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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

Interaction of CAPN3 with SERCA1, SERCA2 and sAnk1 in human skeletal muscle. (a)Immunoprecipitation (IP) of CAPN3 with a goat polyclonal antibody (pIS2C) in avastus lateralis muscle from a healthy donor. Both, SERCA1 andSERCA2 are detected in the CAPN3 IP. White lines indicate noncontiguous lanes run onthe same gel. Input: protein extract. (b) SERCA1 and SERCA2 were immunoprecipitated(IP) with specific monoclonal antibodies in the same muscle. sAnk1 is detected inSERCA1 IP but not in SERCA2 IP. (c) Longitudinal sections from a dorsal human muscleco-immunostained for CAPN3-SERCA1, CAPN3-SERCA2 and CAPN3-sAnk1, showing similardistribution pattern of CAPN3 (green), SERCA1 (red), SERCA2 (red) and sAnk1 (red) inthe panels labelled “Merge”. Scale bar: 10 µm. (d) Co-localisation analysis ofCAPN3, with SERCA1, SERCA2 and sAnk1 in longitudinal sections from human dorsalmuscle using in situ PLA. Red spots represent protein complexes in close proximity(<40 nm). Sections were double-stained for myosin heavy chain (MF20, green).Neg Ctrl, negative control with just one or no primary antibodies. Scale bar:20 µm.
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fig05: Interaction of CAPN3 with SERCA1, SERCA2 and sAnk1 in human skeletal muscle. (a)Immunoprecipitation (IP) of CAPN3 with a goat polyclonal antibody (pIS2C) in avastus lateralis muscle from a healthy donor. Both, SERCA1 andSERCA2 are detected in the CAPN3 IP. White lines indicate noncontiguous lanes run onthe same gel. Input: protein extract. (b) SERCA1 and SERCA2 were immunoprecipitated(IP) with specific monoclonal antibodies in the same muscle. sAnk1 is detected inSERCA1 IP but not in SERCA2 IP. (c) Longitudinal sections from a dorsal human muscleco-immunostained for CAPN3-SERCA1, CAPN3-SERCA2 and CAPN3-sAnk1, showing similardistribution pattern of CAPN3 (green), SERCA1 (red), SERCA2 (red) and sAnk1 (red) inthe panels labelled “Merge”. Scale bar: 10 µm. (d) Co-localisation analysis ofCAPN3, with SERCA1, SERCA2 and sAnk1 in longitudinal sections from human dorsalmuscle using in situ PLA. Red spots represent protein complexes in close proximity(<40 nm). Sections were double-stained for myosin heavy chain (MF20, green).Neg Ctrl, negative control with just one or no primary antibodies. Scale bar:20 µm.

Mentions: In view of the similar localisation pattern of CAPN3, SERCA1, SERCA2 and sAnk1 around theZ-discs and M-lines (Refs 20, 21), we wanted to assess if these proteins interactedwithin the skeletal muscle. First, we performed co-immunoprecipitation assays usingprotein extracts from the vastus lateralis of a healthy human donor.Indeed, we found that both SERCA1 and SERCA2 proteins co-immunoprecipitated with CAPN3(Fig. 5a), which is in line with previousstudies showing interaction between SERCA1 and Capn3 (Ref. 8). Absence of cross-reactivity between SERCA1 and SERCA2 antibodieswas demonstrated by specific detection of each isoform in the immunoprecipitates (Fig. 5b). Interestingly, we found that sAnk1co-immunoprecipitated with SERCA1 but not with SERCA2 (Fig. 5b). Next, we examined the distribution of CAPN3, SERCA1, SERCA2 and sAnk1in the human skeletal muscle by immunofluorescence and we observed a highly similarlocalisation pattern and a broad co-localisation of CAPN3 with SERCA1, SERCA2 and sAnk1(Fig. 5c). To further validate interaction ofthese proteins, we used the in situ PLA, which reveals protein complexes (<40 nmdistance) at a single molecule resolution within the cellular context (Ref. 22). This analysis showed that CAPN3 interacts withSERCA1, SERCA2 and sAnk1 in the human skeletal muscle (Fig. 5d). Also, co-localisation of SERCA1 and sAnk1 was observed in the humanmuscle. Overall, our results indicate that CAPN3 and SERCA1/2 proteins form molecularcomplexes within the skeletal muscle, with a differential contribution of sAnk1 to thecomplexes formed by SERCA1 or SERCA2. Figure 5.


