Cardiac Myosin Light Chain Research Articles

Heterozygous Mylk3 Knockout Mice Partially Recapitulate Human DCM With Heterozygous MYLK3 Mutations.

Backgrounds: Recent studies identified heterozygous variants in MYLK3 gene that encodes cardiac myosin light chain kinase (cMLCK) are related to familial dilated cardiomyopathy (DCM) for the first time. Autosomal dominant traits suggest that pathogenesis of DCM could be related to heterozygous MYLK3 loss-of-function variants (haploinsufficiency). We previously generated and examined homozygous Mylk3 knockout mice that lead to heart failure. It had yet to be examined whether heterozygous Mylk3 knockout mice represent a DCM-like phenotype.Methods and Results: Heterozygous knockout (Mylk3wild/-) mice were examined regarding cardiac function, heart histology and expression of cMLCK protein and mRNA relative to age-matched wild-type controls (Mylk3wild/wild). At 4 months of age, cardiac contractility in heterozygous knockout mice was reduced with percent fractional shortening of 23.3 ± 1.2% compared to 30.1 ± 1.8% in control (Mylk3wild/- vs. Mylk3wild/wild, n = 9 each). In 4-month-old heterozygous knockout hearts, expression of cMLCK mRNA was expectedly reduced by almost half, however, protein expression was reduced by approximately 75% relative to the control wild-type (Mylk3wild/- vs. Mylk3wild/wild, n = 9 each). Isolated ventricular cardiomyocytes from heterozygous knockout mice were larger with increase of short-axis length relative to the cardiomyocytes from control mice. However, increase of heart failure markers as well as interstitial fibrosis were not evident in heterozygous knockout mice compared to controls.Conclusion: Heterozygous Mylk3 knockout mice show mild reduction of cardiac contractility by 4 months of age, and proteins reduced by approximately 75% relative to the control wild-type mice. These mice partly resemble human with the heterozygous MYLK3 mutation, but the reduction in cardiac contractility was milder.

Hereditary heart disease: pathophysiology, clinical presentation, and animal models of HCM, RCM, and DCM associated with mutations in cardiac myosin light chains.

Genetic cardiomyopathies, a group of cardiovascular disorders based on ventricular morphology and function, are among the leading causes of morbidity and mortality worldwide. Such genetically driven forms of hypertrophic (HCM), dilated (DCM), and restrictive (RCM) cardiomyopathies are chronic, debilitating diseases that result from biomechanical defects in cardiac muscle contraction and frequently progress to heart failure (HF). Locus and allelic heterogeneity, as well as clinical variability combined with genetic and phenotypic overlap between different cardiomyopathies, have challenged proper clinical prognosis and provided an incentive for identification of pathogenic variants. This review attempts to provide an overview of inherited cardiomyopathies with a focus on their genetic etiology in myosin regulatory (RLC) and essential (ELC) light chains, which are EF-hand protein family members with important structural and regulatory roles. From the clinical discovery of cardiomyopathy-linked light chain mutations in patients to an array of exploratory studies in animals, and reconstituted and recombinant systems, we have summarized the current state of knowledge on light chain mutations and how they induce physiological disease states via biochemical and biomechanical alterations at the molecular, tissue, and organ levels. Cardiac myosin RLC phosphorylation and the N-terminus ELC have been discussed as two important emerging modalities with important implications in the regulation of myosin motor function, and thus cardiac performance. A comprehensive understanding of such triggers is absolutely necessary for the development of target-specific rescue strategies to ameliorate or reverse the effects of myosin light chain-related inherited cardiomyopathies.

Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy.

