| Background:Hypertension is a substantial public health problem, the major pathophysiological character is pressure overload and cardiovascular remodeling, including vascular remodeling and cardiac remodeling. A lot of clinical investigations reveal that essential hypertension and consequent left ventricular hypertrophy not only contribute to ventricular systolic and diastolic dysfunction, but also have been recognized as a strong, virtually independent risk factor. So the desirability to prevent and induce regression of LVH by drug treatment is widely understood, and a variety of antihypertensive drugs has been shown to cause regression of LVH in treated hypertensives. However, the advantages and disadvantages of these drugs have been debated and there is an impetus for the development of new chemical entities with a more desirable therapeutic endpoint, decreasing morbidity and mortality of hypertensives.Methods:In vivo: Adult male Sprague–Dawley rats (weight range 180–220 g), after rats were anesthetized, under sterile conditions, the abdominal aorta was exposed through a abdominal incision and constricted at the suprarenal level by a 4-0 silk suture tied around both the aorta and a blunted 22-gauge needle, which was then pulled out. A similar procedure was performed for Sham group except without the ligature.In vitro: Sprague–Dawley rats neonatal ventricular myocytes were isolated and cultured. Cardiomyocytes were exposed to isoproterenol (ISO) for 48 h, and the total protein content of the cardiomyocytes was measured. Laser scanning confocal microscope was used to determine the [Ca2+]i of cardiomyocytes.Partâ… The experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload ratsAnimals were divided into 6 groups:â‘´Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;â‘·IPT 3 mg/kg group;⑸IPT 9 mg/kg group;⑹Lisenopril 15 mg/kg group. The drugs were dissolved in distilled water and administered orally via a gastric tube. Drugs were administered from 3 days after operation through 6 weeks after surgery. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson's stain. RT-PCR method was used to measure the cardiac hypertrophy marker gene, ANP and BNP mRNA expression.Partâ…¡IPT reverse cardiovascular remodeling: Endothelial mechanism1 Role of endothelin system in pressure-overload ratsAnimals were divided into 5 groups:â‘´Sham group;⑵C ontrol group;⑶IPT 1 mg/kg group;â‘·IPT 3 mg/kg group;⑸I PT 9 mg/kg group. Radioimmunoassay was used to determine the serum ET-1 content, RT-PCR method was used to measure the cardiac ET-1 and ECE mRNA expression, immunochemistry method was used to evaluate the cardiac ETA and ETB expression.2 Role of eNOS-NO signal pathway in pressure-overload ratsAnimals were divided into 5 groups:â‘´Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;â‘·L-NAME 50 mg/kg group;⑸L -NAME 50 mg/kg + IPT 3 mg/kg group. At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson's stain. RT-PCR method was used to measure the cardiac eNOS mRNA expression, immunochemistry method was used to evaluate the cardiac eNOS expression, and biochemical method was used to determine serum nitric oxide content.3 Role of PGI2 in pressure-overload ratsAnimals were divided into 5 groups:â‘´Sham group;⑵C ontrol group;⑶IPT 3 mg/kg group;â‘·i ndomethacin 2 mg/kg group (Indo 2);⑸indomethacin 2 mg/kg + IPT 3 mg/kg group(Indo 2+IPT 3). At the end of the experiment, an eight-channel direct-writing oscillograph were used to evaluate the hemodynamics and heart function, the histological changes were investigated by HE and Masson's stain. Radioimmunoassay was used to determine the serum PGI2 content.Partâ…¢Effect of ISO on cultured cardiomyocytes hypertrophy induced by ISO1: Ascertain the appropriate concentration of ISO inducing cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plates for 72 h, 1×105/well, and then divided into 6 groups:â‘´Control group;⑵ISO 1×10-8 mol/L;⑶ISO 1×10-7 mol/L;â‘·I SO 1×10-6 mol/L;⑸ISO 1×10-5 mol/L;⑹I SO 1×10-4 mol/L. After 48 h, Lowry's method was used to determine the cardiomyocytes total protein content. 2: Effect of IPT on cardiomyocytes total protein content in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:â‘´Control group;⑵ISO 1×10-5 mol/L;⑶ISO 1×10-5 mol/L+IPT 1×10-7 ;â‘·ISO 1×10-5 mol/L+IPT 1×10-6;⑸ISO 1×10-5 mol/L+IPT 1×10-5;⑹ISO 1×10-5 mol/L+IPT 1×10-4. After 48 h, Lowry's method was used to determine the cardiomyocytes total protein content. 