Myosin ATPase Activity Research Articles

Characterization of the Cardiac Myosin Inhibitor CK-3773274: a Potential Therapeutic Approach for Hypertrophic Cardiomyopathy

Hypercontractility of the cardiac sarcomere may be essential for the underling pathological hypertrophy and fibrosis in genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility in vitro and in vivo. In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC50:1.26 μM). CK-3773274 selectively targets cardiac myosin, as it reduced myosin ATPase activity in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. Importantly, CK-3773274 did not inhibit the ATPase activity of smooth muscle myosin. In transient kinetic studies, CK-3773274 substantially slowed the rate of actin-activated phosphate release, likely stabilizing myosin conformations that bind weakly to actin. Binding of CK-3773274 to cardiac myosin is mutually exclusive with the nonspecific myosin II inhibitor blebbistatin, suggesting they bind to the same or overlapping sites. Consistent with its biochemical phenotype, CK-3773274 (10 μM) reduced fractional shortening (FS) by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium (Ca2+) transient. The effect of CK-3773274 on cardiac contractility in vivo was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Left ventricular dimensions and FS were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion by 20% to 70% relative to vehicle treatment without any changes to heart rate. In conclusion, CK-3773274 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo. Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.

Factors that Modulate the Stability of the Super Relaxed State of Myosin in Skeletal Muscle Fibers

The Super Relaxed State of myosin (SRX) has an ATPase activity that is 10 times slower than the Disordered Relaxed State (DRX). Factors that destabilize the SRX could be effective in treating obesity and type 2 diabetes. We have explored two such possible factors, binding of calcium and addition of covalently attached glucosamine. The regulatory light chain of myosin contains a site for binding divalent cations, either magnesium or calcium. We found that addition of calcium increased the basal myosin ATPase activity by 60% with a half maximum at pCa = 6.1 ± 0.1 and a Hill coefficient of 1.9 ± 0.6. We conclude that during periods of heavy activation magnesium is replaced by calcium, which destabilizes the SRX. Another control system that is present in all eukaryotic cells involves the reaction of glucosamine (GlcNac) with serines and threonines. Both myosin heavy chain and RLC have been shown to be labeled with GlcNac in skeletal muscle. We found that addition of free GlcNac to rabbit skeletal fibers inhibits the population of the SRX by 29 ± 8%. This inhibition presumably occurs because the free GlcNac competes off endogenous GlcNac thus reversing its effect. This would suggest that attachment of GlcNac to proteins in the fiber stabilizes the SRX. Attempts to add more GlcNac moieties to fibers by incubation in the transferase involved, along with its substrates, had no effect on the population of the SRX but increased the lifetime by 60 ± 8%, again suggesting greater stability of the SRX when GlcNac is attached. These very preliminary results suggest GlcNac increases the stability of the SRX. We suggest that such a system modulates the SRX in skeletal muscle.

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Hypertrophic Cardiomyopathy: Diverse Pathophysiology Revealed by Genetic Research, Toward Future Therapy.

Hypertrophic cardiomyopathy (HCM) is an intractable disease that causes heart failure mainly due to unexplained severe cardiac hypertrophy and diastolic dysfunction. HCM, which occurs in 0.2% of the general population, is the most common cause of sudden cardiac death in young people. HCM has been studied extensively using molecular genetic approaches. Genes encoding cardiac β-myosin heavy chain, cardiac myosin-binding protein C, and troponin complex, which were originally identified as causative genes, were subsequently reported to be frequently implicated in HCM. Indeed, HCM has been considered a disease of sarcomere gene mutations. However, fewer than half of patients with HCM have mutations in sarcomere genes. The others have been documented to have mutations in cardiac proteins in various other locations, including the Z disc, sarcoplasmic reticulum, plasma membrane, nucleus, and mitochondria. Next-generation sequencing makes it possible to detect mutations at high throughput, and it has become increasingly common to identify multiple cardiomyopathy-causing gene mutations in a single HCM patient. Elucidating how mutations in different genes contribute to the disease pathophysiology will be a challenge. In studies using animal models, sarcomere mutations generally tend to increase myocardial Ca2+ sensitivity, and some mutations increase the activity of myosin ATPase. Clinical trials of drugs to treat HCM are ongoing, and further new therapies based on pathophysiological analyses of the causative genes are eagerly anticipated.

