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Published in final edited form as: Int J Cardiol. 2015 Mar 25;187:393–400. doi: 10.1016/j.ijcard.2015.03.353

Estrogen-Related Receptor α (ERRα) is required for adaptive increases in PGC-1 isoform expression during electrically stimulated contraction of adult cardiomyocytes in sustained hypoxic conditions☆☆

Kathryn F Cunningham a, Gyda C Beeson b, Craig C Beeson b, Catalin F Baicu a, Michael R Zile a,c, Paul J McDermott a,c,*
PMCID: PMC12912148  NIHMSID: NIHMS2138248  PMID: 25841134

Abstract

Background and objectives:

In adult myocardium, Estrogen-Related Receptor α (ERRα) programs energetic capacity of cardiomyocytes by regulating expression of target genes required for mitochondrial biogenesis, fatty acid metabolism and oxidative phosphorylation. Transcriptional activation by ERRα is dependent on the α or β isoform of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1). This study utilized a model of continuously contracting adult cardiomyocytes to determine the effects of sustained oxygen reduction (hypoxia) on ERRα target gene expression.

Methods and results:

Adult feline cardiomyocytes in primary culture were electrically stimulated to contract at 1 Hz in either normoxia (21% O2) or hypoxia (0.5% O2). Compared to normoxia, hypoxia increased PGC-1α mRNA and PGC-1β mRNA levels by 16-fold and 14-fold after 24 h. ERRα mRNA levels were increased 3-fold by hypoxia over the same time period. Treatment of cardiomyocytes with XCT-790, an ERRα inverse agonist, caused knockdown of ERRα protein expression. The increases in PGC-1 mRNA levels in response to hypoxia were blocked by XCT-790 treatment, which indicates that expression of PGC-1 isoforms is dependent on ERRα activity. The products of two ERRα target genes required for energy metabolism, Cox6c mRNA and Fabp3 mRNA, increased by 4.5-fold and 3.5 fold after 24 h of hypoxia as compared to normoxic controls. These increases were blocked by XCT-790 treatment of hypoxic cardiomyocytes with a concomitant decrease in ERRα expression.

Conclusions:

ERRα activity is required to increase expression of PGC-1 isoforms and downstream target genes as part of the adaptive response of contracting adult cardiomyocytes to sustained hypoxia.

Keywords: Contraction, Cardiomyocyte, Hypoxia, Estrogen-Related Receptor, Peroxisome Proliferator-Activated Receptor, γ Coactivator-1

1. Introduction

The high energy demands of the myocardium are met largely by mitochondrial respiration in which oxidative phosphorylation serves as the primary mechanism for ATP synthesis [1]. Under normal physiological conditions, oxidative phosphorylation is sufficient to maintain a constant supply of ATP despite the high rate of ATP turnover in the cardiomyocyte. However, when ATP turnover rate exceeds oxidative capacity, cardiomyocytes synthesize ATP from additional energy reserves in order to preserve high phosphorylation potential. For example, the phosphocreatine pool is utilized by creatine kinase to catalyze ATP synthesis and reduce cellular ADP levels, and this reaction occurs at a much faster rate than oxidative phosphorylation [2]. Metabolic stresses that either inhibit ATP synthesis or accelerate the rate of ATP turnover will diminish energy reserves and cause the AMP:ATP ratio to rise via adenylate kinase, an enzyme that uses ADP as a substrate to produce ATP and AMP [3]. The resultant increase in AMP:ATP ratio serves as an energy sensor that enables cardiomyocytes to adapt quickly by activation of AMP kinase (AMPK), which results in stimulation of glucose uptake, glycolysis and fatty acid oxidation [4–6]. Longer term metabolic adaptations involve genetic reprogramming to increase glycolytic capacity in the cardiomyocyte such that ATP production (O2 consumed/ATP produced) becomes more efficient [7].

Estrogen-Related Receptors (ERRs) are classified as orphan nuclear receptors that determine metabolic phenotype through their role as transcription factors. Three ERR isoforms (α, β and γ) regulate a large array of target genes involved in glycolysis, β-oxidation of fatty acids, TCA cycle, oxidative phosphorylation and mitochondrial biogenesis [8]. Although ERR isoforms exhibit sequence similarity to estrogen receptors (ERs), their activation is not dependent on binding to estradiol [9]. Crystallography studies indicate that ERRs adopt a conformation that binds to DNA in the absence of ligand, and thus, are not regulated by ligand availability [10]. ERR isoforms bind as monomers or dimers to consensus ERR response elements (ERREs) in target genes, but they are capable of binding to other consensus sequences such as the ER response element (ERE) [8,11,12]. The coactivator Peroxisome Proliferator-Activated Receptor γ Coactivator-1α (PGC-1α), which functions as a master regulator of metabolic genes, controls expression and activity of ERR isoforms through direct protein:protein interactions [12].

