Original Article
Retinoid X Receptor Agonists Inhibit Hypertension-Induced Myocardial Hypertrophy by Modulating LKB1/AMPK/p70S6K Signaling Pathway Jiang Zhu,1 Ruo-Bing Ning,1 Xiao-Yan Lin,2 Da-Jun Chai,3 Chang-Sheng Xu,3 Hong Xie,3 Jin-Zhang Zeng,4 and Jin-Xiu Lin3
methods WKY rats served as controls. SHRs were randomized into 3 groups at the age of 4 weeks and were treated (once daily for 12 weeks) with either bexarotene (30 or 100 mg/kg body weight) or vehicle alone. Echocardiography was performed to determine cardiac structure and function. Neonatal cardiomyocytes were treated with AngII (10–7 mmol/L) with or without the indicated concentration of RXRα ligand 9-cis-RA. The protein abundances of β-actin, RXRα, LKB1, phosphoLKB1, AMPK, phospho-AMPK, P70S6K, phospho-P70S6K, ACE, and AT1 receptor were measured along with blood pressure, body weight and angiotensin II (Ang II) levels. The effects of LKB1 downregulation by LKB1 small, interfering RNA were examined.
results Treatment of SHRs with bexarotene resulted in significant inhibition of LVH without eliminating hypertension. Immunoblot with heart tissue homogenates from SHRs revealed that bexarotene activated the LKB1/AMPK signaling pathway and inhibited p70S6K. However, the increased Ang II levels in SHR serum and heart tissue were not reduced by bexarotene treatment. Treatment of cardiomyocytes with Ang II resulted in significantly reduced LKB1/AMPK activity and increased p70S6K activity. 9-cis-RA antagonized Ang II–induced LKB1/AMPK and p70S6K activation changes in vitro. conclusions RXR agonists prevent the inhibition of the LKB1/AMPK/p70S6K pathway and regulate protein synthesis to reduce LVH. This antihypertrophic effect of bexarotene is independent of blood pressure. Keywords: blood pressure; hypertension; hypertrophy; liver kinase B1; retinoid X receptor. doi:10.1093/ajh/hpu017
Pathological left ventricular hypertrophy (LVH) is a major risk factor for several serious cardiac events, including ischemia/reperfusion injury, arrhythmias, cardiomyopathies, and sudden death.1 Chronic pressure overload has been proposed to be a major factor initiating cardiac hypertrophy,1 which is associated with neurohormonal2 and metabolic3 changes and the activation of inflammatory signaling.4 Recently, increasing evidence suggests that alterations in signaling pathways also play an important role in the development of cardiac remodeling in conjunction with or in the absence of increased afterload.5 LVH is characterized by increased myocardial cell size. Enlargement of cardiomyocytes has been reported to result from an increase in protein content, and a major regulator of protein synthesis and cell growth is the mammalian target of rapamycin (mTOR)/
p70S6 kinase (p70S6K) pathway, which is regulated by several other kinase cascades.6 AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase. The AMPK-induced inhibition of the tuberous sclerosis complex (TSC)-mTOR-p70S6K pathway leads to the inhibition of mRNA translation by the 40S ribosomal protein S6, which culminates in reduced protein synthesis.7 Liver kinase B1 (LKB1) has recently been identified as an AMPK kinase that phosphorylates AMPK on its activating phosphorylation site (Thr172).8 Increased LKB1 activity in cardiac myocytes can decrease hypertrophy-induced protein synthesis, which suggests that LKB1 activation may be a target for the prevention of pathological cardiac hypertrophy.9 Loss of LKB1 is prohypertrophic and may be involved in the development of pathological cardiac hypertrophy.10
Correspondence: Da-Jun Chai ([email protected]).
1First Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China; 2Echocardiological Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China; 3Cardiovascular Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China; 4School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, Xiamen, China.
Initially submitted September 20, 2013; date of first revision October 14, 2013; accepted for publication January 12, 2014; online publication March 6, 2014.
© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: [email protected]
1112 American Journal of Hypertension 27(8) August 2014
Downloaded from http://ajh.oxfordjournals.org/ at Universite Laval on July 14, 2015
background Retinoid X receptor (RXR) has been demonstrated to play an important role in cardiac development and has been implicated in cardiovascular diseases. This study aimed to examine the effects of RXRα agonist bexarotene on pathological left ventricular hypertrophy (LVH) in a spontaneously hypertensive rat (SHR) model and the underlying mechanism.
Retinoid X Receptor Agonists Inhibit Hypertension-Induced Myocardial Hypertrophy
Activation of the renin-angiotensin-aldosterone system (RAAS) plays a major pathophysiological role in the pathegenesis of hypertension and cardiac hypertrophy.11 Angiotensin II (Ang II), a core effector of the RAAS, is a multifunctional peptide with pleiotropic actions, including modulation of vasomotor tone, cell growth, migration, extracellular matrix deposition, and myocardial hypertrophy.12 Angiotensin-converting enzyme (ACE) is a ratelimiting enzyme in Ang II synthesis from angiotensinogen, and angiotensin receptor 1 (AT1R) is the main receptor that Ang II can react on in the process of cardiac hypertrophy.12 Several studies have indicated that inhibition of Ang II production prevents left ventricular (LV) remodeling, indicating that Ang II plays an important role in the development of cardiac remodeling.13,14 The retinoid X receptor (RXR) is an important member of the steroid/thyroid hormone superfamily of nuclear receptors that predominately function as transcription factors, having roles in development, cell differentiation, metabolism, and cell death.15 RXR is unique among nuclear receptors because it forms heterodimers with many other nuclear receptors, such as peroxisome proliferator-activated receptor, liver X receptor, and retinoic acid receptor (RAR), thereby mediating the biological effects of many hormones, vitamins, and drugs.16 RXR is a transcription factor that acts mainly as an RAR/RXR heterodimer.17 RXR plays a key role in the nuclear signaling pathways involving its dimeric partners. Numerous studies have demonstrated that retinoic acid can suppresses myocardial cell hypertrophy in response to mechanical stretch and Ang II.18,19 RXR signaling is crucial for, and indeed causative of, the differentiation phase of cardiomyogenesis.20 RXRα knockout mice die before birth because of a defect in cardiogenesis.21 Considering the function of RXR implied herein, we speculate that RXR may play an important role in the development of myocardial hypertrophy. In this study, we investigated the effect of RXRα on hypertension-induced myocardial hypertrophy and the underlying mechanism of that effect. Our results demonstrated that RXRα agonist treatment attenuated myocardial hypertrophy in spontaneously hypertensive rats (SHRs) by modulating the activation of the LKB1/AMPK/p70S6K signaling pathway, which occurs independent of blood pressure.
Neonatal rat ventricular myocytes (NRVMs) were prepared from the ventricles of 1–2-day-old Sprague-Dawley rat pups as previously described.23 Cells were cultured in Dulbecco’s modified essential medium (Gibco, Beijing, China) with 10% fetal bovine serum. After a 24-hour serum starvation, the cells were treated for 24 hour with Ang II (10–7 mmol/L; Sigma–Aldrich, St. Louis, MO, USA) with or without the natural RXRα ligand 9-cis-retinoic acid (9-cisRA) (Sigma-Aldrich, St. Louis, MO, USA). 9-cis-RA was added to the cells 1 hour before Ang II administration.
METHODS
Immunoblot analysis
Animal care
Cell homogenates and heart tissue homogenates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in gels containing 10% acrylamide and were transferred to nitrocellulose as previously described.24 The following antibodies were used in this study: mouse monoclonal antibody against β-actin and rabbit monoclonal antibody against RXRα (Santa Cruz, Dallas, TX, USA); rabbit polyclonal antibody against total LKB1 and phosphorylated LKB1 (Ser428), rabbit polyclonal antibody against total AMPK and phosphorylated AMPK, and rabbit polyclonal antibody against total P70S6K and phosphorylated P70S6K (Cell Signaling Technology, Boston, MA, USA); and goat polyclonal antibody against AT1R and ACE (Santa Cruz, Dallas, TX, USA).