Tau interactome analyses in CRISPR-Cas9 engineered neuronal cells reveal ATPase-dependent binding of wild-type but not P301L Tau to non-muscle myosins

Protein interactions of Tau are of interest in efforts to decipher pathogenesis in Alzheimer’s disease, a subset of frontotemporal dementias, and other tauopathies. We CRISPR-Cas9 edited two human cell lines to generate broadly adaptable models for neurodegeneration research. We applied the system to inducibly express balanced levels of 3-repeat and 4-repeat wild-type or P301L mutant Tau. Following 12-h induction, quantitative mass spectrometry revealed the Parkinson’s disease-causing protein DJ-1 and non-muscle myosins as Tau interactors whose binding to Tau was profoundly influenced by the presence or absence of the P301L mutation. The presence of wild-type Tau stabilized non-muscle myosins at higher steady-state levels. Strikingly, in human differentiated co-cultures of neuronal and glial cells, the preferential interaction of non-muscle myosins to wild-type Tau depended on myosin ATPase activity. Consistently, transgenic P301L Tau mice exhibited reduced phosphorylation of regulatory myosin light chains known to activate this ATPase. The direct link of Tau to non-muscle myosins corroborates independently proposed roles of Tau in maintaining dendritic spines and mitochondrial fission biology, two subcellular niches affected early in tauopathies.

Tripeptide Arg-Gly-Asp (RGD) modifies the molecular mechanical properties of the non-muscle myosin IIA in human bone marrow-derived myofibroblasts seeded in a collagen scaffold.

Mesenchymal stem cells (MSCs) were obtained from human bone marrow and amplified in cultures supplemented with human platelet lysate in order to generate myofibroblasts. When MSCs were seeded in solid collagen scaffolds, they differentiated into myofibroblasts that were observed to strongly bind to the substrate, forming a 3D cell scaffold network that developed tension and shortening after KCl stimulation. Moreover, MSC-laden scaffolds recapitulated the Frank-Starling mechanism so that active tension increased in response to increases in the initial length of the contractile system. This constituted a bioengineering tissue that exhibited the contractile properties observed in both striated and smooth muscles. By using the A. F. Huxley formalism, we determined the myosin crossbridge (CB) kinetics of attachment (f1) and detachment (g1 and g2), maximum myosin ATPase activity, molar myosin concentration, unitary CB force and maximum CB efficiency. CB kinetics were dramatically slow, characterizing the non-muscle myosin type IIA (NMMIIA) present in myofibroblasts. When MSCs were seeded in solid collagen scaffolds functionalized with Arg-Gly-Asp (RGD), contractility increased and CB kinetics were modified, whereas the unitary NMMIIA-CB force and maximum CB efficiency did not change. In conclusion, we provided a non-muscle bioengineering tissue whose molecular mechanical characteristics of NMMIIA were very close to those of a non-muscle contractile tissue such as the human placenta.

CaATP prolongs strong actomyosin binding and promotes futile myosin stroke.

Calcium plays an essential role in muscle contraction, regulating actomyosin interaction by binding troponin of thin filaments. There are several buffers for calcium in muscle, and those buffers play a crucial role in the formation of the transient calcium wave in sarcomere upon muscle activation. One such calcium buffer in muscle is ATP. ATP is a fuel molecule, and the important role of MgATP in muscle is to bind myosin and supply energy for the power stroke. Myosin is not a specific ATPase, and CaATP also supports myosin ATPase activity. The concentration of CaATP in sarcomeres reaches 1% of all ATP available. Since 294 myosin molecules form a thick filament, naïve estimation gives three heads per filament with CaATP bound, instead of MgATP. We found that CaATP dissociates actomyosin slower than MgATP, thus increasing the time of the strong actomyosin binding. The rate of the basal CaATPase is faster than that of MgATPase, myosin readily produces futile stroke with CaATP. When calcium is upregulated, as in malignant hyperthermia, kinetics of myosin and actomyosin interaction with CaATP suggest that myosin CaATPase activity may contribute to observed muscle rigidity and enhanced muscle thermogenesis.

