Background
Cardiovascular diseases remain a major cause of death worldwide despite progress in disease outcomes of patients [
1]. Heart failure (HF) is the common end-stage of many cardiovascular disorders, with a prevalence of 5.8 million in the USA and about 23 million worldwide [
2,
3]. Annually, 550,000 new cases of HF arise in the U.S.A. The intricate molecular events resulting in heart failure remain incompletely understood, but enlargement of cardiac contractile cells (cardiomyocyte hypertrophy) in response to various stimuli is central to the progression to heart failure [
4].
Cardiac cells are terminally differentiated cells that respond to increased stress by increasing their size rather than mitotically dividing to increase their number [
5]. Cardiovascular events that increase myocardial stress (workload) chronically induce hypertrophic growth. Pressure overload, myocardial infarction, obesity, pregnancy or exercise can independently trigger molecular mechanisms culminating in increased cardiomyocyte size. Cardiac hypertrophy occurs to normalize the elevated demand on the myocardium, and can be physiological or pathological depending on the source of the initiating stimuli [
6]. Physiological hypertrophy prevails in healthy individuals during increased physical activities or in pregnant women. Pathological hypertrophy results from prolonged elevated blood pressure (pressure overload), ischemia accompanied by changes in Ca
++ handling, or genetic abnormalities. Initially, pathological hypertrophic growth compensates for the decline in contractile function, but ultimately the myocardium becomes decompensated from sustained exposure to the initiating stimuli. Understanding the distinct pathways mediating cardiac hypertrophic development has potential to identify new drug targets for the management of heart failure.
Intracellular pH (pH
i) regulation is paramount in maintaining normal cardiac function [
7,
8]. Plasma membrane transporters involved in maintaining pH
i at physiological levels in the heart include the Na
+/H
+ exchanger (NHE1), Na
+/HCO
3
− co-transporters (NBC), and Cl
−/HCO
3
− exchangers [
9,
10]. Cytosolic acidification or hormonal stimulation activate NHE1, which facilitates electroneutral Na
+/H
+ exchange, to alkalinize the cytosol [
11]. Accumulating evidence suggests that NHE1 expression level and activity increase in hypertrophy [
12,
13]. In the hypertrophied myocardium of the spontaneously hypertensive rats (SHR), there was an increased activation of NHE1 [
14] and NHE1 inhibition reduced cardiac hypertrophy and interstitial fibrosis [
15]. Transgenic mice expressing activated NHE1 exchanger had enlargement of the heart and increased sensitivity to hypertrophic stimulation [
16]. Since NHE1 activation induces acid extrusion, alkalinization should accompany NHE1 activation. NHE1 activation was not, however, accompanied by increased pH
i, although cytosolic Na
+ was elevated [
14]. Moreover, under alkaline conditions, NHE1 activity is self-inhibited, which suggests that an acidifying mechanism running counter to NHE1 is necessary for sustained NHE1 activation [
17‐
20]. Indeed, Cl
−/HCO
3
− exchange mediated by AE3 provides this acidifying pathway [
7,
8,
10].
The heart expresses three Cl
−/HCO
3
− exchanger isoforms: AE1, AE2 and AE3 [
10,
21]. Another cardiac Cl
−/HCO
3
−, SLC26a6, [
22‐
24], may represent the Cl
−/OH
− exchanger (CHE) that has been reported in the heart [
25]. Two AE3 variants, AE3 full length (AE3fl) and cardiac AE3 (AE3c) are expressed in the heart; AE3fl is also expressed in the brain and retina [
26‐
28]. Phenylephrine (PE) and angiotensin II (ANGII), acting on their G-protein-coupled receptors (GPCRs), activate AE3fl via protein kinase C (PKC). Interestingly, PKC can indirectly activate NHE1 via MAPK-dependent mechanisms [
29]. Moreover, carbonic anhydrase II (CAII), another modulator of the PE-dependent hypertrophic growth, interacts with both NHE1 and AE3 to provide their respective transport substrates, H
+ and HCO
3
−[
30,
31].
CAII activation was recently found to be important in the induction of cardiomyocyte hypertrophy. In isolated rat cardiomyocytes, inhibition of CAII catalytic activity reduced phenylephrine (PE) and angiotensin II (ANGII) induced cardiomyocyte hypertrophy [
32]. Additionally, infection of neonatal rat cardiomyocytes with adenoviral constructs encoding catalytically inactive CAII mutant, CAII-V143Y, reduced the response of the cardiomyocytes to hypertrophic stimuli, suggested to arise from a dominant negative mode of action [
33]. Cardiomyocytes from CAII-deficient mice had physiological hypertrophy, but were unresponsive to hypertrophic stimulation [
33]. Finally, expression of CAII and CAIV was elevated in the hypertrophic ventricles from failing human hearts, indicating that elevation of carbonic anhydrases is a feature of heart failure in people [
34]. Taken together, these findings show that CAII plays a role in the development of cardiomyocyte hypertrophy.
