BACKGROUND Familial restrictive cardiomyopathy (FRCM) has a poor prognosis due to diastolic dysfunction and restrictive physiology (RP). Myocardial stiffness, with or without fibrosis, underlie the RP, but the mechanism(s) of restrictive remodeling is unclear. Myopalladin (MYPN) is a messenger molecule that links structural and gene regulatory molecules via translocation from the Z-disk and I-bands to the nucleus in cardiomyocytes. Expression of N-terminal MYPN peptide results in a severe disruption of sarcomere. OBJECTIVES A nonsense MYPN-Q529X mutation previously identified in the FRCM family was studied here in an animal model to explore the molecular and pathogenic mechanisms of FRCM. METHODS Functional (echocardiography, cardiac magnetic resonance [CMR] imaging, electrocardiography), morphohistological, gene expression, and molecular studies were performed in knock-in heterozygote (MypnWT/Q526X) and homozygote mice harboring the human MYPN-Q529X mutation. RESULTS At 12 weeks of age, echocardiographic and CMR imaging signs of diastolic dysfunction with preserved systolic function were identified in MypnWT/Q526X mice. Histology revealed interstitial and perivascular fibrosis without overt hypertrophic remodeling. Truncated MypnQ526X protein was found to translocate to the nucleus. Levels of total and nuclear cardiac ankyrin repeat protein (Carp/Ankrd1) and phosphorylation of Mek1/2, Erk1/2, Smad2, and Akt were reduced. Up-regulation was evident for muscle LIM protein (Mlp), desmin, and heart failure (Nppa, Nppb, and Myh6) and fibrosis (Tgfβ1, αSma, Opn, and Postn) markers. CONCLUSIONS Heterozygote MypnWT/Q526X knock-in mice develop RCM due to persistence of mutant Mypn-Q526X protein in the nucleus. Down-regulation of Carp and up-regulation of Mlp and desmin appear to augment fibrotic restrictive remodeling, and reduced Erk1/2 blunts a hypertrophic response in MypnWT/Q526X hearts.

Arrhythmogenic ventricular cardiomyopathy (AVC) is a frequent underlying cause for arrhythmias and sudden cardiac death especially during intense exercise. The mechanisms involved remain largely unknown. The purpose of this study was to investigate how chronic endurance exercise contributes to desmoplakin (DSP) mutation-induced AVC pathogenesis. Transgenic mice with overexpression of desmoplakin, wild-type (Tg-DSP(WT)), or the R2834H mutant (Tg-DSP(R2834H)) along with control nontransgenic (NTg) littermates were kept sedentary or exposed to a daily running regimen for 12 wk. Cardiac function and morphology were analyzed using echocardiography, electrocardiography, histology, immunohistochemistry, RNA, and protein analysis. At baseline, 4-wk-old mice from all groups displayed normal cardiac function. When subjected to exercise, all mice retained normal cardiac function and left ventricular morphology; however, Tg-DSP(R2834H) mutants displayed right ventricular (RV) dilation and wall thinning, unlike NTg and Tg-DSP(WT). The Tg-DSP(R2834H) hearts demonstrated focal fat infiltrations in RV and cytoplasmic aggregations consisting of desmoplakin, plakoglobin, and connexin 43. These aggregates coincided with disruption of the intercalated disks, intermediate filaments, and microtubules. Although Tg-DSP(R2834H) mice already displayed high levels of p-GSK3-β(Ser9) and p-AKT1(Ser473) under sedentary conditions, decrease of nuclear GSK3-β and AKT1 levels with reduced p-GSK3-β(Ser9), p-AKT1(Ser473), and p-AKT1(Ser308) and loss of nuclear junctional plakoglobin was apparent after exercise. In contrast, Tg-DSP(WT) showed upregulation of p-AKT1(Ser473), p-AKT1(Ser308), and p-GSK3-β(Ser9) in response to exercise. Our data suggest that endurance exercise accelerates AVC pathogenesis in Tg-DSP(R2834H) mice and this event is associated with perturbed AKT1 and GSK3-β signaling. Our study suggests a potential mechanism-based approach to exercise management in patients with AVC.

Background Nebulette (NEBL) is a sarcomeric Z-disc protein involved in mechanosensing and force generation via its interaction with actin and tropomyosin-troponin complex. Genetic abnormalities in NEBL lead to dilated cardiomyopathy (DCM) in humans and animal models. Objectives To determine the earliest preclinical mechanical changes in the myocardium and define underlying molecular mechanisms by which NEBL mutations lead to cardiac dysfunction. Methods and Results We examined cardiac function in 3-month-old non-transgenic (non-Tg) and transgenic (Tg) mice (WT-Tg, G202R-Tg, A592E-Tg) by cardiac magnetic resonance (CMR) imaging. Contractility and calcium transients were measured in isolated cardiomyocytes. A592E-Tg mice exhibited enhanced in vivo twist and untwisting rate compared to control groups. Ex vivo analysis of A592E-Tg cardiomyocytes showed blunted calcium decay response to isoproterenol. CMR imaging of G202R-Tg mice demonstrated reduced torsion compared to non-Tg and WT-Tg, but conserved twist and untwisting rate after correcting for geometric changes. Ex vivo analysis of G202R-Tg cardiomyocytes showed elevated calcium decay at baseline and a conserved contractile response to isoproterenol stress. Protein analysis showed decreased α-actinin and connexin43, increased cardiac troponin I phosphorylation at baseline in G202R-Tg, providing a molecular mechanism for enhanced ex vivo calcium decay. Ultrastructurally, G202R-Tg cardiomyocytes exhibited increased I-band and sarcomere length, desmosomal separation, and enlarged t-tubules. A592E-Tg cardiomyocytes also showed abnormal ultrastructural changes and desmin downregulation. Conclusion This study showed distinct effects of NEBL mutations on sarcomere ultrastructure, cellular contractile function, and calcium homeostasis in preclinical DCM in vivo. We suggest that these abnormalities correlate with detectable myocardial wall motion patterns.

AIMTo investigate the regulation of Myopalladin (Mypn) and identify its gene network involved in restrictive cardiomyopathy (RCM).METHODSGene expression values were measured in the heart of a large family of BXD recombinant inbred (RI) mice derived from C57BL/6J and DBA/2J. The proteomics data were collected from Mypn knock-in and knock-out mice. Expression quantitative trait locus (eQTL) mapping methods and gene enrichment analysis were used to identify Mypn regulation, gene pathway and co-expression networks.RESULTSA wide range of variation was found in expression of Mypn among BXD strains. We identified upstream genetic loci at chromosome 1 and 5 that modulate the expression of Mypn. Candidate genes within these loci include Ncoa2, Vcpip1, Sgk3, and Lgi2. We also identified 15 sarcomeric genes interacting with Mypn and constructed the gene network. Two novel members of this network (Syne1 and Myom1) have been confirmed at the protein level. Several members in this network are already known to relate to cardiomyopathy with some novel genes candidates that could be involved in RCM.CONCLUSIONUsing systematic genetics approach, we constructed Mypn co-expression networks that define the biological process categories within which similarly regulated genes function. Through this strategy we have found several novel genes that interact with Mypn that may play an important role in the development of RCM.