Acetylation of Mitochondrial Proteins in the Heart: The Role of SIRT3

1Post-translational modifications involve the covalent modification of a protein after it has been translated in order to change the enzymatic activity, alter protein–protein interactions, and mediate protein stability. For over 50 years, acetylation has been known to play a role in the regulation of nuclear protein transcription. In 1997, the non-histone protein p53 was the first to be reported to be acetylated. Since the discovery of non-histone protein acetylation, a growing number of studies have revealed that the activity of cytosolic and mitochondrial proteins can be regulated by acetylation.

2Several PTMs such as phosphorylation, ubiquitination, sumoylation, and acetylation have been identified in mammalian cells. Acetylation involves the transfer of the acetyl group of acetyl-CoA to a lysine residue in a protein; therefore resulting in the disruption of the positively charged lysine into a non-charged residue. Two types of protein acetylation have been described: non-reversible N-terminal acetylation which occurs co-translationally, and reversible post-translational acetylation of a 𝜀-amino lysine residue. Deacetylation is regulated by four classes of histone deacetylases (HDACs). Sirtuins (SIRTs; silent mating type information regulation two homologs) are class III HDACs, which require NAD+ as a cofactor. In contrast to class I and class II HDACs that are zinc-dependent and sensitive to trichostatin A inhibition, sirtuins are not sensitive to trichostatin A. In mammals, a total of seven sirtuins isoforms are found, SIRT1-7. Although all sirtuins are NAD+-dependent, they vary in subcellular localization, PTM types, and substrate affinity. SIRT1, SIRT6, and SIRT7 are the main nuclear sirtuins whereas SIRT2 localizes in both, the cytoplasm and nucleus. SIRT3, SIRT4, and SIRT5 are the mitochondrial sirtuins responsible for the regulation of protein acetylation. The main characteristics of sirtuins are given in Table 1. In this review, we summarize and discuss previous studies on the role of mitochondrial sirtuins, particularly SIRT3, in the regulation of mitochondrial metabolism and function in the heart. In addition, we will discuss the role of mitochondrial protein acetylation in the pathogenesis of cardiac diseases including MI, IR, cardiac hypertrophy, and HF. Finally, we will describe potential therapeutic strategies that are currently under development to target mitochondrial protein acetylation for treatment of cardiac diseases.

3In the cell, SIRT2 is localized in the cytoplasm, and it is in contact with chromatin in the nucleus. In addition to its deacetylase activity, SIRT2 possesses demyristoylase activity in vitro. It is most abundant in the central nervous system and has important roles in regulating tubulin acetylation, cell cycle, and glucose metabolism. The myocardial SIRT2 expression is reduced during cardiac hypertrophy. The role of SIRT2 in mammalian cell metabolism is discussed elsewhere.

4Among all sirtuins, SIRT1 has been the most extensively studied in cardiovascular diseases. SIRT1 is the homolog of S. cerevisiae Sir2 and is primarily localized in the nucleus. Studies on adult mouse hearts demonstrated that SIRT1 can be translocated from the nucleus to the cytosol. SIRT1 upregulation is associated with cardioprotection since heart-specific SIRT1 overexpression protects the heart against cardiac dysfunction and cell death, cardiac hypertrophy, and mediates the cardioprotective effects of ischemic preconditioning. One of the mechanisms underlying the cardioprotective effects of SIRT1 is mediated through the nuclear induction of the MnSOD (or SOD2) expression.

5SIRT6 is localized in the nucleus where it is tightly bound to chromatin. In addition to its main role in regulating DNA transcription via histone deacetylation, SIRT6 also possesses ADP-ribosyltransferase activity. SIRT6 has been suggested to be crucial for cell survival as SIRT6 KO mice died within 24 days of birth due to degenerative processes. On the other hand, when SIRT6 KO mice were crossbred, the life-span increased to 1 year, but they spontaneously developed cardiac hypertrophy. Also, SIRT6 expression was downregulated in the failing human hearts as well as in a mouse model of HF. Thus, the high mortality and spontaneous development of hypertrophy in SIRT6 deficient animals suggest that SIRT6 has a crucial role in cardiac function.

6SIRT7 is primarily localized in the nucleus/nucleolus although it was also detected in the cytoplasm of fibroblasts. SIRT7 deacetylase and deacylase activity can be regulated by dsDNA and RNA. Interestingly, SIRT7 has been found to regulate mitochondrial biogenesis through the regulation of nuclear-encoded mitochondrial genes. Additionally, SIRT7 deficient mice exhibited a lower mitochondrial oxygen consumption rate in the heart and a 50% decrease in lifespan which correlated with an increase in cardiac hypertrophy, fibrosis, and apoptosis.

7The expression of SIRT3 is high in the heart, and experimental studies demonstrated that mitochondrial protein acetylation only increases in the absence of SIRT3 but not SIRT4 or SIRT5. SIRT3 has been implicated in various cardiac pathologies and it deacetylates multiple enzymes in mitochondrial metabolism. The heart, along with the brain and skeletal muscle, is the organ most affected by SIRT3 deficiency. SIRT3 KO animals display more than a twofold increase in mitochondrial protein acetylation in these organs, suggesting a critical role for SIRT3 in regulating cardiac mitochondria metabolism. Also, NADH levels are higher in SIRT3 deficient hearts, thus indicating that SIRT3 has a role in the maintenance of mitochondrial redox potential and metabolism. However, compared to other tissues such as the liver, the role of SIRT3 in cardiac mitochondria is not well-characterized. Tissue-specific differences in mitochondrial regulation by SIRT3 further highlight the importance of studies elucidating the role of SIRT3 in the heart. Since SIRT3 is the main sirtuin involved in acetylation/deacetylation of mitochondrial proteins, this review focuses on the contribution of SIRT3 to mitochondrial metabolism and function in the heart.

8Mitochondrial SIRT4 and SIRT5 resemble prokaryotic sirtuins which indicate a bacterial ancestry of the mitochondria. SIRT4 has no deacetylase activity, but it is an ADP-ribosyltransferase and is involved in the regulation of lipid metabolism. In the heart, SIRT4 mediates the detrimental effects of cardiac hypertrophy including ROS production and fibrosis. However, hypoxia reduces the expression of SIRT4, and its overexpression protected H9c2 cells against hypoxia-induced cell death. It is likely that SIRT4 participates in regulating cardioprotective pathways although the mechanisms by which it exerts the beneficial effects remain unknown.

9SIRT5 is a weak deacetylase but has strong desuccinylase, demalonylase, and deglutarylase activities. It has also been reported that SIRT5 can function as a deglutarylate. SIRT5 has been implicated in regulating different aspects of mitochondrial metabolism. In H9c2 cells, the expression of SIRT5 is reduced in response to hydrogen peroxide. Downregulation of SIRT5 reduces cell viability in response to oxidative stress, and SIRT5 overexpression protects these cells. In the heart, SIRT5 deficiency renders mice more sensitive to IR injury. The roles of SIRT4 and SIRT5 in the heart are discussed elsewhere.

