Abstract
ABSTRACTThe cardiac muscle ryanodine receptor-Ca2+ release channel (RyR2) constitutes the sarcoplasmic reticulum (SR) Ca2+ efflux mechanism that initiates myocyte contraction, while cardiac myosin-binding protein-C (cMyBP-C; also known as MYBPC3) mediates regulation of acto-myosin cross-bridge cycling. In this paper, we provide the first evidence for the presence of direct interaction between these two proteins, forming a RyR2–cMyBP-C complex. The C-terminus of cMyBP-C binds with the RyR2 N-terminus in mammalian cells and the interaction is not mediated by a fibronectin-like domain. Notably, we detected complex formation between both recombinant cMyBP-C and RyR2, as well as between the native proteins in cardiac tissue. Cellular Ca2+ dynamics in HEK293 cells is altered upon co-expression of cMyBP-C and RyR2, with lowered frequency of RyR2-mediated spontaneous Ca2+ oscillations, suggesting that cMyBP-C exerts a potential inhibitory effect on RyR2-dependent Ca2+ release. Discovery of a functional RyR2 association with cMyBP-C provides direct evidence for a putative mechanistic link between cytosolic soluble cMyBP-C and SR-mediated Ca2+ release, via RyR2. Importantly, this interaction may have clinical relevance to the observed cMyBP-C and RyR2 dysfunction in cardiac pathologies, such as hypertrophic cardiomyopathy.
Highlights
Quantitative (n≥5) densitometry and statistical analysis indicates that RyR2NT subfragments BT4, BT4D, BT4DL and BT4EL retain significant cardiac myosin-binding protein-C (cMyBP-C) binding, binding was reduced relative to RyR2NT (Fig. 2D). These results suggest multiple determinants within the ryanodine receptor 2 complex (RyR2) N-terminus are necessary for robust interaction with cMyBP-C
Given that dephosphorylated cMyBP-C is released from the sarcomere in pathological conditions (Baker et al, 2015; Decker et al, 2012; Govindan et al, 2012; Kulikovskaya et al, 2007; Kuster et al, 2014), the structural and functional interaction between the SRresident RyR2 and sarcomere-released, soluble cMyBP-C, described in the present study, may constitute an important, and previously unrecognised, feedback regulatory mechanism (Fig. 6)
Human cMyBP-C colocalised with RyR2 in HEK293 cells (Fig. 4), co-immunoprecipitated with human RyR2 (Fig. 3) and modulated spontaneous RyR2 Ca2+ release (Fig. 5)
Summary
Cardiac muscle excitation–contraction coupling, where the electrical depolarisation stimulus of cardiomyocytes plasma membrane triggers mechanical contraction of the heart, is mediated by Ca2+-induced Ca2+-release, where a small influx of. During systole (the contraction phase), plasma membrane electrical excitation causes Ca2+ influx through coordinated opening of voltage-gated Ca2+ channels, leading to a global efflux of Ca2+ from the SR via the SR-located Ca2+ release channel-ryanodine receptor 2 complex (RyR2), which in turn induces sarcomere shortening (Bers, 2002). The force and frequency of muscle contraction is influenced by numerous factors including sarcomere Ca2+ responsiveness, phosphorylation and various modulatory proteins; among the latter is myosin-binding protein C (MyBP-C), an important protein that plays a vital modulatory role (Clark et al, 2002; Moss et al, 2015)
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