The effect of oxygen in Sirt3-mediated myocardial protection: a proof-of-concept study in cultured cardiomyoblasts

  • Philipp Diehl
  • Daniel S. Gaul
  • Jonas Sogl
  • Ulrike Flierl
  • Darren Henstridge
  • Juergen Pahla
  • Heiko Bugger
  • Maximilian Y. Emmert
  • Frank Ruschitzka
  • Christoph Bode
  • Thomas F. Lüscher
  • Martin Moser
  • Christian M. Matter
  • Karlheinz Peter
  • Stephan Winnik
Article
  • 6 Downloads

Abstract

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.

Keywords

Sirt3 Cardiomyoblasts Ischemia Oxygen Mitochondrial function 

Highlights

  • 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

Introduction

Ischemic heart disease remains the leading cause of mortality worldwide [1]. Myocardial ischemia is associated with myocardial damage, culminating in the development of heart failure [2]. 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 [3]. Mitochondrial dysfunction has been associated with chronic heart disease and cardiomyopathies [4].

Sirtuin 3 (Sirt3) is a nuclear-encoded, mitochondrial deacetylase that orchestrates mitochondrial metabolism and oxidative defense [5]. 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 [8]. Concordantly, myocardial adenosine triphosphate (ATP) levels in Sirt3 knockout mice are diminished to < 50% of those in wildtype mice [8]. 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 [12]. Left ventricular ischemia following coronary artery ligation was associated with reduced Sirt3 expression in ischemic compared with non-ischemic myocardium [13]. 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 [14].

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 [15], recovery of cardiac function, following myocardial ischemia–reperfusion in perfused isolated hearts, was unaltered compared to wildtype controls [16]. 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 [17].

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].

Methods

Cell culture

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

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.

SDS-PAGE

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).

Flow cytometry

Cell death was quantified by propodium iodide (PI, Invitrogen™, USA) staining (0.125 µg/ml) as described [22]. 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.

Live-cell imaging

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

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.

Statistics

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.

Results

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).

Fig. 1

Serum deprivation is a stronger trigger of apoptosis in H9c2 cells than hypoxia. H9c2 cells were subjected to 8 h of serum deprivation and/or hypoxia, the combination of which is referred to as simulated ischemia. Cell viability was assessed using PI staining in FACS analyses. Representative scatter plots are shown. Red gate: viable cells; green gate: early apoptotic cells; black gate: apoptotic cells. a Normal growth conditions (control). b Serum deprivation. c Hypoxia. d Hypoxia and serum deprivation (“simulated ischemia”). eh Quantification of % counted events. *p < 0.5, **p < 0.01, ***p < 0.001

Fig. 2

Serum deprivation and simulated ischemia trigger apoptosis of H9c2 cells. H9c2 cells were subjected to 8 h of serum deprivation and/or hypoxia before being stained Annexin V (green) and propidium iodide (PI, red); nuclei were counter-stained with Hoechst (blue). a Phase contrast, normal growth conditions. b Phase contrast, serum deprivation. c Phase contrast, hypoxia and serum deprivation (“simulated ischemia”). Arrows indicate morphologic sings of apoptosis, i.e. membrane blebbing. d Fluorescence imaging, normal growth conditions, Hoechst staining (nuclei), Annexin V (green), and PI staining (red). e Fluorescence imaging, serum deprivation, Hoechst staining (nuclei), Annexin V (green), and PI staining (red). f Fluorescence imaging, serum deprivation and hypoxia (“simulated ischemia”), Hoechst staining (nuclei), Annexin V, and PI staining. gi Merge of phase contrast and fluorescence imaging of above described conditions. Representative micrographs are shown. Scale bars: 30 µm

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).

Fig. 3

Sirt3 deficiency increases early apoptotic damage during serum deprivation. H9c2 cells were transfected with Sirt3 siRNA (siSirt3) or scrambled siRNA (scr) (a, b). Expression analyses using a quantitative PCR, and b Western blot analysis. ch Flow cytometry analyses for propidium iodide (PI) of siSirt3 or scr-transfected H9c2 cells following 8 h of serum deprivation and/or hypoxia. Representative scatter plots are shown. Red gate: viable cells; green gate: early apoptotic cells; black gate: apoptotic cells. a Serum deprivation after transfection with scrambled siRNA (scr). b Serum deprivation after transfection with Sirt3 siRNA (siSirt3). c Serum deprivation and hypoxia (“simulated ischemia”) after transfection with scrambled siRNA (scr). d Serum deprivation and hypoxia (“simulated ischemia”) after transfection with Sirt3 siRNA (siSirt3). ef Quantification of percent gated events. *p < 0.05, **p < 0.01, ****p < 0.0001

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 [23] 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.

