The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts

Abstract

Phosphorylation of the myosin regulatory light chain (RLC) by Ca(2+)-calmodulin-activated myosin light chain kinase (MLCK) is known to be essential for the inotropic function of the heart. In this study, we have examined the effects of MLCK-phosphorylation of transgenic (Tg) mouse cardiac muscle preparations expressing the D166V (aspartic acid to valine)-RLC mutation, identified to cause familial hypertrophic cardiomyopathy with malignant outcomes. Our previous work with Tg-D166V mice demonstrated a large increase in the Ca(2+) sensitivity of contraction, reduced maximal ATPase and force and a decreased level of endogenous RLC phosphorylation. Based on studies demonstrating the beneficial and/or protective effects of cardiac myosin phosphorylation for heart function, we hypothesized that an ex vivo phosphorylation of Tg-D166V cardiac muscle may rescue the detrimental contractile phenotypes observed earlier at the level of single myosin molecules and in Tg-D166V papillary muscle fibres. We showed that MLCK-induced phosphorylation of Tg-D166V cardiac myofibrils and muscle fibres was able to increase the reduced myofibrillar ATPase and reverse an abnormally increased Ca(2+) sensitivity of force to the level observed for Tg-wild-type (WT) muscle. However, in contrast to Tg-WT, which displayed a phosphorylation-induced increase in steady-state force, the maximal tension in Tg-D166V papillary muscle fibres decreased upon phosphorylation. With the exception of force generation data, our results support the notion that RLC phosphorylation works as a rescue mechanism alleviating detrimental functional effects of a disease causing mutation. Further studies are necessary to elucidate the mechanism of this unexpected phosphorylation-induced decrease in maximal tension in Tg-D166V-skinned muscle fibres.

Fig 1

Myofibrillar ATPase activity measured in Tg-WT and Tg-D166V preparations before and after Ca²⁺-CaM MLCK treatment. Maximal ATPase activity was measured in pCa 4 (white bars), whereas basal ATPase activity was measured in pCa 8 (black bars). Inset: Urea/SDS-PAGE of phosphorylated and non-phosphorylated cardiac myofibrils (CMF). CMF were run on 8% polyacrylamide gel in the presence of 8 M urea and the RLC protein was visualized with CT-1 antibodies recognizing the total RLC protein content (Materials and Methods). Under these conditions, the phosphorylated form of RLC in +P-WT or +P-D166V CMF migrated faster than the non-phosphorylated WT or D166V. Note, that due to the D166V mutation, the non-phosphorylated D166V migrates slower than the non-phosphorylated WT. 1⁰ ab: Primary CT-1 antibodies; 2⁰ ab: secondary IR-Red antibodies.

Fig 2

The effect of RLC phosphorylation on the force–pCa relationship in skinned muscle fibers from Tg-WT mice and Tg-D166V. The pCa₅₀ and n_H (Hill coefficient) values were: WT (black): pCa₅₀= 5.60 ± 0.01, n_H= 2.89 ± 0.15; +P-WT (red): pCa₅₀= 5.66 ± 0.02, n_H= 2.47 ± 0.16; D166V (blue): pCa₅₀= 5.83 ± 0.04, n_H= 2.16 ± 0.23; +P-D166V (green): pCa₅₀= 5.63 ± 0.04, n_H= 2.57 ± 0.19.

Fig 3

The effect of RLC phosphorylation on maximal force development in Tg-WT (A) and Tg-D166V (B) papillary muscle fibres. Maximal force was measured in pCa 4 buffer before and after CaM–MLCK treatment. Control WT and control D166V fibres were incubated in pCa 6 buffer with no CaM–MLCK complex. Insets: Western blot of experimental WT and +P-WT fibers (A); D166V and +P-D166V fibres (B). Phosphorylation of RLC in +P-WT or +P-D166V muscle fibres was detected with the +P-RLC antibodies specific for the phosphorylated form of cardiac RLC followed by a secondary goat anti-rabbit antibody conjugated with the fluorescent dye, IR red 800 (red bands). Total ELC, which served as a loading control, was detected with the monoclonal ab680 antibody followed by a secondary goat anti-mouse antibody conjugated with the fluorescent dye, Cy 5.5 (green bands). RLC: regulatory light chain of myosin (red); ELC: essential light chain of myosin (green).

Fig 4

The effect of D166V mutation on RLC phosphorylation in situ. Left ventricles of Tg-WT and Tg-D166V mice were flash frozen in liquid N₂ and subsequently analysed for level of RLC phosphorylation (Materials and Methods). Lanes 1 and 3: Tg-WT; lanes 2 and 4: Tg-D166V. Left: Immunoblotted with +P-RLC (antibody recognizing phosphorylated form of RLC: red). Right: Immunoblotted with CT-1 (antibody recognizing total RLC: red). Circled: D166V protein: phosphorylated (left), and total (right). IR-Red 800 and Cy5.5: secondary fluorescent antibodies.

Fig 5

Representative longitudinal sections from left ventricles of ∼6-month-old Tg-WT and of ∼6-month-old Tg-D166V mice stained with haematoxylin and eosin (H&E) for overall morphology and Masson's trichrome for signs of fibrosis. Scale bar = 20 μm.