Cardiac Myosin Light Chain Kinase Research Articles

Constitutive Phosphorylation of Myosin Regulatory Light Chain (RLC) in vivo is Maintained by Low Kinase and Phosphatase Activities

The importance of RLC phosphorylation in enhancing cardiac myofibrillar contraction is well-known. In anesthetized mice the extent of RLC phosphorylation (45%) was not changed by changes in sympathetic tone with prolonged infusion of the beta-agonist dobutamine or treatment with the beta-blocker propranolol. The goal of this study was to determine if the constitutive RLC phosphorylation in vivo was limited to half of the RLC due to: a) negative cooperativity for phosphorylation of two heads in myosin; or b) due to a steric constraint in myofibrils blocking access of soluble cardiac myosin light chain kinase (cMLCK) to RLC. We measured the kinetic properties of RLC phosphorylation in native myosin filaments and myofibrils. Results showed that RLC was phosphorylated by a pseudo-first order rate in both preparations with maximal phosphorylation over 90%. Thus, RLC for each head in myosin was readily available for phosphorylation. Pacing trabeculae at 1.5 Hz for 30 minutes increased RLC phosphorylation from 20±1 % to 43±3 %. Consistent with biochemical results, RLC phosphorylation increased to 91±3 % when myosin light chain phosphatase activity was inhibited with calyculin A while pacing. Together, these results exclude negative cooperativity and steric blocking as mechanisms limiting RLC phosphorylation. We determined that the heart has a high cMLCK content (2.4±0.1 µM) compared to the MLCK present in fast skeletal muscle (0.5±0.03 µM), but similar to the MLCK content in smooth muscle (3.4±0.2 µM). However cMLCK has a low specific activity compared to the other MLCKs. In conclusion, the extent of RLC phosphorylation in a normally beating heart is limited by cMLCK with its low activity in balance with low myosin light chain phosphatase activity. RLC phosphorylation is insensitive to sympathetic activation or inhibition in vivo.

Abstract 17911: Acute Cardiac Dysfunction by Adult-onset Cardiac Myosin Light Chain Kinase Ablation

Introduction: Under sustained pressure overload, myocytes that initially remodel to become short and thick, eventually become elongated and maladaptive with little force for contraction. Mechanisms underlying this transition are largely unknown. We previously demonstrated that 1 week of transverse aortic constriction in wild-type mice reduced cardiac myosin light chain kinase (cMLCK) levels by ~85%. In cultured cardiomyocytes, cMLCK knockdown led to sarcomere disorganization. Germline Mylk3 (encoding cMLCK) knockout mice demonstrated moderate heart failure. Hypothesis: Acute cMLCK reduction is causative for reduced contractility and remodeling of cardiomyocytes during the transition from compensated to decompensated hypertrophy. Methods: To mimic acute cMLCK reduction, adult inducible Mylk3 gene knockout mice, Mylk3flox/flox/merCremer, were generated and compared to 2 control mice (Mylk3flox/flox and Mylk3+/+/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). We assessed cardiac function (MRI, echocardiogram) in addition to histopathologic (tissue sectioning, immunostaining, and EM), cellular (cell size and cell shortening and intracellular free Ca2+) and molecular analyses. Results: Reduction of cMLCK proteins was evident at day 4 following tamoxifen-injection in inducible Mylk3 knockout mice. At day 7, inducible Mylk3 knockout but not control mice demonstrated heart failure [%FS, 28.0% in flox/flox (n=6), 27.7% in +/+/merCremer (n=6), 19.8% in flox/flox/merCremer (n=8)]. Analysis of inducible Mylk3 knockout mice revealed unique, severely convoluted cell morphology; isolated cardiomyocytes were elongated and thinner with disorganized sarcomere and reduced contractility (%FS in sarcomere length, control 5.1% vs. inducible knockout 2.4%). Adenovirus encoding cMLCK enhanced organized sarcomere structures; this was not prevented by co-infection of adenovirus unphosphorylatable MLC2v(Ser14/15Ala) mutants incorporated into sarcomere. Conclusion: Results of acute cMLCK reduction in a novel mouse model suggest that cMLCK plays a pivotal role in transition from compensated to decompensated hypertrophy via sarcomere disorganization likely independent of MLC2 phosphorylation.

