SIRT4 accelerates Ang II-induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity

Aims

Oxidative stress contributes to the development of cardiac hypertrophy and heart failure. One of the mitochondrial sirtuins, Sirt4, is highly expressed in the heart, but its function remains unknown. The aim of the present study was to investigate the role of Sirt4 in the pathogenesis of pathological cardiac hypertrophy and the molecular mechanism by which Sirt4 regulates mitochondrial oxidative stress.

Methods and results

Male C57BL/6 Sirt4 knockout mice, transgenic (Tg) mice exhibiting cardiac-specific overexpression of Sirt4 (Sirt4-Tg) and their respective controls were treated with angiotensin II (Ang II, 1.1 mg/kg/day). At 4 weeks, hypertrophic growth of cardiomyocytes, fibrosis and cardiac function were analysed. Sirt4 deficiency conferred resistance to Ang II infusion by significantly suppressing hypertrophic growth, and the deposition of fibrosis. In Sirt4-Tg mice, aggravated hypertrophy and reduced cardiac function were observed compared with non-Tg mice following Ang II treatment. Mechanistically, Sirt4 inhibited the binding of manganese superoxide dismutase (MnSOD) to Sirt3, another member of the mitochondrial sirtuins, and increased MnSOD acetylation levels to reduce its activity, resulting in elevated reactive oxygen species (ROS) accumulation upon Ang II stimulation. Furthermore, inhibition of ROS with manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin, a mimetic of SOD, blocked the Sirt4-mediated aggravation of the hypertrophic response in Ang II-treated Sirt4-Tg mice.

Conclusions

Sirt4 promotes hypertrophic growth, the generation of fibrosis and cardiac dysfunction by increasing ROS levels upon pathological stimulation. These findings reveal a role of Sirt4 in pathological cardiac hypertrophy, providing a new potential therapeutic strategy for this disease.

Cardiac hypertrophy, Sirt4, Oxidative stress, MnSOD

Translational perspective

Inhibition of oxidative stress is considered a promising therapeutic strategy for pathological cardiac hypertrophy and heart failure. However, effective and specific targets for suppressing oxidative stress remain to be defined. Here, we demonstrate that Sirt4 promotes oxidative stress upon pathological stimulation, which results in an enhanced hypertrophic response in the heart. Furthermore, ROS inhibition abolishes the Sirt4-mediated aggravation of hypertrophy and rescues cardiac function in mice. Thus, suppression of Sirt4-mediated oxidative stress appears to be a potential therapeutic approach for pathological hypertrophy and heart failure.

Introduction

Oxidative stress plays a key role in the pathogenesis of cardiac hypertrophy and heart failure.¹ As an important component of oxidative stress, mitochondria generate excessive reactive oxygen species (ROS) due to electron leakage associated with decreased complex activity in the electron transport chain.¹ Hypertrophic agonists such as angiotensin II (Ang II) increase mitochondrial ROS levels in cardiomyocytes, and mitochondrial oxidative stress contributes to Ang II-induced cardiac hypertrophy.² To protect cells against oxidative damage, mitochondrial antioxidant enzymes such as manganese superoxide dismutase (MnSOD), glutathione and mitochondrial thioredoxin form a complicated defense system to detoxify mitochondrial ROS.¹ These antioxidant enzymes can protect the heart from oxidative injury and cardiac dysfunction.^3–6 Therefore, elucidation of the biological functions of genes involved in the regulation of antioxidant enzyme activities in the mitochondria could provide a new therapeutic approach for the treatment of cardiac hypertrophy and heart failure.

Sirtuins are highly conserved NAD⁺-dependent deacetylases and ADP-ribosyltransferases involved in many cellular processes, including metabolism, genome stability, stress resistance, and aging.⁷ As mediators of adaptive responses to the cellular environment, sirtuins have attracted considerable attention for regulating oxidative stress.⁸ Three sirtuins (Sirt3, Sirt4, and Sirt5) are primarily located in the mitochondria. Sirt3 directly deacetylates and activates several enzymes (such as MnSOD and isocitrate dehydrogenase 2) to mediate the oxidative stress response.^9,10 Sirt4 also participates in the regulation of mitochondrial ROS production,¹¹ but whether Sirt4 affects the activities of antioxidant enzymes in the mitochondrial matrix is unclear. Notably, Sirt4 is highly expressed in the heart.¹² However, the role of Sirt4 in cardiac disease has not previously been investigated.

