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Enhanced activity of ventricular Na⁺-HCO₃^– cotransport in pressure overload hypertrophy

¹Department of Cardiology and Vascular Regenerative Medicine, and ²Department of Pathology and Cell Regulation, Kyoto Prefectural University of Medicine, Kyoto, Japan; and ³Nora Eccles Harrison Cardiovascular Research and Training Institute, University of Utah, Salt Lake City, Utah

Submitted 6 September 2006 ; accepted in final form 8 March 2007

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The Na⁺-HCO₃^– cotransporter (NBC) plays a key role in intracellular pH (pH_i) regulation in normal ventricular muscle. However, the state of NBC in nonischemic hypertrophied hearts is unresolved. In this study, we examined functional and molecular properties of NBC in adult rat ventricular myocytes. The cells were enzymatically isolated from both normal and hypertrophied hearts. Ventricular hypertrophy was induced by pressure overload created by suprarenal abdominal aortic constriction of 50% for 7 wk. pH_i was measured in single cells using the fluorescent pH indicator 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein. Real-time PCR analysis was used to quantitatively assess expression of NBC-encoding mRNA, including SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1). Our results demonstrate that: 1) mRNA levels of both the electrogenic NBCe1 (SLC4A4) and electroneutral NBCn1 (SLC4A7) forms of NBC were increased by aortic constriction, 2) the onset of NBC upregulation occurred within 3 days after constriction, 3) normal and hypertrophied ventricles displayed regional differences in NBC expression, 4) acid extrusion via NBC (J_NBC) was increased significantly in hypertrophied myocytes, 5) although acid extrusion via Na⁺/H⁺ exchange was also increased in hypertrophied myocytes, the relative enhancement of J_NBC was larger, 6) membrane depolarization markedly increased J_NBC in hypertrophied myocytes, and 7) losartan, an ANG II AT₁ receptor antagonist, significantly attenuated the upregulation of both NBCs induced by 3 wk of aortic constriction. Enhanced NBC activity during hypertrophic development provides a mechanism for intracellular Na⁺ overload, which may render the ventricles more vulnerable to Ca²⁺ overload during ischemia-reperfusion.

cardiac myocytes; intracellular pH regulation; messenger ribonucleic acid; polymerase chain reaction

In addition to mediating acid extrusion, activation of NBC and NHE also elicits Na⁺ influx (13, 14, 42, 47). There is increasing evidence linking intracellular Na⁺ concentration ([Na⁺]_i) accumulation and cardiac hypertrophy (5, 9, 14, 15, 30). NHE is thought to be an important downstream mediator of this Na⁺-induced effect (8, 13, 14, 18, 19). In this regard, cardiac hypertrophy is associated with increased activity of NHE-1 (38), the primary cardiac subtype of NHE. Similarly, blockade of NHE-1 has been shown to prevent development of hypertrophy in various experimental models (1, 8, 13, 19). In addition, NHE-1 inhibition blocks the development of necrosis in the absence of apparent, measurable hypertrophy in the hereditary cardiomyopathic hamster (5). Taken together, these findings suggest that NHE plays an important role in cardiac hypertrophy. The renin-angiotensin system is also a key mediator for pressure overload-induced hypertrophy as revealed by the blocking action of losartan on hypertrophic development and gene expression (26).

Considerable information exists concerning cardiac NBC activity under normal conditions (24, 25, 47). In addition, previous studies have shown that ventricular NBC is activated during ischemia (3, 13, 35, 37, 39, 41), and NBC blockade minimizes reperfusion injury (21). However, little is known concerning the functional properties and molecular expression of NBC in nonischemic hypertrophy. Similarly, the relationship between angiotensin and NBC expression in hypertrophic development is unknown. In this study, we examined these questions in ventricular myocytes isolated from hypertrophied rat hearts subjected to nonischemic pressure overload (aortic constriction).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Hypertrophic rat model. All protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Kyoto Prefectural University of Medicine. Pressure-overloaded rats (male Wistar rats) were created by suprarenal abdominal aortic constriction of 50% with a clamp occluder (MT Giken, Tokyo, Japan) at the age of 8 wk (body wt 250 g). They were fed normal rat chow and water ad libitum. After the surgery (7 wk), the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (35 mg/kg). A Microtip catheter (Millar Instruments, Houston, TX) was inserted from a carotid artery to measure blood pressure and heart rate. The degree of left ventricular (LV) hypertrophy was evaluated as the heart weight-to-body weight ratio. We used gender- and age-matched rats as controls. To assess the possible effects of the surgery itself on mRNA expression and NBC function, we also performed sham operations (surgery but no aortic constriction) on nine animals. Measurements made 7 wk later revealed no significant changes in NBC mRNA or acid extrusion of NBC (J_NBC).

