Ablation of Ventricular Myosin Regulatory Light Chain Phosphorylation in Mice Causes Cardiac Dysfunction in Situ and Affects Neighboring Myofilament Protein Phosphorylation

Sarah B Scruggs,Aaron C Hinken,Ariyaporn Thawornkaiwong,Jeffrey Robbins,Lori A Walker,Pieter P De Tombe,David L Geenen,Peter M Buttrick,R John Solaro

doi:10.1074/jbc.m807414200

Abstract

There is little direct evidence on the role of myosin regulatory light chain phosphorylation in ejecting hearts. In studies reported here we determined the effects of regulatory light chain (RLC) phosphorylation on in situ cardiac systolic mechanics and in vitro myofibrillar mechanics. We compared data obtained from control nontransgenic mice (NTG) with a transgenic mouse model expressing a cardiac specific nonphosphorylatable RLC (TG-RLC(P-). We also determined whether the depression in RLC phosphorylation affected phosphorylation of other sarcomeric proteins. TG-RLC(P-) demonstrated decreases in base-line load-independent measures of contractility and power and an increase in ejection duration together with a depression in phosphorylation of myosin-binding protein-C (MyBP-C) and troponin I (TnI). Although TG-RLC(P-) displayed a significantly reduced response to beta(1)-adrenergic stimulation, MyBP-C and TnI were phosphorylated to a similar level in TG-RLC(P-) and NTG, suggesting cAMP-dependent protein kinase signaling to these proteins was not disrupted. A major finding was that NTG controls were significantly phosphorylated at RLC serine 15 following beta(1)-adrenergic stimulation, a mechanism prevented in TG-RLC(P-), thus providing a biochemical difference in beta(1)-adrenergic responsiveness at the level of the sarcomere. Our measurements of Ca(2+) tension and Ca(2+)-ATPase rate relations in detergent-extracted fiber bundles from LV trabeculae demonstrated a relative decrease in maximum Ca(2+)-activated tension and tension cost in TG-RLC(P-) fibers, with no change in Ca(2+) sensitivity. Our data indicate that RLC phosphorylation is critical for normal ejection and response to beta(1)-adrenergic stimulation. Our data also indicate that the lack of RLC phosphorylation promotes compensatory changes in MyBP-C and TnI phosphorylation, which when normalized do not restore function.

Highlights

Understanding the precise mechanisms by which phosphorylation of RLC affects function of ejecting ventricles is important, because mechanisms downstream of Ca2ϩ fluxes at the level of the sarcomere appear to dominate ejection and to sustain ventricular elastance [15]
Levels as NTG Controls with Dobutamine Treatment; the Only Sarcomeric Difference in Phosphorylation Following ␤1-Adrenergic Stimulation Was at RLC Ser15—We compared the global proteome of TG-RLC(PϪ) and NTG sarcomeres using two-dimensional difference gel electrophoresis
Our studies provide new insights into the significant role of RLC phosphorylation in systolic mechanics in basal conditions and in response to adrenergic stimulation

Summary

Introduction

Understanding the precise mechanisms by which phosphorylation of RLC affects function of ejecting ventricles is important, because mechanisms downstream of Ca2ϩ fluxes at the level of the sarcomere appear to dominate ejection and to sustain ventricular elastance [15]. A, representative time-varying elastance curves throughout a cardiac cycle in NTG and TG-RLC(PϪ) ventricles at base line (left panel) and following 5 min of dobutamine stimulation (right panel). There is a dramatic decrease in maximal elastance (Emax) at base line in TG-RLC(PϪ) that persists following dobutamine stimulation, whereas that of NTG substantially increases as expected.

Results

Conclusion