Abstract
Hypertrophic cardiomyopathy (HCM) is frequently linked to mutations in the protein components of the myosin-containing thick filaments leading to contractile dysfunction and ultimately heart failure. However, the molecular structure-function relationships that underlie these pathological effects remain largely obscure. Here we chose an example mutation (R58Q) in the myosin regulatory light chain (RLC) that is associated with a severe HCM phenotype and combined the results from a wide range of in vitro and in situ structural and functional studies on isolated protein components, myofibrils and ventricular trabeculae to create an extensive map of structure-function relationships. The results can be understood in terms of a unifying hypothesis that illuminates both the effects of the mutation and physiological signaling pathways. R58Q promotes an OFF state of the thick filaments that reduces the number of myosin head domains that are available for actin interaction and ATP utilization. Moreover this mutation uncouples two aspects of length-dependent activation (LDA), the cellular basis of the Frank-Starling relation that couples cardiac output to venous return; R58Q reduces maximum calcium-activated force with no significant effect on myofilament calcium sensitivity. Finally, phosphorylation of R58Q-RLC to levels that may be relevant both physiologically and pathologically restores the regulatory state of the thick filament and the effect of sarcomere length on maximum calcium-activated force and thick filament structure, as well as increasing calcium sensitivity. We conclude that perturbation of thick filament-based regulation may be a common mechanism in the etiology of missense mutation-associated HCM, and that this signaling pathway offers a promising target for the development of novel therapeutics.
Highlights
The cardiac cycle of contraction and relaxation is governed by transient interactions between the actin-containing thin and myosincontaining thick filaments, coupled to the hydrolysis of ATP [1]
We used polarized fluorescence from a bifunctional sulforhodamine (BSR) probe attached to the regulatory light chain (RLC) E-helix (Fig. 1A) to determine the effects of the R58Q mutation on myosin head conformation in demembranated rat right ventricular trabeculae with an endogenously low level of RLC phosphorylation (< 0.05 mol Pi/mol RLC) [30]
Since changes in the conformation of the myosin heads may be involved in length-dependent activation (LDA) in cardiac muscle and the Frank-Starling Law of the heart [7,33,34], and impaired LDA has been linked to heart failure associated with sarcomeric protein missense mutations [35], we investigated the effect of the R58Q mutation on the change in myosin head conformation associated with LDA
Summary
The cardiac cycle of contraction and relaxation is governed by transient interactions between the actin-containing thin and myosincontaining thick filaments, coupled to the hydrolysis of ATP [1]. Electron microscopy reconstructions of isolated thick filaments from mammalian cardiac muscle suggest that myosin heads in the OFF state of the thick filament are folded against their tail domains, reducing their availability for actin-binding, ATP hydrolysis and force production [4,5]. Consistent with these structural data, biochemical studies on isolated cardiac muscle preparations demonstrated the existence of a pool of myosin heads in a state with ultra-low ATPase activity in both relaxing and activating conditions, known as the superrelaxed state (SRX) [6]. Regulatory signaling pathways including posttranslational modifications of RLC [7] and myosin binding protein-C (cMyBP-C) [8], and mechano-sensing [9] have been suggested to regulate cardiac muscle function by modulating the myosin OFF or SRX state and the number of myosin heads available for contraction
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
