doi: 10.1161/CIRCRESAHA.108.173005
© 2008 American Heart Association, Inc.
Editorials |
Cardiac Myosin Light Chain Kinase
A New Player in the Regulation of Myosin Light Chain in the Heart
From the Cardiovascular Research Institute (Y.I.), Departments of Cell Biology & Molecular Medicine and Department of Medicine (Cardiology), New Jersey Medical School-University of Medicine and Dentistry of New Jersey, Newark; and Cardiovascular Research Institute (Y.I., R.K.), Yokohama City University Graduate School of Medicine, Yokohama, Japan.
Correspondence to Yoshihiro Ishikawa, MD, PhD, Cardiovascular Research Institute, Departments of Medicine (Cardiology) and Cell Biology & Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 S Orange Ave, Newark, NJ 07103. E-mail ishikayo{at}umdnj.edu
See related article, pages 571–580
Key Words: myosin light chain kinase • cardiac subtype • cardiac function • regulatory myosin light chain
Muscle myosin, which is the highly conserved molecular motor, contains 1 pair of myosin heavy chains (MHCs) and 2 pairs of myosin light chains (MLCs), the latter of which are referred to as essential and regulatory light chains.1,2 It is well known that muscle myosin is regulated through the phosphorylation of regulatory MLC (MLC2). In smooth muscles, for example, phosphorylation of MLC2 by MLC kinase (MLCK) activates the actin-activated myosin ATPase, leading to muscular contraction. In skeletal muscles, in contrast, the actin-myosin interaction is regulated by the troponin–tropomyosin complex, and MLC2 only modestly regulates rate and magnitude of contractile force generation. Thus, the role of MLC2 and the significance of its phosphorylation by MLCK may differ among tissues.
It is well expected that MLC2 plays a major role in regulating cardiac function as well. Such importance of MLC2 has been suggested in human genetic studies. Mutations in MLC2 have been shown in numerous analyses to be well correlated with the occurrence of certain forms of hypertrophic cardiomyopathy.3 Such MLC2 is phosphorylated by MLCK, and, therefore, the expression and activity of this kinase must play an important role in the heart as well. In accordance with this concept, MLCK phosphorylation in the heart can also regulate muscle contractility by increasing the Ca2+ sensitivity of force and accelerating the stretch activation response,4 and the state of MLCK phosphorylation through the thickness of the ventricular walls indeed is not uniform, but a spatial gradient exists, and this gradient is a major determinant of the overall pattern of cardiac contraction.5,6
In vertebrates, 2 genes for MLCK have been identified: 1 for smooth muscle (mylk1) and the other for skeletal muscle MLCK (mylk2).7 The smooth muscle MLCK gene expresses 3 transcripts in a tissue-specific manner via alternate promoters.8 The short form (130 kDa) is expressed in smooth muscles but also in cardiac muscles at lower levels than those detected in smooth muscles. The long form (210 kDa) contains all of the short form in addition to an N-terminal extension but is not normally expressed in adult smooth muscles and is found in smooth muscle cells in culture, embryonic smooth muscles, and nonmuscle cells.9 Thus the long form has been referred to as an embryonic, nonmuscle, endothelial cell or the 210-kDa MLCK. The third transcript of the smooth muscle MLCK gene contains only the C-terminal immunoglobulin module, which results in the expression of the telokin protein in phasic smooth muscle tissues.10
Skeletal muscle MLCK is predominantly expressed in skeletal muscles but also detected in cardiac muscles6 although abundance of this MLCK in the heart may be controversial.11 Nevertheless, it was originally thought that skeletal muscle MLCK was involved in regulating cardiac MLC2 and thus cardiac phenotype. Despite such a prediction for many years, ablation of the skeletal muscle MLCK gene in mice in a recent study resulted in no changes in cardiac MLC2 phosphorylation or cardiac weight phenotype.11 Skeletal muscle MLCK expression was lost, and no significant increase in MLC2 phosphorylation in response to repetitive electric stimulation was confirmed in skeletal muscles. These findings suggested that skeletal muscle MLCK does not play a major role in regulating cardiac MLC2. Similarly, ablation of the long form of smooth muscle MLCK, as described in another recent study,12 did not affect cardiac function, as determined by systolic blood pressure, heart rate, or echocardiographic measurements. Electrocardiographic analysis showed neither atrio- nor intraventricular conduction or repolarization defects. These findings suggested that the long-form smooth muscle MLCK is not involved in regulating cardiac function.
The report by Kasahara and colleagues appearing in this issue of Circulation Research has given us a new surprise.13 The era of molecular cloning seemed already over in the last century, and, thus, it is rather unexpected that a major molecule involved in regulating cardiac function has remained unidentified. The study, indeed, has identified a new MLCK that is specifically expressed in the heart, although the presence of such a MLCK species is not unreasonable after we have learned from recent knockout studies as described above.11,12 Kasahara and colleagues identified a rodent MLCK during the process of identifying genes regulated by cardiac homeobox protein Nkx2–5. The identified MLCK, which has been named "cardiac MLCK," as it would be expected, encodes 795 aa with a predicted molecular mass of 86 kDa excluding posttranslational modifications. The protein consists of a conserved kinase domain that includes an ATP-binding site at the C terminus, with 58% identity with skeletal muscle MLCK and 44% identity with smooth muscle MLCK. However, the N-terminal domain is very different, with no significant homology to other known proteins, including skeletal or smooth muscle MLCK.