10Mitochondria metabolism and function is highly regulated by protein acetylation. Pioneering studies have reported that 30% of cytosolic and 15% of mitochondrial proteins are acetylated in the rat heart. However, analysis of the mitochondrial acetylome in detail by using advanced technology revealed that over 60% of mitochondrial proteins contain acetylation sites and that most of these proteins are involved in regulating energy metabolism, particularly, fatty acid metabolism, TCA cycle, ETC and OXPHOS (Table 2). Acetylation of mitochondrial proteins occurs through both, enzymatic and non-enzymatic pathways. Enzymatic acetylation involves the GNAT family of acetyltransferases, which include acetyl-CoA acetyltransferase (ACAT1) and GCN5L1. Non-enzymatic protein acetylation relies on the spontaneous transfer of an acetyl group from acetyl-CoA to a lysine residue and therefore, does not rely on the activity of an enzyme. Acetylation of mitochondrial proteins occurs primarily through the non-enzymatic mechanism due to the high abundance of acetyl-CoA in the matrix. Therefore, deacetylation by sirtuins, particularly SIRT3, is the main mechanism regulating protein acetylation in mitochondria.

11Acyl-CoA dehydrogenases catalyze the first reaction of FAO in the mitochondria. Among all acyl-CoA dehydrogenases, the role of SIRT3 in the regulation of the activity of the LCAD has been extensively studied. In vitro studies demonstrated that acetylation of LCAD reduced its activity by 75% and that SIRT3 was able to deacetylate LCAD and increase its activity. Two additional acyl-CoA dehydrogenases (ACADs), medium-chain acyl-CoA dehydrogenases (MCAD), and ACAD9, were found to be SIRT3 substrates, but further studies are necessary to confirm the role of acetylation/deacetylation of these enzymes in FAO activity.

12In SIRT3-/- mice, liver mitochondria have 71% of the proteins involved in FAO with increased acetylation. These mitochondria have higher FAO intermediates suggesting decreased levels of FAO. Indeed, only long-chain, but not medium and short chain, fatty acids accumulated in the plasma and in the liver suggesting that acetylation likely plays a role in regulating LCAD activity. Elucidation of LCAD activity in SIRT3 deficient liver mitochondria demonstrated that LCAD is a target of SIRT3 and that acetylation reduced its activity by 53%. Administration of SIRT3 was able to restore LCAD activity. In addition to the liver, several cell types have demonstrated a role of SIRT3 in LCAD regulation.

13Importantly, several groups have demonstrated the role of LCAD acetylation in the heart. SIRT3 deficient animals demonstrate 33% lower FAO in the heart and increased LCAD acetylation when compared to WT animals suggesting that cardiac LCAD acetylation could have an inhibitory effect on the enzymatic activity. LCAD acetylation has been found in several models of cardiac diseases such as hypertrophy and HF. Additionally, using expression vectors in H9c2 cells, murine LCAD was able to interact with SIRT3 and overexpression of SIRT3 prevented hypertrophy-characterized lipid accumulation, further supporting that acetylation has an inhibitory role on LCAD in the heart. In contrast, in the hearts of animals fed a high-fat diet, LCAD was hyperacetylated, and FAO was increased.

14Conflicting evidence exists on the effect of LCAD acetylation on its enzymatic activity in the heart. These results could represent an additive effect of several PTMs of the enzyme, which could influence its activity differently between pathologies. For example, in cardiac diseases, acetylation could reduce LCAD enzymatic activity due to the combination with another PTM, different from the one observed with a high-fat diet. These data suggest that although acetylation is an important mediator of cardiac FAO metabolism, its effect could vary among pathologies.

15The liver of SIRT3 KO mice has 43% of the proteins involved in the TCA cycle with increased acetylation. Therefore, it is evident that acetylation could have a profound role in regulating energy metabolism in the TCA cycle. In the next subsections, we will discuss the data available on the regulation of the TCA cycle enzymes by SIRT3. The role of SIRT3 in regulating the SDH activity is discussed in complex II.

16The PDH complex is composed of three enzymes which catalyze the rate-limiting step of pyruvate decarboxylation (pyruvate to acetyl-CoA) to enter the TCA cycle. This process is critical as it links glycolysis to aerobic energy production through the TCA cycle. Hence, regulation of the enzymatic activity of PDH determines substrate flux through the ETC coupled with OXPHOS in mitochondria.

17Several studies have revealed that acetylation decreases PDH activity in the liver, skeletal muscle, and MEFs derived from SIRT3 deficient animals. In addition, some of these studies have established that PDH deacetylation is directly regulated by SIRT3. Indeed, SIRT3 deficient animals demonstrated increased accumulation of pyruvate in skeletal muscle mitochondria consistent with inhibition of pyruvate decarboxylation. Importantly, a physical interaction was observed between SIRT3 and PDH suggesting that its enzymatic activity is regulated by SIRT3. However, only a few studies have focused on the role of SIRT3 in regulating PDH activity in the heart. In the heart, increased protein acetylation due to SIRT3 deficiency or angiotensin II treatment was associated with decreased PDH activity. Due to the important role in mitochondrial bioenergetics, elucidating the role of SIRT3 in the regulation of PDH activity in the heart needs further studies.

18Aconitase is an enzyme of TCA cycle that catalyzes the isomerization of citrate to isocitrate. In SIRT3-/- mice-derived MEF, aconitase was significantly hyperacetylated when compared to MEF derived from WT animals. In an experimental model of HF and in human HF samples, an increase in aconitase acetylation was observed when compared to sham. Aconitase hyperacetylation was able to increase its activity in the heart and SIRT3, in the presence of NAD+, was able to deacetylate and reduce its activity. Although acetylation is regarded as an inhibitory PTM, evidence suggests that acetylation of aconitase in the heart increases its activity.

19Isocitrate dehydrogenase catalyzes the formation of α-ketoglutarate (α-KG) from isocitrate by utilizing NADP+ and generating NADPH in the TCA cycle. Acetylation of IDH2 was demonstrated in HeLa cells and liver mitochondria isolated from human and mouse tissues. SIRT3 deficiency increased IDH2 acetylation in MEF and liver mitochondria. Several studies have reported that deacetylation of IDH2 by SIRT3 increases its activity in vitro using purified porcine IDH2 and in HEK293 cells. However, IDH2 activity was only found reduced in the liver but not in the inner ear and brain of SIRT3 KO mice. In contrast, it was recently reported that in vitro acetylation of purified IDH2 from the porcine heart increased its activity. These results suggest that acetylation of IDH2 influences its enzymatic activity in a tissue-dependent manner; deacetylation increases its activity in the kidney and liver but decreases its activity in the heart and has no effect in the inner ear and brain.

