Katarzyna Kazmierczak

Cardiomyopathy-linked myosin regulatory light chain mutations disrupt myosin strain-dependent biochemistry

Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in sarcomeric proteins including the myosin regulatory light chain (RLC). Two such FHC mutations, R58Q and N47K, located near the cationic binding site of the RLC, have been identified from population studies. To examine the molecular basis for the observed phenotypes, we exchanged endogenous RLC from native porcine cardiac myosin with recombinant human ventricular wild type (WT) or FHC mutant RLC and examined the ability of the reconstituted myosin to propel actin filament sliding using the in vitro motility assay. We find that, whereas the mutant myosins are indistinguishable from the controls (WT or native myosin) under unloaded conditions, both R58Q- and N47K-exchanged myosins show reductions in force and power output compared with WT or native myosin. We also show that the changes in loaded kinetics are a result of mutation-induced loss of myosin strain sensitivity of ADP affinity. We propose that the R58Q and N47K mutations alter the mechanical properties of the myosin neck region, leading to altered load-dependent kinetics that may explain the observed mutant-induced FHC phenotypes.

Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice

Transgenic (Tg) mice expressing ~95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg‐D166V mice were examined using a Guth muscle research system. A large increase in the Ca2+ sensitivity of force and ATPase (ΔpCa50>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg‐D166V mice compared with control mice. The cross‐bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/ force) was slightly increased in Tg‐D166V fibers compared with controls. The calculated average force per D166V cross‐bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg‐D166V fibers compared with control fibers. Histopathological examination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation‐dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross‐bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg‐D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V‐positive patients.— Kerrick, W. G. L., Kazmierczak, K., Xu, Y., Wang, Y., Szczesna‐Cordary, D. Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice. FASEB J. 23, 855–865 (2009)

Regulatory light chain mutations associated with cardiomyopathy affect myosin mechanics and kinetics

The myosin regulatory light chain (RLC) wraps around the alpha-helical neck region of myosin. This neck region has been proposed to act as a lever arm, amplifying small conformational changes in the myosin head to generate motion. The RLC serves an important structural role, supporting the myosin neck region and a modulatory role, tuning the kinetics of the actin myosin interaction. Given the importance of the RLC, it is not surprising that mutations of the RLC can lead to familial hypertrophic cardiomyopathy (FHC), the leading cause of sudden cardiac death in people under 30. Population studies identified two FHC mutations located near the cationic binding site of the RLC, R58Q and N47K. Although these mutations are close in sequence, they differ in clinical presentation and prognosis, with R58Q showing a more severe phenotype. We examined the molecular based changes in myosin that are responsible for the disease phenotype by purifying myosin from transgenic mouse hearts expressing mutant myosins and examining actin filament sliding using the in vitro motility assay. We found that both R58Q and N47K show reductions in force compared to the wild type that could result in compensatory hypertrophy. Furthermore, we observed a higher ATPase rate and an increased activation at submaximal calcium levels for the R58Q myosin that could lead to decreased efficiency and incomplete cardiac relaxation, potentially explaining the more severe phenotype for the R58Q mutation.

Diastolic Dysfunction in Familial Hypertrophic Cardiomyopathy Transgenic Model Mice

AIMS Several mutations in the ventricular myosin regulatory light chain (RLC) were identified to cause familial hypertrophic cardiomyopathy (FHC). Based on our previous cellular findings showing delayed calcium transients in electrically stimulated intact papillary muscle fibres from transgenic Tg-R58Q and Tg-N47K mice and, in addition, prolonged force transients in Tg-R58Q fibres, we hypothesized that the malignant FHC phenotype associated with the R58Q mutation is most likely related to diastolic dysfunction. METHODS AND RESULTS Cardiac morphology and in vivo haemodynamics by echocardiography as well as cardiac function in isolated perfused working hearts were assessed in transgenic (Tg) mutant mice. The ATPase-pCa relationship was determined in myofibrils isolated from Tg mouse hearts. In addition, the effect of both mutations on RLC phosphorylation was examined in rapidly frozen ventricular samples from Tg mice. Significantly, decreased cardiac function was observed in isolated perfused working hearts from both Tg-R58Q and Tg-N47K mice. However, echocardiographic examination showed significant alterations in diastolic transmitral velocities and deceleration time only in Tg-R58Q myocardium. Likewise, changes in Ca(2+) sensitivity, cooperativity, and an elevated level of ATPase activity at low [Ca(2+)] were only observed in myofibrils from Tg-R58Q mice. In addition, the R58Q mutation and not the N47K led to reduced RLC phosphorylation in Tg ventricles. CONCLUSION Our results suggest that the N47K and R58Q mutations may act through similar mechanisms, leading to compensatory hypertrophy of the functionally compromised myocardium, but the malignant R58Q phenotype is most likely associated with more severe alterations in cardiac performance manifested as impaired relaxation and global diastolic dysfunction. At the molecular level, we suggest that by reducing the phosphorylation of RLC, the R58Q mutation decreases the kinetics of myosin cross-bridges, leading to an increased myofilament calcium sensitivity and to overall changes in intracellular Ca(2+) homeostasis.

