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. 2023 Sep;22(9):e13930.
doi: 10.1111/acel.13930. Epub 2023 Aug 3.

Alterations of SIRT1/SIRT3 subcellular distribution in aging undermine cardiometabolic homeostasis during ischemia and reperfusion

Jingwen Zhang  1   2 , Hao Wang  1   2 , Lily Slotabec  1   2 , Feng Cheng  3 , Yi Tan  4 , Ji Li  1   2   5
Affiliations

Alterations of SIRT1/SIRT3 subcellular distribution in aging undermine cardiometabolic homeostasis during ischemia and reperfusion

Jingwen Zhang et al. Aging Cell. 2023 Sep.

Abstract

Age-related sensors Sirtuin1 (SIRT1) and Sirtuin3 (SIRT3) play an essential role in the protective response upon myocardial ischemia and/or reperfusion (I/R). However, the subcellular localization and co-regulatory network between cardiac SIRT1 and SIRT3 remain unknown, especially their effects on age-related metabolic regulation during acute ischemia and I/R. Here, we found that defects of cardiac SIRT1 or SIRT3 with aging result in an exacerbated cardiac physiological structural and functional deterioration after acute ischemic stress and failed recovery through reperfusion operation. In aged hearts, SIRT1 translocated into mitochondria and recruited more mitochondria SIRT3 to enhance their interaction during acute ischemia, acting as adaptive protection for the aging hearts from further mitochondria dysfunction. Subsequently, SIRT3-targeted proteomics revealed that SIRT1 plays a crucial role in maintaining mitochondrial integrity through SIRT3-mediated substrate metabolism during acute ischemic and I/R stress. Although the loss of SIRT1/SIRT3 led to a compromised PGC-1α/PPARα-mediated transcriptional control of fatty acid oxidation in response to acute ischemia and I/R, their crosstalk in mitochondria plays a more important role in the aging heart during acute ischemia. However, the increased mitochondria SIRT1-SIRT3 interaction promoted adaptive protection to aging-related fatty acid metabolic disorder via deacetylation of long-chain acyl CoA dehydrogenase (LCAD) during ischemic insults. Therefore, the dynamic network of SIRT1/SIRT3 acts as a mediator that regulates adaptive metabolic response to improve the tolerance of aged hearts to ischemic insults, which will facilitate investigation into the role of SIRT1/SIRT3 in age-related ischemic heart disease.

Keywords: SIRT1; SIRT3; aging; fatty acid oxidation; ischemia/reperfusion.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