20Malate dehydrogenase is one of four dehydrogenases involved in the TCA cycle. It catalyzes the oxidation of malate to oxaloacetate, and the latter re-enters the TCA cycle to produce citrate. Several studies revealed that MDH possesses the ability to be acetylated. MDH2 was found hyperacetylated in SIRT3 deficient mouse liver mitochondria and MEF. A 66-fold increase in acetylation levels at Lys-239 was reported in SIRT3 deficient MEF suggesting that acetylation of this residue might have an important role in regulating its enzymatic activity. Indeed, when the lysine was substituted for glutamine, mimicking the acetylation state, a marked reduction of MDH activity was observed. These studies provide evidence that MDH activity is reduced because of its acetylation. However, reported that acetylation of MDH increased its enzymatic activity in Chang liver (HeLa derivative) and HEK293 cells. Interestingly, neither acetylation nor deacetylation had any effect on the enzymatic activity of purified bovine heart MDH2. These results suggest that, like IDH2, the effects of acetylation on the MDH enzymatic activity are tissue-dependent. In addition, differences in experimental models of acetylation, i.e., acetyl mimetics, in vitro acetylation, or SIRT3 deficiency, could account for variations in results.

21Complex I is composed of 45 subunits including 14 core and 31 supernumerary (accessory) subunits. It transfers electrons from NADH to coenzyme Q10 expelling 4H+ into the intermembrane space thereby contributing to the mitochondrial membrane potential (Ψm). Importantly, complex I is one of the most important contributors to ROS production. It is also involved in the pathogenesis of various cardiovascular diseases and plays a crucial role in mitochondria-mediated cell death.

22In the human cell line HEK293T, treatment with NR, a pyridine-nucleoside form of vitamin B3 that functions as a NAD+ precursor, decreased the acetylation levels of the complex I subunit NDUFA9, possibly through activation of SIRT3. This subunit was found acetylated in SIRT3 deficient liver mitochondria and MEF. Other studies demonstrated a direct interaction between SIRT3 and two subunits of complex I, NDUFA11 and NDUFS8. Furthermore, complex I acetylation decreased its activity by 20% in SIRT3 deficient liver mitochondria. In HeLa cells, treatment with NAM (SIRT inhibitor) increased complex I acetylation, and the immunocaptured complex I was deacetylated in vitro in the presence of exogenous SIRT3 but not SIRT4. In contrast, other studies found that skeletal muscle or liver-specific SIRT3 deletion did not affect complex I activity. Altogether, these results suggest that SIRT3 can interact and regulate complex I activity in various non-cardiac tissues.

23In the heart, cardiac-specific deletion of NDUFS4 reduced complex I activity and mitochondrial respiration by 75% and 45%, respectively. The reduced complex I activity was associated with a 50% decrease in the NAD+/NADH ratio leading to inhibition of SIRT3 activity. Mitochondrial proteins from these hearts, including NDUFA9, were found to be hyperacetylated. Conversely, overexpression of SIRT3 was able to reduce protein acetylation. In contrast, SIRT3-/- hearts have decreased mitochondrial respiration for complex I and increased levels of NADH. These results suggest a strong interdependence of OXPHOS and protein acetylation for the maintenance of mitochondrial function.

24Downregulation of SIRT3 increases the sensitivity of cardiac cells to oxidative and energy stress. IR significantly reduced complex I activity in H9c2 cardiomyoblast cells with SIRT3 knockdown. Likewise, ex vivo IR decreased complex I activity and state 3 respiration in SIRT3 deficient hearts. We found no difference in complex I activity between SIRT3-/- and WT hearts subjected to IR. Likewise, no differences were observed in complex I activity after 7 days of permanent coronary artery ligation. Lastly, several complex I subunits were found to be hyperacetylated in an experimental model of HF and in the failing human heart. Altogether, these studies suggest that complex I acetylation is present during cardiac pathologies thereby portraying SIRT3 as a key player.

25Succinate dehydrogenase or complex II is a key enzyme of the TCA cycle and has two main roles in the mitochondria: (i) catalyzes the oxidation of succinate to fumarate, and (ii) reduces ubiquinone to ubiquinol. It is comprised of four subunits: two catalytic subunits, SDHA and SDHB, attached to the IMM and facing into the matrix, and two membrane-anchored subunits, SDHC and SDHD. The SDHA subunit is mostly involved in the oxidation of succinate to fumarate, while the other three subunits catalyze the reduction of ubiquinone to ubiquinol.

26In liver mitochondria, SIRT3 deficiency increased SDHA acetylation and SIRT3 was able to deacetylate SDHA, but it did not affect its activity. On the other hand, there was no difference in acetylation in immunocaptured complex II from WT and SIRT3 deficient livers. Also, these studies found no interaction between complex II and SIRT3, and SIRT3 deletion did not affect complex II-mediated respiration. In contrast, other groups reported that SDHA hyperacetylation decreased its electron transferring activity. Additionally, SDHA was shown to be a target for SIRT3 in SIRT3 KO embryonic fibroblasts. In the absence of SIRT3, SDH activity was reduced by 25% and succinate accumulation increased suggesting inhibition of the SDHA subunit.

27Similarly, SIRT3 deficient hearts demonstrated an increase in SDH acetylation. Moreover, the complex II activity in SIRT3 KO hearts was lower than WT hearts after ex vivo IR, possibly due to complex II hyperacetylation. In the failing human heart, as well as in mice with HF, SDHA acetylation correlated with decreased complex II-mediated respiration and SDH activity. Thus, acetylation of complex II, like complex I, is associated with a reduced activity in the heart due to a possible downregulation of SIRT3.

28Among ETC complexes, acetylation of complexes III and IV and their possible regulation by SIRT3 has been less studied. Cytochrome c reductase or complex III is composed of 11 subunits and contains two monomeric units. Cytochrome c oxidase or complex IV is the final electron acceptor of the ETC; it transfers electrons from cytochrome c to oxygen, thereby producing water at the expense of oxygen.

29Skeletal muscle- or liver-specific SIRT3 deletion did not affect complex IV activity. Furthermore, 3-day differentiated myoblasts with SIRT3 ablation displayed a decrease in the complex IV activity. In the heart, subunits of complex III and complex IV were differentially acetylated in SIRT3 KO mice. The cytochrome c oxidase subunit 4 isoform 1 was found acetylated in an animal model of HF. Interestingly, several subunits of complexes III and IV were found to be hyperacetylated in the failing human heart. However, there was no difference between the enzymatic activity of complexes III and IV of SIRT3-/- and WT hearts exposed to IR. Although existing studies demonstrate acetylation of complexes III and IV, further studies are required to establish the effect of acetylation on the activity of these complexes in the heart.

30The main function of the F1FO-ATP synthase or complex V is to synthesize ATP from ADP and Pi through the process known as OXPHOS. Indeed, several studies have demonstrated the potential role of SIRT3 in regulating ATP production. In HEK293T and human osteosarcoma cells, SIRT3 downregulation increased the acetylation of the OSCP and alpha subunits of complex V, and this was associated with decreased activity and ATP levels. Several groups have reported that SIRT3 physically interacted with the OSCP subunit in human osteosarcoma cells, HEK293T cells, and HCT116 cells. Furthermore, recent studies suggested that the OSCP-SIRT3 interaction is amongst the most specific SIRT3 interactions. Also, there is evidence that SIRT3 interacts with other subunits of the ATP synthase. SIRT3 deficient MEF produced 30% less ATP and expression of SIRT3 restored the ATP levels. In contrast, skeletal muscle or liver-specific SIRT3 deletion did not affect the levels of ATP. This data demonstrates that SIRT3 not only interacts and deacetylates various subunits of complex V, but it can also regulate ATP production.