The role of the N-terminus of the myosin essential light chain in cardiac muscle contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Delta43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Delta43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Delta43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Delta43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Delta43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.

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

Phosphorylation of the myosin regulatory light chain (RLC) by Ca2+‐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 Ca2+ 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 Ca2+ 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.

Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction

The myosin essential light chain (ELC) is a structural component of the actomyosin cross‐bridge, but its function is poorly understood, especially the role of the cardiac specific N‐terminal extension in modulating actomyosin interaction. Here, we generated transgenic (Tg) mice expressing the A57G (alanine to glycine) mutation in the cardiac ELC known to cause familial hypertrophic cardiomyopathy (FHC). The function of the ELC N‐terminal extension was investigated with the Tg‐Δ43 mouse model, whose myocardium expresses a truncated ELC. Low‐angle X‐ray diffraction studies on papillary muscle fibers in rigor revealed a decreased interfilament spacing (~1.5 nm) and no alterations in cross‐bridge mass distribution in Tg‐A57G mice compared to Tg‐WT, expressing the full‐length nonmutated ELC. The truncation mutation showed a 1.3‐fold increase in I1,1/I1,0, indicating a shift of cross‐bridge mass from the thick filament backbone toward the thin filaments. Mechanical studies demonstrated increased stiffness in Tg‐A57G muscle fibers compared to Tg‐WT or Tg‐Δ43. The equilibrium constant for the cross‐bridge force generation step was smallest in Tg‐Δ43. These results support an important role for the N‐terminal ELC extension in prepositioning the cross‐bridge for optimal force production. Subtle changes in the ELC sequence were sufficient to alter cross‐bridge properties and lead to pathological phenotypes.—Muthu, P., Wang, L., Yuan, C.‐C., Kazmierczak, K., Huang, W., Hernandez, O. M., Kawai, M., Irving, T. C., Szczesna‐Cordary, D. Structural and functional aspects of the myosin essential light chain in cardiac muscle contraction. FASEB J. 25, 4394–4405 (2011). www.fasebj.org

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

Significance Genetic hypertrophic cardiomyopathy (HCM) is a debilitating disease affecting 1 in 500 of the general population, and there is no effective therapy to reverse or prevent its development and/or progression to heart failure. To inhibit a detrimental HCM phenotype induced by the D166V mutation of cardiac myosin regulatory light chain (RLC) in mice that also show reduced phosphorylation of endogenous cardiac RLC, constitutively phosphorylated D166V mutant mice were produced and tested. Our in-depth investigation of heart morphology, structure, and function of S15D-D166V mice provided evidence for the pseudophosphorylation-elicited prevention of the progressive HCM-D166V phenotype. This study is significant for the field of HCM, and our findings may constitute a novel therapeutic modality to battle hypertrophic cardiomyopathy associated with RLC mutations. Myosin light chain kinase (MLCK)-dependent phosphorylation of the regulatory light chain (RLC) of cardiac myosin is known to play a beneficial role in heart disease, but the idea of a phosphorylation-mediated reversal of a hypertrophic cardiomyopathy (HCM) phenotype is novel. Our previous studies on transgenic (Tg) HCM-RLC mice revealed that the D166V (Aspartate166 →Valine) mutation-induced changes in heart morphology and function coincided with largely reduced RLC phosphorylation in situ. We hypothesized that the introduction of a constitutively phosphorylated Serine15 (S15D) into the hearts of D166V mice would prevent the development of a deleterious HCM phenotype. In support of this notion, MLCK-induced phosphorylation of D166V-mutated hearts was found to rescue some of their abnormal contractile properties. Tg-S15D-D166V mice were generated with the human cardiac RLC-S15D-D166V construct substituted for mouse cardiac RLC and were subjected to functional, structural, and morphological assessments. The results were compared with Tg-WT and Tg-D166V mice expressing the human ventricular RLC-WT or its D166V mutant, respectively. Echocardiography and invasive hemodynamic studies demonstrated significant improvements of intact heart function in S15D-D166V mice compared with D166V, with the systolic and diastolic indices reaching those monitored in WT mice. A largely reduced maximal tension and abnormally high myofilament Ca2+ sensitivity observed in D166V-mutated hearts were reversed in S15D-D166V mice. Low-angle X-ray diffraction study revealed that altered myofilament structures present in HCM-D166V mice were mitigated in S15D-D166V rescue mice. Our collective results suggest that expression of pseudophosphorylated RLC in the hearts of HCM mice is sufficient to prevent the development of the pathological HCM phenotype.

