Deletion of murine SMN exon 7 directed to skeletal muscle leads to severe muscular dystrophy

Spinal muscular atrophy (SMA) is characterized by degeneration of motor neurons of the spinal cord associated with muscle paralysis and caused by mutations of the survival motor neuron gene (SMN). To determine whether SMN gene defect in skeletal muscle might have a role in SMA pathogenesis, deletion of murine SMN exon 7, the most frequent mutation found in SMA, has been restricted to skeletal muscle by using the Cre-loxP system. Mutant mice display ongoing muscle necrosis with a dystrophic phenotype leading to muscle paralysis and death. The dystrophic phenotype is associated with elevated levels of creatine kinase activity, Evans blue dye uptake into muscle fibers, reduced amount of dystrophin and upregulation of utrophin expression suggesting a destabilization of the sarcolemma components. The mutant mice will be a valuable model for elucidating the underlying mechanism. Moreover, our results suggest a primary involvement of skeletal muscle in human SMA, which may contribute to motor defect in addition to muscle denervation caused by the motor neuron degeneration. These data may have important implications for the development of therapeutic strategies in SMA.     

Forced expression of dystrophin deletion constructs reveals structure- function correlations

Dystrophin plays an important role in skeletal muscle by linking the cytoskeleton and the extracellular matrix. The amino terminus of dystrophin binds to actin and possibly other components of the subsarcolemmal cytoskeleton, while the carboxy terminus associates with a group of integral and peripheral membrane proteins and glycoproteins that are collectively known as the dystrophin-associated protein (DAP) complex. We have generated transgenic/mdx mice expressing "full-length" dystrophin constructs, but with consecutive deletions within the COOH- terminal domains. These mice have enabled analysis of the interaction between dystrophin and members of the DAP complex and the effects that perturbing these associations have on the dystrophic process. Deletions within the cysteine-rich region disrupt the interaction between dystrophin and the DAP complex, leading to a severe dystrophic pathology. These deletions remove the beta-dystroglycan-binding site, which leads to a parallel loss of both beta-dystroglycan and the sarcoglycan complex from the sarcolemma. In contrast, deletion of the alternatively spliced domain and the extreme COOH terminus has no apparent effect on the function of dystrophin when expressed at normal levels. The proteins resulting from these latter two deletions supported formation of a completely normal DAP complex, and their expression was associated with normal muscle morphology in mdx mice. These data indicate that the cysteine-rich domain is critical for functional activity, presumably by mediating a direct interaction with beta-dystroglycan. However, the remainder of the COOH terminus is not required for assembly of the DAP complex.     

The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

The Role of the N-Terminus of the Myosin Essential Light Chain in Cardiac Muscle Contraction

To study the regulation of cardiac muscle contraction by the myosin essential light chain (ELC) and the physiological significance of its N-terminal extension, we generated transgenic (Tg) mice by partially replacing the endogenous mouse ventricular ELC with either the human ventricular ELC wild type (Tg-WT) or its 43-amino-acid N-terminal truncation mutant (Tg-Δ43) in the murine hearts. The mutant protein is similar in sequence to the short ELC variant present in skeletal muscle, and the ELC protein distribution in Tg-Δ43 ventricles resembles that of fast skeletal muscle. Cardiac muscle preparations from Tg-Δ43 mice demonstrate reduced force per cross-sectional area of muscle, which is likely caused by a reduced number of force-generating myosin cross-bridges and/or by decreased force per cross-bridge. As the mice grow older, the contractile force per cross-sectional area further decreases in Tg-Δ43 mice and the mutant hearts develop a phenotype of nonpathologic hypertrophy while still maintaining normal cardiac performance. The myocardium of older Tg-Δ43 mice also exhibits reduced myosin content. Our results suggest that the role of the N-terminal ELC extension is to maintain the integrity of myosin and to modulate force generation by decreasing myosin neck region compliance and promoting strong cross-bridge formation and/or by enhancing myosin attachment to actin.     

