Restoration of NAD+ homeostasis protects C2C12 myoblasts and mouse levator ani muscle from mechanical stress-induced damage

Abstract

Excessive mechanical traction damages the levator ani muscle (LAM), increasing the incidence of pelvic floor dysfunction (PFD). In this study, we explored the effects of oxidized nicotinamide adenine dinucleotide (NAD+) on the damage to both muscle cells and LAM tissue induced by mechanical stress (MS) at the cellular and animal levels. The cell damage model was established using a four-point bending system. The LAM damage model was established using vaginal distention and traction. Exogenous addition of PJ34, an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1), and the nicotinamide mononucleotide (NMN) precursor of NAD+ increased NAD+ levels. ATP content and mitochondrial membrane potential were measured to assess mitochondrial function. NAD+ levels, cell viability, and PARP-1 activity were detected using commercial kits. DNA damage in cells was detected with immunofluorescence staining, and LAM damage was detected with tissue TUNEL staining. PARP-1 activity and DNA damage of LAM were detected by immunohistochemistry. A small amount of DNA damage and PARP-1 activation did not affect NAD+ levels, while excessive DNA damage and PARP-1 activation led to an imbalance of NAD+ homeostasis. Furthermore, increasing NAD+ levels in vivo and in vitro could rescue mitochondrial dysfunction and damage to both muscle cells and LAM tissue induced by MS. In conclusion, MS can induce damage to both C2C12 cells and LAM tissue. Restoring NAD+ homeostasis can rescue this damage by improving mitochondrial function.

Keywords

• Mechanical stress
• levator ani muscle
• C2C12 myoblast
• Oxidized nicotinamide adenine dinucleotide
• mitochondrial dysfunction