Priya Muthu

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.

Single molecule kinetics in the familial hypertrophic cardiomyopathy D166V mutant mouse heart

One of the sarcomeric mutations associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC) is the D166V point mutation in the ventricular myosin regulatory light chain (RLC) encoded by the MYL2 gene. In this report we show that the rates of myosin cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricles of transgenic (Tg)-D166V and Tg-WT mice. We have derived the myosin cross-bridge kinetic rates by tracking the orientation of a fluorescently labeled single actin molecule. Orientation (measured by polarized fluorescence) oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The rate of cross-bridge attachment during isometric contraction decreased from 3 s(-1) in myofibrils from Tg-WT to 1.4 s(-1) in myofibrils from Tg-D166V. The rate of detachment decreased from 1.3 s(-1) (Tg-WT) to 1.2 s(-1) (Tg-D166V). We also showed that the level of RLC phosphorylation was largely decreased in Tg-D166V myofibrils compared to Tg-WT. Our findings suggest that alterations in the myosin cross-bridge kinetics brought about by the D166V mutation in RLC might be responsible for the compromised function of the mutated hearts and lead to their inability to efficiently pump blood.

Rotation of actin monomers during isometric contraction of skeletal muscle

Cyclic interactions of myosin and actin are responsible for contraction of muscle. It is not self-evident, however, that the mechanical cycle occurs during steady-state isometric contraction where no work is produced. Studying cross-bridge dynamics during isometric steady-state contraction requires an equilibrium time-resolved method (not involving application of a transient). This work introduces such a method, which analyzes fluctuations of anisotropy of a few actin molecules in muscle. Fluorescence anisotropy, indicating orientation of an actin protomer, is collected from a volume of a few attoliters (10(-18) L) by confocal total internal reflection (CTIR) microscopy. In this method, the detection volume is made shallow by TIR illumination, and narrow by confocal aperture inserted in the conjugate image plane. The signal is contributed by approximately 12 labeled actin molecules. Shortening of a myofibril during contraction is prevented by light cross-linking with 1-ethyl-3-[3-dimethylamino)-propyl]-carbodiimide. The root mean-squared anisotropy fluctuations are greater in isometrically contracting than in rigor myofibrils. The results support the view that during isometric contraction, cross-bridges undergo a mechanical cycle.

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.

Monolayers of Silver Nanoparticles Decrease Photobleaching: Application to Muscle Myofibrils

Studying single molecules in a cell has the essential advantage that kinetic information is not averaged out. However, since fluorescence is faint, such studies require that the sample be illuminated with the intense light beam. This causes photodamage of labeled proteins and rapid photobleaching of the fluorophores. Here, we show that a substantial reduction of these types of photodamage can be achieved by imaging samples on coverslips coated with monolayers of silver nanoparticles. The mechanism responsible for this effect is the interaction of localized surface plasmon polaritons excited in the metallic nanoparticles with the transition dipoles of fluorophores of a sample. This leads to a significant enhancement of fluorescence and a decrease of fluorescence lifetime of a fluorophore. Enhancement of fluorescence leads to the reduction of photodamage, because the sample can be illuminated with a dim light, and decrease of fluorescence lifetime leads to reduction of photobleaching because the fluorophore spends less time in the excited state, where it is susceptible to oxygen attack. Fluorescence enhancement and reduction of photobleaching on rough metallic surfaces are usually accompanied by a loss of optical resolution due to refraction of light by particles. In the case of monolayers of silver nanoparticles, however, the surface is smooth and glossy. The fluorescence enhancement and the reduction of photobleaching are achieved without sacrificing the optical resolution of a microscope. Skeletal muscle myofibrils were used as an example, because they contain submicron structures conveniently used to define optical resolution. Small nanoparticles (diameter approximately 60 nm) did not cause loss of optical resolution, and they enhanced fluorescence approximately 500-fold and caused the appearance of a major picosecond component of lifetime decay. As a result, the sample photobleached approximately 20-fold more slowly than the sample on glass coverslips.

Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice

We have examined, for the first time, the effects of the familial hypertrophic cardiomyopathy (HCM)-associated Lys104Glu mutation in the myosin regulatory light chain (RLC). Transgenic mice expressing the Lys104Glu substitution (Tg-MUT) were generated and the results were compared to Tg-WT (wild-type human ventricular RLC) mice. Echocardiography with pulse wave Doppler in 6month-old Tg-MUT showed early signs of diastolic disturbance with significantly reduced E/A transmitral velocities ratio. Invasive hemodynamics in 6month-old Tg-MUT mice also demonstrated a borderline significant prolonged isovolumic relaxation time (Tau) and a tendency for slower rate of pressure decline, suggesting alterations in diastolic function in Tg-MUT. Six month-old mutant animals had no LV hypertrophy; however, at >13months they displayed significant hypertrophy and fibrosis. In skinned papillary muscles from 5 to 6month-old mice a mutation induced reduction in maximal tension and slower muscle relaxation rates were observed. Mutated cross-bridges showed increased rates of binding to the thin filaments and a faster rate of the power stroke. In addition, ~2-fold lower level of RLC phosphorylation was observed in the mutant compared to Tg-WT. In line with the higher mitochondrial content seen in Tg-MUT hearts, the MUT-myosin ATPase activity was significantly higher than WT-myosin, indicating increased energy consumption. In the in vitro motility assay, MUT-myosin produced higher actin sliding velocity under zero load, but the velocity drastically decreased with applied load in the MUT vs. WT myosin. Our results suggest that diastolic disturbance (impaired muscle relaxation, lower E/A) and inefficiency of energy use (reduced contractile force and faster ATP consumption) may underlie the Lys104Glu-mediated HCM phenotype.

In Vitro Rescue Study of a Malignant Familial Hypertrophic Cardiomyopathy Phenotype by Pseudo-Phosphorylation of Myosin Regulatory Light Chain

Pseudo-phosphorylation of cardiac myosin regulatory light chain (RLC) has never been examined as a rescue method to alleviate a cardiomyopathy phenotype brought about by a disease causing mutation in the myosin RLC. This study focuses on the aspartic acid to valine substitution (D166V) in the myosin RLC shown to be associated with a malignant phenotype of familial hypertrophic cardiomyopathy (FHC). The mutation has also been demonstrated to cause severe functional abnormalities in transgenic mice expressing D166V in the heart. To explore this novel rescue strategy, pseudo-phosphorylation of D166V was used to determine whether the D166V-induced detrimental phenotype could be brought back to the level of wild-type (WT) RLC. The S15D substitution at the phosphorylation site of RLC was inserted into the recombinant WT and D166V mutant to mimic constitutively phosphorylated RLC proteins. Non-phosphorylatable (S15A) constructs were used as controls. A multi-faceted approach was taken to determine the effect of pseudo-phosphorylation on the ability of myosin to generate force and motion. Using mutant reconstituted porcine cardiac muscle preparations, we showed an S15D-induced rescue of both the enzymatic and binding properties of D166V-myosin to actin. A significant increase in force production capacity was noted in the in vitro motility assays for S15D-D166V vs. D166V reconstituted myosin. A similar pseudo-phosphorylation induced effect was observed on the D166V-elicited abnormal Ca(2+) sensitivity of force in porcine papillary muscle strips reconstituted with phosphomimic recombinant RLCs. Results from this study demonstrate a novel in vitro rescue strategy that could be utilized in vivo to ameliorate a malignant cardiomyopathic phenotype. We show for the first time that pseudo-RLC phosphorylation can reverse the majority of the mutation-induced phenotypes highlighting the importance of RLC phosphorylation in combating cardiac disease.

Diversity and similarity of motor function and cross-bridge kinetics in papillary muscles of transgenic mice carrying myosin regulatory light chain mutations D166V and R58Q

Mechanical properties of skinned papillary muscle fibers from transgenic mice expressing familial hypertrophic cardiomyopathy associated mutations D166V and R58Q in myosin regulatory light chain were investigated. Elementary steps and the apparent rate constants of the cross-bridge cycle were characterized from the tension transients induced by sinusoidal length changes during maximal Ca(2+) activation, together with ATP, ADP, and Pi studies. The tension-pCa relation was also tested in two sets of solutions with differing Pi and ionic strength. Our results showed that in both mutants the fast apparent rate constant 2πc and the rate constants of the cross-bridge detachment step (k2) were smaller than those of wild type (WT), demonstrating the slower cross-bridge kinetics. D166V showed significantly smaller ATP (K1) and ADP (K0) association constants than WT, displaying weaker ATP binding and easier ADP release, whereas those of R58Q were not significantly different from WT. In tension-pCa study, both D166V and R58Q mutations exhibited increased Ca(2+) sensitivity and less cooperativity. We conclude that, while the two FHC mutations have similar clinical manifestations and prognosis, some of the mechanical parameters of cross-bridges (K0, K1) are differently modified, whereas some others (Ca(2+)-sensitivity, cooperativity, k2) are similarly modified by these two FHC associated mutations.