Carbonic anhydrase inhibition prevents and reverts cardiomyocyte hypertrophy

Abstract

Hypertrophic cardiomyocyte growth contributes substantially to the progression of heart failure. Activation of the plasma membrane Na+-H+ exchanger (NHE1) and Cl- -HCO3- exchanger (AE3) has emerged as a central point in the hypertrophic cascade. Both NHE1 and AE3 bind carbonic anhydrase (CA), which activates their transport flux, by providing H+ and HCO3-, their respective transport substrates. We examined the contribution of CA activity to the hypertrophic response of cultured neonatal and adult rodent cardiomyocytes. Phenylephrine (PE) increased cell size by 37 +/- 2% and increased expression of the hypertrophic marker, atrial natriuretic factor mRNA, twofold in cultured neonatal rat cardiomyocytes. Cell size was also increased in adult cardiomyocytes subjected to angiotensin II or PE treatment. These effects were associated with increased expression of cytosolic CAII protein and the membrane-anchored isoform, CAIV. The membrane-permeant CA inhibitor, 6-ethoxyzolamide (ETZ), both prevented and reversed PE-induced hypertrophy in a concentration-dependent manner in neonate cardiomyocytes (IC50=18 microm). ETZ and the related CA inhibitor methazolamide prevented hypertrophy in adult cardiomyocytes. In addition, ETZ inhibited transport activity of NHE1 and the AE isoform, AE3, with respective EC50 values of 1.2 +/- 0.3 microm and 2.7 +/- 0.3 microm. PE significantly increased neonatal cardiomyocyte Ca2+ transient frequency from 0.33 +/- 0.4 Hz to 0.77 +/- 0.04 Hz following 24 h treatment; these Ca2+ -handling abnormalities were completely prevented by ETZ (0.28 +/- 0.07 Hz). Our study demonstrates a novel role for CA in mediating the hypertrophic response of cardiac myocytes to PE and suggests that CA inhibition represents an effective therapeutic approach towards mitigation of the hypertrophic phenotype.

Figure 1. Effect of phenylephrine and the membrane-permeant carbonic anhydrase inhibitor 6-ethoxyzolamide (ETZ) on cell surface area of cultured neonatal rat ventricular myocytes

A and B, micrograph images of cultured rat neonatal myocytes treated with 100 μm ETZ, 10 μm phenylephrine (PE), or untreated (control). Scale bars are 10 μm. A, cells were treated with ETZ and/or PE at the onset of the experiment in an early intervention protocol (Early). Cells were imaged after 24 h treatment. B, in a late intervention protocol (Late) cells were grown under either control or 10 μm PE-treated conditions for a total of 48 h before the images were collected. ETZ (100 μm) was added to the culture medium at the 24 h point of the experiment in the indicated samples. C, cells were treated for 24 h under the ‘Early’ intervention (blue columns) or ‘Late’ intervention protocol (red columns). Cells were treated with 10 or 100 μm ETZ. Values are mean ±s.e.m., n = 6–7. *P < 0.05, compared with control group. #P < 0.05, compared with PE-treated group.

Figure 2. Effect of hypertrophic factors and carbonic anhydrase inhibitors on adult cardiomyocyte hypertrophy

A, micrograph images of cultured adult mouse myocytes treated with 100 μm ETZ, 1 μm angiotensin II (AngII), or untreated (control, C) (scale bar, 30 μm). Cells were treated with ETZ and/or AngII for 24 h, and the images collected after 24 h treatment. Examples of cells used for cell surface area measurement are indicated by a black dot. B, cells were treated for 24 h with 100 μm ETZ or 100 μm MTZ, or for 24 h with either 1 μm AngII or 10 μm PE, or treated for 24 h with either 1 μm AngII or 10 μm PE in the presence of 100 μm ETZ or in the presence of 100 μm MTZ. Light blue bars and dark blue bars represent ETZ and MTZ treatment groups, respectively. Values are mean ±s.e.m., n = 3–4 trials (total cells analysed in each group, 49–188). *P < 0.05, compared with control group. #P < 0.05, compared with PE-treated group. ‡P < 0.05, compared with AngII-treated group.

