Calpain 8/nCL-2 and Calpain 9/nCL-4 Constitute an Active Protease Complex,G-Calpain,Involved in Gastric Mucosal Defense

Calpains constitute a superfamily of Ca²⁺-dependent cysteine proteases, indispensable for various cellular processes. Among the 15 mammalian calpains, calpain 8/nCL-2 and calpain 9/nCL-4 are predominantly expressed in the gastrointestinal tract and are restricted to the gastric surface mucus (pit) cells in the stomach. Possible functions reported for calpain 8 are in vesicle trafficking between ER and Golgi, and calpain 9 are implicated in suppressing tumorigenesis. These highlight that calpains 8 and 9 are regulated differently from each other and from conventional calpains and, thus, have potentially important, specific functions in the gastrointestinal tract. However, there is no direct evidence implicating calpain 8 or 9 in human disease, and their properties and physiological functions are currently unknown. To address their physiological roles, we analyzed mice with mutations in the genes for these calpains, Capn8 and Capn9. Capn8^−/− and Capn9^−/− mice were fertile, and their gastric mucosae appeared normal. However, both mice were susceptible to gastric mucosal injury induced by ethanol administration. Moreover, the Capn8^−/− stomach showed significant decreases in both calpains 9 and 8, and the same was true for Capn9^−/−. Consistent with this finding, in the wild-type stomach, calpains 8 and 9 formed a complex we termed “G-calpain,” in which both were essential for activity. This is the first example of a “hybrid” calpain complex. To address the physiological relevance of the calpain 8 proteolytic activity, we generated calpain 8:C105S “knock-in” (Capn8^CS/CS) mice, which expressed a proteolytically inactive, but structurally intact, calpain 8. Although, unlike the Capn8^−/− stomach, that of the Capn8^CS/CS mice expressed a stable and active calpain 9, the mice were susceptible to ethanol-induced gastric injury. These results provide the first evidence that both of the gastrointestinal-tract-specific calpains are essential for gastric mucosal defense, and they point to G-calpain as a potential target for gastropathies caused by external stresses.     

Understanding the muscular dystrophy caused by deletion of choline kinase beta in mice

Choline kinase in mice is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneously occurring genomic deletion in murine Chkb results in neonatal bone deformity and hindlimb muscular dystrophy. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, causes hindlimb muscular dystrophy. The biosynthesis of phosphatidylcholine (PC) is impaired in the hindlimbs of Chkb^−/− mice, with an accumulation of choline and decreased amount of phosphocholine. The activity of CTP:phosphocholine cytidylyltransferase is also decreased in the hindlimb muscle of mutant mice. Concomitantly, the activities of PC phospholipase C and phospholipase A₂ are increased. The mitochondria in Chkb^−/− mice are abnormally large and exhibit decreased inner membrane potential. Despite the muscular dystrophy in Chkb^−/− mice, we observed increased expression of insulin like growth factor 1 and proliferating cell nuclear antigen. However, regeneration of hindlimb muscles of Chkb^−/− mice was impaired when challenged with cardiotoxin. Injection of CDP-choline increased PC content of hindlimb muscle and decreased creatine kinase activity in plasma of Chkb^−/− mice. We conclude that the hindlimb muscular dystrophy in Chkb^−/− mice is due to attenuated PC biosynthesis and enhanced catabolism of PC.     

Differential expression of choline kinase isoforms in skeletal muscle explains the phenotypic variability in the rostrocaudal muscular dystrophy mouse

Choline kinase in mammals is encoded by two genes, Chka and Chkb. Disruption of murine Chka leads to embryonic lethality, whereas a spontaneous genomic deletion in murine Chkb results in neonatal forelimb bone deformity and hindlimb muscular dystrophy. Surprisingly, muscular dystrophy isn't significantly developed in the forelimb. We have investigated the mechanism by which a lack of choline kinase β, encoded by Chkb, results in minimal muscular dystrophy in forelimbs. We have found that choline kinase β is the major isoform in hindlimb muscle and contributes more to choline kinase activity, while choline kinase α is predominant in forelimb muscle and contributes more to choline kinase activity. Although choline kinase activity is decreased in forelimb muscles of Chkb^−/− mice, the activity of CTP:phosphocholine cytidylyltransferase is increased, resulting in enhanced phosphatidylcholine biosynthesis. The activity of phosphatidylcholine phospholipase C is up-regulated while the activity of phospholipase A₂ in forelimb muscle is not altered. Regeneration of forelimb muscles of Chkb^−/− mice is normal when challenged with cardiotoxin. In contrast to hindlimb muscle, mega-mitochondria are not significantly formed in forelimb muscle of Chkb^−/− mice. We conclude that the relative lack of muscle degeneration in forelimbs of Chkb^−/− mice is due to abundant choline kinase α and the stable homeostasis of phosphatidylcholine.     