The ERRα isoform is highly expressed in tissues that require large amounts of energy and can be induced in response to stimuli that further increase energy demand such as cold-induced browning of adipose tissue, exercised skeletal muscle, and liver and kidney tissue of fasting animals [13–16]. Cardiac expression of ERRα begins perinatally and parallels that of PGC-1α [17]. Studies in rodent cardiomyocytes have identified a subset of ERRα target genes that are involved in cellular fatty acid transport, mitochondrial and peroxisomal fatty acid oxidation, and mitochondrial respiration [18]. In mice subjected to chronic pressure overload in vivo, expression of ERRα is reduced significantly in the myocardium coincident with decreased left ventricular ejection fraction, increased pulmonary congestion and dysregulation of several genes involved in energy metabolism [19]. In homozygous ERRα knockout mice (ERRα−/−), there is a small reduction in left ventricular mass, but contractile function remains normal. However, ERRα−/− mice subjected to chronic pressure overload exhibit abnormal depletion of phosphocreatine and a reduction in maximal ATP synthesis rates, culminating in a rapid transition to a heart failure phenotype [20]. Lastly, there is a significant correlation between altered expression of ERRα/PGC-1α target genes and diminished left ventricular ejection fraction in human heart failure [21].

In this study, the role of ERRα in regulating expression of genes that determine the metabolic phenotype of cardiomyocytes was investigated during sustained hypoxia. An adult feline cardiomyocyte model of continuous electrically stimulated contraction was utilized to examine time-dependent effects of hypoxia and reoxygenation on ERRα-dependent gene expression. Here we demonstrate that sustained hypoxia increases expression of α and β isoforms of PGC-1 mRNA in adult cardiomyocytes and that ERRα activity is required to produce this adaptive response.

2. Material and methods

2.1. Adult feline cardiomyocyte model of hypoxia

The investigation conforms to the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85–23, revised 1996) and was approved by the Animal Care and Use Committee of the Medical University of South Carolina. Cats were anesthetized with 10–20 mg/kg ketamine, im.; 0.1–0.33 mg/kg acepromazine, im; 2.2 mg/kg meperidine, im., and then placed on isoflurane anesthesia (1–3% in 100% oxygen at 1–1.5 L/min) delivered via nose cone. A ketamine overdose of 20 mg/kg was administered iv. just prior to removal of the heart, a left thoracotomy was performed and euthanasia occurred via exsanguination.

Adult feline cardiomyocytes were isolated from the left ventricular myocardium by a combination of collagenase digestion and mechanical agitation as described previously [22]. The cardiomyocytes were plated onto 4-well culture trays coated with laminin at an initial plating density of 1.25 × 104 cells/cm2 and incubated overnight in serum-free media [23]. Adult feline cardiomyocytes do not contract spontaneously in primary culture and remain in a quiescent state. To initiate contractile activity, cardiomyocytes were electrically stimulated at a frequency of 1 Hz by delivering 5 msec pulses of alternating polarity through the culture medium via carbon electrodes [23]. The voltage was set at a value approximately 10% above the threshold for contraction (120–130 V). Non-stimulated cardiomyocytes were used as quiescent controls. Hypoxia was produced using compressed gas (5% CO2, balanced N2) and a Proox (BioSpherix, Ltd., Lacona, NY, USA) oxygen control system with an incubation chamber that was retrofitted for continuous electrical stimulation of cardiomyocyte contraction (Supplementary Fig. 1). Quiescent and contracting cardiomyocytes were maintained under either normoxic conditions (21% O2) or hypoxic conditions (0.5% O2).