31Recent studies suggest that SIRT3 is involved in the regulation of ATP production in the heart through the deacetylation of mitochondrial proteins. ATP levels were reduced in the heart of SIRT3 deficient mice and the effect remained unchanged after ex vivo IR. HKL, a SIRT3 activator, reduced OSCP acetylation in primary rat cardiomyocytes suggesting that the OSCP subunit is also a SIRT3 target in cardiac cells. Most importantly, in animal models of HF and in the failing human heart, several subunits of the F1FO-ATP synthase displayed an increase in acetylation. Reduced ATP production is one of the hallmarks of HF; therefore, these findings provide strong evidence that increased F1FO-ATP synthase acetylation due to reduced SIRT3 activity could be a key mediator of mitochondria-mediated cardiac dysfunction.

32Overexpression of SIRT3 has been demonstrated to prevent ROS accumulation in cardiomyocytes in response to different stressors such as the anticancer agent triptolide (TP) and the α1-adrenergic receptor agonist phenylephrine. Indeed, SIRT3 deficient cardiomyocytes produced double the levels of ROS than WT cells with and without phenylephrine stimulation. In neonatal rat cardiomyocytes, phenylephrine-induced ROS stimulation was abolished by treatment with the sirtuin activator NAD+. Although the mitochondrial ROS levels were no different between SIRT3-/- and WT hearts, IR induced an increase in ROS levels in SIRT3 deficient mitochondria. Resveratrol, a potent SIRT1 activator, prevented H2O2-induced cell death but this effect was absent in the presence of NAM. Nevertheless, these studies suggest that SIRT3 can regulate mitochondrial ROS levels, at least during cardiac stress, through the regulation of the mitochondrial antioxidant system.

33Mitochondrial DNA is susceptible to oxidative damage when the ROS levels exceed the antioxidant capacity of mitochondria. Oxidative damage to DNA results in the oxidation of guanine (G) to 8-oxo-deoxyguanosine (8-oxo-dG) which leads to an abnormal G:T transversion eventually creating a mutation. This type of lesion is repaired by the BER pathway which is also present in the mitochondria, and oxoguanine glycosylase and apurinic/apyrimidinic endonuclease 1 are the enzymes involved in the BER pathway. Although the BER pathway is not directly involved in ROS reduction, it repairs ROS-induced mtDNA lesions. SIRT3 has been shown to regulate the activity of enzymes involved in BER.

34In rat neonatal cardiomyocytes, the anticancer drug doxorubicin increased mtDNA lesions and oxoguanine glycosylase acetylation. Activation of SIRT3 by HKL reduced 8-oxo-dG lesions in cardiomyocytes. SIRT3 can interact with oxoguanine glycosylase. In the absence of SIRT3, doxorubicin increased the levels of 8-oxo-dG, which was abrogated by SIRT3 overexpression. Cardiac-specific SIRT3 overexpression increased the resistance of cardiomyocytes to doxorubicin-induced mtDNA damage, and conversely, the hearts of SIRT3 deficient mice were more susceptible to doxorubicin-induced mtDNA damage. Thus, SIRT3 is involved in regulating the mitochondrial antioxidant system and the BER pathway to prevent ROS-induced protein oxidation and mtDNA damage. In response to energy and oxidative stress, ROS production likely exceeds the antioxidant capacity of the mitochondria due to acetylation-induced inactivation of mitochondrial antioxidant enzymes.

35A growing number of studies suggest a possible link between SIRT3 and the mitochondrial antioxidant system. SIRT3 deficiency increased superoxide levels in rat myoblastic cells, MEF, and liver tissue. The increased ROS was associated with low activity of MnSOD and its hyperacetylation. SIRT3 expression was able to deacetylate MnSOD and increase its activity in SIRT3-/- MEF and reduce mitochondrial and cellular ROS. Similar results were also found in HEK293T and BREC cells. These results suggest that MnSOD is a direct target of SIRT3. Acetylation of MnSOD renders the enzyme inactive whereas deacetylation by SIRT3 activates it, therefore decreasing superoxide levels. Although confounding evidence suggests that SIRT3 regulates MnSOD, it should be noted that tissue-specific deletion of SIRT3 in the muscle or liver did not result in any difference between mitochondrial MnSOD and catalase activity.

36Studies on the heart also suggested a potential role of SIRT3 in regulating mitochondrial ROS. Under physiological conditions, SIRT3 overexpression increased MnSOD but not catalase activity in H9c2 cardiomyoblastic cells. MnSOD activity was reduced in the heart of SIRT3-/+ mice although other studies were unable to detect a difference in MnSOD activity between SIRT3-/- and WT hearts. Interestingly, these studies revealed low MnSOD activity in SIRT3-/+ and SIRT3-/- hearts after oxidative stress induced by ex vivo IR. The reduced MnSOD activity was associated with increased oxidative damage to mitochondrial proteins. Conversely, treatment with NAD+, a sirtuin activator, recovered MnSOD activity in WT hearts after IR.

37In rat neonatal cardiomyocytes, HKL reduced H2O2-induced MnSOD acetylation which was associated with decreased ROS levels and cell death. These effects of HKL were absent in SIRT3 silenced neonatal cardiomyocytes. Interestingly, HKL treatment or SIRT3 overexpression protected neonatal cardiomyocytes against doxorubicin-induced damage through reduction of ROS levels. Likewise, isoproterenol and angiotensin II diminished both, expression and activity of MnSOD and catalase in cardiomyocytes. However, these effects were not observed in SIRT3 overexpressed cells. SIRT3 overexpression also prevented the decrease in catalase and MnSOD activity in response to TP-induced stress. These studies demonstrate a strong link between SIRT3 activity and MnSOD acetylation; SIRT3 deacetylates and activates MnSOD thereby reducing mitochondrial ROS levels.

38Although IDH is a TCA cycle enzyme, it also serves as a mitochondrial source of NADPH and thus, is the main regulator of the NADPH-dependent glutathione reductase (GSR). GSR is involved in regenerating glutathione (GSH) from glutathione disulfide (GSSG), and therefore in H2O2 detoxification with glutathione peroxidase (Figure 1). Thus, high levels of IDH2 increase the NADPH levels and consequently, the GSH pool, augmenting the mitochondrial detoxification system. Indeed, IDH2 contributes 25% of the total mitochondrial NADPH. As discussed previously (see section “Isocitrate Dehydrogenase”), IDH2 is a target of SIRT3, and its deacetylation increased NADPH levels in HEK293 cells. This increase in NADPH levels protected the cells against H2O2-induced oxidative damage. Interestingly, although CR increased the GSH to GSSH ratio in the WT mouse liver, brain, and inner ear, this effect was not observed in SIRT3 deficient tissues. Thus, a growing body of studies provides evidence in favor of indirect actions of SIRT3 in regulating the glutathione system through IDH as a source of NADPH. However, the precise role of SIRT3 in the glutathione system in the heart remains to be elucidated.

