Disruption of the beta-sarcoglycan gene reveals pathogenetic complexity of limb-girdle muscular dystrophy type 2E

Limb-girdle muscular dystrophy type 2E (LGMD 2E) is caused by mutations in the beta-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscle. beta-sarcoglycan-deficient (Sgcb-null) mice developed severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. The sarcoglycan-sarcospan and dystroglycan complexes were disrupted in skeletal, cardiac, and smooth muscle membranes. epsilon-sarcoglycan was also reduced in membrane preparations of striated and smooth muscle. Loss of the sarcoglycan-sarcospan complex in vascular smooth muscle resulted in vascular irregularities in heart, diaphragm, and kidneys. Further biochemical characterization suggested the presence of a distinct epsilon-sarcoglycan complex in skeletal muscle that was disrupted in Sgcb-null mice. Thus, perturbation of vascular function together with disruption of the epsilon-sarcoglycan-containing complex represents a novel mechanism in the pathogenesis of LGMD 2E.     

Skeletal myosphere-derived progenitor cell transplantation promotes neovascularization in delta-sarcoglycan knockdown cardiomyopathy

Bone marrow cells have been shown to contribute to neovascularization in ischemic hearts, whereas their impaired maturation to restore the delta-sarcoglycan (delta-SG) expression responsible for focal myocardial degeneration limits their utility to treat the pathogenesis of cardiomyopathy. Here, we report the isolation of multipotent progenitor cells from adult skeletal muscle, based on their ability to generate floating-myospheres. Myosphere-derived progenitor cells (MDPCs) are distinguishable from myogenic C2C12 cells and differentiate into vascular smooth muscle cells and mesenchymal progeny. The mutation in the delta-SG has been shown to develop vascular spasm to affect sarcolemma structure causing cardiomyopathy. We originally generated delta-SD knockdown (KD) mice and transplanted MDPCs into the hearts. MDPCs enhanced neoangiogenesis and restored delta-SG expression in impaired vasculatures through trans-differentiation, leading to improvement of cardiac function associated with paracrine effectors secretion. We propose that MDPCs may be the promising progenitor cells in skeletal muscle to treat delta-sarcoglycan complex mutant cardiomyopathy.     

Impaired vascular contractility and blood pressure homeostasis in the smooth muscle alpha-actin null mouse.

The smooth muscle (SM) alpha-actin gene activated during the early stages of embryonic cardiovascular development is switched off in late stage heart tissue and replaced by cardiac and skeletal alpha-actins. SM alpha-actin also appears during vascular development, but becomes the single most abundant protein in adult vascular smooth muscle cells. Tissue-specific expression of SM alpha-actin is thought to be required for the principal force-generating capacity of the vascular smooth muscle cell. We wanted to determine whether SM alpha-actin gene expression actually relates to an actin isoform's function. Analysis of SM alpha-actin null mice indicated that SM alpha-actin is not required for the formation of the cardiovascular system. Also, SM alpha-actin null mice appeared to have no difficulty feeding or reproducing. Survival in the absence of SM alpha-actin may result from other actin isoforms partially substituting for this isoform. In fact, skeletal alpha-actin gene, an actin isoform not usually expressed in vascular smooth muscle, was activated in the aortas of these SM alpha-actin null mice. However, even with a modest increase in skeletal alpha-actin activity, highly compromised vascular contractility, tone, and blood flow were detected in SM alpha-actin-defective mice. This study supports the concept that SM alpha-actin has a central role in regulating vascular contractility and blood pressure homeostasis, but is not required for the formation of the cardiovascular system.     