2.2. Oxygen consumption rates in contracting cardiomyocytes

Oxygen consumption rates (OCRs) in cardiomyocytes were measured using a Seahorse Bioscience XF24 analyzer with laminin-coated V28 cell culture microplates (Seahorse Bioscience, North Billerica, MA, USA). To measure OCR in contracting cardiomyocytes, the microplates were modified for electrical stimulation by inserting platinum electrodes in a subset of the wells. Prior to analysis, the cell medium was supplemented with 1% serum and buffered by adding HEPES to a final concentration of 10 mM. A measurement cycle consisted of 1 min periods each of mix and incubation followed by a 2 min measurement period. In a typical experiment, the first 3 measurement cycles were made in quiescent cardiomyocytes to obtain basal OCR, followed by 3 cycles in which the cardiomyocytes were electrically stimulated to contract. Subsequently, 6 measurement cycles were done after the electrical stimulator was turned off. Maximal OCR was measured following injection of 20 μM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) dissolved in cell media. OCR was calculated from the continuous slope of O2 decrease using a compartmentalization model that accounts for O2 partitioning between plastic, atmosphere, and cellular uptake [24]. For each experimental group, the OCR measurements were made in 6–8 individual wells. Measurements of OCR in quiescent and contracting cardiomyocytes were averaged over 3 cycles. Maximal OCR was measured in the first cycle after FCCP injection.

2.3. Quantification of cardiomyocyte RNA levels

Total RNA was extracted using Trizol Reagent (Ambion, Carlsbad, CA, USA) and precipitated in ethanol. The RNA samples were resuspended in H2O and treated with DNase I prior to quantitation of mRNA levels using a one-step reverse transcription-PCR kit (QuantiTect SYBR Green RT-PCR Kit, Qiagen Sciences, MD, USA) and either a CFX96 Real-Time System C1000 Thermal Cycler or iCycler (BioRad, Hercules, CA, USA). Samples were analyzed in triplicate using the gene-specific primer sets (Supplementary Table). The amount of each specific mRNA in individual samples was extrapolated from a standard curve using a dilution series derived from a sample of total cardiomyocyte RNA. The relative level of mRNA was calculated by normalizing to the corresponding amount of 18S rRNA in each sample.

2.4. Isolation of monosome and polysome fractions

Monosomes (mRNPs and ribosome subunits) and polysomes were isolated by fractionation of cardiomyocyte homogenates on 15% to 50% (wt./vol.) linear sucrose gradients as described before [25]. RNA was extracted from individual gradient fractions by the Trizol method. The amount of each specific mRNA was quantified by real-time RT-PCR and divided by 18S rRNA to normalize for ribosome recovery.

2.5. Analysis of ERRα protein expression

Aliquots of total cardiomyocyte protein were electrophoresed using NuPAGE 4–12% Bis-Tris gels (Novex, Carlsbad, CA, USA) followed by Western blotting as described previously [26]. The primary antibodies were ERRα (Millipore 04–1134) and β-actin (Abcam ab8227). After 1 h incubation with secondary antibody (Vector Laboratories, Burlingame, CA, USA), membranes were treated with a chemiluminescence HRP substrate (PerkinElmer, Waltham, MA, USA) and the bands detected by autoradiography. The films were scanned and the optical densities of the bands were quantified by digital image analysis using ImageJ analysis software.

2.6. Statistical analysis

Statistical analyses were performed using SigmaStat software version 3.5 (San Jose, CA, USA). Gene expression at multiple time points was evaluated using a one-way ANOVA. Gene expression at multiple time points comparing DMSO and XCT-790-treated samples was evaluated using a two-way ANOVA. Expression data for ERRα, PGC-1α, PGC-1β, Cox6c and VEGF treated with DMSO or XCT-790 failed the normality test. Therefore, these data underwent a log transformation prior to two-way ANOVA analysis. Post-hoc multiple pairwise comparisons were made via the Holm–Sidak method. Values for fold change in mRNA levels in Figs. 2 and 4 were compared to normoxia controls using the Wilcoxon signed-rank test for paired data. A p value of 0.05 was considered to be a statistically significant difference between means.

Fig. 2.

Fig. 2.

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Effects of hypoxia on gene expression in quiescent and contracting cardiomyocytes. Changes in relative abundance after 24 h of hypoxia or normoxia were calculated by measuring the amount of each mRNA by real-time RT-PCR and dividing by the corresponding amount of 18S rRNA in the same sample. Norm-Q: normoxia-quiescent; Norm-ES: normoxia-electrically stimulated contraction; Hypox-Q: hypoxia-quiescent; Hypox-ES: hypoxia-electrically stimulated contraction. The fold change in abundance of each mRNA was calculated relative to quiescent cardiomyocytes in normoxia. Values are the mean + SE, n = 8 experiments for PGC-1α, ERRα and VEGF or n = 6 experiments for MHC and GAPDH; *p < 0.01, #p < 0.05 compared to the normoxia quiescent group. Each experiment was done using a different isolation of adult feline cardiomyocytes.

