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(Circulation. 1997;95:1007-1014.)
© 1997 American Heart Association, Inc.

Articles

Hypertension Increases Connexin43 in a Tissue-Specific Manner

Jacques-Antoine Haefliger, PhD; Einar Castillo, MS; Gerard Waeber, MD; Gabriela E. Bergonzelli, PhD; Jean-Francois Aubert, MS; Esther Sutter; Pascal Nicod, MD; Bernard Waeber, MD; Paolo Meda, MD

the Department of Internal Medicine B (J.-A.H., E.C., G.W., G.E.B., P.N.), Division of Hypertension (J.-F.A., B.W.), University Hospital, CHUV-1011 Lausanne, Switzerland; and the Department of Morphology (E.S., P.M.), University of Geneva, CMU-1211 Geneve 4, Switzerland.

Correspondence to J.-A. Haefliger, PhD, Department of Internal Medicine, Laboratory of Molecular Biology 19-135, Centre Hospitalier Universitaire Vaudois, CHUV-1011 Lausanne, Switzerland. E-mail jhaeflig@chuv.hospvd.ch.

	Abstract

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Background Connexin43 (Cx43), a membrane protein involved in the control of cell-to-cell communication, is thought to play a role in the contractility of the vascular wall and in the electrical coupling of cardiac myocytes. The aim of this study was to investigate the effects of experimental hypertension on Cx43 expression in rat aorta and heart.

Methods and Results Rats were made hypertensive after one renal artery was clipped (two kidney, one-clip renal model) or after the administration of deoxycorticosterone and salt (DOCA-salt model). After 4 weeks, all rats showed a similar increase in intra-arterial mean blood pressure and in the thickness of both the aortic wall and the heart. Northern blot analysis of aorta mRNA and immunolabeling for Cx43 showed that hypertensive rats expressed twice as much Cx43 in aorta as the control animals. In contrast, no difference in Cx43 mRNA or in the immunolabeled protein was observed in heart.

Conclusions The results show that rats exhibiting a similar degree of blood pressure elevation, as the result of different mechanisms, feature a comparable increase in Cx43 gene expression, which was observed in the aortic but not in the cardiac muscle. These data suggest that localized mechanical forces induced by hypertension are major tissue-specific regulators of Cx43 expression.

Key Words: vasculature • aorta • heart • hypertension • blood pressure

	Introduction

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Specialized channels located at gap junctions represent one way by which vertebrate cells communicate.¹ Gap junctions ensure the electrical and mechanical coupling of different types of muscle cells.^{2 3} In the myocardium, this coupling is implicated in action potential propagation. This propagation, which is driven by electrochemical gradients of current-carrying ions, diffuses from one cell to the next, thereby synchronizing electrical activity.⁴ In vessels, gap junctions also provide a pathway for modulation of the contractile activity of smooth muscle cells.^{5 6 7 8 9 10} Gap junctions have also been involved in the control of cell growth¹¹ and in the regulation of glandular secretion.¹²

Gap junction channels are formed by two hemichannels, called connexons, which are present in the membrane of adjacent cells.¹³ Each connexon surrounds a pore of 1.5-nm diameter that allows for the passage of ions as well as for the exchange of metabolites and second messengers of 1 kD.¹⁴ Connexons are formed by the hexameric assembly of membrane-spanning proteins, known as connexins, which in mammals belong to a family of 13 proteins.¹⁵ Four of these proteins, referred to as Cx43, Cx45, Cx40, and Cx37, have been identified in the cardiovascular system.^{16 17} However, little is known about their role and their possible changes in cardiovascular diseases.