3: Effect of IPT on cardiomyocytes [Ca2+]i in ISO-induced cardiomyocytes hypertrophy. Cardiomyocytes were cultured in 24 well tissue culture plate for 72 h, 1×105/well, then divided into 6 group:â‘´Control group;⑵I SO 1×10-5 mol/L;⑶I SO 1×10-5 mol/L+IPT 1×10-7 ;â‘·ISO 1×10-5 mol/L+IPT 1×10-6;⑸ISO 1×10-5 mol/L+IPT 1×10-5;⑹ISO 1×10-5 mol/L+IPT 1×10-4. After 48 h, laser scanning confocal microscope was used to determine [Ca<sup>2+]i of cardiomyocytes, 20 cells were selected in each group.Results:Partâ… The experimental therapeutic effects of iptakalim on cardiovascular protection in pressure-overload ratsBlood pressure: During the time course, tail artery systolic blood pressure(SBP) in Sham group maintain at the baseline level, tail artery SBP in Control group increased significantly with a time-dependent manner, IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of tail artery SBP markedly from 2 week. At the end of 6 week, carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP increased significantly with a time-dependent manner(P<0.01 vs Sham group), IPT 1,3,9 mg/kg/d and Lisenopril 15 mg/kg/d treatment inhibited the increase of carotid aorta SBP, DBP, MBP and transtenosis gradient of SBP markedly.Heart function: After 42 d of aortic banding, LVSP,±dp/dtmax,Vmp,Vmax increased significantly, while left ventricular LVEDP decreased significantly compared with Sham group (P<0.01). IPT 1, 3, 9 mg/kg/d and Lisenopril 15 mg/kg/d treatment ameliorated left ventricular systolic and diastolic function, augmented cardiac compliance markedly.Cardiovascular remodeling:After 42 d of aortic banding, heart weight/body weight(HW/BW), left ventricular weight/body weight(LVW/BW), right ventricular weight/body weight(RVW/BW) , lung weight/body weight(LW/BW) total area of total aorta(TAA), area of lumen(LA), cross-section area(CSA), CSA/TAA, aorta radium(AR), media(M) and M/L increased significantly, indicating a morphological remodeling of heart and aortic remodeling. Cardiomyocytes cross-section area, CVF, PVCA and cardiac tissue hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01). ANP and BNP mRNA expression increased significantly in Control group rats compared with that in Sham group rats(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, HW/BW, LVW/BW, RVW/BW, LW/BW, cardiomyocyte cross-section area(41.54%,43.59%,44.10%, P<0.01 vs Control group),CVF(35.71%,73.21%,P<0.01 vs Control group), PVCA(34.96%,56.50%,P<0.05 vs Control group),cardiac tissue Hydroxyproline content(56.72%,92.53%,P<0.05 vs Control group) were reversed ,respectively. Cardiac ANP and BNP mRNA expression were downregulated significantly(P<0.05, P<0.01 vs Control group).Partâ…¡IPT reverse cardiovascular remodeling: Endothelial mechanism1 Role of endothelin system in pressure-overload rats After 42 d of aortic banding, plasma ET-1 content was increased (101.97±19.64 pg/ml vs 135.27±36.91 pg/ml,P<0.05 vs Sham group), cardiac tissue ET-1,ECE mRNA and ETA,ETB protein expression were upregulated in Control group(P<0.01). After treatment with IPT 1,3,9 mg/kg/d, plasma ET-1 content was decreased significantly(P<0.01 vs Control group), the upregulation of cardiac tissue ET-1,ECE mRNA and ETA,ETB protein expression were inhibited markedly(P<0.05,P<0.01 vs Control group).2 Role of eNOS-NO signal pathway in pressure-overload ratsBlood pressure: Chronic L-NAME administration caused persistent severe hypertension. The SBP (of the 2nd, 4th, 6th weeks) were 20.6±2.2, 20.8±1.7 and 21.2±1.4 kPa versus 17.9±2.5, 18.7±1.2 and 18.0±1.2 kPa in Control group rats, respectively (P<0.05, P<0.0l). SBP of L-NAME 50 + iptakalim 3 group in the 2nd, 4th, 6th weeks had 10.4%, 15.1%, 10.7% lower than that of rats given L-NAME alone, respectively(P<0.0l).Heart function: After 42 days of NO synthase inhibition, ASBP,LVSP,±dp/dtmax were significantly increased(P<0.05,P<0.01 vs Sham group), while LVEDP decreased markedly (P<0.01 vs Sham group), indicating an enhanced contractility but a reduced diastolic compliance were taken place, and iptakalim could statistically or partially ameliorate the LV function(P<0.05 versus L-NAME 50 group). Interestingly, in L-NAME 50 mg/kg/d rats (whether iptakalim was given or not), the LVW/BW and RVW/BW of rats were not statistically different from those of Sham group, despite of the significant increase in blood pressure.Cardiac remodeling: Histological examination demonstrated that both CVF, PVCA and cardiac tissue hydroxyproline revealed significant difference in both L-NAME 50 mg·kg-1·d-1 and IPT 3 mg·kg-1·d-1+L-NAME 50 mg·kg-1·d-1 compared to that of Sham group, and combined with IPT 3 ameliorated cardiac tissue hydroxyproline compared with that of L-NAME 50 alone. However, the extent of myocyte hypertrophy of rats given IPT 3 mg/kg/d +L-NAME 50 mg/kg/d or given L-NAME 50 mg/kg/d alone were not statistically different from those of Sham group. Ultrastructural examination revealed well-organized myofibrils with mitochondria grouped along the periphery of longitudinally oriented fibers were found in Sham group rats, mitochondria tightly packed, intact horizontally oriented cristae in Sham group rats, well-preserved intercalated