Giulia Mearini

The ubiquitin-proteasome system in cardiac dysfunction.

Since proteins play crucial roles in all biological processes, the finely tuned equilibrium between their synthesis and degradation regulates cellular homeostasis. Controlling the quality of proteome informational content is essential for cell survival and function. After initial synthesis, membrane and secretory proteins are modified, folded, and assembled in the endoplasmic reticulum, whereas other proteins are synthesized and processed in the cytosol. Cells have different protein quality control systems, the molecular chaperones, which help protein folding and stabilization, and the ubiquitin-proteasome system (UPS) and lysosomes, which degrade proteins. It has generally been assumed that UPS and lysosomes are regulated independently and serve distinct functions. The UPS degrades both cytosolic, nuclear proteins, and myofibrillar proteins, whereas the lysosomes degrade most membrane and extracellular proteins by endocytosis as well as cytosolic proteins and organelles via autophagy. Over the last two decades, the UPS has been increasingly recognized as a major system in several biological processes including cell proliferation, adaptation to stress and cell death. More recently, activation or impairment of the UPS has been reported in cardiac disease and recent evidence indicate that autophagy is a key mechanism to maintain cardiac structure and function. This review mainly focuses on the UPS and its various components in healthy and diseased heart, but also summarizes recent data suggesting parallel activation of the UPS and autophagy in cardiac disease.

Nonsense-Mediated mRNA Decay and Ubiquitin–Proteasome System Regulate Cardiac Myosin-Binding Protein C Mutant Levels in Cardiomyopathic Mice

Rationale: Mutations in the MYBPC3 gene encoding cardiac myosin-binding protein (cMyBP)-C are frequent causes of hypertrophic cardiomyopathy, but the mechanisms leading from mutations to disease remain elusive. Objective: The goal of the present study was therefore to gain insights into the mechanisms controlling the expression of MYBPC3 mutations. Methods and Results: We developed a cMyBP-C knock-in mouse carrying a point mutation. The level of total cMyBP-C mRNAs was 50% and 80% lower in heterozygotes and homozygotes, respectively. Surprisingly, the single G>A transition on the last nucleotide of exon 6 resulted in 3 different mutant mRNAs: missense (exchange of G for A), nonsense (exon skipping, frameshift, and premature stop codon) and deletion/insertion (as nonsense but with additional partial retention of downstream intron, restoring of the reading frame, and almost full-length protein). Inhibition of nonsense-mediated mRNA decay in cultured cardiac myocytes or in vivo with emetine or cycloheximide increased the level of nonsense mRNAs severalfold but not of the other mRNAs. By using sequential protein fractionation and a new antibody directed against novel amino acids produced by the frameshift, we showed that inhibition of the proteasome with epoxomicin via osmotic minipumps increased the level of (near) full-length mutants but not of truncated proteins. Homozygotes exhibited myocyte and left ventricular hypertrophy, reduced fractional shortening, and interstitial fibrosis; heterozygotes had no major phenotype. Conclusions: These data reveal (1) an unanticipated complexity of the expression of a single point mutation in the whole animal and (2) the involvement of both nonsense-mediated mRNA decay and the ubiquitin–proteasome system in lowering the level of mutant proteins.

Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms

AIMS Familial hypertrophic cardiomyopathy (FHC) is frequently caused by cardiac myosin-binding protein C (cMyBP-C) gene mutations, which should result in C-terminal truncated mutants. However, truncated mutants were not detected in myocardial tissue of FHC patients and were rapidly degraded by the ubiquitin-proteasome system (UPS) after gene transfer in cardiac myocytes. Since the diversity and specificity of UPS regulation lie in E3 ubiquitin ligases, we investigated whether the muscle-specific E3 ligases atrogin-1 or muscle ring finger protein-1 (MuRF1) mediate degradation of truncated cMyBP-C. METHODS AND RESULTS Human wild-type (WT) and truncated (M7t, resulting from a human mutation) cMyBP-C species were co-immunoprecipitated with atrogin-1 after adenoviral overexpression in cardiac myocytes, and WT-cMyBP-C was identified as an interaction partner of MuRF1 by yeast two-hybrid screens. Overexpression of atrogin-1 in cardiac myocytes decreased the protein level of M7t-cMyBP-C by 80% and left WT-cMyBP-C level unaffected. This was rescued by proteasome inhibition. In contrast, overexpression of MuRF1 in cardiac myocytes not only reduced the protein level of WT- and M7t-cMyBP-C by >60%, but also the level of myosin heavy chains (MHCs) by >40%, which were not rescued by proteasome inhibition. Both exogenous cMyBP-C and endogenous MHC mRNA levels were markedly reduced by MuRF1 overexpression. Similar to cardiac myocytes, MuRF1-overexpressing (TG) mice exhibited 40% lower levels of MHC mRNAs and proteins. Protein levels of cMyBP-C were 29% higher in MuRF1 knockout and 34% lower in TG than in WT, without a corresponding change in mRNA levels. CONCLUSION These data suggest that atrogin-1 specifically targets truncated M7t-cMyBP-C, but not WT-cMyBP-C, for proteasomal degradation and that MuRF1 indirectly reduces cMyBP-C levels by regulating the transcription of MHC.

Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: Mechanisms and therapeutic opportunities

Cardiac myosin-binding protein C (cMyBP-C) is a component of the thick filaments of the sarcomere. Understanding the structural and functional role of cMyBP-C in the heart is clinically relevant since cMyBP-C gene mutations are a widely recognized cause of hypertrophic cardiomyopathy (HCM), which affects 0.2% of the general population. Nonsense and frameshift mutations are common in cMyBP-C and their expressions are regulated by three quality control systems, the nonsense-mediated mRNA decay, ubiquitin-proteasome system, and autophagy, which contribute to minimize the production of potential poison mutant proteins. This review discusses the structural and regulatory functions of cMyBP-C, the molecular mechanisms involved in cMyBP-C-related HCM, as well as potential causative therapies for HCM.

Mybpc3 gene therapy for neonatal cardiomyopathy enables long-term disease prevention in mice

Homozygous or compound heterozygous frameshift mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C) cause neonatal hypertrophic cardiomyopathy (HCM), which rapidly evolves into systolic heart failure and death within the first year of life. Here we show successful long-term Mybpc3 gene therapy in homozygous Mybpc3-targeted knock-in (KI) mice, which genetically mimic these human neonatal cardiomyopathies. A single systemic administration of adeno-associated virus (AAV9)-Mybpc3 in 1-day-old KI mice prevents the development of cardiac hypertrophy and dysfunction for the observation period of 34 weeks and increases Mybpc3 messenger RNA (mRNA) and cMyBP-C protein levels in a dose-dependent manner. Importantly, Mybpc3 gene therapy unexpectedly also suppresses accumulation of mutant mRNAs. This study reports the first successful long-term gene therapy of HCM with correction of both haploinsufficiency and production of poison peptides. In the absence of alternative treatment options except heart transplantation, gene therapy could become a realistic treatment option for severe neonatal HCM.

Rescue of cardiomyopathy through U7snRNA-mediated exon skipping in Mybpc3-targeted knock-in mice

Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral‐mediated AON transfer in a Mybpc3‐targeted knock‐in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var‐4) deleted of exons 5–6 in wild‐type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON‐5 and AON‐6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno‐associated virus (AAV‐U7‐AON‐5+6). Transduction of cardiac myocytes or systemic administration of AAV‐U7‐AON‐5+6 increased Var‐4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof‐of‐concept study paves the way towards a causal therapy of HCM.

Endothelin‐1 Induces Myofibrillar Disarray and Contractile Vector Variability in Hypertrophic Cardiomyopathy–Induced Pluripotent Stem Cell–Derived Cardiomyocytes