Degradation of cardiac myosin light chain kinase by matrix metalloproteinase-2 contributes to myocardial contractile dysfunction during ischemia/reperfusion

Although ischemia/reperfusion (I/R)-induced myocardial contractile dysfunction is associated with a prominent decrease in myofilament Ca2+ sensitivity, the underlying mechanisms have not yet been fully clarified. Phosphorylation of ventricular myosin light chain 2 (MLC-2v) facilitates actin–myosin interactions and enhances contractility, however, its level and regulation by cardiac MLC kinase (cMLCK) and cMLC phosphatase (cMLCP) in I/R hearts are debatable. In this study, the levels and/or effects of MLC-2v phosphorylation, cMLCK, cMLCP, and proteases during I/R were determined. Global myocardial I/R-suppressed cardiac performance in isolated rat hearts was concomitant with decreases of MLC-2v phosphorylation, myofibrillar Ca2+-stimulated ATPase activity, and cMLCK content, but not cMLCP proteins. Consistently, simulated I/R in isolated cardiomyocytes inhibited cell shortening, Ca2+ transients, MLC-2v phosphorylation, and myofilament sensitivity to Ca2+. These observations were reversed by cMLCK overexpression, while the specific cMLCK knockdown by short hairpin RNA (shRNA) had the opposite effect. Moreover, the inhibition of matrix metalloproteinase-2 (MMP-2, a zinc-dependent endopeptidase) reversed IR-decreased cMLCK, MLC-2v phosphorylation, myofibrillar Ca2+-stimulated ATPase activity, myocardial contractile function, and myofilament sensitivity to Ca2+, while the inhibition or knockdown of cMLCK by ML-9 or specific shRNA abolished MMP-2 inhibition-induced cardioprotection. Finally, the co-localization in cardiomyocytes and interaction in vivo of MMP-2 and cMLCK were observed. Purified recombinant rat cMLCK was concentration- and time-dependently degraded by rat MMP-2 in vitro, and this was prevented by the inhibition of MMP-2. These findings reveal that the I/R-activated MMP-2 leads to the degradation of cMLCK, resulting in a reduction of MLC-2v phosphorylation, and myofibrillar Ca2+-stimulated ATPase activity, which subsequently suppresses myocardial contractile function through a decrease of myofilament Ca2+ sensitivity.

Protective role of neuregulin-1 toward doxorubicin-induced myocardial toxicity.

The aim of this study was to investigate the role of the rat neuregulin-1 (NRG-1) protein in reducing doxorubicin (DOX)-induced myocardial toxicity and its underlying mechanism. The prokaryotic expression of the NRG-1 protein and the CCK8-determined activity of rat primary myocardial cells were evaluated under different DOX concentrations. Myocardial cells were divided into three groups: the control group, the 5 μM DOX (DOX5) group, and the DOX5+NRG-1 group. Western blotting was used to determine the Na+-Ca2+ exchanger (NCX-1) and cardiac myosin light-chain kinase (cMLCK) protein expression levels and real-time quantitative polymerase chain reaction methods were used to determine the mRNA expression levels. The prokaryotic expression of NRG-1 in the DOX5 group produced toxicity in the rat myocardial cells, and cell activity was significantly restored with the addition of NRG-1. The protective effect of NRG-1 was limited at higher DOX concentrations (DOX10), and the degree of cellular activity restoration was positively correlated with NRG-1 concentration. The addition of NRG-1 to DOX5 intervention inhibited NCX-1 protein and mRNA expression, and increased cMLCK protein and mRNA expression. In conclusion, DOX-induced toxicity in rat myocardial cells could be protected by NRG-1, and the mechanism may be related to the role of NRG-1 in up-regulating the cMLCK expression level and down-regulating the NCX-1 expression level.