Several reports revealed that CAII physically and functionally interacts with Cl
−/HCO
3
− anion exchangers to enhance the transport activity of anion exchangers forming a bicarbonate transport metabolon [
31,
35‐
37], although some reports have questioned the physiological relevance of this physical and functional linkage [
38‐
40]. CAII also interacts physically and functionally with NHE1 to increase the exchange activity [
30,
41]. These observations suggest that simultaneous activation of AE3, CAII and NHE1 occurs upon pro-hypertrophic stimulation by the PKC-coupled agonists, PE, ANGII or endothelin I (ET-I). This pathological activated complex has been termed the hypertrophic transport metabolon (HTM) [
34].
Accumulating evidence suggests a significant role of AE3 in cardiac function. AE3 Cl
−/HCO
3
− exchange activity is involved in cardiac contractility by altering cardiac Ca
++ handling [
42]. Moreover, disruption of the
ae3 gene in mice resulted in an exacerbated cardiac function and precipitated heart failure in hypertrophic cardiomyopathy mice [
43]. Pacing of AE3 null hearts abrogated frequency-dependent inotropy, which, suggests that AE3 is required in mediating force-frequency response induced by acute biochemical stress [
44]. Taken together, these findings suggest that the AE3 Cl
−/HCO
3
− exchanger is critical in heart growth and function but the exact mechanism remains unknown.
In the present study, we examined the role of AE3 in cardiomyocyte hypertrophy, using AE3-deficient (ae3
−/−
) mice. Cardiac growth parameters and fetal gene reactivation were measured in the presence of pro-hypertrophic stimulation in cardiomyocytes from ae3
−/−
mice. We also examined the role of AE3 in cardiomyocyte steady state pHi, using the ae3
−/−
mice. Our results indicate that ae3 deletion prevents cardiomyocyte hypertrophy and reduces the rate of pHi recovery in cardiomyocytes, reinforcing the importance of AE3 in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy.
Methods
Animal care and use
All procedures involving animals were performed in accordance with the guidelines established by the Canadian Council on Animal Care and the University of Alberta Animal Care and Use Committee.
ae3 null mice
Experiments were performed using
ae3 null mice in a C57BL/6 background. The
ae3 null strain has been previously described and characterized [
42]. Age-matched WT mice from separate breedings were used as controls.
Heart weight to body weight ratio
Mice were weighed and anesthetized with sodium pentobarbital (50 mg/kg) by intraperitoneal injection. Upon reaching surgical plane, hearts were removed after performing midsection thoracotomy and rinsed in 4°C phosphate buffer saline (PBS: 140 mM NaCl, 3 mM KCl, 6.5 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). Ventricles were separated from atria and blood vessels, blotted dry and the ventricular weight was measured. Heart weight to body weight ratio (HW/BW) was then calculated by dividing the weight of the ventricles by the weight of the whole animal.
Hematoxylin-eosin staining of heart sections
Hematoxylin and eosin (HE) staining was performed on longitudinal and transverse sections of wildtype and
ae3 null adult mouse heart, using previously described protocols [
45,
46]. Briefly, paraffin-embedded hearts were sectioned into 3 μm slices, which were trimmed and floated onto a water bath at 42°C, containing 50 mg/l of gelatin while gently stretching the cut sections to avoid wrinkles. Poly-L-lysine coated microscope slides were dipped under the meniscus of the water bath and a tissue slice was carefully mounted onto it. Sections were then air-dried for 16 h at 20°C, after which the slides were placed on edge in an oven and baked for 15 min at 60°C. Sections were deparaffinized by successively immersing them for 5 min with agitation in xylene, 100% ethanol and 70% ethanol, and rehydrated in Tris-EDTA buffer (1 mM EDTA, 0.05% Tween 20, 10 mM Tris, pH 9.0) for 1 min. Slides were rinsed in distilled water for 1 min with agitation. Slides were agitated for 30 s in Mayer’s hematoxylin solution (1.0 g/l hematoxylin (Sigma), sodium iodate (0.2 g/l), aluminum ammonium sulfate · 12 H
2O (50 g/l), chloral hydrate (50 g/l) and citric acid (1 g/l) and rinsed in water for 1 min. Slides were stained in 1% eosin Y solution (1% eosin Y aqueous solution, Fisher) for 30 s with agitation. Sections were dehydrated by successively immersing it twice in 95% ethanol and twice in 100% ethanol for 30 s each. Ethanol was extracted twice in xylene, followed by addition of two drops of mounting medium (Canada Balsam, Sigma), after which the sections were covered with a coverslip. Images of transverse and longitudinal sections were collected, using a Nikon digital camera (DXM 200) mounted on top of a Nikon Eclipse E600 microscope.
