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Resistance to cardiomyocyte hypertrophy in ae3 −/− mice, deficient in the AE3 Cl−/HCO3 −exchanger
© Sowah et al.; licensee BioMed Central Ltd. 2014
Received: 24 March 2014
Accepted: 16 July 2014
Published: 21 July 2014
Cardiac hypertrophy is central to the etiology of heart failure. Understanding the molecular pathways promoting cardiac hypertrophy may identify new targets for therapeutic intervention. Sodium-proton exchanger (NHE1) activity and expression levels in the heart are elevated in many models of hypertrophy through protein kinase C (PKC)/MAPK/ERK/p90RSK pathway stimulation. Sustained NHE1 activity, however, requires an acid-loading pathway. Evidence suggests that the Cl−/HCO3 − exchanger, AE3, provides this acid load. Here we explored the role of AE3 in the hypertrophic growth cascade of cardiomyocytes.
AE3-deficient (ae3 −/− ) mice were compared to wildtype (WT) littermates to examine the role of AE3 protein in the development of cardiomyocyte hypertrophy. Mouse hearts were assessed by echocardiography. As well, responses of cultured cardiomyocytes to hypertrophic stimuli were measured. pH regulation capacity of ae3 −/− and WT cardiomyocytes was assessed in cultured cells loaded with the pH-sensitive dye, BCECF-AM.
ae3 −/− mice were indistinguishable from wild type (WT) mice in terms of cardiovascular performance. Stimulation of ae3 −/− cardiomyocytes with hypertrophic agonists did not increase cardiac growth or reactivate the fetal gene program. ae3 −/− mice are thus protected from pro-hypertrophic stimulation. Steady state intracellular pH (pHi) in ae3 −/− cardiomyocytes was not significantly different from WT, but the rate of recovery of pHi from imposed alkalosis was significantly slower in ae3 −/− cardiomyocytes.
These data reveal the importance of AE3-mediated Cl−/HCO3 − exchange in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is an attractive approach in the treatment of cardiac hypertrophy.
Cardiovascular diseases remain a major cause of death worldwide despite progress in disease outcomes of patients . 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 .
Cardiac cells are terminally differentiated cells that respond to increased stress by increasing their size rather than mitotically dividing to increase their number . 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 . 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 (pHi) regulation is paramount in maintaining normal cardiac function [7, 8]. Plasma membrane transporters involved in maintaining pHi at physiological levels in the heart include the Na+/H+ exchanger (NHE1), Na+/HCO3 − co-transporters (NBC), and Cl−/HCO3 − exchangers [9, 10]. Cytosolic acidification or hormonal stimulation activate NHE1, which facilitates electroneutral Na+/H+ exchange, to alkalinize the cytosol . 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  and NHE1 inhibition reduced cardiac hypertrophy and interstitial fibrosis . Transgenic mice expressing activated NHE1 exchanger had enlargement of the heart and increased sensitivity to hypertrophic stimulation . Since NHE1 activation induces acid extrusion, alkalinization should accompany NHE1 activation. NHE1 activation was not, however, accompanied by increased pHi, although cytosolic Na+ was elevated . 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−/HCO3 − exchange mediated by AE3 provides this acidifying pathway [7, 8, 10].
The heart expresses three Cl−/HCO3 − exchanger isoforms: AE1, AE2 and AE3 [10, 21]. Another cardiac Cl−/HCO3 −, SLC26a6, [22–24], may represent the Cl−/OH− exchanger (CHE) that has been reported in the heart . 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 . 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 HCO3 −[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 . 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 . Cardiomyocytes from CAII-deficient mice had physiological hypertrophy, but were unresponsive to hypertrophic stimulation . 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 . 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−/HCO3 − 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) .
Accumulating evidence suggests a significant role of AE3 in cardiac function. AE3 Cl−/HCO3 − exchange activity is involved in cardiac contractility by altering cardiac Ca++ handling . Moreover, disruption of the ae3 gene in mice resulted in an exacerbated cardiac function and precipitated heart failure in hypertrophic cardiomyopathy mice . 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 . Taken together, these findings suggest that the AE3 Cl−/HCO3 − 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.
