CACNA1H downregulation induces skeletal muscle atrophy involving endoplasmic reticulum stress activation and autophagy flux blockade

Abstract

Multiple vaginal delivery (MVD) is an important factor for pelvic floor muscle (PFM) function decline and pelvic floor dysfunction (PFD). PFD is common in middle-aged and elderly women, but its pathogenesis is not clear. In this study, we found that the expression of CACNA1H was lower in the PFM of old mice after MVD compared with old or adult mice. In in-vitro studies, we found that treatment with the T-type Ca²⁺ channel (T-channel) inhibitor NNC-55 or downregulation of the CACNA1H gene by siRNA intervention promoted myotube atrophy and apoptosis. Mechanistically, we revealed that NNC-55 increased the expression of GRP78 and DDIT3 in myotubes, indicating endoplasmic reticulum stress (ERS) activation, and that the IRE1 and PERK pathways might be involved in this effect. NNC-55 induced the formation of autophagosomes but inhibited autophagy flux. Moreover, rapamycin, an autophagy activator, did not rescue myotube atrophy or apoptosis induced by NNC-55, and the autophagy inhibitors 3-MA and HCQ accelerated this damage. Further studies showed that the ERS inhibitors 4-PBA and TUDAC relieved NNC-55-induced damage and autophagy flux blockade. Finally, we found multisite muscle atrophy and decreased muscle function in Cacna1h^-/- (TH-null) mice, as well as increased autophagy inhibition and apoptotic signals in the PFM of old WT mice after MVD and TH-null mice. Taken together, our results suggest that MVD-associated PFD is partially attributed to CACNA1H downregulation-induced PFM atrophy and that ERS is a potential therapeutic target for this disease.

Fig. 1. The expression of T-channel in PFM, TA and C2C12 myotubes.

a, b The CACNA1G and CACNA1H mRNA expression were detected in PFM (a) and TA (b) of 4-month-old WT mice (n = 7) by qRT-PCR. c CACNA1H mRNA expression was examined during the differentiation of C2C12 by qRT-PCR. d, e The expression of CACNA1H in PFM of the adult virgin (4-month old, n = 7), aged virgin (12-month old, n = 7) and aged MVD (12-month old, n = 7) group mice was analyzed by qRT-PCR (d) and western blotting (e). These data are presented as (mean ± SD). PFM, pelvic floor muscle. TA, anterior tibial muscle. CON, control. Aged MVD, aged multiple vaginal-delivery. NS, no significance. *P < 0.05, **P < 0.01, ***P < 0.001. a, b Unpaired two-tailed Student’s t test. c–e One-way analysis of variance (ANOVA).

Fig. 2. T-channel inhibitor induces myotube atrophy and injury.

a Schematic diagram illustrates the in vitro experimental process. b–e Myotubes were incubated with various concentrations (up to 10 μM) of NNC-55 for 48 h, and the myotubes status were analyzed by anti-MyHC immunofluorescence staining (b), and western blotting (c). Apoptotic myotubes were detected with the annexin V-PE/7-AAD kit (d) and mitochondrial permeability were detected by mPTP kit (e) and then analyzed by flow cytometry. These data are presented as the (mean ± SD) for three independent experiments. GM, growth medium. DM, differential medium. CON, control. NS, no significance. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA).

Fig. 3. T-channel inhibitor induces ERS and intracellular Ca²⁺ disorder in myotubes.

a C1C12 myotubes were incubated with various concentrations (up to 10 μM) of NNC-55 for 48 h, and the Ca²⁺ ion concentration in myotubes was indicated by Fluo-3AM and analyzed with flow cytometry. b, c Myotubes were incubated with NNC-55 (10 μM) for various time (up to 48 h). GRP78, DDIT3 protein expression were analyzed by western blotting (b) and ATF6, IRE1 and PERK gene expression were detected by qRT-PCR (c). These data are presented as the (mean ± SD) for three independent experiments. NC, negative control. CON, control. NS, no significance. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA).

Fig. 4. T-channel inhibitor promotes autophagosome formation but blocks autophagy flux in myotubes.

a Myotubes were incubated with NNC-55 (10 μM) for various time (up to 48 h) and the expression of SQSTM1, Beclin-1, LC3-II transition were analyzed by western blotting. β-actin was included as a loading control. b Working mechanism of tandem mRFP-GFP-LC3 adenovirus reporting system. c C2C12 myotubes over expressed mRFP-GFP-LC3 were treated with 10 μM NNC-55 or DMSO (control) for the indicated time and then subjected to confocal microscopy. The average numbers of green and red LC3 dots per cell in each condition were quantified, and over 30 cells were counted in each condition. d Ultrastructural features of myotubes treated with NNC-55 (10 μM) for 24 and 48 h were analyzed by electron microscopy. Typical images of the nucleus (N), endoplasmic reticulum (ER), and autophagosomes (yellow arrows) and autolysosome (green arrows) are shown at high magnification. These data are presented as the (mean ± SD) for three independent experiments. CON, control. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA).

