Abstract
UNC-45 is a UCS (UNC-45/CRO1/She4P) class chaperone necessary for myosin folding and/or accumulation, but its requirement for maintaining cardiac contractility has not been explored. Given the prevalence of myosin mutations in eliciting cardiomyopathy, chaperones like UNC-45 are likely to be equally critical in provoking or modulating myosin-associated cardiomyopathy. Here, we used the Drosophila heart model to examine its role in cardiac physiology, in conjunction with RNAi-mediated gene silencing specifically in the heart in vivo. Analysis of cardiac physiology was carried out using high-speed video recording in conjunction with movement analysis algorithms. unc-45 knockdown resulted in severely compromised cardiac function in adults as evidenced by prolonged diastolic and systolic intervals, and increased incidence of arrhythmias and extreme dilation; the latter was accompanied by a significant reduction in muscle contractility. Structural analysis showed reduced myofibrils, myofibrillar disarray, and greatly decreased cardiac myosin accumulation. Cardiac unc-45 silencing also dramatically reduced life-span. In contrast, third instar larval and young pupal hearts showed mild cardiac abnormalities, as severe cardiac defects only developed during metamorphosis. Furthermore, cardiac unc-45 silencing in the adult heart (after metamorphosis) led to less severe phenotypes. This suggests that UNC-45 is mostly required for myosin accumulation/folding during remodeling of the forming adult heart. The cardiac defects, myosin deficit and decreased life-span in flies upon heart-specific unc-45 knockdown were significantly rescued by UNC-45 over-expression. Our results are the first to demonstrate a cardiac-specific requirement of a chaperone in Drosophila, suggestive of a critical role of UNC-45 in cardiomyopathies, including those associated with unfolded proteins in the failing human heart. The dilated cardiomyopathy phenotype associated with UNC-45 deficiency is mimicked by myosin knockdown suggesting that UNC-45 plays a crucial role in stabilizing myosin and possibly preventing human cardiomyopathies associated with functional deficiencies of myosin.
Highlights
The myosin superfamily consists of at least 35 classes of actin-based molecular motors
Analysis of heart function in 1 week old Hand.UAS-unc-45 RNAi flies revealed severe heart defects compared to controls (Figure 1B, Movie S1): unc-45 KD hearts were significantly dilated in both the conical chamber (CC) and the third abdominal segment heart and exhibited dramatically arrhythmic beating patterns, compared to control hearts
This confirms our previous finding that unc-45 null mutants were embryonic lethal and deficient in myosin accumulation [17]
Summary
The myosin superfamily consists of at least 35 classes of actin-based molecular motors. These motors are critical for cellular processes such as cytokinesis, vesicle transport, cell motility, and muscle movement [1]. Chaperones are involved with multiple pathways including myosin folding and are critically required for maintaining cardiac function [11,12,13,14]. A growing body of evidence suggests that folding and stability of myosin is dependent upon UNC-45 levels [15,16,17,18,19,20,21,22]. UNC-45 is present in both invertebrates and vertebrates and is important for myosin maturation, thick filament assembly and muscle function [18,24,25,26]
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