The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts
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Sirtuin 3 is a nicotinamide adenine dinucleotide dependent mitochondrial deacetylase that governs mitochondrial metabolism and oxidative defense. The demise in myocardial function following myocardial ischemia has been associated with mitochondrial dysfunction. Sirt3 maintains myocardial contractile function and protects from cardiac hypertrophy. The role of Sirt3 in ischemia is controversial. Our objective was to understand, under what circumstances Sirt3 is protective in different facets of ischemia, using an in vitro proof-of-concept approach based on simulated ischemia in cultured cardiomyoblasts. Cultured H9c2 cardiomyoblasts were subjected to hypoxia and/or serum deprivation, the combination of which we refer to as simulated ischemia. Apoptosis, as assessed by Annexin V staining in life-cell imaging and propidium-iodide inclusion in flow cytometry, was enhanced following simulated ischemia. Interestingly, serum deprivation was a stronger trigger of apoptosis than hypoxia. Knockdown of Sirt3 further increased apoptosis upon serum deprivation, whereas no such effect occurred upon additional hypoxia. Similarly, only upon serum deprivation but not upon simulated ischemia, silencing of Sirt3 led to a deterioration of mitochondrial function in extracellular flux analysis. In the absence of oxygen these Sirt3-dependent effects were abolished. These data indicate, that Sirt3-mediated myocardial protection is oxygen-dependent. Thus, mitochondrial respiration takes center-stage in Sirt3-dependent prevention of stress-induced myocardial damage.
KeywordsSirt3 Cardiomyoblasts Ischemia Oxygen Mitochondrial function
Serum deprivation is a stronger trigger of apoptosis than hypoxia in cultured cardiomyoblasts
Loss of Sirt3 exacerbates apoptotic damage and mitochondrial dysfunction of cultured cardiomyoblasts upon serum deprivation
Additional hypoxia during simulated ischemia blunts these protective effects of Sirt3
Future research is needed to translate this concept of a differential, oxygen-dependent role of Sirt3 from a cellular to an in vivo level
Ischemic heart disease remains the leading cause of mortality worldwide . Myocardial ischemia is associated with myocardial damage, culminating in the development of heart failure . Myocardial function is closely related to mitochondrial function, including mitochondrial energy and reactive oxygen species (ROS) homeostasis and, ultimately, mitochondrial induction of apoptosis. Myocardial apoptosis leads to myocardial scarring, the extent of which predicts post-infarction mortality . Mitochondrial dysfunction has been associated with chronic heart disease and cardiomyopathies .
Sirtuin 3 (Sirt3) is a nuclear-encoded, mitochondrial deacetylase that orchestrates mitochondrial metabolism and oxidative defense . All mammalian sirtuins share a conserved binding motif for the oxidized form of NAD+, defining them as class III histone deacetylases and confining their activity to times of increased energy demands [6, 7]. With the heart relying almost exclusively on mitochondria for energy supply, it is not surprising that myocardial expression levels of Sirt3 are among the highest compared to other tissues in the body . Concordantly, myocardial adenosine triphosphate (ATP) levels in Sirt3 knockout mice are diminished to < 50% of those in wildtype mice . Sirt3 improves mitochondrial respiration and attenuates oxidative stress, thereby protecting cardiomyocytes from stress-induced damage [9, 10, 11]. Sirt3 deficiency exacerbated high-fat diet-induced negative cardiac remodeling and the deterioration of left ventricular ejection fraction . Left ventricular ischemia following coronary artery ligation was associated with reduced Sirt3 expression in ischemic compared with non-ischemic myocardium . Along this line, post-myocardial infarction cardiac repair was reported to be Sirt3-dependent: Sirt3 deficiency was associated with hampered angiogenic capacity, aggravated ROS formation and increased apoptosis of bone marrow-derived endothelial progenitor cells .
However, in the context of myocardial ischemia the role of Sirt3 remains highly controversial: Though both myocardial contractile and mitochondrial function of Sirt3-deficient mice were reduced compared with wildtype controls , recovery of cardiac function, following myocardial ischemia–reperfusion in perfused isolated hearts, was unaltered compared to wildtype controls . On the other hand, ischemic post-conditioning-mediated prevention of myocardial reperfusion injury was Sirt3-dependent, which was associated with a Sirt3-mediated reduction of mitochondria-dependent apoptosis .
