Maternal smoking during pregnancy aggravated muscle phenotype in FHL1 offspring mice similar to congenital clubfoot through P2RX7-mediated pyroptosis.

Congenital clubfoot (CCF) is a common birth defect. Maternal smoking during pregnancy increases the risk of CCF. In previous research, we found muscle phenotypes similar to CCF in four and a half LIM domain protein 1 (FHLI) offspring mice (FHL1). However, the role of P2RX7-mediated pyroptosis in the effect of cigarette smoke (CS) on the skeletal muscle of FHL1 mice during pregnancy is unclear. In the present study, pregnant mice at 11 days of gestation were exposed to CS and male offspring of wild-type (WT) and FHL1 mice were divided into four groups (Control-WT, Control-KO, CS-WT, CS-KO). The histomorphology of lower limb muscles was examined using hematoxylin and eosin (H&E) staining. P2RX7, indicators of pyroptosis (NLRP3, ASC, cleaved-caspase 1, IL-1β), and cytoskeletal proteins (MYBPC2, LDB3) were also detected using immunoblotting. CS exposure during pregnancy aggravated the muscle phenotype similar to CCF in FHL1 offspring mice. FHL1 gene knockout (KO) or CS exposure during pregnancy each activated the expression of P2RX7, cell pyroptosis-related proteins (NLRP3, ASC, cleaved-caspase 1, IL-1β), a muscle injury marker (MYOD1), and cytoskeletal proteins (MYBPC2, LDB3); these two factors had an additive effect. The results showed maternal smoking during pregnancy aggravated muscle phenotype similar to CCF in FHL1 offspring mice through P2RX7-mediated pyroptosis.