Vinculin functions as a molecular clutch that organizes leading edge F-actin, generates traction, and promotes focal adhesion formation and turnover but not adhesion growth.

Vinculin is a ubiquitously expressed multiliganded protein that links the actin cytoskeleton to the cell membrane. In myocytes, it is localized in protein complexes which anchor the contractile apparatus to the sarcolemma. Its function in the myocardium remains poorly understood. Therefore, we developed a mouse model with cardiac-myocyte-specific inactivation of the vinculin (Vcl) gene by using Cre-loxP technology. Sudden death was found in 49% of the knockout (cVclKO) mice younger than 3 months of age despite preservation of contractile function. Conscious telemetry documented ventricular tachycardia as the cause of sudden death, while defective myocardial conduction was detected by optical mapping. cVclKO mice that survived through the vulnerable period of sudden death developed dilated cardiomyopathy and died before 6 months of age. Prior to the onset of cardiac dysfunction, ultrastructural analysis of cVclKO heart tissue showed abnormal adherens junctions with dissolution of the intercalated disc structure, expression of the junctional proteins cadherin and ␤1D integrin were reduced, and the gap junction protein connexin 43 was mislocalized to the lateral myocyte border. This is the first report of tissue-specific inactivation of the Vcl gene and shows that it is required for preservation of normal cell-cell and cell-matrix adhesive structures.

Vinculin and its muscle splice variant metavinculin link focal adhesions and cell-to-cell contact sites to the actin cytoskeleton. We hypothesized that normal expression of vinculin isoforms would be essential for integrity of cardiomyocytes and preservation of normal cardiac function. We studied heterozygous vinculin knockout mice (Vin؉/؊) that develop and breed normally. The Vin؉/؊ mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abnormal electrocardiograms, intercalated disks, and ICD-related protein distribution; 3) increased mortality following acute hemodynamic stress imposed by transverse aortic constriction (TAC); 4) cardiac dysfunction by 6 weeks post-TAC; and 5) misalignment of ␣-actinin containing Z-lines and abnormal myocardial ultrastructure despite preserved cardiac function. Decreased expression of vinculin/metavinculin leads to abnormal myocyte structure without baseline physiological evidence of cardiac dysfunction. These structural changes predispose to stress-induced cardiomyopathy. Vinculin (Vin) is a 117-kd ubiquitously expressed, membrane-associated protein that links focal adhesions (cellsubstrate contacts), adherens junctions (cell-to-cell contacts), and costameres (subsarcolemmal adhesion plaques) to the actin cytoskeleton.1-3 This linkage occurs since vinculin binds to F-actin, ␣-actinin, ␣-catenin, and talin. 4 -6 In cardiac myocytes, vinculin is detected at the lateral sarcolemma, in transverse ribs called costameres, in Z-lines, as well as at cellular attachment sites termed intercalated disks (ICDs). 7-9Dilated cardiomyopathy has been associated with mutations in a number of structural proteins, and many such studies have shown that the link from sarcomere to cell membrane is critical for preservation of normal myocardial function and integrity. 10 Relevant to the focus of the current examination is that a 124-kd splice-variant isoform of vinculin, termed metavinculin (Mvin), is expressed only in cardiac and smooth muscle.11 Alterations in metavinculin have been recently related to human cardiomyopathy. 12,13 Homozygous inactivation of the murine vinculin gene caused embryonic lethality by E10.5. The embryos displayed a thin-walled myocardium and cerebral abnormalities including incomplete fusion of the neural folds.14 The heterozygous knockout mice (Vinϩ/Ϫ) develop and breed normally and are the focus of the current study. We hypothesized that reduced expression of vinculin/metavinculin in the intact Vinϩ/Ϫ mouse heart would lead to impaired cardiac function either in the basal condition or following hemodynamic stress. The strategy used to inactivate the vinculin gene in these mice leads to impaired production of vinculin as well as metavinculin.14 We found that the Vinϩ/Ϫ mice displayed: 1) a 58% reduction of vinculin and a 63% reduction of metavinculin protein levels versus wild-type littermates; 2) normal basal cardiac function and histology but abn...

Vinculin (Vcl) links actin filaments to integrin-and cadherin-based cellular junctions. Zonula occludens-1 (ZO-1, also known as TJP1) binds connexin-43 (Cx43, also known as GJA1), cadherin and actin. Vcl and ZO-1 anchor the actin cytoskeleton to the sarcolemma. Given that loss of Vcl from cardiomyocytes causes maldistribution of Cx43 and predisposes cardiomyocyte-specific Vcl-knockout mice with preserved heart function to arrhythmia and sudden death, we hypothesized that Vcl and ZO-1 interact and that loss of this interaction destabilizes gap junctions. We found that Vcl, Cx43 and ZO-1 colocalized at the intercalated disc. Loss of cardiomyocyte Vcl caused parallel loss of ZO-1 from intercalated dics. Vcl coimmunoprecipitated Cx43 and ZO-1, and directly bound ZO-1 in yeast two-hybrid studies. Excision of the Vcl gene in neonatal mouse cardiomyocytes caused a reduction in the amount of Vcl mRNA transcript and protein expression leading to (1) decreased protein expression of Cx43, ZO-1, talin, and b1D-integrin, (2) reduced PI3K activation, (3) increased activation of Akt, Erk1 and Erk2, and (4) cardiomyocyte necrosis. In summary, this is the first study showing a direct interaction between Vcl and ZO-1 and illustrates how Vcl plays a crucial role in stabilizing gap junctions and myocyte integrity.