Mice lacking skeletal muscle actin show reduced muscle strength and growth deficits and die during the neonatal period

All four of the muscle actins (skeletal, cardiac, vascular, and enteric) in higher vertebrates show distinct expression patterns and display highly conserved amino acid sequences. While it is hypothesized that each of the muscle isoactins is specifically adapted to its respective tissue and that the minor variations among them have developmental and/or physiological relevance, the exact functional and developmental significance of these proteins remains largely unknown. In order to begin to assess these issues, we disrupted the skeletal actin gene by homologous recombination. All mice lacking skeletal actin die in the early neonatal period (day 1 to 9). These null animals appear normal at birth and can breathe, walk, and suckle, but within 4 days, they show a markedly lower body weight than normal littermates and many develop scoliosis. Null mice show a loss of glycogen and reduced brown fat that is consistent with malnutrition leading to death. Newborn skeletal muscles from null mice are similar to those of wild-type mice in size, fiber type, and ultrastructural organization. At birth, both hemizygous and homozygous null animals show an increase in cardiac and vascular actin mRNA in skeletal muscle, with no skeletal actin mRNA present in null mice. Adult hemizygous animals show an increased level of skeletal actin mRNA in hind limb muscle but no overt phenotype. Extensor digitorum longus (EDL) muscle isolated from skeletal-actin-deficient mice at day 2 to 3 showed a marked reduction in force production compared to that of control littermates, and EDL muscle from hemizygous animals displayed an intermediate force generation. Thus, while increases in cardiac and vascular smooth-muscle actin can partially compensate for the lack of skeletal actin in null mice, this is not sufficient to support adequate skeletal muscle growth and/or function.     

Disruption of muscle architecture and myocardial degeneration in mice lacking desmin

Desmin, the muscle specific intermediate filament (IF) protein encoded by a single gene, is expressed in all muscle tissues. In mature striated muscle, desmin IFs surround the Z-discs, interlink them together and integrate the contractile apparatus with the sarcolemma and the nucleus. To investigate the function of desmin in all three muscle types in vivo, we generated desmin null mice through homologous recombination. Surprisingly, desmin null mice are viable and fertile. However, these mice demonstrated a multisystem disorder involving cardiac, skeletal, and smooth muscle. Histological and electron microscopic analysis in both heart and skeletal muscle tissues revealed severe disruption of muscle architecture and degeneration. Structural abnormalities included loss of lateral alignment of myofibrils and abnormal mitochondrial organization. The consequences of these abnormalities were most severe in the heart, which exhibited progressive degeneration and necrosis of the myocardium accompanied by extensive calcification. Abnormalities of smooth muscle included hypoplasia and degeneration. The present data demonstrate the essential role of desmin in the maintenance of myofibril, myofiber, and whole muscle tissue structural and functional integrity, and show that the absence of desmin leads to muscle degeneration.     

epsilon-sarcoglycan replaces alpha-sarcoglycan in smooth muscle to form a unique dystrophin-glycoprotein complex.

The sarcoglycan complex has been well characterized in striated muscle, and defects in its components are associated with muscular dystrophy and cardiomyopathy. Here, we have characterized the smooth muscle sarcoglycan complex. By examination of embryonic muscle lineages and biochemical fractionation studies, we demonstrated that epsilon-sarcoglycan is an integral component of the smooth muscle sarcoglycan complex along with beta- and delta-sarcoglycan. Analysis of genetically defined animal models for muscular dystrophy supported this conclusion. The delta-sarcoglycan-deficient cardiomyopathic hamster and mice deficient in both dystrophin and utrophin showed loss of the smooth muscle sarcoglycan complex, whereas the complex was unaffected in alpha-sarcoglycan null mice in agreement with the finding that alpha-sarcoglycan is not expressed in smooth muscle cells. In the cardiomyopathic hamster, the smooth muscle sarcoglycan complex, containing epsilon-sarcoglycan, was fully restored following intramuscular injection of recombinant delta-sarcoglycan adenovirus. Together, these results demonstrate a tissue-dependent variation in the sarcoglycan complex and show that epsilon-sarcoglycan replaces alpha-sarcoglycan as an integral component of the smooth muscle dystrophin-glycoprotein complex. Our results also suggest a molecular basis for possible differential smooth muscle dysfunction in sarcoglycan-deficient patients.     

Cardiac myosin light chain kinase is necessary for myosin regulatory light chain phosphorylation and cardiac performance in vivo

In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca(2+)/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo.     

