Altered Crossbridge Kinetics in the αMHC ^403/+ Mouse Model of Familial Hypertrophic Cardiomyopathy

Abstract

A mutation in the cardiac beta-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the alpha-myosin heavy chain (alphaMHC403/+). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the alphaMHC403/+ hearts. In resting strips (pCa 8), dynamic stiffness was approximately 40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was approximately 3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing (b) and work-absorbing (c) steps of the crossbridge were less in alphaMHC403/+ strips than in wild-type strips (b=11+/-1 versus 15+/-1 Hz; c= 58+/-3 versus 66+/-3 Hz; 27 degrees C). At maximal calcium activation, strip oscillatory power was reduced (0. 53+/-0.25 versus 1.03+/-0.18 mW/mm3; 27 degrees C), which is partly attributable to the reduced frequency b, at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.

The Essential Light Chain N-terminal Extension Alters Force and Fiber Kinetics in Mouse Cardiac Muscle

“…The right ventricle was trimmed, and the papillary muscles from the left ventricle were removed. Papillary muscles were dissected to yield at least three thin strips (ϳ100 -140 m in diameter and ϳ600 m in length) consisting of longitudinally oriented bundles of myocytes as described previously (29,30). Strips were skinned for 2 h at 22°C and stored at Ϫ20°C for no more than 1 week.…”

“…Papillary muscle strips were isolated from 1v⌬5-14 and NTG mice at 24 (young) and 58 (old) weeks, skinned, and T-clipped as described above. A strip was mounted between a piezoelectric motor (Physik Instrumente, Auburn, MA) and a strain gauge (SensorNor, Horten, Norway), lowered into a 30-l droplet of relaxing solution maintained at 27°C, and incrementally stretched to (and maintained at) 2.2-m sarcomere length as determined using a filar micrometer (30).…”

“…At each calcium concentration, sinusoidal perturbations of amplitude 0.125% strip length were applied at 42 discrete frequencies (0.125-100 Hz). Complex stiffness (expressed as the amplitude ratio and the phase shift between the force response and length perturbation) was calculated at each frequency (30,32,33). Complex modulus (i.e.…”

Speckle tracking imaging in hypertrophic cardiomyopathy

“…Myocardial twist has been associated with myocardial fibrosis and the type of hypertrophy in HCM [4]. In particular, untwist alterations have been observed in HCM mutation carriers [5,6], implying that distortion of ventricular mechanics in HCM is not entirely due to myocardial disarray but begins primarily as a result of impaired actin-myosin interaction kinetics [7]. The latter can result from altered intracellular calcium handling, leading to decreased calcium re-uptake in the sarcoplasmic reticulum and, thus, establishing the link with increased arrhythmogenicity [8].…”

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation

“…We are currently studying the functional difference of α-Tm, β-Tm, and their phosphorylated form (Lu et al 2006). In the future, any mutant protein of the thin filament origin that causes familial hypertrophic cardiomyopathy (Blanchard et al 1999;Wolska and Wieczorek 2003) or dilative cardiomyopathy (Chang and Potter 2005) can be studied to elucidate the structure-function relationship of these diseases. Schematic diagram illustrating the removal and reconstitution protocol of the thin filament.…”