPSC-CMs and caused intracellular myofibrillar disorganization, which was similar to the hypertrophic cardiomyocytes of HCM. Mechanistically, Tuft1 was identified as the key target gene of SP1. The hypertrophic phenotypes induced by Sp1 knockdown in both hiPSC-CMs and mice could be rescued by TUFT1 (tuftelin 1) overexpression. Furthermore, SP1 overexpression suppressed the development of HCM in the mutant allele of Myh6 R404Q/+ mice and also reversed the hypertrophic phenotype of HCM hiPSC-CMs. CONCLUSIONS: Our study demonstrates that SP1 deficiency leads to HCM. SP1 overexpression exhibits significant therapeutic effects on both HCM mice and HCM hiPSC-CMs, suggesting that SP1 could be a potential intervention target for HCM.

Profiling cardiomyocytes at single cell resolution reveals COX7B could be a potential target for attenuating heart failure in cardiac hypertrophy.

Cardiac hypertrophy can develop to end-stage heart failure (HF), which inevitably leading to heart transplantation or death. Preserving cardiac function in cardiomyocytes (CMs) is essential for improving prognosis in hypertrophic cardiomyopathy (HCM) patients. Therefore, understanding transcriptomic heterogeneity of CMs in HCM would be indispensable to aid potential therapeutic targets investigation. We isolated primary CM from HCM patients who had extended septal myectomy, and obtained transcriptomes in 338 human primary CM with single-cell tagged reverse transcription (STRT-seq) approach. Our results revealed that CMs could be categorized into three subsets in nonfailing HCM heart: high energy synthesis cluster, high cellular metabolism cluster and intermediate cluster. The expression of electron transport chain (ETC) was up-regulated in larger-sized CMs from high energy synthesis cluster. Of note, we found the expression of Cytochrome c oxidase subunit 7B (COX7B), a subunit of Complex IV in ETC had trends of positively correlation with CMs size. Further, by assessing COX7B expression in HCM patients, we speculated that COX7B was compensatory up-regulated at early-stage but down-regulated in failing HCM heart. To test the hypothesis that COX7B might participate both in hypertrophy and HF progression, we used adeno associated virus 9 (AAV9) to mediate the expression of Cox7b in pressure overload-induced mice. Mice in vivo data supported that knockdown of Cox7b would accelerate HF and Cox7b overexpression could restore partial cardiac function in hypertrophy. Our result highlights targeting COX7B and preserving energy synthesis in hypertrophic CMs could be a promising translational direction for HF therapeutic strategy.

Vps4a Regulates Autophagic Flux to Prevent Hypertrophic Cardiomyopathy.

Autophagy has stabilizing functions for cardiomyocytes. Recent studies indicate that an impairment in the autophagy pathway can seriously affect morphology and function, potentially leading to heart failure. However, the role and the underlying mechanism of the endosomal sorting complex required for transport (ESCRT) family protein, in particular the AAA-ATPase vacuolar protein sorting 4a (Vps4a), in regulating myocardial autophagy remains unclear. In the present study, cardiomyocyte-specific Vps4a knockout mice were generated by crossing Vps4aflox/flox (Vps4afl/fl) with Myh6-cre transgenic mice. As a result, we observed a partially dilated left ventricular (LV) chamber, a significant increase in heart weight to body weight ratio (HW/BW), and heart weight to tibial length ratio (HW/TL), hypertrophic cardiomyopathy and early lethality starting at 3 months of age. Hematoxylin-eosin (HE), immunofluorescence assay (IFA), and Western blot (WB) revealed autophagosome accumulation in cardiomyocytes. A transcriptome-based analysis and autophagic flux tracking by AAV-RFP-GFP-LC3 showed that the autophagic flux was blocked in Vps4a knockout cardiomyocytes. In addition, we provided in vitro evidence demonstrating that Vps4a and LC3 were partially co-localized in cardiomyocytes, and the knockdown of Vps4a led to the accumulation of autophagosomes in cardiomyocytes. Similarly, the transfection of cardiomyocytes with adenovirus (Adv) mCherry-GFP-LC3 further indicated that the autophagic flux was blocked in cells with deficient levels of Vps4a. Finally, an electron microscope (EM) showed that the compromised sealing of autophagosome blocked the autophagic flux in Vps4a-depleted cardiomyocytes. These findings revealed that Vps4a contributed to the sealing of autophagosomes in cardiomyocytes. Therefore, we demonstrated that Vps4a deletion could block the autophagic flux, leading to the accumulation of degradation substances and compromised cardiac function. Overall, this study provides insights into a new theoretical basis for which autophagy may represent a therapeutic target for cardiovascular diseases.