Figure 3. Expression of atrial natriuretic factor (ANF) and carbonic anhydrase II (CAII) transcripts in neonatal rat ventricular cardiomyocytes

Rat neonatal cardiomyocytes were treated for 24 h under the ‘Early’ intervention (blue bars) or for 48 h in the ‘Late’ intervention protocol (red bars) with ETZ (100 μm), sham (control, C) or PE (10 μm), as indicated. A, RT-PCR analysis of ANF and CAII mRNA expression in control cardiomyocytes. Amplicons were analysed on 1% agarose–EtBr gels. PCR primers amplified ANF (1, 3) and CAII (2, 4). Template was either reverse transcribed control myocyte mRNA (1, 2) or mRNA prepared without reverse transcriptase (3, 4). B, ANF and CAII expression were quantified by real-time quantitative RT-PCR. Data were corrected for variation using GAPDH expression and results expressed as transcript expression normalized to GAPDH. Values are mean ±s.e.m., n = 4 for each treatment group. *P < 0.05, compared with control group.

Figure 4. Effect of phenylephrine and 6-ethoxyzolamide on transporter and carbonic anhydrase protein levels, in neonate cardiomyocytes

A, cellular lysates were prepared from neonatal rat ventricular myocytes, which were untreated controls (C), treated with 10 μm ETZ (E), treated with 10 μm phenylephrine (P, PE) or treated with PE and ETZ (P + E). Immunoblots of the myocardial lysates were probed with antibody against NHE1, AE3, Slc26a6, CAII, CAIV and β-actin antibody, as indicated. B, expression levels of transporters and carbonic anhydrases, quantified by densitometry, were normalized to β-actin expression. Cells were treated for 24 h under the ‘Early’ intervention (blue bars) or for 48 h in the ‘Late’ intervention protocol (red bars) with ETZ (100 μm), sham (control) or PE (10 μm). Values are mean ±s.e.m., n = 4–7. *P < 0.05, compared to control.

Figure 5. Effect of phenylephrine and 6-ethoxyzolamide on carbonic anhydrase and ANF protein levels in adult cardiomyocytes

A, cellular lysates were prepared from adult mouse ventricular myocytes, which were untreated (controls, C), treated with 100 μm 6-ethoxyzolamide (E, ETZ), treated with 10 μm phenylephrine (P, PE) or treated with phenylephrine and 6-ethoxyzolamide (P + E). Immunoblots of the cardiomyocyte lysates were probed with antibody against CAII, CAIV and ANF, as indicated. C, expression levels of carbonic anhydrases and ANF were quantified by densitometry. Cells were treated for 24 h with ETZ (100 μm), sham (control, C) or PE (10 μm). Values are mean ±s.e.m., n = 4. *P < 0.05, compared to control.

Figure 6. Effect of phenylephrine treatment on Cl⁻–HCO₃⁻ exchange activity

HEK293 cells were cotransfected with α_1a-adrenergic receptor, and with AE3fl (A) or SLC26A6 (B) cDNAs. Forty-eight hours after transfection, Cl⁻–HCO₃⁻ exchange assays were performed on these cells before (dark trace) and 10 min after (light trace) exposure to PE (10 μm). Initial rates of change of pH_i during the first 100 s were estimated from the slope of the line fitted by the least squares method before and after treatment. Traces were superimposed to compare slopes. C, summary of the effect of PE (10 μm, 10 min) on transport activity of HEK293 cells co-expressing α_1a-adrenergic receptor with AE3fl (n = 3), or SLC26A6 (n = 4). *P < 0.05, compared with control group.

