A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila

Abstract

An “invariant proline” separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138–43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc5, an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.

Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila

“…Our fly model thus provides a platform for readily validating putative human DCM myosins as leading to phenotypes consistent with DCM and for employing a genetic background that obviates modifier gene effects often observed in human DCM families. Combining our current results with our previous studies on hypertrophic and restrictive cardiomyopathy mutations ( Achal et al. , 2016 ; Kronert et al.…”

“…6 A-B). Cardiac myofibrils (MF) are oriented mainly perpendicular to the anteriorposterior axis and contain discontinuous Z-disks (arrows), as standardly observed in this tissue (Melkani et al, 2013;Kaushik et al, 2015;Achal et al, 2016;Bhide et al, 2018;Kronert et al, 2018). The ultrastructural properties of 4-day-old S532P homozygous mutant hearts are similar to controls, with no obvious signs of myofibrillar disorganization, assembly defects, or degeneration.…”

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy

“…A layer of structurally supportive ventral-longitudinal skeletal muscle is located ventral to the cardiomyocyte layer ( Figure 6 , VL). In heterozygous controls ( PwMhc2 / + ), both young 1-week-old and aged 3-week-old flies showed intact myofibrils with characteristic discontinuous Z-lines ( Achal et al, 2016 ) ( Figure 6 left). In contrast, R146N-15/+ heterozygotes displayed areas of myofibrillar discontinuity, where filamentous fields appear to have been pulled apart ( Figure 6 center, arrows) and these defects worsened with aging.…”

“…For cardiac electron microscopy, heart samples at 1 or 3 weeks of age (three per age per genotype) were surgically exposed in an oxygenated artificial hemolymph solution and fixed using a modified procedure as described previously ( Achal et al, 2016 ). Rhythmic beating was observed to confirm structural integrity of samples.…”

Drosophila Heart as a Model for Cardiac Development and Diseases

“…The Drosophila model has helped to identify novel genes and pathways involved in arrhythmias [47], atrial fibrillation [80], and channelopathies [81]. It has also been applied to modeling cardiac aging [51], and cardiomyopathies including dilated [82][83][84][85][86][87], hypertrophic [88][89][90], and restrictive cardiomyopathy [82,91,92], in which heart failure could occur [43]. Finally, the developed Drosophila models of muscular diseases with cardiac symptoms such as DM1 have been instrumental in dissecting the gene deregulations underlying heart defects and in identifying new therapeutic strategies.…”