Echocardiography
Echocardiographic assessment of cardiac performance in male WT and ae3
−/−
mice was performed by the Cardiovascular Research Centre Core Facility (University of Alberta). Mice were subjected to mild anesthesia by isoflurane inhalation and echocardiography parameters were measured using a Vevo 770 High-Resolution Imaging System with a 30-MHz transducer (RMV-707B; Visual Sonics, Toronto). M-mode images and a four chamber view allowed for the calculation of wall measurements, ejection fraction, fractional shortening, and mitral velocities (E and A). Mitral valve tissue motion (E’) was measured, by tissue Doppler echocardiography of the mitral septal annulus.
Blood pressure measurements
Non-invasive blood pressure measurements were performed by the Cardiovascular Research Centre Core Facility (University of Alberta). Mice were comfortably restrained in a 26°C warming chamber (IITC Life Science) for ~15 min prior to taking blood-pressure (BP) measurements. Tail-cuff sensors were secured on the tail to occlude the blood flow, and connected to the recording device. Systolic and diastolic pressure, heart rate, blood volume and flow were obtained, using CODA6 software (Kent Scientific Corporation, Connecticut, USA).
Isolation and culture of adult mouse cardiomyocytes
Cardiomyocytes from adult male mouse hearts were isolated and cultured with modifications of published protocols [
21,
47]. Briefly, adult mice were euthanized with sodium pentobarbital (50 mg/kg body mass) by intraperitoneal injection. Upon reaching surgical plane, midsection thoracotomy was performed and hearts were quickly excised and placed in 4°C perfusion solution, containing in mM: 120 NaCl, 5.4 KCl, 1.2 MgSO
4, 5.6 glucose, 10 2,3-butanedione monoxime (BDM) (Sigma), 5 taurine (Sigma), 1.2 NaH
2PO
4, 10 HEPES, pH 7.4. Extra-cardiac tissues were removed and hearts were subjected to retrograde perfusion with perfusion solution to remove excess blood. Perfusion was switched for 15 min to perfusion solution at 37°C, supplemented with 0.5 mg/ml collagenase type B (Roche), 0.5 mg/ml collagenase type D (Roche), 0.02 mg/ml protease XIV (Roche) and 50 μM CaCl
2. Ventricles, partially digested at this stage, were removed, cut into several pieces, and digested further in the same enzyme digestion solution by gentle trituration with a transfer pipette. Once the ventricles were completely digested, enzymatic digestion was terminated by addition of Digestion stop buffer I (perfusion solution, containing 10% (v/v) fetal bovine serum (FBS) (GIBCO) and 50 μM CaCl
2). Lysates settled under gravity for 10 min at room temperature and pellets were resuspended in myocyte stopping buffer II (perfusion solution, containing 5% (v/v) FBS and 50 μM CaCl
2). Samples were transferred to 60 mm tissue culture dish and calcium levels were increased by addition of CaCl
2 to obtain final concentrations of 62, 112, 212, 500 and 1000 μM, sequentially at 4 min intervals at 20°C. Cells were transferred to a 14 ml culture tube and allowed to sediment for 10 min under gravity. Cells were resuspended in myocyte culture medium (Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12-Ham (Sigma), supplemented with 10 mM BDM, 5% (v/v) FBS, 1% penicillin (GIBCO), 10 mM BDM, and 2 mM L-glutamine (GIBCO)). Myocytes were plated at a density of (0.5-1) x 10
4 cells/cm
2 onto 35 mm culture dishes, pre-coated for 2 h with 10 μg/ml mouse laminin (Invitrogen) in PBS. Cells were incubated at 37°C in a 5% CO
2 incubator for 1 h at which point medium was replaced with Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F12-Ham, containing 10 mM BDM, 1% penicillin, 2 mM L-glutamine, 0.1 mg/ml bovine serum albumin, and 1x ITS Liquid Media Supplement (Sigma). The entire culture procedure was carried out in a sterile laminar flow hood.
