Cardiac sarcoplasmic reticulum characteristics in hypertrophic hearts from spontaneously hypertensive rats

Summary Sarcoplasmic reticulum (SR) from left ventricles of rats that developed spontaneous hypertension was studied in vitro. Similar increases of left ventricular mass were found when grouping the animals into mild and severe hypertensives (average systolic arterial pressure of 168±4 and 202±6 mm Hg, respectively). The amount of SR protein (mg/g of left ventricle) was higher when obtained from hypertrophic ventricles of both hypertensive groups than from ventricles of the control group. The result agreed with the enhanced Ca²⁺ uptake exhibited by left ventricular homogenates of the hypertensive groups. Consequently, Ca²⁺ uptake in SR microsomes isolated per gram of left ventricle (nmol Ca²⁺/g muscle) was 51.62±10.06 and 64.99±12.84 in mildly and severely hypertensive groups vs. 17.37±5.79 in the control group (P<0.05). The SR microsomes obtained from ventricles of hypertensive rats showed an enhanced Ca²⁺ activated ATPase activity that was not accompanied by increased Ca²⁺ uptake at saturating calcium concentrations, but by increased affinity for calcium (K'_app of 1.09±0.28 and 2.67±0.16 M in SR microsomes of hypertrophic and control ventricles respectively; P<0.05). The rats of calcium loss, measured in SR vesicles passively loaded with⁴⁵Ca, were similar when assayed in SR obtained from ventricles of both hypertensive and normal rats. These results enable us to suggest that in hearts of rats presenting spontaneous hypertension, the function of the SR system could account for a normal handling of cytosolic calcium. They might support the absence of mechanical alterations described in hearts of young rats of the SHR strain.This work was supported by grants from the Consejo nacional de Investigaciones Científicas y Tecnicas de la República Argentina (CONICET).     

血管紧张素转换酶抑制剂和血管紧张素II受体1拮抗剂对组织醛固酮逃逸现象的实验研究

目的　观察血管紧张素转换酶抑制剂 (ACEI)及血管紧张素II受体 1拮抗剂长期治疗是否存在心血管组织的醛固酮逃逸。方法　用肠系膜动脉、心脏离体灌注 ,反向高效液相分离纯化和放免检测ACEI及血管紧张素II受体 1拮抗剂对自发性高血压大鼠 (SHR)心脏、血管局部合成醛固酮的影响 ,用RT PCR证实对心脏、血管表达醛固酮合成酶基因CYP11B2mRNA的作用。结果　治疗 5个月后依那普利未明显降低SHR心脏、血管合成醛固酮及CYP11B2mRNA的表达 ;培哚普利抑制血管局部醛固酮的合成及CYP11B2mRNA的表达 ,但还不能完全抑制心脏合成醛固酮及CYP11B2mRNA的表达 ,虽然与依那普利比较有显著性差异。氯沙坦长期治疗后 ,虽然引起血浆血管紧张素II水平显著增高 ,但不引起血浆醛固酮逃逸现象 ,能显著抑制心脏、血管醛固酮合成及其基因表达至正常Wistar鼠水平。结论　长期ACEI治疗存在组织水平的醛固酮逃逸 ,培哚普利较依那普利导致的逃逸程度轻。氯沙坦长期治疗未发现血浆及组织水平的醛固酮逃逸。     

血管紧张素转换酶抑制剂和血管紧张素Ⅱ受体1拮抗剂对组织醛固酮逃逸现象的实验研究

目的 观察血管紧张素转换酶抑制剂（ACEI）及血管紧张素Ⅱ受体1拮抗剂长期治疗是否存在心血管组织的醛固酮逃逸。方法 用肠系膜动脉、心脏离体灌注,反向高效液相分离纯化和放免检测ACEI及血管紧张素Ⅱ受体1拮抗剂对自发性高血压大鼠（SHR）心脏、血管局部合成醛固酮的影响,用RT－PCR证实对心脏、血管表达醛固酮合成酶基因CYP11B2 mRNA的作用。结果 治疗5个月后依那普利未明显降低SHR心脏、血管合成醛固酮及CYP11B2 mRNA的表达;培哚普利抑制血管局部醛固酮的合成及CYP11B2mRNA的表达,但还不能完全抑制心脏合成醛固酮及CYP11B2 mRNA的表达,虽然与依那普利比较有显性差异。氯沙坦长期治疗后,虽然引起血浆血管紧张素Ⅱ水平显增高,但不引起血浆醛固酮逃逸现象,能显抑制心脏、血管醛固酮合成及其基因表达至正常Wistar鼠水平。结论 长期ACEI治疗存在组织水平的醛固酮逃逸,培哚普利较依那普利导致的逃逸程度轻。氯沙坦长期治疗未发现血浆及组织水平的醛固酮逃逸。     

