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Calcium Influx Is Sufficient to Induce Muscular Dystrophy Through a TRPC-Dependent Mechanism

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Calcium Influx Is Sufficient to Induce Muscular Dystrophy Through a TRPC-Dependent Mechanism Calcium influx is sufficient to induce muscular dystrophy through a TRPC-dependent mechanism Douglas P. Millaya, Sanjeewa A. Goonasekeraa, Michelle A. Sargenta, Marjorie Mailleta, Bruce J. Aronowa, and Jeffery D. Molkentina,b,1 aDepartment of Pediatrics, Cincinnati Children’s Hospital Medical Center, and bHoward Hughes Medical Institute, University of Cincinnati, Cincinnati, OH 45229 Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved September 16, 2009 (received for review June 12, 2009) Muscular dystrophy is a general term encompassing muscle disorders calcium influx truly initiates/mediates myofiber degeneration in that cause weakness and wasting, typically leading to premature MD, nor has definitive proof emerged implicating TRPC chan- death. Membrane instability, as a result of a genetic disruption within nels as mediators of this disease in vivo. the dystrophin-glycoprotein complex (DGC), is thought to induce myofiber degeneration, although the downstream mechanism Results whereby membrane fragility leads to disease remains controversial. Generation of TRPC3 Skeletal Muscle-Specific TG Mice. Here we One potential mechanism that has yet to be definitively proven in generated transgenic mice with TRPC3 overexpression specifically vivo is that unregulated calcium influx initiates disease in dystrophic in skeletal muscle using the skeletal ␣-actin promoter as a means of myofibers. Here we demonstrate that calcium itself is sufficient to elevating calcium influx (Fig. 1A). Multiple founder lines were cause a dystrophic phenotype in skeletal muscle independent of generated and one was selected for analysis that showed approxi- membrane fragility. For example, overexpression of transient recep- mately 6-fold TRPC3 protein overexpression in skeletal muscle, but tor potential canonical 3 (TRPC3) and the associated increase in not in other organs or tissues (Fig. 1B). Immunohistochemistry calcium influx resulted in a phenotype of muscular dystrophy nearly from quadriceps of TRPC3 transgenic mice confirmed that the identical to that observed in DGC-lacking dystrophic disease models, overexpressed protein was appropriately localized to the sarco- including a highly similar molecular signature of gene expression lemma, similar to endogenous TRPC3 in wild-type (WT) muscle changes. Furthermore, transgene-mediated inhibition of TRPC chan- (Fig. 1C). To directly assess TRPC3 activity we measured calcium CELL BIOLOGY nels in mice dramatically reduced calcium influx and dystrophic entry after store depletion in flexor digitorum brevis (FDB) fibers disease manifestations associated with the mdx mutation (dystrophin from 2-month-old WT and TRPC3 TG mice. Fura-2 loaded fibers gene) and deletion of the ␦-sarcoglycan (Scgd) gene. These results were first perfused with calcium-free buffer, and then treated with demonstrate that calcium itself is sufficient to induce muscular dys- EGTA and CPA (SERCA inhibitor) to deplete intracellular cal- trophy in vivo, and that TRPC channels are key disease initiators cium over several minutes. Caffeine was also applied to further downstream of the unstable membrane that characterizes many ensure store depletion. The perfusate was changed to a buffer again types of muscular dystrophy. containing calcium so that store-operated calcium entry (SOCE) could be monitored. A relatively small amount of SOCE was fibrosis ͉ necrosis ͉ degeneration ͉ skeletal muscle detectable in all WT fibers (Fig. 1 D and F). In contrast, FDB fibers from TRPC3 TG mice exhibited a dramatic enhancement in SOCE (Fig. 1 E and F). These results demonstrate the successful gener- number of diverse genetic mutations result in muscular ation of transgenic mice with enhanced TRPC3-dependent calcium dystrophy (MD), although most occur in proteins that either A entry in skeletal muscle. reside within or are associated with the dystrophin-glycoprotein complex (DGC) (1). For example, mutations in the dystrophin TRPC3 TG Mice Develop Severe MD-Like Disease. MD has been gene in Duchenne or Becker MD or mutations in any one of the associated with increased expression and activation of TRPC sarcoglycans can cause destabilization of the muscle cell mem- channels in cultured myofibers or myotubes (18–21), although their brane and a dystrophic phenotype (1, 2). The DGC is a large potential role in vivo as disease mediators has never been examined. protein complex that spans the sarolemma and affixes the basal Remarkably, TRPC3 TG mice showed profound wasting of all lamina outside the cell to the contractile proteins within the cell skeletal muscles examined at 2 months of age by direct measure- and