Myocyte isolation. After completion of the hemodynamic measurements, the heart was attached to an aortic cannula and perfused for 5 min with a Ca²⁺-free solution containing (in mM): 126 NaCl, 4.4 KCl, 5.0 MgCl₂, 1.0 NaH₂PO₄, 20 taurine, 5.0 creatine, 5.0 sodium pyruvate, 24 HEPES, and 22 dextrose (pH 7.2 adjusted with NaOH). This was followed by 8–9 min of perfusion with the same solution containing 0.05 mM Ca²⁺ along with collagenase (type H, 0.5 mg/ml; Sigma) and protease (type 14, 0.1 mg/ml; Sigma). The heart was then perfused for 5 min with 0.1 mM Ca²⁺ solution to wash out the enzymes. All solutions were bubbled with 100% O₂ and kept at 37°C. After the perfusion, the free wall (FW) of the LV was separated, and only cardiomyocytes isolated from the midmural layer were used for measurement of pH_i. The midmural tissue was minced in the 0.1 mM Ca²⁺ solution and gently shaken at 37°C for 10 min. Cells were stored at room temperature in 1.0 mM Ca²⁺ solution and used within 6 h after cell isolation for experiments.

Myocyte superfusion bath chamber. The flow-through cell bath was mounted on the stage of an inverted epifluorescence microscope (Nikon ECLIPSE TE2000-U; Nikon, Tokyo, Japan). The clear glass bottom of the bath was coated with laminin to improve cell adhesion. Bathing solutions were held at 36–37°C as they flowed continuously through the bath at 3–4 ml/min. Complete replacement of a new solution required 5 s.

Experimental solutions. The following two systems were used to buffer H⁺ in the myocyte bathing solutions: HEPES buffered with no added CO₂/HCO₃^– and CO₂/HCO₃^– buffered with no added HEPES. The pH of all bathing solutions was adjusted to 7.4. The normal HEPES-buffered solution contained (in mM): 126 NaCl, 11 dextrose, 4.4 KCl, 1.0 MgCl₂, 1.08 CaCl₂, and 24 HEPES, with pH adjusted with 12.9 mM NaOH. The normal CO₂/HCO₃^–-buffered solutions were continuously gassed with 5.0% CO₂-95.0% O₂ and contained (in mM) 120 NaCl, 11 dextrose, 4.4 KCl, 1.0 MgCl₂, 1.08 CaCl₂, and 18.5 NaHCO₃.

We used hyperkalemia (44.4 mM extracellular K⁺ concentration) to depolarize myocytes and activate NBCe (47). The normal (4.4 mM)-K⁺ CO₂/HCO₃^–-buffered solution contained (in mM) 100 NaCl, 11 dextrose, 4.4 KCl, 1.0 MgCl₂, 40 N-methyl-D-glucamine (NMDG), 1.08 CaCl₂, and 18.5 NaHCO₃ (gassed with 5.0% CO₂-95.0% O₂, pH adjusted to 7.4 with HCl). The normal (4.4 mM)-K⁺ HEPES-buffered solution contained (in mM) 100 NaCl, 11 dextrose, 4.4 KCl, 1.0 MgCl₂, 40 NMDG, 1.08 CaCl₂, 24 HEPES, and 12.9 NaOH (pH adjusted to 7.4 with HCl). For high (44.4 mM)-K⁺ solution, 40 mM NMDG was replaced with the same concentration of KCl. Using these solutions, the switch from 4.4 to 44.4 mM K⁺ occurred with extracellular Na⁺ and Cl^– concentration, and osmolarity held constant (47). To inhibit depolarization-induced cell contraction, 10 µM nifedipine was included in the normal- and high-K⁺ solutions to block L-type Ca²⁺ current.

The pipette filling solution used for capacitance measurements contained (in mM) 125 potassium asparatate, 25 KCl, 5 Na₂ATP, 1 MgCl₂, 5 HEPES, and 10 EGTA (pH adjusted to 7.3 with KOH). Cells were bathed with the normal HEPES-buffered solution.

Measurement of cell size and membrane capacitance. Myocyte dimensions were evaluated in normal bathing solution using a laser-scanning confocal microscope (FV-1000; Olympus) equipped with a x60 objective (PlanApo N, oil, numeric aperture 1.42). We simultaneously obtained both the differential interference contrast (DIC) image and membrane-selective fluorescent dye (di-4-ANEPPS)-stained confocal image of the cells (excitation 488 nm, emission 520 nm). The width and length of the cells were measured from DIC images. Myocyte thickness was determined as the distance between the top and bottom margins of each cell's confocal image.

