Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-014-1616-x
INTEGRATIVE PHYSIOLOGY
Role of smooth muscle cell mineralocorticoid receptor in vascular tone Antoine Tarjus & Ekaterina Belozertseva & Huguette Louis & Soumaya El Moghrabi & Carlos Labat & Patrick Lacolley & Frédéric Jaisser & Guillaume Galmiche
Received: 18 February 2014 / Revised: 15 September 2014 / Accepted: 16 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MRSMKO), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MRSMKO mice, contractions induced by potassium chloride and calcium (Ca2+) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca2+ suggests that the VSMC-specific deficiency of the MR modifies VSM Ca2+ signaling but without altering the intracellular Ca2+ store handling. The relaxation induced by acetylcholine is
not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MRSMKO mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca2+ signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca2+ signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.
Frédéric Jaisser and Guillaume Galmiche equally contributed to this study.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1616-x) contains supplementary material, which is available to authorized users.
The mineralocorticoid receptor (MR) is known to participate in blood pressure (BP) regulation through the control of renal sodium homeostasis and extracellular volume [9]. Thereby, MR antagonists (MRA) have been proved to be an effective treatment for hypertension in experimental models [23] and in patients [8, 23]. Recently, the mechanism of action of MR on BP has been questioned. A meta-analysis of clinical trials demonstrated that BP reduction with MR antagonist does not correlate with changes in plasma electrolytes and, by extension, with renal MR activation [14]. The results are coherent with the growing evidence suggesting that BP could be regulated via vascular tone and not only the kidney [16]. Consistently with this new picture, it has been found that, besides its classical expression in the aldosteronesensitive distal nephron (ASDN), MR is also expressed in
A. Tarjus : S. El Moghrabi : F. Jaisser (*) : G. Galmiche Centre de Recherche des Cordeliers, Inserm U1138 Team 1, 15 rue de l'Ecole de Médecine, Paris Cedex 06 75270, France e-mail: [email protected] A. Tarjus : S. El Moghrabi : F. Jaisser : G. Galmiche Université Pierre et Marie Curie, Paris, France E. Belozertseva : H. Louis : C. Labat : P. Lacolley Faculté de Médecine, INSERM U961, 9 Avenue de la Fôret de Haye, Vandoeuvre-lès-Nancy 54500, France F. Jaisser Centre for Clinical Investigation, INSERM U9501, CHU Brabois, Vandoeuvre-lès-Nancy, France
Keywords Aldosterone . Aorta . Contractility . Relaxation . Transgenic mice
Pflugers Arch - Eur J Physiol
the vasculature, both in the endothelium [19] and in vascular smooth muscle cells (VSMC) [15]. These results raise the question of the implication of MR in the vascular function and particularly in vascular tone and arterial stiffness. Targeted inactivation of MR in VSM was recently achieved using two animal models with conditional MR inactivation in the VSMCs [11, 15]. VSM MR inactivation blunts the age-induced raise of BP [15], without affecting sodium homeostasis in the kidney of adult mice [11]. Moreover, VSM MR inactivation underlined the critical role of MR expressed in VSM for arterial stiffness: VSMC MR expression is necessary for increased arterial stiffness in response to aldosterone and high salt by modulation of cell-matrix attachment proteins independent of major vascular structural changes [11]. The role of VSM in vascular tone has been explored in mesenteric arteries using inducible VSM KO mice underlying a role of VSM MR in modulating Ca2+ channel expression. However, how VSM MR affects the contractile machinery remains unknown. In the present study, we investigated the mechanisms by which MR expressed in SMC affects the vascular response, focusing on endothelial-dependent and endothelial-independent pathways affecting relaxation-contraction coupling.