Chronic hypertension in humans and animal models is associated with cardiac and vascular hypertrophy caused by an increase in both blood pressure and growth factors.¹⁸ The present study was aimed to assess whether the expression of Cx43, the physiologically predominant connexin of myocardial cells⁴ and aortic smooth muscle cells,^{10 19} is altered during chronic hypertension. To this end, we investigated two rat models characterized by a similar degree of hypertension and hypertrophy of both aorta and heart. In the 2K,1C model, hypertension is produced by clipping one renal artery, leading to stimulation of renin secretion and to an Ang II–dependent elevation of blood pressure.^{20 21} In the DOCA-salt model, hypertension results from sodium retention, and renin secretion is suppressed.^{22 23 24}

We show that in these two models, hypertensive rats feature increased levels of Cx43 in the aortic wall but not in heart. Similar changes were observed regardless of the mechanism that caused hypertension, indicating that altered connexin expression is closely, if not causally, related to altered functioning of smooth muscle cells in the aorta. These observations extend to different locales of the cardiovascular system the notion that Cx43 may be differentially regulated in distinct organs of a same animal.²⁵ Our data demonstrate that this differential regulation takes place in smooth muscle cells that feature a comparable degree of hypertension-associated hypertrophy.

	Methods

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Models of Hypertension
Procedures for rat care, surgery, and euthanasia were approved by our institutional review committee for animal experiments. Male normotensive Wistar rats (Iffa Credo) that weighed 140 to 180 g were used. For surgical procedures, all rats were anesthetized with halothane (Arovet AG).

2K,1C
Thirty rats had the left renal artery constricted by a solid U-shaped silver clip of 0.2-mm internal diameter. Thirty sham-operated rats were exposed to the same surgical manipulations, except the clipping. After return to their cage, all rats were maintained on a regular diet with free access to water for 4 weeks.

DOCA-Salt
Twenty-five rats underwent a left nephrectomy and were then injected subcutaneously once a week with 50 mg/kg deoxycorticosterone acetate (DOCA; Steuli and Co AG). Throughout this 4-week treatment, rats were maintained on water supplemented with 1% NaCl and had free access to standard rat chow. Twenty-five rats that underwent a left nephrectomy but did not receive DOCA or salt served as controls.

All rats were housed at constant temperature and humidity and under standard light/dark cycles. Twenty-four hours before they were killed, the animals were instrumented with a catheter in the right internal iliac artery and a catheter in the right femoral vein. Both catheters (PE-10; Portex) were exteriorized between scapulae and filled with an heparinized 0.9% NaCl solution before rats were placed in individual cages. On the day of the experiment, the rats were placed in a plastic tube to allow continuous recording of intra-arterial pressure and heart rate with a data-acquisition system.²⁶ After an initial 2-hour period, during which hemodynamic parameters reached baseline values, blood pressure was measured for 10 minutes. The animals were then killed with an overdose of methohexital. Immediately afterward, 50 mL diethyl pyrocarbonate (Sigma)/PBS were rapidly infused through the left ventricle to wash blood cells and to avoid RNA degradation. Heart and aorta were then removed; the hearts were weighed; and all tissues were rapidly frozen in liquid nitrogen.

RNA Isolation and Northern Blot Analysis
Hearts and aortas were homogenized in 9 mL of 4 mol/L guanidine hydrothiocyanate buffer with the use of a Kinametic Polytron blender (Kriens) and layered onto a cushion of 4 mL of 5.7 mol/L CsCl. Total RNA was pelleted by a 20-hour ultracentrifugation at 33 000 rpm in a 50 Ti rotor (Beckman).

Yields were evaluated by absorbance at 260 nm. Ten micrograms of total mRNA was size-fractionated on 1% agarose gels containing 8% formaldehyde (Fluka) and 1x MOPS buffer (1x contains 20 mmol/L MOPS, 1 mmol/L EDTA, and 5 mmol/L sodium acetate; Fluka). RNAs were transferred overnight to GeneScreen membranes (EI DuPont de Nemours GmbH, NEN Division) through capillary transfer in the presence of 10x SSC (1x contains 150 mmol/L NaCl [Merck] and 15 mmol/L sodium citrate [Merck]). Membranes were UV cross-linked and vacuum-baked for 2 hours at 80°C. After prehybridization, total mRNA levels were determined by hybridization with random primed (Boehringer-Mannheim) cDNA probes that were specific for Cx43, GAPDH, and skeletal actin- and were labeled with [-³²P]dCTP (Amersham). Hybridizations were performed overnight at 42°C in the presence of 5x SSPE (1x contains 150 mmol/L NaCl [Fluka], 10 mmol/L sodium phosphate monobasic [Sigma], and 1 mmol/L EDTA [Fluka], 50% formamide, 5x Denhardt's solution (1x contains 0.02% ficoll 400 [Pharmacia], 0.02% polyvinylpyrrolidone [molecular weight, 360 000; Sigma], and 0.02% albumin [bovine fraction V; Sigma], 5% SDS, and 100 µg/mL purified salmon sperm DNA. Blots were washed three times for 10 minutes at 42°C in 2x SSC/1% SDS and three times for 20 minutes in 0.1x SSC containing 1% SDS. Exposure times of all membranes to radiographic film (X-Omat AR) were chosen to optimize the signals under conditions preventing their saturation. To normalize signal levels, the same filters were rehybridized with probes for the ubiquitously expressed GAPDH.