We also measured membrane capacitance (pF) as an index of cell size using the whole cell patch-clamp technique. Pipettes were pulled from borosilicate glass (GD-1.5; Narishige, Tokyo, Japan) and had resistances of 3–5 M after filling. They were connected to an Axopatch-200A patch-clamp system (Axon Instruments). Membrane capacitance was measured by applying hyperpolarizing 5-mV steps (25-ms duration) from a holding potential of 0 mV. Signals were filtered at 2 kHz and digitized at 10 kHz. Myocyte surface area was estimated from capacitance assuming a specific capacitance of 1 µF/cm². All measurement was performed at room temperature.

Whole cell fluorescence measurement of pH_i. pH_i was measured in single myocytes with an epifluorescence system (Ion Optix, Milton, MA) using acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)5-(6)carboxyfluorescein (BCECF-AM) as the fluorescent pH indicator. All pH_i measurements done on hypertrophied myocytes were from animals subjected to 7 wk of aortic constriction. Myocytes were incubated at room temperature for 30 min in the 13 µM of BCECF-AM-containing solution. Dye-loaded cells were bathed in the normal solution (HEPES or CO₂/HCO₃^– buffered) for at least 20 min before pH_i measurements began. Dual excitation (440 and 495 nm) was provided by a 75-watt Xenon arc lamp and transmitted to the myocytes via a x20 objective. Emitted fluorescence was collected with a photomultiplier tube equipped with a bandpass filter centered at 535 ± 5 nm. The 495-to-440 nm fluorescence ratio was digitized at 5–10 kHz (ION WIZARD fluorescence analysis software). At the end of every experiment, the fluorescence ratio was converted to pH by calibration as previously described (47) using solutions of varying pH that also contained 10 µM nigericin and 15 mM of 2,3-butanedione monoxime.

Determination of intracellular buffering capacity. The intrinsic (non-CO₂) buffering capacity (_int) of rat myocytes was measured by exposing cells to varying concentrations of NH₄Cl in bathing solutions that contained no added HCO₃^–, CO₂, Ca²⁺, or Na⁺ and buffering with HEPES (25, 37, 48). pH_i was allowed to stabilize in Na⁺-free solution before application of NH₄Cl. _int (mM/pH) was calculated according to a previously described procedure (48). Intracellular buffering because of CO₂ (_CO2) was calculated as _CO2= 2.3[HCO₃^–]_i, where intracellular HCO₃^– concentration ([HCO₃^–]_i) = [HCO₃^–]_o10^pH_i–pH_o ([HCO₃^–]_o is extracellular HCO₃^– concentration and pH_o is extracellular pH). Total buffering capacity (_T) is equal to the sum of _int and _CO2. It was assumed that _T = _int when myocytes were bathed in HEPES-buffered solutions containing no added CO₂/HCO₃^–.

Determination of efflux of acid equivalents via NBC and NHE. We activated NBC and NHE by inducing intracellular acidosis while holding pH_o constant at 7.4. Intracellular acid loads were created with two different techniques as follows: 1) rapid switching of the bathing solution from HEPES buffered to CO₂/HCO₃^– buffered and 2) brief exposure to ammonium chloride (10–15 mM), i.e., ammonium prepulse. In the absence of NHE inhibition with cariporide, the first technique activates both NHE and NBC. Similarly, ammonium prepulses activate both transporters when the cells are bathed in CO₂/HCO₃^–-buffered solution. In contrast, only NHE is activated when the prepulse is applied to myocytes bathed in HEPES-buffered solution containing no added CO₂/HCO₃^–. The net influx of HCO₃^– that occurs via NBC (J_NBC) during pH_i recovery from acid loading is equivalent to extrusion of H⁺ and was calculated at successive values of pH_i as _T x dpH_i/dt. Acid extrusion via NHE (J_NHE) was calculated as _int x dpH_i/dt.

