SIRT2 inhibition protects against cardiac hypertrophy and heart failure

Sirtuins (SIRT) exhibit deacetylation or ADP-ribosyltransferase activity and regulate a wide range of cellular processes in the nucleus, mitochondria and cytoplasm. The role of the only sirtuin that resides in the cytoplasm, SIRT2, in the development of heart failure (HF) and cardiac hypertrophy is not known. In this paper, we show that the hearts of mice with deletion of Sirt2 (Sirt2-/-) display improved cardiac function after ischemia-reperfusion (I/R) and pressure overload (PO), suggesting that SIRT2 exerts maladaptive effects in the heart in response to stress. Similar results were obtained in mice with cardiomyocyte-specific Sirt2 deletion. Mechanistic studies suggest that SIRT2 modulates cellular levels and activity of nuclear factor (erythroid-derived 2)-like 2 (NRF2), which results in reduced expression of antioxidant proteins. Deletion of Nrf2 in the hearts of Sirt2-/- mice reversed protection after PO. Finally, treatment of mouse hearts with a specific SIRT2 inhibitors reduces cardiac size and attenuates cardiac hypertrophy in response to PO. These data indicate that SIRT2 has detrimental effects in the heart and plays a role in the progression of HF and cardiac hypertrophy, which makes this protein a unique member of the SIRT family. Additionally, our studies provide a novel approach for treatment of cardiac hypertrophy by targeting SIRT2 pharmacologically, providing a novel avenue for the treatment of this disorder.


INTRODUCTION
Sirtuin (SIRT) family of proteins comprise class III of histone deacetylases. SIRTs require NAD + to carry out their enzymatic reaction, and have been implicated in a wide range of cellular processes including aging, apoptosis, response to stress and inflammation, control of energy efficiency, circadian clocks and mitochondrial biogenesis (1,2). In mammals, seven sirtuins (SIRT1-7) have been identified, which are categorized according to their subcellular localization to the nucleus (SIRT1, 6, and 7), mitochondria (SIRT3, 4, and 5), and cytoplasm (SIRT2). SIRT1-3 have a robust deacetylation activity, while SIRT4 is reported to display ADP-ribosyltransferase activity. SIRT5 may function as a protein desuccinylase and demalonylase, and SIRT6 and SIRT7 display weak deacetylase activity (3)(4)(5)(6).
A number of SIRTs have been studied in the heart (for review, please see (7)). The effects of SIRT1 in the heart are complex. Sirt1 deletion protects against pressure overload (PO)-induced cardiac hypertrophy (8,9); however, low-level overexpression of SIRT1 in the heart attenuates age-associated cardiac hypertrophy, fibrosis and cardiac dysfunction, while high level overexpression of SIRT1 increases these pathological effects (10). In the setting of ischemiareperfusion (I/R), SIRT1 exerts protective effects: Sirt1 knockout (KO) in the heart increases I/Rinduced injury, while its overexpression protects against I/R-induced injury (11). Thus, it appears that the effects of SIRT1 on cardiac response to stress are dependent on its expression levels as well as the context of injury. SIRT3 has also been studied in the heart and shown to protect against both cardiac hypertrophy and I/R injury (12,13), while SIRT6 KO mice exhibit cardiac hypertrophy (14). Recent studies have assessed the role of SIRT2 in the heart. One study showed that deletion of Sirt2 reduces AMPK activation and increases age-related and Angiotensin II-mediated cardiac hypertrophy (15), while another showed that advanced glycation end products (AGEs) and its receptor promote diabetic cardiomyopathy through suppression of SIRT2, however, knockout mice were not used for these studies (16). Another study showed that Sirt2 deficiency increases nuclear localization of NFATc2 and its transcription activity, and that NFAT inhibition rescues the cardiac dysfunction in mice with Sirt2 deletion (17).
NRF2 is a transcription factor that activates a number of cytoprotective genes, including antioxidative enzymes (18). Under normal conditions, NRF2 resides in the cytoplasm, and is degraded primarily through its interaction with Keap1 (kelch-like ECH-associated protein 1), which also serves as a bridge between NRF2 and cullin 3-ubiquitination complex (18). Under oxidative stress, NRF2 escapes degradation, translocates into the nucleus, and binds to antioxidant response elements (ARE) in the promoter of a number of genes (19). NRF2 acetylation is decreased with SIRT1 overexpression (20); however, an association between SIRT1 and NRF2 has not been demonstrated, and the functional consequences of NRF2 deacetylation have not been studied. NRF2 KO mice developed cardiac hypertrophy and heart failure (HF) after transaortic constriction (TAC) (21), indicating the NRF2 is protective against cardiac stress. We recently showed that SIRT2 mediates NRF2 deacetylation in the liver cells and its translocation in the nucleus to regulate anti-oxidant genes (22).
In this paper, we show that SIRT2 plays a detrimental role in the heart in response to injury, in contrast to a previously published report (15). Mechanistically, deletion of Sirt2 is protective through stabilization and increased nuclear translocation of NRF2, leading to increased expression of antioxidant genes. Finally and most importantly, we show that pharmacological inhibition of SIRT2 protects the heart against the development of cardiac hypertrophy, opening potential treatment for this disorder.