Abstract 332: Pharmacologic Characterization of the Cardiac Myosin Inhibitor, CK-3773274: A Potential Therapeutic Approach for Hypertrophic Cardiomyopathy

Hypercontractility of the cardiac sarcomere appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. Here, we characterize the small molecule, CK-3773274, as a novel cardiac myosin inhibitor that decreases contractility in vitro and in vivo . In bovine cardiac myofibrils, CK-3773274 decreased myosin ATPase activity in a concentration-dependent fashion (IC 50 :1.26 μM). CK-3773274 specifically inhibited myosin activity, as it reduced myosin ATPase activity in a concentration-dependent manner in the absence of other sarcomere proteins, including actin, troponin, and tropomyosin. CK-3773274 (10 μM) reduced fractional shortening by 84% in electrically paced, isolated adult rat cardiomyocytes relative to control without any effect on the calcium transient. The effect of CK-3773274 on cardiac contractility in vivo was assessed in healthy male Sprague Dawley (SD) rats using single oral doses ranging from 0.5 to 4 mg/kg. Fractional shortening (FS) and left ventricular dimensions were determined by echocardiography at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced fractional shortening in a dose-related fashion by 20-70% relative to vehicle treatment (FS %: vehicle: 47.9± 1%; 0.5 mg/kg: 39 ± 2%; 4 mg/kg: 15 ± 4%; mean ±SEM, p<0.05 vehicle vs. all doses) without any changes to heart rate. Lastly, the effect of CK-3773274 was evaluated by echocardiography in healthy beagle dogs. Left ventricular ejection fraction (LVEF) was evaluated following single oral doses ranging from 0.75-3 mg/kg over a 48 hour period. 2 hours after dosing, CK-3773274 decreased LVEF in a dose-related fashion by approximately 15-50% relative to vehicle treatment (LVEF vehicle: 74.6 ± 3 %; 0.75 mg/kg: 62.5 ± 3%; 2 mg/kg: 44.9± 3%; 3 mg/kg: 36.8 ± 2%; mean ±SEM, p<0.05 vehicle vs. all doses). In conclusion, CK-3773274 is a novel, small molecule, cardiac myosin inhibitor that reduces cardiac contractility in vitro and in vivo . Cardiac myosin inhibition may be a viable approach to treat the underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.

Downregulation of calponin 2 contributes to the quiescence of lung macrophages.

Calponin 2 is an actin cytoskeleton-associated regulatory protein that inhibits the activity of myosin-ATPase and cytoskeleton dynamics. Recent studies have demonstrated that deletion of calponin 2 restricts the proinflammatory activation of macrophages in atherosclerosis and arthritis to attenuate the disease progression in mice. Here we demonstrate that the levels of calponin 2 vary among different macrophage populations, which may reflect their adaptation to specific tissue microenvironment corresponding to specific functional states. Interestingly, lung resident macrophages express significantly lower calponin 2 than peritoneal resident macrophages, which correlates with decreased substrate adhesion and reduced expression of proinflammatory cytokines and a proresolution phenotype. Deletion of calponin 2 in peritoneal macrophages also decreased substrate adhesion and downregulated the expression of proinflammatory cytokines. Providing the first line of defense against microbial invasion while receiving constant exposure to extrinsic antigens, lung macrophages need to maintain a necessary level of activity while limiting exaggerated inflammatory reaction. Therefore, their low level of calponin 2 may reflect an important physiological adaption. Downregulation of calponin 2 in macrophages may be targeted as a cytoskeleton-based novel mechanism, possibly via endoplasmic reticulum stress altering the processing and secretion of cytokines, to regulate immune response and promote quiescence for the treatment of inflammatory diseases.