EGFR/IGF1R Signaling Modulates Relaxation in Hypertrophic Cardiomyopathy.

BACKGROUND: Diastolic dysfunction is central to diseases such as heart failure with preserved ejection fraction and hypertrophic cardiomyopathy (HCM). However, therapies that improve cardiac relaxation are scarce, partly due to a limited understanding of modulators of cardiomyocyte relaxation. We hypothesized that cardiac relaxation is regulated by multiple unidentified proteins and that dysregulation of kinases contributes to impaired relaxation in patients with HCM. METHODS: We optimized and increased the throughput of unloaded shortening measurements and screened a kinase inhibitor library in isolated adult cardiomyocytes from wild-type mice. One hundred fifty-seven kinase inhibitors were screened. To assess which kinases are dysregulated in patients with HCM and could contribute to impaired relaxation, we performed a tyrosine and global phosphoproteomics screen and integrative inferred kinase activity analysis using HCM patient myocardium. Identified hits from these 2 data sets were validated in cardiomyocytes from a homozygous MYBPC3c.2373insG HCM mouse model. RESULTS: Screening of 157 kinase inhibitors in wild-type (N=33) cardiomyocytes (n=24 563) resulted in the identification of 17 positive inotropes and 21 positive lusitropes, almost all of them novel. The positive lusitropes formed 3 clusters: cell cycle, EGFR (epidermal growth factor receptor)/IGF1R (insulin-like growth factor 1 receptor), and a small Akt (α-serine/threonine protein kinase) signaling cluster. By performing phosphoproteomic profiling of HCM patient myocardium (N=24 HCM and N=8 donors), we demonstrated increased activation of 6 of 8 proteins from the EGFR/IGFR1 cluster in HCM. We validated compounds from this cluster in mouse HCM (N=12) cardiomyocytes (n=2023). Three compounds from this cluster were able to improve relaxation in HCM cardiomyocytes. CONCLUSIONS: We showed the feasibility of screening for functional modulators of cardiomyocyte relaxation and contraction, parameters that we observed to be modulated by kinases involved in EGFR/IGF1R, Akt, cell cycle signaling, and FoxO (forkhead box class O) signaling, respectively. Integrating the screening data with phosphoproteomics analysis in HCM patient tissue indicated that inhibition of EGFR/IGF1R signaling is a promising target for treating impaired relaxation in HCM.

Clinical Trials in Hypertrophic Cardiomyopathy Therapy: A Comprehensive Analysis of Trials Registered in Global Clinical Databases.

