Samantha Harris

Mutations in genes that encode for muscle sarcomeric proteins have been identified in humans and two breeds of domestic cats with hypertrophic cardiomyopathy (HCM). This article reviews the history, genetics, and pathogenesis of HCM in the two species in order to give veterinarians a perspective on the genetics of HCM. Hypertrophic cardiomyopathy in people is a genetic disease that has been called a disease of the sarcomere because the preponderance of mutations identified that cause HCM are in genes that encode for sarcomeric proteins (Maron and Maron, 2013). Sarcomeres are the basic contractile units of muscle and thus sarcomeric proteins are responsible for the strength, speed, and extent of muscle contraction. In people with HCM, the two most common genes affected by HCM mutations are the myosin heavy chain gene (MYH7), the gene that encodes for the motor protein β-myosin heavy chain (the sarcomeric protein that splits ATP to generate force), and the cardiac myosin binding protein-C gene (MYBPC3), a gene that encodes for the closely related structural and regulatory protein, cardiac myosin binding protein-C (cMyBP-C). To date, the two mutations linked to HCM in domestic cats (one each in Maine Coon and Ragdoll breeds) also occur in MYBPC3 (Meurs et al., 2005, 2007). This is a review of the genetics of HCM in both humans and domestic cats that focuses on the aspects of human genetics that are germane to veterinarians and on all aspects of feline HCM genetics.

Myosin binding protein-C (MyBP-C) is a thick-filament protein whose precise function within the sarcomere is not known. However, recent evidence from cMyBP-C knock-out mice that lack MyBP-C in the heart suggest that cMyBP-C normally slows cross-bridge cycling rates and reduces myocyte power output. To investigate possible mechanisms by which cMyBP-C limits cross-bridge cycling kinetics we assessed effects of recombinant N-terminal domains of MyBP-C on the ability of heavy meromyosin (HMM) to support movement of actin filaments using in vitro motility assays. Here we show that N-terminal domains of cMyBP-C containing the MyBP-C "motif," a sequence of approximately 110 amino acids, which is conserved across all MyBP-C isoforms, reduced actin filament velocity under conditions where filaments are maximally activated (i.e. either in the absence of thin filament regulatory proteins or in the presence of troponin and tropomyosin and high [Ca2+]). By contrast, under conditions where thin filament sliding speed is submaximal (i.e. in the presence of troponin and tropomyosin and low [Ca2+]), proteins containing the motif increased filament speed. Recombinant N-terminal proteins also bound to F-actin and inhibited acto-HMM ATPase rates in solution. The results suggest that N-terminal domains of MyBP-C slow cross-bridge cycling kinetics by reducing rates of cross-bridge detachment.

Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction in mice and that these domains have little if any impact on cross bridge kinetics in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.

Cardiac myosin-binding protein C (cMyBP-C) is an accessory protein of striated muscle sarcomeres that is vital for maintaining regular heart function. Its 4 N-terminal regulatory domains, C0-C1-m-C2 (C0C2), influence actin and myosin interactions, the basic contractile proteins of muscle. Using neutron contrast variation data, we have determined that C0C2 forms a repeating assembly with filamentous actin, where the C0 and C1 domains of C0C2 attach near the DNase I-binding loop and subdomain 1 of adjacent actin monomers. Direct interactions between the N terminus of cMyBP-C and actin thereby provide a mechanism to modulate the contractile cycle by affecting the regulatory state of the thin filament and its ability to interact with myosin.

The M-domain is the major regulatory subunit of cardiac myosin-binding protein-C (cMyBP-C) that modulates actin and myosin interactions to influence muscle contraction. However, the precise mechanism(s) and the specific residues involved in mediating the functional effects of the M-domain are not fully understood. Positively charged residues adjacent to phosphorylation sites in the M-domain are thought to be critical for effects of cMyBP-C on cross-bridge interactions by mediating electrostatic binding with myosin S2 and/or actin. However, recent structural studies revealed that highly conserved sequences downstream of the phosphorylation sites form a compact tri-helix bundle. Here we used site-directed mutagenesis to probe the functional significance of charged residues adjacent to the phosphorylation sites and conserved residues within the tri-helix bundle. Results confirm that charged residues adjacent to phosphorylation sites and residues within the tri-helix bundle are important for mediating effects of the M-domain on contraction. In addition, four missense variants within the tri-helix bundle that are associated with human hypertrophic cardiomyopathy caused either loss-of-function or gain-of-function effects on force. Importantly, the effects of the gain-of-function variant, L348P, increased the affinity of the M-domain for actin. Together, results demonstrate that functional effects of the M-domain are not due solely to interactions with charged residues near phosphorylatable serines and provide the first demonstration that the tri-helix bundle contributes to the functional effects of the M-domain, most likely by binding to actin.