Abstract 1254: Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data

Abstract Tubulin binding drugs are approved for the treatment of many cancer types, without the use of molecular markers to select patients likely to respond. Plinabulin binds β-tubulin in a differentiated pocket and is being tested in a Phase 3 clinical study for the treatment of NSCLC. Additional indications are being considered for plinabulin and an algorithm for selecting especially responsive cancers and patient subgroups would be of significant value. With this in mind, Affymetrix HG-U133 Plus 2.0 array mRNA expression data for 43 human breast, lung, prostate, ovarian or CNS cancer cell lines were utilized to develop mathematical models to predict in vitro plinabulin potency against the same cell lines. Cells were treated for only 24 hours with plinabulin and then cultured for another 48 hours without plinabulin. Viable cell number was then measured with a Cell Titer-Blue Assay, and the plinabulin concentration causing a 70% reduction in viable cells (IC70) versus vehicle treated controls was derived. Cell lines were clearly separable into plinabulin Active (21 cell lines with IC70<1.0 μM) and Inactive (IC70>9.5 μM) groups. Log2 transformed Affymetrix gene probeset signal values, preprocessed with the GeneChip robust multi-array average analysis algorithm, were selected and ranked as predictors of plinabulin activity with a bootstrap forest partitioning technique, utilizing JMP 14.1 statistical software. 56 HIT probesets were identified that also had significantly different expression in responding versus non-responding cell lines (p<0.01, uncorrected t-test). For probesets with gene annotation, only the probeset for each gene with the highest Jetset score was utilized (Li et al., 2011). Top HIT predictor genes include CTNNB1 (β-catenin; oncogene), CALD1 (caldesmon; inhibits myosin ATPase activity), ERI1 (RNA processing), LGR5 (adult stem cell biomarker), SECISBP2L (SLAN; prolongs mitosis), and TRAK1 (mitochondrial/endosome trafficking). Models were constructed from HIT gene probesets in JMP to identify plinabulin responding cell lines, utilizing either one-layer TanH multimode fit neural networks or binary logistic regression. Surprisingly, models incorporating approximately 4-10 probeset values were derived that perfectly predicted plinabulin activity. Importantly, the cell lines tested in the above analyses were not those known to express high levels of multi-drug resistant (MDR) transporters. The importance of MDR status was therefore evaluated separately. Plinabulin activity, unlike that of taxanes, was not significantly affected by the MDR transporter inhibitor verapamil (10 μM), in ovarian cancer cell lines with a known MDR phenotype. To conclude, our work provides novel algorithms that may be of value in selecting cancer patients with tumor cells that are particularly susceptible to the direct cytotoxic effects of plinabulin. Citation Format: James R. Tonra, Hagen Klett, Chenghao Shen, Gerhard Kelter, Ramon W. Mohanlal, G. Kenneth Lloyd, Lan Huang. Predictive models for tumor cell targeting with plinabulin, derived from in vitro screening and Affymetrix mRNA expression data [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1254.

Assessment of Na+/K+ ATPase Activity in Small Rodent and Human Skeletal Muscle Samples.

In skeletal muscle, the Na/K ATPase (NKA) plays essential roles in processes linked to muscle contraction, fatigue, and energy metabolism; however, very little information exists regarding the regulation of NKA activity. The scarcity of information regarding NKA function in skeletal muscle likely stems from methodological constraints, as NKA contributes minimally to total cellular ATP utilization, and therefore contamination from other ATPases prevents the assessment of NKA activity in muscle homogenates. Here we introduce a method that improves accuracy and feasibility for the determination of NKA activity in small rodent muscle samples (5-10 mg) and in human skeletal muscle. Skeletal muscle homogenates from mice (n = 6) and humans (n = 3) were used to measure NKA and sarcoplasmic reticulum Ca ATPase (SERCA) activities with the addition of specific ATPase inhibitors to minimize "background noise." We observed that myosin ATPase activity was the major interfering factor for estimation of NKA activity in skeletal muscle homogenates, as the addition of 25 μM of blebbistatin, a specific myosin ATPase inhibitor, considerably minimized "background noise" (threefold) and enabled the determination of NKA maximal activity with values three times higher than previously reported. The specificity of the assay was demonstrated after the addition of 2 mM ouabain, which completely inhibited NKA. On the other hand, the addition of blebbistatin did not affect the ability to measure SERCA function. The coefficient of variation for NKA and SERCA assays were 6.2% and 4.4%, respectively. The present study has improved the methodology to determine NKA activity. We further show the feasibility of measuring NKA and SERCA activities from a common muscle homogenate. This methodology is expected to aid in our long-term understanding of how NKA affects skeletal muscle metabolic homeostasis and contractile function in diverse situations.

