Ana I. Rojas

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.

Remodeling of the heart in hypertrophy in animal models with myosin essential light chain mutations.

Cardiac hypertrophy represents one of the most important cardiovascular problems yet the mechanisms responsible for hypertrophic remodeling of the heart are poorly understood. In this report we aimed to explore the molecular pathways leading to two different phenotypes of cardiac hypertrophy in transgenic mice carrying mutations in the human ventricular myosin essential light chain (ELC). Mutation-induced alterations in the heart structure and function were studied in two transgenic (Tg) mouse models carrying the A57G (alanine to glycine) substitution or lacking the N-terminal 43 amino acid residues (Δ43) from the ELC sequence. The first model represents an HCM disease as the A57G mutation was shown to cause malignant HCM outcomes in humans. The second mouse model is lacking the region of the ELC that was shown to be important for a direct interaction between the ELC and actin during muscle contraction. Our earlier studies demonstrated that >7 month old Tg-Δ43 mice developed substantial cardiac hypertrophy with no signs of histopathology or fibrosis. Tg mice did not show abnormal cardiac function compared to Tg-WT expressing the full length human ventricular ELC. Previously reported pathological morphology in Tg-A57G mice included extensive disorganization of myocytes and interstitial fibrosis with no abnormal increase in heart mass observed in >6 month-old animals. In this report we show that strenuous exercise can trigger hypertrophy and pathologic cardiac remodeling in Tg-A57G mice as early as 3 months of age. In contrast, no exercise-induced changes were noted for Tg-Δ43 hearts and the mice maintained a non-pathological cardiac phenotype. Based on our results, we suggest that exercise-elicited heart remodeling in Tg-A57G mice follows the pathological pathway leading to HCM, while it induces no abnormal response in Tg-Δ43 mice.

The R21C Mutation in Cardiac Troponin I Imposes Differences in Contractile Force Generation between the Left and Right Ventricles of Knock-In Mice

We investigated the effect of the hypertrophic cardiomyopathy-linked R21C (arginine to cysteine) mutation in human cardiac troponin I (cTnI) on the contractile properties and myofilament protein phosphorylation in papillary muscle preparations from left (LV) and right (RV) ventricles of homozygous R21C+/+ knock-in mice. The maximal steady-state force was significantly reduced in skinned papillary muscle strips from the LV compared to RV, with the latter displaying the level of force observed in LV or RV from wild-type (WT) mice. There were no differences in the Ca2+ sensitivity between the RV and LV of R21C+/+ mice; however, the Ca2+ sensitivity of force was higher in RV-R21C+/+ compared with RV-WT and lower in LV- R21C+/+ compared with LV-WT. We also observed partial loss of Ca2+ regulation at low [Ca2+]. In addition, R21C+/+-KI hearts showed no Ser23/24-cTnI phosphorylation compared to LV or RV of WT mice. However, phosphorylation of the myosin regulatory light chain (RLC) was significantly higher in the RV versus LV of R21C+/+ mice and versus LV and RV of WT mice. The difference in RLC phosphorylation between the ventricles of R21C+/+ mice likely contributes to observed differences in contractile force and the lower tension monitored in the LV of HCM mice.

Myosin Regulatory Light Chain Phosphorylation Rescues Cardiac Dysfunction Caused by Familial Hypertrophic Cardiomyopathy-Linked Mutations

In this report, we compared the role of cardiac myosin regulatory light chain (RLC) phosphorylation on cardiac function in skinned muscle preparations containing two RLC mutations, R58Q (arginine to glutamine) and D166V (aspartic acid to valine), both linked with a malignant disease phenotype. Previous studies on D166V-transgenic mice showed that the myosin light chain kinase (MLCK)-induced phosphorylation of D166V mouse myocardium was able to alleviate detrimental functional effects caused by this mutation. In this study, we used recombinant phosphomimetic S15D (serine to aspartate) mutation in the D166V background exchanged into porcine cardiac myosin and investigated the effect of constitutively phosphorylated RLC proteins on the actomyosin interaction. The actin-activated myosin ATPase activity, which was decreased in D166V- exchanged myosin, was partially rescued in S15D-D166V exchanged myosin reaching the level observed for WT-reconstituted myosin. Similarly, myosin reconstituted with S15D-D166V mutant showed an increase in the binding to fluorescently labeled actin compared to D166V-reconstituted myosin, with Kd=1.9 μM and Kd=41 μM, respectively. To further investigate whether MLCK-phosphorylation could rescue the phenotype associated with the R58Q mutation, we studied force development in transgenic R58Q-mouse papillary muscle fibers. Compared to Tg-WT, a drastic reduction in maximal force and myofibrillar ATPase activity was observed in samples carrying the R58Q mutation. However, MLCK induced phosphorylation of R58Q muscle fibers resulted in significantly increased maximal force and myofibrillar ATPase activity. These results suggest that RLC phosphorylation plays an important role not only in the physiological performance of the heart, but also helps to maintain normal cardiac function in the diseased myocardium. Our findings may contribute to the development of targeted cellular therapeutic approaches to limit FHC related cardiac dysfunction. Supported by AHA-10POST3420009 (PM) and NIH- HL071778 and HL090786 (DSC).

The HCM-Linked Ala13thr Mutation in the Cardiac Myosin Regulatory Light Chain Increases Isometric Force Production

The myosin regulatory light chain (RLC) is attached to the α-helical neck region of the myosin head, the so called lever arm, which connects the catalytic and actin binding domains with the thick filament backbone thus participating in the transmission of external forces to the myosin active site. It is understandable that mutations in the RLC associated with hypertrophic cardiomyopathy (HCM) may lead to alterations in force generation affecting cardiac muscle performance. Here, we studied the physiological consequences of an Alanine to Threonine (A13T) mutation in the N-domain of myosin RLC, found in population studies to cause HCM with a specific disease phenotype characterized by mid-ventricular obstruction. We observed an A13T-induced 30-50% increase in maximal force measured in skinned cardiac muscle fibers from transgenic Tg-A13T mice compared to control, Tg-WT and non-Tg littermates. Furthermore, a mutation-mediated 1.3-fold decrease in Vmax and a 1.5-fold increase in Km were observed in the actin-activated myosin ATPase activity compared with myosin from the healthy controls. The binding of Tg-myosin to pyrene-actin was similar for all groups of mice. No changes in the maximal myofibrillar ATPase or in the Ca2+-sensitivity were noted. The same was true for the force-pCa relationship and the mutation did not introduce any alterations in the Ca2+-sensitivity of force development. Gross morphological evaluation revealed enlarged inter-ventricular septa and left ventricles in the hearts from Tg-A13T mice, a phenotype observed in patients harboring the A13T mutation. Our results indicate that the A13T mutation may result in a hypertrophic response through abnormally increased force that may exceed the tolerance of a healthy myocardium. A decreased rate of cross-bridge turnover further demonstrates inadequate energy generation in Tg-A13T mice adding to impaired sarcomeric function. Supported by NIH-HL071778 (DSC).