Background: With the disappointing results associated with the use of cardiac myosin inhibitors in the treatment of hypertrophic cardiomyopathy (HCM), the development of new therapies in clinical trials for HCM has rapidly increased. We assessed the characteristics of therapeutic intervention in HCM registered on ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP). Methods: We conducted a cross-sectional, descriptive study of clinical trials for therapeutic intervention in HCM registered on ClinicalTrials.gov and ICTRP. Results: This study analyzed 137 registered trials. Regarding study designs of these trials, 77.37% were purpose of treatment, 59.12% were randomized, 50.36% were parallel assignment, 45.26% were performed with masking, 48.18% recruited less than 50 participants, and 27.74% were Phase 2 trials. In total, 67 trials were new drug trials, of which 35 drugs were tested in these trials, and 13 trials involved treatment with mavacamten. Of these 67 clinical drug trials, 44.78% of trials involved the study of amines, and 16.42% involved 1-ring heterocyclic compounds. Regarding the NCI Thesaurus Tree, 23.81% of trials involved myosin inhibitors, 23.81% of trials involved drugs belonging to agents affecting the cardiovascular system, and 20.63% were involved in testing cation channel blockers. The drug-target network showed that myosin-7, potassium voltage-gated channel subfamily h member 2, beta-1 adrenergic receptor, carnitine o-palmitoyltransferase 1, and liver isoform were the most targeted pathways of the clinical trials analyzed in the drug-target network. Conclusion: The number of clinical trials investigating therapeutic interventions for HCM has increased in recent years. Ultimately, recent HCM therapeutic clinical trials generally did not incorporate either randomized controlled trials or masking and were small studies recruiting fewer than 50 participants. Although recent research has focused on targeting myosin-7, the molecular signaling mechanisms involved in the pathogenesis of HCM have the potential to elucidate novel target pathways.

Molecular and cellular evidence for the impact of a hypertrophic cardiomyopathy-associated RAF1 variant on the structure and function of contractile machinery in bioartificial cardiac tissues.

Noonan syndrome (NS), the most common among RASopathies, is caused by germline variants in genes encoding components of the RAS-MAPK pathway. Distinct variants, including the recurrent Ser257Leu substitution in RAF1, are associated with severe hypertrophic cardiomyopathy (HCM). Here, we investigated the elusive mechanistic link between NS-associated RAF1S257L and HCM using three-dimensional cardiac bodies and bioartificial cardiac tissues generated from patient-derived induced pluripotent stem cells (iPSCs) harboring the pathogenic RAF1 c.770 C > T missense change. We characterize the molecular, structural, and functional consequences of aberrant RAF1-associated signaling on the cardiac models. Ultrastructural assessment of the sarcomere revealed a shortening of the I-bands along the Z disc area in both iPSC-derived RAF1S257L cardiomyocytes and myocardial tissue biopsies. The aforementioned changes correlated with the isoform shift of titin from a longer (N2BA) to a shorter isoform (N2B) that also affected the active force generation and contractile tensions. The genotype-phenotype correlation was confirmed using cardiomyocyte progeny of an isogenic gene-corrected RAF1S257L-iPSC line and was mainly reversed by MEK inhibition. Collectively, our findings uncovered a direct link between a RASopathy gene variant and the abnormal sarcomere structure resulting in a cardiac dysfunction that remarkably recapitulates the human disease.

The lncRNA ADAMTS9-AS1/miR-185-5p/KAT7 ceRNA network inhibits cardiomyocyte hypertrophy in hypertrophic obstructive cardiomyopathy.

Hypertrophic obstructive cardiomyopathy (HOCM) is a well-recognized inherited cardiac disease. This study was conducted to explore the role of lncRNA ADAMTS9 antisense RNA 1 (ADAMTS9-AS1) in HOCM-induced cardiomyocyte hypertrophy. The serum of HOCM patients was collected. AC16 cells were treated with isoproterenol (ISO) and transfected with oe-ADAMTS9-AS1 vector, miR-185-5p mimic, and lysine acetyltransferase 7 (KAT7) specific small interfering RNA. lncRNA ADAMTS9-AS1, miR-185-5p, KAT7, brain natriuretic peptide (BNP), and atrial natriuretic peptide (ANP) in the serum or cells were determine by qRT-PCR or Western blot assay. Cell surface area was observed by Texas Red-Phalloidin staining. Subcellular localization of lncRNA ADAMTS9-AS1 was tested by nuclear/cytoplasmic fractionation assay, with RNA pull-down and dual-luciferase assay to validate gene interactions. lncRNA ADAMTS9-AS1 was downregulated in the serum of HOCM patients and ISO-treated AC16 cells. lncRNA ADAMTS9-AS1 overexpression inhibited ISO-induced cardiomyocyte hypertrophy and reduced levels of ANP and BNP. lncRNA ADAMTS9- AS1 was located in cytoplasm and inhibited miR-185-5p expression through targeted binding. miR-185-5p bound to KAT7 3'UTR and inhibited KAT7 expression. miR-185-5p overexpression and KAT7 knockdown both neutralized the inhibitory role of lncRNA ADAMTS9-AS1 in cardiomyocyte hypertrophy. Overall, lncRNA ADAMTS9-AS competitively bound to miR-185-5p to up-regulate KAT7 and thus inhibited cardiomyocyte hypertrophy.