SUN-547 Primary Autoimmune Hypothyroidism Presenting with Sleep Apnea and Recurrent Episodes of Rhabdomyolysis in a Pediatric Patient: A Case Report

Background: Hypothyroid myopathy has different presentations including rhabdomyolysis, Hoffman’s syndrome, acute compartment syndrome and Kocher-Debre-Semelaigne syndrome, with most cases being reported in adults. Rarity of this condition in pediatric population, leads to delay in diagnosis of underlying hypothyroidism in children with myopathy. Pathophysiology is not fully understood but is known to occur due to deposition of glycosaminoglycans, poor contractility of actin-myosin units, low myosin ATPase activity, and low ATP turnover in skeletal muscle [1].Treatment with thyroxine replacement results in resolution of myopathy. Clinical Case: A 13-year-old female presented to the emergency department with 4 weeks of intermittent abdominal pain. She reported history of strenuous exercise but denied any exposure to medications. Workup revealed elevated creatine kinase of 2411 units/L ( 21-232 units/L) and creatinine 1.2 mg/dL (0.3-1.0 mg/dL). Creatinine kinase level came down to 1891 units/L after two days of IV hydration and she was discharged with diagnosis of viral or exercise induced rhabdomyolysis. Four weeks later patient returned due to worsening edema and six pound weight gain. Workup revealed elevated creatine kinase 7936 units/L. Thyroid function studies obtained showed an elevated TSH 318 microIU/mL (0.40-5.34 microIU/mL), low Free T4 level 0.13 ng/dL (0.65-2.30 ng/dL), low total T4 0.5 mcg/dL (5.6-14.4 mcg/dL), positive titers of thyroid peroxidase antibody 642 IU/ml (0.0-9.0 IU/mL) and thyroglobulin antibody 2.3 IU/mL (0.0-1.0 IU/mL). Further history revealed fatigue, academic decline and polysomnograpy confirmed obstructive sleep apnea a couple of months prior to presentation. Levothyroxine replacement therapy was initiated at 25 mcg and dose was slowly increased over several weeks. 3 months later TSH level was 38.76 microIU/mL, Free T4 level normalized at 0.9 ng/dL and creatine kinase level normalized at 91 units/L (<143 units/L). Patient reported resolution of her myopathic symptoms and sleep apnea. Conclusion: This is first pediatric case report of rhabdomyolysis and sleep apnea due to hypothyroidism. Hypothyroidism should be considered in the differential diagnosis of rhabdomyolysis, especially if etiology of myopathy is unknown. Prompt thyroxine replacement can prevent further episodes of rhabdomyolysis and in this case also prevented long term complications and morbidity associated with sleep apnea.

Effects of Actin-Binding Compounds on the ATPase Activity of Myosin from Skeletal and Cardiac Muscle

Effects of Actin-Binding Compounds on the ATPase Activity of Myosin from Skeletal and Cardiac Muscle

Electrostatic Interactions Within Loop 1 and the Force Generation Region of Human Cardiac Myosin Affect the Rate of Actomyosin Dissociation and ADP Release

We have studied the kinetics of ATP induced actomyosin dissociation and ADP release in two mutants of beta isoform of the human cardiac myosin S1. The mutations were introduced in the Loop 1 (D208Q: K450L) and in the force-generating region (R694N) of the myosin head. Both mutants support myosin ATPase activity. We introduced these mutations to remove electrostatic interactions between charged residues in the specified domains of myosin S1. Our computational analysis showed that in the wild-type myosin, permanent salt bridges are present in the specified regions of the beta isoform. Those salt bridges are absent in alpha isoform. Since the kinetics of alpha isoform is an order of magnitude faster than the kinetics of beta isoform, we hypothesize that electrostatic interactions in the specified regions play a crucial role in myosin kinetics regulation. Obtained experimental data were analyzed using two models, assuming either rapid equilibrium between actomyosin and nucleotide or slow dissociation of ADP from actomyosin. We discuss the effect of mutations on the retention of the nucleotide by actomyosin and the potential role of electrostatic interactions in the Loop1 and in the force-generating region of human cardiac myosin on its regulation. We conclude that these salt bridges are essential for the proper functioning of beta isoform despite their absence in the alpha isoform.

