Samantha Harris

Thin filament length (TFL) is an important determinant of the force-sarcomere length (SL) relation of cardiac muscle. However, the various mechanisms that control TFL are not well understood. Here we tested the previously proposed hypothesis that the actin-binding protein nebulin contributes to TFL regulation in the heart by using a cardiac-specific nebulin cKO mouse model (αMHC Cre Neb cKO). Atrial myocytes were studied because nebulin expression has been reported to be most prominent in this cell type. TFL was measured in right and left atrial myocytes using deconvolution optical microscopy and staining for filamentous actin with phalloidin and for the thin filament pointed-end with an antibody to the capping protein Tropomodulin-1 (Tmod1). Results showed that TFLs in Neb cKO and littermate control mice were not different. Thus, deletion of nebulin in the heart does not alter TFL. However, TFL was found to be ~0.05μm longer in the right than in the left atrium and Tmod1 expression was increased in the right atrium. We also tested the hypothesis that the length of titin's spring region is a factor controlling TFL by studying the Rbm20(ΔRRM) mouse which expresses titins that are ~500kDa (heterozygous mice) and ~1000kDa (homozygous mice) longer than in control mice. Results revealed that TFL was not different in Rbm20(ΔRRM) mice. An unexpected finding in all genotypes studied was that TFL increased as sarcomeres were stretched (~0.1μm per 0.35μm of SL increase). This apparent increase in TFL reached a maximum at a SL of ~3.0μm where TFL was ~1.05μm. The SL dependence of TFL was independent of chemical fixation or the presence of cardiac myosin-binding protein C (cMyBP-C). In summary, we found that in cardiac myocytes TFL varies with SL in a manner that is independent of the size of titin or the presence of nebulin.

In the 20 years since the discovery of the first mutation linked to familial hypertrophic cardiomyopathy (HCM), an astonishing number of mutations affecting numerous sarcomeric proteins have been described. Among the most prevalent of these are mutations that affect thick filament binding proteins, including the myosin essential and regulatory light chains and cardiac myosin binding protein (cMyBP)-C. However, despite the frequency with which myosin binding proteins, especially cMyBP-C, have been linked to inherited cardiomyopathies, the functional consequences of mutations in these proteins and the mechanisms by which they cause disease are still only partly understood. The purpose of this review is to summarize the known disease-causing mutations that affect the major thick filament binding proteins and to relate these mutations to protein function. Conclusions emphasize the impact that discovery of HCM-causing mutations has had on fueling insights into the basic biology of thick filament proteins and reinforce the idea that myosin binding proteins are dynamic regulators of the activation state of the thick filament that contribute to the speed and force of myosin-driven muscle contraction. Additional work is still needed to determine the mechanisms by which individual mutations induce hypertrophic phenotypes.

The two genes most commonly associated with mutations linked to hypertrophic or dilated cardiomyopathies are β-myosin and cardiac myosin binding protein-C (cMyBP-C). Both of these proteins interact with cardiac actin (ACTC). Currently there are 16 ACTC variants that have been found in patients with HCM or DCM. While some of these ACTC variants exhibit protein instability or polymerization-deficiencies that might contribute to the development of disease, other changes could cause changes in protein-protein interactions between sarcomere proteins and ACTC. To test the hypothesis that changes in ACTC disrupt interactions with cMyBP-C, we examined the interactions between seven ACTC variants and the N-terminal C0C2 fragment of cMyBP-C. We found there was a significant decrease in binding affinity (increase in Kd values) for the A331P and Y166C variants of ACTC. These results suggest that a change in the ability of cMyBP-C to bind actin filaments containing these ACTC protein variants might contribute to the development of disease. These results also provide clues regarding the binding site of the C0C2 fragment of cMyBP-C on F-actin.

Myosin binding protein C (MyBP-C) is a component of the thick filament of striated muscle. The importance of this protein is revealed by recent evidence that mutations in the cardiac gene are a major cause of familial hypertrophic cardiomyopathy. Here we investigate the distribution of MyBP-C in the A-bands of cardiac and skeletal muscles and compare this to the A-band structure in cardiac muscle of MyBP-C-deficient mice. We have used a novel averaging technique to obtain the axial density distribution of A-bands in electron micrographs of well-preserved specimens. We show that cardiac and skeletal A-bands are very similar, with a length of 1.58+/-0.01 mum. In normal cardiac and skeletal muscle, the distributions are very similar, showing clearly the series of 11 prominent accessory protein stripes in each half of the A-band spaced axially at 43-nm intervals and starting at the edge of the bare zone. We show by antibody labelling that in cardiac muscle the distal nine stripes are the location of MyBP-C. These stripes are considerably suppressed in the knockout mouse hearts as expected. Myosin heads on the surface of the thick filament in relaxed muscle are thought to be arranged in a three-stranded quasi-helix with a mean 14.3-nm axial cross bridge spacing and a 43 nm helix repeat. Extra "forbidden" meridional reflections, at orders of 43 nm, in X-ray diffraction patterns of muscle have been interpreted as due to an axial perturbation of some levels of myosin heads. However, in the MyBP-C-deficient hearts these extra meridional reflections are weak or absent, suggesting that they are due to MyBP-C itself or to MyBP-C in combination with a head perturbation brought about by the presence of MyBP-C.

Mutations in MYBPC3, the gene encoding cardiac myosin binding protein C (cMyBP-C), are a major cause of hypertrophic cardiomyopathy (HCM). While most mutations encode premature stop codons, missense mutations causing single amino acid substitutions are also common. Here we investigated effects of a single proline for alanine substitution at amino acid 31 (A31P) in the C0 domain of cMyBP-C, which was identified as a natural cause of HCM in cats. Results using recombinant proteins showed that the mutation disrupted C0 structure, altered sensitivity to trypsin digestion, and reduced recognition by an antibody that preferentially recognizes N-terminal domains of cMyBP-C. Western blots detecting A31P cMyBP-C in myocardium of cats heterozygous for the mutation showed a reduced amount of A31P mutant protein relative to wild-type cMyBP-C, but the total amount of cMyBP-C was not different in myocardium from cats with or without the A31P mutation indicating altered rates of synthesis/degradation of A31P cMyBP-C. Also, the mutant A31P cMyBP-C was properly localized in cardiac sarcomeres. These results indicate that reduced protein expression (haploinsufficiency) cannot account for effects of the A31P cMyBP-C mutation and instead suggest that the A31P mutation causes HCM through a poison polypeptide mechanism that disrupts cMyBP-C or myocyte function.