Mitigation of muscular dystrophy in mice by SERCA overexpression in skeletal muscle (original) (raw)
Generation of SERCA1 Tg mice. Increased ryanodine receptor (RyR) Ca2+ leak and reduced SR Ca2+ cycling are thought to contribute to MD pathogenesis (16, 18–20). Moreover, diminished SERCA1 activity characteristic of dystrophic muscle should exacerbate a Ca2+ overload problem associated with an unstable sarcolemma (3, 21, 22). In an attempt to restore and enhance SERCA1 activity and SR Ca2+ reuptake, we generated a series of Tg mice using the skeletal muscle–specific skeletal α-actin promoter to drive SERCA1. While 12 lines were generated and partially analyzed for protein expression and phenotypic effects, 1 line was selected for in-depth analysis. Western blotting from 1 line showed 2- to 4-fold overexpression of SERCA1 protein across the quadriceps, gastrocnemius, diaphragm, and soleus, while no expression was observed in the heart (Figure 1A). H&E- and Masson’s trichrome–stained histological sections showed no pathological features in the muscles of SERCA1 Tg mice compared with non-Tg littermates, although fiber cross-sectional areas were slightly decreased (Figure 1B). Next, electrically evoked Ca2+ transients were assessed in acutely isolated flexor digitorum brevis (FDB) fibers to determine whether SERCA1 overexpression promotes SR loading and enhanced Ca2+ clearance. As predicted, peak Ca2+ transient amplitudes were significantly increased and the time to decay of the Ca2+ transient was reduced (Figure 1, C, E, and F). Interestingly, the resting Ca2+ ratio was also significantly decreased in FDB fibers isolated from SERCA1 Tg mice compared with non-Tg littermates (Figure 1D). Taken together, these results demonstrate that overexpression of SERCA1 in skeletal muscle enhances Ca2+ clearance and cycling. However, expression levels of critical SR Ca2+ handling proteins were not significantly changed by the SERCA1 transgene, as assessed by quantitative Western blotting (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI43844DS1).
Overexpression of SERCA1 in skeletal muscle enhances Ca2+ cycling during EC coupling. (A) Western blot analysis for SERCA1 expression in different muscle groups isolated from non-Tg (NTg) and SERCA1 Tg (Tg) mice at 3 months of age. Quad, quadriceps; Gas, gastrocnemius; Diaph, diaphragm. (B) H&E and Masson’s trichrome sections of quadriceps. Original magnification, ×200. (C) Representative traces of F340/F380 fluorescence ratio recordings from single FDB myofibers isolated from NTg and SERCA1 Tg mice in response to electrical stimulation. (D) Resting Ca2+ ratio, (E) time constant of decay (τ), and (F) peak Ca2+ transient amplitude in isolated myofibers from the indicated genotypes. *P < 0.05 compared with NTg mice; n = total number of fibers recorded from 4 animals in each genotype shown in the graphs, D–F.
SERCA1 overexpression ameliorates MD in δ-sarcoglycan null mice. To assess the potential beneficial effect of SERCA1 overexpression in the context of MD, we crossed the SERCA1 transgene into the δ-sarcoglycan null (Sgcd–/–) genetic background, the latter of which is a mouse model with fulminant dystrophic disease (23, 24). The presence of the SERCA1 transgene resulted in a remarkable reduction in histopathology in Sgcd–/– mice at 6 weeks, 3 months, and 6 months of age, showing severe reductions in myofibers with centrally located nuclei and overall rates of fibrosis compared with Sgcd–/– only controls (Figure 2, A–F). In addition, serum creatine kinase (CK) levels were significantly reduced in Sgcd–/– mice with the SERCA1 transgene compared with Sgcd–/– littermates (Figure 2H). Associated with this improvement in histopathology, Sgcd–/– mice with the SERCA1 transgene showed a restoration in their ability to run on a treadmill comparable to WT control mice, while Sgcd–/– mice alone were severely compromised (Figure 2G). We also investigated whether sarcolemmal rupture rates and subsequent Evans blue dye (EBD) uptake were affected by the SERCA1 transgene in Sgcd–/– mice. Normally, muscle fibers from Sgcd–/– mice show uptake of EBD after systemic injection due to membrane breaks, which was observed in the quadriceps, gastrocnemius, tibialis anterior (TA), and diaphragm from control null mice only (Figure 2, I and J). However, the presence of the SERCA1 transgene uniformly reduced total EBD uptake in Sgcd–/– mice by 3- to 5-fold (Figure 2, I and J). We interpret this reduction in EBD uptake with the SERCA1 transgene to result from enhanced Ca2+ reuptake, which likely reduces membrane and mitochondrial reactive oxygen species generation that could secondarily diminish membrane stress and a presumed feed-forward mechanism of disease (see discussion). The soleus muscle was not differentially affected, presumably because it is a postural muscle that is always under tension/use (Figure 2J).