Fig. 5. Further inhibition of autophagy enhances the damage induced by T-channel inhibitor, but autophagy activator does not rescue the damage.

C2C12 myotubes were pre-treated with inhibitor (3-MA or HCQ) or activator (Ra) of autophagy for 1 h and then exposed to NNC-55 (10 μM) for another 24 h. a, b Western blotting analysis of SQSTM1, LC3, DDIT3 and Cyt-c expression levels in myotubes. β-actin was used as a loading control. c, d Apoptotic myotubes were detected with the annexin V-PE/7-AAD kit (c) and mitochondrial permeability were detected by mPTP kit (d) and then analyzed by flow cytometry. e The myotubes status were analyzed by anti-MyHC immunofluorescence staining. These data are presented as the (mean ± SD) for three independent experiments. CON, control. 3-MA + N, 3-MA + NNC-55. HCQ + N, HCQ + NNC-55. Ra + N, Rapamycin + NNC-55. NS, no significance. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA).

Fig. 6. ERS inhibitors alleviate the damage and autophagy inhibition induced by T-channel inhibitor.

C2C12 myotubes were pre-treated with ER stress inhibitors (4-PBA or TUDCA) for 1 h and then exposed to NNC-55 (10 μM) for another 24 h. a Western blotting analysis of GRP78, DDIT3, SQSTM1 and LC3 expression levels in myotubes. β-actin was used as a loading control. b Myotubes over expressed mRFP-GFP-LC3 were treated with 10 μM NNC-55 in combination with 4-PBA or TUDCA, and the cells with mRFP-GFP-LC3 punctate dots were examined by confocal microscope. c The myotubes status were analyzed by anti-MyHC immunofluorescence staining. d, e Apoptotic myotubes were detected with the annexin V-PE/7-AAD kit (d) and mitochondrial permeability were detected by mPTP kit (e) and then analyzed by flow cytometry. These data are presented as the (mean ± SD) for three independent experiments. CON, control. TU + N, TUDCA + NNC-55. 4-PBA + N, 4-PBA + NNC-55. *P < 0.05, **P < 0.01, ***P < 0.001. One-way analysis of variance (ANOVA).

Fig. 7. CACNA1H gene silence promotes myotubes atrophy and apoptosis and TH-null mice exhibit impaired muscle function.

a–c To observe the function of CACNA1H, C2C12 myotubes were treated with the CACNA1H siRNA and the effectiveness was verified by western blotting (a). The myotubes status were analyzed by anti-MyHC immunofluorescence staining (b) and apoptotic myotubes were detected with the annexin V-PE/7-AAD kit and then analyzed by flow cytometry (c). d–i 4-month-old female WT and TH-null mice were used for muscle feature analysis (n = 7). The distribution of CSA of the PFM, TA and SOL was evaluated by HE staining (d). Ultrastructural features of PFM were analyzed by electron microscopy (e). The isometric twitch force of SOL was measured through the tension transducer and PowerLab data acquisition system (f). Single myofibers were extracted from the extensor digitorum longus (EDL) muscle and then fixed with formaldehyde for LC3 immunostaining (g). The expression of SQSTM1 in PFM was evaluated by immunofluorescence (h). CTSB, CTSD, LAMP2, MURF1 and Antrogin1 gene expression in PFM were detected by qRT-PCR (i). j, k The PFM of the youth (Youth, 1-month old), aged virgin (Aged, 12-momth old) and aged multiple vaginal-delivery (Aged MVD, 12-momth old) group, WT and TH-null mice (n = 7) were used for CSA analysis by HE staining (j), and SQSTM1, LC3, DDIT3 and Cyt-c protein expression measurement by western blotting (k). These data are presented as the (mean ± SD). CON, control. NC, negative control. Aged MVD, aged multiple vaginal-delivery. PFM, pelvic floor muscle. TA, anterior tibial muscle. SOL, soleus muscle. *P < 0.05, **P < 0.01, ***P < 0.001. a–c, f, h, i, j Unpaired two-tailed Student’s t-test. k One-way analysis of variance (ANOVA).

Fig. 8. Schematic diagram depicts how CACNA1H downregulation induces PFD.

The expression of CACNA1H reduces in PFM of elderly MVD mice. The inhibition of T-channel induces ERS and autophagy flux inhibition and ultimately results in myofiber atrophy and apoptosis, which is the possible mechanism for PFM function impairment and PFD induced by CACNA1H downregulation. ERS, endoplasmic reticulum stress. UPR, unfolding protein response. MVD, multiple vaginal delivery. PFM, pelvic floor muscle. PFD, pelvic floor dysfunction.