Increased expression of deltaCaMKII isoforms in skeletal muscle regeneration: Implications in dystrophic muscle disease

The expression of delta isoforms of calcium-calmodulin/dependent protein kinase II (CaMKII) has been reported in mammalian skeletal muscle; however, their functions in this tissue are largely unknown. This study was conducted to determine if deltaCaMKII expression was altered during regeneration of skeletal muscle fibers in two distinct models. In the first model, necrosis and regeneration were induced in quadriceps of normal mice by intramuscular administration of 50% glycerol. Immunostaining and confocal microscopy revealed that deltaCaMKII expression was clearly enhanced in fibers showing centralized nuclei. The second model was the mdx mouse, which undergoes enhanced muscle necrosis and regeneration due to a mutation in the dystrophin gene. sern blot analysis of hind leg extracts from 4 to 6 week old mdx mice revealed that deltaCaMKII content was decreased when compared to age-matched control mice. This loss in delta kinase content was seen in myofibrillar and membrane fractions and was in contrast to unchanged deltaCaMKII levels in cardiac and brain extracts from dystrophic mice. Confocal microscopy of mdx quadriceps and tibialis muscle showed that deltaCaMKII expression was uniformly decreased in most fibers from dystrophic mice; however, enhanced kinase expression was observed in regenerating muscle fibers. These data support a fundamental role for deltaCaMKII in the regeneration process of muscle fibers in normal and mdx skeletal muscle and may have important implications in the reparative process following muscle death.     

Skeletal muscle reprogramming by activation of calcineurin improves insulin action on metabolic pathways

The protein phosphatase calcineurin is a signaling intermediate that induces the transformation of fast-twitch skeletal muscle fibers to a slow-twitch phenotype. This reprogramming of the skeletal muscle gene expression profile may have therapeutic applications for metabolic disease. Insulin-stimulated glucose uptake in skeletal muscle is both impaired in individuals with type II diabetes mellitus and positively correlated with the percentage of slow- versus fast-twitch muscle fibers. Using transgenic mice expressing activated calcineurin in skeletal muscle, we report that skeletal muscle reprogramming by calcineurin activation leads to improved insulin-stimulated 2-deoxyglucose uptake in extensor digitorum longus (EDL) muscles compared with wild-type mice, concomitant with increased protein expression of the insulin receptor, Akt, glucose transporter 4, and peroxisome proliferator-activated receptor-gamma co-activator 1. Transgenic mice exhibited elevated glycogen deposition, enhanced amino acid uptake, and increased fatty acid oxidation in EDL muscle. When fed a high-fat diet, transgenic mice maintained superior rates of insulin-stimulated glucose uptake in EDL muscle and were protected against diet-induced glucose intolerance. These results validate calcineurin as a target for enhancing insulin action in skeletal muscle.     

Mutations of N-terminal regions render the retinoblastoma protein insufficient for functions in development and tumor suppression.

To assess biological roles of the retinoblastoma protein (RB), four independent transgenic mouse lines expressing human RB with different deletions in the N-terminal region (RBdeltaN) were generated and compared with mice expressing identically regulated, full-length RB. Expression of both RB and RBdeltaN caused developmental growth retardation, but the wild-type protein was more potent. In contrast to wild-type RB, the RBdeltaN proteins were unable to rescue Rb-/- mice completely from embryonic lethality. Embryos survived until gestational day 18.5 but displayed defects in the terminal differentiation of erythrocytes, neurons, and skeletal muscle. In Rb+/- mice, expression of the RBdeltaN transgenes failed to prevent pituitary melanotroph tumors but delayed tumor formation or progression. These results strongly suggest that N-terminal regions are crucial for embryonic and postnatal development, tumor suppression, and the functional integrity of the entire RB protein. Furthermore, these transgenic mice provide models that may begin to explain human families with low-penetrance retinoblastoma and mutations in N-terminal regions of RB.     

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring k_tr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased k_tr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.     

Myocilin interacts with syntrophins and is member of dystrophin-associated protein complex

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.     

Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.     

Lower skeletal muscle mass in male transgenic mice with muscle-specific overexpression of myostatin

