Papers by Melissa Spencer
Human Molecular Genetics, 2011
Skeletal muscle, 2016
Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wa... more Mutations in CAPN3 cause limb girdle muscular dystrophy type 2A (LGMD2A), a progressive muscle wasting disease. CAPN3 is a non-lysosomal, Ca-dependent, muscle-specific proteinase. Ablation of CAPN3 (calpain-3 knockout (C3KO) mice) leads to reduced ryanodine receptor (RyR1) expression and abnormal Ca2+/calmodulin-dependent protein kinase II (Ca-CaMKII)-mediated signaling. We previously reported that Ca(2+) release measured by fura2-FF imaging in response to single action potential stimulation was reduced in old C3KO mice; however, the use of field stimulation prevented investigation of the mechanisms underlying this impairment. Furthermore, our prior studies were conducted on older animals, whose muscles showed advanced muscular dystrophy, which prevented us from establishing whether impaired Ca(2+) handling is an early feature of disease. In the current study, we sought to overcome these matters by studying single fibers isolated from young wild-type (WT) and C3KO mice using a low a...
Human molecular genetics, May 8, 2018
Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3)... more Mutations in CAPN3 cause autosomal recessive limb girdle muscular dystrophy 2A. Calpain 3 (CAPN3) is a calcium dependent protease residing in the myofibrillar, cytosolic and triad fractions of skeletal muscle. At the triad, it colocalizes with calcium calmodulin kinase IIβ (CaMKIIβ). CAPN3 knock out mice (C3KO) show reduced triad integrity and blunted CaMKIIβ signaling, which correlates with impaired transcriptional activation of myofibrillar and oxidative metabolism genes in response to running exercise. These data suggest a role for CAPN3 and CaMKIIβ in gene regulation that takes place during adaptation to endurance exercise. To assess whether CAPN3- CaMKIIβ signaling influences skeletal muscle remodeling in other contexts, we subjected C3KO and WT mice to hindlimb unloading and reloading and assessed CaMKIIβ signaling and gene expression by RNA sequencing. After induced atrophy followed by 4 days of reloading, both CaMKIIβ activation and expression of inflammatory and cellular st...
Muscle & …, 2003
Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Altho... more Calpain 3 (C3) is the only muscle-specific member of the calcium-dependent protease family. Although neither its physiological function nor its in vivo substrates are known, C3 must be an important protein for normal muscle function as mutations in the C3 gene result in limb-girdle muscular dystrophy type 2A. Previous reports have shown that the ubiquitous calpains ( and m) proteolyze filamins in nonmuscle cells. This observation suggests that the muscle-specific filamin C (FLNC) is a good candidate substrate for C3. Binding studies using recombinant proteins establish that recombinant C3 and native FLNC can interact. When these two proteins are translated in vitro and incubated together, C3 cleaves the C-terminal portion of FLNC. Cleavage is specific as C3 fails to cleave FLNC lacking its C-terminal hinge and putative dimerization domains. Cotransfection experiments in COS-7 cells confirm that C3 can cleave the C-terminus of FLNC in live cells. The C-terminus of FLNC has been shown to bind the cytoplasmic domains of both ␦and ␥-sarcoglycan. Removal of the last 127 amino acids from FLNC, a protein that mimics FLNC after C3 cleavage, abolishes this interaction with the sarcoglycans. These studies confirm that C3 can cleave FLNC in vitro and suggest that FLNC may be an in vivo substrate for C3, functioning to regulate protein-protein interactions with the sarcoglycans. Thus, calpain-mediated remodeling of cytoskeletal-membrane interactions, such as those that occur during myoblast fusion and muscle repair, may involve regulation of FLNC-sarcoglycan interactions.