Therefore, we aimed to understand, whether Sirt3 is protective in myocardial ischemia. For this purpose, we used the simplest possible design: To allow for strict control of available oxygen and nutrient supply—the key elements in an ischemia setting—we chose an in vitro approach. To maximize the stability of culture conditions, we used immortalized cardiomyoblasts (H9c2) that were specifically chosen for their functional similarity to primary cardiomyocytes, particularly with regard to energy metabolism, mitochondrial function and sensitivity to hypoxia [18, 19].
H9c2 cells (immortalized embryogenic rat cardiomyoblasts, ATCC, USA) are an established model for cardiomyocytes [20, 21] and were cultured to a confluency of ca. 80% (37 °C, 5% CO2 in Dulbecco’s modified eagle media (DMEM) with 10% (v/v) fetal bovine serum (FBS), 1% (v/v) non-essential amino acids, 1% (v/v) l-glutamine and 1% (v/v) penicillin/streptomycin).
Transfection of H9c2 with Sirt3 small interfering RNA (siRNA, siSirt3) or scrambled siRNA (scr, both Thermo Scientific, USA) were performed by lipofection (DharmaFECT®, Dharmacon, USA). Transfection efficacy was assessed by quantitative real-time PCR (qRT-PCR) and Western blot analyses.
RNA isolation and quantitative PCR
RNA was extracted (TRIzol® Reagent, Invitrogen™, USA) and cDNA synthesis was performed (TaqMan® Reverse Transcription Reagents, Invitrogen™, USA) according to the manufacturer’s instructions. Differential gene expression was assessed by qRT-PCR (SYBR® Green, Life Technologies™, Germany) with commercially available primers for rat Sirt3 (QT01761585, Qiagen, Germany) and rat hypoxanthine phosphoribosyltransferase-1 (rHPRT1: sense 5′-GGT CCA TTC CTA TGA CTG TAG ATT TT-3′, anti-sense 5′-CAA TCA AGA CGT TCT TTC CAG TT-3′) that served as housekeeping gene. Data were analyzed using the ΔΔCT method.
Hypoxia and serum deprivation (simulated ischemia)
H9c2 cells were grown in 12-well plates (105cells/ml, 37 °C, 24 h, 5% CO2) and subjected to hypoxia in a hypoxia chamber (Billups-Rothenberg, USA), which was flushed with 100% gaseous nitrogen for 10 min before incubation at 37 °C. After 8 h the cells were analyzed. To simulate reduced energy supply in the context of ischemia, experiments were carried out in presence (10% v/v) or absence (0% v/v) of fetal bovine serum (FBS). The combination of hypoxia and serum deprivation is referred to as simulated ischemia.
Cells were lysed using radioimmunoprecipitation assay buffer (4 °C, 1 h) supplemented with protease inhibitor (Roche®). Samples were centrifuged (16,000 g, 4 °C, 15 min) and proteins in the supernatant were separated using SDS-PAGE and blotted on a nitrocellulose membrane (PVDF-Membrane, Millipore Corp, USA). Antibodies were: anti-SIRT3 monoclonal antibody (1:1.000) and anti-rabbit horseradish peroxidase (both Cell Signaling™, USA). Signals were revealed using SuperSignal® (Thermo Fisher Scientific, USA) and protein bands were visualized using X-ray films (Hyperfilm™, GE Healthcare, USA).
Cell death was quantified by propodium iodide (PI, Invitrogen™, USA) staining (0.125 µg/ml) as described . After staining, cells were fixed (CellFixTM-solution, BD Bioscience, USA) and subjected to FACS analysis (FACSCalibur, BD Bioscience, USA). A total of 5000 events per sample were acquired. Results are expressed as “percent [counted] events”, if not indicated otherwise.
Cell morphology, integrity and apoptosis-related changes of H9c2 cells upon hypoxia and/or serum deprivation were assessed using fluorescence microscopy. Therefore, experiments were carried out on chamber slides. Following transient knockdown of Sirt3, cells were incubated with PI (1 µg/ml), Annexin V (Fc 50 µg/ml) and Hoechst 33,342 (Fc 2 µg/ml) for 15 min in the dark. Thereafter, microscopy was performed using an Olympus IX71 Inverted Microscope.