The Effects of Neuregulin on Cardiac Myosin Light Chain Kinase Gene-Ablated Hearts

BackgroundActivation of ErbB2/4 receptor tyrosine kinases in cardiomyocytes by neuregulin treatment is associated with improvement in cardiac function, supporting its use in human patients with heart failure despite the lack of a specific mechanism. Neuregulin infusion in rodents increases cardiac myosin light chain kinase (cMLCK) expression and cardiac myosin regulatory light chain (RLC) phosphorylation which may improve actin-myosin interactions for contraction. We generated a cMLCK knockout mouse to test the hypothesis that cMLCK is necessary for neuregulin-induced improvement in cardiac function by increasing RLC phosphorylation.Principal FindingsThe cMLCK knockout mice have attenuated RLC phosphorylation and decreased cardiac performance measured as fractional shortening. Neuregulin infusion for seven days in wildtype mice increased cardiac cMLCK protein expression and RLC phosphorylation while increasing Akt phosphorylation and decreasing phospholamban phosphorylation. There was no change in fractional shortening. In contrast, neuregulin infusion in cMLCK knockout animals increased cardiac performance in the absence of cMLCK without increasing RLC phosphorylation. In addition, CaMKII signaling appeared to be enhanced in neuregulin-treated knockout mice.ConclusionsThus, Neuregulin may improve cardiac performance in the failing heart without increasing cMLCK and RLC phosphorylation by activating other signaling pathways.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Rationale: Alterations to the cardiac regulatory light chain (RLC) phosphorylation state correlate with many types of cardiac disease including familial hypertrophic cardiomyopathies (FHC's) and heart failure (HF) post myocardial infarction (MI). However, little is known regarding the role of RLC phosphorylation in regulating cardiac contraction mechanics during shortening. Objective: Determine the mechanical effect of altered cRLC phosphorylation on the force-velocity characteristics of cardiac muscle. Methods and Results: SDS-PAGE electrophoresis determined the effects of heart failure on cRLC phosphorylation on human heart failure (HF) samples and a rat chronic-MI model. We report an increase in RLC phosphorylation in HF progression. Recombinant cRLC was cloned and expressed. Recombinant RLC was differentially phosphorylated in-vitro using cardiac myosin light chain kinase (cMLCK) and ZIP kinase. Recombinant RLC species were exchanged into permeabilised cardiac trabeculae to perform mechanical measurements during shortening (slack test and force-velocity experimentation protocols). We observed a decrease in peak power, velocity at peak power (VPP) and peak isometric force of contraction when reducing RLC phosphorylation. Conversely, enriching RLC phosphorylation increased peak power, VPP and peak maximal unloaded shortening velocity (VMAX) in comparison to control. Conclusions: Altering cRLC phosphorylation has a significant impact on the mechanical function of cardiac muscle and is dramatically altered during HF progression. These results highlight the importance of understanding thick filament regulation and specifically the ability of cRLC phosphorylation to regulate cardiac muscle function.

Myosin Light Chain Phosphorylation Is Critical for Adaptation to Cardiac Stress

Cardiac hypertrophy is a common response to circulatory or neurohumoral stressors as a mechanism to augment contractility. When the heart is under sustained stress, the hypertrophic response can evolve into decompensated heart failure, although the mechanism(s) underlying this transition remain largely unknown. Because phosphorylation of cardiac myosin light chain 2 (MLC2v), bound to myosin at the head-rod junction, facilitates actin-myosin interactions and enhances contractility, we hypothesized that phosphorylation of MLC2v plays a role in the adaptation of the heart to stress. We previously identified an enzyme that predominantly phosphorylates MLC2v in cardiomyocytes, cardiac myosin light-chain kinase (cMLCK), yet the role(s) played by cMLCK in regulating cardiac function in health and disease remain to be determined. We found that pressure overload induced by transaortic constriction in wild-type mice reduced phosphorylated MLC2v levels by ≈40% and cMLCK levels by ≈85%. To examine how a reduction in cMLCK and the corresponding reduction in phosphorylated MLC2v affect function, we generated Mylk3 gene-targeted mice and transgenic mice overexpressing cMLCK specifically in cardiomyocytes. Pressure overload led to severe heart failure in cMLCK knockout mice but not in mice with cMLCK overexpression in which cMLCK protein synthesis exceeded degradation. The reduction in cMLCK protein during pressure overload was attenuated by inhibition of ubiquitin-proteasome protein degradation systems. Our results suggest the novel idea that accelerated cMLCK protein turnover by the ubiquitin-proteasome system underlies the transition from compensated hypertrophy to decompensated heart failure as a result of reduced phosphorylation of MLC2v.