Fig. 4.

Fig. 4.

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Effects of hypoxia and reoxygenation on distribution of mRNAs in contracting cardiomyocytes. NORMOX: normoxia; HYPOX: 24 h of hypoxia; REOX-30: 24 h of hypoxia followed by 30 min of reoxygenation; REOX-120: 24 h of hypoxia followed by 120 min of reoxygenation. Each of the indicated mRNAs in monosome and polysome fractions were quantified by real-time RT-PCR and divided by the corresponding amount of 18S rRNA in each fraction to correct for ribosome recovery. Values are expressed as the fold change relative to contracting cardiomyocytes in normoxia; mean + SE, n = 5 or more experiments using different isolations of adult feline cardiomyocytes. *p < 0.05 versus normoxia group.

3. Results

Adult feline cardiomyocytes in primary culture can be electrically stimulated to contract by delivering 5 msec square wave electrical pulses of alternating polarity through the culture medium [23]. This system was utilized to examine how continuously contracting cardiomyocytes adapt to hypoxia and reoxygenation by measuring changes in expression of genes involved in cardiac energy metabolism. After placement of cardiomyocytes in the hypoxia chamber, real-time measurements confirmed that pO2 decreased in the culture medium from approximately 120 mm Hg to 60 mm Hg within 60 min of incubation in 0.5% O2, and that reduced pO2 was sustained over 24 h (Supplementary Fig. 2). When these experiments were repeated at 0.1% O2, pO2 of the culture medium remained at 60 mm Hg and did not drop any lower over 24 h. Contractile function was preserved as there were no significant differences in fractional shortening of cardiomyocytes maintained in hypoxic versus normoxic conditions (Supplementary Fig. 3).

Mitochondrial function was analyzed in both quiescent and contracting cardiomyocytes by modifying the electrical stimulation system to measure oxygen consumption rates (OCR) using a Seahorse Bioscience Extracellular Flux Analyzer (Fig. 1). In cardiomyocytes maintained in media containing a physiological KCl concentration of 4 mM, electrically stimulated contraction caused a rapid increase in OCR as compared to the quiescent (non-stimulated) control. Subsequently, when the electrical stimulator was turned off, cardiomyocytes became quiescent again and OCR returned to basal values. To verify that the increase in OCR in response to electrical stimulation was generated by contractile activity, a subset of cardiomyocytes were depolarized in 50 mM KCl to prevent excitation–contraction coupling. Basal OCR values in 50 mM KCl were higher as compared to cardiomyocytes in 4 mM KCl, an effect most likely caused by an increase in Na+/K+ ATPase activity [27]. Cardiomyocytes depolarized in 50 mM KCl did not contract in response to electrical stimulation as confirmed by visual observation and OCR did not increase. These results confirmed that the rise in OCR produced by electrical stimulation was due to increased ATP utilization required for contractility.

Fig. 1.

Fig. 1.

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Effects of contractile activity on oxygen consumption rates (OCRs) in cardiomyocytes. OCR was measured in cardiomyocytes that were either non-stimulated (Quiescent) or electrically stimulated to contract (Elec Stim). Experimental manipulations are labeled by the vertical lines. A: depolarization in 50 mM KCl; B: electrical stimulator turned on; C: electrical stimulator turned off; D: injection of mitochondrial uncoupling agent FCCP.

In Fig. 2, changes in cardiomyocyte gene expression in hypoxic conditions were examined by measuring mRNA levels relative to the quiescent, normoxic group. After 24 h of hypoxia, PGC-1α mRNA was increased by 5.2- and 11.2-fold in quiescent and contracting cardiomyocytes, respectively. Hypoxia also triggered a smaller, but significant increase in ERRα mRNA of 2-fold in contracting cardiomyocytes. Vascular endothelial growth factor (VEGF) mRNA levels increased approximately 20-fold in both quiescent and contracting cardiomyocytes in hypoxic conditions. Given that a robust increase in VEGF mRNA is a characteristic response to oxygen deprivation, these results confirmed that the cardiomyocytes were exposed to a sustained hypoxic environment. Fig. 2 further shows that constitutive expression of β-myosin heavy chain (MHC) mRNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA did not change significantly relative to cardiomyocytes in normoxia.