Here, we report that Sirt4 is a critical regulator of pathological cardiac hypertrophy. Sirt4 deficiency significantly suppresses myocardial hypertrophy and fibrosis following Ang II infusion, whereas Sirt4 overexpression accelerates Ang II-induced cardiac remodeling. We further provide evidence that Sirt4 promotes the development of cardiac hypertrophy by reducing MnSOD activity, thus enhancing oxidative stress, and that mimicking MnSOD activity using manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin (MnTBAP) abrogates the Sirt4-mediated effect on the hypertrophic response.

Methods

All methods are available in Supplementary material.

Results

Sirt4 is a positive regulator of the Ang II-induced hypertrophic response in cardiomyocytes

To study the role of Sirt4 in the heart, we first examined the effect of Sirt4 on the hypertrophic response in cultured neonatal rat cardiomyocytes (NRCMs). To this end, we generated an Sirt4-knockdown recombinant adenovirus (Ad-shSirt4) (Figure 1A). Immunostaining of NRCMs for α-actinin indicated that Sirt4 knockdown dramatically attenuated the increase in cell growth induced by Ang II (Figure1B and C). Ad-shSirt4 infection also prevented the induction of atrial natriuretic peptide (ANP) mRNA levels by Ang II in NRCMs (Figure 1D). NRCMs were also infected with an Sirt4-expressing recombinant adenovirus (Ad-Sirt4) (Figure 1A). As shown in Figure 1B and E, Sirt4 overexpression promoted the hypertrophic growth of cardiomyocytes in response to Ang II. In addition, Sirt4 overexpression significantly increased Ang II-induced ANP expression (Figure 1F). These data suggest that Sirt4 enhances the hypertrophic response in Ang II-treated NRCMs.

Figure 1

Sirt4 enhances the angiotensin II-induced hypertrophic response in cultured cardiomyocytes. (A) Western blotting analysis of Sirt4 in neonatal rat cardiomyocytes infected with the indicated adenovirus. Adenovirus carrying green fluorescent protein is the control adenovirus of Ad-Sirt4 while Ad-U6 is the control adenovirus of Ad-shSirt4. (B) Representative images of neonatal rat cardiomyocytes infected with the indicated adenovirus and then treated with phosphate-buffered saline or Ang II for 48 h. α-Actinin staining was performed to identify cells. Scale bars, 30 μm. (C) Quantification of cell area. Neonatal rat cardiomyocytes were infected with Ad-U6 or Ad-shSirt4 and then treated with phosphate-buffered saline or Ang II. Thirty cells were quantified for each treatment. (D) Atrial natriuretic peptide mRNA levels in neonatal rat cardiomyocytes treated as in (C). (E) Quantification of cell area. Neonatal rat cardiomyocytes were infected with adenovirus carrying green fluorescent protein or Ad-Sirt4 and then treated with phosphate-buffered saline or Ang II. Thirty cells were quantified for each treatment. (F) Atrial natriuretic peptide mRNA levels in neonatal rat cardiomyocytes treated as in (E).

Sirt4 deficiency ameliorates Ang II-induced cardiac hypertrophy and remodeling

To investigate the potential function of Sirt4 during cardiac hypertrophy in vivo, we subjected Sirt4 global knockout mice (Sirt4-KO; C57BL/6 background), which exhibited no Sirt4 protein in the heart (Supplementary material online, Figure S1A), to chronic infusion of Ang II for 4 weeks. At baseline, the heart weight to body weight (HW/BW) ratio of the Sirt4-KO mice was indistinguishable from that of wild-type (WT) mice (Figure 2A). However, Ang II infusion resulted in an ∼28.3% increase in the HW/BW ratio in WT mice, whereas Sirt4-KO mice showed only an 8.9% increase (Figure 2A). A similar effect was observed for the heart weight to tibia length (HW/TL) ratio (Figure 2A). Furthermore, after 4 weeks of Ang II treatment, WT mice exhibited a compensatory increase in the ejection fraction (EF) and fractional shortening (FS), whereas the sensitivity of cardiac performance to Ang II was blunted by Sirt4 knockout (Figure 2B). However, both the heart rate and blood pressure were indistinguishable between Sirt4-KO and WT mice (Supplementary material online, Table S1). Histological analysis with haematoxylin and eosin (H&E) and wheat germ agglutinin staining revealed that the cardiomyocyte hypertrophy induced by Ang II was markedly ameliorated in the Sirt4-KO mice (Figure 2C and D). The evaluation of fibrotic areas also revealed less fibrosis in the hearts of Ang II-treated Sirt4-KO mice compared with WT mice (Figure 2E and F). Consistent with these data, Sirt4 deficiency significantly inhibited the Ang II-induced up-regulation of ANP mRNA levels (Figure 2G). These results demonstrate that Sirt4 deficiency protects against pathological cardiac hypertrophy.