Dietary genistein enhances phosphorylation of regulatory myosin light chain in the myocardium of ovariectomized mice

There is evidence that isoflavones, such as genistein, can directly or indirectly improve lipid profile and lower blood pressure and hence exert cardiovascular protection. It is further believed, that genistein attenuates vascular contraction and thus vascular tone and blood pressure through altering the phosphorylation of the regulatory myosin light chain (MLC) probably via the myosin light chain kinase (MLCK) or the RhoA pathway. However, the direct role of genistein in the myocardium is poorly reviewed. In this study, we investigated the impact of genistein on the cardiac proteome in ovariectomized female mice using a 2DE-MS approach. Dietary genistein intake considerably changed the abundance of several cytoskeletal and contractile proteins and enhanced the phosphorylation of MLC. The MLC phosphorylation was mediated through increased abundance of MLCK and inhibition of myosin light chain phosphatase latest known to be inversely regulated by RhoA. Contrary to others, in our model genistein did neither inhibit the cardiac MLCK, nor the cardiac RhoA pathway in vivo.

Overexpression of microRNA-1 impairs cardiac contractile function by damaging sarcomere assembly

The purpose of the present study was to evaluate the effects of overexpression of microRNA-1 (miR-1) on cardiac contractile function and the potential molecular mechanisms. Transgenic (Tg) mice (C57BL/6) for cardiac-specific overexpression of miR-1 driven by the α-myosin heavy chain promoter were generated and identified by real-time reverse-transcription polymerase chain reaction with left ventricular samples. We found an age-dependent decrease in the heart function in Tg mice by pressure-volume loop analysis. Histological analysis and electron microscopy displayed short sarcomeres with the loss of the clear zone and H-zone as well as myofibril fragmentation and deliquescence in Tg mice. Further studies demonstrated miR-1 post-transcriptionally down-regulated the expression of calmodulin (CaM) and cardiac myosin light chain kinase (cMLCK) proteins by targeting the 3'UTRs of MYLK3, CALM1, and CALM2 genes, leading to decreased phosphorylations of myosin light chain 2v (MLC2v) and cardiac myosin binding protein-C (cMyBP-C). Knockdown of miR-1 by locked nucleic acid-modified anti-miR-1 antisense (LNA-antimiR-1) mitigated the adverse changes of cardiac function associated with overexpression of miR-1. miR-1 induces adverse structural remodelling to impair cardiac contractile function. Targeting cMLCK and CaM likely underlies the detrimental effects of miR-1 on structural components of muscles related to the contractile machinery. Our study provides the first evidence that miRNAs cause adverse structural remodelling of the heart.

Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation

Human congenital heart disease linked to mutations in the homeobox transcription factor, NKX2-5, is characterized by cardiac anomalies, including atrial and ventricular septal defects as well as conduction and occasional defects in contractility. In the mouse, homozygous germline deletion of Nkx2-5 gene results in death around E10.5. It is, however, not established whether Nkx2-5 is necessary for cardiac development beyond this embryonic stage. Because human NKX2-5 mutations are related to septum secundum type atrial septal defects (ASD), we hypothesized that Nkx2-5 deficiency during the processes of septum secundum formation may cause cardiac anomalies; thus, we analysed mice with tamoxifen-inducible Nkx2-5 ablation beginning at E12.5 when the septum secundum starts to develop. Using tamoxifen-inducible Nkx2-5 gene-targeted mice, this study demonstrates that Nkx2-5 ablation beginning at E12.5 results in embryonic death by E17.5. Analysis of mutant embryos at E16.5 shows arrhythmias, contraction defects, and cardiac malformations, including ASD. Quantitative measurements using serial section histology and three-dimensional reconstruction demonstrate growth retardation of the septum secundum and enlarged foramen ovale in Nkx2-5-ablated embryos. Functional cardiac defects may be attributed to abnormal expression of transcripts critical for conduction and contraction, including cardiac voltage-gated Na(+) channel pore-forming α-subunit (Na(v)1.5-α), gap junction protein connexin40, cardiac myosin light chain kinase, and sarcolipin within 4 days after tamoxifen injection. Nkx2-5 is necessary for survival after the mid-embryonic stage for cardiac function and formation by regulating the expression of its downstream target genes.