血管紧张素Ⅱ-1型受体的shRNA对自发性高血压大鼠血压影响的实验研究

目的观察以重组腺病毒为载体的血管紧张素Ⅱ-1型受体的shRNA（AdS—AT1R—shRNA）对自发性高血压大鼠（SHR）血压的影响及对组织血管紧张素Ⅱ-1型受体（AT1R）基因表达的影响。方法在293细胞内扩增已构建好的荧光蛋白标记的携带AT1R shRNA的重组腺病毒（AdS—AT1R—shRNA）,TCID50法测定重组腺病毒滴度。22只SHR随机分为2组,实验组（n=11）和高血压对照组（n=11）,另设11只Wistar—Kyoto（WKY）大鼠为正常血压对照组,实验组SHR经鼠尾静脉单次注射Ad5—AT1R—shRNA,Ad5—AT1R—shRNA经TCID50法测定感染性滴度为1．7×10^9TCID50／ml,高血压对照组和正常血压对照组经鼠尾静脉单次注射对照重组复制缺陷型腺病毒（Ad5—EGFP）,感染性滴度为7.9×10^9TCID50／ml。注射前及注射后每天定时监测血压及心率,于血压出现明显下降时处死部分动物,取出心脏、肝脏、肾脏、主动脉及肾上腺组织,在荧光显微镜下观察他们对Ad5—AT1R—shRNA的吸收情况,采用荧光定量PCR检测肝脏、肾脏及主动脉组织AT1R mRNA的表达情况。结果实验开始24h后,实验组收缩压[（163±7）mmHg,1mmHg=0．133kPa]出现明显下降,最大降压幅度达29mmHg,与SHR组[（182±8）mmHg]比较差异有统计学意义（P〈0．05）,此后降压作用可持续5天,最长可持续7天。SHR组和WKY组血压均未见明显下降,SHR组有的血压可见继续升高。3组动物的心率变化不明显,肾脏、心脏、肝脏、主动脉及肾上腺组织在荧光显微镜下可见大量荧光表达。实验组肾脏及主动脉AT1R的mRNA表达量（分别为0．086±0．014,0,051±0．023）明显低于SHR组（分别为0．362±0．042,0．463±0．045）,P〈0,01。结论AdS—AT1R—shRNA经静脉注射后可被许多重要脏器吸收,且对SHR的AT1R起到RNA干扰的作用,在mRNA水平抑制AT1R的基因表达。AdS—AT1R·shRNA通过阻抑AT1R生成对SHR起到明显且持久的降压作用。     

老年高血压病合并冠心病、脑梗塞患者AT1R基因多态性的研究

目的 探讨血管紧张素Ⅱ-Ⅰ型受体（AT1R）基因A1166／C多态性与老年原发性高血压病的关系，并探讨原发性高血压病的发病机制。方法 应用聚合酶链式反应、限制性内切酶酶解（PCR－RFLP）的方法检测40例健康人和92例原发性高血压病患者（其中31例合并冠心病，37例合并脑梗塞患者）的AT1R基因型；生化技术测定血脂水平。结果 原发性高血压病组、合并冠心病组及合并脑梗塞组的C等位基因频率14．6％、14．5％和10．8％，分别显著高于正常对照组的3．7％（P＜0．05）；带有C等位基因的原发性高血压病患者无论是否合并冠心病、脑梗塞，其血浆LP（a)水平均增高。结论 提示AT1R基因可能是老年原发性高血压病的重要遗传因素，AT1R基因可能参与脂质的调节。     

Estradiol increases angiotensin II type 1 receptor in hearts of ovariectomized rats