AimsCardiac myosin light chain kinase (cMLCK) phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization. However, few data exist regarding the relationship between cMLCK mutations and MLC2v phosphorylation, particularly in terms of developing familial dilated cardiomyopathy (DCM) in whom cMLCK gene mutations were identified. The purpose of the present study was to investigate functional consequences of cMLCK mutations in DCM patients.Methods and resultsThe diagnosis of DCM was based on the patients' history and on echocardiography. We screened cMLCK gene mutations in DCM probands with high resolution melting analysis. Known DCM‐causing genes mutations were excluded by exome sequencing of family members. MLC2v phosphorylation was analysed by Phos‐tag sodium dodecyl sulfate–polyacrylamide gel electrophoresis assays. We also performed ADP‐Glo assays for determining the total amount of adenosine triphosphate used in the kinase reaction. Unrelated DCM probands (109 males and 40 females) were enrolled in this study, of which 16 were familial and 133 sporadic. By mutation screening, a truncation variant of c1915‐1 g>t (p.Pro639Valfs*15) was identified, which was not detected in 400 chromosomes of 200 healthy volunteers; it is listed in the Human Genetic Variation Database with an allele frequency < 0.001. In the proband, the presence of mutations in known DCM‐causing genes was excluded with exome analysis. Familial analysis identified a 19‐year‐old male carrier who manifested slight left ventricular dilation with preserved systolic function. Phosphorylation assays analysed by Phos‐tag SDS‐PAGE revealed that the identified p.Pro639Valfs*15 mutation results in a complete lack of kinase activity, although it did not affect wild‐type cMLCK activity. ADP‐Glo assays confirmed that the mutant cMLCK had no kinase activity, whereas wild‐type cMLCK had a Km value of 5.93 ± 1.47 μM and a V max of 1.28 ± 0.03 mol/min/mol kinase.ConclusionsThese results demonstrate that a truncation mutation in the cMLCK gene p.Pro639Valfs*15 can be associated with significant impairment of MLC2v phosphorylation and possibly with development of DCM, although a larger study of DCM patients is required to determine the prevalence of this mutation and further strengthen its association with disease development.

The effects of N-acetylcysteine on cisplatin induced cardiotoxicity.

Recent studies reported that oxidative stress is an important mechanism that contributes to cisplatin induced cardiotoxicity. In the present study, the effects of N-acetylcysteine (NAC), which is an antioxidant, on cisplatin induced cardiotoxicity were investigated in a rat model. Thirty two rats were separated into 4 equal groups: Control, NAC-250, CP (cisplatin), CP+NAC. Rats in the experimental groups were treated with a single dose of cisplatin intraperitoneally (ip) (10 mg/kg) and NAC (ip, 250 mg/kg) for 3 consecutive days. At the end of the experiment, cardiotoxicity was determined from plasma CK-MB, LDH, cTnI and cardiac myosin light chain-1 (CMLC-1) levels. In the tissue samples, total oxidant capacity (TOC), total antioxidant capacity (TAC), lipid hydroperoxide (ROOH) and thiol levels were measured. The hearts were also analyzed histopathologically. It was determined that cisplatin increased the tissue TOC, ROOH levels and decreased TAC and thiol levels. NAC administration after cisplatin treatment was observed to have ameliorated histological and functional changes in heart. In conclusion, the results of this experimental study suggested that oxidative stress had a serious effect on cisplatin cardiotoxicity, and NAC could be used as a therapeutic agent in addition to standard cisplatin treatment protocols (Tab. 3, Fig. 1, Ref. 35).

Cyanidin ameliorates endotoxin-induced myocardial toxicity by modulating inflammation and oxidative stress through mitochondria and other factors

