The effect of myosin light chain 2 dephosphorylation on Ca2+-sensitivity of force is enhanced in failing human hearts

Objective: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1±2.0%) compared to donor (31.9±4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. Methods: Cooperative effects in isometric force and rate of force redevelopment (Ktr) were studied in single Triton-skinned human cardiomyocytes at various [Ca2+] before and after protein phosphatase-1 (PP-1) incubation. Results: Maximum force and Ktr values did not differ between failing and donor hearts, but Ca2+-sensitivity of force (pCa50) was significantly higher in failing myocardium (ΔpCa50=0.17). Ktr decreased with decreasing [Ca2+], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa50 to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute Ktr values was found after PP-1 in failing and donor myocytes, while the shape of the Ktr-Ca2+ relationships remained unaltered. Conclusions: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca2+-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca2+-sensitivity and impaired Ca2+-handling on diastolic function in human heart failure

Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins

Objective: The alterations in contractile proteins underlying enhanced Ca2+-sensitivity of the contractile apparatus in end-stage failing human myocardium are still not resolved. In the present study an attempt was made to reveal to what extent protein alterations contribute to the increased Ca2+-responsiveness in human heart failure. Methods: Isometric force and its Ca2+-sensitivity were studied in single left ventricular myocytes from non-failing donor (n=6) and end-stage failing (n=10) hearts. To elucidate which protein alterations contribute to the increased Ca2+-responsiveness isoform composition and phosphorylation status of contractile proteins were analysed by one- and two-dimensional gel electrophoresis and Western immunoblotting. Results: Maximal tension did not differ between myocytes obtained from donor and failing hearts, while Ca2+-sensitivity of the contractile apparatus (pCa50) was significantly higher in failing myocardium (ΔpCa50=0.17). Protein analysis indicated that neither re-expression of atrial light chain 1 and fetal troponin T (TnT) nor degradation of myosin light chains and troponin I (TnI) are responsible for the observed increase in Ca2+-responsiveness. An inverse correlation was found between pCa50 and percentage of phosphorylated myosin light chain 2 (MLC-2), while phosphorylation of MLC-1 and TnT did not differ between donor and failing hearts. Incubation of myocytes with protein kinase A decreased Ca2+-sensitivity to a larger extent in failing (ΔpCa50=0.20) than in donor (ΔpCa50=0.03) myocytes, abolishing the difference in Ca2+-responsiveness. An increased percentage of dephosphorylated TnI was found in failing hearts, which significantly correlated with the enhanced Ca2+-responsiveness. Conclusions: The increased Ca2+-responsiveness of the contractile apparatus in end-stage failing human hearts cannot be explained by a shift in contractile protein isoforms, but results from the complex interplay between changes in the phosphorylation status of MLC-2 and TnI