Background Despite the accumulating genetic and molecular investigations into hypertrophic cardiomyopathy (HCM), it remains unclear how this condition develops and worsens pathologically and clinically in terms of the genetic–environmental interactions. Establishing a human disease model for HCM would help to elucidate these disease mechanisms; however, cardiomyocytes from patients are not easily obtained for basic research. Patient‐specific induced pluripotent stem cells (iPSCs) potentially hold much promise for deciphering the pathogenesis of HCM. The purpose of this study is to elucidate the interactions between genetic backgrounds and environmental factors involved in the disease progression of HCM. Methods and Results We generated iPSCs from 3 patients with HCM and 3 healthy control subjects, and cardiomyocytes were differentiated. The HCM pathological phenotypes were characterized based on morphological properties and high‐speed video imaging. The differences between control and HCM iPSC‐derived cardiomyocytes were mild under baseline conditions in pathological features. To identify candidate disease‐promoting environmental factors, the cardiomyocytes were stimulated by several cardiomyocyte hypertrophy‐promoting factors. Interestingly, endothelin‐1 strongly induced pathological phenotypes such as cardiomyocyte hypertrophy and intracellular myofibrillar disarray in the HCM iPSC‐derived cardiomyocytes. We then reproduced these phenotypes in neonatal cardiomyocytes from the heterozygous Mybpc3‐targeted knock in mice. High‐speed video imaging with motion vector prediction depicted physiological contractile dynamics in the iPSC‐derived cardiomyocytes, which revealed that self‐beating HCM iPSC‐derived single cardiomyocytes stimulated by endothelin‐1 showed variable contractile directions. Conclusions Interactions between the patients genetic backgrounds and the environmental factor endothelin‐1 promote the HCM pathological phenotype and contractile variability in the HCM iPSC‐derived cardiomyocytes.

Repair of Mybpc3 mRNA by 5′-trans-splicing in a Mouse Model of Hypertrophic Cardiomyopathy

RNA trans-splicing has been explored as a therapeutic option for a variety of genetic diseases, but not for cardiac genetic disease. Hypertrophic cardiomyopathy (HCM) is an autosomal-dominant disease, characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C) is frequently mutated. We evaluated the 5′-trans-splicing strategy in a mouse model of HCM carrying a Mybpc3 mutation. 5′-trans-splicing was induced between two independently transcribed molecules, the mutant endogenous Mypbc3 pre-mRNA and an engineered pre-trans-splicing molecule (PTM) carrying a FLAG-tagged wild-type (WT) Mybpc3 cDNA sequence. PTMs were packaged into adeno-associated virus (AAV) for transduction of cultured cardiac myocytes and the heart in vivo. Full-length repaired Mybpc3 mRNA represented up to 66% of total Mybpc3 transcripts in cardiac myocytes and 0.14% in the heart. Repaired cMyBP-C protein was detected by immunoprecipitation in cells and in vivo and exhibited correct incorporation into the sarcomere in cardiac myocytes. This study provides (i) the first evidence of successful 5′-trans-splicing in vivo and (ii) proof-of-concept of mRNA repair in the most prevalent cardiac genetic disease. Since current therapeutic options for HCM only alleviate symptoms, these findings open new horizons for causal therapy of the severe forms of the disease.

Cardiac myosin-binding protein C (MYBPC3) in cardiac pathophysiology.

More than 350 individual MYPBC3 mutations have been identified in patients with inherited hypertrophic cardiomyopathy (HCM), thus representing 40–50% of all HCM mutations, making it the most frequently mutated gene in HCM. HCM is considered a disease of the sarcomere and is characterized by left ventricular hypertrophy, myocyte disarray and diastolic dysfunction. MYBPC3 encodes for the thick filament associated protein cardiac myosin-binding protein C (cMyBP-C), a signaling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of contraction and relaxation. This review aims to provide a succinct overview of how mutations in MYBPC3 are considered to affect the physiological function of cMyBP-C, thus causing the deleterious consequences observed inHCM patients. Importantly, recent advances to causally treat HCM by repairing MYBPC3 mutations by gene therapy are discussed here, providing a promising alternative to heart transplantation for patients with a fatal form of neonatal cardiomyopathy due to bi-allelic truncating MYBPC3 mutations.

Prevention of Myofilament Dysfunction by β-Blocker Therapy in Postinfarct Remodeling

Background—Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether &bgr;-blocker therapy could restore myofilament contractility. Methods and Results—In pigs with a MI induced by ligation of the left circumflex coronary artery, &bgr;-blocker therapy (bisoprolol, MI+&bgr;) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (Fmax) was lower, whereas Ca2+ sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca2+ sensitivity was abolished by treatment of cells with the &bgr;-adrenergic kinase, protein kinase A. &bgr;-blocker therapy partially reversed Fmax and Ca2+ sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca2+ sensitivity in MI+&bgr; compared with untreated myocardium, the protein kinase A induced reduction in Ca2+ sensitivity was largest in cardiomyocytes from myocardium treated with &bgr;-blockers. Phosphorylation of &bgr;-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. &bgr;-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions—&bgr;-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of &bgr;-blockade on global left ventricular function after MI.