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

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.


| INTRODUC TI ON
Ischemic heart disease (IHD) accounts for the most serious cardiac issues (Akhtar, 2006;Benjamin et al., 2019). Compared to adult hearts, IHD carries the greatest burden for the older population (Chen et al., 2022). Numerous structural and functional alterations during aging render the heart more vulnerable to various stressors and culminate in the increasing risk of developing IHD (Christoffersen et al., 2014;Madhavan et al., 2018). Fatty acid β-oxidation in mitochondria is a major energy source for the myocardium (Fukushima & Lopaschuk, 2016b) and the aged heart exhibits impaired metabolic flexibility caused by impaired mitochondrial oxidative phosphorylation (OXPHOS) (Lesnefsky et al., 2016). Changes in transcriptional and posttranslational control of fatty acid oxidative enzymes are the potential mechanism contributing to the ischemia-induced shift in cardiac energy metabolism (Fukushima & Lopaschuk, 2016a;Rosano et al., 2008).
The mammalian silent information regulators (sirtuins) are a family of nicotinamide adenine dinucleotide (NAD + )-dependent deacetylases that are closely related to the extension of lifespan (Imai & Guarente, 2014). Moreover, the absence of mammalian sirtuins plays an essential role in multiple crucial cellular processes to combat myocardial ischemia/reperfusion (I/R), including cell survival, DNA repair, inflammation, and metabolism (Zhang et al., 2020).
These modifications controlled by sirtuins are associated with the transcriptional regulation of related genes and lysine acetylation of critical enzymes. In addition, the sirtuin family members localize in distinct subcellular compartments (Kwon et al., 2017;Tong et al., 2013) and appear to be changed in a tissue-specific manner in some stress situations (Bao et al., 2010;Iwahara et al., 2012;Parodi-Rullan et al., 2018). Sirtuin1 (SIRT1) is the closest mammalian homolog to the yeast Sir2 protein in sequence and is expressed in a wide range of tissues such as the heart, liver, and muscle of mice (Imai & Guarente, 2010;Lavu et al., 2008). The nucleocytoplasmic shuttling of SIRT1 during cardiac development and physiological or pathological stimuli affects different deacetylated targets and transcription factors, further influencing its function. SIRT1 is exclusively expressed in the nucleus in the cardiomyocytes of mouse embryos but expressed in both the cytoplasm and nucleus in the adult heart (Tanno et al., 2007). Our previous research showed compromised SIRT1 nuclear shuttling in the aged hearts, which was worsened under ischemia and I/R stress (Tong et al., 2013). Moreover, the activation of SIRT1 in the young hearts upon acute ischemia and short-time I/R stress failed in the aged upon ischemia. (Tong et al., 2013). However, the nucleocytoplasmic shuttling pattern of SIRT1 and its role in age-related cardiac impaired metabolic flexibility during acute ischemia and long-term I/R stress remain unclear.
The huge impact of Sirtuin3 (SIRT3) in the regulation of lifespan, cardiac function, and mitochondrial biology has been evaluated in numerous studies (Alrob et al., 2014;Hirschey et al., 2010;Koentges et al., 2015;Sundaresan et al., 2008). In mice, loss of SIRT3 in the aging heart results in decreased intolerance to I/R stress (Parodi-Rullan et al., 2017). However, the different subcellular localization of SIRT3 and its activity has been a subject of considerable debate.
Human SIRT3 (hSIRT3) was located in the nucleus with its unique Nterminal mitochondrial localization sequence (MLS) and translocated into mitochondria upon cellular stress (Iwahara et al., 2012;Scher et al., 2007). These studies were opposite to Cooper and Spelbrink, who demonstrated that endogenous hSIRT3 was expressed predominately in the mitochondria, while overexpression of hSIRT3 lacking MLS resulted in the expression both in the cytoplasm and nucleus.