The tissue distribution of cardiac MLCK showed a distinct pattern from the other known MLCK; cardiac MLCK was expressed only in the heart and in both atrium and ventricle. Kasahara and colleagues also demonstrated, by the use of a specific antibody raised against the N terminus of cardiac MLCK, that cardiac MLCK was expressed 9- to 18-fold more abundantly than smooth muscle MLCK in the neonatal heart. The intracellular distribution was relatively diffuse in the cytoplasm in cardiac myocytes, whereas, in some areas, a striated pattern was demonstrated. Interestingly, it was not colocalized with MLC2v, the ventricular MLC2 isoform and a potential and major substrate of MLCK, although it was colocalized with actin.
The obvious question was whether this new MLCK can phosphorylate MLC2v and whether such phosphorylation may occur in a Ca2+/calmodulin-dependent manner.14 Kasahara and colleagues demonstrated that, indeed, cardiac MLCK phosphorylated MLC2v but, unexpectedly, not in a Ca2+/calmodulin-dependent manner, as demonstrated by the comparison with skeletal muscle MLCK. Kinetic analysis of this enzyme revealed that cardiac MLCK had a high affinity and relatively low catalytic efficiency to MLC2v, suggesting that this enzyme may serve to maintain basal MLCK activity in the heart. This finding was somewhat different from those shown by Kitakaze and colleagues, who also identified cardiac MLCK almost simultaneously but from human.15 Kitakaze and colleagues found a human cDNA during microarray analysis of failing human hearts and demonstrated that human cardiac MLCK could phosphorylate MLC2v in a Ca2+/calmodulin-dependent manner. Other properties of the MLCK studied by Kitakaze and colleagues were similar to those of rat MLCK. This apparent difference in Ca2+/calmodulin dependence may simply be attributable to species differences; however, such in vitro assessments of the function of MLCK may not be sufficient to understand the exact role of MLCK in regulating function. Thus, we must await studies from genetically manipulated animals such as cardiac MLCK deficient or overexpressed mice.
Nevertheless, overexpression of cardiac MLCK in cells led to enhanced phosphorylation of MLC2v almost to its saturation level, whereas downregulation of cardiac MLCK decreased the steady-state level of MLC2v phosphorylation, supporting the idea that cardiac MLCK plays an important role in, at least, maintaining the basal phosphorylation of MLC2v. Because MLCK itself could be phosphorylated, the potential kinase that can phosphorylate this enzyme may also be involved in regulating its enzyme activity, which needs to be identified.
Then, what is the role of this MLCK in regulating the function of cardiac myocytes? To answer this question, Kasahara and colleagues compared the effect of overexpression and downregulation of cardiac MLCK on myocyte morphology.13 The overexpression of MLCK induced sarcomere organization, whereas downregulation caused disturbance in peripheral structure, the latter of which occurred up to 96 hours after infection of cardiac MLCK harboring adenovirus. Such changes in the cardiac myocyte morphology were in agreement with those reported by Kitakaze and colleagues, who also cloned the zebrafish ortholog, z-cardiac MLCK, and demonstrated that knockdown of z-cardiac-MLCK expression by the use of morpholino antisense oligonucleotides resulted in immature sarcomere structure and dilated cardiac ventricles.16 These findings suggest that cardiac MLCK plays an important role in maintaining sarcomere structure in the heart. Cardiac contractility was increased by the overexpression of cardiac MLCK and was decreased by downregulation in cardiac myocytes.13 Equivalent findings were obtained in zebrafish with cardiac knockdown of cardiac MLCK as well.15 It should be noted that the zebrafish ortholog of cardiac MLCK amino acid sequence is highly homologous to those of other vertebrate orthologs, especially within the C-terminal kinase domain.
Accordingly, it has now been demonstrated, in 2 independent studies, that cardiac MLCK plays an important role in both structure and function of cardiac myocytes. It is possible that the unique N-terminal domain of cardiac MLCK may be responsible for regulation of this kinase activity through other intracellular signals or the interaction with other molecules involved in regulating cardiac function.17 Such regulatory mechanisms may provide cardiac MLCK with a different role(s) from other known MLCKs.
The role played by cardiac MLCK under various cardiac pathophysiological conditions needs to be examined carefully and in detail in future studies. In particular, changes in cardiac MLCK expression under cardiac pathophysiology may not be similar between mRNA and protein, as implicated in the preliminary studies.13,15 The robust finding of these studies is to suggest, however, that there is still much work to be conducted to understand the regulation of MLC in the heart.
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Acknowledgments |
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Sources of Funding
This study was supported in part by grants from the Japan Space Forum, the Japanese Ministry of Education, Culture, Sports, Science, and Technology, the Kitsuen Kagaku Research Foundation, and NIH grants GM-067773 and HL-059139.
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2008 102: 571-580.
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