Mitochondrial function of H9c2 cells was assessed based on direct oxygen consumption rate (OCR) measurements during the XF Cell Mito Stress Test using an XF24-3 Extracellular Flux Analyzer (both Seahorse Bioscience, USA). Serial addition of different modulators of the electron transport chain (ETC) to the Seahorse XF DMEM assay media (1 mM sodium pyruvate, 25 mM glucose) allowed the quantification of ATP production, basal and maximal mitochondrial and non-mitochondrial respiration. The following ETC modulators were added: oligomycin (1 µM) (inhibits ATP synthase/complex V), the consecutive absolute OCR decrease correlates with mitochondrial respiration (cellular ATP turnover); carbonyl-cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) (1 µM) (uncoupling agent, that disrupts the mitochondrial membrane potential), consequently electron flow is inhibited and a maximum of oxygen is consumed, the absolute OCR increase compared with the initial basal respiration correlates with the mitochondrial spare respiratory capacity, an indicator of the capacity to respond to increased energy demands; rotenone (1 µM) (a complex I inhibitor) and antimycin A (1 µM) (complex III inhibitor), a combination that inhibits mitochondrial respiration and allows quantification of non-mitochondrial respiration. 20.000 cells per well were investigated.
Metric variables were assessed for distribution using Kolmogorov–Smirnov tests. Variances were compared using F-tests. Different groups were compared using Students t-tests or Mann–Whitney U tests for the comparison of the means of two groups. Interactions between the effects of two or more independent variables on a dependent continuous variable were assessed using two-way analyses of variance. Significance was accepted at p < 0.05. All p-values are two-sided. At least three independent experiments in biological triplicates were performed. Results are displayed as mean ± SEM. Statistical analyses were performed using Graphpad Prism Version 4.
Simulated ischemia induces apoptosis in H9c2 cardiomyoblasts
To simulate myocardial ischemia, H9c2 cells were subjected to an 8-h period of hypoxia in combination with serum deprivation, serum deprivation alone or hypoxia alone. Cell damage was assessed using PI inclusion, which was quantified by flow cytometry and visualized by live-cell imaging. Following hypoxia alone, the number of PI-including, non-viable cells (black gate) increased only slightly by 1.5-fold (p = 0.2908) compared with normoxic controls (Fig. 1a, c, f). Similarly, the number of intermediately PI-positive cells (green gate), representing early apoptotic cells, increased only marginally by 1.6-fold (p = 0.2268) following hypoxia (Fig. 1c). Serum deprivation alone led to a more pronounced increase in early apoptotic and apoptotic cells by 3.8-fold (p = 0.0076) and 2.6-fold (p = 0.0075), respectively (Fig. 1b, e). Thus, the detrimental effect of serum deprivation exceeded the effect of mere hypoxia (Fig. 1g). The combination of hypoxia and serum deprivation, mimicking myocardial ischemia following coronary artery occlusion, led to the strongest cellular damage, with an increase in early apoptotic and apoptotic cells of 5.8-fold (p = 0.0004) and 3.6-fold (p = 0.0185), respectively, compared with normoxic, non-serum-deprived conditions (Fig. 1d, h). To visualize apoptotic cell damage following serum deprivation alone or simulated ischemia, H9c2 cells were subjected to PI- and Annexin V staining. Life-cell imaging in phase-contrast revealed morphologic signs of apoptosis, i.e. “membrane blebbing”, an effect that occurred already upon serum deprivation, but was more pronounced following simulated ischemia (Fig. 2a–c). Accordingly, serum-deprived cardiomyoblasts stained positive for Annexin V while PI was still excluded (Fig. 2d, e, g, h). When additionally subjected to hypoxia in simulated ischemia, cardiomyoblasts showed PI-inclusion, indicating cell membrane break down, a sign of advanced apoptosis (Fig. 2c, f, i).
Sirt3 knockdown exacerbates early apoptosis in H9c2 cardiomyoblasts upon serum deprivation
To assess the effect of Sirt3 on cellular damage in different facets of ischemia, we used a loss-of-function approach based on an siRNA-mediated Sirt3 knockdown in H9c2 cells. Knockdown efficiency was 74.2% compared with scrambled siRNA transfected controls, as exhibited by qRT-PCR and Western blot (Fig. 3a, b). During serum deprivation alone, under normoxic conditions, silencing of Sirt3 led to a substantial increase, specifically in early as opposed to late apoptosis, compared with controls (Fig. 3c, d, g). However, upon additional hypoxia, during simulated ischemia, knockdown of Sirt3 was associated with only a slight increase of both early apoptotic and late apoptotic cells (Fig. 3e, f, h). Of note, neither under normal growth conditions (normoxia, normal serum supplementation), nor under hypoxia alone (normal serum supplementation), we observed a significant difference between Sirt3 knockdown and scrambled controls (Fig S1). Interestingly, increased Sirt3 mRNA transcription in H9c2 cells, which we observed upon serum deprivation, did not translate into increased protein levels of Sirt3 (Fig S2).