Figure 2

Sirt4 knockout mice are protected from Ang II-induced cardiac hypertrophy. (A) The ratios of heart weight to body weight and heart weight/tibia length in wild-type and Sirt4 knockout mice following saline or Ang II infusion for 4 weeks. n = 12–13. (B) Echocardiographic assessment of the ejection fraction and fractional shortening in saline- and Ang II-treated wild-type and Sirt4 knockout mice. n = 12–13. (C) Histological analysis of heart sections from wild-type and Sirt4 knockout mice following saline or Ang II infusion. Heart cross-sections were stained with haematoxylin-eosin to analyze hypertrophic growth (top row; scale bars, 1 mm); wheat germ agglutinin staining was performed to determine cell boundaries (bottom row; scale bars, 40 μm). (D) Quantification of the cardiomyocyte cross-sectional area in saline- and Ang II-treated wild-type and Sirt4 knockout mice. n = 12–13. (E) Picrosirius red staining to detect fibrosis in wild type and Sirt4 knockout mice following saline or Ang II infusion. Scale bars, 50 μm. (F) Quantification of the fibrotic area in saline- and Ang II-treated wild type and Sirt4 knockout mice. n = 9–14. LV, left ventricle. (G) Atrial natriuretic peptide mRNA levels in the hearts of saline- and Ang II-treated wild-type and Sirt4-KO mice. n = 6–7.

Myocardial Sirt4 overexpression promotes hypertrophic growth and cardiac dysfunction in response to Ang II

To further assess the effect of Sirt4 on cardiac hypertrophy, we generated transgenic (Tg) mice expressing human Sirt4 in the heart under the control of the α-myosin heavy chain promoter (Supplementary material online, Figure S2A). Three independent lines of Sirt4-Tg mice (C57BL/6 background) were obtained (Supplementary material online, Figure S2B). These lines developed normally, with no detectable differences in cardiac size or structure. Among the three established lines of Sirt4-Tg mice, line 1 expressed moderate levels of Sirt4 protein in the heart, was used for further experiments (Supplementary material online, Figure S2B). Western blotting analysis revealed that Sirt4 was robustly expressed in the heart and was located predominantly in the mitochondria (Supplementary material online, Figure S2C and D). We also subjected Sirt4-Tg mice and their non-Tg (N-Tg) littermates to chronic infusion of Ang II. After 4 weeks of Ang II infusion, significant increases in both the HW/BW and HW/TL ratios were detected in Sirt4-Tg mice compared with N-Tg mice (Figure 3A). Furthermore, Sirt4-Tg mice exhibited declines in EF and FS following Ang II administration compared with N-Tg mice (Figure 3B), and there was no significant difference in heart rate or blood pressure between the Sirt4-Tg and N-Tg mice (Supplementary material online, Table S2). Consistently, we observed larger cardiomyocytes, more myocardial fibrosis and higher ANP levels in Ang II-treated Sirt4-Tg mice compared with N-Tg mice (Figure 3C–G). Taken together, these results indicate that Sirt4-Tg mice are hypersensitive to Ang II-induced cardiac hypertrophy.

Figure 3

Sirt4 overexpression promotes the development of pathological cardiac hypertrophy. (A) The heart weight to body weight and heart weight/tibia length ratios in non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. n = 15. (B) Echocardiographic assessment of ejection fraction and fractional shortening in non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. n = 10–12. (C) Histological analysis of heart sections from non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. Heart cross-sections were stained with haematoxylin-eosin (top row; scale bars, 1 mm) and wheat germ agglutinin (bottom row; scale bars, 40 μm). (D) Quantification of the cardiomyocyte cross-sectional area in saline- and Ang II-treated non-transgenic and Sirt4-transgenic mice. n = 12. (E) Picrosirius red staining of heart sections from non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. Scale bars, 50 μm. (F) Quantification of the fibrotic area in saline- and Ang II-treated non-transgenic and Sirt4-transgenic mice. n = 12. (G) Atrial natriuretic peptide mRNA levels in the hearts of saline- and Ang II-treated non-transgenic and Sirt4-transgenic mice. n = 8–15.