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)

Interaction of CAPN3 with SERCA1, SERCA2 and sAnk1 in human skeletal muscle. (a)Immunoprecipitation (IP) of CAPN3 with a goat polyclonal antibody (pIS2C) in avastus lateralis muscle from a healthy donor. Both, SERCA1 andSERCA2 are detected in the CAPN3 IP. White lines indicate noncontiguous lanes run onthe same gel. Input: protein extract. (b) SERCA1 and SERCA2 were immunoprecipitated(IP) with specific monoclonal antibodies in the same muscle. sAnk1 is detected inSERCA1 IP but not in SERCA2 IP. (c) Longitudinal sections from a dorsal human muscleco-immunostained for CAPN3-SERCA1, CAPN3-SERCA2 and CAPN3-sAnk1, showing similardistribution pattern of CAPN3 (green), SERCA1 (red), SERCA2 (red) and sAnk1 (red) inthe panels labelled “Merge”. Scale bar: 10 µm. (d) Co-localisation analysis ofCAPN3, with SERCA1, SERCA2 and sAnk1 in longitudinal sections from human dorsalmuscle using in situ PLA. Red spots represent protein complexes in close proximity(<40 nm). Sections were double-stained for myosin heavy chain (MF20, green).Neg Ctrl, negative control with just one or no primary antibodies. Scale bar:20 µm.
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Related In: Results  -  Collection

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

fig05: Interaction of CAPN3 with SERCA1, SERCA2 and sAnk1 in human skeletal muscle. (a)Immunoprecipitation (IP) of CAPN3 with a goat polyclonal antibody (pIS2C) in avastus lateralis muscle from a healthy donor. Both, SERCA1 andSERCA2 are detected in the CAPN3 IP. White lines indicate noncontiguous lanes run onthe same gel. Input: protein extract. (b) SERCA1 and SERCA2 were immunoprecipitated(IP) with specific monoclonal antibodies in the same muscle. sAnk1 is detected inSERCA1 IP but not in SERCA2 IP. (c) Longitudinal sections from a dorsal human muscleco-immunostained for CAPN3-SERCA1, CAPN3-SERCA2 and CAPN3-sAnk1, showing similardistribution pattern of CAPN3 (green), SERCA1 (red), SERCA2 (red) and sAnk1 (red) inthe panels labelled “Merge”. Scale bar: 10 µm. (d) Co-localisation analysis ofCAPN3, with SERCA1, SERCA2 and sAnk1 in longitudinal sections from human dorsalmuscle using in situ PLA. Red spots represent protein complexes in close proximity(<40 nm). Sections were double-stained for myosin heavy chain (MF20, green).Neg Ctrl, negative control with just one or no primary antibodies. Scale bar:20 µm.
Mentions: In view of the similar localisation pattern of CAPN3, SERCA1, SERCA2 and sAnk1 around theZ-discs and M-lines (Refs 20, 21), we wanted to assess if these proteins interactedwithin the skeletal muscle. First, we performed co-immunoprecipitation assays usingprotein extracts from the vastus lateralis of a healthy human donor.Indeed, we found that both SERCA1 and SERCA2 proteins co-immunoprecipitated with CAPN3(Fig. 5a), which is in line with previousstudies showing interaction between SERCA1 and Capn3 (Ref. 8). Absence of cross-reactivity between SERCA1 and SERCA2 antibodieswas demonstrated by specific detection of each isoform in the immunoprecipitates (Fig. 5b). Interestingly, we found that sAnk1co-immunoprecipitated with SERCA1 but not with SERCA2 (Fig. 5b). Next, we examined the distribution of CAPN3, SERCA1, SERCA2 and sAnk1in the human skeletal muscle by immunofluorescence and we observed a highly similarlocalisation pattern and a broad co-localisation of CAPN3 with SERCA1, SERCA2 and sAnk1(Fig. 5c). To further validate interaction ofthese proteins, we used the in situ PLA, which reveals protein complexes (<40 nmdistance) at a single molecule resolution within the cellular context (Ref. 22). This analysis showed that CAPN3 interacts withSERCA1, SERCA2 and sAnk1 in the human skeletal muscle (Fig. 5d). Also, co-localisation of SERCA1 and sAnk1 was observed in the humanmuscle. Overall, our results indicate that CAPN3 and SERCA1/2 proteins form molecularcomplexes within the skeletal muscle, with a differential contribution of sAnk1 to thecomplexes formed by SERCA1 or SERCA2. Figure 5.

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