AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice
Jiayang Li, Ranganath Mamidi, Chang Yoon Doh, Joshua B. Holmes, Nikhil Bharambe, Rajesh Ramachandran, Julian E. Stelzer
Jiayang Li, Ranganath Mamidi, Chang Yoon Doh, Joshua B. Holmes, Nikhil Bharambe, Rajesh Ramachandran, Julian E. Stelzer
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Research Article Cardiology

AAV9 gene transfer of cMyBPC N-terminal domains ameliorates cardiomyopathy in cMyBPC-deficient mice

Abstract

Decreased cardiac myosin-binding protein C (cMyBPC) expression due to inheritable mutations is thought to contribute to the hypertrophic cardiomyopathy (HCM) phenotype, suggesting that increasing cMyBPC content is of therapeutic benefit. In vitro assays show that cMyBPC N-terminal domains (NTDs) contain structural elements necessary and sufficient to modulate actomyosin interactions, but it is unknown if they can regulate in vivo myocardial function. To test whether NTDs can recapitulate the effects of full-length (FL) cMyBPC in rescuing cardiac function in a cMyBPC-null mouse model of HCM, we assessed the efficacy of AAV9 gene transfer of a cMyBPC NTD that contained domains C0C2 and compared its therapeutic potential with AAV9-FL gene replacement. AAV9 vectors were administered systemically at neonatal day 1, when early-onset disease phenotypes begin to manifest. A comprehensive analysis of in vivo and in vitro function was performed following cMyBPC gene transfer. Our results show that a systemic injection of AAV9-C0C2 significantly improved cardiac function (e.g., 52.24 ± 1.69 ejection fraction in the C0C2-treated group compared with 40.07 ± 1.97 in the control cMyBPC–/– group, P < 0.05) and reduced the histopathologic signs of cardiomyopathy. Furthermore, C0C2 significantly slowed and normalized the accelerated cross-bridge kinetics found in cMyBPC–/– control myocardium, as evidenced by a 32.41% decrease in the rate of cross-bridge detachment (krel). Results indicate that C0C2 can rescue biomechanical defects of cMyBPC deficiency and that the NTD may be a target region for therapeutic myofilament kinetic manipulation.

Authors

Jiayang Li, Ranganath Mamidi, Chang Yoon Doh, Joshua B. Holmes, Nikhil Bharambe, Rajesh Ramachandran, Julian E. Stelzer

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Figure 1

Structure and function of cMyBPC and C0C2.

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Structure and function of cMyBPC and C0C2. (A) Schematic representation ...
(A) Schematic representation of full-length cardiac myosin-binding protein C (cMyBPC) showing N-terminal (C0C2) regulatory domains and C-terminal (C8C10) anchoring domains. (B) Illustrated representation of myofilament organization with yellow bars highlighting the C zones where endogenous cMyBPC localize. (C) Illustration of C0C2 N-terminal domains showing possible interactions with thin filament (blue), tropomyosin (red), and thick filament (orange) that have been demonstrated in in vitro assays. It has been proposed that C0C2 can directly bind to the thin filament and displace tropomyosin toward an “open” state, exposing adjacent myosin binding sites to allow cross-bridge formation and effectively activating the thin filament (black arrow) (18). C0C2 has also been implicated in direct binding to the S1 and S2 regions of myosin (purple arrow), shifting some myosin heads into “super-relaxed” states toward the thick filament backbone and rendering them unavailable for cross-bridge formation (20, 72). A reduction in cMyBPC expression has been proposed to cause hypercontractility in HCM by freeing these myosin heads out of the “sequestered” state. Another proposed mechanism is the binding of cMyBPC to myosin heads once they are in the strongly bound state (gray arrow). This has been suggested to provide biomechanical stability to the cross-bridge and increase cross-bridge lifetime (29).