SERCA1 mitigates biochemical and histological features of MD in Sgcd–/– mice. (A) H&E- and Masson’s trichrome–stained sections of quadriceps from WT, Sgcd–/–, and _Sgcd–/–_-SERCA1 Tg mice at 3 months of age. Original magnification, ×200. (B and D) Percentage of myofibers with centrally located nuclei in Sgcd–/– and _Sgcd–/–_-SERCA1 Tg mice at 6 weeks and 3 months of age. (C, E, and F) Interstitial fibrosis in muscle histological sections analyzed using metamorph analysis software in Sgcd–/– and _Sgcd–/–_-SERCA1 Tg mice at 6 weeks, 3 months, and 6 months of age. Number of mice used for quantitation is shown in the graphs. (G) Time to fatigue in minutes with forced treadmill running in the indicated groups of mice. (H) Quantitation of serum CK levels in Sgcd–/– and _Sgcd–/–_-SERCA1 Tg mice at 3 months of age. (I) Representative immunofluorescence images of EBD uptake in histological sections from quadriceps of 3-month-old mice subjected to running for 2 days in the presence of EBD. Membranes are stained green while EBD-positive fibers are in red. Original magnification, ×100. (J) Quantitation of total EBD fibers in quadriceps, gastrocnemius, TA, diaphragm, and soleus from at least 3 mice in each genotype. *P < 0.05 versus Sgcd–/–. Number of mice used is shown in each of the graphs.
SERCA1 overexpression ameliorates MD in mdx mice. The mouse model of Duchenne MD with defective dystrophin expression results from the mdx mutation. This model, which corresponds to the most prevalent form of human MD, is typically less severe, and mice can live a near normal lifespan compared with Sgcd–/– mice, which manifest more severe disease with premature lethality (23, 24). Here we crossed the SERCA1 transgene into the mdx genetic background, and similarly to what we observed in the Sgcd–/– background, both histological and biochemical markers of MD were dramatically diminished (Figure 3A). At both 6 weeks and 3 months of age, myofiber central nucleation was profoundly reduced in the quadriceps, diaphragm, and soleus (Figure 3, B and D). In fact, at 6 weeks of age, myofiber central nucleation was nearly extinguished in all 3 muscles analyzed (Figure 3B). While fibrosis typically only becomes prominent in older mdx mice, even the mild degree of tissue fibrosis that begins to develop at 6 weeks and 3 months of age was significantly reduced in skeletal muscle of SERCA1/mdx mice (Figure 3, C and E). Finally, serum CK levels at 3 months of age were also reduced by approximately 4-fold in SERCA1/mdx mice when compared with mdx mice (Figure 3F). These results further support the conclusion that enhanced SERCA1 expression can attenuate histopathology and biochemical features of MD in mouse models of disease.
