Altered regional cardiac wall mechanics are associated with differential cardiomyocyte calcium handling due to nebulette mutations in preclinical inherited dilated cardiomyopathy

Nebulette (NEBL) is a sarcomeric Z-disk 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. The objectives of this study are to determine the earliest preclinical mechanical changes in the myocardium and define underlying molecular mechanisms by which NEBL mutations lead to cardiac dysfunction. 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, and 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. 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.