SIRT2 is expressed in the heart and its levels are elevated in HF
We first showed that SIRT2 is expressed in the heart (Figure 1-figure supplement 1A) and in H9c2 cardiomyoblasts (Figure 1-figure supplement 1B) at relatively high levels. We also found that SIRT2 expression was higher in the hearts of mice 4 weeks after TAC compared to sham (Figure 1A), while the levels of other sirtuin family members with major deacetylation activity that have been studied in the heart (i.e., SIRT1, SIRT3 and SIRT6) were not different. Additionally, we noted a significant increase in the levels of SIRT2 in the explanted hearts from end-stage HF patients with dilated cardiomyopathy (Figure 1B). We also assessed the levels of SIRT2 in explanted hearts from patients with ischemic cardiomyopathy and showed that SIRT2 is increased in these hearts ( Figure 1C). These results indicate that SIRT2 levels are increased in HF and ischemic injury.

Sirt2 deficiency preserves cardiac function in response to PO and I/R injury
We first used mice with global deletion of Sirt2 KO (Sirt2 -/-) for our studies. We assessed whether Sirt2 deletion affects the levels of other sirtuin family members in the heart. Sirt2 -/hearts displayed no change in other sirtuin family members at the mRNA level, and no change in protein levels of SIRT1, SIRT3 or SIRT6 (sirtuins with major deacetylation activity) was detected ( Figure  2-figure supplement 1). We then assessed whether Sirt2 deletion protects against PO. Sirt2 -/mice displayed normal cardiovascular parameters at baseline and no overt phenotype. However, in response to TAC, Sirt2 -/mice displayed improved cardiac function than littermate controls, as assessed by fractional shortening (FS) and ejection fraction (EF) (Figure 2A, B). Additionally, Sirt2 -/mice displayed evidence of less cardiac hypertrophy, as evidenced by lower interventricular septal (IVS) thickness on echocardiography (Figure 2C), and reduced cardiac size and heart weight to body weight ratio on gross examination (Figure 2D, E). Histological examination of the hearts also showed smaller cardiomyocytes in Sirt2 -/hearts after PO, as assessed by H&E staining (Figure 2F, G). These data indicate that deletion of Sirt2 results in protection of the heart against PO with improved cardiac function and less cardiac hypertrophy.
To better assess the role of SIRT2 in the development of HF and ischemic damage, we then studied the effects of Sirt2 deletion in the heart on the response to I/R. We subjected Sirt2 -/and their littermate wild type (WT) controls to I/R and cardiac function was assessed after 7 and 21 days. At both time points, EF and FS were significantly higher in Sirt2 -/mice compared to controls ( Figure 3A and 3B). Time course of cardiac assessment showed that while FS was comparable between WT and Sirt2 -/on day 3, it quickly deteriorated in WT mice, consistent with transition into HF, while Sirt2 -/mice maintained their cardiac function ( Figure 3C). To further support these findings, we assessed the effects of Sirt2 modulation on cell death in response to H2O2 in neonatal rat cardiomyocytes (NRCMs) treated with control or Sirt2 siRNA by measuring propidium iodide (PI) positive cells. We found that cells with Sirt2 KD displayed improved cell viability in response to H2O2 (Figure 3D, E). Overall, these results indicate that SIRT2 exerts detrimental effects in the heart in response to PO and I/R, and that its deletion leads to protective effects.
The experiments in Figure 2 and 3 were conducted in mice with global deletion of Sirt2. To confirm a role for SIRT2 in cardiomyocyte response to injury, we then generated cardiac specific Sirt2 KO mice (cs-Sirt2 -/-) by crossing Sirt2 floxed mice with αMHC-Cre mice. The cs-Sirt2 -/mice were then subjected to TAC with littermate Cre negative mice as control, and cardiac function (EF and FS) were assessed at 1 and 2 weeks after injury. At both time points, cs-Sirt2 -/mice displayed improved cardiac function compared to WT controls (Figure 4A, B). consistent with these data, HF markers in the heart, including Nppa and Nppb were significantly lower in cs-Sirt2 -/mice after TAC (Figure 4C, D).

