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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 mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100  μm (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (*P < 0.05;**P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls (n = 3,*P < 0.05). (d and e) Ca2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios(F340/F380) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 50 mm in the absence of extracellularCa2+. (e) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.**P < 0.01, n= total number of myotubesrecorded from six different experiments are shown in the graph.
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fig01: Capn3 deficiency in mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100  μm (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (*P < 0.05;**P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls (n = 3,*P < 0.05). (d and e) Ca2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios(F340/F380) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 50 mm in the absence of extracellularCa2+. (e) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.**P < 0.01, n= total number of myotubesrecorded from six different experiments are shown in the graph.

Mentions: To better understand the role of CAPN3 in the Ca2+ homeostasis of skeletalmuscle, we used lentiviral shRNAs to knock down Capn3 expression in mouse C2C12 myotubes.No obvious morphological differences were found between Capn3 knockdown C2C12 myotubes andmyotubes treated with a nonsilencing (NS) shRNA lentivirus (Fig. 1a, Fig. S7A). Infection with Capn3-shRNA lentivirus caused a70% reduction of Capn3 protein in C2C12 differentiated myotubes, as assessed by Westernblot (Fig. 1b, P ≤ 0.01). Capn3knockdown resulted in a significant decrease in the expression levels of several keyCa2+-handling proteins, such as RyR1 (40.9 ± 13.1%), SERCA1 (13 ± 7.9%) andSERCA2 (17.5 ± 2.9%, P ≤ 0.05) compared with controls (100%,n ≥ 3). In contrast, levels of the dihydropyridine receptor DHPR alpha2subunit or the mechanosensitive voltage-independent Ca2+ channel TRPC-1 werefound unchanged. Therefore, our findings support our hypothesis of Capn3 deficiencycausing a reduction of SERCA1/2 expression levels, which in turn may lead to a generaldysregulation of Ca2+ homeostasis. Figure 1.


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 mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100  μm (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (*P < 0.05;**P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls (n = 3,*P < 0.05). (d and e) Ca2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios(F340/F380) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 50 mm in the absence of extracellularCa2+. (e) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.**P < 0.01, n= total number of myotubesrecorded from six different experiments are shown in the graph.
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fig01: Capn3 deficiency in mouse C2C12 myotubes reduces SERCA protein levels and SERCAfunction. (a) C2C12 myotubes treated with NS or Capn3 shRNAs and differentiated for7 days. Scale bar = 100  μm (b) Western blot analysis showing significant decreaseof Capn3 (SPA antibody), SERCA1, SERCA2 and RyR1 levels in Capn3 knockdown myotubes,compared with controls (*P < 0.05;**P < 0.01). Total levels of DHPR, TRPC1, MyHC and actin arenot significantly changed. N = 3 independent experiments run on thesame gel. (c) SERCA-specific ATPase activity determined in homogenates from C2C12myotubes. Capn3-deficient myotubes show a significant reduction of SERCA activitycompared with NS-controls (n = 3,*P < 0.05). (d and e) Ca2+ imaging of C2C12myotubes loaded with Fura2-AM shows delayed Ca2+ clearance from thecytosol in Capn3 knockdown myotubes. (d) Two representative traces of changes inFura2-AM fluorescence ratios(F340/F380) fromCapn3-shRNA and NS-shRNA treated myotubes. Ca2+ transients were elicitedby local stimulation with KCl 50 mm in the absence of extracellularCa2+. (e) Tau, the time constant of the Ca2+ transient decayphase in seconds (s), is significantly increased in Capn3-deficient myotubes.**P < 0.01, n= total number of myotubesrecorded from six different experiments are shown in the graph.
Mentions: To better understand the role of CAPN3 in the Ca2+ homeostasis of skeletalmuscle, we used lentiviral shRNAs to knock down Capn3 expression in mouse C2C12 myotubes.No obvious morphological differences were found between Capn3 knockdown C2C12 myotubes andmyotubes treated with a nonsilencing (NS) shRNA lentivirus (Fig. 1a, Fig. S7A). Infection with Capn3-shRNA lentivirus caused a70% reduction of Capn3 protein in C2C12 differentiated myotubes, as assessed by Westernblot (Fig. 1b, P ≤ 0.01). Capn3knockdown resulted in a significant decrease in the expression levels of several keyCa2+-handling proteins, such as RyR1 (40.9 ± 13.1%), SERCA1 (13 ± 7.9%) andSERCA2 (17.5 ± 2.9%, P ≤ 0.05) compared with controls (100%,n ≥ 3). In contrast, levels of the dihydropyridine receptor DHPR alpha2subunit or the mechanosensitive voltage-independent Ca2+ channel TRPC-1 werefound unchanged. Therefore, our findings support our hypothesis of Capn3 deficiencycausing a reduction of SERCA1/2 expression levels, which in turn may lead to a generaldysregulation of Ca2+ homeostasis. Figure 1.

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