The cDNA clone coding for rat Cx43 (clone G2, 1.6 kb)²⁷ and the 1.1-kb (HindIII/Eco RI) fragment of GAPDH cDNA²⁸ were used. The probe for skeletal actin-²⁹ was obtained through PCR amplification of rat genomic DNA, which was prepared as follows. A 2-cm section of rat tail was cut and digested overnight at 55°C in 0.7 mL Tris-HCl, pH 8.0, containing 100 mmol/L EDTA, 0.5% SDS, and 500 µg/mL proteinase K. DNA was purified by phenol-chloroform extraction and ethanol precipitated. To generate PCR fragments, the sense primer sequence, originating at position 2881, was 5'-GTC CAC CTT CCA GCA GAT GT-3', and the antisense primer, originating at position 3146, was 5'-GTT TTC CAT TTC CTT CCA CA-3'. Both primers were synthesized by MWG-Biotech. PCR reactions were started with 1 µg rat genomic DNA, 20 ng of sense and antisense primers, 200 mmol/L dNTP in 10x PCR buffer, and 1.5 mmol/L MgCl₂ (GIBCO BRL) under the following conditions: 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute. The 265–base-pair product obtained after 30 cycles was gel purified and used as probe.

Immunofluorescence
For indirect immunofluorescence, anesthetized rats (two per group) were perfused with 20 mL PBS, and the heart and aorta were rapidly excised and quickly frozen in 2-methylbutane cooled in liquid nitrogen. Fragments were frozen in OCT medium (Miles Inc) and cryostat sectioned at 5 µm thickness. Sections were rinsed in PBS and incubated 30 minutes in buffer containing 0.5% BSA. Sections were then incubated for 1 hour in the presence of a monoclonal antibody to Cx43 (Zymed Laboratories Inc) diluted 1:250 in PBS. Primary antibodies were detected using anti-mouse immunoglobulins labeled with fluorescein isothiocyanate (Biosystem). Sections were then rinsed in PBS, stained with Evan's blue, and photographed with an Axiophot microscope (Zeiss) with Kodak Tmax 400 films.

For quantitative evaluation, cryosections of unfixed aortas, processed as outline above, were photographed at a magnification of 170x with a constant exposure time. Color slides of each section were projected at a final magnification of 2500x onto a graphic tablet connected to a Quantimet 500+ analyzer (Leica AG). Photographs were used to score the number of immunofluorescent spots decorating smooth muscle cells over a known area of the media layer. Data were expressed as number of immunolabeled spots/1000 µm².

Western Blotting
Immediately after the animals were killed, 2K,1C (n=4) and sham-operated (n=4) animals were infused with 30 mL PBS, and the heart and aorta were excised and rapidly frozen. The organs were homogenized with a Kinametic Polytron blender in 100 mmol/L Tris-HCl, pH 7.4, supplemented with 20 mmol/L EDTA, 1 mg/mL pepstatin A, 1 mg/mL antipain (Merck), 1 mmol/L benzamidin, 40 KIU/mL aprotinin, 2 mmol/L phenylmethylsulfonyl fluoride (Sigma Chemical Co), and 1 mmol/L diisopropyl fluorophosphate (Aldrich Chemical Co). Homogenates were passed through a syringe to break down the DNA and centrifuged at 3000g for 10 minutes to pellet the intact cells and elastic fibers. Supernatants were collected and centrifuged for 60 minutes at 100 000g and 4°C. The crude membranes that were pelleted were resuspended in a buffer containing 62.5 mmol/L Tris, pH 8.0, 20% SDS, and 10 mmol/L EDTA. Protein content was determined with the BCA protein assay reagent kit (Pierce). Samples of crude membranes were fractionated by electrophoresis in a 12.5% polyacrylamide gel and immunoblotted onto Immobilon PVDF membranes (Millipore Co) for 20 hours at a constant voltage of 25 V. Membranes were blocked for 3 hours at room temperature in PBS containing 3% BSA and 0.1% Tween 20 and then incubated for 4 hours with a monoclonal antibody against Cx43 diluted 1:10 000. After repeated rinsing in PBS and PBS plus 0.1% Tween 20, immunoblots were incubated overnight at 4°C with antibodies against mouse immunoglobulin coupled with alkaline phosphatase (Dako Diagnostic AG) and diluted 1:5000. Bands were revealed with use of the BCIP-NBT method (AP development reagent, BioRad Laboratories).