Activation of AKT signaling promotes cell growth and survival in α7β1 integrin-mediated alleviation of muscular dystrophy

Transgenic expression of the α7 integrin can ameliorate muscle pathology in a mouse model of Duchenne muscular dystrophy (mdx/utr^−/−) and thus can compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the α7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remains to be established. The purpose of this study was to determine a role for signaling in the amelioration of muscular dystrophy by α7 integrin. Activation of PI3K, ILK, AKT, mTOR, p70S6K, BAD, ERK, and p38 was measured in the muscle from wild type (WT), mdx/utr^−/− and α7BX2-mdx/utr^−/− mice using in vitro activity assays or phosphospecific antibodies and western blotting. Significant increases in PI3K activity (47%), ILK activity (2.0-fold), mTOR (Ser2448) (57%), p70S6K (Thr389) (11.7-fold), and ERK (Thr202/Tyr204) (66%) were demonstrated in dystrophic mdx/utr^−/− muscle compared to WT. A significant decrease in p38 phosphorylation (2.9-fold) was also observed. Although most of these signaling events were similar in dystrophic mdx/utr^−/− mice overexpressing the α7 integrin, the AKT (Ser473):AKT ratio (2-fold vs. WT) and p70S6K phosphorylation (18-fold vs. WT) were higher in α7BX2-mdx/utr^−/− compared to mdx/utr^−/− mice. In addition, increased phosphorylation of BAD Serine 112 may contribute to the significant reduction in TUNEL⁺ cells observed in α7BX2-mdx/utr^−/− mice. We conclude that the α7β1 integrin confers a protective effect in dystrophic muscle through the activation of the ILK, AKT, p70S6K and BAD signaling to promote muscle cell survival.     

Calpain-3 is autolyzed and hence activated in human skeletal muscle 24 h following a single bout of eccentric exercise.

The function and normal regulation of calpain-3, a muscle-specific Ca(2+)-dependent protease, is uncertain, although its absence leads to limb-girdle muscular dystrophy type 2A. This study examined the effect of eccentric exercise on calpain-3 autolytic activation, because such exercise is known to damage sarcomeric structures and to trigger adaptive changes that help prevent such damage on subsequent exercise. Six healthy human subjects performed a 30-min bout of one-legged, eccentric, knee extensor exercise. Torque measurements, vastus lateralis muscle biopsies, and venous blood samples were taken before and up to 7 days following the exercise. Peak isometric muscle torque was depressed immediately and at 3 h postexercise and recovered by 24 h, and serum creatine kinase concentration peaked at 24 h postexercise. The amount of autolyzed calpain-3 was unchanged immediately and 3 h after exercise, but increased markedly (from approximately 16% to approximately 35% of total) 24 h after the exercise, and returned to preexercise levels within 7 days. In contrast, the eccentric exercise produced little autolytic activation of the ubiquitous Ca(2+)-activated protease, mu-calpain. Eccentric exercise is the first physiological circumstance shown to result in calpain-3 activation in vivo.     

Progressive Muscular Dystrophy in α-Sarcoglycan–deficient Mice

Limb-girdle muscular dystrophy type 2D (LGMD 2D) is an autosomal recessive disorder caused by mutations in the α-sarcoglycan gene. To determine how α-sarcoglycan deficiency leads to muscle fiber degeneration, we generated and analyzed α-sarcoglycan– deficient mice. Sgca-null mice developed progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, showed ongoing muscle necrosis with age, a hallmark of the human disease. Sgca-null mice also revealed loss of sarcolemmal integrity, elevated serum levels of muscle enzymes, increased muscle masses, and changes in the generation of absolute force. Molecular analysis of Sgca-null mice demonstrated that the absence of α-sarcoglycan resulted in the complete loss of the sarcoglycan complex, sarcospan, and a disruption of α-dystroglycan association with membranes. In contrast, no change in the expression of ε-sarcoglycan (α-sarcoglycan homologue) was observed. Recombinant α-sarcoglycan adenovirus injection into Sgca-deficient muscles restored the sarcoglycan complex and sarcospan to the membrane. We propose that the sarcoglycan–sarcospan complex is requisite for stable association of α-dystroglycan with the sarcolemma. The Sgca-deficient mice will be a valuable model for elucidating the pathogenesis of sarcoglycan deficient limb-girdle muscular dystrophies and for the development of therapeutic strategies for this disease.     