SERCA1 mitigates biochemical and histological features of MD in mdx mice. (A) H&E- and Masson’s trichrome–stained histological sections from quadriceps of WT, mdx, and _mdx_-SERCA1 Tg mice at 3 months of age. Original magnification, ×200. (B and D) Percentage of myofibers with centrally located nuclei in mdx and _mdx_-SERCA1 Tg mice at 6 weeks and 3 months of age. (C and E) Interstitial fibrosis in muscle histological sections analyzed using metamorph analysis software in mdx and _mdx_-SERCA1 Tg mice at 6 weeks and 3 months of age. Number of mice used for quantitation is shown in the graphs. (F) Quantitation of serum CK levels in mdx and _mdx_-SERCA1 Tg mice at 3 months of age. *P < 0.05 versus mdx. Number of mice used is shown in the graphs.
SERCA1 overexpression ameliorates MD in TRPC3-overexpressing Tg mice. We demonstrated that skeletal muscle–specific overexpression of the ion channel TRPC3 was sufficient to induce skeletal muscle disease that was entirely consistent with MD (10). TRPC channels are thought to contribute to Ca2+ influx in MD associated with destabilization of the sarcolemma. Indeed, overexpression of a dominant negative TRPC6 mutant protein in skeletal muscle blocked Ca2+ leak activity of the sarcolemma and significantly reduced MD disease manifestations in mdx and Sgcd–/– mice (10). Here we intercrossed TRPC3- and SERCA1-overexpressing Tg mice to more directly examine the “Ca2+ hypothesis,” since the only disease-inducing effect associated with the TRPC3 transgene is Ca2+ influx. As predicted, H&E staining of quadriceps and soleus muscle showed dramatically less tissue histopathology at 3 months of age in SERCA1/TRPC3 Tg mice compared with TRPC3 Tg mice (Figure 4A). For example, quantitation of centrally located myofibers showed significant reductions in SERCA1/TRPC3 Tg mice compared with TRPC3 Tg mice (Figure 4B). These results further support the hypothesis that SERCA1 overexpression is protective by augmenting SR Ca2+ handling and counteracting the effect of increased membrane Ca2+ leak in MD.
SERCA1 mitigates histological features of MD in TRPC3 Tg mice. (A) Representative histological H&E stain of quadriceps from TRPC3 Tg and TRPC3/SERCA1 double-Tg at 3 months of age. Original magnification, ×200. (B) Percentage of myofibers with centrally located nuclei in quadriceps and soleus from TRPC3 Tg and TRPC3/SERCA1 double-Tg mice. At least 3 mice from each genotype were used. *P < 0.05 versus TRPC3 TG. Number of mice used is shown in the graph.
Enhanced SR Ca2+ uptake in SERCA1-overexpressing mice. To further investigate the mechanism whereby SERCA1 overexpression can protect against MD, we directly measured SR Ca2+ uptake in lysates from muscle of the relevant genotypes. As predicted, SR Ca2+ uptake was approximately 2-fold higher in WT (Sgcd+/+) mice containing the SERCA1 transgene compared with mice without the SERCA1 transgene (Figure 5, A and C). Consistent with past observations in dystrophic myofibers/myotubes, maximal SR Ca2+ uptake was significantly reduced in the Sgcd–/– mice compared with WT controls (Sgcd+/+) (Figure 5C). More importantly, SR Ca2+ uptake in Sgcd–/– Tg mice was 3-fold greater than in Sgcd–/– mice and activity was restored to levels higher than even that in WT controls (Figure 5, B and C). The calculated EC50 value (pCa) was not significantly different in any of the groups, suggesting that increased expression of SERCA1 did not change its affinity for Ca2+ (data not shown). Collectively, these results indicate that SERCA1 overexpression dramatically enhances SR Ca2+ reuptake capacity and corrects a deficit observed in Sgcd–/– myofibers.
SERCA1 enhances SR Ca2+ uptake in Sgcd–/– mice. (A) Average mean values of SR Ca2+ uptake in Sgcd+/+ (WT) and _Sgcd+/+_-SERCA1 Tg mice as a function of pCa2+. (B) Average mean values of SR Ca2+ uptake in Sgcd–/– and _Sgcd–/–_-SERCA1 Tg mice as a function of pCa2+. (C) Mean maximal velocity determined for each genotype in the SR Ca2+ uptake measurements. *P < 0.05 compared with Sgcd+/+; #P < 0.05 compared with Sgcd–/–. Number of mice used is shown in the graph.