Morphometry
Abdominal aorta was pressurized at 80 mm Hg for 15 minutes by a continuous perfusion of 4% paraformaldehyde in PBS. The fixed aorta was excised, and fragments were frozen in OCT medium, sectioned at a thickness of 8 µm, and stained with hemalun-erythrosin. Sections were dehydrated and mounted with Eukitt (O. Kindler and Co). Measurements were performed with a laser-scanned confocal system (MRC 50, BioRad) attached to an inverted microscope (Diaphot, Nikon). Intima-plus-media thickness and internal diameter of the aorta were measured at a 40x magnification. Measurements were made on two sections per animal (Table 1), and the mean of six fields was calculated. Intima-media cross-sectional area (CSA) was determined according to the following formula³⁰ : CSA=[(lumen radius+media-intima thickness)²—(lumen radius)²].

View this table: [in this window] [in a new window]   Table 1. Characteristics of Rats on Experimental Day

To evaluate the number and size of smooth muscle cells, photographs were taken at four points around the circumference of each sectioned aorta. After projection at the final magnification of 2300x, these photographs were semiautomatically analyzed with use of a Quantimet 500+ system to evaluate the surface of intima and media layers, the number of smooth muscle cells, and their profile areas (only profiles showing a nucleus were scored). From these data, we calculated the numerical density (number of cells divided by area of the aortic wall) and the average size of smooth muscle cells (sum of the profile areas divided by the number of cells scored).

For histology, rats were perfused with PBS, and the hearts were rapidly excised and fixed by immersion in Bouin's solution. After fixation, hearts were transversally cut at half the length of the left ventricle, paraffin embedded, sectioned at a thickness of 12 µm, and stained with hemalun-erythrosin.

Statistical Analysis
Densitometric analyses of Western and Northern blots were performed with a Molecular Dynamics scanner. Signals of specific transcripts were related to the corresponding GAPDH signals and expressed relative to the lowest control ratio, which was assigned the arbitrary value of 1. Data were expressed as mean±SEM. Mean values of blood pressure, heart rate, body weight, relative gene expression, density, and areas of smooth muscle cells were compared between groups using one-way ANOVA and Scheffe's test or Fisher's least significant difference. Scheffe's test was also used to compare immunolabeled Cx43 of aortic smooth muscle cells.

	Results

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Characteristics of Hypertensive Rats
The characteristics of the rats on the day of the experiments are given in Table 1. There was no significant difference in body weight between 2K,1C (n=11) and sham-operated (n=11) rats. In contrast, body weight of DOCA-salt hypertensive rats (n=10) was significantly lower than that of corresponding control animals (n=9). Mean intra-arterial blood pressure of both 2K,1C and DOCA-salt animals was higher (P<.001) than that observed in normotensive control animals. 2K,1C and DOCA-salt rats exhibited a comparable elevation of mean blood pressure. The four groups of animals did not significantly differ in heart rate.