RT-PCR assay of NBC. Up to 100 mg of total RNA were extracted from homogenized midlayer LV tissues of normal and hypertrophic hearts using 1 ml TRIzol regent according to the manufacturer's instruction (Invitrogen, Tokyo, Japan). After addition of 0.2 ml chloroform and then 0.5 ml 2-propanol, the purified RNA-contained sample was pelleted by repeated ultracentrifugation (12,000 rpm) for 10–15 min. After RNA was washed with 70% ethanol, the purified RNA material was dissolved in diethyl pyrocarbonate (DEPC)-water. The concentration of RNA was quantified by densitometric measurement of ultraviolet absorption at 260/280 nm. The RNA sample was diluted with DEPC-water to final a concentration of 2 mg/ml. The RT-PCR method was used to detect mRNA for NBC isoforms. Total RNA of 5 µg was reverse transcribed into first-strand cDNA using the SuperScript III First-Strand synthesis system (Invitrogen), and PCR amplification was carried out with 33 cycles of denaturation at 95°C for 30 s, annealing at 58.2°C for 30 s, and extension at 72°C for 60 s. Sense and antisense primers used in RT-PCR were 5'-AGGAATCTGACATCCTCCAGTCTC-3' and 5'-CAGTTCTCTGTAGTTCTTCACAGTCA3' for SLC4A4; 5'-CAAAGCACCAGCTATGGTCATCT-3' and 5'-ATCAGCTCCTCCCCAATTTC-3' for SLC4A5; and 5'-ACCCCAGAACAGTCCTCCTT-3' and 5'-GGATGCCTCAGCTCCTGTAG-3' for SLC4A7. After the completion of PCR, the products were analyzed by 2% agarose gel electrophoresis containing 0.05% ethidium bromide and photographed under ultraviolet illumination.

Real-time quantitative PCR analysis of NBC. The expression level of each NBC isoform was measured using real-time quantitative PCR analysis. To obtain cardiac samples for real-time quantitative PCR analysis, right ventricle (RV), apex, interventricular septum, and FW of the LV were individually dissected out from whole hearts. We assessed transmural distribution of NBC mRNA in another set of experiments by dividing the LV FW into three portions (i.e., endo-, mid-, and epicardium). Before dissection, blood was thoroughly washed from a heart with Ca²⁺-free solution using Langendorff perfusion for at least 5 min. cDNA synthesized with 250 ng of total RNA was analyzed by kinetic real-time PCR using the Light Cycler system (Roche) with Platinum SYBR Green qPCR SuperMix (Invitrogen). Glyceraldehyde-3-phosphate dehydrogenase was used for normalization between samples, and the comparative threshold method was used to assess the relative abundance of the targets. The primers used for SLC4A4 and SLC4A7 were the same as those for RT-PCR analysis.

mRNA levels of the classical markers of hypertrophy [atrial natriuretic peptide (ANP), transforming growth factor-₁ (TGF-₁), -myosin heavy chain (-MHC), and -myosin heavy chain (-MHC)] were also measured by real-time quantitative PCR analysis. The primers used for these markers were commercially available [TGF-₁ was purchased from Qiagen (Tokyo, Japan) and ANP, -MHC and -MHC were from Nippon (Tokyo, Japan)].

Early time course of hypertrophic development and NBC expression. In this protocol, animals were studied at 1, 3, and 5 days after aortic constriction. The measured parameters included systemic blood pressure, body weight, heart weight, and LV weight. We also used real-time quantitative PCR to measure mRNA levels of NBC and the classical indexes of hypertrophy (ANP, TGF-₁, -MHC, and -MHC).

Role of ANG II in NBC hypertrophic expression. In this protocol, aortic-constricted rats were randomly assigned to one of the following two groups: 1) operation only (n = 6) or 2) operation plus intraperitoneal administration of 30 mg·kg^–1·day^–1 losartan, an ANG II AT₁ receptor inhibitor (n = 6). Later (3 wk), systemic blood pressure, body weight, heart weight, and LV weight of aortic-constricted rats were measured, along with NBC mRNA levels from LV FW using real-time quantitative PCR.

Statistics. Summarized results are expressed as means ± SE. A paired Student's t-test was used to test significance between results obtained with each cell serving as its own control. An unpaired t-test was used to test significance between results obtained from different cells. P < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Characteristics of normal and aortic-constricted rats. Table 1 summarizes the general characteristics of normal rats and those subjected to 7 wk of pressure overload. There were no significant differences in either heart rate or body weight between the two groups. In contrast, aortic-constricted rats displayed significant increases in systolic and diastolic blood pressure, heart weight, the heart weight-to-body weight ratio, and the LV weight-to-body weight ratio. These increases are consistent with ventricular hypertrophy. Pressure-overloaded rats had neither pleural nor peritoneal effusion, and no significant difference was observed in the lung weight between the pressure-overloaded and the normal rats, indicating that the pressure-overloaded rat hearts were not failing.