Cardiac Myosin Light Chain Kinase is Essential for Myosin Regulatory Light Chain Phosphorylation and Normal Cardiac Function in vivo

In contrast to studies on skeletal and smooth muscles, protein kinases that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) in the heart are not known. Expression of a Ca2+/calmodulin-activated myosin light chain kinase found only in cardiac muscle (cMLCK) was ablated in mice. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Lack of cMLCK and RLC phosphorylation led to (1) ventricular hypertrophy demonstrated by increased heart weight to tibial length ratios, (2) myocyte hypertrophy and (3) histological evidence of necrosis and fibrosis. Loss of MLC2v phosphorylation led to compromised cardiac function evidenced by echocardiography showing a proportional decrease in systolic performance assessed as percent fractional shortening from 71% in wildtype mice to 34% for hearts with no cMLCK. Declines in cardiac function were associated with ventricular dilation and progressive increases in left ventricular end-systolic and end-diastolic dimensions. Hearts from female mice showed similar responses to loss of cMLCK including diminished RLC phosphorylation and significant ventricular hypertrophy. Prolonged isoproterenol infusion elicited hypertrophic cardiac responses in wildtype mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation which is required for normal physiological cardiac performance in vivo. Supported by NIH NHLBI.

Cardiac Myosin Light Chain Kinase Is Necessary for Myosin Regulatory Light Chain Phosphorylation and Cardiac Performance in Vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.

Cardiac functional improvement in rats with myocardial infarction by up-regulating cardiac myosin light chain kinase with neuregulin

Recombinant human neuregulin-1 (rhNRG-1) improves cardiac function in experimental heart failure models, but the underlying mechanism remains largely unknown. In this study, we evaluated whether rhNRG-1 could improve cardiac function via the cardiac myosin light chain kinase/myosin light chain 2 ventricular (cMLCK/MLC-2v) pathway in rats with myocardial infarction (MI). Rats with MI were intravenously infused with rhNRG-1 (5 µg/kg/h) for 7 days through osmotic pumps. The mechanism of action of rhNRG-1 was investigated by assaying the non-infarcted myocardium with gene chips. The cMLCK expression, phosphorylated MLC-2v and cardiac function were significantly up-regulated, as assessed by real-time PCR, Western blot and echocardiography, in those animals treated with rhNRG-1. Moreover, the restoration of rhNRG-1-induced sarcomeric organization in serum-free cultured neonatal rat cardiomyocytes with rhNRG-1 was inhibited by cMLCK RNA interference or ML-7, an inhibitor of MLCKs. Adenovirus containing the rat cMLCK coding region was injected into non-infarcted myocardium, and cardiac function was monitored using echocardiography and a haemodynamic machine. The dP/dt and fractional shortening decreasing significantly after MI, and improved by 15.7 and 32.1%, respectively, following local cMLCK application (all P < 0.05). Our results suggest that cMLCK is a downstream effector of rhNRG-1 involved in rhNRG-1-induced cardiac function improvement, and that myocardial cMLCK up-regulation can improve cardiac function in rats with MI.

