Filamin c (FLNc) is a large dimeric actin-binding protein located at premyofibrils, myofibrillar Z-discs and myofibrillar attachment sites of striated muscle cells, where it is involved in mechanical stabilization, mechanosensation and intracellular signaling. Mutations in the gene encoding FLNc give rise to skeletal muscle diseases and cardiomyopathies. Here, we demonstrate by fluorescence recovery after photobleaching that a large fraction of FLNc is highly mobile in cultured neonatal mouse cardiomyocytes and in cardiac and skeletal muscles of live transgenic zebrafish embryos. Analysis of cardiomyocytes from Xirp1 and Xirp2 deficient animals indicates that both Xin actin-binding repeat-containing proteins stabilize FLNc selectively in premyofibrils. Using a novel assay to analyze myofibrillar microdamage and subsequent repair in cultured contracting cardiomyocytes by live cell imaging, we demonstrate that repair of damaged myofibrils is achieved within only 4 hours, even in the absence of de novo protein synthesis. FLNc is immediately recruited to these sarcomeric lesions together with its binding partner aciculin and precedes detectable assembly of filamentous actin and recruitment of other myofibrillar proteins. These data disclose an unprecedented degree of flexibility of the almost crystalline contractile machinery and imply FLNc as a dynamic signaling hub, rather than a primarily structural protein. Our myofibrillar damage/repair model illustrates how (cardio)myocytes are kept functional in their mechanically and metabolically strained environment. Our results help to better understand the pathomechanisms and pathophysiology of early stages of FLNc-related myofibrillar myopathy and skeletal and cardiac diseases preceding pathological protein aggregation.

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The lack of a mutant phenotype in homozygous mutant individuals’ due to compensatory gene expression triggered upstream of protein function has been identified as genetic compensation. Whilst this intriguing process has been recognized in zebrafish, the presence of homozygous loss of function mutations in healthy human individuals suggests that compensation may not be restricted to this model. Loss of skeletal ∞-actin results in nemaline myopathy and we have previously shown that the pathological symptoms of the disease and reduction in muscle performance are recapitulated in a zebrafish antisense morpholino knockdown model. Here we reveal that a genetic actc1b mutant exhibits mild muscle defects and is unaffected by injection of the actc1b targeting morpholino. We further show that the milder phenotype results from a compensatory transcriptional upregulation of an actin paralogue providing a novel approach to be explored for the treatment of actin myopathy. Our findings provide further evidence that genetic compensation may influence the penetrance of disease-causing mutations. Many healthy individuals carry loss of function mutations in essential genes that would normally be deleterious for survival. Intriguingly, it may be the presence of the genomic lesion itself in these individuals that triggers the compensatory pathways. It is not known how widespread this phenomenon is in vertebrate populations and how genetic compensation is activated. We have shown that knockdown of actin causes nemaline myopathy as indicated by the formation of nemaline bodies within the skeletal muscle and reduced muscle function which, remarkably, we did not observe in an actin genetic mutant. We have identified that protection from the disease phenotype results from transcriptional upregulation of an actin paralogue restoring actin protein in the skeletal muscle. This study demonstrates that genetic compensation may be more prevalent than previously anticipated and highlights phenotypic differences resulting from genetic mutations versus antisense knockdown approaches. Furthermore, we suggest that activating compensatory pathways may be exploited as a potential novel therapeutic approach for human disorders caused by loss of function mutations.

Myofibrillar myopathy is a progressive muscle disease characterized by the disintegration of muscle fibers and formation of protein aggregates. Causative mutations have been identified in nine genes encoding Z-disk proteins, including the actin binding protein filamin C (FLNC). To investigate the mechanism of disease in FLNC^W2710X myopathy we overexpressed fluorescently tagged FLNC or FLNC^W2710X in zebrafish. Expression of FLNC^W2710X causes formation of protein aggregates but surprisingly, our studies reveal that the mutant protein localizes correctly to the Z-disk and is capable of rescuing the fiber disintegration phenotype that results from FLNC knockdown. This demonstrates that the functions necessary for muscle integrity are not impaired, and suggests that it is the formation of protein aggregates and subsequent sequestration of FLNC away from the Z-disk that results in myofibrillar disintegration. Similar to those found in patients, the aggregates in FLNC^W2710X expressing fish contain the co-chaperone BAG3. FLNC is a target of the BAG3-mediated chaperone assisted selective autophagy (CASA) pathway and therefore we investigated its role, and the role of autophagy in general, in clearing protein aggregates. We reveal that despite BAG3 recruitment to the aggregates they are not degraded via CASA. Additionally, recruitment of BAG3 is sufficient to block alternative autophagy pathways which would otherwise clear the aggregates. This blockage can be relieved by reducing BAG3 levels or by stimulating autophagy. This study therefore identifies both BAG3 reduction and autophagy promotion as potential therapies for FLNC^W2710X myofibrillar myopathy, and identifies protein insufficiency due to sequestration, compounded by impaired autophagy, as the cause.