Sirt3 maintains mitochondrial function in H9c2 cardiomyoblasts during serum deprivation
The cellular damage upon serum deprivation, along with previous reports on serum deprivation-induced apoptosis through mitochondrial pathways  and Sirt3, governing mitochondrial function, prompted us to investigate the effect of Sirt3 on mitochondrial function in H9c2 cells upon serum deprivation. Assessment of direct oxygen consumption revealed a prominent deterioration of mitochondrial respiration upon serum deprivation (Fig. 4b). Though knockdown of Sirt3 did not affect basal respiration and ATP turnover upon serum deprivation, spare respiratory capacity, correlating with the cellular response capacity to increased energy demands, was reduced by half upon silencing of Sirt3 (Fig. 4c). Similarly, maximal mitochondrial respiration, as deduced from the difference between FCCP-induced respiration and non-mitochondrial respiration following rotenone and antimycin A-mediated inhibition of mitochondrial respiration, was significantly impaired upon Sirt3 knockdown (Fig. 4c). Interestingly, upon additional hypoxia, during simulated ischemia, no significant effect of Sirt3 was observed (Fig. 4d). Moreover, no significant interaction between the effects of growth conditions (serum deprivation vs. simulated ischemia) and of the presence or absence of Sirt3 was observed.
In the present proof-of-principle study we shed light on the role of Sirt3 in different components of ischemia, applying an in vitro approach in cultured cardiomyoblasts. Serum deprivation alone was a more potent trigger of apoptosis in cardiomyoblasts than hypoxia alone. However, the combination of serum deprivation and hypoxia, in simulated ischemia, was the strongest trigger of apoptosis. Importantly, silencing of Sirt3 exacerbated cardiomyocyte apoptosis only under normoxic serum deprivation, but not upon additional hypoxia, during simulated ischemia. Along this line, loss of Sirt3 hampered mitochondrial function during serum deprivation, whereas this effect was abolished upon additional hypoxia, during simulated ischemia. These data indicate a differential role of myocardial Sirt3 during ischemia.
Sirt3 appears indispensable to maintain mitochondrial function at times of decreased nutrient supply or increased energy demand. However, upon additional hypoxia, when mitochondrial respiration cannot take place, Sirt3 is no longer capable to protect from mitochondrial dysfunction and myocardial apoptosis. Therefore, mitochondrial respiration likely takes center-stage in Sirt3-mediated myocardial protection. These basic observations underscore the putative importance of Sirt3 in myocardial energy homeostasis and may provide an explanation for the controversial data about Sirt3 in myocardial ischemia in vivo.
Added value in the context of previous studies
Sirt3 has been implied in diverse contexts of myocardial protection [11, 17, 24]. Sundaresan et al. were the first identifying Sirt3 as a stress-responsive deacetylase, protecting primary cardiomyocytes from oxidative stress-induced cell death . In this study, anti-apoptotic properties were attributed to the Sirt3-mediated inhibition of the mitochondrial translocation of the pro-apoptotic protein Bax. Later on, the authors observed that Sirt3-mediated anti-oxidative capacities also protect from cardiac hypertrophy induced by external stressors . In addition to anti-oxidative and anti-apoptotic effects  Sirt3 also plays a crucial role in mitochondrial metabolism and energy homeostasis, by driving oxidative phosphorylation, the tricarboxylic acid cycle as well as fatty acid oxidation . Beating around 100,800 times a day, at the cost of approximately 30 kg of adenosine triphosphate a day, the human heart heavily depends on sufficient energy supply and thereby relies almost exclusively on mitochondrial oxidative phosphorylation . Recently, a number of studies investigated the role of Sirt3 in myocardial function in different settings [9, 10]. However, data on the role of Sirt3 in acute myocardial ischemia, representing an immense metabolic challenge, involving increased oxidative stress and often culminating in myocardial breakdown, are highly controversial: While we observed impaired mitochondrial and myocardial function in aged Sirt3-deficient mice  recovery of cardiac function following experimental ischemia was unaltered . Yet, heterozygous constitutive deletion of Sirt3 was reported to be associated with an exacerbation of ischemia–reperfusion injury and increased infarct size . Along this line, the beneficial effects of ischemic post-conditioning, which targets viable cardiomyocytes in the non-ischemic border zone of infarction [28, 29] was abolished upon Sirt3 deletion.