Cyanidin, an anthocyanin pigment, demonstrates anti-oxidant and anti-inflammatory properties. Here, we examined the mechanistic role of cyanidin in endotoxin induced myocardial injury in inflammation and oxidative stress. In lipopolysaccharide (LPS) induced myocardial injury model, cyanidin ameliorated cardiac injury (Lactate dehydrogenase or LDH, Creatine Kinase or CK, cardiac troponin I or cTnI and cardiac myosin light chains 1 or cMLC1), cell death (caspase 3 activity and PARP activity), and improved cardiac function (ejection fraction or EF and end diastolic left ventricular inner dimension or LVID). Cyanidin also attenuated endotoxin induced myocardial injury by modulating inflammatory cytokines (Tumor necrosis factor alpha or TNFα, Interleukin-1 beta or IL-1β, macrophage inflammatory protein 2 or MIP-2 and chemokine (C-C motif) ligand 2 also known as monocyte chemoattractant protein 1 or MCP1) and oxidative stress (protein nitration). Cyanidin modulated redox homeostasis through intracellular oxidized/reduced glutathione. The most striking properties of cyanidin in endotoxin induced mediated myocardial injury was the modulation of mitochondria, its oxidative damage and associated factor Opa1 and Trx1. Thus, our study demonstrated that cyanidin as a constituent of our food chain may be beneficial and has therapeutic potential in sepsis treatment or other myocardial oxidative and/or inflammation induced injuries.

Identification of MYLK3 mutations in familial dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a primary cause of heart failure, life-threatening arrhythmias, and cardiac death. Pathogenic mutations have been identified at the loci of more than 50 genes in approximately 50% of DCM cases, while the etiologies of the remainder have yet to be determined. In this study, we applied whole exome sequencing in combination with segregation analysis to one pedigree with familial DCM, and identified a read-through mutation (c.2459 A > C; p.*820Sext*19) in the myosin light chain kinase 3 gene (MYLK3). We then conducted MYLK3 gene screening of 15 DCM patients (7 familial and 8 sporadic) who were negative for mutation screening of the previously-reported cardiomyopathy-causing genes, and identified another case with a MYLK3 frameshift mutation (c.1879_1885del; p.L627fs*41). In vitro experiments and immunohistochemistry suggested that the MYLK3 mutations identified in this study result in markedly reduced levels of protein expression and myosin light chain 2 phosphorylation. This is the first report that MYLK3 mutations can cause DCM in humans. The clinical phenotypes of DCM patients were consistent with MYLK3 loss-of-function mouse and zebrafish models in which cardiac enlargement and heart failure are observed. Our findings highlight an essential role for cardiac myosin light chain kinase in the human heart.

Changes in serum cardiac myosin light chain 1 levels in children with fulminant myocarditis during continuous blood purification.

To investigate the changes in serum cardiac myosin light chain 1 (CMLC-1) levels in children with fulminant myocarditis (FM) during continuous blood purification (CBP), as well as to analyze its correlation with other laboratory indexes. Twenty-four (24) children with FM who underwent CBP were enrolled. Before and during treatment (48 and 72 hours after treatment, or death), the optical density value of serum CMLC-1 was measured using enzyme-linked immunosorbent assay, and then the serum CMLC-1 concentration was calculated. The correlations between CMLC-1 OD value change and laboratory indexes including creatine kinase-MB (CK-MB), troponin, myohemoglobin and N-terminal pro-brain natriuretic peptide (NT-proBNP) were analyzed. The serum CMLC-1 concentration significantly increased in the children with FM and decreased obviously during CBP therapy. In the same period, the change of CMLC-1 concentration were positively correlated with creatine kinase-MB (r=0.528), troponin (r=0.726), myohemoglobin (r=0.702), and NT-proBNP levels (r=0.589). The serum CMLC-1 concentration increases significantly in children with FM, but CBP therapy can effectively control this increase.

Apigenin Alleviates Endotoxin-Induced Myocardial Toxicity by Modulating Inflammation, Oxidative Stress, and Autophagy.