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 . 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 H2O (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.
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 MgSO4, 5.6 glucose, 10 2,3-butanedione monoxime (BDM) (Sigma), 5 taurine (Sigma), 1.2 NaH2PO4, 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 CaCl2. 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 CaCl2). 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 CaCl2). Samples were transferred to 60 mm tissue culture dish and calcium levels were increased by addition of CaCl2 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 104 cells/cm2 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% CO2 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)
Sequences of primers used in qRT-PCR
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 , 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) , or rabbit polyclonal anti-NHE1 antibody (1:1000)  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 . 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 H2O) 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 HCO3 − Ringer’s buffer solution (in mM: 128.3 NaCl, 4.7 KCl, 1.35 CaCl2, 20.23 NaHCO3, 1.05 MgSO4 and 11 glucose, pH 7.4) was initiated at 3.5 ml/min. Solutions were bubbled with 5% CO2-balanced air. Intracellular alkalosis was induced by switching to a HCO3 − Ringer’s solution, containing, 20 mM trimethylamine (TMA) (Sigma) and perfusion was continued for 3 min. Perfusion was switched back to the HCO3 − Ringer’s buffer solution. pHi 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, pHi was clamped by the high K+/Nigericin technique  in calibration solutions containing, 140 mM KCl, 1 mM MgCl2, 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 pHi was measured from the pHi value prior to induction of alkalosis. Rate of pHi recovery was measured by linear regression from first min of recovery from imposed alkalosis.
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.
Cardiac development in ae3 −/− mice
Blood pressure and echocardiography
Echocardiographic and blood pressure analysis of WT and ae3 −/− mice
Ejection fraction, %
65 ± 3
67 ± 7
Fractional shortening, %
35 ± 3
37 ± 7
0.70 ± 0.01
0.75 ± 0.04
3.5 ± 0.1
3.4 ± 0.1
0.67 ± 0.03
0.70 ± 0.03
1.08 ± 0.03
1.11 ± 0.09
2.3 ± 0.1
2.1 ± 0.3
1.05 ± 0.07
1.05 ± 0.12
MV E Velocity, mm.s−1
730 ± 50
710 ± 50
MV A Velocity, mm.s−1
390 ± 30
520 ± 40
MV E/A ratio
1.9 ± 0.1
1.4 ± 0.1
24 ± 3
21 ± 4
16 ± 0
17 ± 2
0.63 ± 0.03
0.61 ± 0.02
Systolic pressure, mm Hg
102 ± 7
100 ± 9
Diastolic pressure, mm Hg
66 ± 6
64 ± 8
11 ± 1
10 ± 1
Volume of blood, ml
36 ± 6
31 ± 5
Heart rate, beats.min−1
699 ± 1
721 ± 49
Cardiomyocyte growth upon pro-hypertrophic stimulation
Expression of hypertrophic marker genes
Expression of HTM genes in mouse cardiomyocytes
Since the baseline expression level of CAII was elevated in ae3 −/− cardiomyocytes and was further enhanced by hypertrophic stimulants in WT cardiomyocytes, we evaluated CAII protein expression. Cardiomyocytes isolated from ae3 −/− and WT male adult mice hearts were probed for CAII on immunoblots (Additional file 1: Figure S1A). CAII migrated at ~27 kDa, consistent with the expected molecular weight of CAII. Both the steady-state level of CAII protein (Additional file 1: Figure S1B) and mRNA level for CAII (Figure 6B) were higher in ae3 −/− cardiomyocytes than WT cardiomyocytes. Importantly, however, there was a large quantitative difference in the response, where CAII protein level rose by about 50%, whereas CAII message rose was about eight-fold higher in the ae3 −/− mice than WT. PE and ANGII increased CAII levels in the WT cardiomyocytes. Contrastingly, CAII levels in ae3 −/− cardiomyocytes were not significantly affected by pro-hypertrophic stimulation (Additional file 1: Figure S1B).
