ABSTRACT
Disruption of the sarcoglycan complex leads to muscle membrane instability and muscular dystrophy in humans and mice. Through the dystrophin glycoprotein complex, sarcoglycan participates in connecting the internal cytoskeleton to the membrane and the extracellular matrix. Integrin alpha7beta1 is also a transmembrane protein of skeletal and cardiac muscle that similarly links the cytoskeleton to the extracellular matrix. Mice lacking integrin alpha7 develop mild muscle degeneration, while sarcoglycan mutant mice display overt muscle degeneration and muscular dystrophy. In sarcoglycan-deficient muscle, integrin alpha7 protein was upregulated at the plasma membrane. To ascertain whether integrin alpha7 upregulation compensates for the loss of the transmembrane sarcoglycan linkage in sarcoglycan-deficient muscle, we generated mice lacking both integrin alpha7 and gamma-sarcoglycan (gxi). These double-mutant gxi mice exhibit profound, rapid muscle degeneration leading to death before one month of age consistent with a weakened cellular attachment to the extracellular matrix. The regenerative capacity of gxi muscle was intact with increased embryonic myosin heavy chain expression, myofiber central nucleation and normal in vivo myoblast differentiation. Therefore, upregulation of integrin alpha7beta1 compensates as a transmembrane muscle cell attachment for sarcoglycan consistent with overlapping roles for sarcoglycan and integrins in mediating cytoskeletal-membrane-extracellular matrix interaction.
Subject(s)
Integrins/genetics , Integrins/physiology , Muscles/metabolism , Sarcoglycans/genetics , Sarcoglycans/physiology , Animals , Antigens, CD/genetics , Cell Differentiation , Cell Membrane/metabolism , Cell Nucleus/metabolism , Coloring Agents/pharmacology , Cytoskeleton/metabolism , Dystroglycans/metabolism , Evans Blue/pharmacology , Extracellular Matrix/metabolism , Immunoblotting , Immunohistochemistry , In Situ Nick-End Labeling , Integrin alpha Chains/genetics , Integrins/metabolism , Laminin/metabolism , Mice , Microsomes/metabolism , Muscle, Skeletal/metabolism , Mutation , Phenotype , Regeneration , Reverse Transcriptase Polymerase Chain Reaction , Sarcoglycans/metabolism , Time Factors , Up-RegulationABSTRACT
Genetic defects in the plasma membrane-associated sarcoglycan complex produce cardiomyopathy characterized by focal degeneration. The infarct-like pattern of cardiac degeneration has led to the hypothesis that coronary artery vasospasm underlies cardiomyopathy in this disorder. We evaluated the coronary vasculature of gamma-sarcoglycan mutant mice and found microvascular filling defects consistent with arterial vasospasm. However, the vascular smooth muscle sarcoglycan complex was intact in the coronary arteries of gamma-sarcoglycan hearts with perturbation of the sarcoglycan complex only within the adjacent myocytes. Thus, in this model, coronary artery vasospasm derives from a vascular smooth muscle-cell extrinsic process. To reduce this secondary vasospasm, we treated gamma-sarcoglycan-deficient mice with the calcium channel antagonist verapamil. Verapamil treatment eliminated evidence of vasospasm and ameliorated histological and functional evidence of cardiomyopathic progression. Echocardiography of verapamil-treated, gamma-sarcoglycan-null mice showed an improvement in left ventricular fractional shortening (44.3 +/- 13.3% treated versus 37.4 +/- 15.3% untreated), maximal velocity at the aortic outflow tract (114.9 +/- 27.9 cm/second versus 92.8 +/- 22.7 cm/second), and cardiac index (1.06 +/- 0.30 ml/minute/g versus 0.67 +/- 0.16 ml/minute/g, P < 0.05). These data indicate that secondary vasospasm contributes to the development of cardiomyopathy and is an important therapeutic target to limit cardiomyopathy progression.
Subject(s)
Cardiomyopathies/etiology , Cardiomyopathies/pathology , Coronary Vasospasm/complications , Coronary Vasospasm/physiopathology , Animals , Calcium Channel Blockers/pharmacology , Cardiomyopathies/drug therapy , Coronary Vasospasm/drug therapy , Cytoskeletal Proteins/deficiency , Cytoskeletal Proteins/genetics , Disease Models, Animal , Disease Progression , Echocardiography , Fluorescent Antibody Technique , Heart/drug effects , Heart Function Tests/drug effects , Immunoblotting , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Mutant Strains , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , Myocardium/pathology , Sarcoglycans , Verapamil/pharmacologyABSTRACT
Cellular shape change and movement are central to biologic processes that range from normal embryonic development to inflammatory diseases and cancer. Quantitative visual phenotyping of dynamic cellular behaviors creates unique challenges for image capture, analysis and storage. Despite substantial technological advances in molecular biology, biochemistry, genomics and proteomics, investigating cellular processes remains tremendously challenging and labor-intensive. We have developed algorithms and software implementations that allow for fully-automated analysis of experiments designed to investigate a range of cellular and organismal behaviors. By enabling cellular phenotyping, this automated approach creates a unique opportunity for investigators to perform large-scale experiments designed to determine gene function or to screen for small molecule modulators of important cellular behaviors.