Abstract
Muscle contraction, which is initiated by Ca2+, results in precise sliding of myosin-based thick and actin-based thin filament contractile proteins. The interactions between myosin and actin are finely tuned by three isoforms of myosin binding protein-C (MyBP-C): slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C and cMyBP-C, respectively), each with distinct N-terminal regulatory regions. The skeletal MyBP-C isoforms are conditionally coexpressed in cardiac muscle, but little is known about their function. Therefore, to characterize the functional differences and regulatory mechanisms among these three isoforms, we expressed recombinant N-terminal fragments and examined their effect on contractile properties in biophysical assays. Addition of the fragments to in vitro motility assays demonstrated that ssMyBP-C and cMyBP-C activate thin filament sliding at low Ca2+. Corresponding 3D electron microscopy reconstructions of native thin filaments suggest that graded shifts of tropomyosin on actin are responsible for this activation (cardiac > slow-skeletal > fast-skeletal). Conversely, at higher Ca2+, addition of fsMyBP-C and cMyBP-C fragments reduced sliding velocities in the in vitro motility assays and increased force production in cardiac muscle fibers. We conclude that due to the high frequency of Ca2+ cycling in cardiac muscle, cardiac MyBP-C may play dual roles at both low and high Ca2+. However, skeletal MyBP-C isoforms may be tuned to meet the needs of specific skeletal muscles.
Highlights
Myosin binding protein-C (MyBP-C) is a striated muscle protein that regulates contraction and consists of three isoforms known as slow-skeletal, fast-skeletal, and cardiac, encoded by MYBPC1, MYBPC2 and MYBPC3, respectively[1,2,3]
C0C2 and fsC1C2 increase in Ca2+-sensitivity resulted in increased submaximal force generation (Supplemental Figure S1D), suggesting that these isoforms regulate some aspect of myosin-actin interaction
While no change was observed in ktr at maximal activation, we show that fsC1C2 and C0C2 increased ktr relative to controls at submaximal activation levels (Fig. 1F), suggesting that these fragments promote thin filament activation and/or cross-bridge cycling to regulate Ca2+-sensitivity (Fig. 1D)
Summary
Myosin binding protein-C (MyBP-C) is a striated muscle protein that regulates contraction and consists of three isoforms known as slow-skeletal, fast-skeletal, and cardiac (ssMyBP-C, fsMyBP-C, and cMyBP-C), encoded by MYBPC1, MYBPC2 and MYBPC3, respectively[1,2,3]. The ssMyBP-C isoform has only one phosphorylation site in its M domain and three in its PA region[21] and no known phosphorylation sites have been found in fsMyBP-C Sequence comparisons within this region suggest unique N-terminal structural differences (Supplemental Table S1), prompting us to determine the impact of the three major MyBP-C isoforms on muscle contractility, with a focus on heart muscle. Our objective is to parse out the differences among the N-terminal regulatory regions (up to and including the C2 domain) of each individual MyBP-C isoform (Fig. 1A and Supplemental Table S1) How these isoforms vary in their interactions with the thin filament may explain why three distinct isoforms are expressed, and the answers may lay the groundwork for future studies in the skeletal MyBP-C field. These data are the first to reveal differences in the underlying molecular and cellular regulatory functionality of all three MyBP-C N-termini
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