The familial hypertrophic cardiomyopathy‐associated myosin mutation R403Q accelerates tension generation and relaxation of human cardiac myofibrils

Abstract

The R403Q mutation in β-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC). In spite of extensive work on the functional sequelae of this mutation, the mechanism by which the mutant protein causes the disease has not been definitely identified. Here we directly compare contraction and relaxation mechanics of single myofibrils from left ventricular samples of one patient carrying the R403Q mutation to those from a healthy control heart. Tension generation and relaxation following sudden increase and decrease in [Ca 2+ ] were much faster in the R403Q myofibrils with relaxation rates being the most affected parameters. The results show that the R403Q mutation leads to an apparent gain of protein function but a greater energetic cost of tension generation. Increased energy cost of tension generation may be central to the FHC disease process, help explain some unresolved clinical observations, and carry significant therapeutic implications. The R403Q mutation in the β-myosin heavy chain was the first mutation to be identified as responsible for familial hypertrophic cardiomyopathy (FHC) (Geisterfer-Lowrance et al. 1990), a primary disease of the cardiac sarcomere that is the most commonly identified cause of cardiac sudden death in young people. The functional sequelae of the R403Q mutation have been extensively investigated using a variety of experimental models and approaches (Cuda et al. 1993;Lankford et al. 1995;Sata & Ikebe, 1996;Geisterfer-Lowrance et al. 1996;Marian et al. 1999;Tyska et al. 2000;Lowey, 2002;Keller et al. 2004) but the cardiac sarcomeres of affected individuals have never been directly examined. Here we compare contraction and relaxation of left ventricular myofibrils from one patient carrying the R403Q mutation to those from a healthy control heart. To investigate sarcomere mechanics we use previously This paper has online supplemental material. published techniques to measure and control the force and length of single myofibrils activated and relaxed by fast solution switching (Tesi et al. 2002;Piroddi et al. 2007). One advantage of this approach was that we could probe the acto-myosin transduction cycle while keeping the native structured sarcomere environment of the mutant protein. Preliminary report of this work has been published in abstract form ). Methods PatientsThe investigation conforms with the principles outlined in the Declaration of Helsinki and had been approved by the local Ethics Committee. Informed consent was given for both mutational analysis and mechanical experiments.A 24-year-old man, with a severe family history of premature cardiac death, diagnosed at 13 with FHC, and

Effects of Chronic Atrial Fibrillation on Active and Passive Force Generation in Human Atrial Myofibrils

“…All solutions contained a MgATP-regenerating system and a cocktail of protease inhibitors. 12,13 To avoid the effects on myofibril force and force kinetics of variable levels of contaminant inorganic phosphate (P i ) in the solutions, [Pi] was reduced to less than 5 mol/L using a P i scavenging system. 12,13 …”

“…12 Techniques for mechanical measurements in human cardiac myofibrils were as previously described. [12][13][14] Briefly, myofibrils were transferred to a temperature controlled chamber filled with relaxing solution (pCa8, 15°C). The selected myofibril was horizontally mounted (initial sarcomere length 2.2 to 2.3 m) between a cantilever force probe and a glass needle mounted on the lever arm of a length control motor.…”

“…Mechanical measurements of myofibril force combined with rapid perfusion switching techniques have been developed recently to investigate fast kinetic events related to cross-bridge action and regulation in human cardiac myofibrils. [12][13][14] In this study, sarcomere mechanisms underlying passive and active tension generation and relaxation of atrial myocardium are dissected and compared in myofibrils from surgical samples of cAF and control (SR) patients. In the same samples, we also determined the myosin heavy chain (MHC) and titin isoform expression and the phosphorylation level of several myofilament proteins.…”

“…12,13 Single myofibrils are the smallest units of the contractile apparatus that retain the organized myofilament lattice and its entire ensemble of associated proteins. Mechanical measurements of myofibril force combined with rapid perfusion switching techniques have been developed recently to investigate fast kinetic events related to cross-bridge action and regulation in human cardiac myofibrils.…”

Genetic Testing for Hypertrophic Cardiomyopathy: Ongoing Voyage from Exploration to Clinical Exploitation

“…[21][22][23] In a recent paper describing four HCM patients with triple myofilament gene mutations, representing rare and extreme paradigms of complex genotype, two had a history of resuscitated cardiac arrest or appropriate defibrillator intervention, and three progressed to end-stage HCM by the fourth decade, requiring cardiac biventricular pacing or transplantation 24 ( Figure 2). These adverse consequences likely reflect more profound derangement of sarcomere mechanics and cardiomyocyte energetics caused by multiple mutations, 25 but may also be mediated by greater impairment of coronary microvascular function, selectively caused by sarcomere gene defects via unknown molecular pathways. 26 In the specific HCM patient subsets, genetic screening may thus, provide important additional clues to risk stratification, and potentially indicate the need for differential surveillance strategies based on genotype.…”