Smooth Muscle LDL Receptor-Related Protein-1 Deletion Induces Aortic Insufficiency and Promotes Vascular Cardiomyopathy in Mice

Valvular disease is common in patients with Marfan syndrome and can lead to cardiomyopathy. However, some patients develop cardiomyopathy in the absence of hemodynamically significant valve dysfunction, suggesting alternative mechanisms of disease progression. Disruption of LDL receptor-related protein-1 (Lrp1) in smooth muscle cells has been shown to cause vascular pathologies similar to Marfan syndrome, with activation of smooth muscle cells, vascular dysfunction and aortic aneurysms. This study used echocardiography and blood pressure monitoring in mouse models to determine whether inactivation of Lrp1 in vascular smooth muscle leads to cardiomyopathy, and if so, whether the mechanism is a consequence of valvular disease. Hemodynamic changes during treatment with captopril were also assessed. Dilation of aortic roots was observed in young Lrp1-knockout mice and progressed as they aged, whereas no significant aortic dilation was detected in wild type littermates. Diastolic blood pressure was lower and pulse pressure higher in Lrp1-knockout mice, which was normalized by treatment with captopril. Aortic dilation was followed by development of aortic insufficiency and subsequent dilated cardiomyopathy due to valvular disease. Thus, smooth muscle cell Lrp1 deficiency results in aortic dilation and insufficiency that causes secondary cardiomyopathy that can be improved by captopril. These findings provide novel insights into mechanisms of cardiomyopathy associated with vascular activation and offer a new model of valvular cardiomyopathy.     

Dystrophin predominantly localizes to the transverse tubule/Z-line regions of single ventricular myocytes and exhibits distinct associations with the membrane

Dystrophin is a high molecular weight protein present at low abundance in skeletal, cardiac and smooth muscle and in trace amounts in brain. In skeletal muscle, dystrophin is uniformly distributed along the inner surface of the plasma membrane. Biochemical fractionation studies have shown that all detectable skeletal muscle dystrophin is tightly associated with a complex of wheat germ agglutinin (WGA)-binding and concanavalin A (Con A) binding sarcolemmal glycoproteins. Absence of dystrophin is the primary biochemical defect in patients with Duchenne muscular dystrophy and leads to segmental necrosis of their skeletal myofibers. Although present in similar amounts in normal cardiac and skeletal muscle, the absence of dystrophin from cardiac muscle has less severe effects on the survival of cardiac cells. We have therefore examined whether there are differences in the properties of cardiac and skeletal dystrophin. We report that in contrast to skeletal muscle, cardiac dystrophin is distributed between distinct pools: a soluble cytoplasmic pool, a membrane-bound pool not associated with WGA-binding glycoproteins and a membrane-bound pool associated with WGA-binding glycoproteins. Cardiac dystrophin was not associated with any Con A binding glycoproteins. Immunohistochemical localization studies in isolated ventricular myocytes reveal a distinct punctate staining pattern for dystrophin, approximating to the level of the transverse tubule/Z-line and contrasting with the uniform sarcolemmal staining reported for skeletal muscle fibers. The distinct properties of cardiac dystrophin suggest unique roles for this protein in cardiac versus skeletal muscle function.Abbreviations Dys Dystrophin - T-tubule Transverse tubule - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - WGA Wheat Germ Agglutinin - Con A Concanavalin A - DHP Dihydropyridine receptor - FITC Fluorescein Isothiocyanate Conjugate - NAG N-Acetyl-D-Glucosamine - NP-40 NONIDET P-40 - PBS Phosphate-Buffered Saline - TBST Tris Buffered Saline-Tween     

Smooth muscle myosin light chain kinase expression in cardiac and skeletal muscle

The purpose of this study was to characterize myosin light chain kinase (MLCK) expression in cardiac and skeletal muscle. The only classic MLCK detected in cardiac tissue, purified cardiac myocytes, and in a cardiac myocyte cell line (AT1) was identical to the 130-kDa smooth muscle MLCK (smMLCK). A complex pattern of MLCK expression was observed during differentiation of skeletal muscle in which the 220-kDa-long or "nonmuscle" form of MLCK is expressed in undifferentiated myoblasts. Subsequently, during myoblast differentiation, expression of the 220-kDa MLCK declines and expression of this form is replaced by the 130-kDa smMLCK and a skeletal muscle-specific isoform, skMLCK in adult skeletal muscle. These results demonstrate that the skMLCK is the only tissue-specific MLCK, being expressed in adult skeletal muscle but not in cardiac, smooth, or nonmuscle tissues. In contrast, the 130-kDa smMLCK is ubiquitous in all adult tissues, including skeletal and cardiac muscle, demonstrating that, although the 130-kDa smMLCK is expressed at highest levels in smooth muscle tissues, it is not a smooth muscle-specific protein.     