SIRT2 deacetylates NRF2 resulting in decreased transcriptional activity in the heart
We previously showed that SIRT2 deacetylates NRF2 protein in the liver and alters iron release from hepatocytes (22). Since deacetylation of NRF2 leads to protein destabilization and NRF2 regulates the expression of many anti-oxidant genes, we hypothesized that the mechanism for the protective effects of Sirt2 deletion in response to stress is through decreased NRF2 deacetylation and degradation, resulting in increased expression of antioxidant proteins. To test this hypothesis, we first assessed whether there is physical interaction between SIRT2 and NRF2 in the heart. Co-immunoprecipitation (IP) experiments showed that SIRT2 interacts with NRF2 in the heart of WT mice ( Figure 5A).
We then measured acetylation levels of NRF2 in WT and Sirt2 -/hearts, and showed that NRF2 acetylation levels are increased within Sirt2 -/hearts ( Figure 5B). We then assessed whether deacetylation of NRF2 alters its levels in the cardiac cells, as shown before in the liver (22). Treatment of NRCMs with Sirt2 siRNA resulted in increased NRF2 protein levels compared with control siRNA, indicating that SIRT2 leads to a reduction in the levels of NRF2 protein ( Figure  5C). Since NRF2 protein levels are higher in Sirt2 -/hearts, we next assessed whether SIRT2 alters the stability of NRF2 protein. NRF2 levels were significantly lower starting at 60 minutes after treatment with the protein synthesis inhibitor cycloheximide (CHX), leading to almost complete degradation at 120 minutes in cells treated with control siRNA. However, we noted no change in NRF2 protein levels in cells treated with Sirt2 siRNA ( Figure 5D). These data indicate that SIRT2 binds to NRF2, and its deacetylation leads to the instability and degradation of NRF2.
NRF2 is a transcription factor and upon activation, translocates into the nucleus to exert its transcriptional activity (23). Thus, we measured nuclear level of NRF2 and found it to be increased in NRCMs with Sirt2 KD (Figure 5E). Since the increase in nuclear levels of NRF2 suggests possibly higher transcriptional activity of the protein, we next assessed the effects of Sirt2 modulation on NRF2 transcriptional activity in H9c2 cells treated with lentivirus expressing either control or SIRT2 lentivirus. Consistent with its increased nuclear levels, SIRT2 overexpression in H9c2 cells resulted in lower levels of known NRF2 target genes ( Figure 5F-H). However, the mRNA levels of non-NRF2 targeted anti-oxidant genes were not affected by SIRT2 overexpression (Figure 5-figure supplement 1).
Since our data suggest a role for SIRT2 in the regulation of NRF2-mediated expression of antioxidant genes, we next assessed whether SIRT2 has an effect on reactive oxygen species (ROS) production. NRCMs treated with Sirt2 siRNA displayed less ROS levels after treatment with H 2O2 (Figure 5-figure supplement 2), further supporting a role of for SIRT2 in regulating oxidative state of cardiomyocytes.

Sirt2/Nrf2 double KO mice display more cardiac damage after I/R compared to Sirt2 -/mice
Our results thus far demonstrate that NRF2 is a target of SIRT2 and that SIRT2 regulates NRF2 acetylation and protein levels. To determine whether the protective effects of SIRT2 are mediated through NRF2, we generated Sirt2/Nrf2 double KO mice and subjected the mice to I/R injury. The Sirt2/Nrf2 double KO mice displayed reduced EF and FS compared to Sirt2 -/mice ( Figure 6A,B), indicating that deletion of Nrf2 reverses the protective effects of SIRT2.