39Caloric restriction, or a reduction of caloric intake of 30% or more without malnutrition, has been linked to lifespan extension. It has been shown to induce changes in mitochondrial protein acetylation. However, the heart, along with the kidney and brain, demonstrates only subtle changes in mitochondrial protein acetylation after CR in comparison to the liver and brown adipose tissue. Interestingly, changes in acetylation in response to CR vary among tissues, where, in contrast to the liver, CR reduced mitochondrial protein acetylation in the brown adipose tissue. Although the heart, skeletal muscle, and brain contain fewer acetylation sites in mitochondrial proteins in comparison to the liver and kidney, these sites are more dynamic in response to 48 h of fasting suggesting an essential role of CR-mediated acetylation in mitochondrial metabolism.

40Since SIRT3 is a NAD+-dependent deacetylase, the increase in mitochondrial acetylation observed with aging is probably due to low NAD+ levels and/or reduced SIRT3 expression. However, CR increased SIRT3 expression in liver, skeletal muscle, and in white and brown adipose tissue but not in the heart, kidney or brain. Therefore, it would be equally important to measure, in addition to SIRT3 expression, NAD+ availability for SIRT3 activity during CR.

41It has been reported that aging decreased the activity of NAM phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ synthesis, and NAD+ levels in the mitochondria suggesting that the anti-aging effects observed with CR could also be mediated through increasing mitochondrial NAD+ levels. Interestingly, rats that underwent fasting for 48 h displayed enhanced mitochondrial NAMPT expression associated with increased NAD+ levels in liver mitochondria. Most importantly, CR was able to increase SIRT3 activity in cardiac mitochondria. Although SIRT3 is essential in the prevention of age-related hearing loss, there is no data on the role of SIRT3 in mediating the cardioprotective effects of CR. Studies in humans suggest that SIRT3 can be involved in the mediation of lifespan extension. Since cardiovascular diseases, particularly HF, are age-associated diseases, it is important to study the potential mechanism by which SIRT3 can mediate the cardiac-related effects of CR on lifespan.

42Mitochondrial biogenesis is a mechanism of mitochondrial turnover, primarily driven by the expression of nuclear-encoded proteins such as the peroxisome PGC-1α. PGC-1α is a member of the nuclear receptor superfamily that mediates the assembly of transcriptional machinery directed to specific DNA sequences. PGC-1α is highly expressed in the skeletal muscle and in the heart. It regulates, through the mitochondrial transcriptional network, expression of genes encoding mitochondrial proteins that are involved in FAO, the TCA cycle, and OXPHOS (reviewed in ). It also regulates the nuclear respiratory factor 1 and 2 (NRF1 and NRF2) which interact with mitochondrial transcription factor A (TFAM). The latter regulates the expression of mtDNA-encoded genes by binding to the light and heavy strand mtDNA promoters. The transcriptional network regulating mitochondrial biogenesis is reduced in cardiac diseases including MI (IR) and HF. Expression of PGC-1α and its downstream factors were diminished in animal models of HF induced by aortic banding and post-MI as well as in patients with HF.

43Several studies demonstrated crosstalk between mitochondrial biogenesis and SIRT3 in cardiac pathologies, particularly in cardiac remodeling induced by a hypertrophic response. PGC-1α has been shown to regulate the expression of oxidative stress protective genes through the interaction with the transcriptional activator protein FOXO3a, which triggers apoptosis. Interestingly, FOXO3a binding sites have been found in the PGC-1α promoter suggesting that FOXO3a can regulate the expression of PGC-1α. Studies on SIRT3 deficient mice revealed that SIRT3 plays a cardioprotective role in cardiac hypertrophy by suppressing ROS production through the induction of FOXO3a. Interestingly, oxidative stress in vitro did not increase acetylation of PGC-1α in H9c2 cardioblastic cells. However, PGC-1α deficiency resulted in a reduction of SIRT3 gene expression in mice muscle and liver cells. The same study showed that SIRT3 plays an important role in the regulation of ROS detoxifying enzymes such as MnSOD and glutathione peroxidase, which are also associated with PGC-1α. Studies on human umbilical vein endothelial cells revealed that SIRT3 mediates the deacetylation of FOXO3, inducing its translocation to the nucleus where it upregulates key proteins for mitochondrial biogenesis and quality control such as PGC-1α, mitochondrial transcription factor A, Drp1, Fis1, and Mfn2. The activation of SIRT3 by HKL treatment increased mRNA levels of PGC-1α, reduced ROS production, and improved mitochondrial respiration in a mouse model of pressure overload hypertrophy. These findings demonstrate a central role of SIRT3 in regulating mitochondrial biogenesis, reducing oxidative stress and protecting mitochondrial metabolism. Additionally, in SIRT3 deficient mice, inhibition of the interaction between SIRT3, FOXO3a, and Parkin caused a reduction in mitophagy leading to the development of diabetic cardiomyopathy.

44Mitochondrial dynamics plays an important role in regulating mitochondrial quality control and it consists of two main processes: (i) fusion: the combination of two mitochondria to maintain their functional and structural integrity through content exchange such as mtDNA, matrix proteins, and essential phospholipids; and (ii) fission: mitochondrial division into two smaller daughter organelles, sometimes referred to as fragmentation.

45Mitofusins 1 (Mfn1) and 2 are proteins localized in the OMM that participate in the fusion process (the merging of two individual mitochondria). Studies on Mfn1 and Mfn2 double KO mice demonstrated that the heart became dilated by 5 weeks and developed HF by 7–8 weeks. The OPA1 protein, which is embedded in the IMM, enables fusion of the IMM through the formation of trans-complexes. In addition to fusion, OPA1 is involved in remodeling and maintaining the cristae structure in the IMM. Altogether, fusion proteins play a vital role in maintaining cardiac and contractile functions as well as in late cardiac development.

46Mitochondrial fission is mediated by the GTPase protein Drp1 and the adapter protein Fis1 on the OMM. Drp1 is a cytosolic GTPase that is recruited to the OMM during fission. Drp1 homo-oligomerizes to form a constraining chain around the mitochondrion to divide it into two daughter organelles. Mitochondrial fission factor, an OMM protein, functions as a Drp1 receptor that regulates the mitochondrial fission process.

47Alterations in mitochondrial fusion-fission processes are involved in the pathogenesis of cardiac diseases, particularly, MI and HF. Both in vitro and in vivo models of cardiac hypertrophy and HF decreased OPA1 and Mfn2 expression and increased mitochondrial fragmentation. Reduced expression of fusion proteins and increased mitochondrial fission in response to post-infarction remodeling was associated with mitochondrial dysfunction in rats. Recent studies using a double KO of Mfn1 and Mfn2 and triple KO of Mfn1, Mfn2, and Drp1 mouse model demonstrated that the complete ablation of mitochondrial dynamics results in cardiac hypertrophy and HF due to diminished mitophagy. Also, alterations in mitochondrial dynamics accelerated mitochondrial senescence.