DNA methylome and transcriptome profiling reveal key electrophysiology and immune dysregulation in hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is the most common inherited heart disease. However, a detailed DNA methylation (DNAme) landscape has not yet been elucidated. Our study combined DNAme and transcriptome profiles for HCM myocardium and identify aberrant DNAme associated with altered myocardial function in HCM. The transcription of methylation-related genes did not significantly differ between HCM and normal myocardium. Nevertheless, the former had an altered DNAme profile compared with the latter. The hypermethylated and hypomethylated sites in HCM tissues had chromosomal distributions and functional enrichment of correlated genes differing from those of their normal tissue counterparts. The GO analysis of network underlying the genes correlated with DNAme alteration and differentially expressed genes (DEGs) shows functional clusters centred on immune cell function and muscle system processes. In KEGG analysis, only the calcium signalling pathway was enriched either by the genes correlated with changes in DNAme or DEGs. The protein-protein interactions (PPI) underlying the genes altered at both the DNAme and transcriptional highlighted two important functional clusters. One of these was related to the immune response and had the estrogen receptor-encoding ESR1 gene as its node. The other cluster comprised cardiac electrophysiology-related genes. Intelliectin-1 (ITLN1), a component of the innate immune system, was transcriptionally downregulated in HCM and had a hypermethylated site within 1500 bp upstream of the ITLN1 transcription start site. Estimates of immune infiltration demonstrated a relative decline in immune cell population diversity in HCM. A combination of DNAme and transcriptome profiles may help identify and develop new therapeutic targets for HCM.

Multi-Omic Architecture of Obstructive Hypertrophic Cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular hypertrophy. Currently, hypertrophy pathways responsible for HCM have not been fully elucidated. Their identification could serve as a nidus for the generation of novel therapeutics aimed at halting disease development or progression. Herein, we performed a comprehensive multi-omic characterization of hypertrophy pathways in HCM. METHODS: Flash-frozen cardiac tissues were collected from genotyped HCM patients (n=97) undergoing surgical myectomy and tissue from 23 controls. RNA sequencing and mass spectrometry-enabled deep proteome and phosphoproteomic assessment were performed. Rigorous differential expression, gene set enrichment, and pathway analyses were performed to characterize HCM-mediated alterations with emphasis on hypertrophy pathways. RESULTS: We identified transcriptional dysregulation with 1246 (8%) differentially expressed genes and elucidated downregulation of 10 hypertrophy pathways. Deep proteomic analysis identified 411 proteins (9%) that differed between HCM and controls with strong dysregulation of metabolic pathways. Seven hypertrophy pathways were upregulated with antagonistic upregulation of 5 of 10 hypertrophy pathways shown to be downregulated in the transcriptome. Most upregulated hypertrophy pathways encompassed the rat sarcoma-mitogen-activated protein kinase signaling cascade. Phosphoproteomic analysis demonstrated hyperphosphorylation of the rat sarcoma-mitogen-activated protein kinase system suggesting activation of this signaling cascade. There was a common transcriptomic and proteomic profile regardless of genotype. CONCLUSIONS: At time of surgical myectomy, the ventricular proteome, independent of genotype, reveals widespread upregulation and activation of hypertrophy pathways, mainly involving the rat sarcoma-mitogen-activated protein kinase signaling cascade. In addition, there is a counterregulatory transcriptional downregulation of the same pathways. Rat sarcoma-mitogen-activated protein kinase activation may serve a crucial role in hypertrophy observed in HCM.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy.