Protein synthesis in pro-hypertrophically-stimulated cardiomyocytes
pHiregulation in mouse cardiomyocytes
Expression of pHi regulators in ae3 −/− cardiomyocytes
We next assessed the expression level of the other pHi regulatory transporters at the protein level. On immunoblots the proteins migrated at the expected sizes, NHE1 at ~100 kDa, and SLC26a6 at ~80 kDa (Additional file 2: Figure S2 and Additional file 3: Figure S3). Consistent with a previous study , expression of NHE1 was elevated in ae3 −/− cardiomyocytes compared to WT, but remained unchanged in the heterozygotes (Additional file 2: Figure S2A-B). Expression of SLC26a6 protein was not affected by deletion of the ae3 gene (Additional file 3: Figure S3A-B).
Pathological cardiac hypertrophy renders the heart susceptible to cardiac failure. Accumulating evidence implicates NHE1 as a key candidate mediating pathological hypertrophy. Prolonged NHE1 activation produces intracellular alkalinization. Sustained NHE1 activation can only occur in the presence of a counter acidifying mechanism. The present study examined the possibility that the Cl−/HCO3 − anion exchange mediated by AE3 is responsible for the acidification mechanism. Our studies, using AE3 deficient mice, support a role for AE3 in cardiovascular pH regulation and the development of hormonally-induced cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is thus a possible therapeutic direction in the prevention of maladaptive cardiac hypertrophy.
Role of AE3 in cardiomyocyte hypertrophy
The role of AE3 in cardiac physiology is incompletely characterized, but several lines of evidence suggest that AE3 Cl−/HCO3 − exchange is required to maintain pHi homeostasis [10, 61]. Consistent with this, we found that the rate of recovery from an alkaline load was reduced in ae3 −/− cardiomyocytes, relative to WT.
The hypertrophic transport metabolon is a proposed pathological pathway in which AE3, NHE1 and CAII are coordinately activated and promote hypertrophic growth [32, 33]. Specifically, pro-hypertrophic agonists, including PE, ANGII and endothelin are coupled to PKC activation. NHE1 and AE3 are both activated by agonists coupled to PKC activation [59, 62, 63]. Co-activation of these respective alkalinizing and acidifying transporters has the net effect of loading the cell with NaCl, with no change of cytosolic pH. Elevated cytosolic Na+ in turn reduces the efficacy of Ca++-efflux by the plasma membrane Na+/Ca++ exchanger, resulting in a rise in cytosolic Ca++. Ca++ is a pro-hypertrophic second messenger [64, 65] and also may promote hypertrophy in a feed-forward cascade by stimulation of PKC . Accumulating evidence suggests that both NHE1 and AE3 activities are influenced by physical and functional interactions with CAII [32, 41], which provides substrates for these transporters. That said, very recent data suggest that intracellular carbonic anhydrase does not activate cardiomyocyte Na+/H+ exchange . Prevention of PE-induced cardiomyocyte hypertrophy upon CAII blockade [32, 33] may thus arise from reduced AE3-mediated cytosolic acidification, thereby decreasing driving force for NHE-mediated alkalinization. We propose that CAII, NHE1 and AE3 form a hypertrophic transport metabolon, where hypertrophy is promoted by the pathological activation of AE3 and NHE1, stimulated by interactions with CAII.
The functional relationship between AE3, CAII and NHE1 was further supported by our analysis of protein expression in ae3 −/− mice. Increased CAII transcript abundance and protein expression in ae3 −/− mice compared to WT mice suggest that there is compensation for a loss of AE3. This finding parallels results in retinal tissue from ae3 −/− mice, where there was increased CAII expression . Functional complementarity of AE3 and CAII is further supported by the significant increase in AE3 transcript abundance in Car2 (caii −/− ) mice, compared to WT mice . The upregulation of NHE1 transcript abundance and increased protein expression provide further support for the HTM. Taken together, these data support a functional interaction between AE3 and CAII, where there is compensation of one for the loss of the other.