48Several studies have elucidated the role of protein acetylation in mitochondrial dynamics. In human fibroblasts, treatment with NAM enhanced mitochondrial fragmentation and increased the expression of Drp1, Fis1, and Mfn1, though acetylation of these proteins and the mechanisms of their upregulation were not examined. OPA1 has been shown to contain acetylation sites, and SIRT3 can target OPA1 and regulate its activity. Hyperacetylation of OPA1 inhibits its GTPase activity, while SIRT3 prevents OPA1 acetylation and preserves its activity. In favor of this, electron microscopy imaging of SIRT3 deficient cardiac mitochondria displayed a continuous (fused) OMM between two mitochondria, but weakly bound (not fused) IMM, indicating dysfunctional fusion dynamics.

49Tert-butyl hydroperoxide (t-BHP), an organic peroxide, decreased SIRT3 expression associated with OPA1 acetylation in H9c2 cardiomyoblasts. The acetylation of OPA1 induced its cleavage, inevitably provoking apoptosis. Similarly, doxorubicin reduced SIRT3 expression, which was associated with a decrease in OPA1 and Mfn1 expression in mice. Treatment with SIRT3 activator HKL prevented doxorubicin-induced myocardial injury and preserved the expression levels of SIRT3, OPA1, and Mfn1. These studies indicate the importance of maintaining SIRT3 functional activity to prevent the downregulation of mitochondrial fusion proteins.

50Mitophagy is the process of removing damaged, dysfunctional, or abnormal mitochondria through autophagy to maintain proper mitochondrial function in the cell. Under physiological conditions, a membrane potential-dependent protein known as presenilin-associated rhomboid-like protein (PARL) induces cleavage of the mitochondria-localized serine/threonine-protein kinase PTEN-induced putative kinase 1 (PINK1). Under stress, the ability of PARL to cleave PINK1 decreases due to diminished mitochondrial membrane potential. Recent studies also suggested a role of ATP depletion in decreased PINK1 cleavage. As a result, uncleaved, full-length PINK1 recruits Parkin, an E3 ubiquitin ligase which regulates ubiquitination of target proteins such as Nip3-like protein X (NIX). In mammalian cells, the ubiquitination of the latter has been shown to serve as a receptor for mitophagy. Other proteins such as Bcl2/adenovirus EB interacting protein 3 (BNIP3), cardiolipin, and FUN14 domain-containing protein-1 (FUNDC1) participate in mitophagy by binding to LC3.

51One of the first indicators of the mitophagy-sirtuin interplay came from NAM-treated primary human fibroblasts. The study found that the sirtuin inhibitor, NAM, induced a decrease in mitochondrial mass and an increase in membrane potential by activating mitophagy to eliminate damaged mitochondria. Furthermore, the effects of NAM on the mitochondrial quality could be mediated through an increase in the [NAD+]/[NADH] ratio leading to SIRT1 activation and induction of mitophagy. However, mitochondrial sirtuins, particularly SIRT3, were not assessed in these studies. Studies with SIRT3 deficient and overexpressing mice and primary neonatal mouse cardiomyocytes provided evidence that SIRT3 plays a regulatory role in autophagy and mitophagy through deacetylation of FOXO3a and Parkin. This conclusion was further supported by other studies elucidating the effects of angiotensin II treatment in WT and SIRT3-/- mice where SIRT3 deficiency enhanced the microvascular rarefication (thinning) provoked by angiotensin II. The study concluded that SIRT3 can stimulate mitophagy by the deacetylation of PINK1 and Parkin and thus, inhibiting angiogenesis and endothelial dysfunction. Nevertheless, the role of SIRT3 in mitophagy remains elusive.

52Cardiac hypertrophy involves the enlargement of the myocardium and development of fibrosis. Animal models of cardiac hypertrophy are induced by surgery and pro-hypertrophic agents that increase pressure and volume overload (stress). Post-infarction remodeling, transverse aortic constriction as well as pro-hypertrophic agents such as angiotensin II, phenylephrine, endothelin-1, and isoproterenol are used to develop animal models of cardiac hypertrophy. Increased ROS production and energy deficiency play a crucial role in cardiac remodeling associated with cardiac dysfunction. During the progression of cardiac hypertrophy to HF, Akt signaling is linked to alterations in mitochondrial metabolism leading to mitochondrial dysfunction. This pathway is activated through suppression of metabolic transcription regulators such as FOXO and PGC-1α. Mitochondrial dysfunction reduces metabolic flux thereby reducing NAD+ availability, inactivating SIRT3, and consequently provoking an increase in acetylation of proteins such as FOXO3a. Acetylation enhances the susceptibility of FOXO3a to phosphorylation by Akt, thus activating pathways that lead to mitophagy, autophagy, and cell death. This pathway is involved in the development of the cardiac hypertrophic response which is attenuated by SIRT3 overexpression in mice. In addition, the AMPK/SIRT1 pathway has been shown to mediate hypertrophy-induced mitochondrial dysfunction in H9c2 cardioblast cells and in the myocardium of spontaneously hypertensive rat.

53The role of mitochondrial protein acetylation in cardiac hypertrophy was confirmed in in vitro studies where activation or overexpression of SIRT3 prevented the development of cardiac hypertrophy. Overexpression of SIRT3 was able to prevent cardiac hypertrophy in response to phenylephrine or angiotensin II treatment in H9c2 cardiomyoblast cells and rat neonatal cardiomyocytes. Likewise, pharmacological activation of SIRT3 by treatment with NAD+ or HKL prevented phenylephrine- and angiotensin II-induced increase in cell size and attenuated induction of hypertrophic markers in rat neonatal cardiomyocytes. Furthermore, HKL was found to translocate to the mitochondria where it enhanced the activity and expression of SIRT3, therefore reducing acetylation of mitochondrial proteins. These findings suggest that HKL exerts anti-hypertrophic effects in a SIRT3-dependent manner.

54In vivo studies further confirmed the anti-hypertrophic role of SIRT3 in the heart. In a mouse model of angiotensin II-induced cardiac hypertrophy, NAD+ prevented the development of hypertrophy and induction of hypertrophic markers. Similarly, HKL treatment prevented and also reverted the development of hypertrophy and fibrosis, and the induction of hypertrophic markers, thus indicating that SIRT3 not only protects against cardiac hypertrophy but can also revert it.

55SIRT3 deficient hearts develop hypertrophy and cardiac fibrosis. However, contradicting evidence exists as to whether these animals develop cardiac dysfunction. On and all, when these animals were infused with angiotensin II, isoproterenol, or phenylephrine, the hearts developed twice as much cardiac hypertrophy and cardiac dysfunction as their WT counterparts. In response to transverse aortic constriction, SIRT3 KO mice developed a more severe cardiac hypertrophy accompanied by impaired cardiac function and fibrosis than WT animals. Importantly, the cardioprotective effects of HKL against cardiac hypertrophy were abolished in the absence of SIRT3. Also, cardiac-specific SIRT3 overexpression prevented angiotensin II- and doxorubicin-induced cardiac hypertrophy, fibrosis, and fetal gene expression confirming that SIRT3 has a role in protecting the heart against hypertrophic stimuli.