disk with smooth contours in Sham group rats. The cardiomyocytes in iptakalim 3+L-NAME 50 group showed almost uniform parallel myofibril arrangement. Z-lines dividing the sarcomeres were linear and perpendicular to the myofilaments. Myofibrils alternated with few rows of ovoid mitochondria. However, the number of mithochondria in the latter group seems to be larger. Ultrastructure of the cardiomyocytes from L-NAME-treated rats presented mitochondrial changes, characterized by diffuse mild to moderate increase in size (hypertrophy) and number (hyperplasia), focally forming several rows of enlarged mitochondria separating the myofibrils. Many mitochondria had the appearance of elongated giant mitochondria. Longitudinally oriented cristae, either parallel to the long axis of the mitochondrion or irregular and concentric, were observed in many areas.eNOS-NO: After 42 d of aortic banding, serum NO content was decreased (21.56±8.06μmol/L vs 13.89±1.59μmol/L,P<0.05), treatment with IPT 1,3,9 mg/kg increase serum NO content significantly with a dose-dependent manner, significant negative linear correlations were found between LVW/BW, cardiac tissue hydroxyproline , SBP and serum NO. There was a tendency for decreased immunoreactivity to eNOS and eNOS mRNA expression in Control group rats compared with that in Sham group rats. Immunoreactivity for eNOS and eNOS mRNA expression was obviously enhanced in cardiomyocytes by long-term iptakalim treatment. However, long-term L-NAME administration reduced immunoreactivity for eNOS and eNOS mRNA expression which can be completely improved by iptakalim 3 +L-NAME 50.3 Role of PGI2 in pressure-overload ratsBlood pressure: Chronic indomethacin administration caused time-dependent severe hypertension, increased ASBP and transtenosis blood pressure. Combined with IPT 3 ameliorated indomethacin induced severe hemodynamics partially. There were no statistically difference between these two groups and Control group.Heart function: Prolonged administration indomethacin enhanced cardiac contractility and reduced cardiac diastolic compliance, combined with IPT 3 inhibited the increase of cardiac contractility, ameliorated cardiac compliance.Cardiac remodeling: After 42 d of aortic banding, HW/BW, LVW/BW, RVW/BW and cardiomyocytes cross-section increased significantly in Indo 2 group. Combined with IPT 3 ameliorated cardiac hypertrophy partially but not completely. Long-term treatment with indomethacin cardiac CVF, PVCA and hydroxyproline increased significantly compared with Sham group(P<0.05,P<0.01), combined with IPT 3 inhibited the deterioration of CVF and PVCA(P<0.05,P<0.01 vs Indo 2) ,inhibited the increase of cardiac hydroxyproline(P<0.05 vs Indo 2).PGI2: After 42 d of aortic banding, plasma PGI2 content decreased markedly compared with Sham group(63.87%,P<0.05).Long-term treatment with IPT 1, 3, 9 mg/kg increase plasma PGI2 significantly with a dose-dependent manner. Chronic administration indomethacin decrease plasma PGI2 content significantly, combined with IPT 3 inhibited the decrease of plasma PGI2 content markedly(P<0.05 vs Indo 2). Partâ…¢Effect of ISO on cultured cardiomyocytes hypertrophy induced by ISO 1: The total protein of cardiomyocytes was increased by 24.53% and 31.74% in ISO 1×10-5mol/L group and ISO 1×10-4mol/L group, respectively(P<0.01 vs Control group), and there is no statistically difference between these two group, indicating 1×10-5mol/L ISO can activatedβ-AR completely; 2: The total protein of cardiomyocytes was increased by 39.62% in ISO 1×10-5mol/L group(P<0.01 vs Control group), in IPT 1×10-7mol/L, 1×10-6mol/L, 1×10-5mol/L, 1×10-4mol/L group, the total protein of cardiomyocytes was markedly decreased with a dose-dependent manner and the inhibition rate was 52.38%, 85.71%, 95.24% and 95.24%, respectively; 3: Compared with values in Control group, the fluorescence intensity increased significantly in ISO 1×10-5mol/L group(15.18±10.99 vs 125.82±45.56,P <0.01)which could be inhibited completely in IPT-treated group, and the inhibition rate was 26.36%, 31.82%, 70.91% and 90.91%, respectively.Conclusions1 iptakalim 1, 3, 9 mg/kg can significantly reverse abdominal aorta binding/pressure- overload induced cardiovascular remodeling with a dose-dependent manner;2 iptakalim might possess cardioreparative properties, its mechanism may contribute to binding KATP channel in endothelial cells, ameliorating endothelium cells function, augmenting NO and PGI2 secretion, inhibiting ET-1 biosynthesis, secretion, inhibiting ET-1 system activation;3 In cultured cardiomyocytes in vitro, iptakalim inhibites cardiomyocytes hypertrophy and total protein enhancement, this may contribute to decreasing cell resting membrane potential and inhibiting [Ca2+]i.
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