Calpain-6 Deficiency Promotes Skeletal Muscle Development and Regeneration

Calpains are Ca²⁺-dependent modulator Cys proteases that have a variety of functions in almost all eukaryotes. There are more than 10 well-conserved mammalian calpains, among which eutherian calpain-6 (CAPN6) is unique in that it has amino acid substitutions at the active-site Cys residue (to Lys in humans), strongly suggesting a loss of proteolytic activity. CAPN6 is expressed predominantly in embryonic muscles, placenta, and several cultured cell lines. We previously reported that CAPN6 is involved in regulating microtubule dynamics and actin reorganization in cultured cells. The physiological functions of CAPN6, however, are still unclear. Here, to elucidate CAPN6''s in vivo roles, we generated Capn6-deficient mice, in which a lacZ expression cassette was integrated into the Capn6 gene. These Capn6-deficient mouse embryos expressed lacZ predominantly in skeletal muscles, as well as in cartilage and the heart. Histological and biochemical analyses showed that the CAPN6 deficiency promoted the development of embryonic skeletal muscle. In primary cultured skeletal muscle cells that were induced to differentiate into myotubes, Capn6 expression was detected in skeletal myocytes, and Capn6-deficient cultures showed increased differentiation. Furthermore, we found that CAPN6 was expressed in the regenerating skeletal muscles of adult mice after cardiotoxin-induced degeneration. In this experimental system, Capn6-deficient mice exhibited more advanced skeletal-muscle regeneration than heterozygotes or wild-type mice at the same time point. These results collectively showed that a loss of CAPN6 promotes skeletal muscle differentiation during both development and regeneration, suggesting a novel physiological function of CAPN6 as a suppressor of skeletal muscle differentiation.     

Rbfox1 Downregulation and Altered Calpain 3 Splicing by FRG1 in a Mouse Model of Facioscapulohumeral Muscular Dystrophy (FSHD)

Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD–like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6–) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.     

DNA damage, somatic aneuploidy, and malignant sarcoma susceptibility in muscular dystrophies

Albeit genetically highly heterogeneous, muscular dystrophies (MDs) share a convergent pathology leading to muscle wasting accompanied by proliferation of fibrous and fatty tissue, suggesting a common MD-pathomechanism. Here we show that mutations in muscular dystrophy genes (Dmd, Dysf, Capn3, Large) lead to the spontaneous formation of skeletal muscle-derived malignant tumors in mice, presenting as mixed rhabdomyo-, fibro-, and liposarcomas. Primary MD-gene defects and strain background strongly influence sarcoma incidence, latency, localization, and gender prevalence. Combined loss of dystrophin and dysferlin, as well as dystrophin and calpain-3, leads to accelerated tumor formation. Irrespective of the primary gene defects, all MD sarcomas share non-random genomic alterations including frequent losses of tumor suppressors (Cdkn2a, Nf1), amplification of oncogenes (Met, Jun), recurrent duplications of whole chromosomes 8 and 15, and DNA damage. Remarkably, these sarcoma-specific genetic lesions are already regularly present in skeletal muscles in aged MD mice even prior to sarcoma development. Accordingly, we show also that skeletal muscle from human muscular dystrophy patients is affected by gross genomic instability, represented by DNA double-strand breaks and age-related accumulation of aneusomies. These novel aspects of molecular pathologies common to muscular dystrophies and tumor biology will potentially influence the strategies to combat these diseases.     