56In conclusion, SIRT3 has a crucial role in the development and progression of cardiac hypertrophy. The pleiotropic effects of SIRT3 activation in the prevention of mitochondrial ROS could be a crucial pathway to the inhibition of cardiac hypertrophy. However, in addition to ROS, other SIRT3-mediated mechanisms could be involved in preserving mitochondrial and cardiac function in response to hypertrophic stimuli.

57Myocardial infarction, or ischemia, is the most common type of cardiovascular disease. Standard treatment after MI is to restore the blood flow to the previously ischemic area, also known as reperfusion. Although restoration of blood flow to the ischemic area is crucial, reperfusion contributes up to 50% of additional damage to the final infarct size; a process also termed as IR injury.

58Emerging evidence implicating SIRT3 in a variety of cardioprotective mechanisms led to the postulation that SIRT3 could be implicated in IR injury. H9c2 cells silenced for SIRT3 expression were more susceptible to IR as evidenced by the enhanced release of lactate dehydrogenase, a marker of cellular damage. Indeed, the absence of one or both SIRT3 alleles in the heart (SIRT3-/+ or SIRT3-/-) led to a marked susceptibility to ex vivo IR. These hearts demonstrated reduced cardiac recovery, and increased infarct size and cell damage after IR. Furthermore, intravenous administration of NAD+ before ischemia protected the rat heart from IR injury. These animals demonstrated a significant reduction in infarct size, cardiac troponin levels, and apoptotic markers suggesting the potential role of sirtuins, such as SIRT3, in protecting the heart against IR. However, the causative link between SIRT3 and cardioprotection remains to be elucidated. In contrast, another group found no difference between SIRT3-/- and WT hearts in the susceptibility to IR. These discrepancies between studies could be explained by differences in heart preparation and duration of ischemia and reperfusion periods. Indeed, the same study demonstrated that in vivo coronary artery ligation in the absence of SIRT3 did not render the hearts more prone to cardiac dysfunction than their WT counterparts at 7 days post-MI. Nevertheless, the mechanisms mediating cardiac dysfunction are completely different in reperfused and non-reperfused ischemic hearts. SIRT3 activity is affected differently depending on the severity of both, ischemia and reperfusion injury in the heart. Furthermore, pathological mPTP induction can play a central role in mediating the adverse effects of protein acetylation.

59The mPTP is a non-selective pore that forms in the mitochondria and allows the non-selective passage of ions, solutes, and water up to 1.5 kDa into the mitochondrial matrix. As a result, the increase in IMM permeability induces mitochondrial swelling and rupture of the OMM leading to cell death. The mPTP opening is induced by high matrix Ca2+, ROS, and loss of mitochondrial membrane potential; all of these conditions occur at reperfusion (reviewed in;; ). The detailed molecular structure of the mPTP remains unknown. The adenine nucleotide translocase (ANT) and the voltage-dependent anion channel (VDAC) have been proposed as the core mPTP components, however, genetic studies revealed that these proteins play a regulatory rather than a structural role in mPTP induction. Several studies suggested that dimerization of the F1FO-ATPase (ETC complex V) and the c-subunit ring in the FO domain of the ATP synthase are involved in the mPTP. Most recent findings challenged the pore-forming role of the peripheral stalk subunits and the subunit c thus, further studies are required to establish the potential role of the F1FO-ATPase in mPTP formation. Cyclophilin-D (CyP-D), a matrix located peptidyl-prolyl cis–trans isomerase, is considered a key mPTP regulator. In addition to mPTP regulation, CyP-D plays an important role in regulating mitochondrial metabolism and bioenergetics under physiological conditions, thereby raising a question whether it is a viable target for cardioprotection.

60The discovery of the acetylation capacity of CyP-D hinted toward a possible involvement of SIRT3 in modulating mPTP formation. Cardiac-specific deletion of the NDUFS4 subunit of complex I decreased the NAD+/NADH ratio, and this was associated with increased mitochondrial protein acetylation and high mPTP sensitivity, thus providing evidence that acetylation could be a mediator of mPTP formation. Also, an increase in mitochondrial protein acetylation and reduced SIRT3 expression correlated with increased mPTP opening in a rodent model of post-MI HF. SIRT3 was shown to interact and deacetylate CyP-D and absence of SIRT3 increased CyP-D acetylation and induced mPTP opening. However, acute oxidative stress (10–30 min) in H9c2 cells did not affect CyP-D acetylation but, instead, further reduced acetyl-CyP-D levels after 60 min of incubation with hydrogen peroxide. Interestingly, mitochondria isolated from SIRT3 deficient hearts demonstrated high basal swelling in the absence of mPTP inducers (IR and Ca2+). However, another group reported that absence of SIRT3 did not affect the sensitivity of cardiac mitochondria to mPTP. The difference observed in the sensitivity of mitochondria to Ca2+ in the absence of SIRT3 can be explained by a possible cardioprotective role of hypothermia that was applied to the heart before IR. Interestingly, acetylation of CyP-D, and the oligomycin sensitivity conferring protein (OSCP) subunit of the F1FO-ATP synthase in animals subjected to transverse aortic constriction promoted their interaction inducing mPTP formation, and the interaction was reverted by NAD+, thus proposing that SIRT3 deacetylation can modulate mPTP induction.

61Overexpression of SIRT3 prevented mitochondrial membrane depolarization in triptolide-treated cardiomyocytes, a consequence of mPTP induction. Thus, some studies propose that CyP-D acetylation can sensitize mitochondria to mPTP induction and conversely, CyP-D deacetylation by SIRT3 desensitizes mitochondria to mPTP induction, therefore protecting cardiac mitochondria.

62Heart failure is the final stage after a progressive adaptation of the heart to increased workload induced by MI, hypertension, cardiomyopathy, etc. The failing heart is unable to pump sufficient amount of oxygen and substrates to the organs and tissues, which results in maladaptive secondary alterations. Mice with SIRT3 deficiency display signs of cardiac hypertrophy and interstitial fibrosis by 8 weeks of age. In response to hypertrophic stimuli, SIRT3 deficient mice developed severe cardiac hypertrophy, in contrast to SIRT3-overexpressing mice that did not respond to these stimuli. In animal models of HF, SIRT3 expression was reduced and acetylation of mitochondrial proteins was increased. In addition, the failing hearts contain decreased levels of NAD+.

63Indeed, several proteins have been found to be acetylated in animal models of HF. Protein acetylation was associated with downregulation of their enzymatic activity, and metabolic alterations in the heart. During normal cardiac physiology, the mitochondria primarily utilizes FAO (up to 90%) as the source of ATP. However, during HF, FAO is reduced, and glucose oxidation is increased, but as HF progresses, the body becomes insulin resistant further compromising cardiac energy metabolism. Impaired fuel utilization is accompanied by energy depletion, with a drop in ATP levels leading to contractile dysfunction (reviewed in ). In HF, LCAD was found to be more acetylated which has been associated with decreased activity, and that correlates with the reduction in FAO observed in HF. Other groups have reported that SDH activity, respiration rates, and ATP levels were significantly reduced in failing hearts. Indeed, various enzymes involved in ATP production have been found acetylated in HF. Also, in cardiomyocytes from guinea-pigs that developed HF, ROS levels were elevated which could be due to a reduction in the activity of the primary antioxidant in the mitochondria, MnSOD, which has been extensively studied as a SIRT3 target.