View larger version (32K): [in this window] [in a new window]   Fig. 1. Representative confocal images of a normal ventricular myocyte (left) and one from a heart subjected to 7 wk of suprarenal abdominal aortic constriction of 50% (right). Cells were isolated from the midmural region of the left ventricular (LV) free wall (FW) and loaded with di-4-ANEPPS.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Qualitative assessment of Na+-HCO3– cotransporter (NBC) mRNA expression in normal rat tissues using RT-PCR. Both SLC4A4 (encoding electrogenic NBC, NBCe1) and SLC4A7 (electroneutral NBC, NBCn1) were detectable in the heart (midmural layer of LV). SLC4A5 was not detectable in heart muscle. NBCs were amplified in 33 cycles with the specific primers described in MATERIALS AND METHODS.

View larger version (9K): [in this window] [in a new window]   Fig. 3. Regional expression of NBC in normal and hypertrophied ventricles measured with the real-time PCR method. In all regions examined, both SLC4A4 (left) and SLC4A7 (right) were increased significantly in hypertrophied hearts after 7 wk of aortic constriction (filled bar, n = 6 samples from 6 rats) compared with normal (open bar, n = 6 samples from 6 rats). Results are expressed as means ± SE. P < 0.05, statistically significant compared with normal (*), statistically significant compared with normal apex (#), and statistically significant compared with hypertrophied apex (+).

View larger version (8K): [in this window] [in a new window]   Fig. 4. Transmural expression of NBC in the LV measured with real-time PCR analysis. Compared with normal (open bar, n = 7 samples from 7 rats), NBC was upregulated in all regions of hypertrophied hearts 7 wk after aortic constriction (filled bar, n = 7 samples from 7 rats). Results are expressed as means ± SE. P < 0.05, statistically significant compared with normal (*) and statistically significant compared with normal epicardium (#).

1

_int in normal and hypertrophic myocytes. It is necessary to determine _int to quantitatively assess H⁺ flux (J_H⁺) via NBC and NHE. It is also important to directly measure _int since it may be altered by cardiac hypertrophy. Previous reports, however, demonstrated that ventricular _int was not significantly altered by either pressure-overloaded (16) or volume-overloaded (37) hypertrophy. Our results confirm this finding for pressure-overload hypertrophy (data not shown). For normal cells, the _int-pH_i relationship is given by: _int = –11,135 + 5,058.4 x (pH_i) – 754.98 x (pH_i)² + 37.182 x (pH_i)³. The relationship for hypertrophied myocytes is: _int = –21,458 + 9,779.0 x (pH_i) – 1,468.9 x (pH_i)² + 72.922 x (pH_i)³.

J_NBC in normal and hypertrophied myocytes. Intracellular acidosis is a key activator of both NBC and NHE. Figure 5, A and B, presents, respectively, examples of NBC activation by intracellular acidosis (pH_o = 7.4) induced by 1) switching from HEPES-buffered to CO₂/HCO₃^–-buffered solution and 2) an ammonium prepulse. Cariporide (30 µM) was present in all solutions to block NHE. The relationship between J_NBC and pH_i generated by these two techniques is summarized in Fig. 5, C and 5D. Both acid-loading techniques revealed that J_NBC in hypertrophied myocytes was significantly higher than normal at any given value of pH_i.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Effect of 7 wk of aortic constriction on acid extrusion via NBC (JNBC) in isolated myocytes from midmural layer of LV FW. A and B: time courses of pHi recovery from intracellular acidosis induced by switching HEPES buffer to CO2/HCO3– buffer (A) and the application and removal of NH4Cl (15 mM) in CO2/HCO3– buffer (B). All bathing solutions contained NHE inhibitor, cariporide (30 µM). C and D: summary of the relationship between JNBC and pHi derived from the experimental protocols shown in A and B. C: switching HEPES buffer to CO2/HCO3– buffer. Data have been pooled from several cells and averaged for the following 0.05 pHi ranges starting at pHi 6.6, with sample sizes, normal: n = 10, 14, 15, 6, 5, 8, 8, 5, 10, 10, 12, and 6 cells from a total of 10 rats; hypertrophy: n = 18, 16, 18, 12, 10, 24, 13, 13, 25, 20, 16, and 6 cells from a total of 8 rats. JNBC was significantly increased in hypertrophied myocytes. *P < 0.05. D: ammonium prepulse. JNBC has been averaged over successive 0.1 unit pHi bins, starting for the range 6.2–6.3, and from pHi 6.4 and above, data have been averaged over successive 0.05 pHi ranges; normal: n = 5, 7, 6, 7, 7, 7, 13, 14, 15, 12, 17, 9, 10, 10, 7, 9, 7, and 5 cells from a total of 7 rats; hypertrophy: n = 7, 11, 6, 7, 9, 11, 16, 19, 17, 17, 16, 14, 16, 16, 11, 11, 11, and 6 cells from a total of 7 rats. P < 0.05 at all points.