We tested the hypothesis that 17beta-estradiol (E(2)) has dual effects on the heart, increasing levels of proteins thought to have beneficial cardiovascular effects (e.g. endothelial nitric oxide (NO) synthase (eNOS)) as well as those thought to have detrimental cardiovascular effects (e.g. type 1 angiotensin II (AngII) receptor (AT(1)R)). Ovariectomized Wistar rats consuming a high-sodium diet received one of four treatments (n=7 per group): group 1, placebo pellets; group 2, E(2) (0 x 5 mg/pellet, 21-day release); group 3, NOS inhibitor, N(omega)-nitro-L-arginine-methyl-ester (L-NAME; 40 mg/kg per day for 14 days) plus Ang II (0 x 225 mg/kg per day on days 11-14); group 4, E(2) plus L-NAME/Ang II. E(2) increased cardiac levels of estrogen receptors ESR1 and ESR2, an ESR-associated membrane protein caveolin-3, eNOS, and phosphorylated (p)eNOS, thus, exerting potentially beneficial cardiovascular effects on NO. However, E(2) also increased cardiac levels of proteins associated with cardiovascular injury and inflammation including, AT(1)R, protein kinase C delta (PRKCD), phosphorylated PRKC, and phosphorylated extracellular signal regulated kinase (pMAPK)3/1, plasminogen activator inhibitor-1 (PAI-1), osteopontin and ED-1, a monocyte/macrophage-specific protein. E(2) treatment led to similar protein changes in the hearts of L-NAME/Ang II-treated rats except that the increase in peNOS was prevented, and L-NAME/Ang II and E(2) had additive effects in increasing cardiac PRKCD and PAI-1. Thus, the highest levels of cardiac PAI-1 and PRKCD occurred in L-NAME/Ang II-treated rats receiving E(2). In summary, E(2) treatment increased cardiac expression of AT(1)R as well as the expression of pro-inflammatory and prothrombotic factors.     

Postischemic apoptosis and functional recovery after angiotensin II type 1 receptor blockade in isolated working rat hearts

OBJECTIVE: To determine whether chronic angiotensin (AngII) type I receptor (AT1R) blockade inhibits cardiomyocyte (CM) apoptosis and attenuates left ventricular (LV) dysfunction after ischemia-reperfusion (IR) in the isolated working rat heart. METHODS: Postischemic recovery of LV developed pressure, the apoptotic index (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP in situ nick end labeling or TUNEL assay), and changes in expression of apoptotic markers Bcl-2, Bax, p53 and caspase-3 (Western immunoblots) were measured after IR (50 min aerobic perfusion; 25 min global ischemia; 40 min reperfusion) in working rat hearts that were randomized to five groups of six each along 1 week or 3 week pretreatment arms: sham (no drug, no perfusion); no drug, aerobic perfusion; and oral AT1R blockers losartan (30 mg/kg per day) or UP269-6 (3 mg/kg per day), or no drug before IR. RESULTS: Compared to the no drug group after IR, losartan (not UP269-6) preserved functional recovery in 1 and 3 week groups. However, both losartan and UP269-6 reduced the apoptotic index and normalized the increase in Bax, decrease in Bcl-2 and increase in p53 and caspase-3 after IR. A bell-shaped relation between apoptosis and functional recovery after IR was flattened by AT1R blockade. CONCLUSION: The results indicate that IR is associated with LV dysfunction and CM apoptosis involving activation of p53, caspase-3, and increased Bax/Bcl-2 ratio in the working rat heart. Importantly, chronic AT1R blockade inhibited the apoptosis and changes in expression of the markers without improving functional recovery, implying that decrease in apoptosis does not necessarily translate into decreased LV dysfunction.     

Delivery of angiotensin II type 1 receptor antisense inhibits angiotensin action in neurons from hypertensive rat brain.