Pharmacological inhibition of SIRT2 protects the heart against ischemic damage
Since a reduction in SIRT2 levels led to protection against the development of heart failure and cardiac hypertrophy, we next studied whether pharmacological inhibition of SIRT2 also exerts protective effects in the heart in response to PO. For these studies, we used AGK2, a selective SIRT2 inhibitor (24)(25)(26). Eight-week-old C57B6 mice were underwent TAC and one day later, they were randomized to treatment with 40 mg/Kg of AGK2 or vehicle intraperitoneally twice a week for 4 weeks. At the conclusion of the study, their cardiac function and heart chamber size were assessed using echocardiography ( Figure 6C). Treatment with AGK2 did not change the systolic function of the heart, as assessed by EF, FS (Figure 6D-F). However, measures of cardiac size, as assessed by LV diameter during diastole and systole (LVDd and LVDs, respectively) were increased, while measures of left ventricular (LV) wall diameter, as assessed by IVSd and posterior wall thickness during diastole (PWTd) were reduced (Figure 6G-J). These results indicate that pharmacological inhibition of SIRT2 can protect the heart against cardiac hypertrophy and improve cardiac remodeling in response to pressure overload.

DISCUSSION
Sirtuins play a major role in post-translational modification of proteins, and their deletion have been shown to lead to a number of physiological changes and pathological conditions (27)(28)(29). Although multiple sirtuins have been investigated in the context of cardiovascular diseases (15)(16)(17), it is not known whether SIRT2 has a role in protection against HF and cardiac hypertrophy. In this paper, we used genetic models to show that SIRT2 has detrimental effects in the heart in the setting of cardiac insults and demonstrate that the deleterious effects of SIRT2 is through increased NRF2 deacetylation and its degradation and eventual reduction in the levels of antioxidant genes. We also show that deletion of Nrf2 reverses the protective effects of Sirt2 deletion. Finally, we provide a clinical significance for our findings and show that treatment of mice with AGK2, a selective SIRT2 inhibitor, results in protection against cardiac hypertrophy in response to PO.
There are limited published studies on SIRT2 in the heart. Two studies have shown protective effects of SIRT2 in the heart, with one study showing that deletion of Sirt2 increases age-related and Angiotensin II-mediated cardiac hypertrophy (15), while another study showing that Sirt2 deficiency leads to cardiac dysfunction and cardiac hypertrophy (17). The reason for this discrepancy in our data is not clear, however, we used both global and cardiac specific KO of Sirt2, while these studies have used mice with global deletion of the gene. The genetic background of the mice and the different gene targeting strategy might have also contributed to the difference. Additionally, these studies used either angiotensin-or isoproterenol-induced models to cause cardiac hypertrophy and other potential effects of these drugs may explain the differences in our results. However, we used two different genetic models (global and cardiac specific KO of Sirt2) and a pharmacological approach to test of our hypothesis, all of which produced similar results. Finally, we provide a mechanism for the deleterious effects of SIRT2 in the heart through its regulation of anti-oxidant proteins by NRF2 protein.
NRF2 plays a major role in the regulation of genes involved in oxidative stress, metabolic processes, drug metabolism and stress response, among others. Thus, its activation has been studied extensively in a number of diseases, however, these studies have not led to an effective therapy. It is possible that direct activation of NRF2 may have unwanted side effects. Our studies provide a proof of concept that targeting SIRT2 and indirect activation of NRF2 by altering its posttranslational protein modification may prove to be a more effective therapeutic strategy for a number of diseases.
Cardiac hypertrophy is a major complication of hypertrophy and metabolic disorders in this country (30)(31)(32), however, our treatment options are limited. We generally treat the underlying cause without directly targeting cardiac function and remodeling. Our data indicate that targeting SIRT2 with AGK2 would improve cardiac remodeling and cardiac hypertrophy in response to PO, potentially providing a novel therapy for these disorders. However, systemic inhibition of SIRT2 may have unwanted side effects, which would need to be investigated further.
In summary, our data demonstrate that SIRT2 has deleterious effects in the heart through its post-translational modification of NRF2. We show that SIRT2 binds to NRF2 and that Sirt2 deletion leads to an increase in NRF2 stability and nuclear levels, resulting in higher production of antioxidant genes. Additionally, deletion of Nrf2 reverses the protective effects of Sirt2 deletion. Finally, our results provide a potential therapy for cardiac hypertrophy by using AGK2, a specific inhibitor of SIRT2.

