Comparison of elementary steps of the cross-bridge cycle in rat papillary muscle fibers expressing α- and β-myosin heavy chain with sinusoidal analysis.

Abstract

In mammalian ventricles, two myosin heavy chain (MHC) isoforms have been identified. Small animals express α-MHC, whereas large animals express β-MHC, which contribute to a large difference in the heart rate. Sprague-Dawley rats possessing~99% α-MHC were treated with propylthiouracil to result in 100% β-MHC. Papillary muscles were skinned, dissected into small fibers, and used for experiments. To understand the functional difference between α-MHC and β-MHC, skinned-fibers were activated under the intracellular ionic conditions: 5mM MgATP, 1mMMg2+, 8mM Pi, 200mM ionic strength, pH 7.00 at 25°C. Small amplitude sinusoidal length oscillations were applied in the frequency range 0.13-100Hz (corresponding time domain: 1.6-1200ms), and effects of Ca2+, Pi, and ATP were studied. The results show that Ca2+ sensitivity was slightly less (10-15%) in β-MHC than α-MHC containing fibers. Sinusoidal analysis at pCa 4.66 (full Ca2+ activation) demonstrated that, the apparent rate constants were 2-4×faster in α-MHC containing fibers. The ATP study demonstrated that, in β-MHC containing fibers, K 1 (ATP association constant) was greater (1.7×), k 2 and k -2 (cross-bridge detachment and its reversal rate constants) were smaller (×0.6). The Pi study demonstrated that, in β-MHC containing fibers, k 4 (rate constant of the force-generation step) and k -4 were smaller (0.75× and 0.25×, respectively), resulting in greater K 4 (3×). There were no differences in active tension, rigor stiffness, or K 2 (equilibrium constant of the cross-bridge detachment step). Our study further demonstrated that there were no differences in parameters between fibers obtained from left and right ventricles, but with an exception in K 5 (Pi association constant).