View larger version (13K): [in this window] [in a new window]   Fig. 6. Comparison between JNBC and acid extrusion via NHE (JNHE) in normal and hypertrophied myocytes after 7 wk of aortic constriction. A: JNBC and JNHE as a function of pHi in normal myocytes. Flux values for NHE have been averaged over successive 0.05 pHi ranges starting (left to right) with 6.50–6.55; n = 13, 18, 19, 21, 25, 28, 26, 26, 16, 13, 9, 6, 5, and 5 cells from a total of 9 rats. The JNBC results were taken from Fig. 5D. B: JNBC and JNHE as a function of pHi in hypertrophied myocytes. JNHE have been averaged over successive 0.05 pHi ranges starting with 6.50–6.55; n = 5, 5, 13, 15, 23, 25, 24, 27, 26, 25, 20, 12, 12, and 5 cells from a total of 9 rats. The JNBC results were obtained from Fig. 5D. P < 0.05 at all points in A and B. C: acid efflux ratio [JNBC/(JNHE + JNBC)] in normal and hypertrophied myocytes calculated from the results in A and B. Compared with JNHE, JNBC displayed a proportionally larger increase in hypertrophied cells. Ammonia prepulses were used to acid load cells in all experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 7. Effect of membrane depolarization on JNBC during recovery from intracellular acidosis. A and B: comparison of JNBC-pHi curves from normal (A) and hypertrophied (B) myocytes at normal resting membrane potential [4.4 mM extracellular K+ concentration ([K+]o)] and in cells depolarized with 44.4 mM [K+]o during pHi recovery from an acid load (induced by switching HEPES buffer to HCO3– buffer). Depolarization significantly stimulated JNBC in both normal and hypertrophied myocytes. All bathing solutions used were buffered with HCO3– and contained 30 µM of cariporide. Data have been averaged over successive 0.05 pHi ranges. 4.4 mM K+ normal: n = 6, 5, 8, 8, 5, 10, 10, 12, and 6 cells from a total of 9 rats; hypertrophy: n = 10, 24, 13, 13, 25, 20, 16, and 6 cells from a total of 7 rats. 44.4 mM K+ normal: n = 5, 4, 5, 7, 6, 7, 8, 7, and 15 cells from a total of 8 rats; hypertrophy: n = 4, 8, 6, 8, 10, 9, 9, and 12 cells from a total of 6 rats. *Statistically significant (P < 0.05).

View larger version (7K): [in this window] [in a new window]   Fig. 8. Effect of losartan on NBC expression. The upregulation of NBCe1 (SLC4A4) and NBCn1 (SLC4A7) induced by 3 wk of aortic constriction was attenuated significantly by chronic treatment with losartan (30 mg·kg–1·day–1). Real-time PCR was used to quantify mRNA levels. SLC4A4: n = 6 normal, 6 operated, and 6 losartan rats; SLC4A7: n = 6 normal, 6 operated, and 6 losartan rats. *Statistically significant compared with operated.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Expression of NBCs. The SLC4 family of transporters consists of membrane proteins that mediate transmembrane flux of bicarbonate (or CO₃^2–) coupled to Na⁺ and/or Cl^– (32). As such, they play a major role in the regulation of pH_i in a variety of organs, including heart. Two cardiac NBCs have been identified as electrogenic. They include products of the SLC4A4 gene, NBCe1-B or hhNBC (6), and the SLC4A5 gene, NBCe2-C or NBC4c (31, 32, 34, 44). In addition, one cardiac NBC is reported to be electroneutral, NBCn1(-B), encoded by the SLC4A7 gene (7, 32).

Previous expression studies have revealed both NBCe1 and NBCn1 in adult rat ventricle (7, 21). In contrast, the presence of NBCe2 has not been identified in rat heart although it is reported to express in other mammalian hearts, including humans (31, 34, 44). We were unable to detect SLC4A5 mRNA expression in rat heart but did find it in lung and kidney tissue. Thus ventricular SLC4A5 mRNA levels may be below our measurement resolution.

