The importance of the epicardium for myocardial and valvuloseptal development has been well established; perturbation of epicardial development results in cardiac abnormalities, including thinning of the ventricular myocardial wall and malformations of the atrioventricular valvuloseptal complex. To determine the spatiotemporal contribution of epicardially derived cells to the developing fibroblast population in the heart we have used a mWt1/IRES/GFP-Cre mouse to trace the fate of EPDCs from embryonic day (ED)10 until birth. EPDCs begin to populate the compact ventricular myocardium around ED12. The migration of epicardially-derived fibroblasts toward the interface between compact and trabecular myocardium is completed around ED14. Remarkably, epicardially-derived fibroblasts do not migrate into the trabecular myocardium until after ED17. Migration of EPDCs into the atrioventricular cushion mesenchyme commences around ED12. As development progresses, the number of EPDCs increases significantly, specifically in the leaflets which derive from the lateral atrioventricular cushions. In these developing leaflets the epicardially-derived fibroblasts eventually largely replace the endocardially-derived cells. Importantly, the contribution of EPDCs to the leaflets derived from the major AV cushions is very limited. The differential contribution of EPDCs to the various leaflets of the atrioventricular valves provides a new paradigm in valve development and could lead to new insights into the pathogenesis of abnormalities that preferentially affect individual components of this region of the heart. The notion that there is a significant difference in the contribution of epicardially and endocardially derived cells to the individual leaflets of the atrioventricular valves has also important pragmatic consequences for the use of endocardial and epicardial cre-mouse models in studies of heart development.

Abstract-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 2ϩ /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. (Circ Res. 2008;102:571-580.)Key Words: kinase Ⅲ transcription Ⅲ contraction P hosphorylation of both myosin heavy chain and myosin light chain (MLC) affects motor activity and thick filament assembly. 1 In smooth muscle cells, phosphorylation of MLC2 by smooth muscle MLCK is thought to be responsible for the initiation of contraction. 2 In skeletal and cardiac muscles, however, initiation of muscle contraction depends on voltage-gated L-type Ca 2ϩ channels in the plasma membrane and T-tubules. Increased local Ca 2ϩ concentrations allow the sarcoplasmic reticulum to release large amounts of Ca 2ϩ , which bind to troponin C followed by myosin-actin cross-bridge formation. During this process, MLCK potentiates peak tension in skeletal muscle 1,3 and the force and rate of cross-bridge recruitment in cardiac myocytes. 4,5 To date, smooth muscle (encoded by mylk1 gene) and skeletal (encoded by mylk2 gene) MLCKs have been characterized. 3 Mouse skeletal muscle MLCK is predominantly expressed in skeletal muscle, and mouse smooth muscle MLCK is expressed in several tissues but predominantly in smooth muscle. 6,7 Mutations in human skeletal MLCK on human chromosome 20 have been mapped to a disease locus for familial cardiac hypertrophy (Online Mendelian Inheritance in Man no. 606566), suggesting that abnormal function of skeletal MLCK stimulates cardiac hypertrophy. 8 However, the abundance of skeletal MLCK expression in the heart is controversial, 8 -10 and gene-targeted mice for skeletal MLCK appear to have normal cardiac function. 10 Short-form (130-kDa) smooth muscle MLCK is expressed in the heart at lower levels than those detected in smooth muscle-rich organs such as gut, ...