Limits...
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

SERCA expression and localisation in muscle biopsies from patients with musculardystrophy. (a) Analysis of CAPN3 (SPA), SERCA1, SERCA2, slow (sMyHC) and totalmyosin heavy chain (MyHC) expression in muscle samples from 4 LGMD2A patients (P1–4)and 4 controls (C1–C4). All muscles from LGMD2A patients show absence of SERCA2protein. Left panel shows representative western blot signals. Right panel depictsoptical density values of proteins normalised to MyHC in LGMD2A patients andexpressed as fold change over controls. *P < 0.01 versuscontrol samples. Statistical significance was determined using unpaired, 2-tailedStudent's t test. (b) Western blot analysis of dystrophin,dysferlin, SERCA1, SERCA2, CAPN3 and MyHC in two controls (C1-C2) and 5 patientswith other muscular dystrophies: fascioscapulohumeral muscular dystrophy (FSH2),Duchenne (DMD), LGMD2B (LG2B); myotonic dystrophy (DM1). Baseline represents averageof control sample levels C1 and C2. None of these dystrophic patients show deficientexpression of SERCA2 in the muscle. (c) Cross-sections from 1 control (C2) and 2LGMD2A (P2, P4) human muscles co-immunostained for CAPN3, SERCA1 and SERCA2.Immunofluorescence analysis showed reduced expression of CAPN3 in LGMD2A musclefibres. SERCA2 levels appear reduced in the LGMD2A samples and showed a preferentiallocalisation near the sarcolemma. Scale bar: 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4836212&req=5

fig03: SERCA expression and localisation in muscle biopsies from patients with musculardystrophy. (a) Analysis of CAPN3 (SPA), SERCA1, SERCA2, slow (sMyHC) and totalmyosin heavy chain (MyHC) expression in muscle samples from 4 LGMD2A patients (P1–4)and 4 controls (C1–C4). All muscles from LGMD2A patients show absence of SERCA2protein. Left panel shows representative western blot signals. Right panel depictsoptical density values of proteins normalised to MyHC in LGMD2A patients andexpressed as fold change over controls. *P < 0.01 versuscontrol samples. Statistical significance was determined using unpaired, 2-tailedStudent's t test. (b) Western blot analysis of dystrophin,dysferlin, SERCA1, SERCA2, CAPN3 and MyHC in two controls (C1-C2) and 5 patientswith other muscular dystrophies: fascioscapulohumeral muscular dystrophy (FSH2),Duchenne (DMD), LGMD2B (LG2B); myotonic dystrophy (DM1). Baseline represents averageof control sample levels C1 and C2. None of these dystrophic patients show deficientexpression of SERCA2 in the muscle. (c) Cross-sections from 1 control (C2) and 2LGMD2A (P2, P4) human muscles co-immunostained for CAPN3, SERCA1 and SERCA2.Immunofluorescence analysis showed reduced expression of CAPN3 in LGMD2A musclefibres. SERCA2 levels appear reduced in the LGMD2A samples and showed a preferentiallocalisation near the sarcolemma. Scale bar: 50 µm.

Mentions: We analysed expression of SERCA1/2 proteins in human skeletal muscle samples from controland LGMD2A dystrophic patients, in order to determine if our results obtained in vitrowere relevant for this disease. For this analysis we used muscle biopsies from 4 LGMD2Apatients (P1–4) and 4 sex- and age-matched controls (C1–4). Clinical information ofcontrol and dystrophic patients used in this study are shown in Table 1. Western blot analysis showed that the four LGMD2A patientsanalysed had significantly lower amount of CAPN3 protein compared with control samples,when normalised with total myosin heavy chain (MyHC) (30 ± 14%;P < 0.05, Fig. 3a). SERCA1protein levels were not significantly different in control versus LGMD2A samples, althoughP3 and P4 samples, which had the lowest CAPN3 levels among LGMD2A samples, showed adistinct reduction of SERCA1 levels compared with control samples (P3 = 1%; P4 = 31%). Onthe other hand, SERCA2 protein was undetectable in all the LGMD2A samples, while it wasnormally expressed in control samples. Absence of SERCA2 in LGMD2A samples was not becauseof lower numbers of slow muscle fibres in these samples, as demonstrated by the similarlevels of slow myosin heavy chain (sMyHC) observed in control and LGMD2A samples (Fig. 3a). Figure 3.