Transthoracic echocardiography was performed on mildly anesthetized rats with a 10S-(5.4–11.8 MHz) transducer (Vivid 7; GE Healthcare, Diegem, Belgium). Short-axis view and M-mode tracings were used to determine end-diastolic LV internal diameter, LV posterior wall thickness, interventricular septal thickness, and ejection fractions. Noninvasive blood pressure measurements were made with the Softron BP98A tail cuff system (Softron, Tokyo, Japan). Analysis of heart tissue and blood serum
Body weight (BW) was monitored at the end of 16th week. Then animals were killed with isoflurane (2% in oxygen). Blood samples were collected, the heart was removed, and the left ventricles were separated, blotted, and weighed. Hypertrophy was evaluated using the left ventricle weight to BW ratio. Hematoxylin and eosin staining was performed and then imaged. Myocyte cross-sectional area (CSA) was calculated by Image-Proplus software version 6.0 (Media Cybernetics, Bethesda, MD, USA). Samples (20 mg) of the left ventricle were homogenized, and 20 μg of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot analysis. Ang II concentrations were determined by radio immunoassay.22 Serum alanine aminotransferase and aspartate aminotransferase were measured with continuous monitoring method (Roche, Shanghai, China). Serum creatinine and blood urea nitrogen were detected by enzymatic method and urease Berthelot method, respectively (Roche, Shanghai, China). Cell culture
American Journal of Hypertension 27(8) August 2014 1113
Downloaded from http://ajh.oxfordjournals.org/ at Universite Laval on July 14, 2015
The Fujian Medical University Animal Policy and Welfare Committee adhere to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences. All SHRs (male) and Wistar Kyoto (WKY) rats (male) were obtained from Vital River Laboratories (Beijing, China). All SHRs were randomized into 3 groups (n = 10 rats/group) at the age of 4 weeks and were treated by oral gavage once daily for 12 weeks with either the synthetic RXRα agonist bexarotene (obtained from Eisai, Tokyo, Japan) at doses of 30 or 100 mg/ kg body weight or with vehicle alone.
Echocardiographic and hemodynamic measurements
Zhu et al. Transfection of small interfering RNA
Predesigned small interfering RNAs (siRNAs) against rat LKB1 and control scrambled siRNAs were synthesized by Sigma (St Louis, MO). The sequences of the dsRNAs of LKB1 used in our experiments were as follows: sense: 5ˊ-GGG UAC UUC CGC CAG CUG AdTdT-3-ˊ; antisense: 5ˊ-UCA GCU GGC GGA AGU UAC CCdTdT-3ˊ. NRVMs were transfected with 25 nmol/L oligonucleotide using Transpass R2 Transfection Reagent (New England BioLabs, San Diego, CA, USA) under serum-free conditions and according to the manufacturer’s protocol. The control NRVMs were treated with the noncoding RNA. Target gene knockdown was assessed using Western blotting. Statistical analysis
