Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2

Abstract

A role for myosin phosphorylation in modulating normal cardiac function has long been suspected, and we hypothesized that changing the phosphorylation status of a cardiac myosin light chain might alter cardiac function in the whole animal. To test this directly, transgenic mice were created in which three potentially phosphorylatable serines in the ventricular isoform of the regulatory myosin light chain were mutated to alanines. Lines were obtained in which replacement of the endogenous species in the ventricle with the nonphosphorylatable, transgenically encoded protein was essentially complete. The mice show a spectrum of cardiovascular changes. As previously observed in skeletal muscle, Ca 2؉ sensitivity of force development was dependent upon the phosphorylation status of the regulatory light chain. Structural abnormalities were detected by both gross histology and transmission electron microscopic analyses. Mature animals showed both atrial hypertrophy and dilatation. Echocardiographic analysis revealed that as a result of chamber enlargement, severe tricuspid valve insufficiency resulted in a detectable regurgitation jet. We conclude that regulated phosphorylation of the regulatory myosin light chains appears to play an important role in maintaining normal cardiac function over the lifetime of the animal.The roles of the regulatory myosin light chains (RLCs) 1 and the reversible post-translational modifications they undergo in striated muscle are beginning to be defined. In skeletal muscle, a serine at the amino end of the protein can be phosphorylated by a sarcoplasmic kinase (1), and it is now clear that RLCs in the different striated muscles are phosphorylated to differing degrees, leading presumably to different physiological effects. In smooth muscle, RLC phosphorylation by myosin light chain kinase (MLCK), which is activated by a Ca 2ϩ /calmodulin-dependent pathway, is responsible for initiating muscle contraction (2, 3). However, in skeletal and cardiac muscle, in which the thin, rather than thick, filament mediates control of contraction, RLC phosphorylation does not activate contraction but appears to play a modulatory role. In skinned skeletal muscle fibers, RLC phosphorylation increases sensitivity to activating Ca 2ϩ such that there is a significant leftward shift in the force-Ca 2ϩ relationship (4 -6). Increased RLC phosphorylation in skeletal muscle is also associated with potentiation of isometric twitch tension with repeated activation and inactivation of contraction (7, 8), rate of force production (9 -11), and maximum Ca 2ϩ -stimulated MgATPase activity (12). The mechanistic basis for the effects of RLC phosphorylation in striated muscle is hypothesized to be a lessening of the weak interaction of the myosin head with the myosin backbone and is probably due to a net charge change in a critical region of the protein (13). Upon phosphorylation, the myosin heads move away from the backbone to a position closer to actin, which presumably increases the rate at which myosin-actin interaction...

Keratin binding to 14-3-3 proteins modulates keratin filaments and hepatocyte mitotic progression

“…This approach is likely to be useful for studying the function of the posttranslational modifications (e.g., phosphorylation and glycosylation) of other IF proteins and has also been used recently in studying the function of non-IF protein phosphorylation (32)(33)(34). The approach may entail overexpression of the mutant or WT keratin (as done herein to generate a dominant-negative phenotype), expression of mutant or WT proteins in null backgrounds, or gene replacement.…”

Examining the in Vivo Role of the Amino Terminus of the Essential Myosin Light Chain

“…The polymerase chain reaction product was sequenced, and the fragment was linked to the mouse ␣-MyHC promoter. The final constructs containing a deleted ELC1v from position 5-14 (ELC1v⌬5-14) and a deleted ELC1a at the same region (ELC1a⌬5-14) were digested free of vector sequence with NotI, purified from agarose, and used to generate TG mice as described previously (20). Miscellaneous Methods-Transcript analysis was performed by RNA blots with transcript-specific probes as described previously (20).…”

“…The final constructs containing a deleted ELC1v from position 5-14 (ELC1v⌬5-14) and a deleted ELC1a at the same region (ELC1a⌬5-14) were digested free of vector sequence with NotI, purified from agarose, and used to generate TG mice as described previously (20). Miscellaneous Methods-Transcript analysis was performed by RNA blots with transcript-specific probes as described previously (20). For myofibrillar protein analysis, the left ventricular apex and atrial flaps were obtained from euthanized ELC1v, ELC1a, ELC1v⌬5-14 TG, ELC1a⌬5-14 TG, and nontransgenic (NTG) mice.…”

Myosin light chain phosphorylation enhances contraction of heart muscle via structural changes in both thick and thin filaments

“…cRLC is partially phosphorylated in vivo under basal conditions (8)(9)(10)(11)(12), and changes in its phosphorylation level are linked to heart disease (8,11,13,14). cRLC mutations associated with hypertrophic cardiomyopathy abolish cRLC phosphorylation in vitro (15), and mice expressing nonphosphorylatable cRLCs show severe cardiac dysfunction (10,16). In the vertebrate heart, cRLCs are phosphorylated almost exclusively by the cardiac isoform of myosin light chain kinase (cMLCK) (17,18), and cMLCK gene ablation leads to severe cardiac hypertrophy (19).…”

“…cRLC is at the thick filament end of the myosin head, where it joins the coiled-coil tail that forms the thick filament backbone. cRLC is partially phosphorylated in vivo under basal conditions (8)(9)(10)(11)(12), and changes in its phosphorylation level are linked to heart disease (8,11,13,14). cRLC mutations associated with hypertrophic cardiomyopathy abolish cRLC phosphorylation in vitro (15), and mice expressing nonphosphorylatable cRLCs show severe cardiac dysfunction (10,16).…”