Sirt4 promotes oxidative stress in Ang II-induced cardiac hypertrophy

Oxidative stress is well known to play a crucial role in cardiac hypertrophy and heart failure,¹ and a previous report indicated that Sirt4 promotes ROS production in HeLa cells.¹¹ Thus, we determined whether Sirt4 could also regulate ROS levels in the presence of hypertrophic stimuli. To this end, we assessed ROS levels in the hearts of Sirt4-KO and Tg mice. The results showed that Ang II induced an increase in total ROS levels (superoxide measured by dihydroethidium (DHE) staining) in the hearts of WT mice, whereas this effect was significantly suppressed in Sirt4-KO mice (Figure 4A and B). Given that Sirt4 is located in the mitochondria, we also examined the effect of Sirt4 on mitochondrial superoxide production through mitoSOX staining. Consistent with the results of DHE staining, Sirt4-KO mice exhibited less mitochondrial ROS generation in response to Ang II treatment than WT mice (Figure 4A and B). In Sirt4-Tg mice, much higher levels of ROS were detected after Ang II infusion compared with their littermates (Figure 4C and D). Sirt4 knockdown also inhibited Ang II-induced ROS production in NRCMs, and cardiomyocytes overexpressing Sirt4 exhibited much higher levels of ROS after Ang II treatment compared with the controls (Supplementary material online, Figure S3A–D). Collectively, these results indicate that Sirt4 is capable of increasing ROS levels during cardiac hypertrophy.

Figure 4

Sirt4 increases reactive oxygen species levels during cardiac hypertrophy. (A) Measurement of reactive oxygen species levels in the hearts of wild-type and Sirt4-knockout mice following saline or Ang II infusion. Dihydroethidium staining (top row) and mitoSOX staining (bottom row) were performed to assess total cellular reactive oxygen species and mitochondrial reactive oxygen species, respectively. Scale bars, 50 μm. (B) Quantification of reactive oxygen species levels in the hearts of wild-type and Sirt4-KO mice following saline or Ang II infusion based on measurement of the intensity of fluorescence. n = 5–8. (C) Dihydroethidium (top row) and mitoSOX (bottom row) staining to detect reactive oxygen species levels in the hearts of non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. Scale bars, 50 μm. (D) Quantification of reactive oxygen species levels in the hearts of non-transgenic and Sirt4-transgenic mice following saline or Ang II infusion. n = 9–14. (E) Representative western blotting showing the expression of the indicated kinases in heart samples from wild-type and Sirt4-knockout mice following saline or Ang II infusion. (F) Quantitative data in (E). n = 4. (G) Heart extracts prepared from saline or Ang II-treated Sirt4-transgenic mice and non-transgenic mice were analysed by western blotting with the indicated antibodies. (H) Quantitative data in (G). n = 4.

Because ROS can activate multiple signaling cascades during cardiac hypertrophy,¹³ we analyzed the phosphorylation levels of hypertrophy signaling kinases. The phosphorylation levels of cRaf, MEK, and ERK were increased by Ang II in WT mice, and this effect was blunted in Sirt4-KO mice (Figure 4E and F). In addition, Sirt4-Tg mice exhibited enhanced Ang II-mediated activation of the MAPK–ERK pathway compared with the N-Tg controls (Figure 4G and H). However, Sirt4 did not affect the activities of the MAPK-P38 and PI3K-AKT signaling pathways in Ang II-induced cardiac hypertrophy (data not shown). Similar effects were observed in Ang II-treated NRCMs (Supplementary material online, Figure S3E and F). These data indicate that Sirt4 elevates the activity of the MAPK–ERK signaling pathway in response to hypertrophic stimuli.

