Loss of cardiac mitochondrial complex I persulfidation impairs NAD+ homeostasis in aging

Abstract

Protein persulfidation is a significant post-translational modification that involves addition of a sulfur atom to the cysteine thiol group and is facilitated by sulfide species. Persulfidation targets reactive cysteine residues within proteins, influencing their structure and/or function across various biological systems. This modification is evolutionarily conserved and plays a crucial role in preventing irreversible cysteine overoxidation, a process that becomes prominent with aging. While, persulfidation decreases with age, its levels in the aged heart and the functional implications of such a reduction in cardiac metabolism remain unknown. Here we interrogated the cardiac persulfydome in wild-type adult mice and age-matched mice lacking the two sulfide generating enzymes, namely cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST). Our findings revealed that cardiac persulfidated proteins in wild type hearts are less abundant compared to those in other organs, with a primary involvement in mitochondrial metabolic processes. We further focused on one specific target, NDUFB7, which undergoes persulfidation by both CSE and 3MST derived sulfide species. In particular, persulfidation of cysteines C80 and C90 in NDUFB7 protects the protein from overoxidation and maintains the complex I activity in cardiomyocytes. As the heart ages, the levels of CSE and 3MST in cardiomyocytes decline, leading to reduced NDUFB7 persulfidation and increased cardiac NADH/NAD⁺ ratio. Collectively, our data provide compelling evidence for a direct link between cardiac persulfidation and mitochondrial complex I activity, which is compromised in aging.

Keywords: 3MST; CSE; Cardiac aging; NDUFB7; Persulfidation.

Graphical abstract

Fig. 1

Reduced persulfidation in the hearts of CSE and 3MST knock out mice affects NDUFB7 persulfidation and NADH/NAD⁺ ratio. (A-B) Volcano plots showing persulfidated cysteines in proteins, in hearts from wild type (WT) compared to (A) CSE or (B) 3MST knock out (KO) mice and respective GO enrichment analysis of the modified proteins. n = 4/group. Unpaired Student's t-test. Red/Green: persulfidated cysteines in the respective proteins with a p < 0.1 enriched in the WT samples, blue: persulfidated proteins with a p < 0.1 enriched in the CSE (A) or the 3MST (B) samples. (C) Persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF in whole hearts from mice as in panels A and B. n = 6/group. one-way ANOVA, Tukey's multiple comparisons test. (D) NADH/NAD⁺ ratio in whole heart homogenates from mice as in panels A and B. n = 4/group. Kruskal-Wallis, Dunn's multiple comparisons test. (E) Representative Western blot analysis for HA tagged NDUFB7, GAPDH and persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF in HEK293 cells transfected with wild type (WT) NDUFB7, or NDUFB7 mutated at Cys80 to alanine (C80A), Cys90 to alanine (C90A) or double mutated and co-transfected with pcDNA or a CSE and a 3MST expressing plasmid. Dithiothreitol (DTT) was used as a negative control for the removal of endogenous persulfidation. n = 6/group. two-way ANOVA, Tukey's multiple comparisons test. (F) Representative Western blot analysis for CSE, 3MST, HA tagged NDUFB7, Vinculin and persulfidated NDUFB7 S-SH detected as the relative fluorescence intensity of Daz2:Cy5/NBF in murine HL-1 cardiomyocytes infected with lentiviruses expressing a wild type (WT) NDUFB7, or NDUFB7 mutated at Cys80 to alanine (C80A), Cys90 to alanine (C90A) or double mutated. DTT was used as a negative control. n = 6/group. one-way ANOVA, Tukey's multiple comparisons test.

Fig. 2

CSE and 3MST-triggered persulfidation preserves mitochondrial complex I activity and NADH/NAD⁺ levels. (A-B) Oxygen consumption rate (OCR) in (A) HEK293 cells co-transfected with CSE and 3MST as well as wild type (WT) NDUFB7, or NDUFB7 mutated at Cys80 to alanine (C80A), Cys90 to alanine (C90A) or double mutated (B) murine HL-1 cardiomyocytes infected with lentiviruses expressing a WT or mutated NDUFB7. Complex I oxygen consumption rate was evaluated in response to 10 μmol/L Rotenone. n = 6–14/group. one-way ANOVA, Tukey's multiple comparisons test. (C) NADH/NAD⁺ ratio in HEK293 cells as in panel A. (D) NADH/NAD⁺ ratio in HL-1 cardiomyocytes as in panel B. Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) was used as a disulfide bond breaker. n = 6/group. two-way ANOVA, Tukey's multiple comparisons test (C). one-way ANOVA, Tukey's multiple comparisons test (D). (E) NADH/NAD⁺ ratio in HEK293 cells transfected with NDUFB7 and either pcDNA or CSE and 3MST plasmids and treated with solvent (Sol.) or 500 μmol/L of H₂O₂ for up to 72 h n = 6/group. one-way ANOVA, Tukey's multiple comparisons test. (F) SIRT3 activity in HEK293 cells transfected with CSE and 3MST as well as a WT or mutated NDUFB7. n = 6/group. one-way ANOVA, Tukey's posthoc analysis.

Fig. 3

Reduced NDUFB7 persulfidation during aging impairs NADH/NAD⁺ ratio. (A) Representative immunoblotting analysis for CSE and 3MST and relative densitometric analysis normalized to the DNA content in primary murine isolated cardiomyocytes from 3 or 18 month old wild type mice. n = 4/group, Unpaired Student's t-test. (B) Representative confocal images and quantification showing the results of the dimedone switch method for the detection of persulfidation plotted as the intensity of the Daz2:Cy5 persulfidation signal (blue to red, upper panel) and DAPI (grey), NBF–Cl (red) and a-actinin (blue) in the lower panel in samples as in A. n = 4–6/group, Unpaired Student's t-test. (C) Representative immunobloting and respective quantification for NDUFB7 and VINCULIN for samples as in panel A. n = 4–5/group. (D-E) Persulfidated NDUFB7 S-SH detected with a dimedone switch method and plotted as the relative fluorescence intensity of Daz2:Cy5/NBF (D) and NADH/NAD ratio (E) in samples as in panel A, treated for 10 min with solvent (Sol.) or 100 μmol/L NaHS. n = 6/group. two-way ANOVA, Bonferroni's posthoc analysis.