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

Suting Li,Menglei Hao,Li Hong,Ming Hu,Jie Min,Jianming Tang,Bingshu Li,Mao Chen,Shasha Hong,Jue Chen

doi:10.1038/s41419-020-2484-2

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.

Highlights

Skeletal muscle is one of the most important organs in the body, comprising approximate 50% of the total body weight
Our results suggest that Multiple vaginal delivery (MVD)-associated pelvic floor dysfunction (PFD) is partially attributed to CACNA1H downregulation-induced pelvic floor muscle (PFM)
We found that the GRP78 and DDIT3 proteins were upregulated in the NNC-55-treated group compared with the control group, and this increase was evident after 24 h of treatment, while there is no difference in the control groups at different time points (Fig. 3b)

Summary

Materials and methods

Other reagent sources are listed below: FBS, horse serum, trypsin/EDTA solution, DMEM (GIBCO-BRL, Gaithersburg, MD, USA); 3-Methyladenine (3-MA), hydroxychloroquine (HCQ), 4-phenylbutyric acid (4-PBA), Tauroursodeoxycholate (TUDCA) and collagenase (MCE, Monmouth Junction, USA); Annexin VPE/7-AAD Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA); Mitochondrial Permeability Transition Pore (mPTP) Assay Kit (YEASEN, Shanghai, China); Fluo-3AM (Sigma-Aldrich, 39294). Immunofluorescence staining Extensor digitorum longus from adult mice were digested in type I collagenase at 37 °C for 2-3 h, and dissociated into single fibers These single fibers and myotubes fixed with 4% paraformaldehyde for 15 min at 4 °C and performed as described previously[36]. Flow cytometric analysis of Ca2+ concentration Myotubes were harvested, incubated with Fluo-3AM (10 μM) for 30 min in the incubator and performed as described previously[36]. Statistical analysis was performed with GraphPad Prism software version 7 (San Diego, CA, USA)

Result

Findings

Discussion