Despite the loss of the N-terminus compared to hSIRT3, the mouse SIRT3 (mSIRT3) still had two forms (Pillai et al., 2010). Both were enzymatically active and detected in the nuclear and cytoplasmic, and mitochondria fractions of H9cC2 cells, while their abundance in each fraction was disparate (Bao et al., 2010). In addition, a previous study showed localization of SIRT3 protein change from mitochondria to the nucleus is associated with the co-expression with sirtuin (SIRT5).
Interestingly, it has been recognized that liver SIRT1 mediated the SIRT3 activity via deacetylation during the obesity and aging process (Kwon et al., 2017). Till now, the subcellular localization, activity, and co-regulatory network of cardiac SIRT3 are still uncertain, especially their effects on age-related metabolic regulation during acute ischemia and long-term I/R stress.
The present study investigated the altered distribution of short-form SIRT1 and SIRT3 in young and aged hearts during acute ischemia and long-term I/R stress. We attempted to elucidate the interaction between SIRT1 and SIRT3 under physiological and pathological conditions, as well as explored their effects on maintaining cardiac adaptive metabolic response. Our study demonstrated that age-related SIRT1 and SIRT3 deficiency contributed to the mitochondrial metabolic homeostasis out of balance in the heart, resulting in exacerbated myocardial cell death and cardiac dysfunction upon acute ischemia stress, and cannot be recovered after 6 h reperfusion treatment. Aging-related SIRT1 inactivation during ischemia led to an adaptive response of SIRT1 mitochondria relocation rather than nuclear. Our results provide novel mechanistic insights into the roles of the SIRT1/ SIRT3 subcellular regulatory network in protecting against cardiac acute ischemia and the recovery from I/R operation in the aging heart.

| SIRT1 and SIRT3 deficiency with aging causes cardiac histological damage and augments cardiac sensitivity to ischemic insults
To investigate the critical role of SIRT1 and SIRT3 in age-related ischemic heart disease, the protein expression of SIRT1 and SIRT3 was first detected in young and aged mouse heart's left ventricle under sham operations, myocardial acute ischemia of 30 min and 30 min ischemia followed by 6 h reperfusion. The results demonstrated that SIRT1 and SIRT3 protein levels are decreased with aging, and both were downregulated in response to acute ischemia and I/R stress in the young left ventricle (Figure 1a). The confirmatory results were obtained by analyzing SIRT1 and SIRT3 protein expression levels in the area at risk (AAR) of young and aged mice heart's left ventricle by immunofluorescent staining (Shihan et al., 2021). Lower levels of SIRT1 and SIRT3 proteins were observed in aged AAR as compared to young AAR under sham conditions (Figure 1b,c). In addition, acute ischemic stress significantly decreased SIRT1 and SIRT3 total protein levels and enhanced the nuclear translocation of SIRT1 in young AAR, whereas blunted in aged AAR (Figure 1b,c). However, there is no significant change after I/R treatment in both young and aged AAR. These results indicate that cardiac SIRT1 and SIRT3 are decreased with aging, and there could be differences in the SIRT1 and SIRT3 expression between AAR and infarct area.
To assess the effects of age-related deficiency in SIRT1 and SIRT3 on cardiac histology, mouse hearts were subjected to TTC and Evens Blue staining, HE, and Masson staining. The myocardial infarction measurements demonstrated that the infarct size was significantly larger in aged hearts as compared to young hearts after I/R surgery ( Figure 1d). The deletion of cardiomyocyte SIRT1 (icSIRT1 −/− ) versus SIRT1 f/f mice also showed a larger infarction size after myocardial I/R stress ( Figure 1d). Similarly, cSIRT3 −/− versus SIRT3 f/f mouse hearts are more sensitive to I/R stress as shown with larger myocardial infarction size ( Figure 1d). Thus, age-related absence of cardiac SIRT1/ SIRT3 could be a factor causing vulnerability of the heart to ischemic insults. Moreover, the histopathologic evaluation demonstrated that the cardiomyocytes in young sham groups were orderly arranged, and dense, and the cardiac muscle fascicles were complete in shape.  indicate that the deficiency of SIRT1 and SIRT3 with aging can cause alterations in mitochondrial morphology in response to acute ischemia. However, due to the distinct subcellular locales (Hollander et al., 2014), the three mitochondrial have different work in regulating cardiac mitochondrial homeostasis upon I/R stress. The absence of cardiac SIRT1 seems to have a greater effect on PNM during I/R treatment, while the absence of cardiac SIRT3 seems to have a greater effect on SSM.