Apigenin, a component in daily diets, demonstrates antioxidant and anti-inflammatory properties. Here, we intended to explore the mechanism of apigenin-mediated endotoxin-induced myocardial injury and its role in the interplay among inflammation, oxidative stress, and autophagy. In our lipopolysaccharide- (LPS-) induced myocardial injury model, apigenin ameliorated cardiac injury (lactate dehydrogenase (LDH) and creatine kinase (CK)), cell death (TUNEL staining, DNA fragmentation, and PARP activity), and tissue damage (cardiac troponin I (cTnI) and cardiac myosin light chain-1 (cMLC1)) and improved cardiac function (ejection fraction (EF) and end diastolic left ventricular inner dimension (LVID)). Apigenin also alleviated endotoxin-induced myocardial injury by modulating oxidative stress (nitrotyrosine and protein carbonyl) and inflammatory cytokines (TNF-α, IL-1β, MIP-1α, and MIP-2) along with their master regulator NFκB. Apigenin modulated redox homeostasis, and its anti-inflammatory role might be associated with its ability to control autophagy. Autophagy (determined by LAMP1, ATG5, and p62), its transcriptional regulator transcription factor EB (TFEB), and downstream target genes including vacuolar protein sorting-associated protein 11 (Vps11) and microtubule-associated proteins 1A/1B light chain 3B (Map1lc3) were modulated by apigenin. Thus, our study demonstrated that apigenin may lead to potential development of new target in sepsis treatment or other myocardial oxidative and/or inflammation-induced injuries.

Heart Failure Induced by Perinatal Ablation of Cardiac Myosin Light Chain Kinase.

Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK) causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7) as opposed to 31.4 ± 1.0% (n = 11) in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12) relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11) and 5.81 ± 0.33 (wild/wild/Cre, n = 5), accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320) as opposed to 87.1 ± 1.6 μm (n = 360) in the controls.Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing from the compensatory hypertrophy shown in the germline knockout, and the cardomyocyte thinning shown in adult-inducible knockout.

Role of myosin light chain phosphatase in cardiac physiology and pathophysiology

Maintenance of contractile performance of the heart is achieved in part by the constitutive 40% phosphorylation of myosin regulatory light chain (RLC) in sarcomeres. The importance of this extent of RLC phosphorylation for optimal cardiac performance becomes apparent when various mouse models and resultant phenotypes are compared. The absence or attenuation of RLC phosphorylation results in poor performance leading to heart failure, whereas increased RLC phosphorylation is associated with cardiac protection from stresses. Although information is limited, RLC phosphorylation appears compromised in human heart failure which is consistent with data from mouse studies. The extent of cardiac RLC phosphorylation is determined by the balanced activities of cardiac myosin light chain kinases and phosphatases, the regulatory mechanisms of which are now emerging. This review thusly focuses on kinases that may participate in phosphorylating RLC to make the substrate for cardiac myosin light chain phosphatases, in addition to providing perspectives on the family of myosin light chain phosphatases and involved signaling mechanisms. Because biochemical and physiological information about cardiac myosin light chain phosphatase is sparse, such studies represent an emerging area of investigation in health and disease.

Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities

The well-known, muscle-specific smooth muscle myosin light chain kinase (MLCK) (smMLCK) and skeletal muscle MLCK (skMLCK) are dedicated protein kinases regulated by an autoregulatory segment C terminus of the catalytic core that blocks myosin regulatory light chain (RLC) binding and phosphorylation in the absence of Ca(2+)/calmodulin (CaM). Although it is known that a more recently discovered cardiac MLCK (cMLCK) is necessary for normal RLC phosphorylation in vivo and physiological cardiac performance, information on cMLCK biochemical properties are limited. We find that a fourth uncharacterized MLCK, MLCK4, is also expressed in cardiac muscle with high catalytic domain sequence similarity with other MLCKs but lacking an autoinhibitory segment. Its crystal structure shows the catalytic domain in its active conformation with a short C-terminal "pseudoregulatory helix" that cannot inhibit catalysis as a result of missing linker regions. MLCK4 has only Ca(2+)/CaM-independent activity with comparable Vmax and Km values for different RLCs. In contrast, the Vmax value of cMLCK is orders of magnitude lower than those of the other three MLCK family members, whereas its Km (RLC and ATP) and KCaM values are similar. In contrast to smMLCK and skMLCK, which lack activity in the absence of Ca(2+)/CaM, cMLCK has constitutive activity that is stimulated by Ca(2+)/CaM. Potential contributions of autoregulatory segment to cMLCK activity were analyzed with chimeras of skMLCK and cMLCK. The constitutive, low activity of cMLCK appears to be intrinsic to its catalytic core structure rather than an autoinhibitory segment. Thus, RLC phosphorylation in cardiac muscle may be regulated by two different protein kinases with distinct biochemical regulatory properties.