Loss of AE3 prevents cardiomyocyte hypertrophy
This study lends support to the idea that AE3 is the Cl−/HCO3 − exchanger isoform working in conjunction with NHE1 to promote cardiomyocyte hypertrophy. Non-specific inhibition of Cl−/HCO3 − exchangers, using stilbene derivatives, prevented hypoxia-induced acidification in rat ventricular myocytes, as well as increases in intracellular Cl− and Ca++ concentrations [68, 69], suggesting a role of Cl−/HCO3 − exchangers in cardiac pathology. When subjected to ischemia and reperfusion, hearts isolated from ae3 −/− mice revealed no effect on cardiac performance demonstrated as contractility, ventricular developed pressure or end diastolic pressure relative to wildtype . Double knock-out ae3/nkcc1 (Na+-K+-2Cl− co-transporter) mice, however, had elevated ischemia/reperfusion injury, which resulted in impaired cardiac contractility and overall cardiac performance . These findings were attributed to impaired Ca++ handling in the double knock-out cardiomyocytes  compared to the single mutants. In a hypertrophic cardiomyopathy mouse model carrying a Glu180Gly mutation in α-tropomyosin (TM180), disruption of ae3 did not prevent or reverse the hypertrophic phenotype . The TM180/ae3 double knockout mice had reduced cardiac function and compromised Ca++ regulation, which accounted for the rapid decline to heart failure .
Taken together, these two studies suggest that AE3 loss is not cardioprotective, which contrasts with findings of the present study, which found that loss of AE3 renders cardiomyocytes less susceptible to pro-hypertrophic stimulation. Our data showed that the marked rise in cell surface area, protein synthesis, and fetal gene reactivation observed in response to hypertrophic stimulation in cardiomyocytes from WT mice was not present in ae3 −/− mice. In the context of cardiomyocyte hypertrophy mediated by hormonal stimuli, our data demonstrate that ablation of AE3 affords protection against cardiomyocyte hypertrophy.
The discrepancy could arise from the model of hypertrophy and the cardiac pathology being investigated. Hypertrophic cardiomyopathy is a genetic disorder, which occurs as a result of mutations in the genes that encode cardiac contractile proteins . This anomaly manifests as sudden cardiac death, arrhythmias, hypertrophy and heart failure . Overall, hypertrophic cardiomyopathy results in impairment of Ca++ sensitivity by the myofibrils. In the present study however, we employed a model of hypertrophy induced by PE or ANGII, which involves interaction of these ligands with their cell surface receptors, GPCR . The resultant intracellular response leads to increased cytosolic Ca++ overload, which mediates a cascade of signaling pathway involving activation of PKC, which ultimately induces cardiomyocyte hypertrophy .
One other possible explanation for the discrepancy between the present findings and earlier studies of ae3 −/− mice is the assay of cardiac hypertrophy. Earlier investigations probed whole animal physiology, whereas we studied isolated cardiomyocytes in culture. Isolated cardiomyocytes are an established model, which may be more sensitive in detecting alterations of heart cell growth than assessments of functional alterations of heart function sued in earlier studies.