| Acute ischemia stress affects the subcellular distribution and SIRT1-SIRT3 interaction
To verify the difference between SIRT1 and SIRT3 localization in cardiomyocytes under physiological and pathological conditions, the immunogold double labeling of the AAR in hearts' left ventricle from young and aged C57BL/6J mice was performed (Enger, 2017).
In the nucleus, positive staining showed that there was an increased distribution of SIRT1 and SIRT3 in response to acute ischemic stress in young AAR, whereas this response was blunted in aged AAR ( Figure 3a). The nuclear SIRT1-SIRT3 colocalization followed the same pattern, which was significantly increased in young AAR upon acute ischemic stress, and significantly decreased in aged AAR ( Figure 3a). Moreover, although the abundance of SIRT1 or SIRT3 in their nuclear interaction have no significant changes under those stress, a light increase pattern of SIRT1 in young AAR and SIRT3 in Aged AAR under acute ischemia was observed. These results suggest the increased nuclear shuttling of SIRT1 increases its activity (Tong et al., 2013) and results in the enhanced colocalization between SIRT1 and SIRT3. These changes served as an adaptive response under ischemic conditions in young AAR, and this adaptive response is alleviated in aged AAR. In response to both acute ischemia and I/R stress, the mitochondrial SIRT1 was reduced versus sham conditions in young AAR. Interestingly, mitochondrial SIRT1 decreased in aged AAR under sham conditions versus the young group, while there were more SIRT1 translocated into mitochondria in aged hearts upon acute ischemia (Figure 3b). The mitochondria SIRT1-SIRT3 colocalization was significantly increased in young AAR upon acute ischemic stress, while blunted in aged AAR, which is like the alterations in the nucleus ( Figure 3b). Notably, the amount of SIRT1 in mitochondria SIRT1-SIRT3 colocalization was increased in young but not in aged AAR in response to acute ischemia ( Figure 3b).
However, the amount of SIRT3 in mitochondria SIRT1-SIRT3 colocalization was augmented in mitochondria in aged but not in young hearts during acute ischemia ( Figure 3b). These findings indicate that the deficiency and impaired nuclear shuttling of SIRT1 in aging under acute ischemia cause the increased abundance of mitochondria SIRT1. This adaptive response could maintain the stability of the SIRT1-SIRT3 complex in aged mitochondria during acute ischemia, which could be a protection for SIRT3 and its mediated mitochondria regulation.
We also performed the immunogold double labeling on human cardiomyocytes from the donor's heart to further validate the association between SIRT1 with SIRT3 ( Figure 3c). Remarkably, the SIRT1-SIRT3 colocalization was observed in both nucleus and mitochondria of human cardiomyocytes (Figure 3c), suggesting that the association between SIRT1 and SIRT3 could also play an important role in human hearts under physiological and pathological conditions.