Effect of hydrogen sulfide on cardiac myosin light chain kinase expression in diabetic rats

To investigate the effect of hydrogen sulfide (H2S) on cardiac myosin light chain kinase (MLCK) expression in diabetic rats. A total of 32 male SD rats were randomly divided into a normal control group (NC group), a diabetic control group (DM), a NaHS treatment group (DM+NaHS) and a NaHS group (NaHS) (n=8 in each group). Intraperitoneal injection of streptozotocin was utilized to establish Type 1 diabetic rat model. The diabetic rats in the DM+NaHS and NaHS groups were intraperitoneally injected with 28 μmol/kg NaHS solution. Eight weeks later, the ventricular hemodynamic parameters, the ratio of heart weight/body weight (HW/BW ratio), the levels of lactate dehydrogenase (LDH) and creatine kinase MB isozyme (CK-MB) in serum were determined. The ultrastructures of myocardium were observed under electron microscopy. The expressions of MLCK mRNA and protein level in myocardium were detected by RT-PCR and Western blot, respectively. Compared with the NC group, there was no significant difference in the various indexes in the NaHS group (all P>0.05). The function of left ventricular contract and relaxation were decreased obviously in diabetic rats, while the HW/BW ratio was increased (all P<0.01). The levels of LDH and CK-MB were increased (both P<0.01) in serum, while the levels of MLCK mRNA and protein were decreased significantly (both P<0.01) in myocardial tissues. Compared with the DM group, the left ventricular hemodynamic parameters and myocardial ultrastructure damage were improved in the DM+NaHS group, while the HW/BW ratio was decreased (all P<0.05). The levels of LDH and CK-MB were decreased (both P<0.01), while the levels of MLCK mRNA and protein were increased significantly (both P<0.01). H2S can protect myocardium in diabetic rats, which may be associated with upregulation of cardiac MLCK.

Acute heart failure with cardiomyocyte atrophy induced in adult mice by ablation of cardiac myosin light chain kinase.

Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. To mimic acute cMLCK reduction in adult hearts, the floxed-Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3(flox/flox)/merCremer mice (Mylk3-KO), and compared with two control mice (Mylk3(flox/flox) and Mylk3(+/+)/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization.

Non-neural and cardiac differentiating properties of Tbx6-expressing mouse embryonic stem cells

T-box transcription factors play important roles in vertebrate mesoderm formation. Eomesodermin is involved in the initial step of the prospective mesodermal cells recruited near the primitive streak. Then T or Brachyury gene is responsible for general and axial mesodermal development. Tbx6, on the other hand, promotes paraxial mesodermal development while suppressing neural differentiation. Here, we studied differentiative properties of mouse ES cells (mESCs) with its Tbx6 expression regulated under the Tet-off system. mESCs were treated with noggin to promote neural differentiation. When Tbx6 was simultaneously turned on, later neural differentiation of these cells hardly occurred. Next, mESCs were subjected to formation of the embryoid bodies (EBs). When Tbx6 was turned on during EB formation, the rate of later cardiac troponin T (cTnT)-positive cells increased. If the cells were further treated with a wnt inhibitor KY02111 after EB formation, a synergistic increase of cTnT-positive cells occurred. Tbx6 expression in mESCs influenced the constituent ratio of the cardiac myosin light chain types, such that atrial species markedly increased over ventricular ones. These results are coincident with the function of Tbx6 in normal development, in that Tbx6 strongly suppressed neural differentiation while promoting cardiac development in a cooperative manner with wnt inhibition.

