β-adrenergic activation may promote myosin light chain kinase degradation through calpain in pressure overload-induced cardiac hypertrophy

Background/Aims: Angiotensin II (Ang II) has been shown to promote cardiac remodeling during the process of hypertrophy. Myosin light chain kinase (MLCK), a specific kinase for the phosphorylation of myosin light chain 2 (MLC2), plays an important role in regulating cardiac muscle contraction and hypertrophy. However, whether Ang II could facilitate cardiac hypertrophy by altering the expression of MLCK remains unclear. This study aimed to investigate this effect and the underlying mechanisms. Methods: Cardiac hypertrophy was induced via pressure overload in rats, which were then evaluated via histological and biochemical measurements and echocardiography. Angiotensin-converting enzyme inhibitor (ACEI) was used to inhibit Ang II. Neonatal rat cardiomyocytes were stimulated with Ang II to induce hypertrophy and were treated with a matrix metalloproteinase 9 (MMP9) inhibitor. Myocyte hypertrophy was evaluated using immunofluorescence and qRT-PCR. Degradation of recombinant human MLCK by recombinant human MMP9 was tested using a cleavage assay. The expression levels of MLCK, MLC2, phospho-myosin light chain 2 (p-MLC2), myosin phosphatase 2 (MYPT2), and calmodulin (CaM) were measured using western blotting. Results: ACEI improved cardiac function and remodeling and increased the levels of MLCK and p-MLC2 as well as reduced the expression of MMP9 in pressure overload-induced cardiac hypertrophy. Moreover, the MMP9 inhibitor alleviated myocyte hypertrophy and upregulated the levels of MLCK and p-MLC2 in Ang II-induced cardiomyocyte hypertrophy. Recombinant human MLCK was concentration- and time-dependently degraded by recombinant human MMP9 in vitro, and this process was prevented by the MMP9 inhibitor. Conclusion: Our results suggest that Ang II is involved in the degradation of MLCK in pressure overload-induced cardiac hypertrophy and that this process was mediated by MMP9.

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

Circulation ◽

10.1161/circ.116.suppl_16.ii_221-a ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Jason Y Chan ◽

Morihiko Takeda ◽

Laura E Briggs ◽

Jonathan T Lu ◽

Nobuo Horikoshi ◽

...

Keyword(s):

Heart Failure ◽

Skeletal Muscle ◽

Smooth Muscle ◽

Cardiac Hypertrophy ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Contractile Function ◽

Severe Heart Failure ◽

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.

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Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro

Nature Medicine ◽

10.1038/72287 ◽

2000 ◽

Vol 6 (2) ◽

pp. 183-188 ◽

Cited By ~ 105

Author(s):

Hiroki Aoki ◽

Junichi Sadoshima ◽

Seigo Izumo

Keyword(s):

Cardiac Hypertrophy ◽

Light Chain ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain

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

Circulation ◽

10.1161/circ.130.suppl_2.17911 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Michael Massengill ◽

Eileen Chang ◽

Hasssan Ashraf ◽

Stephen Chrzanowski ◽

Rajib Chowdhury ◽

...

Keyword(s):

Heart Failure ◽

Light Chain ◽

Knockout Mice ◽

Myosin Light Chain Kinase ◽

Myosin Light Chain ◽

Gene Knockout ◽

Pressure Overload ◽

Cardiac Myosin ◽

Moderate Heart Failure ◽

Gene Knockout Mice

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.

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