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(Circulation. 2009;119:1473-1483.)
© 2009 American Heart Association, Inc.

Molecular Cardiology

Cardiac Myosin-Binding Protein C Mutations and Hypertrophic Cardiomyopathy

Haploinsufficiency, Deranged Phosphorylation, and Cardiomyocyte Dysfunction

Sabine J. van Dijk, MSc; Dennis Dooijes, PhD; Cris dos Remedios, PhD; Michelle Michels, MD, PhD; Jos M.J. Lamers, PhD; Saul Winegrad, PhD; Saskia Schlossarek, PhD; Lucie Carrier, PhD; Folkert J. ten Cate, MD, PhD; Ger J.M. Stienen, PhD; Jolanda van der Velden, PhD

From the Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, the Netherlands (S.J.v.D., G.J.M.S., J.v.d.V.); Clinical Genetics (D.D.), Thorax Center (M.M., F.J.t.C.), and Department of Biochemistry (J.M.J.L.), Erasmus Medical Center Rotterdam, Rotterdam, the Netherlands; Muscle Research Unit, Institute for Biomedical Research, University of Sydney, Sydney, Australia (C.d.R.); Department of Physiology, University of Philadelphia School of Medicine, Philadelphia, Pa (S.W.); Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg-Eppendorf, Germany (S.S., L.C.); and INSERM U582, U974-CNRS UMR 7215, UPMC University of Paris, Paris, France (L.C.).

Correspondence to Jolanda van der Velden, PhD, Laboratory for Physiology, VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, the Netherlands. E-mail j.vandervelden{at}vumc.nl

Received June 13, 2008; accepted December 29, 2008.

Background— Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3_mut).

Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3_mut. Protein expression of cMyBP-C was significantly reduced in MYBPC3_mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3_mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the β-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3_mut (20.2±2.7 kN/m²) compared with donor (34.5±1.1 kN/m²). Moreover, Ca²⁺ sensitivity was higher in MYBPC3_mut (pCa₅₀=5.62±0.04) than in donor (pCa₅₀=5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic β-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca²⁺ sensitivity between MYBPC3_mut (pCa₅₀=5.46±0.03) and donor (pCa₅₀=5.48±0.02).

Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca²⁺ sensitivity in MYBPC3_mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

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