Sirt4 inhibits Sirt3-mediated manganese superoxide dismutase deacetylation during cardiac hypertrophy

In the mitochondrial matrix, several antioxidative enzymes (e.g. MnSOD, catalase, thioredoxin reductase [TrxR], gluthathione reductase [GSR], and glutathione peroxidase [GPx]) are critical for determining ROS levels (Supplementary material online, Figure S4A) and maintaining cardiac function.^4,13 As Sirt4 is also located in the mitochondrial matrix and has the ability to affect protein activity,^12,14 we next examined whether Sirt4 modulates the activity of these enzymes during cardiac hypertrophy. The enzyme activity analysis showed that Sirt4 knockdown significantly inhibited the decline in MnSOD activity induced by Ang II in NRCMs, whereas Sirt4 overexpression had the opposite effect (Supplementary material online, Figure S4B and C). However, Sirt4 was unable to affect the activity of catalase, TrxR, GSR, or GPx at baseline or in the presence of Ang II in NRCMs (Supplementary material online, Figure S4D–G). We also found that Ang II reduced MnSOD activity in the hearts of both WT and N-Tg mice (Figure 5A). This reduction of MnSOD activity was prevented in Ang II-treated Sirt4-KO mice, whereas cardiac Sirt4 overexpression promoted the Ang II-induced decrease of MnSOD activity (Figure 5A). These findings indicate that Sirt4 predominantly inhibits the activity of MnSOD. We further found that Sirt4 did not affect MnSOD expression (Figure 5B), suggesting that Sirt4 may influence the levels of post-translational modifications of MnSOD to regulate its activity. The acetylation of MnSOD directs its enzymatic activity.¹⁰ Therefore, we tested MnSOD acetylation levels in the hearts of Sirt4-KO and Sirt4-Tg mice. Ang II stimulation resulted in a decrease in MnSOD acetylation in the hearts of WT mice, and the acetylation levels of MnSOD were further reduced in Sirt4-KO mice (Figure 5C), whereas MnSOD acetylation was maintained well in the hearts of Sirt4-Tg mice (Figure 5D). Similarly, Sirt4 knockdown dramatically reduced MnSOD acetylation levels, whereas Sirt4 overexpression increased MnSOD acetylation levels in Ang II-treated NRCMs (Supplementary material online, Figure S5A and B), thus, confirming a role for Sirt4 in the regulation of MnSOD acetylation upon Ang II stimulation.

Figure 5

Sirt4 inhibits Sirt3-mediated manganese superoxide dismutase deacetylation. (A) Enzymatic activity of manganese superoxide dismutase in the indicated groups of mice. Mitochondria prepared from the hearts of Sirt4-knockout (left) and Sirt4-transgenic (right) mice and their respective controls were used for the manganese superoxide dismutase activity assay. n = 6–19. (B) Western blotting analysis of manganese superoxide dismutase and Sirt3 levels in the hearts of Sirt4-knockout (left) and Sirt4-transgenic (right) mice following saline or Ang II infusion. (C and D) Acetylation levels of manganese superoxide dismutase in the hearts of Sirt4-knockout (C) and Sirt4-transgenic mice (D) following saline or Ang II infusion. Endogenous manganese superoxide dismutase was isolated by immunoprecipitation with an anti-manganese superoxide dismutase antibody, followed by western blotting with an anti-acetyl-lysine antibody. (E) In vitro deacetylation assay. Endogenous manganese superoxide dismutase was immunopurified from 293T cells, followed by incubation with recombinant human Sirt4 or/and Sirt3. Manganese superoxide dismutase acetylation levels were assessed by western blotting with an anti-acetyl-lysine antibody. (F) Acetylation levels of manganese superoxide dismutase in the indicated 293T cells. Manganese superoxide dismutase was cotransfected with pcDNA4, Sirt4, or/and Sirt3 into 293T cells. The acetylation levels of immunopurified manganese superoxide dismutase were determined by western blotting with an anti-acetyl-lysine antibody. (G) The interaction between Sirt3 and manganese superoxide dismutase in the indicated 293T cells. Cells were transfected with manganese superoxide dismutase, pcDNA4, Sirt4 or/and Sirt3, as indicated. Cell lysates were harvested, and immunoprecipitation was performed with an antibody specific for Sirt3. (H) The interaction between Sirt3 and manganese superoxide dismutase in the indicated neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes infected with an adenovirus expressing Sirt4 or GFP were stimulated with Ang II, then immunopurified and analyzed by western blotting with anti-manganese superoxide dismutase and anti-Sirt3 antibodies. (I) Quantification of mitochondrial reactive oxygen species levels in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were infected with Ad-U6, Ad-shSirt4, Ad-shMnSOD, or Ad-shSirt4 + Ad-shMnSOD, and then treated with Ang II for 4 h. Mitochondrial reactive oxygen species level was determined based on measurement of the intensity of fluorescence of mitoSOX staining. (J) Quantification of cell area in neonatal rat cardiomyocytes. Neonatal rat cardiomyocytes were infected with Ad-U6, Ad-shSirt4, Ad-shMnSOD or Ad-shSirt4 + Ad-shMnSOD, and then treated with Ang II for 48 h. Thirty cells were quantified for each treatment.