RESULTS RXRα agonist bexarotene reduces LVH in hypertensive rats
After 12 weeks of bexarotene treatment, SHRs remained hypertensive. There were no significant differences in
Table 1. Physical and cardiac parameters of WKY rats and spontaneously hypertensive rats (SHRs) treated with vehicle or bexarotene
Parameter
Body weight, g
WKY
299.0 ± 1.87
SHR
305.7 ± 5.71
SHR + bexarotene
SHR + bexarotene
(30 mg/kg)
(100 mg/kg)
294.3 ± 3.68
Heart weight, g
0.91 ± 0.027
1.19 ± 0.042*
1.01 ± 0.026*,**
LV weight, g
0.69 ± 0.012
0.90 ± 0.038*
0.78 ± 0.023*,**
2.95 ± 0.104*
2.66 ± 0.064*,**
LVW/BW, g/kg
2.31 ± 0.039
296.0 ± 4.0 0.88 ± 0.024** 0.67 ± 0.012** 2.28 ± 0.027*
Systolic pressure, mm Hg
120.5 ± 4.98
191.8 ± 3.12*
185.6 ± 7.27*
178.7 ± 7.35*
Diastolic pressure, mm Hg
89.6 ± 3.24
132.7 ± 5.54*
118.5 ± 10.79*
115.5 ± 6.19*
LVPWd, mm
1.78 ± 0.086
2.14 ± 0.065*
1.87 ± 0.081*
1.84 ± 0.068* 1.90 ± 0.055*,**
IVSd, mm
1.66 ± 0.04
2.09 ± 0.055*
1.90 ± 0.058*,**
LVIDd, mm
6.40 ± 0.26
6.54 ± 0.15
6.57 ± 0.17
6.36 ± 0.19
Ejection fraction, %
84.88 ± 1.23
80.03 ± 2.20
78.63 ± 2.30
78.47 ± 1.47
ALT, U/L
9.8 ± 3.58
12.6 ± 6.23
11.67 ± 5.15
13.8 ± 3.48
AST, U/L
25.2 ± 4.03
26.4 ± 7.79
27.33 ± 10.93
28.2 ± 11.54
BUN, mmol/L
1.74 ± 0.38
1.66 ± 0.25
1.68 ± 0.23
2.31 ± 0.95
Scr, mmol/L
2.74 ± 0.52
3.24 ± 0.58
3.45 ± 0.99
3.04 ± 0.69
Data are mean + SEM. Abbreviations: ALT, glutamic-pyruvic transaminase; AST, glutamic oxalacetic transaminase; BUN, blood urea nitrogen; IVSd, diastolic intraventricular septal wall thickness; LVIDd, diastolic left ventricular internal diameter; LVPWd, diastolic left ventricular posterior wall thickness; Scr, serum creatinine. *P
Retinoid X Receptor Agonists Inhibit Hypertension-Induced Myocardial Hypertrophy by Modulating LKB1/AMPK/p70S6K Signaling Pathway Jiang Zhu,1 Ruo-Bing Ning,1 Xiao-Yan Lin,2 Da-Jun Chai,3 Chang-Sheng Xu,3 Hong Xie,3 Jin-Zhang Zeng,4 and Jin-Xiu Lin3
methods WKY rats served as controls. SHRs were randomized into 3 groups at the age of 4 weeks and were treated (once daily for 12 weeks) with either bexarotene (30 or 100 mg/kg body weight) or vehicle alone. Echocardiography was performed to determine cardiac structure and function. Neonatal cardiomyocytes were treated with AngII (10–7 mmol/L) with or without the indicated concentration of RXRα ligand 9-cis-RA. The protein abundances of β-actin, RXRα, LKB1, phosphoLKB1, AMPK, phospho-AMPK, P70S6K, phospho-P70S6K, ACE, and AT1 receptor were measured along with blood pressure, body weight and angiotensin II (Ang II) levels. The effects of LKB1 downregulation by LKB1 small, interfering RNA were examined.
