Hamster Embryonic Fibroblasts

GPX4 Activity is Broadly Required for Cold Tolerance in Primary Cells Across Evolutionarily Distant Mammalian Species

Hibernators survive core-temperature drops to 4 °C, yet the genetic basis of mammalian cold tolerance is unknown. Unbiased genome-wide screens in hamster (hibernator) and human (non-hibernator) cells at 4 °C converged on glutathione peroxidase 4 (GPX4) and its selenoprotein network.

Given that the studies had largely employed transformed cell lines, Lam et al. extended their investigation to primary cells and additional hibernating species. They obtained embryonic fibroblasts from human, mouse, rat, and three distantly related hibernators (Syrian hamster, 13-lined ground squirrel, and horseshoe bat). While hibernator-derived fibroblasts showed robust, uniform cold tolerance, non-hibernator primary cells exhibited surprising variability (Fig. 1a), with human dermal fibroblasts achieving 79 ± 4.93% viability after 7 days at 4°C-comparable to hamster BHK-21 cells. To examine GPX4's role in primary cell cold resistance, they generated GPX4 knockout human kidney fibroblasts (Fig. 1b). Consistent with cell line findings, GPX4 loss significantly reduced cold viability, which was largely rescued by ferrostatin-1. RSL3 treatment similarly decreased wild-type human kidney fibroblast viability in the cold (Fig. 1c, d), with effects dependent on GPX4 inhibition-GPX4 knockout cells were unaffected by RSL3. Testing fibroblasts from six mammalian species revealed that GPX4 activity broadly contributes to cold tolerance across both hibernators and non-hibernators, as RSL3-induced cell death was rescued by ferrostatin-1 (Fig. 1e, f). These findings extended to non-fibroblast cell types, with primary cortical neurons from neonatal mice and hamsters showing similar patterns (Fig. 1g, h). Together, their data indicate that primary cells are strongly sensitive to GPX4 loss in the cold, with differential cold sensitivities reflecting varying levels of GPX4-mediated protection.