Effects of Hypertension on Aorta
The animals used for morphometry displayed biological characteristics similar to those shown in Table 1 (Table 2). Thus, the body weight of DOCA-salt hypertensive rats was significantly (P<.001) less than that of control animals, and the mean blood pressure was equally increased (P<.001) in the two groups of hypertensive rats. Hypertensive animals showed a thickening of aortic wall (Fig 1), which resulted in a 30% increase (P<.001) in the cross-sectional area of aorta despite a constant luminal radius (Table 2). Smooth muscle cells of aortic media were enlarged (P<.001) in both types of hypertensive rats compared with those of normotensive animals (Table 2). Morphometry also revealed that the numerical density of these cells was reduced (P<.001) in the aorta of the two types of hypertensive rats studied. Northern blot analysis showed that the expression of skeletal actin-, a marker of smooth muscle cells, was also increased twofold in the aorta of all hypertensive rats (Fig 1).

View this table: [in this window] [in a new window]   Table 2. Morphometrical Characteristics of Aorta

View larger version (168K): [in this window] [in a new window]   Figure 1. Thickness of the aortic wall is increased in hypertensive rats. Left, Four weeks after surgery, the aorta of hypertensive 2K,1C rats (B) had a significantly thicker wall than that of normotensive, sham-operated animals (A), despite a similar internal diameter. Hypertensive rats also showed smooth muscle cells that were hypertrophic (D) compared with those of control animals (C). L indicates lumen. Bar represents 300 µm in A and B and 40 µm in C and D. Right, Northern blots revealed that expression of skeletal actin- (ASA) was significantly increased over control values (solid and dark shaded columns) in the aorta of hypertensive rats of both the 2K,1C (open column) and the DOCA-salt (slightly shaded column) models. In contrast, levels of GAPDH were not significantly altered in the same RNA samples (not shown). **P<.01.

Quantitative assessment of Northern blots (Fig 2) showed significantly (P<.01) higher levels of Cx43 mRNA in the aorta of 2K,1C and DOCA-salt (4.9±0.8 and 5.4±0.8 arbitrary units [AU], respectively) than in control animals (2.5±0.4 AU for sham-operated rats and 2.8±0.4 AU for control rats). Cx43 was easily immunolocated on smooth muscle cells of the aortic media of both hypertensive and normotensive rats (Fig 3) but was barely detectable on the endothelial cells of the same vessels. In the hypertrophied aorta of 2K,1C animals, the number of immunoreactive spots/1000 µm² (11.6±0.7) was increased (P<.001) by 65% compared with that observed in the aorta of sham-operated animals (7.6±0.3). Also, crude membranes from aortas of hypertensive rats contained two immunoreactive bands; the average intensity of the bands increased 61% (P<.05 in three separate experiments) compared with controls. In the same blots, Cx43 consistently appeared to be less phosphorylated in aortas of sham-operated rats than in aortas of hypertensive rats (Fig 3).

View larger version (96K): [in this window] [in a new window]   Figure 2. Expression of Cx43 gene is increased in aortas of hypertensive rats. Left, Northern blots revealed that Cx43 mRNA, which was mostly contributed to by smooth muscle cells, was increased in the aorta of hypertensive rats in the two models we investigated. In the same samples, levels of GAPDH mRNA were not modified. Each lane shows a sample from a different rat. Right, Quantitative assessment of 9 to 11 measurements (1 measurement per rat) confirmed that Cx43 mRNA levels were increased approximately twofold over control values (solid and dark shaded columns) in the aorta of hypertensive rats of both the 2K,1C (open column) and DOCA-salt (slightly shaded column) models. All lanes were loaded with 10 µg total RNA. Values represent ratios of densitometric measurements of Cx43 and GAPDH mRNAs and are expressed as mean±SEM. **P<.01.

View larger version (91K): [in this window] [in a new window]   Figure 3. Expression of Cx43 in rat aorta. Left, Immunofluorescence labeling with specific mouse monoclonal antibodies located Cx43 at discrete spots dispersed throughout the media of the aortic wall. No obvious difference in the number and pattern of these spots was noticed between sham-operated (A) and control (B) rats. In contrast, a modest but sizable increase in the number of immunofluorescent spots was observed in the thickened media of hypertensive rats from both the 2K,1C (C) and the DOCA-salt (D) models. Because of the focus plane, the limited amount of Cx43 expressed by endothelial cells is not detectable in these figures. L indicates lumen. Bar represents 27 µm. Right, Western blots revealed that the levels of Cx43, which was immunodetected as two bands of 45 and 43 kD, were higher in the aorta of hypertensive 2K,1C rats than in that of sham-operated animals. This difference was not apparent in the hearts of the same animals. Liver served as a negative control. Lanes with aorta samples were loaded with 50 µg protein. Lanes with heart and liver samples were loaded with 20 µg protein.