Animal models
All animals were maintained and handled in accordance with the Northwestern Animal Care and Use Committee. Sirt2 -/and Sirt2 floxed mice were obtained from Dr. David Gius. Nrf2 -/mice were purchased from Jackson labs. Sirt2 -/-/Nrf2 -/mice were generated by crossing the Sirt2 -/with Nrf2 -/mice. All animals were kept in accordance with standard animal care requirements and maintained in a 22°C room with a 12-hour light/dark cycle, and received food and drinking water ad libitum.

Study approval
All animal studies were approved by the Institutional Animal Care and Use Committee at Northwestern University (Chicago, Illinois) and were performed in accordance with guidelines from the National Institutes of Health. The approval number of the animal protocol currently associated with this activity is IS00006808.

Human heart samples
Non-failing and cardiomyopathy cardiac tissue samples were obtained from the Human Heart Tissue Collection at the Cleveland Clinic. Informed consent was obtained from all the transplant patients and from the families of the organ donors before tissue collection. Protocols for tissue procurement were approved by the Institutional Review Board of the Cleveland Clinic (Cleveland, Ohio, USA), which is AAHRPP accredited. The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.

Animal surgeries
Cardiac surgeries were performed as previously described (33,34). Briefly, mice of 10-12 weeks old were anesthetized with 2% isoflurane. The animals were placed in a supine position and ECG leads were attached. The body temperature was monitored using a rectal probe and was maintained at 37°C with heating pads throughout the experiment. A catheter was inserted into the trachea and was then attached to the mouse ventilator via a Y-shaped connector. The mice were ventilated at a tidal volume of 200μl and a rate of 105 breaths/min using a rodent ventilator. Chronic pressure overload was induced by TAC as described. A 7-0 silk suture was placed around the transverse aorta between the origin of the right innominate and left common carotid arteries against an externally positioned 27 gauge needle to yield a narrowing 0.4 mm in diameter when the needle was removed after ligation. The sham procedure was identical except that the aorta was not ligated. For cardiac I/R injury, a 1 mm section of PE-10 tubing was placed on top of left anterior descending artery (LAD), and a knot was tied on the top of the tubing to occlude the coronary artery with an 8-0 silk suture. Ischemia was verified by pallor of the anterior wall of the left ventricle and by ST-segment elevation and QRS widening on the ECG. After occlusion for 45 minutes, reperfusion occurred by cutting the knot on top of the PE-10 tubing. Animals were given buprenorphine for post-operative pain.

Echocardiography
Parasternal short-and long-axis views of the heart were obtained using a Vevo 770 highresolution imaging system with a 30 MHz scan head. 2D and M-mode images were obtained and analyzed. Ejection fraction was calculated from M-mode image using Teichholtz equation, and fractional shortening was directly calculated from end-systolic and end-diastolic chamber size from M-mode images.

Histological analysis
Hearts were fixed in 10% formalin (PBS buffered), dehydrated, and embedded in paraffin. Heart architecture was determined from transverse 5μm deparaffinized sections stained with H&E. Fibrosis was detected with Masson's trichrome staining.

Cell culture and reagents
NRCMs were isolated from 1-to 2-day-old Sprague-Dawley rats as previously described (35). Cardiomyocytes were cultured in DMEM, supplemented with 5% FBS, 1.5 mM vitamin B12 and 1 mM penicillin-streptomycin (Gibco). To prevent proliferation of non-myocytes, 100 µM bromodeoxyuridine (BrdU) was added to the culture media. To induce oxidative stress, cells were exposed to hydrogen peroxide (H2O2, VWR) for 4 hours. Cycloheximide (Sigma) was used to check protein stability. H9C2 line were grown on DMEM, supplemented with 10% FBS. All cells were maintained in a 37 °C incubator with 5% CO2 and 6% oxygen and were 70-90% confluent when collected for various analyses unless otherwise noted.

