For myosin light chain phosphatase, a very small subunit can make very big differences in the heart

The idea that myosin phosphorylation was involved in contraction first emerged in the early 1960s (6), and by the mid-1970s a prevailing view was that myosin phosphorylation was prerequisite for the thick filament regulation of contraction in smooth muscle but not for the thin filament regulation of skeletal and cardiac muscle contraction (8). The nearly concurrent discoveries that 20-kDa myosin light chain (MLC) was the site of regulatory phosphorylation by highly specific MLC kinases, and dephosphorylation by MLC phosphatases (MLCPs), attracted widespread attention to the function of the these enzymes, and by the early 2000s it was firmly established that modulation of MLCP activity was a primary mechanism whereby myofilament calcium sensitivity was regulated in smooth muscle (9). In parallel with the intense focus on the role of MLCP in smooth muscle contraction, the concept that MLCP might also be important in the heart began to gain momentum in the 1980s. This view was supported by observations that MLCs were expressed and could be phosphorylated in the heart (3) and dephosphorylated by specific cardiac phosphatases (7). Somewhat unexpectedly, inhibition of MLCPs also produced significant alterations in cardiac electrophysiology, suggesting that perhaps these phosphatases served functions beyond those involved in contraction (5). Subsequent work has revealed that the targeting subunit of MLCP is highly organ specific (4), and, in 2001, Arimura et al. (2) reported that cardiac tissues express a heart-specific small subunit of MLCP. Arimura and colleagues have since reported a series of studies detailing the physiology and biochemistry of this heart-specific subunit, and their most recent study (1), recently published in the American Journal of Physiology-Heart and Circulatory Physiology, offers new evidence of the involvement of this small subunit in pathways modulating cardiac electrophysiology and gene expression.

To explore the cardiac function of hHS-M₂₁, a heart-specific small subunit of MLCP that facilitates targeting of Rho-associated coiled-coil forming kinase (ROCK), Arimura et al. created multiple lines of transgenic mice that exhibited cardiac-specific overexpression of the hHS-M₂₁ subunit, as validated using custom-made antibodies directed against hHS-M₂₁. Overexpression of hHS-M₂₁ caused multiple indications of contractile dysfunction including sinus bradycardia and atrioventricular conduction disturbances (Fig. 1). The effects of overexpression on contractility, but not on conduction, were ameliorated by long-term treatment with fasudil given between 2 and 8 mo of age to block the influence of ROCK. Structurally, overexpression caused ventricular enlargement, as measured by transthoracic echocardiography, and interstitial fibrosis, as determined via histology. Overexpression also increased expression of myosin phosphatase target subunit 1 and ROCK2 and decreased both RhoA and MLC2 phosphorylation. Paradoxically, these effects were associated with increased cardiac calcium sensitivity measured in membrane skinned fibers from left ventricular papillary muscle. Overexpression also altered the expression of >3,000 genes, including some involved with cardiac remodeling and inflammation, as quantified via microarray and validated with PCR. Together, these results strongly suggest that overexpression of hHS-M₂₁ negatively affected cardiac structure and electrophysiology, in addition to its expected direct effects on cardiac contractility.

Fig. 1.

Overexpression of hHS-M₂₁, a small, heart-specific subunit of cardiac myosin light chain phosphatase (MLCP), produced multiple effects independent of the level of phosphorylation of cardiac myosin light chain (MLC2). These included dramatic changes in cardiac gene expression, conduction abnormalities such as atrioventricular (AV) block and bradycardia and depressed ventricular function typified by increased fibrosis, left ventricular (LV) dilation, and decreased ejection fraction. Despite these structural and functional changes, myofilament calcium sensitivity was increased. Long-term treatment with the Rho-associated coiled-coil forming kinase (ROCK) inhibitor fasudil ameliorated the effects of hHS-M₂₁ overexpression on cardiac structure and function but did not improve conduction abnormalities. How hHS-M₂₁ overexpression caused changes in cardiac gene expression remains unknown and may involve both primary and secondary effects elicited by structural and functional changes in the heart. CaM, calmodulin; MHC, myosin heavy chain; MLCK, MLC kinase; MYPT, targeting subunit of MLCP; PP1cδ, the catalytic subunit of MLCP.

Whereas the new study by Arimura et al. clearly advances understanding of, and interest in, hHS-M₂₁, many important questions remain. The effects of hHS-M₂₁ overexpression on patterns of gene expression were impressive, but it remains unclear which gene changes were a primary response to hHS-M₂₁ overexpression and which were a secondary response to the cardiac abnormalities caused by this overexpression. Similarly, it is uncertain what compensations were initiated by long-term inhibition of ROCK with fasudil. Furthermore, from the results of this study, it is difficult to ascertain the physiological functions of hHS-M₂₁ at nominal levels of expression.

A key finding in this study was that the overall phosphorylation of MLC2 was not altered in transgenic mice despite a small but significant increase in myofilament calcium sensitivity. Owing to the dynamic nature of MLC2 phosphorylation, it remains possible that peak MLC2 phosphorylation measured precisely during maximal contractile activation may have been greater in transgenic than wild-type mice, even though basal levels of MLC2 phosphorylation were similar in the two groups. Alternatively, it is also possible that overexpression of hHS-M₂₁ elevated apparent myofilament calcium sensitivity independent of MLC2 phosphorylation, perhaps by activating a pathway of calcium-independent contraction. Such mechanisms have been proposed to operate in smooth muscle, in part through an enhanced ability of myosin cross bridges to interact with the cytoskeleton (10). In turn, this pattern of “calcium sensitization” implies the interactions of multiple potential kinase pathways with thin filament regulatory proteins such as caldesmon, calponin, heat shock proteins, and many others. Consistent with this general idea, calcium-independent mechanisms of force regulation in cardiac myocytes have long been recognized (11), although the molecular basis for this regulation remains unidentified. Perhaps the ability of hHS-M₂₁ to modulate myofilament calcium sensitivity independent of MLC2 phosphorylation can finally provide an opportunity to explore how calcium sensitivity is regulated in the heart.

From a translational perspective, the new results from Arimura et al. (1) may illuminate a new mechanism for cardiomyopathy with bradycardia and fibrosis. Correspondingly, it may be useful to search for possible mutations in hHS-M₂₁ in such patients. Therapeutically, it may be productive to consider fasudil as a treatment option for such cases. That said, many more preclinical studies are needed, particularly in light of the possibility that hHS-M₂₁ may interact with unknown targets beyond the traditional contractile apparatus. Are these extra-contractile effects of hHS-M₂₁ dependent on phosphatase activity or can the subunit act independently of the fully assembled phosphatase? Is the inflammation associated with hHS-M₂₁ overexpression prerequisite for its effects on cardiac gene expression? What molecular pathway couples overexpression of hHS-M₂₁ to changes in gene expression? Are such effects mediated by changing the phosphorylation levels of key transcription factors or through effects further upstream from nuclear translocation events? This abundance of questions related to the diverse effects of hHS-M₂₁ overexpression is a testament to the importance of these new findings and their potential to lead to better understanding of MLCP function in the heart.

GRANTS

This work was supported by National Institutes of Health Grants P01-NS-082184, P01-HD-083132, and R01-NS-076945.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

W.J.P. drafted manuscript; W.J.P. edited and revised manuscript; W.J.P. approved final version of manuscript.