Role of TFEB Mediated Autophagy, Oxidative Stress, Inflammation, and Cell Death in Endotoxin Induced Myocardial Toxicity of Young and Aged Mice

Elderly patients are susceptible to sepsis. LPS induced myocardial injury is a widely used animal model to assess sepsis induced cardiac dysfunction. The age dependent mechanisms behind sepsis susceptibility were not studied. We analyzed age associated changes to cardiac function, cell death, inflammation, oxidative stress, and autophagy in LPS induced myocardial injury. Both young and aged C57BL/6 mice were used for LPS administration. The results demonstrated that LPS induced more cardiac injury (creatine kinase, lactate dehydrogenase, troponin I, and cardiac myosin-light chains 1), cardiac dysfunction (left ventricular inner dimension, LVID, and ejection fraction (EF)), cell death, inflammation, and oxidative stress in aged mice compared to young mice. However, a significant age dependent decline in autophagy was observed. Translocation of Transcription Factor EB (TFEB) to nucleus and formation of LC3-II were significantly reduced in LPS administered aged mice compared to young ones. In addition to that, downstream effector of TFEB, LAMP-1, was induced in response to LPS challenge in young mice. The present study newly demonstrates that TFEB mediated autophagy is crucial for protection against LPS induced myocardial injury particularly in aging senescent heart. Targeting this autophagy-oxidative stress-inflammation-cell death axis may provide a novel therapeutic strategy for cardioprotection in the elderly.

New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response.

Background & AimsCardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.Methods and ResultsBy Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15–30%.ConclusionsWe identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.

Hypertrophic remodelling in cardiac regulatory myosin light chain (MYL2) founder mutation carriers.

Phenotypic heterogeneity and incomplete penetrance are common in patients with hypertrophic cardiomyopathy (HCM). We aim to improve the understanding in genotype-phenotype correlations in HCM, particularly the contribution of an MYL2 founder mutation and risk factors to left ventricular hypertrophic remodelling. We analysed 14 HCM families of whom 38 family members share the MYL2 c.64G > A [p.(Glu22Lys)] mutation and a common founder haplotype. In this unique cohort, we investigated factors influencing phenotypic outcome in addition to the primary mutation. The mutation alone showed benign disease manifestation with low penetrance. The co-presence of additional risk factors for hypertrophy such as hypertension, obesity, or other sarcomeric gene mutation increased disease penetrance substantially and caused HCM in 89% of MYL2 mutation carriers (P = 0.0005). The most prominent risk factor was hypertension, observed in 71% of mutation carriers with HCM and an additional risk factor. The MYL2 mutation c.64G > A on its own is incapable of triggering clinical HCM in most carriers. However, the presence of an additional risk factor for hypertrophy, particularly hypertension, adds to the development of HCM. Early diagnosis of risk factors is important for early treatment of MYL2 mutation carriers and close monitoring should be guaranteed in this case. Our findings also suggest that the presence of hypertension or another risk factor for hypertrophy should not be an exclusion criterion for genetic studies.

Regular exercise improves cardiac contractile activation by modulating MHC isoforms and SERCA activity in orchidectomized rats.

Data from the trial known as Testosterone in Older Men with Mobility Limitations (TOM) has indicated an association between testosterone administration and a greater risk for adverse cardiovascular events. We therefore propose that regular exercise is a cardioprotective alternative that prevents detrimental changes in contractile activation when a deficiency in male sex hormones exists. Ten-week-old orchidectomized (ORX) rats were subjected to a 9-wk treadmill running program at moderate intensity starting 1 wk after surgery. Although exercise-induced cardiac hypertrophy was observed both in rats that underwent ORX and sham surgery, regular exercise enhanced cardiac myofilament Ca(2+) sensitivity and myosin light-chain 2 phosphorylation only in rats that underwent a sham operation. Although the rats that had sham surgery and and given exercise exhibited no change in maximum developed tension, regular running prevented the suppression of maximum active tension in the hearts of ORX rats. Regular exercise also prevented a shift in myosin heavy chain (MHC) isoforms toward β-MHC, a reduction in sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) activity, and an increase in SERCA sensitivity in the hearts of ORX rats. Neither SERCA content nor its modulating component, phospholamban (PLB), was altered by exercise in either sham-operated or ORX rats. However, decreases in the phosphorylated Thr(17) form of PLB and the phosphorylated Thr(287) form of Ca(2+)/calmodulin-dependent kinase II in the hearts of ORX rats were abolished after regular exercise. These results thus support the use of regular running as a cardioprotective alternative to testosterone replacement in hypogonadal conditions.