Methods Generation of VSMC-specific MR inactivation The generation of the MRSMKO (MRfl/fl;SM22-Cre) allowing VSMC-specific MR inactivation in the vasculature has been described previously [11]. For all studies, males MRSMKO and their littermates, MRfl/fl mice (control mice, CTL), were phenotyped at age 4–5 months. All animal breeding, housing, and protocols were performed in accordance with the ethical guidelines of INSERM for the care and use of laboratory animals. Vascular reactivity analysis Vascular contractile and relaxing responses were assessed in isolated abdominal aortas from male MRSMKO and CTL mice as previously described [10]. Briefly, aortas were excised, cut into 2.5-mm segments and mounted in a Mulvany-Halpern wire myograph [17] (DMT, Aarhus, DK) in a 8-mL organ bath containing physiological salt solution (PSS, pH 7.4) continuously bubbled with carbogen (95 % O2, 5 % CO2) and maintained at 37 °C. When required by the experimental protocol, the endothelial layer was removed by gently rubbing the intimal surface of the vessels with the myograph wires. Rings were allowed to equilibrate for 30 min at a resting tension of 1 g, with the bath medium changed every 10 min. Administration of 10−5 mol/L phenylephrine (PE) was used to
test arterial viability, and the presence of intact endothelium was verified by a relaxing dose of acetylcholine (Ach, 10−4 mol/L). The α-adrenergic pathway was assessed via the contraction evoked by PE (10−9 to 10−5 mol/L), while the response to calcium (Ca2+) channel activation was determined by direct depolarization of the cell membrane using potassium chloride (KCl) cumulative dose-response curves (10 to 80 mmol/L). In order to assess the extracellular calcium response, aortic rings were first preincubated in Ca2+-free solution (containing 80 mmol/L KCl instead of an equimolar amount of NaCl) with 2 mmol/L EGTA added for 20 min and then in Ca2+-free solution containing no EGTA. CaCl2 was then added from a stock solution to obtain the requested concentrations (10−5 to 10−2 mol/L), and the effect of each Ca2+ concentration was recorded. The calcium release from sarcoplasmic reticulum via ryanodine receptor channels was assessed by a bolus of caffeine (10−2 mol/L). To evaluate endothelium-dependent relaxation, the doseresponse to Ach (10−9 to 10−6 mol/L) was determined in intact rings precontracted by 10−5 mol/L of PE. The endotheliumdependent relaxation induced by endogenous prostacyclin (PGI2) was assessed by the dose-response to Ach in the presence of Nω-nitro-L-arginine (10−4 mol/L, L-NNA), a nitric oxide (NO) inhibitor, (Sigma-Aldrich, St. Quentin Fallavier, France). The endothelium-dependent relaxation induced by endogenous NO was assessed by the dose-response to Ach in the presence of indomethacin (Indo, 10−5 mol/L), an inhibitor of cyclooxygenase (COX) 1 and 2 enzymes. The VSMC sensitivity (endothelium-independent relaxation) to NO and PGI2 pathways was also assessed in endothelium-denuded rings precontracted with PE (10−5 mol/L) by dose-responses to a NO donor sodium nitroprussiate (SNP, 10−9 to 10−5 mol/L) and to treprostinil, a synthetic analog of PGI2 (Trep, 10−9 to 10−5 mol/L), respectively. VSMC sensitivity to cGMP was assessed by a single dose of cGMP analog 8-Br-cGMP (10−4 mol/L) in endothelium-denuded rings precontracted with PE (10−5 mol/L). The maximal response (Emax) and the concentration of agonist inducing 50 % of maximal responses (EC50) were interpolated from the individual concentrationeffect curves. These latter values were transformed into pD2 values, that is, negative logarithms of EC50 values. Western blotting analysis Western blot analysis was performed on protein collected from the aorta from control and MRSMKO mice that were immediately homogenized after dissection using a 1 % SDS homogenization buffer with protease and phosphatase inhibitors (Roche Diagnostics, Meylan, France). Extracts were centrifuged at 13,200 rpm for 20 min at 4 °C and supernatants were frozen. Twenty micrograms of total proteins diluted with Laemmli buffer (2×, Sigma-Aldrich, St. Quentin Fallavier, France) and heated at 95 °C for 5 min were loaded on
