Degradation Of Sarcomeric Proteins Research Articles

New cardiac and skeletal protein aggregate myopathy associated with combined MuRF1 and MuRF3 mutations.

Protein aggregate myopathies (PAMs) define muscle disorders characterized by protein accumulation in muscle fibres. We describe a new PAM in a patient with proximal muscle weakness and hypertrophic cardiomyopathy, whose muscle fibres contained inclusions containing myosin and myosin-associated proteins, and aberrant distribution of microtubules. These lesions appear as intact A- and M-bands lacking thin filaments and Z-discs. These features differ from inclusions in myosin storage myopathy (MSM), but are highly similar to those in mice deficient for the muscle-specific RING finger proteins MuRF1 and MuRF3. Sanger sequencing excluded mutations in the MSM-associated gene MYH7 but identified mutations in TRIM63 and TRIM54, encoding MuRF1 and MuRF3, respectively. No mutations in other potentially disease-causing genes were identified by Sanger and whole exome sequencing. Analysis of seven family members revealed that both mutations segregated in the family but only the homozygous TRIM63 null mutation in combination with the heterozygous TRIM54 mutation found in the proband caused the disease phenotype. Both MuRFs are microtubule-associated proteins localizing to sarcomeric M-bands and Z-discs. They are E3 ubiquitin ligases that play a role in degradation of sarcomeric proteins, stabilization of microtubules and myogenesis. Lack of ubiquitin and the 20S proteasome subunit in the inclusions found in the patient suggested impaired turnover of thick filament proteins. Disruption of microtubules in cultured myotubes was rescued by transient expression of wild-type MuRF1. The unique features of this novel myopathy point to defects in homeostasis of A-band proteins in combination with instability of microtubules as cause of the disease.

O.5 Mutations in MuRF1 and MuRF3 cause a novel protein aggregate myopathy and cardiomyopathy

MuRF1 and MuRF3 are microtubule-associated proteins that localize to the sarcomeric M-band and Z-line of striated muscle. They are E3 ubiquitin ligases that mediate degradation of sarcomeric proteins and participate in diverse biological functions including stabilisation of microtubules and myogenesis. Double knock-out mice for MuRF1 and MuRF3 developed cardiomyopathy and skeletal myopathy. Morphological features included myosin accumulation and aggregates of fragmented sarcomeres with preserved A-bands and M-lines. We studied a family with three affected members suffering from cardiomyopathy or cardioskeletal myopathy. We performed sequencing analysis of TRIM63 and TRIM54 encoding MuRF1 and MuRF3, respectively. Transfection of wild-type MuRF1 in cultured patient myoblast was performed to assess the rescue assay. To characterize the behaviour of TRIM54 mutation, control myoblasts were transfected with wild-type or mutant MuRF3. We identified a homozygous MuRF1 null mutation and heterozygous MuRF3 mutation in the patient with cardioskeletal myopathy. Myopathological features were highly reminiscent of that of MuRF1−/−/MuRF3−/− mice. Cultured myotubes from patient showed perturbed myofibrillogenesis and abnormal organization of the microtubule network. Transfection of wild-type MuRF1 in cultured patient myocytes rescued the phenotype. Transfection of control myoblasts with mutant MuRF3 induced the formation of irregular filamentous structures demonstrating the pathogenic effect of the MuRF3 mutation. We describe a novel protein aggregate myopathy and cardiomyopathy due to MuRF1 deficiency and a deleterious MuRF3 missense mutation. Unique morphological features including myosin accumulation, disrupted microtubule structures and fragmented sarcomeres with preserved A-bands and M-lines in the absence of thin filaments and Z-lines characterize the disease associated with combined MuRF1 and MuRF3 deficiency.

The myosin-interacting protein SMYD1 is essential for sarcomere organization

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.

The myosin-interacting protein SMYD1 is essential for sarcomere organization

Assembly, maintenance and renewal of sarcomeres require highly organized and balanced folding, transport, modification and degradation of sarcomeric proteins. However, the molecules that mediate these processes are largely unknown. Here, we isolated the zebrafish mutant flatline (fla), which shows disturbed sarcomere assembly exclusively in heart and fast-twitch skeletal muscle. By positional cloning we identified a nonsense mutation within the SET- and MYND-domain-containing protein 1 gene (smyd1) to be responsible for the fla phenotype. We found SMYD1 expression to be restricted to the heart and fast-twitch skeletal muscle cells. Within these cell types, SMYD1 localizes to both the sarcomeric M-line, where it physically associates with myosin, and the nucleus, where it supposedly represses transcription through its SET and MYND domains. However, although we found transcript levels of thick filament chaperones, such as Hsp90a1 and UNC-45b, to be severely upregulated in fla, its histone methyltransferase activity - mainly responsible for the nuclear function of SMYD1 - is dispensable for sarcomerogenesis. Accordingly, sarcomere assembly in fla mutant embryos can be reconstituted by ectopically expressing histone methyltransferase-deficient SMYD1. By contrast, ectopic expression of myosin-binding-deficient SMYD1 does not rescue fla mutants, implicating an essential role for the SMYD1-myosin interaction in cardiac and fast-twitch skeletal muscle thick filament assembly.