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.

Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

BACKGROUND: The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. METHODS: In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. RESULTS: WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. CONCLUSIONS: We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

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.

The association between the clinical severity of heart failure and docosahexaenoic acid accumulation in hypertrophic cardiomyopathy.

OBJECTIVE: Hypertrophic cardiomyopathy (HCM) is a common genetic disease with diverse morphology, symptoms, and prognosis. Hypertrophied myocardium metabolism has not been explored in detail. We assessed the association between myocardium lipid metabolism and clinical severity of heart failure (HF) in HCM using imaging mass spectrometry (IMS). RESULTS: We studied 16 endomyocardial biopsy (EMB) specimens from patients with HCM. Analysis was conducted using desorption electrospray ionization IMS. The samples were assigned into two cohorts according to the period of heart biopsy (cohort 1, n = 9 and cohort 2, n = 7). In each cohort, samples were divided into two groups according to the clinical severity of HF in HCM: clinically severe and clinically mild groups. Signals showing a significant difference between the two groups were analyzed by volcano plot. In cohort 1, the volcano plot identified four signals; the intensity in the clinically severe group was more than twice that of the mild group. Out of the four signals, docosahexaenoic acid (DHA) showed significant differences in intensity between the two groups in cohort 2 (10,575.8 ± 2750.3 vs. 19,839.3 ± 4803.2, P = 0.025). The intensity of DHA was significantly higher in EMB samples from the clinically severe HCM group than in those from the mild group.

LIM domain-wide comprehensive virtual mutagenesis provides structural rationale for cardiomyopathy mutations in CSRP3.

Cardiomyopathies are a severe and chronic cardiovascular burden worldwide, affecting a large cohort in the general population. Cysteine and glycine-rich protein 3 (CSRP3) is one of key proteins implicated in dominant dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM). In this study, we device a rapid in silico screening protocol that creates a mutational landscape map for all possible allowed and disallowed substitutions in the protein of interest. This map provides the structural and functional insights on the stability of LIM domains of CSRP3. Further, the sequence analysis delineates the eukaryotic CSRP3 protein orthologs which complements the mutational map, but provide limited information of amino acid exchanges. Next, we also evaluated the effect of HCM/DCM mutations on these domains. One of highly destabilising mutations-L44P (also disease causing) and a neutral mutation-L44M were further subjected to molecular dynamics (MD) simulations. The results establish that L44P substitution affects the LIM domain structure by altering secondary structure and due to loss of hydrophobic interaction with Phenylananine 35. The present study provides a useful perspective to our understanding of the role of mutations in the CSRP3 LIM domains and their evolution. This study provides a novel computational screening method for quick identification of key mutation sites for specific protein structures that can reduce the burden on experimental research.

Letrozole Accelerates Metabolic Remodeling through Activation of Glycolysis in Cardiomyocytes: A Role beyond Hormone Regulation.

Estrogen receptor-positive (ER+) breast cancer patients are recommended hormone therapy as a primary adjuvant treatment after surgery. Aromatase inhibitors (AIs) are widely administered to ER+ breast cancer patients as estrogen blockers; however, their safety remains controversial. The use of letrozole, an AI, has been reported to cause adverse cardiovascular effects. We aimed to elucidate the effects of letrozole on the cardiovascular system. Female rats exposed to letrozole for four weeks showed metabolic changes, i.e., decreased fatty acid oxidation, increased glycolysis, and hypertrophy in the left ventricle. Although lipid oxidation yields more ATP than carbohydrate metabolism, the latter predominates in the heart under pathological conditions. Reduced lipid metabolism is attributed to reduced ß-oxidation due to low circulating estrogen levels. In letrozole-treated rats, glycolysis levels were found to be increased in the heart. Furthermore, the levels of glycolytic enzymes were increased (in a high glucose medium) and the glycolytic rate was increased in vitro (H9c2 cells); the same was not true in the case of estrogen treatment. Reduced lipid metabolism and increased glycolysis can lower energy supply to the heart, resulting in predisposition to heart failure. These data suggest that a letrozole-induced cardiac metabolic remodeling, i.e., a shift from ß-oxidation to glycolysis, may induce cardiac structural remodeling.

