Hydrogen sulfide increases thermotolerance and lifespan in Caenorhabditis elegans

Abstract

Hydrogen sulfide (H(2)S) is naturally produced in animal cells. Exogenous H(2)S has been shown to effect physiological changes that improve the capacity of mammals to survive in otherwise lethal conditions. However, the mechanisms required for such alterations are unknown. We investigated the physiological response of Caenorhabditis elegans to H(2)S to elucidate the molecular mechanisms of H(2)S action. Here we show that nematodes exposed to H(2)S are apparently healthy and do not exhibit phenotypes consistent with metabolic inhibition. Instead, animals exposed to H(2)S are thermotolerant and long-lived. These phenotypes require SIR-2.1 activity but are genetically independent of the insulin signaling pathway, mitochondrial dysfunction, and caloric restriction. These studies suggest that SIR-2.1 activity may translate environmental change into physiological alterations that improve survival. It is interesting to consider the possibility that the mechanisms by which H(2)S increases thermotolerance and lifespan in nematodes are conserved and that studies using C. elegans may help explain the beneficial effects observed in mammals exposed to H(2)S.

Fig. 1.

H₂S increases thermotolerance in wild-type C. elegans. (A) Animals exposed to H₂S survive longer than untreated controls at high temperature. Nematodes were moved to 35°C in the same gaseous atmosphere in which they had been cultured (SI Fig. 5). The mean survival time of animals grown in H₂S was 65.5 h (solid line; n = 136), compared with 9.1 h (n = 96) for untreated controls (dashed line). (B) Prior exposure to H₂S is required to survive high temperature in H₂S. All animals were grown in room air without H₂S and then moved to 35°C in the presence or absence of 50-ppm H₂S. Animals first exposed to H₂S at high temperature had a mean survival time of 2.1 h (solid line; n = 20), whereas the control group exposed in room air survived for 7.3 h (dashed line; n = 20). (C) The continuous presence of H₂S in the atmosphere is required for increased survival at high temperature. Animals were exposed to 35°C in room air. Animals grown in H₂S before heat shock survived 7.3 h (solid line; n = 20), which is not significantly longer than untreated controls (dashed line; 7.0 h; n = 20). Indicated P values were determined by log-rank analysis.

Fig. 2.

H₂S increases lifespan in C. elegans. (A) Animals grown in H₂S live longer than untreated controls. The lifespan of animals was monitored in the same conditions in which they had developed. The mean lifespan of animals in H₂S was 22.6 ± 1.0 days (solid line; n = 80), compared with 13.0 ± 1.0 days for untreated controls in room air (dashed line; n = 40). Maximum lifespan also was increased. (B) Exposure to H₂S beginning as L4 does not increase lifespan. All animals were from populations grown in room air. The lifespan of animals moved into H₂S-containing environments at the beginning of the lifespan experiment (solid line) is 14.8 ± 0.3 days (n = 73), which is slightly shorter than controls that remained in house air (dashed line; mean lifespan 18.2 ± 0.4 days; n = 48). (C) Increased lifespan requires continuous exposure to H₂S. The lifespan of all animals was monitored in room air. The lifespan of animals raised in H₂S until L4 (solid line; 12.8 ± 0.7 days; n = 52) was indistinguishable from untreated controls (dashed line; 13.2 ± 0.7 days; n = 59). All lifespan experiments were performed at room temperature.

Fig. 3.

Thermotolerance of canonical long-lived mutants is increased by H₂S. Just as H₂S increases thermotolerance of wild-type worms (Fig. 1 and SI Fig. 6), long-lived mutants in canonical pathways that influence lifespan (20) also are more thermotolerant when grown in H₂S. All animals grown in H₂S were challenged with high temperature in H₂S (solid line), whereas the thermotolerance of untreated controls was assayed in room air (dashed line). (A) H₂S effects are genetically independent of IIS. The thermotolerance of daf-2(e1370) animals can be enhanced by exposure to H₂S, and daf-16(m26) mutants, which are defective in IIS, become thermotolerant when grown in H₂S. To facilitate the experiments shown in this figure, strains that show intrinsic thermotolerance, such as daf-2(e1370) (30), were tested at a slightly higher temperature both in room air and H₂S. However, the thermotolerance at 35°C also is increased when the animals are grown in H₂S (data not shown). (B) H₂S-induced thermotolerance is observed in isp-1(gk267) and clk-1(qm30) animals that are long-lived as a result of mitochondrial dysfunction. (C) H₂S-induced thermotolerance is observed in eat-2(ad1116) mutant animals, which have defects in pharyngeal pumping that result in dietary restriction.

Fig. 4.

sir-2.1 is required for increased thermotolerance and lifespan in H₂S. (A) H₂S does not increase thermotolerance of animals that have a deletion in sir-2.1. The mean survival time of sir-2.1(ok434) animals grown in H₂S and exposed to high temperature in H₂S (solid line) is 9.8 ± 0.3 h (n = 20), which is not significantly longer than untreated controls in room air (dashed line; mean survival 9.6 ± 0.3 h; n = 20). (B) H₂S does not increase the lifespan of sir-2.1(ok434) animals. The lifespan of sir-2.1(ok434) animals raised in H₂S is 20.0 ± 1.6 days (solid line; n = 47), which is statistically indistinguishable from control animals in room air (dashed line; 22.2 ± 1.2 days; n = 26). Indicated P values were determined by log-rank analysis.