Pflugers Arch - Eur J Physiol
4–15 % Mini-PROTEAN® TGX™ precast polyacrylamide gels (Bio-Rad, Marnes-la-Coquette, France) and then transferred to PVDF membranes. The antibodies are listed in Online Resource 1 (Table S1). RNA extraction and real-time PCR Total RNA was extracted from aortic arteries by using the TRIzol® reagent (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s protocol. The reverse transcription of messenger RNA (mRNA) (100 ng) was performed with SuperScript II Reverse Transcriptase KIT (Life Technologies Corporation, Carlsbad, CA, USA). Transcript levels of genes were analyzed by real-time PCR (fluorescence detection of SYBR Green) in an iCycler iQ apparatus (Bio-Rad, Marnes-la-Coquette, France). For each sample, mRNA levels normalized the geometric mean of the amount of two housekeeping genes, 18s and hypoxanthine guanine phosphoribosyltransferase (Hprt). The analyzed genes and their specific primer sequences are listed in Online Resource 1 (Table S2). Statistical analysis Values are expressed as mean±standard error of the mean (SEM). mRNA and protein expressions were analyzed using unpaired Student’s t test. Vascular reactivity experiments were analyzed using one-way ANOVA, and the Fisher’s LSD post hoc test was used for intergroup comparisons. An unpaired Student’s t test was used for the relaxations in response to SNP, treprostinil, and Ach in the presence of indomethacin. Differences were considered significant at values of P
INTEGRATIVE PHYSIOLOGY
Role of smooth muscle cell mineralocorticoid receptor in vascular tone Antoine Tarjus & Ekaterina Belozertseva & Huguette Louis & Soumaya El Moghrabi & Carlos Labat & Patrick Lacolley & Frédéric Jaisser & Guillaume Galmiche
Received: 18 February 2014 / Revised: 15 September 2014 / Accepted: 16 September 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Identification of the mineralocorticoid receptor (MR) in the vasculature (i.e., endothelial and smooth muscle cells) raised the question of its role in vascular function and blood pressure control. Using a mouse model with conditional inactivation of MR in vascular smooth muscle cell (VSMC) (MRSMKO), we have recently shown that the VSMC MR is crucial for aldosterone-salt-induced carotid stiffening. In the present study, we have investigated the specific contribution of the VSMC MR in the regulation of vascular tone in large vessels. In MRSMKO mice, contractions induced by potassium chloride and calcium (Ca2+) are decreased in the aorta, whereas contraction is normal in response to phenylephrine and caffeine. The difference in response to Ca2+ suggests that the VSMC-specific deficiency of the MR modifies VSM Ca2+ signaling but without altering the intracellular Ca2+ store handling. The relaxation induced by acetylcholine is
not affected by the absence of MR. However, the relaxation induced by Ach in the presence of indomethacin and the relaxation induced by sodium nitroprussiate are significantly reduced in MRSMKO mice compared to controls. Since endothelial nitric oxide synthase (eNOS) activity is increased in mutant mice, their altered relaxation reflects impairment of the nitric oxide (NO) signaling pathway. In addition to altered NO and Ca2+ signaling, the activity of myosin light chain and its regulators, myosin light chain kinase (MLCK) and myosin phosphatase (MLCP), is reduced. In conclusion, MR expressed in VSMC is required for NO and Ca2+ signaling pathways and contractile protein activity leading to an altered contraction/relaxation coupling.
Frédéric Jaisser and Guillaume Galmiche equally contributed to this study.
Introduction
Electronic supplementary material The online version of this article (doi:10.1007/s00424-014-1616-x) contains supplementary material, which is available to authorized users.
