Kristina L. Bezold

In the Thick of It HCM-Causing Mutations in Myosin Binding Proteins of the Thick Filament

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.

Contribution of the Myosin Binding Protein C Motif to Functional Effects in Permeabilized Rat Trabeculae

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C “motif” or “m-domain” increased Ca2+ sensitivity of tension and increased rates of tension redevelopment (i.e., ktr) at submaximal levels of Ca2+. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca2+ and inhibited maximum Ca2+-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.

Earning stripes: myosin binding protein-C interactions with actin.

Myosin binding protein-C (MyBP-C) was first discovered as an impurity during the purification of myosin from skeletal muscle. However, soon after its discovery, MyBP-C was also shown to bind actin. While the unique functional implications for a protein that could cross-link thick and thin filaments together were immediately recognized, most early research nonetheless focused on interactions of MyBP-C with the thick filament. This was in part because interactions of MyBP-C with the thick filament could adequately explain most (but not all) effects of MyBP-C on actomyosin interactions and in part because the specificity of actin binding was uncertain. However, numerous recent studies have now established that MyBP-C can indeed bind to actin through multiple binding sites, some of which are highly specific. Many of these interactions involve critical regulatory domains of MyBP-C that are also reported to interact with myosin. Here we review current evidence supporting MyBP-C interactions with actin and discuss these findings in terms of their ability to account for the functional effects of MyBP-C. We conclude that the influence of MyBP-C on muscle contraction can be explained equally well by interactions with actin as by interactions with myosin. However, because data showing that MyBP-C binds to either myosin or actin has come almost exclusively from in vitro biochemical studies, the challenge for future studies is to define which binding partner(s) MyBP-C interacts with in vivo.

A gain-of-function mutation in the M-domain of cardiac myosin-binding protein-C increases binding to actin

Background: Cardiac myosin-binding protein C (cMyBP-C) regulates heart muscle contraction by influencing actomyosin interactions. Results: Amino acids within the tri-helix bundle of the M-domain contribute to the functional effects of cMyBP-C. Conclusion: Amino acids outside of phosphorylation sites influence function of the M-domain. Significance: The tri-helix bundle is important to the regulatory role of cMyBP-C, likely through actin-binding interactions. 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.

Functional Differences between the N-Terminal Domains of Mouse and Human Myosin Binding Protein-C

The N-terminus of cMyBP-C can activate actomyosin interactions in the absence of Ca2+, but it is unclear which domains are necessary. Prior studies suggested that the Pro-Ala rich region of human cMyBP-C activated force in permeabilized human cardiomyocytes, whereas the C1 and M-domains of mouse cMyBP-C activated force in permeabilized rat cardiac trabeculae. Because the amino acid sequence of the P/A region differs between human and mouse cMyBP-C isoforms (46% identity), we investigated whether species-specific differences in the P/A region could account for differences in activating effects. Using chimeric fusion proteins containing combinations of human and mouse C0, Pro-Ala, and C1 domains, we demonstrate here that the human P/A and C1 domains activate actomyosin interactions, whereas the same regions of mouse cMyBP-C are less effective. These results suggest that species-specific differences between homologous cMyBP-C isoforms confer differential effects that could fine-tune cMyBP-C function in hearts of different species.

Helix Three (H3) of the M-Domain of Cardiac Myosin Binding Protein-C is not Essential for Functional Effects in Permeabilized Rat Trabeculae

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein involved in the regulation of cardiac muscle contraction. Cardiac specific phosphorylation sites in the M-domain regulate binding of cMyBP-C to myosin and actin. Immediately downstream of the phosphorylation sites is a bundle of three α-helixes that are well conserved across all species and isoforms of MyBP-C, however the functional significance of these helices is unknown. Helix 3 (H3) bears high homology to the inhibitory peptide of troponin I, suggesting it as a potential binding site for interactions with actin. To understand the functional significance of H3, we produced a recombinant N-terminal protein containing domains C1, M and C2 (C1C2) with the H3 sequence deleted (H3KO). We then assessed effects of the mutant H3KO protein on contractile forces in permeabilized rat trabeculae and compared them to effects of the wild-type C1C2. Results showed that 5µM C1C2 significantly increased Ca2+-sensitivity of force (ΔpCa50=0.29±0.03), whereas 5µM H3KO had no effect (ΔpCa50=0.02±0.01) and 10µM H3KO only modestly increased Ca2+-sensitivity (ΔpCa50=0.11±0.02,). Similarly, whereas 10µM C1C2 activated force in the absence of Ca2+ (pCa 9.0), H3KO activated force in the absence of Ca2+ only at concentrations >20µM. Together, these results establish that H3 is not absolutely required for the functional effects of the N-terminal domains of cMyBP-C to either increase Ca2+-sensitivity of force or to activate tension in sarcomeres, but that H3 contributes to the overall efficacy of the N-terminus in mediating these effects. Potentially, H3 could dock the N-terminus of cMyBP-C with actin or extend/stabilize cMyBP-C interactions with actin or other regulatory proteins. This work was supported by NIH HL-080367 (SPH), NDSEG and AHA graduate fellowships (KLB), and AHA undergraduate fellowship (JKK).

Charged Amino Acids in the N-Terminus of Cardiac Myosin Binding Protein-C Contribute to Contractile Effects in Permeabilized Myocytes

Recombinant N-terminal domains of cardiac myosin binding protein C (cMyBP-C) increase calcium-sensitivity of force (pCa50) and the rate of tension redevelopement (ktr) in permeabilized cardiac myocytes. To identify specific amino acids required for these effects, we investigated functional effects of alanine substitution mutations of charged residues near phosphorylation sites within the regulatory M-domain of cMyBP-C. Mutations were introduced into a recombinant protein, C1C2, comprised of the M-domain and its two flanking Ig domains (i.e, C1-M-C2). Compared to wild-type C1C2, a double mutant containing A for R substitutions at positions 270 and 271 upstream of S273 (phosphorlyation site A), reduced the ability of C1C2 to increase force in permeabilized rat cardiac trabeculae. Another double mutant, the 299AA300 substitution for 299KR300 upstream of S302 (phosphorylation site C), also reduced the efficacy of C1C2. A triple mutant that replaced a cluster of three charged residues 298KKR300 nearly abolished the functional effects of C1C2. These results suggest that positively charged amino acids upstream of protein kinase A phosphorylation sites are important for mediating the contractile effects of C1C2 and that these sites may be involved in the in vivo response to -adrenergic stimulation potentially by contributing to binding of the M-domain to actin and/or myosin S2. This work is supported by NIH HL080367 to SPH and a DOD NDSEG fellowship to KLB.