Triadins modulate intracellular Ca(2+) homeostasis but are not essential for excitation-contraction coupling in skeletal muscle

To unmask the role of triadin in skeletal muscle we engineered pan-triadin-null mice by removing the first exon of the triadin gene. This resulted in a total lack of triadin expression in both skeletal and cardiac muscle. Triadin knockout was not embryonic or birth-lethal, and null mice presented no obvious functional phenotype. Western blot analysis of sarcoplasmic reticulum (SR) proteins in skeletal muscle showed that the absence of triadin expression was associated with down-regulation of Junctophilin-1, junctin, and calsequestrin but resulted in no obvious contractile dysfunction. Ca(2+) imaging studies in null lumbricalis muscles and myotubes showed that the lack of triadin did not prevent skeletal excitation-contraction coupling but reduced the amplitude of their Ca(2+) transients. Additionally, null myotubes and adult fibers had significantly increased myoplasmic resting free Ca(2+).[(3)H]Ryanodine binding studies of skeletal muscle SR vesicles detected no differences in Ca(2+) activation or Ca(2+) and Mg(2+) inhibition between wild-type and triadin-null animals. Subtle ultrastructural changes, evidenced by the appearance of longitudinally oriented triads and the presence of calsequestrin in the sacs of the longitudinal SR, were present in fast but not slow twitch-null muscles. Overall, our data support an indirect role for triadin in regulating myoplasmic Ca(2+) homeostasis and organizing the molecular complex of the triad but not in regulating skeletal-type excitation-contraction coupling.     

GLUT4 ablation in mice results in redistribution of IRAP to the plasma membrane

Glucose transporter (GLUT) 4 is the insulin responsive glucose transporter in adipose tissue, skeletal muscle, and heart. Insulin elicits increased glucose uptake by recruiting GLUT4 from a specialized intracellular storage site to the cell surface. Expression of various proteins that colocalize with GLUT4 and/or are involved in insulin-stimulated GLUT4 translocation was examined in adipocytes as well as skeletal and cardiac muscles from GLUT4 null mice. Our data demonstrate that expression of insulin-regulated aminopeptidase (IRAP) is divergently regulated in GLUT4 null tissues, e.g., upregulated 1.6-fold in GLUT4 null adipocytes and downregulated in GLUT4 null skeletal muscle (40%) and heart (60%). IRAP exhibited abnormal subcellular distribution and impaired insulin-stimulated translocation in GLUT4-deficient tissues. We propose the compartment containing IRAP and proteins normally associated with GLUT4 vesicle traffics constitutively to the cell surface in GLUT4 null adipocytes and skeletal muscle.     

Characterization and in vivo functional analysis of splice variants of cypher

Previously, we reported two splice variants of Cypher, a striated muscle-specific PDZLIM domain protein, Cypher1 and Cypher2. We have now characterized four additional splice isoforms, two of which are novel. The six isoforms can be divided into skeletal or cardiac specific classes, based on the inclusion of skeletal or cardiac specific domains. Short and long isoforms share an N-terminal PDZ domain, but the three C-terminal LIM domains are unique to long isoforms. By RNA and protein analysis, we have demonstrated that Cypher isoforms are developmentally regulated in both skeletal and cardiac muscle. We have previously shown that knockout of Cypher is neonatal lethal. To investigate the function of splice variants in vivo, we have performed a rescue experiment of the Cypher null mutant by replacing the endogenous Cypher gene with cDNAs encoding either a short or long skeletal muscle isoform. In contrast to Cypher null mice, a percentage of mice that express only a short or a long skeletal muscle-specific isoform can survive to at least 1 year of age. Although surviving mice exhibit muscle pathology, these results suggest that either isoform is sufficient to rescue the lethality associated with the absence of Cypher.     