Effects of Hypertension on the Heart
The hearts of hypertensive rats were hypertrophied compared with those of normotensive control animals, as indicated by a 30% increase (P<.001) in heart index (Fig 4). Northern blots showed a twofold increase in the expression of skeletal actin- mRNA in the two groups of hypertensive rats (Fig 4). Histological analysis further revealed that the thickness of the left ventricular wall was larger in hypertensive than in normotensive animals (Fig 5).

View larger version (69K): [in this window] [in a new window]   Figure 4. Heart muscle is increased in hypertensive rats. Left, Compared with normotensive sham-operated (solid column) and control (dark shaded column) animals, the 2K,1C (open column) and DOCA-salt (slightly shaded column) hypertensive models showed a 30% increase in heart index. This parameter, which gives the weight of myocardium per unit of animal weight, is expressed as mean±SEM of 9 to 11 measurements (1 measurement per rat). ***P<.001. Right, Northern blots revealed that expression of skeletal actin- was significantly increased over control values (solid and dark shaded columns) in hearts of hypertensive rats of both the 2K,1C (open column) and the DOCA-salt (slightly shaded column) models. Values represent ratios of densitometric measurements of skeletal actin- and GAPDH mRNAs and are expressed as mean±SEM of 9 to 11 measurements (1 measurement per rat). **P<.01.

View larger version (111K): [in this window] [in a new window]   Figure 5. Thickness of left ventricular (LV) wall is increased in hypertensive rats. Left, At the time of death, the thickness of the LV wall was larger in 2K,1C rats (B) than in sham-operated rats (A), whereas no such difference was observed in the right ventricle (RV). Right, Immunofluorescence labeling with mouse monoclonal antibodies located Cx43 at intercalated disks throughout LV myocardium. No obvious difference in the number and pattern of these bands was noticed between sham-operated (C) and 2K,1C (D) rats. Bar represents 1.6 mm in A and B and 27 µm in C and D.

Quantification of Northern blots showed that Cx43 expression was similar in hearts of hypertensive (1.9±0.2 AU for the 2K,1C group and 1.8±0.1 AU for the DOCA-salt group) and control rats (1.8±0.1 and 2.0±0.2 AU, respectively) (Fig 6). Also, the levels and distribution of Cx43 were similar in all groups of animals (Figs 3 and 5).

View larger version (102K): [in this window] [in a new window]   Figure 6. Expression of Cx43 gene is not modified in hearts of hypertensive rats. Left, Northern blots revealed that the levels of Cx43 mRNA were not altered in hearts of the two types of hypertensive rats we investigated. Similarly, no major change in the level of GAPDH mRNA were detected in the same samples. Right, Quantitative assessment of 9 to 11 measurements (1 measurement per rat) confirmed that Cx43 mRNA levels were similar in control (solid and dark shaded columns) and hypertensive (open and slightly shaded columns) rats. All lanes were loaded with 10 µg total RNA. Values represent ratios of densitometric measurements of Cx43 and GAPDH mRNAs and are expressed as mean±SEM.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We examined the effects of chronic experimental hypertension on the expression of Cx43, the major native connexin of the cardiovascular system.^{27 31} To this end, we studied two rat models that feature a comparable increase in blood pressure but differ in the mechanism causing this change.

In the 2K,1C model, development of hypertension results from the constriction of one renal artery and the ensuing activation of the renin-angiotensin system.²¹ This activation is reflected by an increase in both renin mRNA levels in the hypoperfused kidney and in plasma renin activity.³² The further proteolytic cleavage of angiotensinogen by renin and the processing of Ang I by ACE lead to the generation of Ang II, a most potent vasoconstrictor that also induces vascular and cardiac hypertrophy.^{33 34} Therefore, cellular changes observed in this model could result from the increase in blood pressure and angiotensin II levels. To discriminate between these possibilities, we investigated the DOCA-salt hypertension model, in which the renin-angiotensin system is functionally suppressed. In this case, hypertension results from salt retention caused by the combined administration of a mineralocorticoid and a high sodium intake.²²