Discrete effects of A57G-myosin essential light chain mutation associated with familial hypertrophic cardiomyopathy.

The functional consequences of the familial hypertrophic cardiomyopathy A57G (alanine-to-glycine) mutation in the myosin ventricular essential light chain (ELC) were assessed in vitro and in vivo using previously generated transgenic (Tg) mice expressing A57G-ELC mutant vs. wild-type (WT) of human cardiac ELC and in recombinant A57G- or WT-protein-exchanged porcine cardiac muscle strips. Compared with the Tg-WT, there was a significant increase in the Ca²⁺ sensitivity of force (ΔpCa₅₀ ≅ 0.1) and an ~1.3-fold decrease in maximal force per cross section of muscle observed in the mutant preparations. In addition, a significant increase in passive tension in response to stretch was monitored in Tg-A57G vs. Tg-WT strips indicating a mutation-induced myocardial stiffness. Consistently, the hearts of Tg-A57G mice demonstrated a high level of fibrosis and hypertrophy manifested by increased heart weight-to-body weight ratios and a decreased number of nuclei indicating an increase in the two-dimensional size of Tg-A57G vs. Tg-WT myocytes. Echocardiography examination showed a phenotype of eccentric hypertrophy in Tg-A57G mice, enhanced left ventricular (LV) cavity dimension without changes in LV posterior/anterior wall thickness. Invasive hemodynamics data revealed significantly increased end-systolic elastance, defined by the slope of the pressure-volume relationship, indicating a mutation-induced increase in cardiac contractility. Our results suggest that the A57G allele causes disease by means of a discrete modulation of myofilament function, increased Ca²⁺ sensitivity, and decreased maximal tension followed by compensatory hypertrophy and enhanced contractility. These and other contributing factors such as increased myocardial stiffness and fibrosis most likely activate cardiomyopathic signaling pathways leading to pathologic cardiac remodeling.

Deletion of 1–43 amino acids in cardiac myosin essential light chain blunts length dependency of Ca2+ sensitivity and cross-bridge detachment kinetics

The role of cardiac myosin essential light chain (ELC) in the sarcomere length (SL) dependency of myofilament contractility is unknown. Therefore, mechanical and dynamic contractile properties were measured at SL 1.9 and 2.2 μm in cardiac muscle fibers from two groups of transgenic (Tg) mice: 1) Tg-wild-type (WT) mice that expressed WT human ventricular ELC and 2) Tg-Δ43 mice that expressed a mutant ELC lacking 1-43 amino acids. In agreement with previous studies, Ca(2+)-activated maximal tension decreased significantly in Tg-Δ43 fibers. pCa(50) (-log(10) [Ca(2+)](free) required for half maximal activation) values at SL of 1.9 μm were 5.64 ± 0.02 and 5.70 ± 0.02 in Tg-WT and Tg-Δ43 fibers, respectively. pCa(50) values at SL of 2.2 μm were 5.70 ± 0.01 and 5.71 ± 0.01 in Tg-WT and Tg-Δ43 fibers, respectively. The SL-mediated increase in the pCa(50) value was statistically significant only in Tg-WT fibers (P < 0.01), indicating that the SL dependency of myofilament Ca(2+) sensitivity was blunted in Tg-Δ43 fibers. The SL dependency of cross-bridge (XB) detachment kinetics was also blunted in Tg-Δ43 fibers because the decrease in XB detachment kinetics was significant (P < 0.001) only at SL 1.9 μm. Thus the increased XB dwell time at the short SL augments Ca(2+) sensitivity at short SL and thus blunts SL-mediated increase in myofilament Ca(2+) sensitivity. Our data suggest that the NH(2)-terminal extension of cardiac ELC not only augments the amplitude of force generation, but it also may play a role in mediating the SL dependency of XB detachment kinetics and myofilament Ca(2+) sensitivity.