Phosphorylation of myosin regulatory light chain controls myosin head conformation in cardiac muscle

The effect of phosphorylation on the conformation of the regulatory light chain (cRLC) region of myosin in ventricular trabeculae from rat heart was determined by polarized fluorescence from thiophosphorylated cRLCs labelled with bifunctional sulforhodamine (BSR). Less than 5% of cRLCs were endogenously phosphorylated in this preparation, and similarly low values of basal cRLC phosphorylation were measured in fresh intact ventricle from both rat and mouse hearts. BSR-labelled cRLCs were thiophosphorylated by a recombinant fragment of human cardiac myosin light chain kinase, which was shown to phosphorylate cRLCs specifically at serine 15 in a calcium- and calmodulin-dependent manner, both in vitro and in situ. The BSR-cRLCs were exchanged into demembranated trabeculae, and polarized fluorescence intensities measured for each BSR-cRLC in relaxation, active isometric contraction and rigor were combined with RLC crystal structures to calculate the orientation distribution of the C-lobe of the cRLC in each state. Only two of the four C-lobe orientation populations seen during relaxation and active isometric contraction in the unphosphorylated state were present after cRLC phosphorylation. Thus cRLC phosphorylation alters the equilibrium between defined conformations of the cRLC regions of the myosin heads, rather than simply disordering the heads as assumed previously. cRLC phosphorylation also changes the orientation of the cRLC C-lobe in rigor conditions, showing that the orientation of this part of the myosin head is determined by its interaction with the thick filament even when the head is strongly bound to actin. These results suggest that cRLC phosphorylation controls the contractility of the heart by modulating the interaction of the cRLC region of the myosin heads with the thick filament backbone.

Cardiac myosin light chain phosphorylation and inotropic effects of a biased ligand, TRV120023, in a dilated cardiomyopathy model

Therapeutic approaches to treat familial dilated cardiomyopathy (DCM), which is characterized by depressed sarcomeric tension and susceptibility to Ca(2+)-related arrhythmias, have been generally unsuccessful. Our objective in the present work was to determine the effect of the angiotensin II type 1 receptor (AT1R) biased ligand, TRV120023, on contractility of hearts of a transgenic mouse model of familial DCM with mutation in tropomyosin at position 54 (TG-E54K). Our rationale is based on previous studies, which have supported the hypothesis that biased G-protein-coupled receptor ligands, signalling via β-arrestin, increase cardiac contractility with no effect on Ca(2+) transients. Our previous work demonstrated that the biased ligand TRV120023 is able to block angiotensin-induced hypertrophy, while promoting an increase in sarcomere Ca(2+) response. We tested the hypothesis that the depression in cardiac function associated with DCM can be offset by infusion of the AT1R biased ligand, TRV120023. We intravenously infused saline, TRV120023, or the unbiased ligand, losartan, for 15 min in TG-E54K and non-transgenic mice to obtain left ventricular pressure-volume relations. Hearts were analysed for sarcomeric protein phosphorylation. Results showed that the AT1R biased ligand increases cardiac performance in TG-E54K mice in association with increased myosin light chain-2 phosphorylation. Treatment of mice with an AT1R biased ligand, acting via β-arrestin signalling, is able to induce an increase in cardiac contractility associated with an increase in ventricular myosin light chain-2 phosphorylation. AT1R biased ligands may prove to be a novel inotropic approach in familial DCM.