Assessment of cardiomyocyte hypertrophic growth
Cardiomyocytes were isolated and cultured from adult mouse hearts as described above. Following 18 h of culture, myocytes were treated with solvent carrier (control), 10 μM phenylephrine (Sigma) or 1 μM angiotensin II (Sigma) for another 24 h. Hypertrophy was assessed by analysis of cell surface area of cardiomyocytes pre- and post-treatment with the hypertrophic agonists. Images of characteristic rod-shaped cardiomyocytes were collected with a QICAM fast-cooled 12-bit colour camera (QImaging Corporation). Cell surface areas were measured, using Image-Pro Plus software (Media Cybernetics). Each treatment group contained 100–200 cells of ~ ten different experiments. Cell surface area (% relative to control) = Surface area (post-treatment)/Surface area (pre-treatment) X 100.
Real-time quantitative reverse transcription PCR (qRT-PCR)
Cardiomyocytes were prepared and treated as above. At the end of the culture period, medium was aspirated and cardiomyocytes were harvested in 350 μl Buffer RLT (Qiagen). RNA was extracted from the lysates with an RNeasy Plus Mini Kit, as per manufacturer’s instructions (Qiagen). RNA samples (100 ng) were reverse transcribed, following the manufacturer’s instructions for SuperScript II™ reverse transcriptase (Invitrogen). qRT-PCR was performed in a Rotorgene 3000 real time thermal cycler (Corbett Research), using a reaction mix containing: 5 μl template cDNA, 12 μl 2x Rotor-Gene SYBR Green PCR Master Mix (Rotor-Gene SYBR Green PCR Kit, Qiagen), and 1 μM of each primer. Data were obtained and analyzed, using Rotor Gene 6.0.14 software. Cycle threshold (Ct) values were obtained for carbonic anhydrase II (CAII), NHE1, atrial natriuretic peptide (ANP), β-MHC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Primers (Table
1) were designed, using Primer3 (
http://Frodo.wi.mit.edu/primer3/).
Table 1
Sequences of primers used in qRT-PCR
Anp
| F: | 5′-TCCAGGCCATATTGGAGCAAATCC-3′ |
R: | 5′-TCCAGGTGGTCTAGCAGGTTCTTG-3′ |
β-mhc
| F: | 5′-GAGACGGAGAATGGCAAGAC-3′ |
R: | 5′-AAGCGTAGCGCTCCTTGAG-3′ |
Caii
| F: | 5′-CTCTGCTGGAATGTGTGACCT-3′ |
R: | 5′-GCGTACGGAAATGAGACATCTGC-3′ |
Nhe1
| F: | 5′-TTTTCACCGTCTTTGTGCAG-3′ |
R: | 5′-TGTGTGGATCTCCTCGTTGA-3′ |
Gapdh
| F: | 5′-CCTCGTCCCGTAGACAAAAT-3′ |
| R: | 5′-TGATGGCAACAATCTCCACT-3′ |
Immunoblotting
Cardiomyocytes, isolated and cultured from adult mouse hearts, were subjected to drug intervention as described above. Twenty-four h following treatment, medium was aspirated and myocytes were washed with 4°C PBS. Cells were lysed with SDS-PAGE sample buffer (10% (v/v) glycerol, 2% (w/v) SDS, 2% 2-mercaptoethanol, 0.001% (w/v) bromophenol blue, 65 mM Tris, protease inhibitors (1 μg/ml) pH 6.8) and lysates were heated 5 min at 65°C. Protein concentrations were determined by the bicinchoninic acid (Pierce Biotechnology) assay [
48], and 20 μg of protein was resolved by SDS-PAGE on 10% acrylamide gels. Proteins were transferred onto PVDF membranes by electrophoresis for 1 h at 100 V in transfer buffer (10% (v/v) methanol, 25 mM Tris, and 192 mM glycine). PVDF membranes were blocked for 30 min with 5% (w/v) nonfat dry milk/ 0.1% (v/v) Tween 20 in TBS (137 mM NaCl, 20 mM Tris, pH 7.5). Immunoblots were incubated with rabbit polyclonal anti-CAII antibody (Santa Cruz Biotechnology; 1:1000), rabbit anti-human SLC26a6 (1:1000) [
49], or rabbit polyclonal anti-NHE1 antibody (1:1000) [
32] in TBST-M for 16 h at 4°C. Immunoblots were washed with TBST (TBS, containing 0.1% (v/v) Tween 20) and incubated with donkey anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology; 1:2000) or mouse anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology; 1:2000) for 1 h at room temperature. Immunoblots were washed in TBST and visualized, using ECL reagents (Perkin Elmer) and a Kodak Imaging Station 440CF (Kodak, Rochester, NY). Proteins were quantified by densitometry, using Kodak Molecular Imaging software (version 4.0.3; Kodak). Immunoblots were stripped by incubating in 10 ml of stripping buffer (2% (w/v) SDS, 10 mM 2-mercaptoethanol, 62.5 mM Tris, pH 6.8) at 50°C for 10 min with occasional shaking, followed by three washes with TBST. Membranes were incubated with mouse monoclonal anti-β-actin antibody (Santa Cruz Biotechnology; 1:2000) for 1 h at 20°C, washed with TBST, and incubated with sheep anti-mouse IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology; 1:3000) for 1 h. Immunoblots were washed and visualized again, as described above.