Calpain 3 is activated through autolysis within the active site and lyses sarcomeric and sarcolemmal components

Calpain 3 (Capn3) is known as the skeletal muscle-specific member of the calpains, a family of intracellular nonlysosomal cysteine proteases. This enigmatic protease has many unique features among the calpain family and, importantly, mutations in Capn3 have been shown to be responsible for limb girdle muscular dystrophy type 2A. Here we demonstrate that the Capn3 activation mechanism is similar to the universal activation of caspases and corresponds to an autolysis within the active site of the protease. We undertook a search for substrates in immature muscle cells, as several lines of evidence suggest that Capn3 is mostly in an inactive state in muscle and needs a signal to be activated. In this model, Capn3 proteolytic activity leads to disruption of the actin cytoskeleton and disorganization of focal adhesions through cleavage of several endogenous proteins. In addition, we show that titin, a previously identified Capn3 partner, and filamin C are further substrates of Capn3. Finally, we report that Capn3 colocalizes in vivo with its substrates at various sites along cytoskeletal structures. We propose that Capn3-mediated cleavage produces an adaptive response of muscle cells to external and/or internal stimuli, establishing Capn3 as a muscle cytoskeleton regulator.     

Animal Models for Muscular Dystrophy Show Different Patterns of Sarcolemmal Disruption

Genetic defects in a number of components of the dystrophin–glycoprotein complex (DGC) lead to distinct forms of muscular dystrophy. However, little is known about how alterations in the DGC are manifested in the pathophysiology present in dystrophic muscle tissue. One hypothesis is that the DGC protects the sarcolemma from contraction-induced damage. Using tracer molecules, we compared sarcolemmal integrity in animal models for muscular dystrophy and in muscular dystrophy patient samples. Evans blue, a low molecular weight diazo dye, does not cross into skeletal muscle fibers in normal mice. In contrast, mdx mice, a dystrophin-deficient animal model for Duchenne muscular dystrophy, showed significant Evans blue accumulation in skeletal muscle fibers. We also studied Evans blue dispersion in transgenic mice bearing different dystrophin mutations, and we demonstrated that cytoskeletal and sarcolemmal attachment of dystrophin might be a necessary requirement to prevent serious fiber damage. The extent of dye incorporation in transgenic mice correlated with the phenotypic severity of similar dystrophin mutations in humans. We furthermore assessed Evans blue incorporation in skeletal muscle of the dystrophia muscularis (dy/dy) mouse and its milder allelic variant, the dy^2J/dy^2J mouse, animal models for congenital muscular dystrophy. Surprisingly, these mice, which have defects in the laminin α2-chain, an extracellular ligand of the DGC, showed little Evans blue accumulation in their skeletal muscles. Taken together, these results suggest that the pathogenic mechanisms in congenital muscular dystrophy are different from those in Duchenne muscular dystrophy, although the primary defects originate in two components associated with the same protein complex.     

Macrophage ABCA5 deficiency influences cellular cholesterol efflux and increases susceptibility to atherosclerosis in female LDLr knockout mice

Objectives

To determine the role of macrophage ATP-binding cassette transporter A5 (ABCA5) in cellular cholesterol homeostasis and atherosclerotic lesion development.

Methods and results

Chimeras with dysfunctional macrophage ABCA5 (ABCA5^−M/−M) were generated by transplantation of bone marrow from ABCA5 knockout (ABCA5^−/−) mice into irradiated LDLr^−/− mice. In vitro, bone marrow-derived macrophages from ABCA5^−M/−M chimeras exhibited a 29% (P < 0.001) decrease in cholesterol efflux to HDL, whereas a 21% (P = 0.07) increase in cholesterol efflux to apoA-I was observed. Interestingly, expression of ABCA1, but not ABCG1, was up-regulated in absence of functional ABCA5 in macrophages. To induce atherosclerosis, the transplanted LDLr^−/− mice were fed a high-cholesterol Western-type diet (WTD) for 6, 10, or 18 weeks, allowing analysis of effects on initial as well as advanced lesion development. Atherosclerosis development was not affected in male ABCA5^−M/−M chimeras after 6, 10, and 18 weeks WTD feeding. However, female ABCA5^−M/−M chimeras did develop significantly (P < 0.05) larger aortic root lesions as compared with female controls after 6 and 10 weeks WTD feeding.

Conclusions

ABCA5 influences macrophage cholesterol efflux, and selective disruption of ABCA5 in macrophages leads to increased atherosclerotic lesion development in female LDLr^−/− mice.     

The influence of 5-lipoxygenase on cigarette smoke-induced emphysema in mice

Background

Pulmonary emphysema is characterized by the loss of lung architecture. Our hypothesis is that the inhibition of 5-lipoxygenase (5-LO) production may be an important strategy to reduce inflammation, oxidative stress, and metalloproteinases in lung tissue resulting from cigarette smoke (CS)-induced emphysema.

