Loss of membrane integrity drives myofiber death in lipin1‐deficient skeletal muscle

Abstract

Mutations in lipin1 are suggested to be a common cause of massive rhabdomyolysis episodes in children; however, the molecular mechanisms involved in the regulation of myofiber death caused by the absence of lipin1 are not fully understood. Loss of membrane integrity is considered as an effective inducer of cell death in muscular dystrophy. In this study, we utilized a mouse line with selective homozygous lipin1 deficiency in the skeletal muscle (Lipin1 Myf5cKO ) to determine the role of compromised membrane integrity in the myofiber death in lipin1‐deficient muscles. We found that Lipin1 Myf5cKO muscles had significantly elevated proapoptotic factors (Bax, Bak, and cleaved caspase‐9) and necroptotic proteins such as RIPK1, RIPK3, and MLKL compared with WT mice. Moreover, Lipin1 Myf5cKO muscle had significantly higher membrane disruptions, as evidenced by increased IgG staining and elevated uptake of Evans Blue Dye (EBD) and increased serum creatine kinase activity in Lipin1 Myf5cKO muscle fibers. EBD‐positive fibers were strongly colocalized with apoptotic or necroptotic myofibers, suggesting an association between compromised plasma membrane integrity and cell death pathways. We further show that the absence of lipin1 leads to a significant decrease in the absolute and specific muscle force (normalized to muscle mass). Our work indicates that apoptosis and necroptosis are associated with a loss of membrane integrity in Lipin1 Myf5cKO muscle and that myofiber death and dysfunction may cause a decrease in contractile force.

Lipin1 plays complementary roles in myofibre stability and regeneration in dystrophic muscles

“…Our recent publication suggested that lipin1 deficiency alone led to a compromise in muscle membrane integrity and function in skeletal muscle‐specific lipin1 deficient lipin1 Myf5cKO mice (Ramani Sattiraju et al., 2020). EBD, a membrane‐permeable marker, has been widely used to detect membrane damage of skeletal muscle fibres (Matsuda et al., 1995; Straub et al., 1997).…”

“…In situ muscle force measurements were performed on mice anaesthetized with isoflurane using a SomnoSuite low‐flow anaesthesia system (Kent Scientific, Torrington, CT, USA), as described previously (Myers et al., 2021; Ramani Sattiraju et al., 2020; Wang et al., 2020). Body temperature was monitored with a SomnoSuite temperature probe and maintained at ∼35°C using a heat lamp.…”

“…Our previous studies found that lipin1 is important for skeletal muscle regeneration (Jiang et al., 2015) and myoblast differentiation (Jama et al., 2019). Studies have also suggested that lipin1 is important for membrane integrity, which is indicated by Evans blue dye (EBD) leakage in lipin1 deficient skeletal muscle (Ramani Sattiraju et al., 2020; Rashid et al., 2019). Loss of membrane integrity leads to muscle degeneration, as indicated by upregulation of necroptotic markers including RIPK3 and MLKL in lipin1 deficient muscle (Ramani Sattiraju et al., 2020; Schweitzer et al., 2019).…”

“…Studies have also suggested that lipin1 is important for membrane integrity, which is indicated by Evans blue dye (EBD) leakage in lipin1 deficient skeletal muscle (Ramani Sattiraju et al., 2020; Rashid et al., 2019). Loss of membrane integrity leads to muscle degeneration, as indicated by upregulation of necroptotic markers including RIPK3 and MLKL in lipin1 deficient muscle (Ramani Sattiraju et al., 2020; Schweitzer et al., 2019). Therefore, lipin1 and dystrophin may have complementary roles in maintaining the structural and functional integrity of skeletal muscle.…”

Automated Image Analysis Pipeline Development to Monitor Disease Progression in Muscular Dystrophy Using CellProfiler

“…EBD dye injection was performed as what we did previously 16 . Briefly, mice were injected with EBD (10mg/ml stock in sterile saline, 0.1ml/10g body weight) I.P.…”