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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

Disruption of sAnk1 expression and localisation in CAPN3-deficient fibres. (a)Representative Western blots showing reduced sAnk1 protein levels in mouseCapn3-deficient C2C12 myotubes (left) and human CAPN3-deficient LHCN-M2 myotubes(right) compared with controls. Membranes were stained with Ponceau-S forverification of equal total protein loaded. (b) Western blot analysis of sAnk1expression in human muscle samples. (c) Cross sections from a control and two LGMD2Ahuman muscles co-immunostained for sAnk1 and DAPI. Note the higher accumulation ofsAnk1 at the nuclei in the LGMD2A fibres (arrows). Scale bar: 25 µm.
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fig04: Disruption of sAnk1 expression and localisation in CAPN3-deficient fibres. (a)Representative Western blots showing reduced sAnk1 protein levels in mouseCapn3-deficient C2C12 myotubes (left) and human CAPN3-deficient LHCN-M2 myotubes(right) compared with controls. Membranes were stained with Ponceau-S forverification of equal total protein loaded. (b) Western blot analysis of sAnk1expression in human muscle samples. (c) Cross sections from a control and two LGMD2Ahuman muscles co-immunostained for sAnk1 and DAPI. Note the higher accumulation ofsAnk1 at the nuclei in the LGMD2A fibres (arrows). Scale bar: 25 µm.

Mentions: sAnk1, an SR protein, which binds with the giant sarcomeric proteins titin and obscurin,is essential for maintenance of the SR network integrity and its organisation around thecontractile apparatus (Refs 19, 20). Silencing sAnk1 expression in rat myofibresresults in reduced expression of SERCA1 (Ref. 20), which is similar to the effect observed in CAPN3 knockdown myotubes.Therefore, we sought to determine if sAnk1 expression was altered in the absence of CAPN3.For this purpose, we analysed expression of sAnk1 in CAPN3 knockdown myotubes, and foundreduction of sAnk1 protein levels in both mouse and human CAPN3-deficient myotubescompared with NS controls (Fig. 4a). In this line,we found that in human muscle samples sAnk1 was strongly reduced in the LGMD2A sampleswith nondetectable CAPN3 expression (P3, P4; Fig.4b, upper panel), while expression levels of sAnk1 in muscle samples from otherforms of muscular dystrophy were comparable with control levels (Fig. 4b, lower panel). Using immunohistochemistry, we were able todetect expression of sAnk1 in P2 as well as in P4 LGMD2A samples. Interestingly, thisanalysis showed that in both LGMD2A patients, sAnk1 was abnormally accumulated at thenuclei (Fig. 4c). Taken together, our resultsindicate that in the skeletal muscle, CAPN3 may have an impact on sAnk1 expression levelsand on its appropriate localisation to the SR. Further studies will be needed in order todetermine the role of sAnk1 in the nucleus. Figure 4.


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)

Disruption of sAnk1 expression and localisation in CAPN3-deficient fibres. (a)Representative Western blots showing reduced sAnk1 protein levels in mouseCapn3-deficient C2C12 myotubes (left) and human CAPN3-deficient LHCN-M2 myotubes(right) compared with controls. Membranes were stained with Ponceau-S forverification of equal total protein loaded. (b) Western blot analysis of sAnk1expression in human muscle samples. (c) Cross sections from a control and two LGMD2Ahuman muscles co-immunostained for sAnk1 and DAPI. Note the higher accumulation ofsAnk1 at the nuclei in the LGMD2A fibres (arrows). Scale bar: 25 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig04: Disruption of sAnk1 expression and localisation in CAPN3-deficient fibres. (a)Representative Western blots showing reduced sAnk1 protein levels in mouseCapn3-deficient C2C12 myotubes (left) and human CAPN3-deficient LHCN-M2 myotubes(right) compared with controls. Membranes were stained with Ponceau-S forverification of equal total protein loaded. (b) Western blot analysis of sAnk1expression in human muscle samples. (c) Cross sections from a control and two LGMD2Ahuman muscles co-immunostained for sAnk1 and DAPI. Note the higher accumulation ofsAnk1 at the nuclei in the LGMD2A fibres (arrows). Scale bar: 25 µm.
Mentions: sAnk1, an SR protein, which binds with the giant sarcomeric proteins titin and obscurin,is essential for maintenance of the SR network integrity and its organisation around thecontractile apparatus (Refs 19, 20). Silencing sAnk1 expression in rat myofibresresults in reduced expression of SERCA1 (Ref. 20), which is similar to the effect observed in CAPN3 knockdown myotubes.Therefore, we sought to determine if sAnk1 expression was altered in the absence of CAPN3.For this purpose, we analysed expression of sAnk1 in CAPN3 knockdown myotubes, and foundreduction of sAnk1 protein levels in both mouse and human CAPN3-deficient myotubescompared with NS controls (Fig. 4a). In this line,we found that in human muscle samples sAnk1 was strongly reduced in the LGMD2A sampleswith nondetectable CAPN3 expression (P3, P4; Fig.4b, upper panel), while expression levels of sAnk1 in muscle samples from otherforms of muscular dystrophy were comparable with control levels (Fig. 4b, lower panel). Using immunohistochemistry, we were able todetect expression of sAnk1 in P2 as well as in P4 LGMD2A samples. Interestingly, thisanalysis showed that in both LGMD2A patients, sAnk1 was abnormally accumulated at thenuclei (Fig. 4c). Taken together, our resultsindicate that in the skeletal muscle, CAPN3 may have an impact on sAnk1 expression levelsand on its appropriate localisation to the SR. Further studies will be needed in order todetermine the role of sAnk1 in the nucleus. Figure 4.

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