SERCA1 overexpression restores EC-coupling defects observed in dystrophic myofibers. Multiple studies have shown deficits in global EC-coupling in dystrophic muscle fibers (25–27). These include a significant reduction in the peak amplitude of the Ca2+ transient and diminished Ca2+ reuptake into the SR. Here we investigated whether enhancing SR Ca2+ uptake with SERCA1 overexpression could ameliorate the defects in global EC coupling observed in dystrophic myofibers. We assessed electrically evoked Ca2+ transients in acutely isolated individual FDB fibers in Sgcd+/+ and Sgcd–/– mice with and without the SERCA1 transgene. Interestingly, the peak amplitude of the Ca2+ transient was increased in WT (Sgcd+/+) myofibers with the SERCA1 transgene compared with WT alone (Figure 6, A, B, and E). Consistent with results in the literature, the peak amplitude of the Ca2+ transient was reduced in Sgcd–/– myofibers compared with WT fibers, but this decrease was prevented with the SERCA1 transgene (Figure 6, C–E). SR Ca2+ load assessed with 4-chloro-m-cresol (4-CMC, to release SR Ca2+ stores) was not significantly different between Sgcd–/– and WT myofibers (Figure 6G). However, the SERCA1 transgene did significantly elevate SR Ca2+ load in WT myofibers above WT levels, but not in Sgcd–/– myofibers (Figure 6G). More importantly, the reuptake phase of the 4-CMC–induced Ca2+ profile and the decay time of the Ca2+ transient were each significantly prolonged in myofibers from Sgcd–/– mice, and this defect was corrected with the SERCA1 transgene (Figure 6, C, D, and F). Similarly, FDB myofibers from mdx mice also showed a reduction in the amplitude of the Ca2+ transient and an increase in relaxation time, parameters that were again corrected by the SERCA1 transgene (Figure 6, H and I). Taken together, these results demonstrate that dystrophic myofibers have significant defects in SR Ca2+ handling, which can be corrected by SERCA1 expression.
SERCA1 overexpression enhances EC coupling and Ca2+ clearance in Sgcd–/– and mdx myofibers. (A–D) Representative traces of changes in ratiometric fluorescence ratios in acutely isolated FDB fibers from Sgcd–/– mice in response to electrical and chemical stimulation over time. The brackets in C and D show the Ca2+ reuptake characteristics. (E) Amplitude of the Ca2+ transient after electrical twitch stimulation in the indicated genotypes shown in the legend next to F (applies to E–G). (F) Time constant of decay (τ) and (G) maximal response to 4-CMC in the indicated genotypes as assessed with Fura-2 ratio analysis. *P < 0.05 versus Sgcd+/+; #P < 0.05 versus Sgcd–/–. Number of fibers used in E–G is shown in the graphs. (H) Amplitude of the Ca2+ transient after electrical twitch stimulation in FDB myofibers from mdx and control mice as indicated. *P < 0.05 versus WT; #P < 0.05 versus mdx. (I) Time constant of decay (τ) of the Ca2+ transient in FDB myofibers in the indicated mdx and control groups. *P < 0.05 versus WT; #P < 0.05 versus mdx.
