Big mitogen-activated protein kinase 1 (BMK1), also known as ERK5, is a member of the MAPK family. Genetic ablation of BMK1 in mice leads to embryonic lethality, precluding the exploration of pathophysiological roles of BMK1 in adult mice. We generated a BMK1 conditional mutation in mice in which disruption of the BMK1 gene is under the control of the inducible Mx1-Cre transgene. Ablation of BMK1 in adult mice led to lethality within 2-4 weeks after the induction of Cre recombinase. Physiological analysis showed that the blood vessels became abnormally leaky after deletion of the BMK1 gene. Histological analysis revealed that, after BMK1 ablation, hemorrhages occurred in multiple organs in which endothelial cells lining the blood vessels became round, irregularly aligned, and, eventually, apoptotic. In vitro removal of BMK1 protein also led to the death of endothelial cells partially due to the deregulation of transcriptional factor MEF2C, which is a direct substrate of BMK1. Additionally, endothelial-specific BMK1-KO leads to cardiovascular defects identical to that of global BMK1-KO mutants, whereas, surprisingly, mice lacking BMK1 in cardiomyocytes developed to term without any apparent defects. Taken together, the data provide direct genetic evidence that the BMK1 pathway is critical for endothelial function and for maintaining blood vessel integrity. 1138The Nonstandard abbreviations used: big mitogen-activated protein kinase-1 (BMK1); BMK1 flox/flox Mx1-Cre mice treated with pIpC (BMK1-CKO); cardiomyocyte-specific BMK1-KO (BMK1-cmKO); EC-specific BMK1-KO (BMK1-ecKO); embryonic day (E); endothelial cell (EC); endothelial cell growth supplement (ECGS); extracellular signalregulated kinases-1 and -2 (ERK1/2); lacZ/alkaline phosphatase (Z/AP); MAPK kinase (MEK); MAPK kinase kinase (MEKK); mouse lung capillary endothelial cell (MLCEC); myocyte enhancer factor-2 (MEF2); polyinosinic-polycytidylic acid (pIpC).

. Targeted deletion of MMP-2 attenuates early LV rupture and late remodeling after experimental myocardial infarction. Am J Physiol Heart Circ Physiol 285: H1229-H1235, 2003. First published May 29, 2003 10.1152/ ajpheart.00207.2003.-Matrix metalloproteinase-2 (MMP-2) is prominently overexpressed both after myocardial infarction (MI) and in heart failure. However, its pathophysiological significance in these conditions is still unclear. We thus examined the effects of targeted deletion of MMP-2 on post-MI left ventricular (LV) remodeling and failure. Anterior MI was produced in 10-to 12-wk-old male MMP-2 knockout (KO) and sibling wild-type (WT) mice by ligating the left coronary artery. By day 28, MI resulted in a significant increase in mortality in association with LV cavity dilatation and dysfunction. The MMP-2 KO mice had a significantly better survival rate than WT mice (56% vs. 85%, P Ͻ 0.05), despite a comparable infarct size (50 Ϯ 3% vs. 51 Ϯ 3%, P ϭ not significant), heart rate, and arterial blood pressure. The KO mice had a significantly lower incidence of LV rupture (10% vs. 39%, P Ͻ 0.05), which occurred within 7 days of MI. The KO mice exerted less LV cavity dilatation and improved fractional shortening after MI by echocardiography. The LV zymographic MMP-2 level significantly increased in WT mice after coronary artery ligation; however, this was completely prevented in KO mice. In contrast, the increase in the LV zymographic MMP-9 level after MI was similar between KO and WT mice. MMP-2 activation is therefore considered to contribute to an early cardiac rupture as well as late LV remodeling after MI. The inhibition of MMP-2 activation may therefore be a potentially useful therapeutic strategy to manage post-MI hearts. matrix metalloproteinase; cardiac rupture; heart failure; myocyte; extracellular matrix; mouse MYOCARDIAL INFARCTION (MI) leads to complex structural alterations (remodeling) involving both the infarcted and noninfarcted left ventricular (LV) myocardium (21). Early remodeling as LV cavity dilatation occurs during the early phase of MI, which is likely due to wall thinning in the infarct region. This might lead to a cardiac rupture, thereby accounting for the 5-30% of in-hospital mortality after acute MI (1). During the first several days, LV enlargement follows, and, thereafter, a progressive dilatation of the noninfarcted LV occurs over weeks (21). These progressive changes in LV geometry contribute to the development of depressed cardiac function, clinical heart failure, and increased mortality. Accordingly, it is of critical importance to explore the mechanisms of LV remodeling and develop therapeutic strategies that will effectively inhibit this deleterious process.The dynamic synthesis and breakdown of extracellular matrix (ECM) proteins play an important role in post-MI LV remodeling. In particular, the increased expression and activation of matrix metalloproteinases (MMPs) have been implicated in this process (4,5). Several studies have demonstrated that MMPs are involved not...