when defective, can lead to membrane damage after con- ment of muscle weights (Fig. 2A), reminiscent of the type of MD traction (3, 4). A defect in the stability of the sarcolemma is observed in Lama2Ϫ/Ϫ mice that also lack a pseudohypertrophy widely accepted as the initiating stimulus underlying most forms phase. However, the soleus muscle showed a mild pseudohyper- of MD, although the next step in the process leading to myofiber trophy phase at 2 months of age that was eventually lost by 6 months degeneration has long been debated. One long-standing hypoth- of age (Fig. 2 A and B). By 6 months of age all other skeletal muscles esis is that defects in the membrane permit calcium entry (5, 6), examined showed even more wasting, while the heart was unaf- thus activating calpains and/or mitochondrial swelling (7–9). fected (Fig. 2B). Histological analysis of muscle from TRPC3 TG However, that intracellular calcium levels are elevated in dys- mice showed profound disease at 2 months of age, characterized by trophic fibers, or even within the subsarcolemmal space, remains increased central nucleation of fibers (due to degeneration/ unproven and controversial (10–14). Another point of conten- regeneration cycles), increased numbers of smaller myofibers, tion relating to the calcium hypothesis, is the route of entry. fibrosis, and infiltration of inflammatory cells (Fig. 2 C, E, and F, Whether calcium enters through ‘‘microtears’’ in the sarco- lemma, or by a more active process involving ion channels remains to be resolved. Indeed, calcium leak channels, stretch- Author contributions: D.P.M. and J.D.M. designed research; D.P.M., S.A.G., M.A.S., and activated channels, receptor-operated channels, and store- M.M. performed research; B.J.A. analyzed data; and D.P.M. and J.D.M. wrote the paper. operated calcium channels have been implicated as calcium The authors declare no conflict of interest. influx pathways in MD (15–18). While the identity of these This article is a PNAS Direct Submission. putative channels has been elusive, the transient receptor po- 1To whom correspondence should be addressed. E-mail: [email protected]. tential canonical (TRPC) family of nonselective cation channels This article contains supporting information online at www.pnas.org/cgi/content/full/ has been proposed. However, it remains unknown if unregulated 0906591106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906591106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 WT TG A skeletal -actin promoter B TRPC3 pA TRPC3 C WT TRPC3 TG GAPDH D 2mM Ca2+ 6 2+ ) 0 Ca EGTA, CPA 5 0 Ca2+ 380 /F 4 Caffeine 340 3 WT 2 1 Ratio (F 0 F Time WT (n=5) 2mM Ca2+ ) 3.5 E TRPC3 TG (n=4) 5 0 Ca2+ EGTA, CPA ) 380 3.0 * 0 Ca2+ /F 2.5 380 4 Caffeine /F 340 2.0 3 1.5 340 TRPC3 TG 2 1.0 ratio (F ratio 0.5 1 0 Ratio (F 0 Time Fig. 1. TRPC3 transgenesis enhances calcium influx. (A) Schematic of the construct used to make TRPC3 TG mice. pA represents polyA sequence. (B) Western blotting for TRPC3 protein in wild-type (WT) and TRPC3 TG mice from quadriceps at 2 months of age. GAPDH was used as a loading control. (C) Immunohis- tochemistry showing TRPC3 localization to the sarcolemma in WT and TRPC3 TG quadriceps. (D and E) A representative trace of fura-2 fluoresence (F340/F380) for store-operated calcium entry (SOCE) from a WT or TRPC3 TG FDB myofiber. The bars above the tracings show the different perfusates that were used in the 3-different stages (see Methods). (F) Quantitation of fura-2 ratio changes in WT and TRPC3 TG myofibers from a total of 5 WT and 4 TRPC3 TG mice were assayed (5–10 fibers analyzed per mouse). *, P Ͻ 0.05 vs. WT. and see Fig. S1 A and B). By 6 months of age histological analysis area distributions, and prominent macrophage infiltration (Fig. 2D showed even more disease characterized by fatty tissue replacement and Fig. S1 C–F). All these features suggest that TRPC3 overex- throughout the muscle, increased fibrosis, greater variations in fiber pression induces dystrophy and not myopathy, although we also A 2 months B 10 6 months WT G WT TRPC3 TG -/- 8 TRPC3 TG 8 C3 TG 6 TRP Scgd WT * 6 * 4 * 4 * 2 2 6 8 MW/TL (mg/mm) 6 9 MW/TL (mg/mm) 0 * 0 Gas. Quad. Sol. Heart Gas. Quad. Sol. Heart C WT TG D WT TG 2 months 6 months E F 50 50 WT WT * TRPC3 TG 40 TRPC3 TG 40 30 * 30 20 * 20 * * % of fibers 10 10 6 8 % central nuclei 6 8 0 0 <300 300-600 600-800 800-1000 >1000 Sol. Gas. Quad. Fiber area (µm2) Fig. 2. TRPC3-dependent calcium influx induces MD-like disease. (A and B) Muscle weights normalized to tibia length from the gastrocnemius (Gas.), quadriceps (Quad.), soleus (sol.), and heart in WT and TRPC3 TG mice at 2 and 6 months of age. *, P Ͻ 0.05 versus WT. Number of mice analyzed is shown in the bars. (C and D) H&E-stained sections of the quadriceps from 2- and 6-month-old WT and TRPC3 TG mice. Images are shown at 400ϫ magnification.
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