Verapamil extends lifespan in Caenorhabditis elegans by inhibiting calcineurin activity and promoting autophagy

Abstract

Previous evidence has revealed that increase in intracellular levels of calcium promotes cellular senescence. However, whether calcium channel blockers (CCBs) can slow aging and extend lifespan is still unknown. In this study, we showed that verapamil, an L-type calcium channel blocker, extended the Caenorhabditis elegans (C. elegans) lifespan and delayed senescence in human lung fibroblasts. Verapamil treatment also improved healthspan in C. elegans as reflected by several age-related physiological parameters, including locomotion, thrashing, age-associated vulval integrity, and osmotic stress resistance. We also found that verapamil acted on the α1 subunit of an L-type calcium channel in C. elegans. Moreover, verapamil extended worm lifespan by inhibiting calcineurin activity. Furthermore, verapamil significantly promoted autophagy as reflected by the expression levels of LGG-1/LC3 and the mRNA levels of autophagy-related genes. In addition, verapamil could not further induce autophagy when tax-6, calcineurin gene, was knocked down, indicating that verapamil-induced lifespan extension is mediated via promoting autophagy processes downstream of calcineurin. In summary, our study provided mechanistic insights into the anti-aging effect of verapamil in C. elegans.

Keywords: Caenorhabditis elegans; anti-aging; autophagy; cell senescence; verapamil.

Figure 1

Verapamil extends lifespan and improves healthspan in C. elegans. (A) Around 1,386 FDA-approved drugs were screened, and C. elegans were used as the model for lifespan evaluation. Finally, verapamil was selected as a hit anti-aging compound. (B) Verapamil extended the lifespan of wildtype C. elegans (N2) at 100 μM (^***P < 0.001) and 400 μM (^**P < 0.01). (C) Verapamil (100 μM and 400 μM) did not reduce bacterial growth. Multiple t-tests were used to calculate the P-values and error bars represent SEM. (D) Verapamil increased the crawling speed of worms on day 2 (100 μM, ^****P < 0.0001; 400 μM, ^****P < 0.0001), but had no influence in late life. (E) Verapamil significantly increased the number of body bends on day 8 (400 μM, ^*P < 0.05) and day 12 (100 μM, ^*P < 0.05; 400 μM, ^**P < 0.01). A two-way ANOVA along with Sidak multiple comparisons test was used to calculate P-values, and error bars represent SEM in (D) and (E). (F) Total Avid was significantly decreased by verapamil (100 μM, ^**P < 0.01; 400 μM, ^**P < 0.01). An unpaired t-test was used to calculate the P-values and error bars represent SEM. (G) Verapamil specifically improved the resistant to osmotic stress (400 μM, ^**P < 0.01), but had no effect at 100 μM. The log-rank (Mantel-Cox) test was used to assess the P-values in (B) and (G).

Figure 2

Verapamil enhances cell viability and delays cellular senescence. (A) Viability of MRC-5 cells in the absence (Ctrl) or presence of verapamil (Ver) at different concentrations. (B) SA-β-Gal staining of MRC-5 cells and quantification of SA-β-Gal-positive cells at a late passage (P31). Verapamil (3 μM) delayed the senescence of MRC-5 cells (^*P < 0.05). Metformin (100 μM) was used as positive control (^*P < 0.05). An unpaired t-test was used to calculate the P-values and error bars represent SEM.

Figure 3

Verapamil acts on the α1 subunit of an L-type calcium channel in C. elegans. (A) Verapamil (100 μM and 400 μM) did not extend the lifespan of egl-19 mutant worms expressing a defective L-type Ca²⁺ channel α1 subunit. (B) Verapamil (400 μM) decreased the pharyngeal pumping rate, especially at day 3 and 6; however, 100 μM verapamil had no effect on the pumping rate. A two-way ANOVA along with Sidak multiple comparisons test was used to calculate the P-values and error bars represent SEM. (C) Verapamil (400 μM) extended the lifespan even under bacterial dilution conditions (^*P < 0.05). The log-rank (Mantel-Cox) test was used to assess the P-value in (A) and (C).

Figure 4

Verapamil extends lifespan by inhibiting the activity of calcineurin. (A) Verapamil (100 μM) reduced the activity of calcineurin in C. elegans (^***P < 0.001). Cyclosporin A (CsA) was used as positive control (^***P < 0.001). An unpaired t-test was used to calculate the P-values and error bars represent SEM. (B) Verapamil (100 μM) did not extend the lifespan of worms treated with tax-6 RNAi. The log-rank (Mantel-Cox) test was used to assess the P-values.

Figure 5

Verapamil extends lifespan through activating autophagy in C. elegans. (A, B) The degree of autophagy induced by verapamil (3 μM and 15 μM) was evaluated by assessing the LC3-II/LC3-I ratio in MRC-5 cells. (C) GFP::LGG-1 levels were evaluated in the seam cells during the L3 stage (^****P < 0.0001) to assess the degree of verapamil (100 μM)-induced autophagy. (D) Verapamil (100 μM) significantly activated the expression of autophagy-related genes. Multiple t-tests were used to evaluate the P-values and error bars represent SEM. (E) Verapamil (100 μM) did not extend the lifespan of worms in which bec-1 expression was downregulated. The log-rank (Mantel-Cox) test was used to calculate the P-value.

Figure 6

Verapamil facilitates autophagy downstream of calcineurin in C. elegans. tax-6 RNAi induced autophagy in worms expressing GFP::LGG-1 (^****P < 0.0001); however, verapamil (100 μM) did not facilitate autophagy under tax-6 RNAi treatment. An unpaired t-test was used to calculate the P-values and error bars represent SEM.