To further characterize the effects of hypoxia and reoxygenation on expression of PGC-1 and ERR isoforms, changes in mRNA levels were measured in contracting cardiomyocytes as a function of time. Fig. 3 shows that PGC-1α mRNA and PGC-1β mRNA levels rose in parallel and were increased by 16-fold and 14-fold after 24 of hypoxia, respectively. ERRα mRNA levels increased significantly by 3-fold after 24 of hypoxia, but not at earlier time points. Taken together, increases in PGC-1 isoforms were more robust and preceded any significant changes in ERRα expression. The ERRγ isoform, which is expressed abundantly in adult heart, functions coordinately with ERRα through formation of heterodimers and by sharing a common set of promoters in target genes [28,29]. In adult feline cardiomyocytes, relative levels of ERRγ mRNA were lower than ERRα mRNA. Fig. 3 further shows that ERRγ mRNA levels did not change significantly over 24 h of hypoxia.

Fig. 3.

Fig. 3.

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Time-dependent effects of hypoxia and reoxygenation on expression of ERR and PGC-1 mRNA isoforms in contracting cardiomyocytes. The shaded area starting at 24 h indicates the period of reoxygenation. Changes in relative abundance were calculated by measuring the amount of mRNA by real-time RT-PCR and dividing by the corresponding amount of 18S rRNA in the same sample. The fold change in abundance of each mRNA was calculated relative to contracting cardiomyocytes in normoxia (dotted line). Values are the mean ± SE, n = 5 or more experiments per time point. *p < 0.05 compared to contracting cardiomyocytes at the initial hypoxia time point, 4 h hypoxia. Each experiment was done using a different isolation of adult feline cardiomyocytes.

Reoxygenation was initiated after 24 h of hypoxia by returning the cardiomyocytes to normoxic conditions. Fig. 3 shows that expression of PGC-1 isoforms decreased and returned to baseline values by 8 h of reoxygenation. In contrast, the increase in ERRα mRNA levels was sustained over 24 h of reoxygenation. These findings suggest that ERRα mRNA either has a longer half-life than PGC-1 mRNA isoforms or that its stability is enhanced during hypoxia.

To determine whether hypoxia altered translational efficiency in cardiomyocytes, the distribution of PGC-1α mRNA and ERRα mRNA were measured in monosome and polysome fractions isolated on linear sucrose gradients (Fig. 4). Levels of each mRNA were normalized to 18S rRNA in the corresponding fraction to correct for differences in ribosome recovery. After 24 h of hypoxia, PGC-1α mRNA levels increased in both monosome and polysome fractions as compared to contracting cardiomyocytes in normoxia, and the magnitude of these increases were similar to PGC-1α mRNA measured in total cellular RNA. PGC-1α mRNA in both monosomes and polysomes remained elevated after 120 min of reoxygenation. Fig. 4 further shows that levels of ERRα mRNA and VEGF mRNA increased proportionally in monosome and polysome fractions during hypoxia and reoxygenation of contracting cardiomyocytes while MHC mRNA remained constant. The differential effects on ERRα mRNA, VEGF mRNA and MHC mRNA levels were consistent with the results obtained using total cellular RNA as measured after 24 h of hypoxia (compare Figs. 2 and 4). These data demonstrate that neither hypoxia nor reoxygenation altered the ability of the cardiomyocytes to initiate translation and thereby mobilize expanding pools of mRNA into polysomes for protein synthesis. Consistent with these findings, rates of total protein synthesis were maintained in contracting cardiomyocytes in hypoxia relative to controls in normoxia (Supplementary Fig. 4).

To determine whether ERRα activity is required for hypoxia-induced gene expression, contracting cardiomyocytes were treated with XCT-790, an inverse agonist with high specificity for the ERRα isoform [30]. XCT-790 treatment resulted in knockdown of ERRα mRNA (Fig. 5) and protein (Fig. 6) as compared to vehicle-treated controls. Fig. 5 further shows that time-dependent changes in PGC-1α and PGC-1β mRNA levels in response to hypoxia and reoxygenation were blocked by XCT-790, but not in vehicle-treated controls. These data indicate that ERRα was required to enhance expression of PGC-1 isoforms in hypoxic conditions. In contrast, XCT-790 treatment did not block the ability of hypoxia to increase in VEGF mRNA levels or the subsequent decrease after 2 h of reoxygenation. These findings differ from skeletal muscle cells in which stimulation of VEGF expression by hypoxia was partly dependent on coactivation of ERRα by PGC-1α [31]. Residual activity of the ERRγ isoform could provide an alternative pathway for hypoxia-induced increases in VEGF mRNA levels in cardiomyocytes treated with XCT-790 given that both ERRα and ERRγ bind to VEGF gene promoter [29].

