Effects of low-level α-myosin heavy chain expression on contractile kinetics in porcine myocardium

Abstract

Myosin heavy chain (MHC) isoforms are principal determinants of work capacity in mammalian ventricular myocardium. The ventricles of large mammals including humans normally express ∼10% α-MHC on a predominantly β-MHC background, while in failing human ventricles α-MHC is virtually eliminated, suggesting that low-level α-MHC expression in normal myocardium can accelerate the kinetics of contraction and augment systolic function. To test this hypothesis in a model similar to human myocardium we determined composite rate constants of cross-bridge attachment (f(app)) and detachment (g(app)) in porcine myocardium expressing either 100% α-MHC or 100% β-MHC in order to predict the MHC isoform-specific effect on twitch kinetics. Right atrial (∼100% α-MHC) and left ventricular (∼100% β-MHC) tissue was used to measure myosin ATPase activity, isometric force, and the rate constant of force redevelopment (k(tr)) in solutions of varying Ca(2+) concentration. The rate of ATP utilization and k(tr) were approximately ninefold higher in atrial compared with ventricular myocardium, while tension cost was approximately eightfold greater in atrial myocardium. From these values, we calculated f(app) to be ∼10-fold higher in α- compared with β-MHC, while g(app) was 8-fold higher in α-MHC. Mathematical modeling of an isometric twitch using these rate constants predicts that the expression of 10% α-MHC increases the maximal rate of rise of force (dF/dt(max)) by 92% compared with 0% α-MHC. These results suggest that low-level expression of α-MHC significantly accelerates myocardial twitch kinetics, thereby enhancing systolic function in large mammalian myocardium.

Fig. 1.

Myosin heavy chain (MHC) composition of pig atrial and ventricular myocardium. MHC isoform expression was determined with 6% SDS-PAGE. This representative gel shows pig ventricular myocardium (lane 1) expressing ∼100% β-MHC and pig atrial myocardium expressing ∼100% α-MHC (lane 4). Lanes 2 and 3 show combinations of atria and ventricle at various dilutions.

Fig. 2.

Determination of total protein content and basal phosphorylation in porcine atrial and ventricular myocardium. A: myofibrillar protein phosphorylation was measured with SYPRO-Ruby staining for total protein content (left) and Pro-Q Diamond staining for protein phosphorylation (right), as shown in this representative SDS-PAGE at loading concentrations of 3 and 4 μl. Volumes of 3, 4, 6, and 8 μl were loaded in successive lanes for each sample preparation. cMyBP-C, cardiac myosin binding protein-C; TnT, troponin T; TnI, troponin I; ALC-1 and VLC-1, atrial and ventricular essential light chain, respectively; ALC-2 and VLC-2, atrial and ventricular regulatory light chain, respectively. B–E: levels of protein phosphorylation in atrial and ventricular porcine myocardium were compared for MyBP-C (B), TnT (C), TnI (D), and myosin light chain 2 (MLC-2) (E). Regression lines for optical band intensity vs. protein load were determined for both Pro-Q Diamond- and SYPRO-Ruby (not shown)-stained gels, and the ratio of regression line slopes was calculated. AU, arbitrary units.

Fig. 3.

Rate constant of force redevelopment (k_tr) in porcine atrial and ventricular myocardium. A representative trace of k_tr as measured in maximal Ca²⁺ concentration ([Ca²⁺]) (pCa 4.5) in pig atrial (∼100% α-MHC, 22.4 ± 1.2 s⁻¹) and ventricular (∼100% β-MHC, 2.5 ± 0.7 s⁻¹) myocardium. Data are means ± SE. P, submaximal force; P_o, maximal force.

Fig. 4.

Rate of ATP utilization in porcine atrial and ventricular myocardium. The rate of ATP utilization was measured in skinned myocardium from atrial (●, 100% α-MHC, n = 10) and ventricular (○, 0% β-MHC, n = 10) preparations and plotted against relative force (P/P_o). Forces recorded at submaximal free Ca²⁺ (P) were expressed relative to the maximal force measured at pCa 4.5 (P_o). Data are means ± SE.

Fig. 5.

Tension cost in porcine atrial and ventricular myocardium. The rate of ATP utilization was measured in skinned myocardium from atrial (●, 100% α-MHC, n = 10) and ventricular (○, 0% α-MHC, n = 10) preparations and plotted against Ca²⁺-activated isometric force. The slope of the line is equal to tension cost. Force and ATPase were measured simultaneously in solutions of varying free [Ca²⁺] (pCa 4.5, 5.4–6.2). Data are means ± SE.

Fig. 6.

A: model predictions of twitch time courses in myocardium with variable ratios of α-MHC and β-MHC. y-Axis indicates the fraction of cross bridges bound; x-axis indicates time in seconds. Indigo line indicates the Ca²⁺ pulse (arbitrary units) that was used in each of the simulations. Traces represent 100% β-MHC (0% α-MHC), 90% β-MHC (10% α-MHC), 80% β-MHC (20%-α MHC), and 70% β-MHC (30% α-MHC) of the total MHC content. B: model predictions of normalized twitch time courses in myocardium with variable ratios of α-MHC and β-MHC. Force simulations are expressed relative to the maximal force calculated in A. Indigo line indicates the Ca²⁺ pulse (arbitrary units) that was used in each of the simulations. Traces represent 100% β-MHC (0% α-MHC), 90% β-MHC (10% α-MHC), 80% β-MHC (20% α-MHC), and 70% β-MHC (30% α-MHC) of the total MHC content.

Fig. 7.

Model predictions for the rates of force development and relaxation in myocardium with variable expression of α-MHC. Maximal rate of force development (dF/dt_max) is shown on y-axis; x-axis is time in seconds. Traces represent 100% β-MHC (0% α-MHC), 90% β-MHC (10% α-MHC), 80% β-MHC (20% α-MHC), and 70% β-MHC (30% α-MHC) of the total MHC content.

Fig. 8.

Effect of α-MHC expression on the time required to reach peak force in mammalian myocardium. Data were obtained from model predictions of twitch time courses in myocardium with variable ratios of α-MHC and β-MHC. Each data point is taken from a single time value obtained from the derived time required to reach peak force; therefore no error bars are present. Fold decrease in time to peak force from 0% α-MHC was computed as the ratio of the time to peak force from 10%, 20%, and 30% α-MHC to 0% α-MHC. Porcine myocardium (●) is shown compared with values from rat myocardium (○) taken from previous work by the authors (31).