SERCA1 overexpression reverses mitochondrial swelling in dystrophic muscle. We recently demonstrated that mitochondria isolated from dystrophic skeletal muscle of Sgcd–/– mice are swollen at baseline and that this swelling can lead to myofiber necrosis (14). Indeed, prevention of Ca2+-mediated mitochondrial swelling and myofiber necrosis by deletion of cyclophilin D or use of Debio-025 in mice (both of which inhibit MPTP) significantly reduced MD in Sgcd–/– and mdx mice (14). Here we hypothesized that the SERCA1 transgene would reduce Ca2+ overload and spare mitochondria from swelling and induction of myofiber necrosis. Mitochondrial swelling was measured in purified preparations in vitro after isolation from muscle; thereafter swelling/shrinking was assessed with exogenous Ca2+ or PEG3350, respectively. As we have previously observed, mitochondria from Sgcd–/– muscle showed a significantly reduced capacity to swell upon Ca2+ addition compared with Sgcd+/+ mitochondria given their already swollen state, although shrinkage was enhanced (Figure 7, A–C). As predicted, this swollen state of mitochondria from Sgcd–/– muscle was rescued by the SERCA1 Tg (Figure 7, A–C). Ca2+ overload in dystrophic skeletal muscle is associated with calpain activation, which can lead to myofiber degeneration and cellular necrosis (12). We observed an increase in calpain enzymatic activity in muscle from Sgcd–/– mice, and importantly, this increase was significantly reduced by the presence of the SERCA1 transgene (Figure 7D). Collectively, these results again demonstrate that the SERCA1 transgene corrects Ca2+ overload, which likely protects the myofibers from cell death by diminishing mitochondrial swelling and calpain activation, reducing myofiber necrosis.
SERCA1 overexpression protects against Ca2+-induced disease indexes in skeletal muscle of Sgcd–/– mice. (A) Mitochondrial swelling after Ca2+ addition (arrow) as a function of increased light scattering (decreased absorbance) from muscle purified mitochondria from Sgcd+/+, _Sgcd+/+_-SERCA1 Tg, Sgcd–/–, and _Sgcd–/–_-SERCA1 Tg mice. (B) Average maximal change in absorbance in response to external Ca2+ in purified mitochondria from the indicated groups. *P < 0.05 compared with Sgcd+/+; #P < 0.05 versus Sgcd–/–. (C) Change in absorbance in response to PEG to show degree of mitochondrial shrinkage in purified mitochondria. *P < 0.05 compared with Sgcd+/+. (D) Calpain enzymatic activity in skeletal muscle of the indicated groups *P < 0.05 compared with Sgcd+/+; #P < 0.05 versus Sgcd–/–. Number of mice used is shown in the bars of each panel.
SERCA2a gene therapy in Sgcd–/– mice mitigates dystrophic disease. The results presented to this point suggest that SERCA overexpression could serve as a “universal” therapy for many diverse forms of MD that are associated with a Ca2+ imbalance or a leaky plasma membrane. Hence, a gene therapeutic strategy to overexpress SERCA protein in dystrophic skeletal muscle could be an exciting new approach to consider to treat patients. Here we injected 3-day-old Sgcd–/– pups with adeno-associated virus 9–SERCA2a (AAV9-SERCA2a) in the left gastrocnemius or AAV9-GFP (control) in the right gastrocnemius with 1010 viral particles each. The AAV9 serotype yields robust expression in muscle, and the SERCA2a protein functions very similarly to SERCA1. Mice were then harvested 6 weeks later for full histological assessment of muscle disease and expression of GFP. GFP expression was observed throughout the gastrocnemius of Sgcd–/– mice (greater than 90% of the fibers; data not shown), and tissue pathology was fulminant in the control injected muscle, as is typically observed (Figure 8A). However, expression of SERCA2a by AAV-mediated gene therapy dramatically attenuated dystrophic disease in the injected gastrocnemius, showing 8-fold less central nucleation and preserved cellular architecture with little signs of necrosis (Figure 8, A and B). Thus, AAV-SERCA gene therapy is an exciting new avenue to potentially pursue for treating human dystrophic or myopathic diseases.
AAV9-SERCA2a gene therapy mitigates disease in Sgcd–/– gastrocnemius. (A) Representative histological H&E stain of gastrocnemius from injected neonates taken 6 weeks later given control AAV9-GFP or experimental AAV9-SERCA2a. Original magnification, ×200. (B) Quantitation of myofiber central nucleation in the 2 groups taken from 4 mice analyzed. *P < 0.05 versus GFP.