Protein stability assay
For protein stability studies, 100 μg/ml of CHX (Sigma) was added to H9c2 cells, and samples were isolated at 0, 15, 30, 60, 90, and 120 minutes after the addition of CHX. Samples were then run on a gel for Western blot analysis.

RNA isolation and qRT-PCR
RNA was isolated using RNA-STAT60 (Tel-Test) according to the manufacturer's instructions and subjected to DNAse I (Ambion) digestion to remove residual DNA. Purified RNA was then reverse transcribed with random hexamer and oligo-dT(16) (Applied Biosystems) and amplified on a 7500 Fast Real-Time PCR system using Fast SYBR Green PCR Master Mix (Applied Biosystems). The sequences for primers are included in Appendix Table S2. mRNA levels were calculated based on the difference of threshold Ct values in the target gene and average Ct values of 18s, Actb, B2m, and Hprt in the same sample.

Cell death studies
Permeability to PI (Sigma-Aldrich) was used as a fluorescent signal for cell death (38). NRCMs were treated with H2O2 for 4hours, washed one time with HBSS and co-stained with PI and Hoescht. After several steps of wash, images were taken using Zeiss AxioObserver.Z1 fluorescent microscope. Data were analyzed with Image J (NIH).

Measurement of reactive oxygen species (ROS)
Intracellular ROS levels were determined using dihydroethidium (DHE) assay. Briefly, NRCMs were treated with H2O2. After 4 hours of incubation, cells were washed and loaded with 10 µM DHE and Hoechst for 30min. After two washing steps, Fluorescence images were acquired with the Zeiss AxioObserver.Z1.

Isolation of nuclei
Nuclei were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce).

Western blot and immunoprecipitation (IP)
20-40 µg of protein were resolved on SDS-PAGE gels and transferred to nitrocellulose membranes (Invitrogen, CA). The membranes were probed with antibodies against SIRT1, SIRT2 (Sigma-Aldrich), SIRT3, SIRT6 (Cell Signaling Technology), NRF2 (Abcam, cell signaling), HPRT, GAPDH (Proteintech), TBP, and β-actin(Abcam). HRP-conjugated donkey anti-rabbit and donkey anti-mouse were used as secondary antibodies (Jackson ImmunoResearch) and visualized by Pierce Super Signal Chemiluminescent Substrates. For IP, cells or tissue were lysed using IP buffer (25 mM Tris-HCl pH7.5, 150 mM NaCl, 1 mM EDTA, 0.1% NP-40 and 5% glycerol), and cell extracts were incubated overnight with appropriate antibodies followed by incubation with protein A or G agarose beads for 4 h at 4 o C. After washing five times with IP buffer, immunocomplexes were resolved using SDS-PAGE and analyzed by western blot.

Treatment of WT mice with AGK2
AGK2 administration to C57BL6J mice which underwent TAC surgery by intraperitoneal injection was started 1 day after the surgery with the dose of 40 mg/Kg. The injection was performed twice a week for 4 weeks, and then their cardiac function was assessed by echocardiography. AGK2 (Selleckchem) was dissolved in DMSO, and administered to C57BL6-J mice 2 hours before TAC surgery and then twice a week at a dose of 40mg/kg started on day 1 after the surgery and continued for two weeks. At the end of studies, cardiac function was assessed by echocardiography.

Statistical analysis
For sample size, based on our previous experience with similar studies, we estimated at least 4-6 animals per group is needed to detect significant functional difference. However, the sample size was not pre-determined. Animals were randomized into sham versus control group. Surgical operator was blinded regarding animal's genotype. For AGK2 treatment study, mice were randomized into control versus AGK2 group. Data analysis was not masked. The replicate number for in vitro experiment and animal numbers for in vivo experiments were based on our prior experience of studying gene expression and cardiac function after insult. All reported replicates for in vitro experiments are technical replicates (unless otherwise specifically noted).
All data are expressed as mean ± SEM. Exclusion criteria were not pre-established. No sample or data points were omitted from analysis. Statistical significance was assessed with two-tailed unpaired t-test for two group comparison or with ANOVA for data with more than two groups. Post hoc Tukey's test was performed for multiple-group comparison if ANOVA reached statistical significance. Kolmogorov-Smirnov test was used to test for normal distribution. Levene's test was used to evaluate equal variance among groups. A P-value of < 0.05 was considered statistically significant.