Increased brain angiotensin II (AII) type 1 receptor (AT1R) expression has been implicated in the hyperactive brain angiotensin system and the development and maintenance of hypertension in the genetically spontaneously hypertensive (SH) rat. Neuronal cells in primary culture from the cardioregulatory-relevant brain areas (hypothalamus/brainstem) mimic increased brain AT1R gene expression and AT1R function of the adult SH rat. They have been utilized in the present study to determine whether cellular actions of AII could be regulated by the transfer of AT1R antisense (AT1R-AS) with the use of a retroviral-mediated gene delivery system developed for the central nervous system cultures. AII stimulates norepinephrine (NE) uptake in neuronal cultures of both normotensive (Wistar Kyoto) and SH rat brains. This neuromodulatory action is mediated by the AT1R subtype, is significantly higher in SH neurons, and is associated with a parallel stimulation of mRNAs for c-fos and NE transporter. Infection of neuronal cultures with a retrovirus vector that contains AT1R-AS (LNSV-AT1R-AS) results in an inhibition of AT1R-mediated stimulation of both c-fos and NE transporter mRNA, as well as NE uptake in both strains of rats; however, the inhibition is more pronounced in SH neurons compared with Wistar Kyoto rat brain neurons. The higher sensitivity of the SH rat brain neurons is further supported by our observation that a certain dose of LNSV-AT1R-AS that fails to induce inhibition of cellular actions of AII in WKY neurons causes a significant inhibition of AII actions in SH neurons. These observations show that retrovirally mediated delivery of AT1R-AS could be used to selectively control the actions of AII in primary neuronal cultures from SH rat brain.     

Angiotensin II type 1 receptor antagonist downregulates nonmuscle myosin heavy chains in spontaneously hypertensive rat aorta

The aim of this study was to clarify the differences between the angiotensin II type 1 (AT1) receptor antagonist and the angiotensin-converting enzyme (ACE) inhibitor on smooth muscle and nonmuscle myosin heavy chain isoforms in aortic smooth muscle cells of Wistar-Kyoto rats and spontaneously hypertensive rats. All 4 myosin heavy chain isoforms are heterogeneously expressed in the smooth muscle cells of the aortic tunica media in 20-week-old rats, and the contractile-type myosin heavy chains are highly expressed in smooth muscle cells of the aortic tunica media compared with the synthetic-type myosin heavy chains. Both the AT1 receptor antagonist and the ACE inhibitor had the same effects on hemodynamics, smooth muscle cell hypertrophy and proliferation, fibrosis, and vascular remodeling in spontaneously hypertensive rats. However, the AT1 receptor antagonist had a more potent effect on the downregulation of the synthetic-type myosin heavy chains than the ACE inhibitor in spontaneously hypertensive rat aortic tunica media. In contrast, these effects of the AT1 receptor antagonist and the ACE inhibitor on hemodynamics, morphology, fibrosis, and expression of myosin heavy chain isoforms in smooth muscle cells of the aortic tunica media were not observed in Wistar-Kyoto rats. Thus, within 6 weeks, the AT1 receptor antagonist might modulate the cellular composition of myosin heavy chain isoforms in smooth muscle cells more efficiently than the ACE inhibitor, without morphological changes in the spontaneously hypertensive rat aorta.     

Role of angiotensin II type 2 receptor during regression of cardiac hypertrophy in spontaneously hypertensive rats

We previously reported that the AT₁ receptor antagonist valsartan and the angiotensin converting enzyme (ACE) inhibitor enalapril decrease DNA synthesis and stimulate apoptosis in interstitial fibroblasts and epicardial mesothelial cells during regression of ventricular hypertrophy in spontaneously hypertensive rats (SHR). To examine the role of the AT₂ receptor in this model, we studied hearts from SHR treated with valsartan or enalapril either alone or combined with the AT₂ antagonist PD123319 for 1 or 2 weeks. Apoptosis was evaluated by quantification of DNA fragmentation or by TUNEL labeling. At 1 week, valsartan significantly increased ventricular DNA fragmentation, increased apoptosis in epicardial mesothelial cells, and decreased DNA synthesis. At 2 weeks, ventricular DNA content and cardiomyocyte cross-sectional area were significantly reduced. These valsartan-induced changes were attenuated by PD123319 co-administration. However, valsartan-induced increases in apoptosis of left ventricular interstitial non-cardiomyocytes was unaffected by the AT₂ blocker. Enalapril-induced changes were similar to those observed with valsartan but were not affected by co-treatment with PD123319. These results demonstrate that AT₁ and AT₂ receptors act in a coordinated yet cell-specific manner to regulate cell growth and apoptosis in the left ventricle of SHR during AT₁ receptor blockade but not ACE inhibition.     

