Abstract 3845: Cardiac Myosin Light Chain Kinase Regulates Cardiac Contractility and the Level of Phosphorylation of Myosin Light Chain in vivo
Background: Phosphorylation of myosin light chain 2 (MLC2) is one regulatory mechanism to enhance cardiac contraction. Myosin light chain kinase (MLCK) that is specifically expressed in the heart was recently identified as a downstream target of the transcription factor Nkx2–5, and was named cardiac MLCK. Cardiac MLCK (cMLCK) phosphorylates MLC2v and 2a in vitro and in cardiomyocytes. In cultured rat neonatal cardiomyocytes, reduction of cardiac MLCK by adenoviral shRNA resulted in disorganized sarcomere structure and a reduction of cardiomyocytes contraction. Its function in vivo remains to be elucidated.
Methods and Results: We generated a conditional null allele of cMLCK by introducing loxP sites spanning exon 5 through homologous recombination in ES cells. This deletion eliminates the first coding exon of the catalytic domain and results in the frame-shift of the subsequent downstream exons. Mice heterozygous for this cMLCK allele were bred to mice expressing ACTA1-Cre transgene, resulting in a cMLCK+/− allele in germ line, followed by intercrossing to generate cMLCK−/−mice. Northern blotting demonstrated that cMLCK mRNA expression was below the level of detection in cMLCK−/− hearts using the cDNA probe recognizing the upstream from exon 5, indicating that cMLCK−/− mice do not express stable cMLCK mRNA. cMLCK−/− mice were born at the expected Mendelian ratios; however they demonstrated an approximately 20% increase of heart weight/body weight at postnatal day 2 (wild-type, n=16; −/−, n=10). cMLCK−/− mice survive through adulthood despite the mild increase in heart weight/body weight accompanied by reduced cardiac contraction; %FS, 35% in wild-type (n=8) vs. 26% in cMLCK−/− (n=8) at 14 weeks of age. The phosphorylated-form of MLC2v was below the level of detection in 2D electrophoresis followed by Western blotting with anti-MLC antibody (n=5), while the relative amounts of phosphorylated MLC2v to the total MLC2v was approximately 28% in wild-type (n=4).
Conclusion: Our results demonstrate that loss of cMLCK in gene-targeted mice resulted in reduced cardiac contraction accompanied by reduction of phosphorylation of MLC2v, suggesting that cardiac MLCK is a predominant player for phosphorylation of MLC2v and is important for cardiac contraction.