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

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 ␤ 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 ␤ 1 -adrenergic stimulation, a mechanism prevented in TG-RLC(P؊), thus providing a biochemical difference in ␤ 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 ␤ 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.Phosphorylation of sarcomeric proteins tunes the intensity and dynamics of cardiac contraction and relaxation independent of membrane Ca 2ϩ fluxes to meet physiologic demands (1, 2). We focus here on ventricular myosin regulatory light chain, which is phosphorylated in vivo (3-5) but whose functional role in control of cardiac dynamics has remained unclear. The identification of RLC 2 mutations linked to familial hypertrophic cardiomyopathy (6) underscores the importance of understanding its action as a regulator of contraction. Functionally, in vitro cardiac RLC phosphorylation by MLCK produces a sensitizing shift in the force-Ca 2ϩ relation in skinned fibers (7-11). Moreover, studies show that RLC phosphorylation manifests as a gradient across the wall of the heart, which may be important for both normalizing wall stress and for generation of torsion about the long axis of the ejecting heart (12-14). Yet there remains a lack of understanding of the in situ functional effects of RLC phosphorylation and whether phosphorylation of RLC influences other sarcomeric sites as substrates for kinases and phosphatases.Understanding the precise mechanisms by w...

Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice

“…Phosphorylation of Ser15 has been recognized to play an important role in cardiac muscle contraction under normal and disease conditions (9). Significantly reduced RLC phosphorylation was reported in patients with heart failure (10, 11) and observed in animal models of cardiac disease (5, [12][13][14]. Attenuation of RLC phosphorylation in cardiac MLCK knockout mice led to ventricular myocyte hypertrophy, with histological evidence of necrosis and fibrosis, and to mild dilated cardiomyopathy (15,16).…”

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).…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Interestingly, following β1-adrenergic stimulation, non-transgenic controls were significantly phosphorylated at RLC Ser-15, which was abrogated in Tg-RLC(P-). The study showed the importance of RLC phosphorylation in maintenance of normal ejection fraction and β 1 -adrenergic responsiveness (Scruggs et al 2009). These reports indicate that maintaining the physiological levels of RLC phosphorylation is critical for the normal function of the heart and suggest that the myocardium containing dephosphorylated myosin, the molecular motor of muscle contraction, has a reduced ability to produce force and maintain cardiac function at physiological levels.…”

“…This claim of a spatial gradient of RLC phosphorylation, however, has been challenged by the Stull group and quantitative measurements have revealed no evidence for its existence in the hearts of WT and transgenic mice overexpressing cMLCK (Huang et al 2008;Kamm and Stull 2011). Significantly depressed Ser-15 RLC phosphorylation was reported in HF patients (van der Velden et al 2003a, b) and was also observed in experimental animal models of cardiac disease (Scruggs et al 2009;Sheikh et al 2012;Yuan et al 2015). Studies in mice showed that reduced RLC phosphorylation resulted in abnormal heart performance, presumably through morphological and/or myofibrillar functional alterations (e.g., change in force, myofilament calcium sensitivity, ATPase activity, cross-bridge kinetics) (Huang et al 2008;Kerrick et al 2009b;Warren et al 2012).…”

“…Likewise, attenuation of RLC phosphorylation in cMLCK knock-out mice was demonstrated to cause ventricular hypertrophy, fibrosis and dilated cardiomyopathy (Ding et al 2010). Scruggs et al (2009) determined the effects of RLC phosphorylation on the in situ cardiac systolic mechanics and in vitro myofibrillar mechanics using a transgenic mouse model expressing a cardiac specific non-phosphorylatable RLC [Tg-RLC(P-)]. The group showed a decrease in base-line load-independent measures of contractility and power as well as an increase in ejection duration together with a depression in phosphorylation levels of MyBP-C and TnI.…”