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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

Increased protein degradation of SERCA1 and SERCA2 in CAPN3-deficient LHCN-M2 humanmyotubes. (a) CAPN3 and SERCA mRNA levels were analysed in human myotubes byquantitative RT–PCR. Statistical analysis showed significant decrease in CAPN3expression (19.3 ± 2.6%) in CAPN3 knockdown myotubes as compared to matched controls(100 ± 12.8%). N = 3; *P < 0.005. Nosignificant changes were observed in SERCA1, SERCA2 or SERCA3 mRNA expressionlevels. (b) SERCA1 and (c) SERCA2 ubiquitination and sumoylation were analysed incontrol and CAPN3-deficient human myotubes through SERCA1/2 immunoprecipitation withspecific mouse monoclonal antibodies. Pools of control and CAPN3-deficient cultureswere used for immunoprecipitation assays. N = 2 andN = 3 independent experiments were performed for SERCA1 and SERCA2,respectively. White lines indicate noncontiguous lanes run on the same gel.Ubiquitination of SERCA1 and SERCA2 in CAPN3-deficient myotubes was increased 3.30and 4.35-fold, respectively, compared with controls. No SUMO1 specific sumoylationof SERCA1 and SERCA2 proteins was detected in controls or CAPN3-deficientmyotubes.
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fig06: Increased protein degradation of SERCA1 and SERCA2 in CAPN3-deficient LHCN-M2 humanmyotubes. (a) CAPN3 and SERCA mRNA levels were analysed in human myotubes byquantitative RT–PCR. Statistical analysis showed significant decrease in CAPN3expression (19.3 ± 2.6%) in CAPN3 knockdown myotubes as compared to matched controls(100 ± 12.8%). N = 3; *P < 0.005. Nosignificant changes were observed in SERCA1, SERCA2 or SERCA3 mRNA expressionlevels. (b) SERCA1 and (c) SERCA2 ubiquitination and sumoylation were analysed incontrol and CAPN3-deficient human myotubes through SERCA1/2 immunoprecipitation withspecific mouse monoclonal antibodies. Pools of control and CAPN3-deficient cultureswere used for immunoprecipitation assays. N = 2 andN = 3 independent experiments were performed for SERCA1 and SERCA2,respectively. White lines indicate noncontiguous lanes run on the same gel.Ubiquitination of SERCA1 and SERCA2 in CAPN3-deficient myotubes was increased 3.30and 4.35-fold, respectively, compared with controls. No SUMO1 specific sumoylationof SERCA1 and SERCA2 proteins was detected in controls or CAPN3-deficientmyotubes.

Mentions: Next, we sought to determine if CAPN3 deficiency affected transcription of ATP2A1 andATP2A2 genes using real-time quantitative RT–PCR. Interestingly, we found that mRNA levelsof SERCA1, SERCA2 and SERCA3 in CAPN3-deficient myotubes were comparable with mRNAexpression in control samples, while CAPN3 mRNA levels were significantly reduced to 20%of control levels (Fig. 6a). This indicates thatthe reduced SERCA protein levels found in CAPN3-deficient myotubes are most likely becauseof protein degradation. Figure 6.


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)

Increased protein degradation of SERCA1 and SERCA2 in CAPN3-deficient LHCN-M2 humanmyotubes. (a) CAPN3 and SERCA mRNA levels were analysed in human myotubes byquantitative RT–PCR. Statistical analysis showed significant decrease in CAPN3expression (19.3 ± 2.6%) in CAPN3 knockdown myotubes as compared to matched controls(100 ± 12.8%). N = 3; *P < 0.005. Nosignificant changes were observed in SERCA1, SERCA2 or SERCA3 mRNA expressionlevels. (b) SERCA1 and (c) SERCA2 ubiquitination and sumoylation were analysed incontrol and CAPN3-deficient human myotubes through SERCA1/2 immunoprecipitation withspecific mouse monoclonal antibodies. Pools of control and CAPN3-deficient cultureswere used for immunoprecipitation assays. N = 2 andN = 3 independent experiments were performed for SERCA1 and SERCA2,respectively. White lines indicate noncontiguous lanes run on the same gel.Ubiquitination of SERCA1 and SERCA2 in CAPN3-deficient myotubes was increased 3.30and 4.35-fold, respectively, compared with controls. No SUMO1 specific sumoylationof SERCA1 and SERCA2 proteins was detected in controls or CAPN3-deficientmyotubes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4836212&req=5

fig06: Increased protein degradation of SERCA1 and SERCA2 in CAPN3-deficient LHCN-M2 humanmyotubes. (a) CAPN3 and SERCA mRNA levels were analysed in human myotubes byquantitative RT–PCR. Statistical analysis showed significant decrease in CAPN3expression (19.3 ± 2.6%) in CAPN3 knockdown myotubes as compared to matched controls(100 ± 12.8%). N = 3; *P < 0.005. Nosignificant changes were observed in SERCA1, SERCA2 or SERCA3 mRNA expressionlevels. (b) SERCA1 and (c) SERCA2 ubiquitination and sumoylation were analysed incontrol and CAPN3-deficient human myotubes through SERCA1/2 immunoprecipitation withspecific mouse monoclonal antibodies. Pools of control and CAPN3-deficient cultureswere used for immunoprecipitation assays. N = 2 andN = 3 independent experiments were performed for SERCA1 and SERCA2,respectively. White lines indicate noncontiguous lanes run on the same gel.Ubiquitination of SERCA1 and SERCA2 in CAPN3-deficient myotubes was increased 3.30and 4.35-fold, respectively, compared with controls. No SUMO1 specific sumoylationof SERCA1 and SERCA2 proteins was detected in controls or CAPN3-deficientmyotubes.
Mentions: Next, we sought to determine if CAPN3 deficiency affected transcription of ATP2A1 andATP2A2 genes using real-time quantitative RT–PCR. Interestingly, we found that mRNA levelsof SERCA1, SERCA2 and SERCA3 in CAPN3-deficient myotubes were comparable with mRNAexpression in control samples, while CAPN3 mRNA levels were significantly reduced to 20%of control levels (Fig. 6a). This indicates thatthe reduced SERCA protein levels found in CAPN3-deficient myotubes are most likely becauseof protein degradation. Figure 6.

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