To further investigate the mechanism by which Sirt4 affects MnSOD acetylation, an in vitro deacetylation assay was performed. The results showed that MnSOD acetylation levels were unchanged following incubation with the Sirt4 recombinant protein alone (Figure 5E), suggesting that Sirt4 did not directly modulate MnSOD acetylation in vitro. Sirt3 has been reported to directly deacetylate MnSOD,¹⁰ and Sirt4 was demonstrated to interact with Sirt3 in HEK293 cells,¹⁵ which was confirmed in NRCMs (Supplementary material online, Figure S6A). Therefore, we speculated that Sirt4 might affect MnSOD acetylation through the regulation of Sirt3. Indeed, Sirt3 reduced MnSOD acetylation in vitro, but Sirt4 blocked the deacetylation effect of Sirt3 on MnSOD (Figure 5E). Moreover, Sirt4 overexpression inhibited Sirt3-mediated MnSOD deacetylation in 293T cells (Figure 5F).

As Sirt3 directs MnSOD deacetylation through physical binding, Sirt4 might influence the interaction between Sirt3 and MnSOD. In 293T cells, Sirt4 overexpression dramatically suppressed the binding of Sirt3 to MnSOD (Figure 5G). In NRCMs, Ang II stimulation increased the interaction between Sirt3 and MnSOD, which was inhibited by Sirt4 overexpression (Figure 5H), but Sirt4 did not affect the protein level and activity of Sirt3 (Figure 5B; Supplementary material online, Figure S7A and B). In addition, we found that the catalytic mutant H161Y of Sirt4 (Sirt4H161Y) could also inhibit Sirt3-MnSOD interaction and Sirt3-mediated MnSOD deacetylation (Supplementary material online, Figure S8A and B). Likewise, overexpression of Sirt4H161Y inhibited MnSOD activity and elevated mitochondrial ROS level (Supplementary material online, Figure S8C and D). Therefore, Sirt4 functions indirectly. Taken together, these results demonstrate that Sirt4 inhibits Sirt3-MnSOD interaction and thus suppresses Sirt3-mediated MnSOD deacetylation to decrease MnSOD activity in cardiomyocytes upon Ang II stimulation.

Furthermore, we investigated whether MnSOD essentially contributes to the effects of Sirt4 on mitochondrial ROS accumulation and hypertrophy in cardiomyocytes. We knocked down MnSOD with adenovirus-mediated shRNA (Supplementary material online, Figure S9A) and found that MnSOD knockdown promoted Ang II-induced mitochondrial ROS production and hypertrophy in NRCMs (Figure 5I and J; Supplementary material online, Figure S9B and C). However, the protective role of Sirt4 knockdown was blocked when MnSOD was knocked down (Figure 5I and J; Supplementary material online, Figure S9B and C). These results implicate that Sirt4 regulates Ang II-induced ROS generation and cardiomyocyte hypertrophy, at least in part, through inhibiting MnSOD activity.

Inhibition of reactive oxygen species by manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin abrogates the Sirt4-related effect on the hypertrophic response

To evaluate the contribution of oxidative stress to the phenotype of the Sirt4-Tg mice in response to Ang II, we performed a rescue experiment using a superoxide dismutase mimetic, manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin (MnTBAP). MnTBAP treatment indeed mimicked MnSOD activity and prevented ROS production in the hearts of Ang II-treated Sirt4-Tg and N-Tg mice (Supplementary material online, Figure S10A–C). Notably, the administration of MnTBAP to Ang II-treated Sirt4-Tg mice reversed the HW/BW and HW/TL ratios to the same extent as in N-Tg mice (Figure 6A). Furthermore, Sirt4-Tg mice that received both Ang II and MnTBAP exhibited a similar cardiac function to N-Tg mice (Figure 6B). Additionally, MnTBAP reduced blood pressure in N-Tg mice in response to Ang II, but there was no difference in blood pressure between N-Tg and Sirt4-Tg mice (Supplementary material online, Table S3). The effects of Sirt4 on the hypertrophic response, measured according to the cross-sectional area, fibrosis, and ANP expression, were completely abolished by MnTBAP (Figure 6C–E; Supplementary material online, Figure S10D). MnTBAP also inhibited ROS accumulation in Ang II-treated NRCMs (Supplementary material online, Figure S11A and B), and importantly, the hypertrophic response regulated by Sirt4 was markedly inhibited by MnTBAP treatment in NRCMs (Supplementary material online, Figure S11C–E). Collectively, these data indicate that enhanced oxidative stress caused by inhibition of MnSOD activity plays a key role in the Sirt4-mediated pro-hypertrophic effect.