Hypertrophied myocytes displayed significantly increased expression of both NBCe1 and NBCn1 (Figs. 3 and 4). The onset of NBC upregulation was rapid and occurred within 3 days after constriction. It coincided with increases in the LV weight-to-body weight ratio, the heart weight-to-body weight ratio, as well as ANP and -MHC expression (Table 3). There is no previous study showing the early time course of NBC expression during pressure overload. Our observation that NBC upregulation did not precede the rise in all hypertrophic markers (Table 3) raises the possibility that augmented NBC activity may not contribute directly to hypertrophic development. However, we cannot exclude this possibility given the close early temporal relationship between NBC upregulation and hypertrophy. Previous studies have also reported increased mRNA expression of NBC associated with cardiac pathology. For example, Sandmann et al. (33) reported that mRNA levels of NBC-1 (NBCe) were increased twofold in the rat LV FW after myocardial infarction. In human cardiomyopathic hearts (i.e., ischemic and dilated cardiomyopathy), Khandoudi et al. (21) demonstrated that hhNBC (NBCe1) mRNA expression increased, whereas mRNA level of NBCn remained unchanged. The discrepancy between this study's NBCn mRNA findings and ours may be related to the differences in species and/or disease state.

Our mRNA measurements were obtained from ventricular tissue and thus may contain message from cells other than ventricular myocytes. In this regard, a recent immunohistochemical study of normal rat ventricle reported no NBCn1 in ventricular myocytes but high levels in endothelial cells of ventricular capillaries (11). Similarly, NBCn1 has recently been found in the vascular smooth muscle of normal mouse coronary arteries (4). Thus we cannot rule out contributions from these nonmyocyte sources to our NBCn1 message levels. However, our recent functional analysis of NBC suggests that both electrogenic and electroneutral forms of NBC are operational in ventricular myocytes from rat, rabbit, and guinea pig (24, 47). Additional molecular and functional studies are required to fully resolve this issue. Regardless of its exact spatial distribution, our results demonstrate for the first time that NBCn1 upregulation occurs in the ventricles during chronic pressure overload. If the NBCn1 we measured was solely vascular in origin, perhaps its increase in hypertrophied hearts represents an adaptive response of the coronary vasculature to increased afterload. Regulation of pH_i is known to have important effects on vascular smooth muscle function, including contractility (4, 46).

Spatial distribution of NBC expression. Regional heterogeneity in ventricular action potentials, ion channels, and transporters is well established (9, 49), and hypertrophy can alter their spatial distribution (36, 40, 45). Compared with normal, increased NBC expression occurred throughout the hypertrophied heart. We also found transmural differences in NBC mRNA levels within the LV wall of normal hearts such that epi > endo. This gradient was not present in hypertrophied LV (Fig. 4). Thus hypertrophic growth appears to be associated with disappearance of the transmural heterogeneities in NBC expression that normally occurs in the ventricles. It is interesting that the variation in mRNA expression level in normal and hypertrophied hearts showed a quite similar pattern between SLC4A4 and SLC4A7 (Figs. 3 and 4). The complex intervention of factors, including the elevated wall stress, myocyte hyperplasia, arterial wall thickness, altered blood flow regulation, and the enhanced cardioactive hormones (e.g., ANG II and endothelin-1), may be involved in the altered state of NBC expression in hypertrophy.

The physiological significance of regional differences in NBC expression in normal hearts is unclear. However, because pH_i and [Na⁺]_i have major effects on contractility, perhaps it provides a mechanism for maintaining contractility in the face of regional differences in local acid production. In this regard, [Na⁺]_i in normal rabbit LV was found to be higher in epicardial than endocardial myocytes (10). Our finding that NBC mRNA levels are higher in epicardium than endocardium suggests that NBC activity may contribute to this [Na⁺]_i gradient.

NHE and NBC activity during intracellular acidosis. Ventricular myocytes respond to intracellular acid loads by mediating acid-equivalent efflux via NHE (J_NHE) and NBC (J_NBC) (25). Although J_NHE and J_NBC were both significantly increased in hypertrophied myocytes, the relative contribution of J_NBC to total acid efflux was larger in hypertrophied cells (Fig. 6C). Thus, compared with normal myocytes, hypertrophied cells displayed a proportionally greater reliance on NBC for translocation of acid equivalents across the sarcolemma. To our knowledge, this is the first report of NBC upregulation in hypertrophied ventricular myocytes from hearts subjected to nonischemic pressure overload. This functional enhancement of NBC activity is in accord with the augmented NBC mRNA expression we observed in hypertrophied cells. Thus, through a combined enhancement of both NBC and NHE, hypertrophied myocytes have a strikingly increased ability to extrude acid in response to acute intracellular acid loads.