Global Gene Expression Profiling in the Failing Myocardium

Heart failure (HF) is a syndrome that involves multiple cellular mechanisms leading to a common phenotype of reduced ventricular contraction and cardiac chamber dilation. To clarify the mechanisms, a number of microarray analyses of the failing myocardium have been conducted. Gene expression profiles are usually compared between opposing pairs of samples, such as non-failing vs failing hearts, ischemic vs non-ischemic hearts, male vs female failing hearts or atria vs ventricles of failing hearts. Apart from these conventional methods, a different novel approach identified cardiac myosin light chain kinase (MLCK) as a HF-related gene by the comprehensive search for the genes that had an expression level that strongly correlated with the severity of HF; further investigations proved the important role of cardiac MLCK in HF. Moreover, a robust gene expression signature composed of 27 genes was revealed on analysis of 4 independent microarray data sets from the failing myocardium of dilated cardiomyopathy. The authors newly demonstrate 107 HF-related genes that were listed in 2 or more of 7 microarray data sets previously reported. Among these genes, many were observed to be involved in mitochondrial dysfunction and oxidative phosphorylation and 3 extracellular molecules, including periostin, pleiotrophin, and SERPINA3, which might become novel diagnostic and therapeutic targets for HF. These novel strategies warrant the new identification of specific genes that are linked to the pathophysiology of HF.

Identification of Cardiac-Specific Myosin Light Chain Kinase

Two myosin light chain (MLC) kinase (MLCK) proteins, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCK, have been shown to be expressed in mammals. Even though phosphorylation of its putative substrate, MLC2, is recognized as a key regulator of cardiac contraction, a MLCK that is preferentially expressed in cardiac muscle has not yet been identified. In this study, we characterized a new kinase encoded by a gene homologous to mylk1 and -2, named cardiac MLCK, which is specifically expressed in the heart in both atrium and ventricle. In fact, expression of cardiac MLCK is highly regulated by the cardiac homeobox protein Nkx2-5 in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK; however, the amino terminus is quite unique, without significant homology to other known proteins, and its catalytic activity does not appear to be regulated by Ca(2+)/calmodulin in vitro. Cardiac MLCK is phosphorylated and the level of phosphorylation is increased by phenylephrine stimulation accompanied by increased level of MLC2v phosphorylation. Both overexpression and knockdown of cardiac MLCK in cultured cardiomyocytes revealed that cardiac MLCK is likely a new regulator of MLC2 phosphorylation, sarcomere organization, and cardiomyocyte contraction.

Abstract 1102: Cardiac-Specific Myosin Light Chain Kinase (MLCK), a Third Member of MLCK Family, Potentiates Sarcomere Formation and Cardiac Contraction

Background: Two myosin light chain kinase (MLCK) proteins, skeletal (encoded by mylk2 gene) and smooth muscle MLCK (encoded by mylk1 gene) have been shown to be expressed in mammals. Human mylk2 has been mapped as a disease locus for familial cardiac hypertrophy (OMIM 606566 ), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. While phosphorylation of the putative substrate of skeletal MLCK, myosin light chain 2 (MLC2), is recognized as a key regulator of cardiac contraction, the abundance of skeletal MLCK in the heart is controversial, suggesting the existence of an additional MLCK that is preferentially expressed in cardiac muscle. Methods and Results: We characterized a new kinase named cardiac MLCK that is encoded by a gene homologous to mylk1 and 2 and is specifically expressed in the heart in both atrium and ventricle. Expression of cardiac MLCK was highly regulated by the cardiac homeobox transcription factor, Nkx2.5, in neonatal cardiomyocytes. The overall structure of cardiac MLCK protein is conserved with skeletal and smooth muscle MLCK including putative catalytic and adjacent Ca2+/calmodulin binding domains at the carboxyl-terminus. The amino-terminus is unique without significant homology to other known proteins. Cardiac MLCK phosphorylated MLC2v with a catalytic value of Km=4.3 micro M (Lineweaver-Burk analysis) indicating high affinity of cardiac MLCK to MLC2v, similar to the affinity of skeletal muscle MLCK to skeletal muscle MLC2 and smooth muscle MLCK to smooth muscle MLC2. Adenoviral-mediated overexpression of cardiac MLCK and knockdown of cardiac MLCK using RNAi in cultured cardiomyocytes revealed that cardiac MLCK regulates MLC2v phosphorylation, sarcomere organization and cardiac myocyte contraction. Expression of cardiac MLCK protein was significantly decreased in severe heart failure in vivo (post-myocardial infarction heart failure mouse model). Conclusion: Cardiac MLCK is a new key regulator of cardiac contraction and sarcomere organization. Reduction of cardiac MLCK function leading to decreased phosphorylation of MLC2v may contribute to compromised contractile function in the failing heart.

