Muscle weakness in TPM3-myopathy is due to reduced Ca²⁺-sensitivity and impaired acto-myosin cross-bridge cycling in slow fibres

Dominant mutations in TPM3, encoding a-tropomyosin_slow, cause a congenital myopathy characterized by generalized muscle weakness. Here, we used a multidisciplinary approach to investigate the mechanism of muscle dysfunction in 12 TPM3-myopathy patients.We confirm that slow myofibre hypotrophy is a diagnostic hallmark of TPM3-myopathy, and is commonly accompanied by skewing of fibre-type ratios (either slow or fast fibre predominance). Patient muscle contained normal ratios of the three tropomyosin isoforms and normal fibre-type expression of myosins and troponins. Using 2D-PAGE, we demonstrate that mutant a-tropomyosin_slow was expressed, suggesting muscle dysfunction is due to a dominant-negative effect of mutant protein on muscle contraction. Molecular modelling suggested mutant a-tropomyosin_slow likely impacts actin-tropomyosin interactions and, indeed, co-sedimentation assays showed reduced binding of mutant a-tropomyosin_slow (R168C) to filamentous actin. Single fibre contractility studies of patient myofibres revealed marked slow myofibre specific abnormalities. At saturating [Ca²⁺] (pCa 4.5), patient slow fibres produced only 63% of the contractile force produced in control slow fibres and had reduced acto-myosin cross-bridge cycling kinetics. Importantly, due to reduced Ca²⁺-sensitivity, at sub-saturating [Ca²⁺] (pCa 6, levels typically released during in vivo contraction) patient slow fibres produced only 26% of the force generated by control slow fibres. Thus, weakness in TPM3-myopathy patients can be directly attributed to reduced slow fibre force at physiological [Ca²⁺], and impaired acto-myosin cross-bridge cycling kinetics. Fastmyofibres are spared; however, they appear to be unable to compensate for slow fibre dysfunction. Abnormal Ca²⁺-sensitivity in TPM3-myopathy patients suggests Ca²⁺-sensitizing drugs may represent a useful treatment for this condition.