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)

SERCA expression and localisation in muscle biopsies from patients with musculardystrophy. (a) Analysis of CAPN3 (SPA), SERCA1, SERCA2, slow (sMyHC) and totalmyosin heavy chain (MyHC) expression in muscle samples from 4 LGMD2A patients (P1–4)and 4 controls (C1–C4). All muscles from LGMD2A patients show absence of SERCA2protein. Left panel shows representative western blot signals. Right panel depictsoptical density values of proteins normalised to MyHC in LGMD2A patients andexpressed as fold change over controls. *P < 0.01 versuscontrol samples. Statistical significance was determined using unpaired, 2-tailedStudent's t test. (b) Western blot analysis of dystrophin,dysferlin, SERCA1, SERCA2, CAPN3 and MyHC in two controls (C1-C2) and 5 patientswith other muscular dystrophies: fascioscapulohumeral muscular dystrophy (FSH2),Duchenne (DMD), LGMD2B (LG2B); myotonic dystrophy (DM1). Baseline represents averageof control sample levels C1 and C2. None of these dystrophic patients show deficientexpression of SERCA2 in the muscle. (c) Cross-sections from 1 control (C2) and 2LGMD2A (P2, P4) human muscles co-immunostained for CAPN3, SERCA1 and SERCA2.Immunofluorescence analysis showed reduced expression of CAPN3 in LGMD2A musclefibres. SERCA2 levels appear reduced in the LGMD2A samples and showed a preferentiallocalisation near the sarcolemma. Scale bar: 50 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: SERCA expression and localisation in muscle biopsies from patients with musculardystrophy. (a) Analysis of CAPN3 (SPA), SERCA1, SERCA2, slow (sMyHC) and totalmyosin heavy chain (MyHC) expression in muscle samples from 4 LGMD2A patients (P1–4)and 4 controls (C1–C4). All muscles from LGMD2A patients show absence of SERCA2protein. Left panel shows representative western blot signals. Right panel depictsoptical density values of proteins normalised to MyHC in LGMD2A patients andexpressed as fold change over controls. *P < 0.01 versuscontrol samples. Statistical significance was determined using unpaired, 2-tailedStudent's t test. (b) Western blot analysis of dystrophin,dysferlin, SERCA1, SERCA2, CAPN3 and MyHC in two controls (C1-C2) and 5 patientswith other muscular dystrophies: fascioscapulohumeral muscular dystrophy (FSH2),Duchenne (DMD), LGMD2B (LG2B); myotonic dystrophy (DM1). Baseline represents averageof control sample levels C1 and C2. None of these dystrophic patients show deficientexpression of SERCA2 in the muscle. (c) Cross-sections from 1 control (C2) and 2LGMD2A (P2, P4) human muscles co-immunostained for CAPN3, SERCA1 and SERCA2.Immunofluorescence analysis showed reduced expression of CAPN3 in LGMD2A musclefibres. SERCA2 levels appear reduced in the LGMD2A samples and showed a preferentiallocalisation near the sarcolemma. Scale bar: 50 µm.
Mentions: We analysed expression of SERCA1/2 proteins in human skeletal muscle samples from controland LGMD2A dystrophic patients, in order to determine if our results obtained in vitrowere relevant for this disease. For this analysis we used muscle biopsies from 4 LGMD2Apatients (P1–4) and 4 sex- and age-matched controls (C1–4). Clinical information ofcontrol and dystrophic patients used in this study are shown in Table 1. Western blot analysis showed that the four LGMD2A patientsanalysed had significantly lower amount of CAPN3 protein compared with control samples,when normalised with total myosin heavy chain (MyHC) (30 ± 14%;P < 0.05, Fig. 3a). SERCA1protein levels were not significantly different in control versus LGMD2A samples, althoughP3 and P4 samples, which had the lowest CAPN3 levels among LGMD2A samples, showed adistinct reduction of SERCA1 levels compared with control samples (P3 = 1%; P4 = 31%). Onthe other hand, SERCA2 protein was undetectable in all the LGMD2A samples, while it wasnormally expressed in control samples. Absence of SERCA2 in LGMD2A samples was not becauseof lower numbers of slow muscle fibres in these samples, as demonstrated by the similarlevels of slow myosin heavy chain (sMyHC) observed in control and LGMD2A samples (Fig. 3a). Figure 3.

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