Cross-Bridge Kinetics in Rat Papillary Muscle Fibers that Carry α-MHC and β-MHC by Sinusoidal Analysis

Abstract

In mammalian heart muscles, two isoforms of MHC were identified. Small animals carry primarily α-MHC (fast), whereas large animals (e.g., bovines and humans) carry mostly β-MHC (slow), resulting in a large difference in the heart rate. Sprague-Dawley rats, which possess 99% α-MHC in their ventricles, were treated with propylthiouracil to result in 100% β-MHC, as demonstrated by SDS-PAGE. Papillary muscles were dissected, skinned, split into small fibers (90-110 µm diameter), and mounted on experimental apparatus. To understand the functional difference between α-MHC and β-MHC, fibers were activated under intracellular ionic conditions of cardiomyocytes: 5mM ATP, 1mM Mg2+, 8mM phosphate (Pi), 200mM ionic strength, and pH 7.0. These measurements were carried out at 25°C. When steady tension developed, small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200 ms), and the effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (12%) in β-MHC than α-MHC, while cooperativity was not statistically different. Sinusoidal analysis demonstrated that, in β-MHC containing fibers, K1 (ATP association constant) was greater (1.7x), k2 and k-2 (cross-bridge detachment and its reversal rate constants) were smaller (0.6x); k4 (rate constant of the force-generation step) and k-4 (its reversal step) were smaller (0.75x and 0.25x, respectively), which resulted in greater (3x) K4 (equilibrium constant of the force generation step) than those of α-MHC. There were no differences in K2 (equilibrium constant of the cross-bridge detachment step), K5 (Pi association constant), active tension, or rigor stiffness. Our study also demonstrated that there were no differences in the above mentioned parameters between fibers obtained from left and right ventricles, indicating that there is no difference at the myofilament level in both ventricles.