results Treatment of SHRs with bexarotene resulted in significant inhibition of LVH without eliminating hypertension. Immunoblot with heart tissue homogenates from SHRs revealed that bexarotene activated the LKB1/AMPK signaling pathway and inhibited p70S6K. However, the increased Ang II levels in SHR serum and heart tissue were not reduced by bexarotene treatment. Treatment of cardiomyocytes with Ang II resulted in significantly reduced LKB1/AMPK activity and increased p70S6K activity. 9-cis-RA antagonized Ang II–induced LKB1/AMPK and p70S6K activation changes in vitro. conclusions RXR agonists prevent the inhibition of the LKB1/AMPK/p70S6K pathway and regulate protein synthesis to reduce LVH. This antihypertrophic effect of bexarotene is independent of blood pressure. Keywords: blood pressure; hypertension; hypertrophy; liver kinase B1; retinoid X receptor. doi:10.1093/ajh/hpu017
Pathological left ventricular hypertrophy (LVH) is a major risk factor for several serious cardiac events, including ischemia/reperfusion injury, arrhythmias, cardiomyopathies, and sudden death.1 Chronic pressure overload has been proposed to be a major factor initiating cardiac hypertrophy,1 which is associated with neurohormonal2 and metabolic3 changes and the activation of inflammatory signaling.4 Recently, increasing evidence suggests that alterations in signaling pathways also play an important role in the development of cardiac remodeling in conjunction with or in the absence of increased afterload.5 LVH is characterized by increased myocardial cell size. Enlargement of cardiomyocytes has been reported to result from an increase in protein content, and a major regulator of protein synthesis and cell growth is the mammalian target of rapamycin (mTOR)/
p70S6 kinase (p70S6K) pathway, which is regulated by several other kinase cascades.6 AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase. The AMPK-induced inhibition of the tuberous sclerosis complex (TSC)-mTOR-p70S6K pathway leads to the inhibition of mRNA translation by the 40S ribosomal protein S6, which culminates in reduced protein synthesis.7 Liver kinase B1 (LKB1) has recently been identified as an AMPK kinase that phosphorylates AMPK on its activating phosphorylation site (Thr172).8 Increased LKB1 activity in cardiac myocytes can decrease hypertrophy-induced protein synthesis, which suggests that LKB1 activation may be a target for the prevention of pathological cardiac hypertrophy.9 Loss of LKB1 is prohypertrophic and may be involved in the development of pathological cardiac hypertrophy.10
Correspondence: Da-Jun Chai ([email protected]).
1First Clinical Medical College, Fujian Medical University, Fuzhou, Fujian, China; 2Echocardiological Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China; 3Cardiovascular Department, First Affiliated Hospital, Fujian Medical University, Fuzhou, Fujian, China; 4School of Pharmaceutical Sciences and Institute for Biomedical Research, Xiamen University, Xiamen, China.
Initially submitted September 20, 2013; date of first revision October 14, 2013; accepted for publication January 12, 2014; online publication March 6, 2014.
© American Journal of Hypertension, Ltd 2014. All rights reserved. For Permissions, please email: [email protected]
1112 American Journal of Hypertension 27(8) August 2014
Downloaded from http://ajh.oxfordjournals.org/ at Universite Laval on July 14, 2015
background Retinoid X receptor (RXR) has been demonstrated to play an important role in cardiac development and has been implicated in cardiovascular diseases. This study aimed to examine the effects of RXRα agonist bexarotene on pathological left ventricular hypertrophy (LVH) in a spontaneously hypertensive rat (SHR) model and the underlying mechanism.
Retinoid X Receptor Agonists Inhibit Hypertension-Induced Myocardial Hypertrophy
Activation of the renin-angiotensin-aldosterone system (RAAS) plays a major pathophysiological role in the pathegenesis of hypertension and cardiac hypertrophy.11 Angiotensin II (Ang II), a core effector of the RAAS, is a multifunctional peptide with pleiotropic actions, including modulation of vasomotor tone, cell growth, migration, extracellular matrix deposition, and myocardial hypertrophy.12 Angiotensin-converting enzyme (ACE) is a ratelimiting enzyme in Ang II synthesis from angiotensinogen, and angiotensin receptor 1 (AT1R) is the main receptor that Ang II can react on in the process of cardiac hypertrophy.12 Several studies have indicated that inhibition of Ang II production prevents left ventricular (LV) remodeling, indicating that Ang II plays an important role in the development of cardiac remodeling.13,14 The retinoid X receptor (RXR) is an important member of the steroid/thyroid hormone superfamily of nuclear receptors that predominately function as transcription factors, having roles in development, cell differentiation, metabolism, and cell death.15 RXR is unique among nuclear receptors because it forms heterodimers with many other nuclear receptors, such as peroxisome proliferator-activated receptor, liver X receptor, and retinoic acid receptor (RAR), thereby mediating the biological effects of many hormones, vitamins, and drugs.16 RXR is a transcription factor that acts mainly as an RAR/RXR heterodimer.17 RXR plays a key role in the nuclear signaling pathways involving its dimeric partners. Numerous studies have demonstrated that retinoic acid can suppresses myocardial cell hypertrophy in response to mechanical stretch and Ang II.18,19 RXR signaling is crucial for, and indeed causative of, the differentiation phase of cardiomyogenesis.20 RXRα knockout mice die before birth because of a defect in cardiogenesis.21 Considering the function of RXR implied herein, we speculate that RXR may play an important role in the development of myocardial hypertrophy. In this study, we investigated the effect of RXRα on hypertension-induced myocardial hypertrophy and the underlying mechanism of that effect. Our results demonstrated that RXRα agonist treatment attenuated myocardial hypertrophy in spontaneously hypertensive rats (SHRs) by modulating the activation of the LKB1/AMPK/p70S6K signaling pathway, which occurs independent of blood pressure.
