Matrix stiffness induces Drp1-mediated mitochondrial fission through Piezo1 mechanotransduction in human intervertebral disc degeneration

Background: Extracellular matrix stiffness is emerging as a crucial mechanical cue that drives the progression of various diseases, such as Cancer, fibrosis, and inflammation. The matrix stiffness of the nucleus pulposus (NP) tissues increase gradually during intervertebral disc degeneration (IDD), while the mechanism through which NP cells sense and react to matrix stiffness remains unclear. In addition, mitochondrial dynamics play a key role in various cellular functions. An in-depth investigation of the pathogenesis of IDD can provide new insights for the development of effective therapies. In this study, we aim to investigate the effects of matrix stiffness on mitochondrial dynamics in IDD.

Methods: To build the gradient stiffness model, NP cells were cultured on polystyrene plates with different stiffness. Western blot analysis, and immunofluorescence staining were used to detect the expression of mitochondrial dynamics-related proteins. Flow cytometry was used to detect the mitochondrial membrane potential and intracellular Ca²⁺ levels. Apoptosis related proteins, ROS level, and TUNEL staining were performed to assess the effect of substrate stiffness on NP cells.

Results: Stiff substrate increased phosphorylation of dynamin-related protein 1 (Drp1) at Ser616 by activating extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which promoted mitochondrial fission and Apoptosis in NP cells. Furthermore, Piezo1 activation was involved in the regulation of the post-translational modifications of Drp1 and mitochondrial fission caused by matrix stiffness. Inhibition of Piezo1 and ERK1/2 can effectively reduce stiffness-induced ROS elevation and Apoptosis in NP cells.

Conclusions: Our results revealed that stiff substrate causes Piezo1 activation and Ca²⁺ influx, results in ERK1/2 activation and phosphorylation of Drp1 at S616, and finally leads to mitochondrial fission and Apoptosis in NP cells. These findings reveal a new mechanism of mechanotransduction in NP cells, providing novel insights into the development of therapies for treating IDD.

Intervertebral disc degeneration; Matrix stiffness; Mechanotransduction; Mitochondrial fission; Nucleus pulposus; Piezo1.