64Likewise, patients with end-stage HF exhibited decreased expression of SIRT3 and increased differentiation of fibroblasts to myofibroblasts, a critical step in developing tissue fibrosis. Indeed, SIRT3 KO mice developed tissue fibrosis in the heart, liver, kidney, and lungs in an age-dependent manner and this was associated with increased TGF-β1 expression and hyperacetylation of glycogen synthase kinase 3β (GSK-3β). Conversely, SIRT3 overexpression blocked aging-associated tissue fibrosis in mouse cardiac fibroblasts through SIRT3-induced deacetylation and activation of GSK3β which, in turn, inhibited TGF-β1 signaling and prevented tissue fibrosis. Additionally, in accord to animal models, failing human hearts also displayed increased acetylation of mitochondrial proteins, particularly the enzymes involved in energy metabolism such as IDH and MDH, associated with reduced NAD+ levels.

65Thus, SIRT3 acts as a negative regulator of cardiac hypertrophy and aging-associated tissue fibrosis. It is tempting to speculate that the mitochondrial metabolic alterations observed in severe hypertrophy and HF are, at least in part, the result of hyperacetylation of mitochondrial proteins due to reduced activity of SIRT3.

66A growing number of studies portraying SIRT3 as a key regulator of mitochondrial metabolism and energetics highlight the necessity to develop new pharmacological compounds and conditional approaches targeting SIRT3 for treatment of cardiovascular diseases. Therapeutically, the main goal would be to enhance SIRT3 activity and expression. One of the most important therapeutic approaches is aimed at increasing the mitochondrial NAD+ levels or other SIRT3 activators (Figure 2).

67Consumption of tryptophan or niacin in the diet stimulates de novo NAD+ biosynthesis. A diet rich in meat products, diary, and eggs can increase the NAD+ availability, thereby activating SIRT3. Another approach to enhance NAD+ synthesis is through the salvage pathway. Since NAMPT converts NAM to nicotinamide mononucleotide (NMN) for NAD+ biosynthesis in mammalian cells, supplementation with NMN or overexpressing NAMPT would increase NAD+ levels. Indeed, cardiac-specific NAMPT overexpression improved cardiac function and prevented hypertrophy and the progression of HF induced by transverse aortic constriction or isoproterenol treatment in animals. Treatment with NMN recovered the NAD+/NADH ratio and decreased the sensitivity of cardiac mitochondria to Ca2+-induced stress in mice with complex I deficiency. In mice with a high-fat diet (HFD)-induced type 2 diabetes, NMN improved glucose tolerance and enhanced hepatic insulin sensitivity by restoring NAD+ levels. Also, the expression of genes involved in oxidative stress, inflammation, and circadian rhythm was restored by NMN treatment. The beneficial effects of NMN were abrogated by EX527, a SIRT1-specific inhibitor, thereby confirming that SIRT1 is involved in mediating the effects of NMN. A 1-week treatment with NMN reversed the metabolic changes observed during age-related muscle pathology such as increased lactate, reduced ATP levels, and decreased expression of mtDNA-encoded OXPHOS genes. However, the effects of NMN were not observed in SIRT1 or NMN adenylyl transferase-1 KO mice. According to the mechanism proposed by the authors, NMN increases SIRT1 activity which then stimulated mitochondrial biogenesis through the deacetylation of PGC-1α. The activity or expression of mitochondrial sirtuins was not analyzed in these studies.

68Also, NAM supplementation for treatment of pellagra (niacin deficiency) could serve as a therapeutic target. Although NAM is mostly used as a SIRT inhibitor, its inhibitory effects are short-lasting, most of the NAM is rapidly converted to NAD+. Also, the NAD+ precursor, NR, was able to increase the mitochondrial NAD+ levels. Currently, NR has been recommended as a nutritional supplement to maintain the high NAD+/NADH ratio. Intriguingly, exogenous NAD+ seems to be of equal significance; daily NAD+ treatment maintained intracellular levels of NAD+ and prevented phenylephrine-induced in vitro hypertrophy in cultured cardiomyocytes and angiotensin II-induced cardiac hypertrophy in vivo in mice. Similarly, intravenous administration of NAD+ before ischemia protected the rat heart from IR injury.

69Additionally, several chemical compounds demonstrated the capability to activate SIRT3. SIRT3 activity has been shown to be markedly increased by HKL, a compound isolated from trees in the Magnolia genus. Also, a recent screening of different compounds led to the discovery of 7-hydroxy-3-(4′-metoxyphenyl) coumarin (C12) which activated SIRT3 in primary cortical astrocytes. Lastly, oral garlic administration could increase SIRT3 expression and activity in the diabetic heart. In addition to chemical compounds and nutrients, dietary interventions such as CR have been proposed to exert its beneficial effects through SIRT3 by increasing its activity or expression. The precise mechanisms of CR have not been fully understood but can be explained, in part, by increases in the NAD+/NADH ratio. Increased NADH levels as a result of overnutrition associated with obesity and diabetes reduces the NAD+/NADH ratio. The latter, in turn, increases protein acetylation in mitochondria due to SIRT3 inactivation. Apparently, similar mechanisms mediate the alterations in the ETC and OXPHOS complexes associated with the decreased NAD+/NADH ratio which then stimulate mitochondrial protein acetylation. Conversely, CR was able to reduce oxidative stress and delay disease onset and mortality in animal models. Although the mechanisms underlying SIRT3 activation remain unclear, several lines of therapeutic strategies aiming at increasing SIRT3 activity are available for clinical consideration.

70Numerous studies have provided strong evidence of the involvement of mitochondrial protein acetylation in cardiac pathology therefore portraying SIRT3 as the main regulator of mitochondrial metabolism and function. The main role of SIRT3 is to maintain mitochondrial bioenergetics by regulating the activity of enzymes involved in FAO, TCA cycle, ETC, and OXPHOS in the heart. The heart is the most energy consuming organ and the mitochondria, the main powerhouse of the cell, occupy 30–35% of total cardiomyocyte volume and supply ∼90% of ATP needed for normal cardiac function. Mitochondria also play a central role in cell death and are involved in the pathogenesis of cardiac diseases, particularly, MI, IR, and HF. Therefore, a careful balance between energetic expenditure and generation is important for maintenance of optimal cardiac function. SIRT3 appears to maintain the balance between cardiac function and energy expenditure by regulating the activity of enzymes involved in ATP production in the mitochondria. Downregulation of SIRT3 due to its inhibition or low expression may lead to the imbalance in mitochondrial bioenergetics associated with mitochondrial dysfunction. Therefore, the maintenance of SIRT3 activity is important for normal mitochondrial metabolism and function and this strategy can be useful for the prevention of pathological consequences of cardiac diseases.

71RP-R reviewed and analyzed the literature, prepared a draft of the manuscript and proofed it. XC-D reviewed and analyzed the literature and participated in writing the manuscript. SJ supervised the project, edited and compiled final version of the manuscript.

72The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MB and handling Editor declared their shared affiliation.