The international journal of biochemistry & cell …, 2000
Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their ... more Calpains are a ubiquitous, well-conserved family of calcium-dependent, cysteine proteases. Their function in muscle has received increased interest because of the discoveries that the activation and concentration of the ubiquitous calpains increase in the mouse model of Duchenne muscular dystrophy (DMD), but null mutations of muscle speci®c calpain causes limb girdle muscular dystrophy 2A (LGMD2A). These ®ndings indicate that modulation of calpain activity contributes to muscular dystrophies by disrupting normal regulatory mechanisms in¯uenced by calpains, rather than through a general, nonspeci®c increase in proteolysis. Thus, modulation of calpain activity or expression through pharmacological or molecular genetic approaches may provide therapies for some muscular dystrophies. #
The Journal of Cell Biology, 2001
ystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (N... more ystrophin-deficient muscles experience large reductions in expression of nitric oxide synthase (NOS), which suggests that NO deficiency may influence the dystrophic pathology. Because NO can function as an antiinflammatory and cytoprotective molecule, we propose that the loss of NOS from dystrophic muscle exacerbates muscle inflammation and fiber damage by inflammatory cells. Analysis of transgenic mdx mice that were null mutants for dystrophin, but expressed normal levels of NO in muscle, showed that the normalization of NO production caused large reductions in macrophage concentrations in the mdx muscle. Expression of the NOS transgene in mdx muscle also prevented the majority of muscle membrane injury that is detectable in vivo, and resulted in D large decreases in serum creatine kinase concentrations. Furthermore, our data show that mdx muscle macrophages are cytolytic at concentrations that occur in dystrophic, NOS-deficient muscle, but are not cytolytic at concentrations that occur in dystrophic mice that express the NOS transgene in muscle. Finally, our data show that antibody depletions of macrophages from mdx mice cause significant reductions in muscle membrane injury. Together, these findings indicate that macrophages promote injury of dystrophin-deficient muscle, and the loss of normal levels of NO production by dystrophic muscle exacerbates inflammation and membrane injury in muscular dystrophy.
Neuromuscular Disorders, 2001
Address all correspondence to James G. Tidball,
Journal of Biological Chemistry, 1995
The Journal of physiology, 2002
The Journal of cell biology, 1993
Address all correspondence to James G. Tidball, Anti-PY immunoblots of cells stimulated with PDGF... more Address all correspondence to James G. Tidball, Anti-PY immunoblots of cells stimulated with PDGF for 0, 10, or 30 min shows polypeptides at ,o180, ,o230, and ,o280 kD that contain PY as a result of PDGF stimulation ). Of these polypeptides, the 180-kD band is the strongest sig-
Human molecular genetics, 2002
Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular d... more Reduced sarcolemmal integrity in dystrophin-deficient muscles of mdx mice and Duchenne muscular dystrophy (DMD) patients has been reported to result in altered calcium homeostasis. Previous studies have shown a correlative relationship between calcium-dependent protease (calpain) activity in dystrophic muscle and muscle necrosis, but have not tested whether calpain activation precedes cell death or is a consequence of it. To test a causal relationship between calpain activation and muscle cell death in dystrophin deficiency, mdx mice were generated that overexpress a calpastatin transgene in muscle. Calpastatin (CS) is a specific, endogenous inhibitor of m-and l-calpains that does not inhibit calpain 3 (p94). CS overexpression on a C57/BL 10 background produced no phenotype. Transgenic (Tg) mice crossed with mdx mice were tested for pathological indicators of necrosis, regeneration and membrane damage. Two lines of mice were examined, with different levels of CS overexpression. Both lines of Tg/mdx mice showed reductions in muscle necrosis at 4 weeks of age. These mice had fewer as well as smaller lesions. In addition, one line of mice had significantly less regeneration, indicating a reduction in previous necrosis. The extent of improvement correlated with the level of CS protein expression. Membrane damage, as assessed by procion orange and creatine kinase assays, was unchanged, supporting the idea that calpains act downstream of the primary muscle defect. These data suggest that calpains play an active role in necrotic processes in dystrophic muscle and that inhibition of calpains might provide a good therapeutic option for treatment of DMD.
Journal of the …, 1997
Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to l... more Antibodies that recognise the muscle-specific calpain 3 (CANP3) were used in a 'blind' study to label blots of skeletal muscle from 12 control subjects and from 12 patients with various muscle diseases. Calpain 3 was clearly detected in all control muscle samples analysed, even though some of the muscle had been at room temperature for over an hour before being dissected and snap-frozen. Calpain 3 was also detected in the muscle biopsies from non-LGMD2A patients, but was absent in samples from 3 patients with LGMD2A. These results show that (i) calpain 3 protein can be detected in whole extracts of human muscle, and (ii) that antibodies can be used to differentiate patients with LGMD2A from those with other muscle diseases. This represents an invaluable diagnostic aid since the limb-girdle dystrophies are very difficult to separate on clinical grounds alone. One possible function that was considered for calpain 3 was the post-translational cleavage of the 97 kDa dystroglycan precursor polypeptide into the mature a-and /3-dystroglycan proteins. The @dystroglycan band was the correct size on blots of LGMD2A muscle, indicating that calpain 3 is probably not involved in the post-translational processingof dystroglycan. O
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Papers by Melissa Spencer