Fig. 4

Sirt3 silencing impairs mitochondrial function during serum deprivation but not upon simulated ischemia. Direct assessment of oxygen consumption rates of H9c2 cells during normal growth conditions, serum deprivation and/or hypoxia, using extracellular flux analysis. a Schematic overview of the experimental setup. b Exemplary effects of normal growth conditions and serum deprivation on oxygen consumption in H9c2 cardiomyoblasts. c Basal respiration, ATP turnover, spare respiratory capacity, and maximal respiration upon knockdown of Sirt3 (siSirt3) or scrambled controls (scr) during serum deprivation. d As in c upon serum deprivation and hypoxia (“simulated ischemia”). *p < 0.05. p interaction (basal respiration) = 0.15, p interaction (ATP turnover) = 0.15, p interaction (spare respiratory capacity) = 0.20, p interaction (maximal mitochondrial respiration) = 0.10

Discussion

Principal findings

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 [9]. 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 [24]. In addition to anti-oxidative and anti-apoptotic effects [10] 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 [25]. 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 [26]. 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 [15] recovery of cardiac function following experimental ischemia was unaltered [16]. Yet, heterozygous constitutive deletion of Sirt3 was reported to be associated with an exacerbation of ischemia–reperfusion injury and increased infarct size [27]. 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 [21] 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 [30]. Likewise, ischemia–reperfusion injury, a phenomenon depending on viable myocardium in the ischemic border zone [29] may yield a Sirt3-mediated protection, while in others, large ischemia zones may obscure Sirt3-dependent effects in surrounding non-ischemic myocardium [16, 17].

Limitations

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.

Conclusion

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.

Outlook

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.

Notes

Funding

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.

Supplementary material

11239_2018_1677_MOESM1_ESM.docx (282 kb)
Supplementary material 1 (DOCX 282 KB)