Founder mutation in myosin-binding protein C with an early onset and a high penetrance in males.

OBJECTIVE: One of the challenges in hypertrophic cardiomyopathy (HCM) is to determine the pathogenicity of genetic variants and to establish genotype/phenotype correlations. This study aimed to: (1) demonstrate that MYBPC3 c.2149-1G>A is a founder pathogenic variant, (2) describe the phenotype and clinical characteristics of mutation carriers and (3) compare these patients with those with the most frequent pathogenic HCM variants: MYBPC3 p.Arg502Trp/Gln. METHODS: We reviewed genetic tests performed in HCM probands at our institution. We carried out transcript analyses to demonstrate the splicing effect, and haplotype analyses to support the founder effect of MYBPC3 c.2149-1G>A. Carriers with this mutation were compared with those from MYBPC3 p.Arg502Trp/Gln in terms of presentation features, imaging and outcomes. RESULTS: MYBPC3 c.2149-1G>A was identified in 8 of 570 probands and 25 relatives. Penetrance was age and sex dependent, 50.0% of the carriers over age 36 years and 75.0% of the carriers over 40 years showing HCM. Penetrance was significantly higher in males: in carriers older than 30 years old, 100.0% of males vs 50.0% of females had a HCM phenotype (p=0.01). Males were also younger at diagnosis (32±13 vs 53±10 years old, p<0.001). MYBPC3 c.2149-1G>A resulted in an abnormal transcript that led to haploinsufficiency and was segregated in two haplotypes. However, both came from one founder haplotype. Affected carriers showed a better functional class and higher left ventricular ejection fraction (LVEF) than patients with MYBPC3 p.Arg502Trp/Gln (p<0.05 for both). Nevertheless, the rate of major adverse outcomes was similar between the two groups. CONCLUSIONS: MYBPC3 c.2149-1G>A splicing variant is a founder mutation. Affected males show an early onset of HCM and with higher penetrance than women. Carriers show better functional class and higher LVEF than MYBPC3 p.Arg502Trp/Gln carriers, but a similar rate of major adverse outcomes.

Direct detection of the myosin super-relaxed state and interacting-heads motif in solution.

The interacting-heads motif (IHM) is a structure of myosin that has been proposed to modulate cardiac output by occluding myosin molecules from undergoing the force-generating cycle. It is hypothesized to be the structural basis for the super-relaxed state (SRX), a low-ATPase kinetic state thought to be cardioprotective. The goal of the present study was to test this hypothesis by determining directly and quantitatively the fractions of myosin in the IHM and SRX under the same conditions in solution. To detect the structural IHM, we used time-resolved fluorescence resonance energy transfer to quantitate two distinct populations. One population was observed at a center distance of 2.0 nm, whereas the other was not detectable by fluorescence resonance energy transfer, implying a distance greater than 4 nm. We confirmed the IHM assignment to the 2.0-nm population by applying the same cross-linking protocol used previously to image the IHM by electron microscopy. Under the same conditions, we also measured the fraction of myosin in the SRX using stopped-flow kinetics. Our results show that the populations of SRX and IHM myosin were similar, unless treated with mavacamten, a drug that recently completed phase III clinical trials to treat hypertrophic cardiomyopathy and is proposed to act by stabilizing both the SRX and IHM. However, we found that mavacamten had a much greater effect on the SRX (55% increase) than on the IHM (4% increase). We conclude that the IHM structure is sufficient but not necessary to produce the SRX kinetic state.