Protein synthesis assays
Cardiomyocytes were prepared and subjected to hypertrophic stimulation as above. Radiolabeled phenylalanine ([
3H]-Phe, 1 μCi/ml, (Perkin Elmer)) was added immediately after drug intervention and cells were incubated for another 24 h. Proteins were precipitated, using trichloroacetic acid (TCA) as described previously with some modifications [
50]. Medium was carefully aspirated and 500 μl of 0.5% (v/v) Triton X-100, containing protease inhibitor cocktail (Roche) were added. Lysates were transferred into 1.5 ml microcentrifuge tubes and TCA (100%: 500 g in 350 ml H
2O) was added to each tube to a final concentration of 40% (v/v). Samples were incubated at 4°C for 30 min, after which proteins were sedimented by centrifugation at 12 682 x g for 15 min at 4°C. Pellets were resuspended by adding 200 μl acetone (−20°C) and sedimented again by centrifugation, as above, for 10 min. The acetone wash was repeated one more time, and the pellets air dried for 20 min at room temperature. Pellets were resuspended in 200 μl of 0.2 M NaOH, 1% (w/v) SDS. Scintillation fluid (Perkin Elmer, 3.5 ml) was added to each sample and the radioactivity of [
3H]-Phe was counted in a Beckman LS6500 liquid scintillation counter.
Measurement of pHiin adult mouse cardiomyocytes
The protocol was as described previously with minor modifications [
51,
52]. Briefly, cardiomyocytes were isolated as described above and cultured on laminin-coated glass coverslips. Approximately 2 h later, cells were loaded with 2 μM BCECF-AM (Sigma-Aldrich, Canada) for 30 min at 37°C. Coverslips were placed in an Attofluor cell chamber (Invitrogen, Canada), then transferred onto the stage of a Leica DMIRB microscope. Perfusion with HCO
3
− Ringer’s buffer solution (in mM: 128.3 NaCl, 4.7 KCl, 1.35 CaCl
2, 20.23 NaHCO
3, 1.05 MgSO
4 and 11 glucose, pH 7.4) was initiated at 3.5 ml/min. Solutions were bubbled with 5% CO
2-balanced air. Intracellular alkalosis was induced by switching to a HCO
3
− Ringer’s solution, containing, 20 mM trimethylamine (TMA) (Sigma) and perfusion was continued for 3 min. Perfusion was switched back to the HCO
3
− Ringer’s buffer solution. pH
i of individual cardiomyocytes was measured by photometry at excitation wavelengths of 502.5 nm and 440 nm with a Photon Technologies International (PTI, Lawrenceville, NJ, USA) Deltascan monochromator. Emission wavelength, 528.7 nm, was selected, using a dichroic mirror and narrow range filter (Chroma Technology Corp., Rockingham, VT, USA) and was measured with a PTI D104 photometer. At the end of experiments, pH
i was clamped by the high K
+/Nigericin technique [
53] in calibration solutions containing, 140 mM KCl, 1 mM MgCl
2, 2 mM EGTA, 11 mM glucose, 20 mM BDM, 10 mM HEPES. Three pH standards spanned a range of 6.5-7.5. Steady-state pH
i was measured from the pH
i value prior to induction of alkalosis. Rate of pH
i recovery was measured by linear regression from first min of recovery from imposed alkalosis.
Statistical analysis
Data are expressed as mean ± S.E.M. Statistical analyses were performed using paired t-tests or ANOVA where appropriate. P < 0.05 was considered significant.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
DS: First author of the manuscript and completed the majority of the experimental procedures. BFB: Assisted with manuscript preparation. AQ: Collected the qRT-PCR data and generated/optimized the respective primers. BVA: Collection of HW: BW data. JRC: Conceived and supervised the experiments and assisted with manuscript preparation. All authors read and approved the final manuscript.