Interestingly, AE3 also forms a complex with CAXIV in the myocardium . In the hypertrophic myocardium of SHR rats, exacerbated AE3/CAXIV activation was proposed to elicit hypertrophic growth of the heart . Thus, the etiology, signaling pathway and pathophysiology of hypertrophic cardiomyopathy are distinct from that mediated by hormonal factors. These differences could account for the disparity between the influence of AE3 on hypertrophy in the present report and that shown in previous models of cardiovascular disease [42, 43]. Since hypertrophic interventions by PE and ANGII failed to induce hypertrophy in the ae3 null cardiomyocytes in our study, the PKC-coupled hypertrophic cascade appears to require AE3. Recently, it was demonstrated that when paced, AE3 null hearts had an impaired force-dependent inotropy characterized by an elevation of protein kinase B (PKB, Akt) phosphorylation and a downregulation of AMPK activity . This observation may provide additional support of the present findings that revealed that ae3 null cardiomyocytes are less susceptible to develop hypertrophy in upon pro-hypertrophic stimulation. Akt phosphorylation is central to the signaling pathway mediated by growth factors that induce physiological hypertrophy . Thus, its activation under stressful conditions may trigger a physiological growth response to counter the pathological signaling cascade stimulated the source of stress. The lack of response by the AE3 knock-out cardiomyocytes to hypertrophic stimulants seen here could arise in part from effects on Akt phosphorylation.
Phenotype of ae3 −/− mice
ae3 −/− mice have been reported to have no apparent defects, and the results of our analysis of the cardiac function of ae3 −/− mice is comparable to these previous studies [42, 43, 74]. Combined analysis of echocardiographic measurements of ventricular wall dimensions, chamber diameter and cardiac function between the two genotypes further suggests that loss of AE3 does not affect hypertrophy or cardiovascular performance. A significant decrease in the HW/BW ratio in ae3 −/− mice is the result of a reduction in heart size, arising from a decrease in cardiomyocyte size. This suggests a critical role for AE3 in heart development.
Role of AE3 in control of cardiomyocyte pHi
Since cardiomyocyte steady-state pH was the same in ae3 −/− and WT mice, loss of Cl−/HCO3 − exchange activity by ae3 deletion is likely compensated for by another protein. The principal Cl−/HCO3 − exchanger of cardiomyocytes is SLC26a6 , making it the most likely acid loading transporter to compensate for loss of AE3. Since we did not see an increase in SLC26a6 expression, SLC26a6 activation may occur through post-translational mechanisms. We did note an increase of CAII expression in ae3 −/− mice. Since CAII increases the rate of CO2/HCO3 − inter-conversion, it increases the rate of observed Cl−/HCO3 − for SLC26a6 . Thus increased CAII expression might in part compensate for loss of AE3. We also noted a much larger increase in CAII message than we saw for CAII protein. Since proteins carry out cellular functions, not mRNA, we consider the protein change to be a more reliable measure of cell response than the mRNA increase. The reason for a muted increase in CAII protein level in comparison to the mRNA rise remains unclear.
The importance of AE3 in intracellular pH regulation was, however, evident in the reduced rate of pHi recovery from imposed intracellular alkalosis in cardiomyocytes from ae3 −/− mice compared to WT. Endothelin 1 stimulation of myocardial Cl−/HCO3 − exchange activity in isolated rat papillary muscle is almost totally attributable to the AE3 isoform, on the basis of inhibition by an anti-AE3 antibody . This provides a possible explanation for the resistance of ae3 −/− mice to pro-hypertrophic stimuli; ae3 −/− cardiomyocytes have reduced acidifying activity to counter enhanced NHE1 activity associated with pro-hypertrophic stimulation.
We explored the role of AE3 in the development of cardiomyocyte hypertrophy and cardiovascular pH regulation, using AE3 deficient mice. Cardiomyocytes from ae3 −/− mice were protected from increases in cell surface area, protein synthesis, and fetal gene reactivation in response to hypertrophic stimulation. Steady-state cardiomyocyte pHi in ae3 −/− mice was comparable to WT, but slower to recover from imposed intracellular alkalosis. Our findings demonstrate that AE3 is important in hypertrophic signaling pathways activated by PE and ANGII, possibly acting through the hypertrophic transport metabolon. Pharmacologically targeting AE3 activity in the event of hypertrophy is an attractive strategy to treat heart failure patients.
We thank Dr. Gary Shull (University of Cincinnati) for providing breeding stock of ae3 null mice. J.R.C. was a Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). BVA is an Established Investigator of CONICET (Argentina). The Heart and Stroke Foundation of Alberta provided operating support for this work.
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