Neonatal rat ventricular myocytes (NRVMs) were prepared from the ventricles of 1–2-day-old Sprague-Dawley rat pups as previously described.23 Cells were cultured in Dulbecco’s modified essential medium (Gibco, Beijing, China) with 10% fetal bovine serum. After a 24-hour serum starvation, the cells were treated for 24 hour with Ang II (10–7 mmol/L; Sigma–Aldrich, St. Louis, MO, USA) with or without the natural RXRα ligand 9-cis-retinoic acid (9-cisRA) (Sigma-Aldrich, St. Louis, MO, USA). 9-cis-RA was added to the cells 1 hour before Ang II administration.
METHODS
Immunoblot analysis
Animal care
Cell homogenates and heart tissue homogenates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis in gels containing 10% acrylamide and were transferred to nitrocellulose as previously described.24 The following antibodies were used in this study: mouse monoclonal antibody against β-actin and rabbit monoclonal antibody against RXRα (Santa Cruz, Dallas, TX, USA); rabbit polyclonal antibody against total LKB1 and phosphorylated LKB1 (Ser428), rabbit polyclonal antibody against total AMPK and phosphorylated AMPK, and rabbit polyclonal antibody against total P70S6K and phosphorylated P70S6K (Cell Signaling Technology, Boston, MA, USA); and goat polyclonal antibody against AT1R and ACE (Santa Cruz, Dallas, TX, USA).
Transthoracic echocardiography was performed on mildly anesthetized rats with a 10S-(5.4–11.8 MHz) transducer (Vivid 7; GE Healthcare, Diegem, Belgium). Short-axis view and M-mode tracings were used to determine end-diastolic LV internal diameter, LV posterior wall thickness, interventricular septal thickness, and ejection fractions. Noninvasive blood pressure measurements were made with the Softron BP98A tail cuff system (Softron, Tokyo, Japan). Analysis of heart tissue and blood serum
Body weight (BW) was monitored at the end of 16th week. Then animals were killed with isoflurane (2% in oxygen). Blood samples were collected, the heart was removed, and the left ventricles were separated, blotted, and weighed. Hypertrophy was evaluated using the left ventricle weight to BW ratio. Hematoxylin and eosin staining was performed and then imaged. Myocyte cross-sectional area (CSA) was calculated by Image-Proplus software version 6.0 (Media Cybernetics, Bethesda, MD, USA). Samples (20 mg) of the left ventricle were homogenized, and 20 μg of protein were used for sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot analysis. Ang II concentrations were determined by radio immunoassay.22 Serum alanine aminotransferase and aspartate aminotransferase were measured with continuous monitoring method (Roche, Shanghai, China). Serum creatinine and blood urea nitrogen were detected by enzymatic method and urease Berthelot method, respectively (Roche, Shanghai, China). Cell culture
American Journal of Hypertension 27(8) August 2014 1113
Downloaded from http://ajh.oxfordjournals.org/ at Universite Laval on July 14, 2015
The Fujian Medical University Animal Policy and Welfare Committee adhere to the principles for biomedical research involving animals developed by the Council for International Organizations of Medical Sciences. All SHRs (male) and Wistar Kyoto (WKY) rats (male) were obtained from Vital River Laboratories (Beijing, China). All SHRs were randomized into 3 groups (n = 10 rats/group) at the age of 4 weeks and were treated by oral gavage once daily for 12 weeks with either the synthetic RXRα agonist bexarotene (obtained from Eisai, Tokyo, Japan) at doses of 30 or 100 mg/ kg body weight or with vehicle alone.