References

  1. 1.
    Moran AE, Oliver JT, Mirzaie M, Forouzanfar MH, Chilov M, Anderson L, Morrison JL, Khan A, Zhang N, Haynes N, Tran J, Murphy A, Degennaro V, Roth G, Zhao D, Peer N, Pichon-Riviere A, Rubinstein A, Pogosova N, Prabhakaran D, Naghavi M, Ezzati M, Mensah GA (2012) Assessing the global burden of ischemic heart disease: part 1: methods for a systematic review of the global epidemiology of ischemic heart disease in 1990 and 2010. Glob Heart 7(4):315–329.  https://doi.org/10.1016/j.gheart.2012.10.004 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ziaeian B, Fonarow GC (2016) Epidemiology and aetiology of heart failure. Nat Rev Cardiol 13(6):368–378.  https://doi.org/10.1038/nrcardio.2016.25 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Roes SD, Kelle S, Kaandorp TA, Kokocinski T, Poldermans D, Lamb HJ, Boersma E, van der Wall EE, Fleck E, de Roos A, Nagel E, Bax JJ (2007) Comparison of myocardial infarct size assessed with contrast-enhanced magnetic resonance imaging and left ventricular function and volumes to predict mortality in patients with healed myocardial infarction. Am J Cardiol 100(6):930–936.  https://doi.org/10.1016/j.amjcard.2007.04.029 CrossRefPubMedGoogle Scholar
  4. 4.
    Wallace DC (2000) Mitochondrial defects in cardiomyopathy and neuromuscular disease. Am Heart J 139(2 Pt 3):S70–S85Google Scholar
  5. 5.
    Winnik S, Auwerx J, Sinclair DA, Matter CM (2015) Protective effects of sirtuins in cardiovascular diseases: from bench to bedside. Eur Heart J 36(48):3404–3412.  https://doi.org/10.1093/eurheartj/ehv290 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Chalkiadaki A, Guarente L (2012) Sirtuins mediate mammalian metabolic responses to nutrient availability. Nat Rev Endocrinol 8(5):287–296.  https://doi.org/10.1038/nrendo.2011.225 CrossRefPubMedGoogle Scholar
  7. 7.
    Houtkooper RH, Pirinen E, Auwerx J (2012) Sirtuins as regulators of metabolism and healthspan. Nat Rev Mol Cell Biol 13(4):225–238.  https://doi.org/10.1038/nrm3293 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ahn BH, Kim HS, Song S, Lee IH, Liu J, Vassilopoulos A, Deng CX, Finkel T (2008) A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis. Proc Natl Acad Sci USA 105(38):14447–14452.  https://doi.org/10.1073/pnas.0803790105 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Sundaresan NR, Samant SA, Pillai VB, Rajamohan SB, Gupta MP (2008) SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70. Mol Cell Biol 28(20):6384–6401.  https://doi.org/10.1128/MCB.00426-08 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Chen CJ, Fu YC, Yu W, Wang W (2013) SIRT3 protects cardiomyocytes from oxidative stress-mediated cell death by activating NF-kappaB. Biochem Biophys Res Commun 430(2):798–803.  https://doi.org/10.1016/j.bbrc.2012.11.066 CrossRefPubMedGoogle Scholar
  11. 11.
    Cheung KG, Cole LK, Xiang B, Chen K, Ma X, Myal Y, Hatch GM, Tong Q, Dolinsky VW (2015) Sirtuin-3 (SIRT3) protein attenuates doxorubicin-induced oxidative stress and improves mitochondrial respiration in H9c2 cardiomyocytes. J Biol Chem 290(17):10981–10993.  https://doi.org/10.1074/jbc.M114.607960 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Zeng H, Vaka VR, He X, Booz GW, Chen JX (2015) High-fat diet induces cardiac remodelling and dysfunction: assessment of the role played by SIRT3 loss. J Cell Mol Med 19(8):1847–1856.  https://doi.org/10.1111/jcmm.12556 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Klishadi MS, Zarei F, Hejazian SH, Moradi A, Hemati M, Safari F (2015) Losartan protects the heart against ischemia reperfusion injury: sirtuin3 involvement. J Pharm Pharm Sci 18(1):112–123CrossRefPubMedGoogle Scholar
  14. 14.
    Zeng H, Li L, Chen JX (2014) Loss of Sirt3 limits bone marrow cell-mediated angiogenesis and cardiac repair in post-myocardial infarction. PLoS ONE 9(9):e107011.  https://doi.org/10.1371/journal.pone.0107011 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Koentges C, Pfeil K, Schnick T, Wiese S, Dahlbock R, Cimolai MC, Meyer-Steenbuck M, Cenkerova K, Hoffmann MM, Jaeger C, Odening KE, Kammerer B, Hein L, Bode C, Bugger H (2015) SIRT3 deficiency impairs mitochondrial and contractile function in the heart. Basic Res Cardiol 110(4):36.  https://doi.org/10.1007/s00395-015-0493-6 CrossRefPubMedGoogle Scholar
  16. 16.
    Koentges C, Pfeil K, Meyer-Steenbuck M, Lother A, Hoffmann MM, Odening KE, Hein L, Bode C, Bugger H (2016) Preserved recovery of cardiac function following ischemia-reperfusion in mice lacking SIRT3. Can J Physiol Pharmacol 94(1):72–80.  https://doi.org/10.1139/cjpp-2015-0152 CrossRefPubMedGoogle Scholar
  17. 17.
    Bochaton T, Crola-Da-Silva C, Pillot B, Villedieu C, Ferreras L, Alam MR, Thibault H, Strina M, Gharib A, Ovize M, Baetz D (2015) Inhibition of myocardial reperfusion injury by ischemic postconditioning requires sirtuin 3-mediated deacetylation of cyclophilin D. J Mol Cell Cardiol 84:61–69.  https://doi.org/10.1016/j.yjmcc.2015.03.017 CrossRefPubMedGoogle Scholar