Figure 6

Manganese 5, 10, 15, 20-tetrakis-(4-benzoic acid) porphyrin abolishes the Sirt4-mediated pro-hypertrophic effect. (A) heart weight to body weight and heart weight/tibia length ratios in non-transgenic and Sirt4-transgenic mice following MnTBAP or/and Ang II infusion for 4 weeks. n = 12. (B) Ejection fraction and fractional shortening of non-transgenic and Sirt4-transgenic mice following MnTBAP or/and Ang II infusion. n = 10–23. (C) Histological analysis of heart sections from non-transgenic and Sirt4-transgenic mice following MnTBAP or/and Ang II infusion. Heart cross-sections were stained with haematoxylin-eosin (top row; scale bars, 1 mm), wheat germ agglutinin (second row; scale bars, 40 μm), and picrosirius red (third and fourth row; scale bars, 50 μm). (D and E) Quantification of the cardiomyocyte cross-sectional area (D) and fibrotic area (E) in non-transgenic and Sirt4-transgenic mice following MnTBAP or/and Ang II infusion. n = 10–15. MnT, MnTBAP.

Discussion

The present work indicates a critical role for the mitochondrial sirtuin, Sirt4, in the development of pathological cardiac hypertrophy. Sirt4 is highly expressed in the heart, kidney, liver, and brain,¹² implicating possible roles for Sirt4 in these tissues. Here, we discovered that Sirt4 effectively regulates heart function during pathological cardiac hypertrophy. Following Ang II stimulation, WT or N-Tg mice exhibited increased cardiac function (elevated EF and FS) compared with saline controls, suggesting a compensatory stage.¹⁶ Strictly, cardiac overexpression of Sirt4 (Sirt4-Tg) dramatically accelerated cardiac decompensation upon Ang II treatment. In contrast, cardiac function of Sirt4-KO mice was preserved and no significant difference was observed between saline and Ang II treatments in Sirt4-KO mice. These findings indicate that Sirt4 mediates the sensitivity of cardiac performance to hypertrophic stress.

Reactive oxygen species affect nearly all of the key features of cardiac maladaptation, including the hypertrophic response, contractile dysfunction, extracellular matrix remodeling, and arrhythmia.¹ Many previous reports have underscored the importance of sirtuins in the regulation of oxidative stress,^17–21 and the present study indicated that Sirt4 promotes ROS accumulation in the myocardium upon hypertrophic stress. Although this study does not rule out other possible mechanisms by which Sirt4 promotes hypertrophy, inhibition of oxidative stress by MnTBAP was sufficient to block the Sirt4-mediated hypertrophic response, indicating that mitochondrial ROS play a major role in this process.

Mitochondria generate ROS during oxidative phosphorylation, and prolonged oxidative stress can damage mitochondria.¹³ To avoid excessive ROS accumulation in the mitochondria, MnSOD catalyzes the production of H₂O₂ from O₂⁻, and H₂O₂ is subsequently converted to H₂O by other antioxidant enzymes, such as catalase.¹³ Targeted inhibition of ROS production in mitochondria holds promise as a therapeutic strategy over the untargeted approach with classical antioxidants.^1,22 In humans, mutations of mitochondrial antioxidants (e.g. MnSOD, catalase, GPx, and TrxR) increase the risk for cardiovascular diseases.^23–26 MnSOD is essential for normal heart function and even a relatively slight reduction in MnSOD activity can result in cardiac dysfunction.⁴ Mutation in MnSOD increases the risk for non-familial idiopathic dilated cardiomyopathy in human,²³ and MnSOD deficiency in mouse causes dilated cardiomyopathy.^27,28 In addition, both the protein level and activity of MnSOD are decreased in murine hypertrophic hearts and human failing myocardia.^21,29 However, it is unclear whether the post-transcriptional modification of MnSOD is altered in response to hypertrophic stress. Our data showed that Ang II treatment reduced MnSOD acetylation, which is an important post-transcriptional modification determining MnSOD enzyme activity. The increase of Sirt3 binding to MnSOD may account for the decrease in MnSOD acetylation in response to Ang II, as MnSOD was reported to be directly deacetylated by Sirt3.¹⁰ However, due to the marked decrease in the protein levels of MnSOD, its overall activity was reduced during hypertrophic stress. Moreover, Sirt3 has been reported to suppress the hypertrophic response by activating forkhead box O3a in the nucleus.²¹ In this context, the effect of Sirt3 in the mitochondria in response to Ang II might also contribute to its anti-hypertrophic function.