Our NHE findings (Fig. 6) confirm those of prior studies demonstrating its increased activity in the setting of pressure and volume overload-induced hypertrophy (1, 5, 19, 20). It is worth noting that the surface-to-/volume ratio (S/V) of LV myocytes from aortic-banded rats is reported to remain normal (12). Thus the observed changes in J_NBC and J_NHE in hypertrophied cells are unlikely to simply reflect changes in S/V, but instead represent an actual increase in transporter density.

A shift of J_H⁺ curves for NBC and NHE in the alkaline direction will promote increased Na⁺ influx even in the absence of significant intracellular acidosis. This may produce increased steady-state [Na⁺]_i. Growing evidence implicates NHE as a key mediator of myocyte hypertrophy (8, 13, 14, 18, 19). Several ligands (e.g., endothelin-1 and ANG II) operating through G protein-coupled receptors reportedly induce hypertrophy partly by NHE activation in the presence or absence of intracellular acidosis (18). One of mechanisms underlying the NHE-induced hypertrophic effect is Na⁺ influx. The ability of pH_i to affect protein synthesis also renders NHE a potentially important regulator of cell proliferation and hypertrophy. Perhaps the enhanced activation of NBC we observed provides an additional stimulus for hypertrophic development, recognizing that upregulation of NBC may also be a consequence of hypertrophic development. A role for ANG II and the AT₁ receptor in NBC upregulation during aortic constriction in rats is strongly suggested by our finding that losartan attenuated the increased expression of both transporters (Table 4 and Fig. 8). This result also raises the possibility the reduced NBC expression may have contributed to the regression of hypertrophy.

Electrogenic NBC. Figure 7 clearly shows that hypertrophied myocytes possess a functional electrogenic NBC, since J_NBC was markedly increased by membrane depolarization. Over the range of pH_i values shown in Fig. 7, the mean change in J_NBC induced by hyperkalemia was 1.47 mM/min in normal cells and 1.65 mM/min in hypertrophied cells, suggestive of NBCe1 upregulation in hypertrophied cells. This is consistent with the augmented message levels of NBCe1 in hypertrophied myocytes (Figs. 3 and 4).

Clinical implications of enhanced NBC activity in cardiac hypertrophy. Upregulation of NBC may have significant physiological consequences for hypertrophied myocytes. For example, it may promote arrhythmias and reperfusion injury as a result of 1) its ability to generate a current in the case of NBCe and 2) [Na⁺]_i accumulation leading to [Ca²⁺]_i overload via sarcolemmal Na⁺/Ca²⁺ exchange. Inhibition of NBCe has been shown to markedly reduce myocardial damage in normal rat ventricle subjected to ischemia (21). In addition, normal ventricular myocytes display a significant rise in [Na⁺]_i when NBC is activated by intracellular acidosis (47). Our results suggest that the rise in [Na⁺]_i will be even more pronounced in hypertrophied myocytes subjected to intracellular acidosis. For example, at a pH_i of 6.53, Na⁺ influx via NBC alone will be approximately three times greater in hypertrophied myocytes (6 vs. 2 mM/min; Fig. 6, A and B). When considering combined Na⁺ entry via both NBC and NHE in hypertrophied myocytes, the total Na⁺ influx at a pH_i of 6.53 will be 2.2 times higher in hypertrophied myocytes (26 vs. 12 mM/min; Fig. 6, A and B). Thus combined inhibition of both transporters may be especially beneficial in hypertrophied hearts for mitigating injury and arrhythmias. Because [Na⁺]_i overload promotes hypertrophic development (15, 22, 43), it also seems possible that chronic attenuation of Na⁺ influx via NBC may reduce remodeling as it does when NHE is blocked (2, 19, 22). Our time course measurements of NBC upregulation (Table 3) and our losartan results (Table 4 and Fig. 8) raise the possibility that hypertrophic development may be mediated in part by NBC upregulation. However, additional studies are required to fully resolve this important issue.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was supported in part by a research grant from Banyu Pharmaceutical (to T. Yamamoto), a MERIT award from the National Heart, Lung, and Blood Institute (5R37HL-042873), and the Nora Eccles Treadwell Foundation (to K. W. Spitzer).

	ACKNOWLEDGMENTS

 
Cariporide was kindly provided by Dr. J. Puenter (Aventis Pharma, Germany). Losartan was kindly provided by Merck (Whitehouse Station, NJ).

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Yamamoto, Dept. of Cardiology and Vascular Regenerative Medicine, Kyoto Prefectural Univ. of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 602-8566, Japan (e-mail: takuy24{at}koto.kpu-m.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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