Phosphorylation of Cardiac Myosin Light Chain 2 by Protein Kinase C and Myosin Light Chain Kinase Increases Ca 2+-Stimulated Actomyosin MgATPase Activity

Myosin light chain 2 (MLC2) phosphorylation in rat cardiac whole myosin by cardiac myosin light chain kinase (MLCK) or by protein kinase C (PKC) resulted in increased actin-stimulated myosin MgATPase activity. The phosphorylation also increased Ca 2+-stimulated myofibrillar MgATPase activity upon substitution of the phosphorylated myosin into myofibrils. In addition, phosphorylation of MLC2 in myofibrils by MLCK increased both the Ca 2+-sensitivity and maximum activity of the myofibrillar Ca 2+-stimulated MgATPase activity. The latter effect was inhibited by PKC-phosphorylation of troponin I, troponin T and C-protein. A role for both PKC and MLCK in regulating cardiac myofibrillar activity, via phosphorylation of various contractile proteins, is indicated.

Differential regulation of calcium-dependent and calcium-independent cyclic nucleotide phosphodiesterases from heart by palmitoylcarnitine and palmitoyl coenzyme A

Regulation of Ca 2+-dependent (peak I) and Ca 2+-independent (peak II) phosphodiesterases from the heart by various fatty acyl esters and phospholipids were studied. DL-Palmitoylcaritine stimulated the basal activity (in the absence of Ca 2+) of peak I enzyme, while non-competitively inhibiting peak II enzyme with respect to cyclic AMP. It had no effect on other species of Ca 2+-independent phosphodiesterases, including cyclic AMP- and cyclic GMP-specific enzymes from the lung, and cyclic CMP enzyme from the liver. Palmitoyl-CoA and phosphatidyl-serine also stimulated the basal activity of peak I enzyme, but they were without effect on peak II enzyme. In comparison, DL-palmitoylcarnitine inhibited Ca 2+-dependent activity of cardiac myosin light chain kinase, whereas phosphatidylserine was without effect. It is conceivable that differential regulation of phosphodiesterases by these lipids could profoundly alter the levels or effects, or both, of cyclic nucleotides and Ca 2+ in the myocardium.

Purification and characterization of bovine cardiac calmodulin-dependent myosin light chain kinase.

Myosin light chain kinase, which is located primarily in the soluble fraction of bovine myocardium, has been isolated and purified approximately 1200-fold with 16% yield by a three-step procedure. The approximate content of soluble myosin light chain kinase in heart is calculated to be 0.63 microM. The isolated kinase is active only as a ternary complex consisting of the kinase, calmodulin, and Ca2+; the apparent Kd for calmodulin is 1.3 nM. The enzyme also exhibits a requirement for Mg2+ ions. Myosin light chain kinase is a monomeric enzyme with Mr = 85,000. The enzyme exhibits a Km for ATP of 175 microM, and a K0.5 for the regulatory light chain of cardiac myosin of 21 microM. The optimum pH is 8.1. Kinase activity is specific for the regulatory light chain of myosin. The specific activity of the isolated enzyme (30 nmol 32P/min/mg of protein) is considerably less than and corresponding values reported for the skeletal and smooth muscle light chain kinases. This is probably due to proteolysis during extraction of the myocardium, a phenomenon which has, as yet, proven impossible to eliminate. In contrast to the smooth muscle enzyme (Adelstein, R.S., Conti, M.A., Hathaway, D.R., and Klee, C.B. (1978) J. Biol. Chem. 253, 8347-8350), the cardiac kinase is not phosphorylated by the catalytic subunit of cAMP-dependent protein kinase.