To determine whether SIRT1 could interact with SIRT3, we used computational prediction of protein-protein interaction to first analyze the potential SIRT1-SIRT3 interaction. The docking structure of SIRT1 and SIRT3 with the largest cluster size output from Cluspro 2.0 was shown in Figure 3d. SIRT1 and SIRT3 are colored green and red. The structure shows that an alpha-helix (from Glu420 to Lys430, highlighted by yellow in Figure 3d) of SIRT1 fitted into the biggest cavity on the SIRT3 surface after binding. Four residues in this alpha-helix (Glu420, Arg424, Lys427, and Lys430) form very strong electrostatic interactions with residues (Arg158, Glu181, Glu177, and Glu323) of SIRT3, respectively. Besides the helix, another two strong electrostatic interactions (Lys375, Arg446 of SIRT1 with Glu296, Glu325 of SIRT3) on both sides of the alpha-helix strengthen the binding of the helix of SIRT1 with SIRT3. Our docking results indicated SIRT1 not only fits the shape of the SIRT3 surface but also forms very strong interactions with SIRT3 to stabilize the binding ( Figure 3d).
To further determine whether there are alterations in SIRT1-SIRT3 interaction in response to acute ischemia, we performed coimmunoprecipitation with SIRT1 antibody in the nuclear fraction and mitochondrial fraction from young/aged hearts' whole left ventricle.
There was no significant change in the nuclear fraction under acute ischemia. However, acute ischemic stress can trigger upregulation of SIRT1/SIRT3 interaction in the mitochondria of young hearts, which was not observed in the aged hearts ( Figure 3e). These data suggest that there is an intracellular nuclear-shuttling of SIRT1 in response to acute ischemic stress in young hearts, but this nuclear-shuttling of SIRT1 changes into mitochondria in the aged hearts during acute ischemia. As aging hearts are characterized by mitochondria dysfunction, the increased mitochondrial SIRT1 stabilizes its interaction with SIRT3 and further maintains SIRT3-mediated mitochondria regulation. These could be one adaptive response to combat the ischemic insults in aging.

| SIRT1 deficiency affects SIRT3-mediated mitochondria substrate metabolic components during acute ischemia and I/R stress
To examine whether SIRT1 is critical for the protection of SIRT3mediated mitochondria function in the aging heart under physiological and pathological conditions, we determined the protein expression levels of SIRT3 in SIRT1 f/f and icSIRT1 −/− mouse hearts F I G U R E 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),

Damaged mitochondria (%)
Young Aged  Figure 4b). However, more upregulated SIRT3-associated proteins showed in aged hearts in response to acute ischemia and I/R stress.
Intriguingly, an increased portion of upregulated SIRT3-targeted proteins also showed in icSIRT1 −/− group as compared to the SIRT1 f/f group during acute ischemia and I/R stress (Figure 4c), indicating that cardiac SIRT1 deficiency in aging alters SIRT3-associated metabolic proteins in response to acute ischemia and I/R stress.

| SIRT3 deficiency causes disturbed metabolic reprogramming in response to acute ischemia and I/R stress
The ex vivo working heart system model was used to measure the substrate metabolism in young/aged wild-type and SIRT3 f/f /  (Figure 5c, lower panel). The relative ATP production from glycolysis, glucose oxidation, and oleate oxidation showed that age-related SIRT3 deficiency plays a dominant role in metabolic remodeling during acute ischemic stress (Figure 5d). In conclusion, our results indicated that SIRT3 is critical to maintain fatty acid metabolism during acute ischemia and I/R stress in the heart, and important to mitochondrial oxidative phosphorylation during acute ischemia. Age-related cardiac SIRT3 deficiency could be a factor leading to maladaptive metabolic remodeling in the aging heart.

| DISCUSS ION
In this study, we showed an adaptive response regarding the SIRT1-   Percentage hearts from further mitochondria dysfunction induced by acute ischemia. The present study evaluated the nuclear and mitochondrial SIRT1/SIRT3 network and pointed out that enhancing mitochondrial sirtuins function is more efficient to protect aging hearts from ischemic insults.
