Abstract-Integrins link the extracellular matrix to the cellular cytoskeleton and serve important roles in cell growth, differentiation, migration, and survival. Ablation of ␤1 integrin in all murine tissues results in peri-implantation embryonic lethality. To investigate the role of ␤1 integrin in the myocardium, we used Cre-LoxP technology to inactivate the ␤1 integrin gene exclusively in ventricular cardiac myocytes. Animals with homozygous ventricular myocyte ␤1 integrin gene excision were born in appropriate numbers and grew into adulthood. These animals had 18% of control levels of ␤1D integrin protein in the heart and displayed myocardial fibrosis. High-fidelity micromanometer-tipped catheterization of the intact 5-week-old ␤1 integrin knockout mice showed depressed left ventricular basal and dobutamine-stimulated contractility and relaxation (LV dP/dt max and LV dP/dt min ) as compared with control groups (nϭ8 to 10 of each, PϽ0.01). Hemodynamic loading imposed by 7 days of transverse aortic constriction showed that the ␤1 integrin knockout mice were intolerant of this stress as they had 53% survival versus 88% in controls (nϭ15 each). Key Words: extracellular matrix Ⅲ homologous recombination Ⅲ Cre recombinase Ⅲ heart Ⅲ positron emission tomography I ntegrins are a large family of heterodimeric cell surface receptors composed of ␣ and ␤ subunits. They function in cell-extracellular matrix (ECM) adhesion and cell-cell adhesion, and signal bidirectionally across the cell membrane. 1,2 Further, they serve as mechanotransducers, converting mechanical signals to biochemical ones. 3 This combination of properties allows integrins to play important roles in cell growth, differentiation, migration, and survival 4 and also makes them attractive candidates for essential roles in the developing and postnatal heart.Our previous work has shown that ␤1 integrins are linked to the hypertrophic response of cultured ventricular myocytes and also that dominant-negative disruption of integrin function in transgenic mice resulted in cardiac fibrosis and abnormal cardiac function. 5-7 Ablation of ␤1 integrin expression in all murine tissues resulted in gastrulation defects and death by E5.5 of the 21-day gestation period. 8,9 Chimeric mice as well as embryoid bodies constructed from ␤1 integrin-null cells showed delayed development and differentiation of ␤1-deficient cells along the cardiac lineage, as well as abnormal sarcomerogenesis of these cardiac-like cells. 10 Although a few ␤1 integrin-null cells were detected in the myocardium of chimeric mice, cellular debris was always detected along with the null cells. These null cells were completely lost from the myocardium of the chimeric mouse heart by 6 months of age.To more specifically evaluate the role of ␤1 integrin in the myocardium, we used a Cre-loxP gene targeting approach. Cre recombinase expression driven by the myosin light chain-2 ventricular (MLC-2v) promoter caused ␤1 integrin gene excision exclusively in ventricular cardiac myocytes. 11 Our results in these ...

Alterations in the extracellular matrix occur during the cardiac hypertrophic process. Because integrins mediate cell-matrix adhesion and beta(1D)-integrin (beta1D) is expressed exclusively in cardiac and skeletal muscle, we hypothesized that beta1D and focal adhesion kinase (FAK), a proximal integrin-signaling molecule, are involved in cardiac growth. With the use of cultured ventricular myocytes and myocardial tissue, we found the following: 1) beta1D protein expression was upregulated perinatally; 2) alpha(1)-adrenergic stimulation of cardiac myocytes increased beta1D protein levels 350% and altered its cellular distribution; 3) adenovirally mediated overexpression of beta1D stimulated cellular reorganization, increased cell size by 250%, and induced molecular markers of the hypertrophic response; and 4) overexpression of free beta1D cytoplasmic domains inhibited alpha(1)-adrenergic cellular organization and atrial natriuretic factor (ANF) expression. Additionally, FAK was linked to the hypertrophic response as follows: 1) coimmunoprecipitation of beta1D and FAK was detected; 2) FAK overexpression induced ANF-luciferase; 3) rapid and sustained phosphorylation of FAK was induced by alpha(1)-adrenergic stimulation; and 4) blunting of the alpha(1)-adrenergically modulated hypertrophic response was caused by FAK mutants, which alter Grb2 or Src binding, as well as by FAK-related nonkinase, a dominant interfering FAK mutant. We conclude that beta1D and FAK are both components of the hypertrophic response pathway of cardiac myocytes.

Integrins are heterodimeric cell-surface receptors that link the extracellular matrix and the intracellular cytoskeleton and function as mechanotransducers. Signaling through integrins is important for cell growth, migration, and survival. Extracellular matrix is altered in the myocardium during hypertrophic induction and the transition to heart failure. The role of integrins in this process is poorly understood. Recently, integrin subunits have been identified that are dominantly expressed in striated muscle. We tested the hypothesis that since integrins are mechanotransducers, their expression and signaling would be modulated with murine left ventricular hemodynamic loading. The acute and chronic effects of pressure overload on changes in the expression of integrins, as well as related integrin-mediated signaling events were studied. Acute pressure loading increased phosphorylation of focal adhesion kinase, p42 and p44 extracellular signal-regulated kinase. Chronic loading:(1) increased expression of α1, α5, and β1 integrin transcripts and (2) increased protein expression of integrin subunits which are dominantly expressed in striated muscle (α7 and β1D) both by western blotting and immunofluorescent microscopy. These results show that adaptive responses of the myocardium to pressure overload include acute modulation of integrin-related signaling molecules and more chronic changes effect expression of integrin subunits, including ones dominantly expressed in muscle.

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