FIGURE 1
FIGURE 1
Deficiency of SIRT1 and SIRT3 with aging leads to cardiac vulnerable to acute ischemia and I/R stress. (a) The protein levels of SIRT1 and SIRT3 from the left ventricle of the male mouse hearts declined in aging, and a blunted response occurred in aged hearts during acute ischemia and I/R stress. (N = 5, values are mean ± SEM from five biological replicates, *p < 0.05 vs. young; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (b) and (c) Upper: Representative immunofluorescence staining images of SIRT1, SIRT3, troponin T, and DAPI in the area at risk (AAR) of young and aged male heart's left ventricle under sham, acute ischemia, or I/R conditions. Lower: Statistical analysis of SIRT1, SIRT3 staining and percentage of nuclear SIRT1 and SIRT3 in young and aged AAR under sham, acute ischemia, and I/R conditions. (N = 5, values are mean ± SEM from five biological replicates, *p < 0.05 vs. young; † p < 0.05 versus sham, respectively, two‐way ANOVA with Tukey's post hoc test). (d) Young (4–6 months)/aged (24–26 months) wild‐type C57BL/6J, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f and cSIRT3 −/− C57BL/6J male mice were subjected to in vivo regional acute ischemia for 30 min only or followed by 6 h of reperfusion. Left: Representative sections of the extent of myocardial infarction were presented. TTC staining showed a larger infarct area in aged versus young, icSIRT1 −/− versus SIRT1 f/f , and cSIRT3 −/− versus SIRT3 f/f hearts, respectively. Right: The ratio of the AAR to the total myocardial area refers to the area affected by ischemia, and the ratio of the infarcted area to AAR is used to access the myocardium injury. (N > =3, values are means ± SEM from at least three biological replicates, *p < 0.05 versus young, SIRT1 f/f , SIRT3 f/f , respectively, two‐way ANOVA with Tukey's post hoc test). (e) Representative H&E‐stained myocardium of young (4–6 months), aged (24–26 months), SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f , and cSIRT3 −/− male mice hearts sham, acute ischemia, and I/R conditions. (f) Upper: Representative images of myocardial fibrosis measured by Masson's trichrome staining in young, aged, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f , and cSIRT3 −/− hearts under sham, acute ischemia, and I/R conditions. Lower: Quantification analysis of Masson's trichrome staining. (N = 5, values are mean ± SEM from five biological replicates, *p < 0.05 versus sham, respectively: † p < 0.05 versus young I/R, SIRT1 f/f I/R, SIRT3 f/f I/R, respectively, two‐way ANOVA with Tukey's post hoc test).
FIGURE 2
FIGURE 2
The morphological alterations in different cardiac mitochondria after acute ischemia and I/R operations. The transmission electronic microscope (TEM) images were obtained from subsarcolemmal mitochondria (a), interfibrillar mitochondria (b), and perinuclear mitochondria (c) in the left ventricle of young/aged C57BL/6J wild type, SIRT1 f/f /icSIRT1 −/− , SIRT3 f/f /cSIRT3 −/− hearts under sham, acute ischemia, and I/R stress. Upper: Representative TEM at a magnification of 60,000; Lower: Quantitative analysis by ImageJ to calculate the percentage of damaged mitochondria (Value are means ± SEM from two biological replicates with five technical repeats at a magnification of 20,000. *p < 0.05 versus young, SIRT1 f/f , SIRT3 f/f , respectively; † p < 0.05 versus sham, respectively, two‐way ANOVA with Tukey's post hoc test).
FIGURE 3
FIGURE 3
Distinct subcellular distribution and interaction of SIRT1 and SIRT3 with cardiac aging after acute ischemia and I/R operations. (a) Immunogold double labeling of SIRT1 and SIRT3 in the nucleus of the left ventricle AAR of young and aged hearts under sham, acute ischemia, and I/R conditions. Upper: Left, representative immunogold labeling images of SIRT1 (labeled with red dots) and SIRT3 (labeled with blue dots) in each group at a magnification of 100,000. Right, the distribution of nuclear SIRT1 and SIRT3 gold particles in the heart's left ventricle AAR. Lower: the colocalization of SIRT1 and SIRT3 in the myocardium nucleus and their percentage ratio involved in the colocalization. (N > =3 nucleus of each replicate, values are means ± SEM form at least three technical replicates at a magnification of 20,000. *p < 0.05 vs. young; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (b) Immunogold double labeling of SIRT1 and SIRT3 in the mitochondria of the left ventricle AAR of young and aged hearts under sham, acute ischemia, and I/R conditions. Upper: Left, representative immunogold labeling images of SIRT1 (labeled with red dots) and SIRT3 (labeled with blue dots) in each group at a magnification of 60,000. Right, the distribution of mitochondrial SIRT1 and SIRT3 gold particles in the heart's left ventricle AAR. Lower: the colocalization of SIRT1 and SIRT3 in the myocardium mitochondria and their percentage ratio involved in the colocalization. (N > =10 mitochondria of each replicate, values are means ± SEM from seven technical replicates at a magnification of 20,000. *p < 0.05 vs. young; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (c) Representative immunogold double labeling images of SIRT1 and SIRT3 at a magnification of 100,000 in the left ventricle of the human heart. (d) The protein–protein docking analysis indicated that SIRT1 and SIRT3 can form strong interactions. (e) The interaction of SIRT1 and SIRT3 in nuclear and mitochondrial portions of the left ventricle of young and aged hearts under sham or acute ischemia. (N = 3, values are means ± SEM from three biological replicates, *p < 0.05 vs. young; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test).
