In this setting, HCM patients with MYH7 were diagnosed at a younger age and had more hypertrophy, but they had no greater frequency of sudden death among first-degree relatives. Although these associations may prove useful for targeted gene screening, caution should be exercised in terms of using pathogenic status in risk stratification.

We generated transgenic mice that overexpressed gamma-(cyto) actin 2000-fold above wild-type levels in skeletal muscle. gamma-(cyto) actin comprised 40% of total actin in transgenic skeletal muscle, with a concomitant 40% decrease in alpha-actin. Surprisingly, transgenic muscle was histologically and ultrastructurally identical to wild-type muscle despite near-stoichiometric incorporation of gamma-(cyto) actin into sarcomeric thin filaments. Furthermore, several parameters of muscle physiological performance in the transgenic animals were not different from wild type. Given these surprising results, we tested whether overexpression of gamma-(cyto) actin could rescue the early postnatal lethality in alpha-(sk) actin-null mice (Acta1(-/-)). By quantitative Western blot analysis, we found total actin levels were decreased by 35% in Acta1(-/-) muscle. Although transgenic overexpression of gamma-(cyto) actin on the Acta1(-/-) background restored total actin levels to wild type, resulting in thin filaments composed of 60% gamma-(cyto) actin and a 40% mixture of cardiac and vascular actin, the life span of transgenic Acta1(-/-) mice was not extended. These results indicate that sarcomeric thin filaments can accommodate substantial incorporation of gamma-(cyto) actin without functional consequences, yet gamma-(cyto) actin cannot fully substitute for alpha-(sk) actin.

Backgroundγ-cytoplasmic (γ-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice.MethodsWe transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG), and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups.ResultsLevels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions.ConclusionsThe data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

α-Dystrobrevin is a component of the dystrophin-glycoprotein complex (DGC) and is thought to have both structural and signaling roles in skeletal muscle. Mice deficient for α-dystrobrevin (adbn−/−) exhibit extensive myofiber degeneration and neuromuscular junction abnormalities. However, the biochemical stability of the DGC and the functional performance of adbn−/− muscle have not been characterized. Here we show that the biochemical association between dystrophin and β-dystroglycan is compromised in adbn−/− skeletal muscle, suggesting that α-dystrobrevin plays a structural role in stabilizing the DGC. However, despite muscle cell death and DGC destabilization, costamere organization and physiological performance is normal in adbn−/− skeletal muscle. Our results demonstrate that myofiber degeneration alone does not cause functional deficits and suggests that more complex pathological factors contribute to the development of muscle weakness in muscular dystrophy.

BackgroundThe X-linked recessive disease Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding the protein dystrophin. Despite its large size, dystrophin is a highly stable protein, demonstrating cooperative unfolding during thermal denaturation as monitored by circular dichroism spectroscopy. In contrast, internal sequence deletions have been associated with a loss of the cooperative unfolding and cause in vitro protein aggregation. Several emerging therapy options for DMD utilize internally deleted micro-dystrophins and multi-exon-skipped dystrophins that produce partially functional proteins, but the stability of such internally truncated proteins has not been investigated.MethodsIn this study, we analyzed the in vitro stability of human dystrophin constructs skipped around exon 45 or exon 51, several dystrophin gene therapy constructs, as well as human full-length and micro-utrophin. Constructs were expressed in insect cells using the baculovirus system, purified by affinity chromatography, and analyzed by high-speed sedimentation, circular dichroism spectroscopy, and differential scanning fluorimetry.ResultsOur results reveal that not all gene therapy constructs display stabilities consistent with full-length human dystrophin. However, all dystrophins skipped in-frame around exon 45 or exon 51 show stability profiles congruent with intact human dystrophin. Similar to previous studies of mouse proteins, full-length human utrophin also displays stability similar to human dystrophin and does not appear to be affected by a large internal deletion.ConclusionsOur results suggest that the in vitro stability of human dystrophin is less sensitive to smaller deletions at natural exon boundaries than larger, more complex deletions present in some gene therapy constructs.