After 1 month, the increase in blood pressure achieved in the two models was comparable. The two groups of hypertensive animals also exhibited a comparable degree of thickening of the aortic wall, which was accounted for by hypertrophy of smooth muscle cells and an accumulation of extracellular materials. Both types of hypertensive rats also featured a comparable increase in the expression of Cx43 in the smooth muscle cells of the aortic media, supporting the view that this change was associated with the elevation of blood pressure and not with alterations in Ang II levels, which differed considerably in the 2K,1C and the DOCA-salt models. This is the first evidence implicating a regulation of aortic Cx43 by increased blood pressure, even though a previous study suggested that Cx43-made gap junctions may be controlled by pressure in myometrial cells.³⁵

The molecular mechanism leading to the pressure-induced increase in expression of the Cx43 gene remains to be elucidated. The presence of multiple promoters in the 5' untranslated region of this gene^{36 37} raises the possibility that distinct transcription factors control its tissue-specific regulation.^{25 38 39 40} The recent findings that transcription of Cx43 gene can be promoted by an increase in the expression of c-fos⁴¹ is of interest because the mRNA coding for this transcription factor accumulates in smooth muscle cells of rat aortas after exposure to Ang II,^{42 43} a vasoconstrictor agent that contributes to hypertension in one (2K,1C) of the two animal models we studied.

The function of Cx43 in the smooth muscle cells as well as the cause and significance of the increase in this protein during chronic hypertension remains to be elucidated. Several studies have suggested that connexins are important in the coordination of the mechanical contraction of smooth muscle cells.^{6 7 35} Along the same lines, it could be speculated that Cx43 is implicated in the mediation and modulation of vasomotor tone in the aortic wall.^{5 6} Certainly, Cx43 can provide an intercellular pathway for the syncytial functioning of distant smooth muscle cells that could be recruited for synchronous contraction through the propagation of gap junction–permeant second messengers.¹⁰ Further studies are required to determine whether increased Cx43 expression is related to the development of the vascular hypertrophy that is observed in hypertensive rats.

The hypertensive animals we studied also featured cardiac hypertrophy. However, in marked contrast with the observations made in aortas, this change was not associated with an alteration in the level of expression and distribution of Cx43. This negative finding, despite the fact that hypertensive animals featured significantly thicker left ventricular walls, suggests that Cx43 is not involved in the myocardial adaptation that accompanies the hypertension-induced increase in heart load. It remains to be shown whether any of the other connexins that colocalize with Cx43 at myocardial gap junctions^{17 44} and in endothelial cells¹⁹ are regulated differently under the same conditions.

In summary, we found that the expression of Cx43 is differentially regulated in the hypertrophic muscle cells of heart and aorta and that this differential regulation takes place in rats made similarly hypertensive via different mechanisms. Elucidation of how the changes in Cx43 expression participate to the adaptive response of the aorta to high blood pressure awaits the availability of novel animal models in which the expression of Cx43, and possibly of other connexins, could be specifically regulated at the level of aortic and heart smooth muscle cells. At this point, our data indicate that Cx43 is a tissue-specific marker associated with hypertensive vascular changes, raising the possibility that this gap junction protein may contribute to the still obscure pathogenesis of several forms of hypertension in humans.

	Selected Abbreviations and Acronyms

 

Ang = angiotensin Cx43 = connexin43 DOCA-salt = deoxycorticosterone-salt hypertension model GAPDH = glyceraldehyde-3-phosphate dehydrogenase 2K,1C = two kidney, one-clip renal hypertension model

	Acknowledgments

 
Dr Haefliger is supported by a career award from the Max Cloetta Foundation. This work was supported by grants from the Swiss National Science Foundation (31-37393.93 to Dr Haefliger, 32-31915.91 and 32-29317.91 to Dr G. Waeber, 32-0338.05 to Dr B. Waeber, 32-43086.95 to Dr Meda) and the European Union (BMH4-CT96-1427 to Dr Meda). We thank G. Centeno for expert technical assistance.

Received July 29, 1996; revision received September 25, 1996; accepted October 7, 1996.

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