Ischemic heart disease, characterized by constriction in the coronary blood vessel, is known to have a higher morbidity and mortality rate in the elderly population (Dong et al., 2020). Both at clinical and experimental levels, aged hearts are more sensitive to ischemic insults and sustain greater damage during acute ischemia and I/R stress (Wu et al., 2018). The histological and metabolic changes due to senescence are known to be a fundamental factor promoting age-related changes in the heart. In this study, we have demonstrated that SIRT1 and SIRT3 deficiency aggravates age-related changes in senescence signaling. In particular, the hallmark of cardiac aging with increased  with aging in hearts and its activity is also limited due to the reduction of NAD + level in aged hearts during myocardial ischemia stress (Tong et al., 2013). Furthermore, SIRT1 administration in aged hearts via AAV delivery increased the tolerance of aged hearts to I/R injury (Wang et al., 2018). The protein expression and activity of SIRT3 are also downregulated with cardiac aging as a result of the decreased NAD + levels (Parodi-Rullan et al., 2018).
The deficiency of SIRT3 in aged hearts increases their tolerance to ischemic insults and I/R injury with increased cardiac reactive oxygen species (ROS) level (Parodi-Rullan et al., 2017). However, the comprehensive mechanism of how SIRT1 and SIRT3 protect the aged hearts from greater damage upon acute ischemia and I/R stress remains unclear. The study first demonstrated that agerelated deficiency of SIRT1 and SIRT3 changed the cardiomyocyte physiological structure and aggravated cardiac dysfunction after acute ischemic stress and failed recovery through reperfusion operation.
Due to its crucial role in cellular functions, mitochondrial dysfunction has long been considered a major factor in the development of the aging heart (Lopez-Otin et al., 2023). Previous studies showed that the three populations of cardiac mitochondria possess distinct functional differences (Hollander et al., 2014). Here, we found that age-related deficiency of SIRT1 and SIRT3 showed more severe damage in all the SSM, IFM and PNM after the acute ischemic operation in the AAR. Notably, only IFM were significantly damaged after I/R stress in the aged AAR verse young group. However, the absence of cardiac SIRT1 seems to have a greater effect on PNM during I/R treatment, while the loss of cardiac SIRT3 seems to have a greater effect on SSM after I/R stress. These data suggest that SIRT1/SIRT3 could work as a complex during acute ischemia, and may control distinct recovery processes during the reperfusion period.
Previous studies revealed age-related mitochondrial alterations in oxidative phosphorylation (OXPHOS) and ROS production appeared to be limited predominantly to the IFM subpopulation (Hollander et al., 2014). We have demonstrated that aged-related SIRT1/SIRT3 deficiency impaired cardiomyocyte contractility and is associated with changes in mitochondrial dynamics, OXPHOS and redox homeostasis (Zhang et al., 2021). However, the subcellular localization, activity, and co-regulatory network of cardiac SIRT1/ SIRT3 in aging are still uncertain during acute ischemia and I/R stress. We first confirmed the nuclear translocation and activation of SIRT1 during acute ischemic stress conditions in young hearts and its weakening in aged hearts, which is consistent with our previous study (Tong et al., 2013). In addition, we also found enhanced SIRT1-SIRT3 colocalization in the nucleus of young hearts at that time.
However, SIRT1 changed to translocate into mitochondria in aged hearts' IFM during acute ischemia stress. Increased mitochondria SIRT1 recruit more mitochondria SIRT3 to enhance their interaction.
These results suggest that the dynamic SIRT1/SIRT3 subcellular distribution could be protection for the mitochondria in aging hearts from further impairment induced by acute ischemia.
Interestingly, it has been recognized that liver SIRT1 mediated the SIRT3 activity via deacetylation during the obesity and aging process (Kwon et al., 2017). We found that the cardiac deletion of SIRT1 caused the downregulation of SIRT3, which suggests that SIRT1 is a critical regulator for SIRT3 activity under physiological or pathological conditions. We then try to underline the mechanism of how the interaction between SIRT1 and SIRT3 protects hearts from ischemic insults in cardiac aging. The proteomics analysis of SIRT3associated proteins in the heart revealed that SIRT3 is closely associated with target proteins involved in the tricarboxylic acid (TCA) cycle, glycolysis, OXPHOS complex, and fatty acid oxidation. Acute ischemia and I/R stress trigger a larger portion of downregulation of SIRT3-related proteins in young hearts, while increasing the portion of upregulated SIRT3-related proteins in aged hearts. Intriguingly, the deletion of cardiomyocyte SIRT1 showed similarly greater upregulated SIRT3-associated proteins in response to acute ischemia and I/R stress. Thus, SIRT1 plays a role in modulating SIRT3-mediated mitochondrial function and substrate metabolism to adapt to myocardial acute ischemia and I/R stress.