Abstract, Costameres, the vinculin-rich, sub-membranous transverse ribs found in many skeletal and cardiac muscle cells (Pardo, J. V., J. D. Siciliano, and S, W, Craig. 1983. Proc. Natl. Acad. Sci. USA. 80:363-367.) are thought to anchor the Z-lines of the myofibrils to the sarcolemma. In addition, it has been postulated that costameres provide mechanical linkage between the cells' internal contractile machinery and the extracellular matrix, but direct evidence for this supposition has been lacking. By combining the flexible silicone rubber substratum technique (Harris, A. K., P. Wild, and D. Stopak. 1980. Science (Wash. DC). 208:177-179.) with the microinjection of fluorescently labeled vinculin and oL-actinin, we have been able to correlate the distribution of costameres in adult rat cardiac myocytes with the pattern of forces these cells exert on the flexible substratum. In addition, we used interference reflection microscopy to identify areas of the cells which are in close contact to the underlying substratum. Our results indicate that, in older cell cultures, costameres can transmit forces to the extracellular environment. We base this conclusion on the following observations: (a) adult rat heart cells, cultured on the silicone rubber substratum for 8 or more days, produce pleat-like wrinkles during contraction, which diminish or disappear during relaxation; (b) the pleat-like wrinkles form between adjacent alpha-actinin-positive Z-lines; (c) the presence of pleat-like wrinkles is always associated with a periodic, "costameric" distribution of vinculin in the areas where the pleats form; and (d) a banded or periodic pattern of dark gray or close contacts (as determined by interference reflection microscopy) has been observed in many cells which have been in culture for eight or more days, and these close contacts contain vinculin. A surprising finding is that vinculin can be found in a costameric pattern in cells which are contracting, but not producing pleat-like wrinkles in the substratum. This suggests that additional proteins or posttranslational modifications of known costamere proteins are necessary to form a continuous linkage between the myofibrils and the extracellular matrix. These results confirm the hypothesis that costameres mechanically link the myofibrils to the extracellular matrix. We put forth the hypothesis that costarneres are composite structures, made up of many protein components; some of these components function primarily to anchor myofibrils to the sarcolemma, while others form transmembrane linkages to the extracellular matrix.C OSTAMERES are vinculin-containing, electron-dense plaques located between the cell membrane and Z-discs in cardiac muscle and certain types of skeletal muscle (Pardo et al., 1983a,b;Koteliansky and Gneushev, 1983; Shear and Bloch, I985). Pardo and colleagues (1983a) used the term "costamere" to define them because of the rib-like appearance of the plaques when immunostained Barbara A. Danowski and Kyoko Imanaka-Yoshida contributed equally to this work.

SUMMARY: Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for ␣-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization. (Lab Invest 2001, 81:1015-1024.T enascins constitute a family of extracellular matrix glycoproteins (Erickson, 1993). The first member found, tenascin-C (TNC), is highly expressed in embryonic tissue during morphogenesis and sparsely expressed in the adult, but reappears during wound healing, regeneration, or cancer invasion (Chiquet-Ehrismann et al, 1986). TNC is transiently expressed in distinct areas in association with active tissue remodeling. Various in vitro findings have indicated that TNC has multiple functions in the regulation of cell migration, proliferation, and apoptosis (Chung et al, 1996;Jones and Jones, 2000). TNC counterbalances cell adhesion to substrata, correlated with cytokinesis and motility, and prevents cells from adhering too tightly to other extracellular matrix proteins (Chiquet-Ehrismann, 1995;Chiquet-Ehrismann et al, 1988;Chung et al, 1996;Murphy-Ullrich et al, 1991;Prieto et al, 1992).In the heart, TNC appears only at very early stages of embryonic development and may play important roles in the differentiation of cardiomyocytes, structural organization of the myocardium, or development of coronary vessels . TNC is not normally expressed in the adult heart but reappears under various pathologic conditions such as dilated cardiomyopathy, myocarditis, or myocardial infarction (Imanaka-Yoshida et al, 1998, 2000Tamura et al, 1996;Willems et al, 1996).To understand its roles in tissue remodeling in the diseased myocardium, we examined the expression of TNC during tissue repair after experimental myocardial infarction in rats. Sequential changes in the localization of the molecule were analyzed by immunohistochemistry, and TNC-producing cells were identified by in situ hybridization (ISH) combined with immunohistochemistry. Additionally, to ...

Tenascin-C (TN-C) is an extracellular matrix molecule that is expressed during wound healing in various tissues. Although not detectable in the normal adult heart, it is expressed under pathological conditions. Previously, using a rat model, we found that TN-C was expressed during the acute stage after myocardial infarction and that alpha-smooth muscle actin (alpha-SMA)-positive myofibroblasts appeared in TN-C-positive areas. In the present study, we examined whether TN-C controls the dynamics of myofibroblast recruitment and wound healing after electrical injury to the myocardium of TN-C knockout (TNKO) mice compared with wild-type (WT) mice. In TNKO mice, myocardial repair seemed to proceed normally, but the appearance of myofibroblasts was delayed. With cultured cardiac fibroblasts, TN-C significantly accelerated cell migration, alpha-SMA expression, and collagen gel contraction but did not affect proliferation. Using recombinant fragments of murine TN-C, the functional domain responsible for promoting migration of cardiac fibroblasts was mapped to the conserved fibronectin type III (FNIII)-like repeats and the fibrinogen (Fbg)-like domain. Furthermore, alternatively spliced FNIII and Fbg-like domains proved responsible for the up-regulation of alpha-SMA expression. These results indicate that TN-C promotes recruitment of myofibroblasts in the early stages of myocardial repair by stimulating cell migration and differentiation.