In addition, we found that Sirt4 inhibits the interaction between Sirt3 and MnSOD to increase MnSOD acetylation levels upon Ang II treatment. Sirt4 has been reported to influence Sirt3 expression in the mouse liver,³⁰ and an interaction between Sirt4 and Sirt3 was detected in HEK293 cells.¹⁵ In the heart, no effect of Sirt4 on Sirt3 expression was detected, but an interaction between Sirt4 and Sirt3 was observed in cardiomyocytes. The interaction of Sirt4 with Sirt3 was decreased in hypertrophic cardiomyocytes, and Sirt4 did not affect the deacetylase activity or protein levels of Sirt3. We, therefore, deduce that Sirt4 competes with MnSOD for binding to Sirt3, and Sirt4 overexpression increases MnSOD acetylation levels during cardiac hypertrophy. However, the effects of Sirt4 on mitochondrial ROS and hypertrophy were blocked by MnSOD knockdown. As the deacetylation effect of Sirt3 on MnSOD increases MnSOD enzymatic activity, we speculate that this may be a compensatory mechanism in the heart in response to stress. Nevertheless, Sirt4 overexpression blocks this adaptive cardiac pathway during stress, thus accelerating cardiac dysfunction and remodeling.

In conclusion, we identified Sirt4 as a novel regulator of pathological cardiac hypertrophy. The hearts of Sirt4-KO mice exhibit improved tolerance to Ang II, showing a blunted hypertrophic response. However, Sirt4-Tg mice display rapid onset of cardiac dysfunction. In response to Ang II, Sirt4 inhibits Sirt3-mediated MnSOD activation to increase ROS levels, thereby promoting the development of pathological cardiac hypertrophy (Supplementary material online, Figure S12). The present study provides a novel mechanistic insight into the likely link between mitochondrial oxidative stress and pathological cardiac hypertrophy, which may have a major impact on the understanding and treatment of pathological cardiac hypertrophy and heart failure.

Limitations

There are still some limitations applied for the present study which need to be considered and addressed in the future. First of all, we used multiple primary cells and cell lines in this study, some of which (H9C2) may not best represent cardiomyocytes. Second, extrapolation of data obtained in genetically modified mice to human is always difficult. Therefore, in order to support our conclusion regarding the translational application of Sirt4 regulators, further experiments on human samples should be considered in the future study.

Supplementary material

Supplementary material is available at European Heart Journal online.

Authors’ contributions

Y.-X.L., X.T. performed statistical analysis; D.-P.L., H.-Z.C. handled funding and supervision; Y.-X.L., X.T., X.-Z.A., X.-M.X., X.-F.C., X.Z., D.-L.H.: Y.-X.L., X.T., H.-Z.C., D.-P.L. acquired the data; Y.-X.L., X.T., H.-Z.C., D.-P.L. conceived and designed the research, drafted the manuscript; Y.-X.L., X.T., X.-Z.A., X.-M.X., X.-F.C., X.Z., D.-L.H., H-Z.C., D.-P.L. made critical revision of the manuscript for key intellectual content.

Funding

This work was supported by grants from the National Natural Science Foundation of China (81422002, 91339201, 31271227, and 31571193) and the National Science and Technology Support Project (2013YQ0309230502 and 2014BAI02B01).

Conflict of interest: none declared.

Author notes

†

These authors contributed equally to this work.

See page 1399 for the editorial comment on this article (doi:10.1093/eurheartj/ehw199)