Figure 7. Effect of carbonic anhydrase inhibition on AE3fl and NHE1 transport activity

A, lysates prepared from HEK293 cells transfected with pcDNA3 (lane 1, empty vector) or AE3fl-HA cDNA (lane 2), or AP1 cells (lane 3), or AP1 cell line stably transfected with the pYN4+ plasmid containing HA-tagged NHE1 (lane 4) were immunoblotted and probed for the expression of AE3fl-HA or NHE1-HA. B and C, dose–response curves of the ETZ effect on AE3fl and NHE1 transport activity. HEK293 cells transfected with AE3fl-HA were subjected to anion exchange assays (B), and AP1 cells expressing NHE1-HA, subjected to NHE1 activity assay (C), in the absence and presence of varied concentrations of ETZ.

Figure 8. Effect of carbonic anhydrase inhibition on basal pH_i in single mouse cardiomyocytes stimulated with the α₁-adrenergic agonist phenylephrine

A, example of a single mouse myocyte used for pH_i measurement experiments. Cell was imaged with differential interference contrast microscopy (DICM). Cardiac myocytes were loaded with BCECF pH-sensitive fluorescent dye to measure pH_i. Scale bar, 30 μm. B, changes in steady-state pH_i (ΔpH_i) observed in myocytes treated with the α₁-adrenergic agonist, phenylephrine (PE, 10 μm; red trace), treated with the CA inhibitor, 6-ethoxyzolamide (ETZ, 100 μm; blue trace), or a combination of phenylephrine and 6-ethoxyzolamide (ETZ + PE, green trace), for 10 min in Ringer buffer solution containing 25 mm NaHCO₃⁻. For control experiments, cardiomyocytes were incubated for 10 min in Ringer solution containing 25 mm NaHCO₃⁻ (black trace). Gray shading highlights period before treatment. C, summary of cardiomyocyte pH unit changes (ΔpH_i) after 10 min of no treatment (control), or 10 min with 10 μm PE, 100 μm ETZ, or 10 μm PE + 100 μm ETZ. *P value < 0.05, versus control group. Colours of columns correspond to colour coding in B. Number of trials is shown in parentheses.

Figure 9. Cytosolic Ca²⁺ and contractile activity in rat neonatal cardiomyocytes

A, representative traces of calcium transient recordings of rat neonatal cardiac myocytes after 24 h treatments with either 10 μm phenylephrine alone or 10 μm phenylephrine with 100 μm ethoxyzolamide. ΔF represents change in 525 nm fluorescence signal, which is a measure of cytosolic [Ca²⁺]. B, frequency of rat neonatal cardiac myocyte contractions after 24 h treatment with either 10 μm phenylephrine (PE), 100 μm ethoxyzolamide (ETZ) or 10 μm phenylephrine with 100 μm ethoxyzolamide (PE + ETZ). *P value < 0.05, control group versus PE group. #P value < 0.05, PE group versus PE + ETZ group. n = 8–9 trials for all treatment groups.

Figure 10. Proposed model for anti-hypertrophic action of 6-ethoxyzolamide

G protein-coupled receptors (GPCR), including receptors for AngII, endothelin-1 and α_1a-R, are coupled to activation of protein kinase C (PKC) in cardiomyocytes. PKC directly activates AE3 by phosphorylation of Ser-67 (Alvarez et al. 2001) and also activates the MAP kinase signalling pathway (MAPK), which in turn activates NHE1. AE3 and NHE1 physically associate with cytosolic carbonic anhydrase II (CAII), which activates their transport activity by supplying HCO₃⁻ and H⁺ for their respective transport functions. Co-activation of AE3 and NHE1 is pathological, since the acid generated by AE3 efflux of bicarbonate is in turn removed by NHE1. Resulting hyperactivity of NHE1 causes cellular sodium loading. Sodium activates PKC (Hayasaki-Kajiwara et al. 1999), resulting in feed-forward activation of PKC. Elevated cytosolic sodium levels reduce activity of the plasma membrane sodium–calcium exchanger, which is normally required to maintain low calcium levels. Activated PKC and elevated cytosolic calcium levels will contribute to hypertrophic growth in the myocardium (Karmazyn, 2001; Frey et al. 2004). 6-Ethoxyzolamide may arrest the hypertrophic programme directly by inhibition of carbonic anhydrase and indirectly by limiting substrates for transport by NHE1 and AE3fl.