Through an isolated working heart system, the oleate was selected as a fatty acid substrate in the ex vivo heart perfusion as palmitate can have potentially toxic effects on the heart under stress conditions (Russell et al., 2004). In the present study, we found that age-related SIRT3 deficiency is critical to fatty acid metabolic disorder during acute ischemia and I/R stress in the heart. Alterations in transcriptional and posttranslational control of substrate metabolic enzymes are the primary potential role contributing to the ischemiainduced shift in mitochondria metabolism in the aged heart. PPARα, as a nutritional sensor, is expressed highly in tissues with high fatty acid oxidation rates such as the liver, heart, and kidney, allowing adaptation of the rates of fatty acid catabolism, and ketone body synthesis under stress conditions (Nakamura et al., 2014). PPARα and its coactivator PGC-1α are transcriptional regulators of genes involved in mitochondrial β-oxidation and fatty acid transport (Nakamura et al., 2014). Moreover, CD36 is a high-affinity receptor for longchain fatty acid that facilities the cellular fatty acid uptake (Luiken et al., 2020). Then the long-chain fatty acids can bind and activate PPARα, thus acting as strong endogenous ligand candidates of PPARα (Nakamura et al., 2014). In this study, we found that the decreased in mitochondria helps cardiomyocytes survive from acute ischemic stress via maintain a sufficient energy supply through fatty acid oxidation.
Our present study still has some limitations. First, we choose the AAR in the present study, as AAR is a major determinant of final infarct size and prognosis (Redfors et al., 2012). However, the SIRT1/ SIRT3 network needs to be further confirmed in the whole left ventricle and an efficient fraction of nuclear/mitochondria is needed.
Second, further analysis of subcellular distribution and interaction of SIRT1 and SIRT3 in PNM and SSM of cardiomyocytes, especially their specific effects and related signaling pathway during I/R stress.
The multi-omics analyses of cardiac IFM, PNM, and SSM are beneficial to understand their division of work under physiological and pathological conditions. Third, more studies are needed to interpret the factors that influence the preference of sirtuins translocation between nuclear and mitochondria. Previous studies have pointed out that the posttranslational modification or mitochondrial/nuclear targeting sequence of sirtuins play important roles in their shuttling (Murugasamy et al., 2022;Tong et al., 2013). Finally, we performed the SIRT1-SIRT3 colocalization in human samples, we advocate studying their role in ischemic heart disease populations to address future clinical usages and therapeutic options.
Taken together, these data revealed that the loss of cardiac SIRT1 and SIRT3 with aging results 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 stress, acting as adaptive protection for the aging hearts from further mitochondria dysfunction. The increased mitochondria SIRT1-SIRT3 interaction maintains mitochondrial fatty acid metabolism to meet the cardiac energy demand via deacetylation and activation of LCAD under acute ischemia stress. Thus, the mitochondrial SIRT1/SIRT3 network is more efficient to protect aging hearts from ischemic insults and is a promising therapeutic target for numerous agingrelated processes.

| E XPERIMENTAL PROCEDURE S
The authors declare that all supporting data are available within the article and the Appendix S1.
Young ( The data, analytic methods, and study materials will be made available to other researchers for the purposes of reproducing the results or replicating the procedures. Expended detailed materials and methods can be founded in the Expanded Materials and Methods in the Appendix S1. Tan and J. Li analyzed data; and J. Zhang and J. Li wrote the paper.