Mutations in the myostatin gene are associated with hypermuscularity, suggesting that myostatin inhibits skeletal muscle growth. We postulated that increased tissue-specific expression of myostatin protein in skeletal muscle would induce muscle loss. To investigate this hypothesis, we generated transgenic mice that overexpress myostatin protein selectively in the skeletal muscle, with or without ancillary expression in the heart, utilizing cDNA constructs in which a wild-type (MCK/Mst) or mutated muscle creatine kinase (MCK-3E/Mst) promoter was placed upstream of mouse myostatin cDNA. Transgenic mice harboring these MCK promoters linked to enhanced green fluorescent protein (EGFP) expressed the reporter protein only in skeletal and cardiac muscles (MCK) or in skeletal muscle alone (MCK-3E). Seven-week-old animals were genotyped by PCR of tail DNA or by Southern blot analysis of liver DNA. Myostatin mRNA and protein, measured by RT-PCR and Western blot, respectively, were significantly higher in gastrocnemius, quadriceps, and tibialis anterior of MCK/Mst-transgenic mice compared with wild-type mice. Male MCK/Mst-transgenic mice had 18-24% lower hind- and forelimb muscle weight and 18% reduction in quadriceps and gastrocnemius fiber cross-sectional area and myonuclear number (immunohistochemistry) than wild-type male mice. Male transgenic mice with mutated MCK-3E promoter showed similar effects on muscle mass. However, female transgenic mice with either type of MCK promoter did not differ from wild-type controls in either body weight or skeletal muscle mass. In conclusion, increased expression of myostatin in skeletal muscle is associated with lower muscle mass and decreased fiber size and myonuclear number, decreased cardiac muscle mass, and increased fat mass in male mice, consistent with its role as an inhibitor of skeletal muscle mass. The mechanism of gender specificity remains to be clarified.     

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.     

Muscle-specific overexpression of the adenovirus primary receptor CAR overcomes low efficiency of gene transfer to mature skeletal muscle

Significant levels of adenovirus (Ad)-mediated gene transfer occur only in immature muscle or in regenerating muscle, indicating that a developmentally regulated event plays a major role in limiting transgene expression in mature skeletal muscle. We have previously shown that in developing mouse muscle, expression of the primary Ad receptor CAR is severely downregulated during muscle maturation. To evaluate how global expression of CAR throughout muscle affects Ad vector (AdV)-mediated gene transfer into mature skeletal muscle, we produced transgenic mice that express the CAR cDNA under the control of the muscle-specific creatine kinase promoter. Five-month-old transgenic mice were compared to their nontransgenic littermates for their susceptibility to AdV transduction. In CAR transgenics that had been injected in the tibialis anterior muscle with AdVCMVlacZ, increased gene transfer was demonstrated by the increase in the number of transduced muscle fibers (433 +/- 121 in transgenic mice versus 8 +/- 4 in nontransgenic littermates) as well as the 25-fold increase in overall beta-galactosidase activity. Even when the reporter gene was driven by a more efficient promoter (the cytomegalovirus enhancer-chicken beta-actin gene promoter), differential transducibility was still evident (893 +/- 149 versus 153 +/- 30 fibers; P < 0.001). Furthermore, a fivefold decrease in the titer of injected AdV still resulted in significant transduction of muscle (253 +/- 130 versus 14 +/- 4 fibers). The dramatic enhancement in AdV-mediated gene transfer to mature skeletal muscle that is observed in the CAR transgenics indicates that prior modulation of the level of CAR expression can overcome the poor AdV transducibility of mature skeletal muscle and significant transduction can be obtained at low titers of AdV.     

Expression of myostatin pro domain results in muscular transgenic mice

Myostatin, a member of the TGF-beta family, negatively regulates skeletal muscle development. Depression of myostatin activity leads to increased muscle growth and carcass lean yield. In an attempt to down-regulate myostatin, transgenic mice were produced with a ribozyme-based construct or a myostatin pro domain construct. Though the expression of the ribozyme was detected, muscle development was not altered by the ribozyme transgene. However, a dramatic muscling phenotype was observed in transgenic mice carrying the myostatin pro domain gene. Expression of the pro domain transgene at 5% of beta-actin mRNA levels resulted in a 17-30% increase in body weight (P < 0.001). The carcass weight of the transgenic mice showed a 22-44% increase compared with nontransgenic littermates at 9 weeks of age (16.05 +/- 0.67 vs. 11.16 +/- 0.28 g in males; 9.99 +/- 0.38 vs. 8.19 +/- 0.19 g in females, P < 0.001). Extreme muscling was present throughout the whole carcass of transgenic mice as hind and fore limbs and trunk weights, all increased significantly (P < 0.001). Epididymal fat pad weight, an indicator of body fat, was significantly decreased in pro domain transgenic mice (P < 0.001). Analysis of muscle morphology indicated that cross-sectional areas of fast-glycolytic fibers (gastrocnemius) and fast-oxidative glycolytic fibers (tibialis) were larger in pro domain transgenic mice than in their controls (P < 0.01), whereas fiber number (gastrocnemius) was not different (P > 0.05). Thus, the muscular phenotype is attributable to myofiber hypertrophy rather than hyperplasia. The results of this study suggest that the over-expression of myostatin pro domain may provide an alternative to myostatin knockouts as a means of increasing muscle mass in other mammals.