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.

Molecular study of electron transfer flavoprotein alpha-subunit deficiency in two Japanese children with different phenotypes of glutaric acidemia type II.

BACKGROUND: Electron transfer flavoprotein is a mitochondrial matrix protein composed of alpha- and beta-subunits (ETF alpha and ETF beta, respectively). This protein transfers electrons between several mitochondrial dehydrogenases and the main respiratory chain via ETF dehydrogenase (ETF-DH). Defects in ETF or ETF-DH cause glutaric acidemias type II (GAII). MATERIALS AND METHODS: We investigated the molecular basis of ETF alpha deficiency in two Japanese children with different clinical phenotypes using expression study. RESULTS: Patient 1 had the severe form of GAII, a compound heterozygote of two mutations: 799G to A (alpha G267R) and nonsense 7C to T (alpha R3X). Patient 2 had the mild form and carried two heterozygous mutations: 764G to T (alpha G255V) and 478delG (frameshift). Both patients had one each of missense mutations in one allele; the others were either nonsense or truncated. Restriction enzyme digestion assay using genomic DNAs from 100 healthy Japanese revealed that these mutations were all novel. No signal for ETF alpha was detected by immunoblotting in cases of missense mutants, while wild-type cDNA resulted in expression of ETF alpha protein. Transfection with wild-type ETF alpha cDNA into cultured cells from both patients elevated incorporation of radioisotope-labelled fatty acids. CONCLUSION: These four mutations were pathogenic for GAII and missense mutations, alpha G255V and alpha G267R were considered anecdotal for mild and severe forms, respectively.