Myosin heavy chains encoded byMYH7andMYH2are among the most abundant proteins in human skeletal muscle. After decades of intense research using a wide range of biophysical and biological approaches, their functions have begun to be elucidated. Despite this, it remains unclear how mutations in these genes and resultant proteins disrupt myosin structure and function, inducing pathological states and skeletal myopathies termed myosinopathies. Here, we have analysed the effects of several commonMYH7andMYH2mutations located in light meromyosin (LMM) using a broad range of approaches. We determined the secondary structure and filament forming capabilities of expressed and purified LMM constructs in vitro, performedin-silicomodelling of LMM constructs, and evaluated the incorporation of eGFP-myosin heavy chain constructs into sarcomeres in cultured myotubes. Using muscle biopsies from patients, we applied Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, X-ray diffraction measurements to estimate myosin head order and myofibre mechanics to investigate contractile function. We found that humanMYH7andMYH2LMM mutations commonly disrupt myosin coiled-coil structure and packing of filamentsin vitro; decrease the myosin super-relaxed statein vivoand increase the basal myosin ATP consumption; but are not associated with myofibre contractile deficits. Altogether, these findings indicate that the structural remodelling resulting from LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help in the design of future therapies for myosinopathies.