A Complete Absence of Missense Mutation in Myosin Regulatory and Essential Light Chain Genes of South Indian Hypertrophic and Dilated Cardiomyopathies

Background: Myosin is a hexameric contractile protein composed of 2 heavy chains associated with 4 light chains of 2 distinct classes – 2 regulatory light chains (MYL2) and 2 essential light chains (MYL3). The myosin light chains stabilize the long alpha helical neck of the myosin head and regulate the myosin ATPase activities. Objectives: Mutations in MYL2 and MYL3 are reported to be associated with cardiomyopathies. However, there is no study available on these genes in Indian cardiomyopathies, and therefore we planned to study them. Method: For the first time we sequenced MYL2 and MYL3 genes in a total of 248 clinically well-characterized cardiomyopathies consisting of 101 hypertrophic and 147 dilated cases along with 207 healthy controls from south India. Results: Our study revealed a total of 10 variations – 7 in MYL2 and 3 in MYL3, of which 3 are novel variations observed exclusively in cases. However, the 15 causative missense mutations previously reported are totally absent in our study, which showed that the sequences of MYL2 and MYL3 are highly conserved in Indian cases/controls. Conclusions: MYL2 and MYL3 mutations are rare and the least cause of cardiomyopathies in Indians.

Essential oil of the leaves of Eugenia sulcata preserve myocardial contractility and does not present immunotoxicity

The essential oil of the leaves of Eugenia sulcata, in the Myrtaceae family, has a demonstrated antihypertensive effect, but its effects on heart muscle and its toxicity have not yet been elucidated. Little chemical or biological data are available for E. sulcata, whether emphasizing the beneficial effects or the pharmacological security of this species. This study aims to evaluate myocardial contractility and to analyze angiotensin converting enzyme (ACE) and myosin ATPase activities associated with use of this essential oil. In addition, we evaluated the immunotoxicity of E. sulcata essential oil. Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were treated daily for 30 days (10 mg/kg of oil) to evaluate the isometric force of the papillary muscle, ACE measured by fluorimetry, and myosin ATPase activities by inorganic phosphate. Lymphocyte cultures were used to evaluate cytotoxicity, DNA damage, and mutagenicity of the essential oil. The results demonstrate that the treatment did not change the cardiac contraction force and did not alter the functioning of the sarcoplasmic reticulum, extrusion of the membrane calcium, or modify the membrane calcium channels or β-adrenergic receptor activity. Tetanic contractions were potentiated in the SHR animals. Myosin ATPase activity was also increased in the SHR animals. Cardiac ACE activity was reduced in both animal strains, and the serum ACE was reduced only in the SHR animals. The essential oil did not cause cytotoxicity or mutagenicity and presented low DNA damage. Our results demonstrated that the essential oil does not change myocardial contractility and does not present relevant immunotoxicity.

Interplay of actin, ADP and Mg2+ interactions with striated muscle myosin: Implications of their roles in ATPase

The effects of Mg2+ on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg2+ inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg2+ were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg2+ bound to a fluorescent ADP analog about five-times tighter than without Mg2+. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg2+. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (Kd) without Mg2+ was more than five-fold smaller than with Mg2+. Further, we examined effects of F-actin, ADP and Mg2+ binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg2+-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg2+ affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.

Phosphomimetic-mediated invitro rescue of hypertrophic cardiomyopathy linked to R58Q mutation in myosin regulatory light chain.

Myosin regulatory light chain (RLC) phosphorylation is important for cardiac muscle mechanics/function as well as for the Ca2+ -troponin/tropomyosin regulation of muscle contraction. This study focuses on the arginine to glutamine (R58Q) substitution in the human ventricular RLC (MYL2 gene), linked to malignant hypertrophic cardiomyopathy in humans and causing severe functional abnormalities in transgenic (Tg) R58Q mice, including inhibition of cardiac RLC phosphorylation. Using a phosphomimic recombinant RLC variant where Ser-15 at the phosphorylation site was substituted with aspartic acid (S15D) and placed in the background of R58Q, we aimed to assess whether we could rescue/mitigate R58Q-induced structural/functional abnormalities invitro. We show rescue of several R58Q-exerted adverse phenotypes in S15D-R58Q-reconstituted porcine cardiac muscle preparations. A low level of maximal isometric force observed for R58Q- versus WT-reconstituted fibers was restored by S15D-R58Q. Significant beneficial effects were also observed on the Vmax of actin-activated myosin ATPase activity in S15D-R58Q versus R58Q-reconstituted myosin, along with its binding to fluorescently labeled actin. We also report that R58Q promotes the OFF state of myosin, both in reconstituted porcine fibers and in Tg mouse papillary muscles, thereby stabilizing the super-relaxed state (SRX) of myosin, characterized by a very low ATP turnover rate. Experiments in S15D-R58Q-reconstituted porcine fibers showed a mild destabilization of the SRX state, suggesting an S15D-mediated shift in disordered-relaxed (DRX)↔SRX equilibrium toward the DRX state of myosin. Our study shows that S15D-phosphomimic can be used as a potential rescue strategy to abrogate/alleviate the RLC mutation-induced phenotypes and is a likely candidate for therapeutic intervention in HCM patients.