Echocardiographic and hemodynamic measurements
Zhu et al. Transfection of small interfering RNA
Predesigned small interfering RNAs (siRNAs) against rat LKB1 and control scrambled siRNAs were synthesized by Sigma (St Louis, MO). The sequences of the dsRNAs of LKB1 used in our experiments were as follows: sense: 5ˊ-GGG UAC UUC CGC CAG CUG AdTdT-3-ˊ; antisense: 5ˊ-UCA GCU GGC GGA AGU UAC CCdTdT-3ˊ. NRVMs were transfected with 25 nmol/L oligonucleotide using Transpass R2 Transfection Reagent (New England BioLabs, San Diego, CA, USA) under serum-free conditions and according to the manufacturer’s protocol. The control NRVMs were treated with the noncoding RNA. Target gene knockdown was assessed using Western blotting. Statistical analysis
RESULTS RXRα agonist bexarotene reduces LVH in hypertensive rats
After 12 weeks of bexarotene treatment, SHRs remained hypertensive. There were no significant differences in
Table 1. Physical and cardiac parameters of WKY rats and spontaneously hypertensive rats (SHRs) treated with vehicle or bexarotene
Parameter
Body weight, g
WKY
299.0 ± 1.87
SHR
305.7 ± 5.71
SHR + bexarotene
SHR + bexarotene
(30 mg/kg)
(100 mg/kg)
294.3 ± 3.68
Heart weight, g
0.91 ± 0.027
1.19 ± 0.042*
1.01 ± 0.026*,**
LV weight, g
0.69 ± 0.012
0.90 ± 0.038*
0.78 ± 0.023*,**
2.95 ± 0.104*
2.66 ± 0.064*,**
LVW/BW, g/kg
2.31 ± 0.039
296.0 ± 4.0 0.88 ± 0.024** 0.67 ± 0.012** 2.28 ± 0.027*
Systolic pressure, mm Hg
120.5 ± 4.98
191.8 ± 3.12*
185.6 ± 7.27*
178.7 ± 7.35*
Diastolic pressure, mm Hg
89.6 ± 3.24
132.7 ± 5.54*
118.5 ± 10.79*
115.5 ± 6.19*
LVPWd, mm
1.78 ± 0.086
2.14 ± 0.065*
1.87 ± 0.081*
1.84 ± 0.068* 1.90 ± 0.055*,**
IVSd, mm
1.66 ± 0.04
2.09 ± 0.055*
1.90 ± 0.058*,**
LVIDd, mm
6.40 ± 0.26
6.54 ± 0.15
6.57 ± 0.17
6.36 ± 0.19
Ejection fraction, %
84.88 ± 1.23
80.03 ± 2.20
78.63 ± 2.30
78.47 ± 1.47
ALT, U/L
9.8 ± 3.58
12.6 ± 6.23
11.67 ± 5.15
13.8 ± 3.48
AST, U/L
25.2 ± 4.03
26.4 ± 7.79
27.33 ± 10.93
28.2 ± 11.54
BUN, mmol/L
1.74 ± 0.38
1.66 ± 0.25
1.68 ± 0.23
2.31 ± 0.95
Scr, mmol/L
2.74 ± 0.52
3.24 ± 0.58
3.45 ± 0.99
3.04 ± 0.69
Data are mean + SEM. Abbreviations: ALT, glutamic-pyruvic transaminase; AST, glutamic oxalacetic transaminase; BUN, blood urea nitrogen; IVSd, diastolic intraventricular septal wall thickness; LVIDd, diastolic left ventricular internal diameter; LVPWd, diastolic left ventricular posterior wall thickness; Scr, serum creatinine. *P