Fig. 5.

Fig. 5.

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Effects of inhibiting ERRα activity on gene expression in contracting cardiomyocytes over 24 h hypoxia and 4 h reoxygenation. Cardiomyocytes were treated with either 10 μM XCT-790 or an equivalent volume of vehicle (DMSO). The shaded portion of the graphs indicates the period of reoxygenation. Changes in relative abundance were calculated by measuring the amount of each indicated mRNA and dividing by the corresponding amount of 18S rRNA in the same sample. Values are expressed as the fold change in mRNA abundance relative to contracting cardiomyocytes in normoxia (dotted line); mean ± SE, n = 5 or more experiments using different isolations of adult feline cardiomyocytes. *p < 0.05 compared to contracting cardiomyocytes at the initial hypoxia time point, 8 h hypoxia; #p < 0.05 compared to DMSO-treated control.

Fig. 6.

Fig. 6.

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Effects of inhibiting ERRα activity on ERRα protein levels. Contracting cardiomyocytes were treated with 10 μM XCT-790 (striped bars) or DMSO (solid bars) in normoxia (N), hypoxia (HYPOX) or hypoxia followed by reoxygenation (H/R). A representative Western blot is shown in the upper panel and summary data from multiple experiments are shown in lower graph. ERRα bands on the blot were quantified and normalized to the β-actin band as a loading control. Changes in ERRα expression were calculated relative to contracting cardiomyocytes in normoxia (dotted line). Values are the mean + SE, n = 4 or more experiments using different isolations of adult feline cardiomyocytes; #p < 0.05 compared to DMSO-treated control.

To verify the identity of the ERRα band detected by Western blotting in Fig. 6, an alternative approach was used to knockdown ERRα expression by infecting cardiomyocytes with Ad-GFP-ERRα-shRNA. The same 53 kD band detected by the ERRα antibody decreased over time in cardiomyocytes expressing ERRα-shRNA (Supplementary Fig. 5).

Previous studies in murine myocardium demonstrated that ERRα and ERRγ isoforms share a consensus ERRE in the promoter of target genes [29]. Two of these target genes, Cox6c and Fabp3, were used as endpoints of ERR activity in contracting cardiomyocytes by measuring changes in their corresponding mRNA levels in response to hypoxia. Cox6c encodes a component of the electron transport chain and Fabp3 encodes a protein involved in fatty acid uptake, metabolism and transport. Fig. 7 shows that levels of Cox6c mRNA and Fabp3 mRNA increased significantly in vehicle-treated controls after 24 h of hypoxia. These increases were blocked by treatment with XCT-790 to knockdown ERRα expression. These data indicate that ERRα activity was necessary, but not sufficient for the hypoxia-induced increases in expression of established target genes linked to energy metabolism in adult cardiomyocytes.

Fig. 7.

Fig. 7.

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Time-dependent effects of hypoxia and reoxygenation on expression of ERRα target genes in contracting cardiomyocytes. Cardiomyocytes were treated with either 10 μM XCT-790 or an equivalent volume of vehicle (DMSO). Changes in relative abundance were calculated by measuring the amount of Cox6c or Fabp3 mRNA by real-time RT-PCR and dividing by the corresponding amount of 18S rRNA in the same sample. Values are expressed as the fold change in mRNA abundance relative to contracting cardiomyocytes in normoxia (dotted line). Values are the mean ± SE, n = 5 or more experiments using different isolations of adult feline cardiomyocytes. *p < 0.05 compared to contracting cardiomyocytes at the initial hypoxia time point, 8 h hypoxia; #p < 0.05 compared to DMSO-treated control.

4. Discussion

Cardiomyocytes can be exposed to either intermittent or sustained hypoxia by multiple pathophysiological conditions of which the most prevalent are coronary artery occlusion that causes myocardial ischemia and pressure overload cardiac hypertrophy that triggers adverse myocardial remodeling with associated perfusion abnormalities [32–34]. The ability of cardiomyocytes to prevent cellular injury and ultimately survive is dependent on multiple variables such as the extent and duration of hypoxia and the deleterious effects produced during reoxygenation of the myocardium (ischemia/reperfusion) [35]. Pathological remodeling associated with heart failure development is characterized by an adaptive shift in myocardial energy metabolism from β-oxidation of fatty acids to glycolysis to produce ATP [33]. In particular, mitochondrial function in cardiomyocytes is impaired by down-regulation of ERR target genes required for fatty acid oxidation, electron transport and oxidative phosphorylation [20]. Thus, changes in PGC-1 expression that occur in adult cardiomyocytes during hypoxia are highly relevant to this shift in energy metabolism during adverse myocardial remodeling because expression of ERR target genes is dependent upon coactivation by PGC-1 isoforms [8].