In the present study, we addressed the role of Sirt3 in different components of ischemia-induced cell death. We therefore, for the first time in this context, applied a stepwise in vitro approach to separately evaluate the relevance of Sirt3 in serum deprivation alone, hypoxia alone or the combination of both, in simulated ischemia. As described previously  hypoxia alone did not yield a significant increase in apoptosis, whereas serum deprivation alone led to a significant demise of cardiomyoblasts. This effect was exacerbated upon the combination of serum deprivation and hypoxia. Silencing of Sirt3 increased apoptosis and diminished mitochondrial function upon serum deprivation under normoxic conditions. However, upon additional hypoxia, during simulated ischemia, both of these Sirt3-dependent effects were abolished. Thus, Sirt3 protects from stress-induced mitochondrial dysfunction and consecutive cell death only in the presence of oxygen. Under hypoxic conditions, when mitochondrial respiration can no longer take place, Sirt3 loses its protective capacities. We therefore postulate that oxidative phosphorylation is a prerequisite for Sirt3-mediated myocardial protection.
These findings may explain Sirt3-dependent protection in models, in which oxidative phosphorylation is maintained, such as chronic heart failure. i.e., regional myocardial function of non-ischemic myocardium was preserved upon resveratrol supplementation in a swine model of metabolic syndrome and ameroid constrictor placement on the proximal left circumflex coronary artery . Likewise, ischemia–reperfusion injury, a phenomenon depending on viable myocardium in the ischemic border zone  may yield a Sirt3-mediated protection, while in others, large ischemia zones may obscure Sirt3-dependent effects in surrounding non-ischemic myocardium [16, 17].
The present study needs to be interpreted in light of the following limitations: To allow for strict control of available oxygen and nutrient supply—the key elements in an ischemia setting—we chose an in vitro approach, limiting direct extrapolation to in vivo settings. To maximize the stability of culture conditions, we used immortalized cardiomyoblasts (H9c2) that were specifically chosen for their functional similarity to primary cardiomyocytes, particularly with regard to energy metabolism, mitochondrial function and sensitivity to hypoxia [18, 19]. Therefore, extrapolation to primary, post-mitotic cardiomyocytes is limited. Moreover, mitochondrial respiration was assessed based on initially generated data on the role of Sirt3 in apoptosis, where the conditions of serum deprivation and “simulated ischemia” (i.e. hypoxia and serum deprivation) were found to be central for the role of Sirt3. Thus, the role of Sirt3 in mitochondrial at hypoxia only or normal growth conditions was not assessed. Though our data are highly suggestive of a differential, oxygen-dependent role of Sirt3 in mitochondrial respiration, no significant interaction between the effects of growth conditions (simulated ischemia vs. serum deprivation) and the presence or absence of Sirt3 on direct mitochondrial oxygen consumption was observed—a drawback that we attribute to the limited size of this proof-of-concept study. In addition, the data presented in this study are based on loss-of-function approaches. Further studies are needed to confirm these results in gain-of-function settings.
Our data suggest a differential role of Sirt3 in myocardial protection from ischemia. Under normoxic conditions, Sirt3 maintains mitochondrial respiration at times of myocardial “caloric restriction”, thus, likely protecting from myocardial demise. Under hypoxic conditions, Sirt3 fails to protect from mitochondrial dysfunction and cell death. Nonetheless, Sirt3-dependent protection in the surrounding non-ischemic myocardium may compensate for ischemic myocardial damage. We therefore postulate, that mitochondrial respiration takes center-stage in Sirt3-mediated myocardial protection, which may provide a potential explanation for the controversial results of previous in vivo studies.
Taken together, in addition to beneficial Sirt3-mediated non-myocardial effects, such as maintaining metabolic flexibility, delaying the onset of the metabolic syndrome, and protecting from oxidative stress, Sirt3 supports myocardial mitochondrial function at times of increased myocardial stress under non-hypoxic conditions.
Future studies in post-mitotic primary cardiomyocytes are needed to confirm the hypotheses put forward in the current proof-of-concept study. Genetic gain-of-function approaches may set the stage for pharmacological manipulation of myocardial Sirt3 in different, clinically relevant settings, such as heart failure.
This work was funded by the National Health and Medical Research Council (NHMRC), the Victorian Government’s Operational Infrastructure Support Program, the Swiss National Science Foundation (310030, 146923), Matching Funds at the University of Zurich and the Zurich Heart House, Zurich, Switzerland. PD was supported by the German Research Foundation. JS was supported by the German Society of Cardiology. KP is a Principal Research Fellow of the NHMRC.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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