Effects of angiotensin II subtype 1 receptor blockade on cardiac fibrosis and sarcoplasmic reticulum Ca2+ handling in hypertensive transgenic rats overexpressing the Ren2 gene

OBJECTIVE: We evaluated the effects of angiotensin II subtype 1 (AT1) receptor antagonism on cardiac fibrosis and sarcoplasmic (SR) Ca2+ handling in a transgenic rat model of renin-dependent left ventricular (LV) hypertrophy (LVH). METHODS: Hypertensive transgenic rats overexpressing the Ren2 gene (TGR(mRen2)27) were treated between 10 and 30 weeks of age with the angiotensin II subtype 1 (AT1) receptor antagonist, eprosartan, in an antihypertensive (Ren2-E60, 60 mg/kg per day) and a non-antihypertensive (Ren2-E6, 6 mg/kg per day) dose applied intraperitoneally via osmotic-mini-pumps. They were compared to age-matched Ren2 and Sprague-Dawley (SD) control rats receiving 0.9% NaCl as vehicle via osmotic mini-pumps (Ren2-Vehicle, SD-Vehicle, respectively). RESULTS: Systolic blood pressure (SBP), LV weight, LV end-diastolic pressure (LVEDP), and cardiac fibrosis were elevated in Ren2-Vehicle, while diastolic function (-dP/dt(max)) and sarcoplasmic reticulum (SR) Ca2+ uptake were decreased in Ren2-Vehicle compared to SD-Vehicle (P < 0.05, respectively). SBP was not altered in Ren2-E6, but reduced to normotensive levels in Ren2-E60 compared to Ren2-Vehicle and SD-Vehicle (P < 0.0001). In both Ren2-E6 and Ren2-E60, LV weights were reduced and LVEDP and -dP/dt(max)normalized compared to Ren2-Vehicle (P < 0.05). SR Ca2+ uptake was normalized in both Ren2-E6 and Ren2-E60. Cardiac fibrosis did not change in Ren2-E6, but perivascular LV fibrosis and hydroxyprolin content were reduced in Ren2-E60 compared to Ren2-Vehicle (P < 0.05, respectively). CONCLUSIONS: Normalization of LV SR Ca2+ uptake is an important mechanism by which AT1 receptor antagonism improves LV diastolic dysfunction independent from a reduction of SBP and cardiac fibrosis in the TGR (mRen2)27 model.     

利尿剂对高血压大鼠细胞色素P450表氧化酶及血管紧张素Ⅱ1型受体基因表达的影响

目的　噻嗪类利尿剂是广泛使用的降血压药物 ,然而其降血压机理仍然不完全清楚。本研究观察了氢氯噻嗪和吲哒帕胺对自发性高血压大鼠 (SHR)细胞色素P4 5 0 (CYP)表氧化酶 2C11及血管紧张素Ⅱ 1型受体 (AT1)基因表达的影响 ,以探讨其降低血压的分子机制。方法　成年雄性SHR随机分为三组 ,分别每日经胃管给予氢氯噻嗪 (10mg/kg)、吲哒帕胺 (0 6 2 5mg/kg)和等量去离子水灌胃 ,并测量血压和 2 4h尿量。 4周后 ,检测主要脏器的CYP表氧化酶 2C11和AT1表达情况及形态学变化。结果　氢氯噻嗪和吲哒帕胺用药 1周后动物血压降低 ,4周时降压幅度达到非常显著水平 ,与对照组比较P <0 0 1。同时在mRNA和蛋白质水平上调肾脏、心脏和主动脉中CYP表氧化酶 2C11和下调AT1基因表达 ;胶原染色显示 ,两种药物均能显著减少心肌胶原沉积 ,减轻高血压所致的肾脏损害。结论　噻嗪类利尿剂氢氯噻嗪和吲哒帕胺可能通过上调CYP表氧化酶及下调AT1降低血压和保护器官。     

Myocardial angiotensin receptor type 1 gene expression in a rat model of cardiac volume overload

