Mitochondria-targeted hydrogen sulfide attenuates endothelial senescence by selective induction of splicing factors HNRNPD and SRSF2

Abstract

Cellular senescence is a key driver of ageing, influenced by age-related changes to the regulation of alternative splicing. Hydrogen sulfide (H₂S) has similarly been described to influence senescence, but the pathways by which it accomplishes this are unclear.We assessed the effects of the slow release H₂S donor Na-GYY4137 (100 µg/ml), and three novel mitochondria-targeted H₂S donors AP39, AP123 and RT01 (10 ng/ml) on splicing factor expression, cell proliferation, apoptosis, DNA replication, DNA damage, telomere length and senescence-related secretory complex (SASP) expression in senescent primary human endothelial cells.All H₂S donors produced up to a 50% drop in senescent cell load assessed at the biochemical and molecular level. Some changes were noted in the composition of senescence-related secretory complex (SASP); IL8 levels increased by 24% but proliferation was not re-established in the culture as a whole. Telomere length, apoptotic index and the extent of DNA damage were unaffected. Differential effects on splicing factor expression were observed depending on the intracellular targeting of the H₂S donors. Na-GYY4137 produced a general 1.9 - 3.2-fold upregulation of splicing factor expression, whereas the mitochondria-targeted donors produced a specific 2.5 and 3.1-fold upregulation of SRSF2 and HNRNPD splicing factors only. Knockdown of SRSF2 or HNRNPD genes in treated cells rendered the cells non-responsive to H₂S, and increased levels of senescence by up to 25% in untreated cells.Our data suggest that SRSF2 and HNRNPD may be implicated in endothelial cell senescence, and can be targeted by exogenous H₂S. These molecules may have potential as moderators of splicing factor expression and senescence phenotypes.

Keywords: AP123; AP39; HS 2; RT01; mitochondria-targeting; persulfide; perthiol; senescence; splicing factors.

Figure 1

H₂S donor treatment is associated with partial rescue from cellular senescence phenotypes. Levels of the senescence-associated total CDKN2A gene expression (A) and levels its alternatively-expressed isoforms p14 and p16 (B) were assessed by qRTPCR in senescent endothelial cells after 24h treatment with H₂S donors (Na-GYY4137 at 100 µg/ml, AP39, AP123, RT01 at 10 ng/ml). Data are expressed relative to stable endogenous control genes GUSB, IDH3B and PPIA, and are given normalised to the levels of the individual transcripts as present in vehicle-only treated control cells. Fold change was calculated for in triplicate for three biological replicates. (C). The proportion of cells staining positive for Senescence Associated β-galactosidase (SA-β-Gal) activity following treatment with H₂S donors was determined by manually counting the percentage of SA-β gal positive cells. (D) The proportion of cells staining positive for H2Ax marker of DNA damage following treatment with H₂S donors was determined by manually counting the percentage of H2Ax positive cells. N = >300 cells for each sample. Statistical significance is indicated by *p<0.05, ** p<0.005, *** p<0.0001 (2 way ANOVA).

Figure 2

Cell proliferation rate is not affected by H₂S donor treatment. (A) Proliferation index was assessed for treated cells as assessed by Ki67 immunofluorescence (>400 nuclei counted per sample). (B) Cell counts following 24h treatment with Na-GYY4137 at 100 µg/ml, AP39, AP123, RT01 at 10 ng/ml. (C) Apoptotic index in senescent cells treated with inhibitors as determined by TUNEL assay. Data are derived from duplicate testing of 3 biological replicates. (D) Telomere length was assessed by qPCR in three biological and 3 technical replicates. (E) BrdU incorporation into cellular DNA. Relative BrdU incorporation was assessed in 3 biological replicates and was calculated by normalization of data to values corresponding to untreated (control) cells and are expressed as % BrdU incorporation. (F) Effect of 24h treatment with Na-GYY4137 at 100 µg/ml, AP39, AP123, RT01 at 10 ng/ml on accumulation of senescent cells over 2 passages in early passage cells (PD = 44). Mean+- SD of three independent experiment is shown. Statistical significance is indicated by *** p<0.001. Error bars represent the standard error of the mean.

Figure 3

H₂S donor treatments affect splicing factor transcript expression. The change in splicing factor mRNA levels in response to 24hr treatment with H₂S donors are given ; Na-GYY4137 at 100 µg/ml, AP39, AP123, RT01 at 10 ng/ml. Green indicates up-regulated genes, red denotes down-regulated genes. The colour scale refers to fold-change in expression. Only statistically significant changes are presented in the heat map.

Figure 4

The cellular and molecular effects of targeted knockdown of HNRPND and SRSF2 genes. (A) Senescent cell load as indicated by SA-β-Gal staining following either HNRPND or SRSF2 gene knockdown. n>300 cells for each sample. (B) Senescent cell load as indicated by CDKN2A gene expression following HNRPND or SRSF2 gene knockdown. Data are expressed relative to stable endogenous control genes GUSB, IDH3B and PPIA, and normalised to the levels of the individual transcripts in vehicle only controls. (C) The effect of HNRPND or SRSF2 gene knockdown on senescent cell load after H₂S donor treatment. Data are derived from duplicate testing of 3 biological replicates. Statistical significance is indicated by *** p<0.001. Error bars represent the standard error of the mean.

Figure 5

The structure of the H2S donor compounds used in this study. The structures of the compounds used in this study are given. (A) AP39 (B) RT01 (C) AP123 (D) Sodium GYY4137.