The mineralocorticoid receptor (MR) is known to participate in blood pressure (BP) regulation through the control of renal sodium homeostasis and extracellular volume [9]. Thereby, MR antagonists (MRA) have been proved to be an effective treatment for hypertension in experimental models [23] and in patients [8, 23]. Recently, the mechanism of action of MR on BP has been questioned. A meta-analysis of clinical trials demonstrated that BP reduction with MR antagonist does not correlate with changes in plasma electrolytes and, by extension, with renal MR activation [14]. The results are coherent with the growing evidence suggesting that BP could be regulated via vascular tone and not only the kidney [16]. Consistently with this new picture, it has been found that, besides its classical expression in the aldosteronesensitive distal nephron (ASDN), MR is also expressed in
A. Tarjus : S. El Moghrabi : F. Jaisser (*) : G. Galmiche Centre de Recherche des Cordeliers, Inserm U1138 Team 1, 15 rue de l'Ecole de Médecine, Paris Cedex 06 75270, France e-mail: [email protected] A. Tarjus : S. El Moghrabi : F. Jaisser : G. Galmiche Université Pierre et Marie Curie, Paris, France E. Belozertseva : H. Louis : C. Labat : P. Lacolley Faculté de Médecine, INSERM U961, 9 Avenue de la Fôret de Haye, Vandoeuvre-lès-Nancy 54500, France F. Jaisser Centre for Clinical Investigation, INSERM U9501, CHU Brabois, Vandoeuvre-lès-Nancy, France
Keywords Aldosterone . Aorta . Contractility . Relaxation . Transgenic mice
Pflugers Arch - Eur J Physiol
the vasculature, both in the endothelium [19] and in vascular smooth muscle cells (VSMC) [15]. These results raise the question of the implication of MR in the vascular function and particularly in vascular tone and arterial stiffness. Targeted inactivation of MR in VSM was recently achieved using two animal models with conditional MR inactivation in the VSMCs [11, 15]. VSM MR inactivation blunts the age-induced raise of BP [15], without affecting sodium homeostasis in the kidney of adult mice [11]. Moreover, VSM MR inactivation underlined the critical role of MR expressed in VSM for arterial stiffness: VSMC MR expression is necessary for increased arterial stiffness in response to aldosterone and high salt by modulation of cell-matrix attachment proteins independent of major vascular structural changes [11]. The role of VSM in vascular tone has been explored in mesenteric arteries using inducible VSM KO mice underlying a role of VSM MR in modulating Ca2+ channel expression. However, how VSM MR affects the contractile machinery remains unknown. In the present study, we investigated the mechanisms by which MR expressed in SMC affects the vascular response, focusing on endothelial-dependent and endothelial-independent pathways affecting relaxation-contraction coupling.
Methods Generation of VSMC-specific MR inactivation The generation of the MRSMKO (MRfl/fl;SM22-Cre) allowing VSMC-specific MR inactivation in the vasculature has been described previously [11]. For all studies, males MRSMKO and their littermates, MRfl/fl mice (control mice, CTL), were phenotyped at age 4–5 months. All animal breeding, housing, and protocols were performed in accordance with the ethical guidelines of INSERM for the care and use of laboratory animals. Vascular reactivity analysis Vascular contractile and relaxing responses were assessed in isolated abdominal aortas from male MRSMKO and CTL mice as previously described [10]. Briefly, aortas were excised, cut into 2.5-mm segments and mounted in a Mulvany-Halpern wire myograph [17] (DMT, Aarhus, DK) in a 8-mL organ bath containing physiological salt solution (PSS, pH 7.4) continuously bubbled with carbogen (95 % O2, 5 % CO2) and maintained at 37 °C. When required by the experimental protocol, the endothelial layer was removed by gently rubbing the intimal surface of the vessels with the myograph wires. Rings were allowed to equilibrate for 30 min at a resting tension of 1 g, with the bath medium changed every 10 min. Administration of 10−5 mol/L phenylephrine (PE) was used to