To investigate the regulation of the angiotensin receptor type 1 (AT1) in different organs in cardiac volume overload, we measured AT1 mRNA content in the atria, left and right ventricle, kidney and liver of rats with an aortocaval shunt, produced by infrarenal aortocaval puncture 4 weeks earlier. For angiotensin receptor mRNA quantitation a novel quantitative PCR procedure based on liquid phase hybridization was used that allowed the determination of absolute AT1 mRNA copy numbers and its comparison in different organs. Glyceraldehydephosphate dehydrogenase (GAPDH) mRNA was measured by RT-PCR to control externally equal mRNA content and quality of RNA extraction in shunt animals and controls. Heart weight was increased in the shunt animals, with the greatest increase in the atria. Blood pressure, plasma renin activity, plasma angiotensin I and II and aldosterone concentrations were not significantly altered. The AT1 mRNA content was significantly increased in the atria (shunt: 1167±350 copies AT1 mRNA/ng RNA vs controls: 803±240; p<0.05). No change was found in the right or left ventricle, in the kidney and liver. The findings document that atrial hypertrophy in cardiac volume overload parallel with a significant increase in atrial AT1 mRNA content.     

Brain angiotensin in the developing spontaneously hypertensive rat

There are several factors in the manifestation of high blood pressure in spontaneously hypertensive rats (SHR) which implicate a central role for brain angiotensin II (Ang II). We have measured levels of angiotensin in the brain of SHR and rats of the Wistar-Kyoto strain (WKY). The experiments were carried out in 2-, 4-, 14- and 20-week-old rats. Areas of brain from rats were homogenized and purified with SepPak C-18 cartridges. The levels were measured by radio-immunoassay whose detection limit was 1.95 pg/tube. Significant differences were found between the different age groups and between SHR and controls. In the hypothalamus, there was a consistent elevation of brain Ang II in SHR as compared to WKY in all age groups. Cerebellum also had higher levels in SHR, especially in rats at 2 and 4 weeks of age. Brainstem levels were significantly higher in SHR only in the 14-week-old age group. Plasma levels during these time periods did not differ significantly between the strains. The results demonstrate changes in brain Ang II with development. At an early age, there are high levels of Ang II in the hypothalamus and cerebellum which do not correlate with hypertension but may be important for the development of hypertension. The higher levels of brain Ang II in SHR support the hypothesis that hypertension in SHR is related to brain Ang II activity.     

Aldosterone breakthrough during angiotensin II receptor antagonist therapy in stroke-prone spontaneously hypertensive rats

Aldosterone breakthrough during ACE inhibitor therapy has been reported. This study investigates changes in plasma aldosterone concentration (PAC) and its mechanism and effects on target organ damage during long-term angiotensin II type 1 (AT1) receptor antagonist (AT1A) therapy in hypertensive rats. An AT1A (candesartan, 1 mg/kg per day PO) was administered in stroke-prone spontaneously hypertensive rats from 4 weeks of age for 34 weeks. PAC was significantly decreased during the first 4 weeks but showed aldosterone breakthrough after 8 weeks of AT1A administration. Plasma angiotensin II concentration was significantly elevated, whereas no change was seen in plasma ACTH or serum potassium. The mechanism(s) of aldosterone breakthrough were investigated by giving high doses of candesartan (3 mg/kg per day PO), dexamethasone (200 microg/kg per day IP), or the AT2 antagonist (PD123319, 10 mg/kg per day SC) during the last week of the 24-week AT1A treatment period. Dexamethasone and AT2 antagonist but not high-dose AT1A produced a significant decrease in PAC, with a larger decrease produced by the AT2 antagonist. To clarify the effects of the residual aldosterone, effects of coadministration of low-dose spironolactone (10 mg/kg per day SC), an aldosterone antagonist, on left ventricular hypertrophy and expression of brain natriuretic peptide mRNA were determined. Low-dose spironolactone further improved left ventricular hypertrophy and brain natriuretic peptide mRNA expression despite no additional depressor effect. These results suggest that aldosterone breakthrough occurs during long-term AT1A therapy, mainly by an AT2-dependent mechanism. Residual aldosterone may attenuate the cardioprotective effects of AT1A.