Methods

5-LO knockout (129S2-Alox5^tm1Fun/J) and wild-type (WT) mice (129S2/SvPas) were exposed to CS for 60 days. Mice exposed to ambient air were used as Controls. Oxidative, inflammatory, and proteolytic markers were analyzed.

Results

The alveolar diameter was decreased in CS 5-LO^−/− mice when compared with the WT CS group. The CS exposure resulted in less pronounced pulmonary inflammation in the CS 5-LO^−/− group. The CS 5-LO^−/− group showed leukotriene B4 values comparable to those of the Control group. The expression of MMP-9 was decreased in the CS 5-LO^−/− group when compared with the CS WT group. The expression of superoxide dismutase, catalase, and glutathione peroxidase were decreased in the CS 5-LO^−/− group when compared with the Control group. The protein expression of nuclear factor (erythroid-derived 2)-like 2 was reduced in the CS 5-LO^−/− group when compared to the CS WT group.

Conclusion

In conclusion, we show for the first time that 5-LO deficiency protects 129S2 mice against emphysema caused by CS. We suggest that the main mechanism of pathogenesis in this model involves the imbalance between proteases and antiproteases, particularly the association between MMP-9 and TIMP-1.General significanceThis study demonstrates the influence of 5-LO mediated oxidative stress, inflammation, and proteolytic markers in CS exposed mice.     

Mutations in GDP-Mannose Pyrophosphorylase B Cause Congenital and Limb-Girdle Muscular Dystrophies Associated with Hypoglycosylation of α-Dystroglycan

Congenital muscular dystrophies with hypoglycosylation of α-dystroglycan (α-DG) are a heterogeneous group of disorders often associated with brain and eye defects in addition to muscular dystrophy. Causative variants in 14 genes thought to be involved in the glycosylation of α-DG have been identified thus far. Allelic mutations in these genes might also cause milder limb-girdle muscular dystrophy phenotypes. Using a combination of exome and Sanger sequencing in eight unrelated individuals, we present evidence that mutations in guanosine diphosphate mannose (GDP-mannose) pyrophosphorylase B (GMPPB) can result in muscular dystrophy variants with hypoglycosylated α-DG. GMPPB catalyzes the formation of GDP-mannose from GTP and mannose-1-phosphate. GDP-mannose is required for O-mannosylation of proteins, including α-DG, and it is the substrate of cytosolic mannosyltransferases. We found reduced α-DG glycosylation in the muscle biopsies of affected individuals and in available fibroblasts. Overexpression of wild-type GMPPB in fibroblasts from an affected individual partially restored glycosylation of α-DG. Whereas wild-type GMPPB localized to the cytoplasm, five of the identified missense mutations caused formation of aggregates in the cytoplasm or near membrane protrusions. Additionally, knockdown of the GMPPB ortholog in zebrafish caused structural muscle defects with decreased motility, eye abnormalities, and reduced glycosylation of α-DG. Together, these data indicate that GMPPB mutations are responsible for congenital and limb-girdle muscular dystrophies with hypoglycosylation of α-DG.     

Prognostic Significance of Capn4 Overexpression in Intrahepatic Cholangiocarcinoma

Background

Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of hepatocellular carcinoma. This study aimed to investigate the role of Capn4 in intrahepatic cholangiocarcinoma (ICC).

Methods

Capn4 expression was measured in 33 ICC tissues by quantitative real-time polymerase chain reaction and western blot. The role of Capn4 in the migration, invasion and proliferation of ICC cells and matrix metalloproteinase 2 (MMP2) expression were assessed after Capn4 depletion by specific small interfering RNA. Capn4 expression was further examined by immunohistochemistry in a tissue microarray consisting of 140 ICC patients and 13 normal liver tissues, and the prognostic role of Capn4 in ICC was evaluated by Kaplan-Meier and Cox regression analyses.