This study is the first to examine the effects of sustained hypoxia in adult cardiomyocytes that were electrically stimulated to contract continuously. This model provides several advantages for determining how cardiomyocytes adapt to hypoxic conditions. First, as compared to prior studies that have utilized either primary, neonatal rat cardiomyocytes or H9c2 cardiomyoblasts, adult cardiomyocytes have greater oxidative capacity in order to meet energy requirements for excitation–contraction coupling [36]. Second, adult cardiomyocytes utilize fatty acids as their primary metabolic fuel source for ATP production, while neonatal cardiomyocytes are more dependent on glucose metabolism [36]. Third, similar to humans, adult feline cardiomyocytes predominantly express the β-myosin heavy chain isoform in the left ventricle [25,37]. The myosin ATPase activity of this isoform is slower than that of the α-myosin heavy chain isoform expressed in adult cardiomyocytes derived from rodent species, and it functions by generating active tension more efficiently with respect to ATP consumption [38]. Fourth, electrically stimulated contraction substantially increases energy demand in cardiomyocytes as measured by OCR. The added energy expenditure required for contraction was not taken into account in prior studies in which quiescent cardiomyocytes were utilized to determine the effects of hypoxia on ERRα and PGC-1 expression.

Adult cardiomyocytes adapted to sustained hypoxia by generating robust increases in PGC-1α and PGC-1β mRNA levels as measured over 24 h. Several lines of evidence support the conclusion that ERRα is necessary, but not sufficient to increase PGC-1 isoform expression in response to hypoxia. First, Fig. 3 shows that increases in PGC-1 mRNA levels preceded the much smaller increase in ERRα mRNA. Second, Fig. 6 shows that ERRα protein levels did not increase significantly over 24 h of hypoxia. Third, Fig. 5 shows that the hypoxia-induced increases in PGC-1 mRNA levels were blocked by treatment of cardiomyocytes with XCT-790, which suggests that ERRα functions as part of an autoregulatory feedback loop. In contrast to adult cardiomyocytes, PGC-1α mRNA levels in neonatal rat cardiomyocytes decreased significantly during the first 12 h of hypoxia as compared to controls in normoxia, followed by recovery in PGC-1α expression back to normoxic levels after 24 h [39]. This initial reduction in PGC-1α mRNA expression was preceded by a decrease in ERRα mRNA of approximately 50%, but changes in PGC-1α mRNA levels were not causally linked to loss of ERRα activity. The decrease in PGC-1α mRNA levels in response to hypoxia could reflect differences in ATP utilization and turnover rates in neonatal rat cardiomyocytes versus adult cardiomyocytes that are contracting continuously by electrical stimulation. This conclusion is supported by the measurements of OCR in Fig. 1, which confirmed that electrically stimulated contraction immediately increases ATP utilization in adult cardiomyocytes as compared to quiescent (non-stimulated) controls.

The ERRα-dependent increases in PGC-1 and downstream target genes during sustained hypoxia indicate that cardiomyocytes were stimulated to increase their oxidative capacity. This type of adaptive response might seem counterintuitive given that the amount of O2 available to the cardiomyocytes was reduced by approximately 50% (Supplemental Fig. 2). Although oxidative phosphorylation is sufficient to maintain high [ATP] under normal conditions, the metabolic stress of hypoxia can reduce the phosphocreatine:ATP ratio in the adult cardiomyocyte [40,41]. The resulting loss of energy reserves in the form of phosphocreatine in sustained hypoxia increases [ADP] and [Pi] and leads to conversion into AMP by the adenylate kinase reaction [42]. Consistent with these findings, hypoxia has been shown to cause a rapid increase in AMPK activity in cardiomyocytes that is sustained by a high AMP:ATP ratio [43]. The activation of AMPK can increase PGC-1α expression as demonstrated previously using cultures of neonatal rat cardiomyocytes depleted of oxygen and glucose [44]. Further evidence indicates that changes in activity of the catalytic subunit, AMPKα2, directly regulate expression of ERRα in neonatal cardiomyocytes [19].