Interactive effects of osmotic stress and burrowing activity on protein metabolism and muscle capacity in the soft shell clam Mya arenaria

Bioturbators such as sediment-dwelling marine bivalves are ecosystem engineers that enhance sediment-water exchange and benthic-pelagic coupling. In shallow coastal areas, bivalves are exposed to frequent disturbance and salinity stress that might negatively affect their activity and physiological performance; however, the mechanisms underlying these effects are not fully understood. We investigated the effects of osmotic stress (low and fluctuating salinity) and repeated burrowing on aerobic and contractile capacity of the foot muscle (assessed by the activity of succinate dehydrogenase and myosin ATPase) as well as the levels of organic osmolytes (free amino acids) and biochemical markers of protein synthesis and proteolysis in key osmoregulatory and energy storing tissues (gills and hepatopancreas, respectively) in a common bioturbator, the soft shell clam Mya arenaria. Osmotic stress and exhaustive exercise altered the foot muscle capacity of soft shell clams and had a strong impact on protein and amino acid homeostasis in tissues not directly involved in locomotion. Acclimation to constant low salinity (5 practical salinity units) depleted the whole-body free amino acid pool and affected protein synthesis but not protein breakdown in the gill. In contrast, fluctuating (5–15) salinity increased protein breakdown rate, suppressed protein synthesis, caused oxidative damage to proteins in the gill and selectively depleted whole-body glycine pool. Clams acclimated to normal salinity (15) increased the aerobic capacity of the foot muscle upon repeated burrowing, whereas acclimation to low and fluctuating salinity reduced this adaptive muscle plasticity. Under the normal and low salinity conditions, exhaustive exercise induced protein conservation pathways (indicated by suppression of protein synthesis and catabolism), but this effect was disrupted by fluctuating salinity. These findings indicate that exhaustive exercise and osmotic stress interactively affect whole-body protein homeostasis and functional capacity of the foot muscle in soft shell clams which might contribute to reduced burrowing activity of bivalve bioturbators in osmotically challenging environments such as estuaries and shallow coastal zones.

Left ventricular mechanoenergetics in excised, cross-circulated rat hearts under hypo-, normo-, and hyperthermic conditions

We investigated the effects of altering cardiac temperature on left ventricular (LV) myocardial mechanical work and energetics using the excised, cross-circulated rat heart model. We analyzed the LV end-systolic pressure–volume relationship (ESPVR) and linear relationship between myocardial oxygen consumption per beat (VO2) and systolic pressure–volume area (PVA; total mechanical energy per beat) in isovolumically contracting rat hearts during hypo- (32 °C), normo- (37 °C), and hyperthermia (42 °C) under a 300-beats per minute pacing. LV ESPVR shifted downward with increasing cardiac temperature. The VO2–PVA relationship was superimposable in these different thermal conditions; however, each data point of VO2–PVA shifted left-downward during increasing cardiac temperature on the superimposable VO2–PVA relationship line. VO2 for Ca2+ handling in excitation–contraction coupling decreased, which was associated with increasing cardiac temperature, during which sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity was suppressed, due to phospholamban phosphorylation inhibition, and instead, O2 consumption for basal metabolism was increased. The O2 cost of LV contractility for Ca2+ also increased with increasing cardiac temperature. Logistic time constants evaluating LV relaxation time were significantly shortened with increasing cardiac temperature related to the acceleration of the detachment in cross-bridge (CB) cycling, indicating increased myosin ATPase activity. The results suggested that increasing cardiac temperature induced a negative inotropic action related to SERCA activity suppression in Ca2+ handling and increased myosin ATPase activity in CB cycling. We concluded that thermal intervention could modulate cardiac inotropism by changing CB cycling, Ca2+ handling, and basal metabolism in rat hearts.