Results

Capn4 expression was significantly higher in the ICC tissues compared to the peritumor tissues. Capn4 down-regulation impaired the migration/invasion ability of HCCC-9810 and QBC939 cells in vitro and decreased MMP2 expression. Capn4 overexpression significantly correlated with the presence of lymphatic metastasis of ICC (p = 0.026) and the tumor-node-metastasis (TNM) stage (p = 0.009). The postoperative 2- and 5-year overall survivals in patients with Capn4^low were higher than those in the Capn4^high group. The cumulative recurrence rate in patients with Capn4^low was much lower than in the Capn4^high group. Multivariate analysis showed that Capn4 overexpression was an independent prognostic marker in ICC.

Conclusions

Capn4 overexpression was implicated in ICC metastasis/invasion, and Capn4 overexpression may be used as a molecular therapeutic target for ICC.     

The muscular dystrophy with myositis (mdm) mouse mutation disrupts a skeletal muscle-specific domain of titin

Muscular dystrophy with myositis (mdm) is a recessive mouse mutation that causes severe and progressive muscular degeneration. Here we report the identification of the mdm mutation as a complex rearrangement that includes a deletion and a LINE insertion in the titin (Ttn) gene. Mutant allele-specific splicing results in the deletion of 83 amino acids from the N2A region of TTN, a domain thought to bind calpain-3 (CAPN3) the product of the human limb-girdle muscular dystrophy type 2A (LGMD2A) gene. The Ttn(mdm) mutant mouse may serve as a model for human tibial muscular dystrophy, which maps to the TTN locus at 2q31 and shows a secondary reduction of CAPN3 similar to that observed in mdm skeletal muscle. This is the first demonstration that a mutation in Ttn is associated with muscular dystrophy and provides a novel animal model to test for functional interactions between TTN and CAPN3.     

m-Calpain is required for preimplantation embryonic development in mice

Background  

μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4 ^-/- mice.     

Contribution of Dysferlin Deficiency to Skeletal Muscle Pathology in Asymptomatic and Severe Dystroglycanopathy Models: Generation of a New Model for Fukuyama Congenital Muscular Dystrophy

Defects in dystroglycan glycosylation are associated with a group of muscular dystrophies, termed dystroglycanopathies, that include Fukuyama congenital muscular dystrophy (FCMD). It is widely believed that abnormal glycosylation of dystroglycan leads to disease-causing membrane fragility. We previously generated knock-in mice carrying a founder retrotransposal insertion in fukutin, the gene responsible for FCMD, but these mice did not develop muscular dystrophy, which hindered exploring therapeutic strategies. We hypothesized that dysferlin functions may contribute to muscle cell viability in the knock-in mice; however, pathological interactions between glycosylation abnormalities and dysferlin defects remain unexplored. To investigate contributions of dysferlin deficiency to the pathology of dystroglycanopathy, we have crossed dysferlin-deficient dysferlin ^sjl/sjl mice to the fukutin-knock-in fukutin ^Hp/− and Large-deficient Large ^myd/myd mice, which are phenotypically distinct models of dystroglycanopathy. The fukutin ^Hp/− mice do not show a dystrophic phenotype; however, (dysferlin ^sjl/sjl: fukutin ^Hp/−) mice showed a deteriorated phenotype compared with (dysferlin ^sjl/sjl: fukutin ^Hp/+) mice. These data indicate that the absence of functional dysferlin in the asymptomatic fukutin ^Hp/− mice triggers disease manifestation and aggravates the dystrophic phenotype. A series of pathological analyses using double mutant mice for Large and dysferlin indicate that the protective effects of dysferlin appear diminished when the dystrophic pathology is severe and also may depend on the amount of dysferlin proteins. Together, our results show that dysferlin exerts protective effects on the fukutin ^Hp/− FCMD mouse model, and the (dysferlin ^sjl/sjl: fukutin ^Hp/−) mice will be useful as a novel model for a recently proposed antisense oligonucleotide therapy for FCMD.     

Na+ Dysregulation Coupled with Ca2+ Entry through NCX1 Promotes Muscular Dystrophy in Mice

Unregulated Ca²⁺ entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na⁺-Ca²⁺ exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd^−/−), Dysf^−/−, and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd^−/− mice. Measured increases in baseline Na⁺ and Ca²⁺ in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca²⁺ influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca²⁺ levels. Indeed, Atp1a2^+/− (encoding Na⁺-K⁺ ATPase α2) mice, which have reduced Na⁺ clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na⁺-K⁺ ATPase inhibitor digoxin. Treatment of Sgcd^−/− mice with ranolazine, a broadly acting Na⁺ channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.