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Calpain 3 deficiency affects SERCA expression and function in the skeletal muscle.

Toral-Ojeda I, Aldanondo G, Lasa-Elgarresta J, Lasa-Fernández H, Fernández-Torrón R, López de Munain A, Vallejo-Illarramendi A - Expert Rev Mol Med (2016)

Bottom Line: In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes.Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes.Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Area,Biodonostia Research Institute,San Sebastian,Spain.

ABSTRACT
Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

No MeSH data available.


Related in: MedlinePlus

CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 μm. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRα2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls (n = 3,*P < 0.05). D-F) Ca2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca2+] anddelayed Ca2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca2+] was measured in the presence of 2 mmCa2+ at 37°C. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca2+] compared with controls(*P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios(F340/F380) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 130 mm in the absence of extracellularCa2+. (f) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.*P < 0.05, n= total number of myotubesrecorded from three different experiments are shown in the graph.
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fig02: CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 μm. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRα2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls (n = 3,*P < 0.05). D-F) Ca2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca2+] anddelayed Ca2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca2+] was measured in the presence of 2 mmCa2+ at 37°C. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca2+] compared with controls(*P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios(F340/F380) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 130 mm in the absence of extracellularCa2+. (f) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.*P < 0.05, n= total number of myotubesrecorded from three different experiments are shown in the graph.

Mentions: Next, we used the human myoblast line LHCN-M2 (Ref. 11) to study the effect of CAPN3 knockdown on SERCA1/2 expression. LHCN-M2myoblasts were treated with a human specific CAPN3-shRNA or the NS-shRNA, and allowed todifferentiate for 9–14 days. We did not observe any difference in proliferation ordifferentiation between myoblasts treated with the different shRNAs (Fig. 2a, Fig. S7). CAPN3-shRNA treated myotubes (CAPN3-sh) showed aclear reduction of CAPN3 protein levels compared with controls (NS-sh), as determined byWestern blot analysis. Similar to C2C12 myotubes, knocking down CAPN3 in human myotubesresulted in decreased levels of SERCA1, SERCA2 and RyR1 proteins, while DHPR, MyHC,α-sarcoglycan and actin protein levels remained unchanged (Fig. 2b, Fig. S2). Figure 2.


Calpain 3 deficiency affects SERCA expression and function in the skeletal muscle.

Toral-Ojeda I, Aldanondo G, Lasa-Elgarresta J, Lasa-Fernández H, Fernández-Torrón R, López de Munain A, Vallejo-Illarramendi A - Expert Rev Mol Med (2016)

CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 μm. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRα2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls (n = 3,*P < 0.05). D-F) Ca2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca2+] anddelayed Ca2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca2+] was measured in the presence of 2 mmCa2+ at 37°C. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca2+] compared with controls(*P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios(F340/F380) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 130 mm in the absence of extracellularCa2+. (f) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.*P < 0.05, n= total number of myotubesrecorded from three different experiments are shown in the graph.
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fig02: CAPN3 deficiency in human myotubes reduces SERCA protein levels and SERCA function.(a) LHCN-M2 myoblasts treated with control NS-shRNA or CAPN3-shRNAs anddifferentiated for 9 days. Scale bar = 50 μm. (b) Representative Western blotanalysis showing decrease of CAPN3 (12A2 mAb), SERCA1, SERCA2 and RyR1 proteinlevels in CAPN3-sh treated myotubes compared with controls. DHPRα2, MyHC and actinlevels remain unaltered. (c) SERCA-specific ATPase activity determined inhomogenates from LHCN-M2 myotubes. CAPN3-deficient myotubes show a significantreduction of SERCA activity compared with NS-controls (n = 3,*P < 0.05). D-F) Ca2+ imaging of LHCN-M2myotubes loaded with Fura2-AM shows increased cytosolic [Ca2+] anddelayed Ca2+ clearance from the cytosol in CAPN3 knockdown myotubes. (d)Resting cytosolic [Ca2+] was measured in the presence of 2 mmCa2+ at 37°C. CAPN3-deficient human myotubes show significantlyincreased resting cytosolic [Ca2+] compared with controls(*P < 0.05; n = 10 experiments). Totalnumbers of myotubes recorded are shown in the graph. (e) Two representative tracesof changes in Fura2-AM fluorescence ratios(F340/F380) fromCAPN3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 130 mm in the absence of extracellularCa2+. (f) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in CAPN3-deficient myotubes.*P < 0.05, n= total number of myotubesrecorded from three different experiments are shown in the graph.
Mentions: Next, we used the human myoblast line LHCN-M2 (Ref. 11) to study the effect of CAPN3 knockdown on SERCA1/2 expression. LHCN-M2myoblasts were treated with a human specific CAPN3-shRNA or the NS-shRNA, and allowed todifferentiate for 9–14 days. We did not observe any difference in proliferation ordifferentiation between myoblasts treated with the different shRNAs (Fig. 2a, Fig. S7). CAPN3-shRNA treated myotubes (CAPN3-sh) showed aclear reduction of CAPN3 protein levels compared with controls (NS-sh), as determined byWestern blot analysis. Similar to C2C12 myotubes, knocking down CAPN3 in human myotubesresulted in decreased levels of SERCA1, SERCA2 and RyR1 proteins, while DHPR, MyHC,α-sarcoglycan and actin protein levels remained unchanged (Fig. 2b, Fig. S2). Figure 2.

Bottom Line: In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes.Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes.Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Neuroscience Area,Biodonostia Research Institute,San Sebastian,Spain.

ABSTRACT
Limb-girdle muscular dystrophy type 2A (LGMD2A) is a form of muscular dystrophy caused by mutations in calpain 3 (CAPN3). Several studies have implicated Ca2+ dysregulation as an underlying event in several muscular dystrophies, including LGMD2A. In this study we used mouse and human myotube cultures, and muscle biopsies in order to determine whether dysfunction of sarco/endoplasmatic Ca2+-ATPase (SERCA) is involved in the pathology of this disease. In CAPN3-deficient myotubes, we found decreased levels of SERCA 1 and 2 proteins, while mRNA levels remained comparable with control myotubes. Also, we found a significant reduction in SERCA function that resulted in impairment of Ca2+ homeostasis, and elevated basal intracellular [Ca2+] in human myotubes. Furthermore, small Ankyrin 1 (sAnk1), a SERCA1-binding protein that is involved in sarcoplasmic reticulum integrity, was also diminished in CAPN3-deficient fibres. Interestingly, SERCA2 protein was patently reduced in muscles from LGMD2A patients, while it was normally expressed in other forms of muscular dystrophy. Thus, analysis of SERCA2 expression may prove useful for diagnostic purposes as a potential indicator of CAPN3 deficiency in muscle biopsies. Altogether, our results indicate that CAPN3 deficiency leads to degradation of SERCA proteins and Ca2+ dysregulation in the skeletal muscle. While further studies are needed in order to elucidate the specific contribution of SERCA towards muscle degeneration in LGMD2A, this study constitutes a reasonable foundation for the development of therapeutic approaches targeting SERCA1, SERCA2 or sAnk1.

No MeSH data available.


Related in: MedlinePlus