FIGURE 4
FIGURE 4
Associate of SIRT1 with SIRT3 in the heart and SIRT3‐associated proteins involved in metabolic regulation in response to acute ischemia and I/R conditions. (a) Western blot analysis of SIRT3 levels in the left ventricle of SIRT1 f/f and icSIRT1 −/− male mice hearts under sham, acute ischemia, and I/R conditions. (N = 5, values are mean ± SEM from five biological replicates, *p < 0.05 vs. SIRT1 f/f ; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (b) Ingenuity pathway analysis (IPA) enrichment analysis of the dynamics of SIRT3‐associated proteins in the left ventricle of young (4–6 months)/aged (24–26 months) C57BL/6J male mice hearts in response to acute ischemia and I/R conditions, respectively. Blue bars represent the percentage of genes in the pathway that were downregulated in response to acute ischemia and I/R in young or aged hearts versus sham conditions, respectively. Red bars represent the percentage of genes in the pathway that were upregulated in response to acute ischemia and I/R in young or aged hearts versus sham conditions, respectively. (c) Ingenuity pathway analysis (IPA) enrichment analysis of the dynamics of SIRT3‐associated proteins in the left ventricle of SIRT1 f/f and icSIRT1 −/− male mouse hearts in response to acute ischemia and I/R conditions, respectively. Green bars represent the percentage of genes in the pathway that were downregulated in response to acute ischemia and I/R in SIRT1 f/f or icSIRT1 −/− hearts versus sham conditions, respectively. Red bars represent the percentage of genes in the pathway that were up‐regulated in response to acute ischemia and I/R in SIRT1 f/f or icSIRT1 −/− hearts versus sham conditions, respectively.
FIGURE 5
FIGURE 5
Alterations in substrate metabolism occurred in aged and cSIRT3 −/− hearts. (a) D‐[5‐3H]‐glucose was used in the ex vivo working heart perfusion system to measure glycolysis rate in young, aged, SIRT3 f/f and cSIRT3 −/− male mouse hearts subjected to 10 min ischemia and 20 min reperfusion. (N = 2–4 time point of each condition, values are mean ± SEM of from four to five biological replicates, *p < 0.05 vs. young, SIRT3 f/f , respectively: † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (b) Glucose oxidation was analyzed by measuring [14C]‐glucose incorporation into 14CO2 in the ex vivo working heart perfusion system of young, aged, SIRT3 f/f and cSIRT3−/− male mouse hearts subjected to 10 min of ischemia and 20 min of reperfusion. (N = 2–4 time point of each condition, values are mean ± SEM of from four to five biological replicates, *p < 0.05 vs. young, SIRT3 f/f , respectively: † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (c) The oleate oxidation was analyzed by measuring the incorporation of [9,10‐3H] oleate into 3H2O. (N = 2–4 time point of each condition, values are mean ± SEM of from four to five biological replicates, *p < 0.05 vs. young, SIRT3 f/f , respectively: † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (d) The relative percentage of ATP production calculated from glycolysis, glucose, and oleate oxidation in young, aged, SIRT3 f/f , and cSIRT3−/− hearts.
FIGURE 6
FIGURE 6
The role of SIRT1 and SIRT3 in regulating fatty acid metabolism mediated by PGC‐1α/PPARα during acute ischemia and I/R stress. (a), (b) and (c) Western blot analysis of PGC‐1α, PPARα, CD36, and CPT1β in young, aged, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f , and cSIRT3 −/− left ventricle of male mice hearts under sham, acute ischemia, and I/R conditions. (N = 4, values are mean ± SEM from four biological replicates, *p < 0.05 vs. young, SIRT1 f/f , SIRT3 f/f , respectively; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (d), (e) and (f) Real‐time PCR analysis of the mRNA levels of CD36, SCAD, MCAD, LCAD, and VLCAD in young, aged, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f , and cSIRT3 −/− left ventricle of male mice hearts under sham, acute ischemia, and I/R conditions. (N = 3 technical repeat of each replicate, values are mean ± SEM from at least three biological replicates, *p < 0.05 vs. young, SIRT1 f/f , SIRT3 f/f , respectively; † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test).
FIGURE 7
FIGURE 7
Aged‐related SIRT1 is critical for SIRT3‐mediated fatty acid oxidation during acute ischemia and I/R stress. (a), (b) and (c) Western blot analysis of LCAD and phosphorylation of PDHE1α in young, aged, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f and cSIRT3 −/− left ventricle of male mice hearts under sham, acute ischemia, and I/R conditions. (N = 5, values are mean ± SEM from five biological replicates, *p < 0.05 vs. young, SIRT1 f/f , SIRT3 f/f , respectively: † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test). (d) Immunoprecipitation analysis of LCAD acetylation and its interaction with SIRT3 in young, aged, SIRT1 f/f , icSIRT1 −/− , SIRT3 f/f and cSIRT3−/− male mice hearts under sham, acute ischemia, and I/R conditions. (N = 3, values are mean ± SEM from three biological replicates, *p < 0.05 vs. young, SIRT1 f/f , SIRT3 f/f , respectively: † p < 0.05 vs. sham, respectively, two‐way ANOVA with Tukey's post hoc test).
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