  18. 18.
    Kuznetsov AV, Javadov S, Sickinger S, Frotschnig S, Grimm M (2015) H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia-reoxygenation. Biochim Biophys Acta 1853(2):276–284.  https://doi.org/10.1016/j.bbamcr.2014.11.015 CrossRefPubMedGoogle Scholar
  19. 19.
    Watkins SJ, Borthwick GM, Arthur HM (2011) The H9C2 cell line and primary neonatal cardiomyocyte cells show similar hypertrophic responses in vitro. In Vitro Cell Dev Biol Anim 47(2):125–131.  https://doi.org/10.1007/s11626-010-9368-1 CrossRefPubMedGoogle Scholar
  20. 20.
    Hescheler J, Meyer R, Plant S, Krautwurst D, Rosenthal W, Schultz G (1991) Morphological, biochemical, and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ Res 69(6):1476–1486CrossRefPubMedGoogle Scholar
  21. 21.
    Bonavita F, Stefanelli C, Giordano E, Columbaro M, Facchini A, Bonafe F, Caldarera CM, Guarnieri C (2003) H9c2 cardiac myoblasts undergo apoptosis in a model of ischemia consisting of serum deprivation and hypoxia: inhibition by PMA. FEBS Lett 536(1–3):85–91CrossRefPubMedGoogle Scholar
  22. 22.
    Walsh GM, Dewson G, Wardlaw AJ, Levi-Schaffer F, Moqbel R (1998) A comparative study of different methods for the assessment of apoptosis and necrosis in human eosinophils. J Immunol Methods 217(1–2):153–163CrossRefPubMedGoogle Scholar
  23. 23.
    Charles I, Khalyfa A, Kumar DM, Krishnamoorthy RR, Roque RS, Cooper N, Agarwal N (2005) Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. Invest Ophthalmol Vis Sci 46(4):1330–1338.  https://doi.org/10.1167/iovs.04-0363 CrossRefPubMedGoogle Scholar
  24. 24.
    Sundaresan NR, Gupta M, Kim G, Rajamohan SB, Isbatan A, Gupta MP (2009) Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice. J Clin Invest 119(9):2758–2771.  https://doi.org/10.1172/JCI39162 PubMedPubMedCentralGoogle Scholar
  25. 25.
    Hirschey MD, Shimazu T, Goetzman E, Jing E, Schwer B, Lombard DB, Grueter CA, Harris C, Biddinger S, Ilkayeva OR, Stevens RD, Li Y, Saha AK, Ruderman NB, Bain JR, Newgard CB, Farese RV Jr, Alt FW, Kahn CR, Verdin E (2010) SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature 464(7285):121–125.  https://doi.org/10.1038/nature08778 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Stride N, Larsen S, Hey-Mogensen M, Sander K, Lund JT, Gustafsson F, Kober L, Dela F (2013) Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction. Eur J Heart Fail 15(2):150–157.  https://doi.org/10.1093/eurjhf/hfs172 CrossRefPubMedGoogle Scholar
  27. 27.
    Porter GA, Urciuoli WR, Brookes PS, Nadtochiy SM (2014) SIRT3 deficiency exacerbates ischemia-reperfusion injury: implication for aged hearts. Am J Physiol Heart Circ Physiol 306(12):H1602–H1609.  https://doi.org/10.1152/ajpheart.00027.2014 CrossRefGoogle Scholar
  28. 28.
    Piper HM, Garcia-Dorado D, Ovize M (1998) A fresh look at reperfusion injury. Cardiovasc Res 38(2):291–300CrossRefPubMedGoogle Scholar
  29. 29.
    Argaud L, Gateau-Roesch O, Raisky O, Loufouat J, Robert D, Ovize M (2005) Postconditioning inhibits mitochondrial permeability transition. Circulation 111(2):194–197.  https://doi.org/10.1161/01.CIR.0000151290.04952.3B CrossRefPubMedGoogle Scholar
  30. 30.
    Robich MP, Chu LM, Burgess TA, Feng J, Han Y, Nezafat R, Leber MP, Laham RJ, Manning WJ, Sellke FW (2012) Resveratrol preserves myocardial function and perfusion in remote nonischemic myocardium in a swine model of metabolic syndrome. J Am Coll Surg 215(5):681–689.  https://doi.org/10.1016/j.jamcollsurg.2012.06.417 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Philipp Diehl
    • 1
    • 2
  • Daniel S. Gaul
    • 3
  • Jonas Sogl
    • 1
    • 2
  • Ulrike Flierl
    • 2
    • 4
  • Darren Henstridge
    • 2
  • Juergen Pahla
    • 5
  • Heiko Bugger
    • 1
  • Maximilian Y. Emmert
    • 6
  • Frank Ruschitzka
    • 3
  • Christoph Bode
    • 1
  • Thomas F. Lüscher
    • 3
    • 5
    • 7
  • Martin Moser
    • 1
  • Christian M. Matter
    • 3
    • 6
    • 7
  • Karlheinz Peter
    • 1
    • 2
  • Stephan Winnik
    • 3
    • 5
  1. 1.Department of Cardiology & Angiology I, Heart Center Freiburg University, Faculty of MedicineUniversity of FreiburgFreiburgGermany
  2. 2.Baker IDI Heart and Diabetes InstituteMelbourneAustralia
  3. 3.Department of Cardiology, University Heart Center ZurichUniversity Hospital ZurichZurichSwitzerland
  4. 4.Hannover Medical SchoolHanoverGermany
  5. 5.Center for Molecular CardiologyUniversity of ZurichZurichSwitzerland
  6. 6.Swiss Center for Regenerative MedicineUniversity of ZurichZurichSwitzerland
  7. 7.Zurich Center for Human Integrative Physiology (ZHIP)University of ZurichZurichSwitzerland

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