test arterial viability, and the presence of intact endothelium was verified by a relaxing dose of acetylcholine (Ach, 10−4 mol/L). The α-adrenergic pathway was assessed via the contraction evoked by PE (10−9 to 10−5 mol/L), while the response to calcium (Ca2+) channel activation was determined by direct depolarization of the cell membrane using potassium chloride (KCl) cumulative dose-response curves (10 to 80 mmol/L). In order to assess the extracellular calcium response, aortic rings were first preincubated in Ca2+-free solution (containing 80 mmol/L KCl instead of an equimolar amount of NaCl) with 2 mmol/L EGTA added for 20 min and then in Ca2+-free solution containing no EGTA. CaCl2 was then added from a stock solution to obtain the requested concentrations (10−5 to 10−2 mol/L), and the effect of each Ca2+ concentration was recorded. The calcium release from sarcoplasmic reticulum via ryanodine receptor channels was assessed by a bolus of caffeine (10−2 mol/L). To evaluate endothelium-dependent relaxation, the doseresponse to Ach (10−9 to 10−6 mol/L) was determined in intact rings precontracted by 10−5 mol/L of PE. The endotheliumdependent relaxation induced by endogenous prostacyclin (PGI2) was assessed by the dose-response to Ach in the presence of Nω-nitro-L-arginine (10−4 mol/L, L-NNA), a nitric oxide (NO) inhibitor, (Sigma-Aldrich, St. Quentin Fallavier, France). The endothelium-dependent relaxation induced by endogenous NO was assessed by the dose-response to Ach in the presence of indomethacin (Indo, 10−5 mol/L), an inhibitor of cyclooxygenase (COX) 1 and 2 enzymes. The VSMC sensitivity (endothelium-independent relaxation) to NO and PGI2 pathways was also assessed in endothelium-denuded rings precontracted with PE (10−5 mol/L) by dose-responses to a NO donor sodium nitroprussiate (SNP, 10−9 to 10−5 mol/L) and to treprostinil, a synthetic analog of PGI2 (Trep, 10−9 to 10−5 mol/L), respectively. VSMC sensitivity to cGMP was assessed by a single dose of cGMP analog 8-Br-cGMP (10−4 mol/L) in endothelium-denuded rings precontracted with PE (10−5 mol/L). The maximal response (Emax) and the concentration of agonist inducing 50 % of maximal responses (EC50) were interpolated from the individual concentrationeffect curves. These latter values were transformed into pD2 values, that is, negative logarithms of EC50 values. Western blotting analysis Western blot analysis was performed on protein collected from the aorta from control and MRSMKO mice that were immediately homogenized after dissection using a 1 % SDS homogenization buffer with protease and phosphatase inhibitors (Roche Diagnostics, Meylan, France). Extracts were centrifuged at 13,200 rpm for 20 min at 4 °C and supernatants were frozen. Twenty micrograms of total proteins diluted with Laemmli buffer (2×, Sigma-Aldrich, St. Quentin Fallavier, France) and heated at 95 °C for 5 min were loaded on
Pflugers Arch - Eur J Physiol
4–15 % Mini-PROTEAN® TGX™ precast polyacrylamide gels (Bio-Rad, Marnes-la-Coquette, France) and then transferred to PVDF membranes. The antibodies are listed in Online Resource 1 (Table S1). RNA extraction and real-time PCR Total RNA was extracted from aortic arteries by using the TRIzol® reagent (Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturer’s protocol. The reverse transcription of messenger RNA (mRNA) (100 ng) was performed with SuperScript II Reverse Transcriptase KIT (Life Technologies Corporation, Carlsbad, CA, USA). Transcript levels of genes were analyzed by real-time PCR (fluorescence detection of SYBR Green) in an iCycler iQ apparatus (Bio-Rad, Marnes-la-Coquette, France). For each sample, mRNA levels normalized the geometric mean of the amount of two housekeeping genes, 18s and hypoxanthine guanine phosphoribosyltransferase (Hprt). The analyzed genes and their specific primer sequences are listed in Online Resource 1 (Table S2). Statistical analysis Values are expressed as mean±standard error of the mean (SEM). mRNA and protein expressions were analyzed using unpaired Student’s t test. Vascular reactivity experiments were analyzed using one-way ANOVA, and the Fisher’s LSD post hoc test was used for intergroup comparisons. An unpaired Student’s t test was used for the relaxations in response to SNP, treprostinil, and Ach in the presence of indomethacin. Differences were considered significant at values of P