Previous studies have identified autoregulatory elements in the promoter region of the ERRα gene that are activated by ERRα/PGC-1α and demonstrated that transcription of the ERRα gene is stimulated by increased expression of PGC-1α via a positive feedback loop [45,46]. Conversely, ERRα has been shown to enhance expression of PGC-1α mRNA in several cell types [47–49]. The promoter of the PGC-1α gene contains a possible consensus ERRE in reverse orientation that is located at 1978 and 2394 bp upstream of the transcription start site in the human and rat gene, respectively [39]. This ERRE functions in neonatal rat cardiomyocytes by maintaining PGC-1α expression in normoxia, but its role in hypoxia remains uncertain because binding of ERRα to this element in the PGC-1α promoter did not change significantly as measured over 12 h of hypoxia [39].

Germline ERRγ null mice die perinatally due to developmental abnormalities that includes an inability to shift to oxidative metabolism, but ERRα null mice develop normally and exhibit a relatively mild phenotype under baseline hemodynamic conditions [20,28]. Despite these differences, ERRα and ERRγ isoforms have a significant amount of functional redundancy by binding to ERREs in an array of target genes including Fabp3 and Cox6c [29]. PGC-1α mRNA and ERRγ mRNA levels increase in the myocardium of ERRα null mice under baseline conditions and gene expression is preserved except for a small subset of ERR target genes [20,29]. These findings suggest that ERRγ compensates for loss of ERRα function to maintain myocardial energy metabolism. However, compensatory increases in PGC-1α mRNA and ERRγ mRNA do not occur in either ERRα null or wild-type mice subjected to LV pressure overload hypertrophy [20]. These findings are consistent with the adaptive response of adult cardiomyocytes to sustained hypoxia in that ERRα activity is required to increase PGC-1α in response to increased energy demands imposed by a metabolic stress.

Treatment of cardiomyocytes with the ERRα inverse agonist XCT-790 resulted in a rapid knockdown of ERRα protein. This reduction was most likely caused by proteasome-dependent degradation of ERRα protein as shown previously in MCF7 human mammary epithelial cells treated with XCT-790 [50]. The corresponding reduction in ERRα mRNA levels is consistent with the presence of an autoregulatory mechanism in which transcription of the ERRα gene is controlled by ERRα/PGC-1 complexes [45]. A limitation of this study is that efficient ERRα knockdown required treatment with XCT-790 rather than a miRNA targeting approach using shRNA or siRNA. It was found that adenoviral-mediated expression of ERRα-shRNA required an extended incubation period of several days to achieve partial knockdown of ERRα prior to electrical stimulation under either normoxic or hypoxic conditions. Consequently, shRNA was not a suitable experimental approach to examine loss of ERRα function on cardiomyocyte gene expression. Steady-state protein synthesis and protein content are significantly lower in non-stimulated (quiescent) cardiomyocytes as measured after 4 days in primary culture as compared to electrically stimulated (contracting) cardiomyocytes [23].

In conclusion, these studies demonstrate that expression of PGC-1 isoforms is induced in contracting adult cardiomyocytes during adaptation to hypoxic conditions. The ERRα isoform is necessary for increased expression of PGC-1 isoforms and downstream target genes as part of the adaptive response of contracting adult cardiomyocytes to sustained hypoxia. By controlling expression of PGC-1 isoforms, the metabolic phenotype of the adult cardiomyocyte can be altered through regulation of ERRα target genes. Thus, the mechanism by which ERRα regulates PGC-1 isoform expression may be relevant to the well documented alterations in cardiomyocyte energy metabolism in ischemic heart disease.

Supplementary Material

supplementary material

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.ijcard.2015.03.353.

Acknowledgments

We wish to thank Daisy Dominick, Dr. Harinath Kasiganesan and Shaun R. Wahl for their excellent technical assistance. This research was supported by a Merit Review Award of the Department of Veterans Affairs (to P.J.M.); NIH Predoctoral Fellowship Training Grant to Improve Cardiovascular Therapies (to K.F.C.); and American Heart Association Predoctoral Fellowship (K.F.C., 12PRE1205007).

Grant support:

This research was supported by a Merit Review Award of the Department of Veterans Affairs (to P.J.M.); NIH Predoctoral Fellowship Training Grant to Improve Cardiovascular Therapies (to K.F.C.); American Heart Association Predoctoral Fellowship (K.F.C., 12PRE1205007).

Footnotes

Conflict of interest

The authors report no relationships that could be construed as a conflict of interest.

☆☆

This author takes responsibility for all aspects of the reliability and freedom from bias of the data presented and their discussed interpretation.

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