Atrial natriuretic peptide and osteopontin are useful markers of cardiac disorders in mice

Biomarkers are not established for cardiovascular phenotyping in mice. We compared the use of echocardiography with the determination of N-terminal propeptide of the atrial natriuretic peptide (Nt-proANP) and osteopontin (Opn). We measured plasma Nt-proANP and Opn levels in (1) the inbred strains C57BL/6, BALB/c, C3H/He, DBA/2, FVB/N, 129S1/Sv; (2) a surgical model of nonischemic myocardial infarction; and (3) delta-sarcoglycan (Sgcd) and calsarcin 1 [also known as myozenin 2 (Myoz2)] knockout models of cardiomyopathy. Left ventricular function was assessed as fractional shortening (FS) by echocardiography in conscious mice. Plasma Nt-proANP exhibited marked variability and ranged from 0.31 +/- 0.19 (C57BL/6 male mice) to 1.34 +/- 0.43 nmol/l (DBA/2 female mice), depending on sex, age, and genetic background. Opn was less variable than Nt-proANP and was decreased significantly in C3H/He and DBA/2 throughout the 16 wk of study. Nt-proANP increased temporarily in mice with myocardial injury. In contrast, Opn increased in both operated and sham-treated mice. Nt-proANP was inversely correlated with FS and distinguished controls from Sgcd and Myoz2 mutants with 100% sensitivity and 71% specificity. Opn was increased in Sgcd mutants, which exhibited only mildly reduced FS but marked myocardial degeneration and fibrosis. Both of these histologic features were absent in Myoz2 mutants. Nt-proANP is an early marker of cardiac disease and is suitable for age- and sex-matched comparisons between groups of transgenic and matched control mice. Opn is useful to detect inflammatory and degenerative myocardial disorders that may be missed by echocardiography.     

Caveolin-3 knock-out mice develop a progressive cardiomyopathy and show hyperactivation of the p42/44 MAPK cascade

A growing body of evidence suggests that muscle cell caveolae may function as specialized membrane micro-domains in which the dystrophin-glycoprotein complex and cellular signaling molecules reside. Caveolin-3 (Cav-3) is the only caveolin family member expressed in striated muscle cell types (cardiac and skeletal). Interestingly, skeletal muscle fibers from Cav-3 (-/-) knock-out mice show a number of myopathic changes, consistent with a mild-to-moderate muscular dystrophy phenotype. However, it remains unknown whether a loss of Cav-3 affects the phenotypic behavior cardiac myocytes in vivo. Here, we present a detailed characterization of the hearts of Cav-3 knock-out mice. We show that these mice develop a progressive cardiomyopathic phenotype. At four months of age, Cav-3 knock-out hearts display significant hypertrophy, dilation, and reduced fractional shortening, as revealed by gated cardiac MRI and transthoracic echocardiography. Histological analysis reveals marked cardiac myocyte hypertrophy, with accompanying cellular infiltrates and progressive interstitial/peri-vascular fibrosis. Interestingly, loss of Cav-3 expression in the heart does not change the expression or the membrane association of the dystrophin-glycoprotein (DG) complex. However, a marker of the DG complex, alpha-sarcoglycan, was specifically excluded from lipid raft domains in the absence of Cav-3. Because activation of the Ras-p42/44 MAPK pathway in cardiac myocytes can drive cardiac hypertrophy, we next assessed the activation state of this pathway using a phospho-specific antibody probe. We show that p42/44 MAPK (ERK1/2) is hyperactivated in hearts derived from Cav-3 knock-out mice. These results are consistent with previous in vitro data demonstrating that caveolins may function as negative regulators of the p42/44 MAPK cascade. Taken together, our data argue that loss of Cav-3 expression is sufficient to induce a molecular program leading to cardiac myocyte hypertrophy and cardiomyopathy.     

Chronic losartan administration reduces mortality and preserves cardiac but not skeletal muscle function in dystrophic mice

Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the mdx mouse model of DMD. Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease.