WKY Hearts Research Articles

Quantitative analysis of hypertrophic myocardium using diffusion tensor magnetic resonance imaging.

Systemic hypertension is a causative factor in left ventricular hypertrophy (LVH). This study is motivated by the potential to reverse or manage the dysfunction associated with structural remodeling of the myocardium in this pathology. Using diffusion tensor magnetic resonance imaging, we present an analysis of myocardial fiber and laminar sheet orientation in ex vivo hypertrophic (6 SHR) and normal (5 WKY) rat hearts using the covariance of the diffusion tensor. First, an atlas of normal cardiac microstructure was formed using the WKY b0 images. Then, the SHR and WKY b0 hearts were registered to the atlas. The acquired deformation fields were applied to the SHR and WKY heart tensor fields followed by the preservation of principal direction (PPD) reorientation strategy. A mean tensor field was then formed from the registered WKY tensor images. Calculating the covariance of the registered tensor images about this mean for each heart, the hypertrophic myocardium exhibited significantly increased myocardial fiber derangement ([Formula: see text]) with a mean dispersion of 38.7deg, and an increased dispersion of the laminar sheet normal ([Formula: see text]) of 54.8deg compared with 34.8deg and 51.8deg, respectively, in the normal hearts. Results demonstrate significantly altered myocardial fiber and laminar sheet structure in rats with hypertensive LVH.

Catestatin increases the expression of anti-apoptotic and pro-angiogenetic factors in the post-ischemic hypertrophied heart of SHR.

BackgroundIn the presence of comorbidities the effectiveness of many cardioprotective strategies is blunted. The goal of this study was to assess in a hypertensive rat model if the early reperfusion with anti-hypertensive and pro-angiogenic Chromogranin A-derived peptide, Catestatin (CST:hCgA352–372; CST-Post), protects the heart via Reperfusion-Injury-Salvage-Kinases (RISK)-pathway activation, limiting infarct-size and apoptosis, and promoting angiogenetic factors (e.g., hypoxia inducible factor, HIF-1α, and endothelial nitric oxide synthase, eNOS, expression).Methods and ResultsThe effects of CST-Post on infarct-size, apoptosis and pro-angiogenetic factors were studied in isolated hearts of spontaneously hypertensive rats (SHR), which underwent the following protocols: (a) 30-min ischemia and 120-min reperfusion (I/R); (b) 30-min ischemia and 20-min reperfusion (I/R-short), both with and without CST-Post (75 nM for 20-min at the beginning of reperfusion). In unprotected Wistar-Kyoto hearts, used as normal counterpart, infarct-size resulted smaller than in SHR. CST-Post reduced significantly infarct-size and improved post-ischemic cardiac function in both strains. After 20-min reperfusion, CST-Post induced S-nitrosylation of calcium channels and phosphorylation of RISK-pathway in WKY and SHR hearts. Yet specific inhibitors of the RISK pathway blocked the CST-Post protective effects against infarct in the 120-min reperfusion groups. Moreover, apoptosis (evaluated by TUNEL, ARC and cleaved caspase) was reduced by CST-Post. Importantly, CST-Post increased expression of pro-angiogenetic factors (i.e., HIF-1α and eNOS expression) after two-hour reperfusion.ConclusionsCST-Post limits reperfusion damages and reverses the hypertension-induced increase of I/R susceptibility. Moreover, CST-Post triggers antiapoptotic and pro-angiogenetic factors suggesting that CST-Post can be used as an anti-maladaptive remodeling treatment.

The beneficial effects of exercise in rodents are preserved after detraining: a phenomenon unrelated to GLUT4 expression

BackgroundAlthough exercise training has well-known cardiorespiratory and metabolic benefits, low compliance with exercise training programs is a fact, and the harmful effects of physical detraining regarding these adaptations usually go unnoticed. We investigated the effects of exercise detraining on blood pressure, insulin sensitivity, and GLUT4 expression in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY).MethodsStudied animals were randomized into sedentary, trained (treadmill running/5 days a week, 60 min/day for 10 weeks), 1 week of detraining, and 2 weeks of detraining. Blood pressure (tail-cuff system), insulin sensitivity (kITT), and GLUT4 (Western blot) in heart, gastrocnemius and white fat tissue were measured.ResultsExercise training reduced blood pressure (19%), improved insulin sensitivity (24%), and increased GLUT4 in the heart (+34%); gastrocnemius (+36%) and fat (+22%) in SHR. In WKY no change in either blood pressure or insulin sensitivity were observed, but there was an increase in GLUT4 in the heart (+25%), gastrocnemius (+45%) and fat (+36%) induced by training. Both periods of detraining did not induce any change in neither blood pressure nor insulin sensitivity in SHR and WKY. One-week detraining reduced GLUT4 in SHR (heart: -28%; fat: -23%) and WKY (heart: -19%; fat: -22%); GLUT4 in the gastrocnemius was reduced after a 2-week detraining (SHR: -35%; WKY: -25%). There was a positive correlation between GLUT4 (gastrocnemius) and the maximal velocity in the exercise test (r = 0.60, p = 0.004).ConclusionsThe study findings show that in detraining, despite reversion of the enhanced GLUT4 expression, cardiorespiratory and metabolic beneficial effects of exercise are preserved.

Effects of a protocol of ischemic postconditioning and/or captopril in hearts of normotensive and hypertensive rats

Brief periods (a few seconds) of cyclic coronary occlusions applied early in reperfusion induce a cardioprotection against infarct size, called postconditioning (PostC) in which B(2)-bradykinin receptors play a pivotal role. Since angiotensin-converting enzyme (ACE) inhibitors reduce degradation of kinins, we studied the effects of PostC on infarct size and postischemic myocardial dysfunction in both normotensive (WKY) and spontaneously hypertensive rats (SHR) acutely or chronically treated with the ACE inhibitor Captopril. Isolated hearts from SHR and WKY rats were subjected to the following protocols: (a) ischemia for 30- and 120-min reperfusion (I/R); (b) I/R + PostC protocol (5-cycles 10-s I/R); (c) pretreatment with Captopril for 4-weeks before to subject the hearts to I/R with or without PostC maneuvers. Some SHR hearts were treated with Captopril during the 20- or 40-min early reperfusion with or without PostC maneuvers. Cardiac function was assessed in vivo with echocardiography. Left ventricular pressure and infarct size were measured ex vivo. Chronic Captopril significantly reduced left ventricular hypertrophy in SHR, and reduced infarct size in both WKY and SHR hearts. PostC maneuvers significantly reduced infarct size in WKY, but not in SHR hearts. Yet, PostC slightly improved postischemic systolic function in untreated SHR. Captopril given in reperfusion was unable to limit I/R injury in SHR hearts. Data show that PostC protection against infarct size is blunted in SHR and that PostC is unable to add its protective effect to those of chronic Captopril, which per se reduces cardiac hypertrophy and heart susceptibility to I/R insult.

Calcineurin inhibition normalizes β-adrenergic responsiveness in the spontaneously hypertensive rat

Calcineurin, a Ca(2+)-regulated protein phosphatase, links myocardial Ca(2+) signaling with hypertrophic gene transcription. Calcineurin abundance increases in pressure-overload hypertrophy and may reduce agonist-mediated phospholamban (PLB) phosphorylation to underlie blunted beta-adrenergic receptor (beta-AR) responsiveness in hypertension. This hypothesis was tested by measuring the effects of calcineurin inhibition on changes in cardiac contractility caused by beta-adrenergic stimulation in spontaneously hypertensive rats (SHR). Female SHR (age: 7 mo) and age-matched female Wistar-Kyoto rats (WKY) were studied. Heart weight-to-body weight ratio (P < 0.01) and systolic blood pressure (P < 0.01) were greater in SHR compared with WKY and were associated with increased myocardial calcineurin mRNA (CnAbeta) and activity (P < 0.05). beta-AR stimulation with isoproterenol (Iso) increased calcineurin activity (P < 0.05) in both WKY and SHR hearts, and this activity was suppressed with cyclosporin A (CsA) treatment. In SHR, CsA improved left ventricular whole heart and isolated myocyte beta-AR responsiveness by normalizing PLB phosphorylation at Ser(16) and Thr(17) (P < 0.05). These CsA-induced, PLB-mediated effects were associated with an augmentation in cardiomyocyte peak Ca(2+) and a reduced rate (time constant of isovolumic pressure relaxation, tau) and magnitude of diastolic Ca(2+) during beta-AR stimulation. In conclusion, CsA normalized the blunted beta-AR responsiveness associated with hypertension, in part, by mitigating calcineurin activity while improving PLB phosphorylation and subsequent sarcoplasmic reticulum Ca(2+) regulation.

P38 MAPK is involved in adenosine receptors' protection against ischemia–reperfusion (I/R) injury of the heart

A1 adenosine receptor (A1R) or A3 adenosine receptor (A3R) activation elicits I/R protection. We investigated (1) the effects of preischemic adenosine A1R (CCPA) or A3R (Cl-IB-MECA) activation on the I/R recovery of normal WKY and hypertrophied SHR hearts. (2) p38 MAPK activation in this process (3) whether p38 phosphorylation occurs upstream or downstream from the opening of mitochondrial KATP channels. WKY and SHR animals were injected 24 h before the experiment with 100 μg/kg CCPA or Cl-IB-MECA. Isolated rat hearts were subjected to I/R (30 min). The intracellular protection mechanism was studied in cultured cardiomyocytes incubated with CCPA, or Cl-IB-MECA or diazoxide (mitochondrial KATP channels opener) with and without SB203580 for 15 min prior to 90 min hypoxia. Both A1R and A3R agonists reduced hypoxia-induced injury in vivo and in vitro. In the WKY hearts, phosphorylated p38 appeared only in hearts pretreated with CCPA or Cl-IB-MECA. In SHR hearts, phosphorylation was observed in all groups, with the additive effects of adenosine treatment, mostly in A3R activation. This protection was prevented when cultured cardiomyocytes were treated with SB203580 together with either CCPA or Cl-IB-MECA, or diazoxide, as revealed by LDH release, ATP content and mitochondrial membrane potentials. CCPA and Cl-IB-MECA reduced I/R damage in normal and particularly hypertrophied hearts. This protection was partially related to the increased phosphorylation of p38 MAPK, which is located downstream from mitochondrial KATP channels opening.

Ischemic preconditioning is lost in aging hypertensive rat heart: Independent effects of aging and longstanding hypertension

Although in experimental hypertension the cardioprotective effects of ischemic preconditioning (PC) appear to be maintained, most studies have examined the short-term hypertension in juvenile animals. However, aging may be an additional factor that influences the effectiveness of PC. The aim of this study was to characterise the effects on PC of LVH and aging simultaneously. Hearts from spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar–Kyoto rats (WKY) were studied. Excised hearts were Langendorff-perfused to give equivalent coronary flow per gram heart weight. The left main coronary artery was occluded for 35 min followed by 120 min reperfusion. Infarct size was determined by tetrazolium staining. Heart size was assessed as left ventricle/body weight ratio (LV/BW). PC was effected with 2 × 5 min periods of global ischemia prior to coronary occlusion. Hearts were studied at 3–4 months (juvenile), 7–8 months (mature) or 12–13 months (aging). LV/BW ratio in SHR increased relative to WKY controls by 20%, 32% and 40% in juvenile, mature and aging hearts, respectively, but ischemic risk zone size was similar in all groups (52–59% of LV). PC was equally effective at limiting infarct size in juvenile and mature SHR and WKY hearts but was ineffective in aging hearts from both WKY and SHR. Since angiotensin-converting enzyme inhibitors enhance sub-threshold PC in normal myocardium, we also examined the action of captopril (Cap) in aging hearts. Additional aging hearts received treatment with Cap 200 μM as an adjunct to PC. Although Cap + PC was able to induce modest protection in aging WKY hearts, this was not seen in aging SHR hearts. We conclude that PC is lost in longstanding hypertension through independent contributions of both hypertension and aging. These findings may have implications for the clinical development of preconditioning-based therapies since elderly patients with longstanding hypertension are at high risk of developing ischemic heart disease.

The protective effect of prior ischemia reperfusion adenosine A1 or A3 receptor activation in the normal and hypertrophied heart

The increased susceptibility to ischemic injury of hypertrophied hearts has long been recognized. The purpose of this study was to investigate the effects of pre-ischemic pharmacological preconditioning (PC) with adenosine A(1) or A(3) receptor activation, on the recovery of the isolated myocardium post cardioplegic ischemia. In addition, we examined the p38 MAPK activation in this process. WKY and SHR hearts were subjected to two different modes of treatment. (1) In the perfusion mode- (the first window of PC) isolated rat hearts were perfused for 10 min with Krebs Henseleit solution and then A(1) receptor agonist (CCPA) or A(3) receptor agonist (Cl-IB-MECA), 10 nM for 20 min, followed by 30 min of warm cardioplegic ischemia and 30 min of reperfusion. (2) In the injection mode (the second window of PC) 100 microg/kg CCPA or Cl-IB-MECA, were administered 24 h before the experiment. Isolated hearts were perfused for 30 min with KH and then subjected to the same protocol as described above. Recovery of hemodynamic parameters was always better in the normal vs. hypertrophied hearts. CCPA improved recovery of left ventricular developed pressure, coronary flow and ATP levels of the hearts (normal and hypertrophied) in both modes of treatment. Cl-IB-MECA was partially beneficial especially in the injected mode. Increased phosphorylation of p38 MAPK relative to baseline, in both early (perfused) and late (injected) modes of treatment especially in the WKY hearts, is demonstrated. CCPA in both modes of treatment and Cl-IB-MECA, especially in the injected mode, were beneficial in protecting the normal and hypertrophied perfused isolated rat heart subjected to normothermic cardioplegic ischemia. This protection was partially related to the increased phosphorylation of p38 MAPK.

Age and hypertrophy alter the contribution of sarcoplasmic reticulum and Na +/Ca 2+ exchange to Ca 2+ removal in rat left ventricular myocytes

Age and hypertension contribute significantly to cardiac morbidity and mortality, however the importance of each during the progression of hypertrophy is unclear. This investigation examined the effect of age and hypertension on Ca 2+ handling in rat ventricular myocytes by comparing a genetic model of hypertension and cardiac hypertrophy (spontaneously hypertensive rat, SHR) with its normotensive control (Wistar–Kyoto rat, WKY) at 5 and 8 months of age. Experiments were performed on single left ventricular myocytes isolated from SHR or WKY hearts. Intracellular Ca 2+ was measured optically using fura-2 or fluo-3. SHR myocytes had a significantly larger cell width and volume and a significantly decreased cell length/width ratio at 5 and 8 months compared to normotensive controls. Age had no effect on cell length, width, volume or the length/width ratio. Ca 2+ transient amplitude, sarcoplasmic reticulum (SR) Ca 2+ content and contraction amplitude were unaffected by age or hypertrophy. However at 8 months the contribution of the SR to Ca 2+ uptake during relaxation decreased, with a concomitant increase in the contribution of Na +/Ca 2+ exchanger (NCX) function to relaxation, in SHR and WKY myocytes. The incidence of non-synchronous SR Ca 2+ release decreased with age but not hypertrophy in SHR and WKY myocytes. These results show that the changes in Ca 2+ handling observed during progression of mild hypertrophy in SHR are the same as those that occur during ageing in normotensive control animals and can, therefore, be ascribed to maturation rather than hypertrophy.

Differential regulation of the nitric oxide–cGMP pathway exacerbates postischaemic heart injury in stroke‐prone hypertensive rats

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO-cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia-reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca(2+)-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca(2+)-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca(2+)-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca(2+)-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.

Expression and activity of the glutamate transporter EAAT2 in cardiac hypertrophy: implications for ischaemia reperfusion injury

The expression and activity of the glutamate transporter, excitatory amino acid transporter 2 (EAAT2), in cardiac hypertrophy were investigated with respect to glutamate's potential as a cardioprotective agent. Sarcolemmal vesicles (SV) isolated from hypertrophic hearts of male spontaneously hypertensive rats (SHR) or normotrophic hearts from age-matched male Wistar Kyoto rats (WKY) were used to measure the relative level of EAAT2 expression by Western blotting and the initial rate of 0-0.3 mM L-[(14)C]glutamate uptake. The effects of 20-min global normothermic ischaemia +/-0.5 mM glutamate on cardiac function were measured in isolated working SHR/WKY hearts. In a separate series of hearts, glutamate, lactate and ATP levels were measured. Both the level of EAAT2 expression and the V (max) for sodium-dependent L-[(14)C]glutamate uptake were significantly greater in SHR SV compared to WKY SV. The reperfusion cardiac output (CO) of SHR hearts was significantly worse than that of the WKY hearts (24.3+/-2.2 ml/min vs 39.8+/-3.3 ml/min, n=7/9+/-SE, p<0.01). The addition of 0.5 mM L-glutamate improved the SHR reperfusion CO to 45.2+/-5 ml/min, (n=6+/-SE, p<0.01) but had no effect on WKYs (46.2+/-3.8 ml/min, n=6+/-SE). SHR with 0.5 mM L-glutamate had higher glutamate levels at the start of ischaemia, plus higher glutamate and ATP levels at the end of ischaemia compared to any other group. These results suggest that increased glutamate transporter expression and activity in the SHR hearts helped facilitate glutamate entry into the SHR cardiomyocytes leading to improved myocardial metabolism during ischaemia and better functional recovery on reperfusion.

Decreased Ca2+extrusion via Na+/Ca2+exchange in epicardial left ventricular myocytes during compensated hypertrophy

Hypertension-induced cardiac hypertrophy alters the amplitude and time course of the systolic Ca2+ transient of subepicardial and subendocardial ventricular myocytes. The present study was designed to elucidate the mechanisms underlying these changes. Myocytes were isolated from the left ventricular subepicardium and subendocardium of 20-wk-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY; control). We monitored intracellular Ca2+ using fluo 3 or fura 2; caffeine (20 mmol/l) was used to release Ca2+ from the sarcoplasmic reticulum (SR), and Ni2+ (10 mM) was used to inhibit Na+/Ca2+ exchange (NCX) function. SHR myocytes were significantly larger than those from WKY hearts, consistent with cellular hypertrophy. Subepicardial myocytes from SHR hearts showed larger Ca2+ transient amplitude and SR Ca2+ content and less Ca2+ extrusion via NCX compared with subepicardial WKY myocytes. These parameters did not change in subendocardial myocytes. The time course of decline of the Ca2+ transient was the same in all groups of cells, but its time to peak was shorter in subepicardial cells than in subendocardial cells in WKY and SHR and was slightly prolonged in subendocardial SHR cells compared with WKY subendocardial myocytes. It is concluded that the major change in Ca2+ cycling during compensated hypertrophy in SHR is a decrease in NCX activity in subepicardial cells; this increases SR Ca2+ content and hence Ca2+ transient amplitude, thus helping to maintain the strength of contraction in the face of an increased afterload.

Effects of Angiotensin II Type 1 Receptor Antagonist on Smooth Muscle Cell Phenotype in Intramyocardial Arteries from Spontaneously Hypertensive Rats

To clarify the precise mechanisms involved in the reduced coronary flow reserve in hypertension, we compared the effects of the angiotensin II type 1 (AT1) receptor antagonist FK-739 with those of the angiotensin-converting enzyme (ACE) inhibitor enalapril for 6 weeks on the smooth muscle (SM) cell phenotype in intramyocardial arteries from male Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Compared with WKY, SHR showed a significant increase in left ventricular (LV) hypertrophy and fibrosis, blood pressure (BP), and vascular remodeling of the intramyocardial arteries, and a significant decrease in endothelial NO synthase and the contractile-type myosin heavy chain isoform SM2 of the intramyocardial arteries as well as calponin 1 and GATA-6. In the hearts of SHR, both drugs equivalently and significantly reduced BP, which was still significantly higher than that in the WKY groups, and also reduced LV hypertrophy and fibrosis, whereas endothelial NO synthase was significantly restored. Although both drugs showed little effect on the vascular remodeling of the intramyocardial arteries in the SHR hearts, FK-739, but not enalapril, significantly restored SM2 and GATA-6 in the SHR hearts to the same levels as those of the vehicle WKY group. The effects of the two drugs on these indices were not observed in the three WKY hearts. Thus, the AT1 receptor antagonist may modulate the SM cell phenotype toward the contractile-type more effectively than the ACE inhibitor before the morphological changes occur in the intramyocardial arteries of the SHR hearts.

Calcium Antagonist Reduces Oxidative Stress by Upregulating Cu/Zn Superoxide Dismutase in Stroke-Prone Spontaneously Hypertensive Rats

Recent studies have suggested that the calcium antagonists have an antiatherogenic antioxidant property. The effects of the calcium antagonists on reactive oxygen species (ROS)-related enzymes, however, remain unknown. We hypothesized that the calcium antagonists inhibit oxidative stress in the hearts of stroke-prone spontaneously hypertensive rats (SHRSP) through the ROS-scavenging enzymes known as superoxide dismutases (SODs). Male 12-week-old Wister-Kyoto rats (WKY) and SHRSP were used for the study. SHRSP were randomized and treated for 6 weeks with a vehicle, amlodipine (5 mg/kg/day), or enalapril (10 mg/kg/day). NAD(P)H oxidase activity was measured by a luminescence assay, and SOD activity was measured spectrophotometrically. Protein expressions were analyzed by immunoblots. Both drugs showed equipotent effects on systolic blood pressure, left ventricular hypertrophy and fibrosis, the wall-to-lumen ratio, the manganese SOD activity, ROS, and the endothelial NO synthase expression in the SHRSP hearts. Furthermore, amlodipine significantly restored copper/zinc-containing SOD (Cu/ZnSOD) expression and its activity in SHRSP hearts to a level equal to that of WKY more effectively than did enalapril (p <0.05), whereas enalapril downregulated NAD(P)H oxidase activity more than did amlodipine (p <0.05) in the SHRSP hearts. Furthermore, amlodipine restored Cu/ZnSOD expression and its activity in SHRSP hearts to a level equal to that in WKY hearts, and this restoration was significantly more effective than that by enalapril (p <0.05); on the other hand, enalapril induced a greater downregulation of NAD(P)H oxidase activity in SHRSP hearts than did amlodipine (p <0.05). Thus, amlodipine may inhibit vascular remodeling and oxidative stress in the SHRSP heart by efficiently upregulating Cu/ZnSOD, suggesting that the calcium antagonist may exhibit an antiatherogenic antioxidative action beyond blood-pressure lowering through the restoration of Cu/ZnSOD activity in the heart in cases of hypertension.

Increased cardiac norepinephrine release in spontaneously hypertensive rats: role of presynaptic alpha-2A adrenoceptors.

An increased sympathoadrenergic activation is thought to contribute to the maintenance of elevated blood pressure levels in hypertension. Therefore, the regulation of cardiac presynaptic sympathetic neurotransmission was investigated in spontaneously hypertensive (SHR) and Wistar-Kyoto rats (WKY). Electrical field stimulation (1 min, 4 Hz) evoked a higher norepinephrine (NE) overflow from isolated perfused SHR than from WKY hearts (171 +/- 78 versus 111 +/- 27 nmol/g; means +/- SD, n = 7, P < 0.05). The difference in stimulation-evoked NE overflow was neither due to increased NE stores nor to a higher density of sympathetic nerve endings in SHR hearts. Furthermore, impairment of cardiac NE re-uptake was ruled out, as pharmacological inhibition of NE re-uptake by desipramine (300 nmol/l) similarly increased NE overflow from SHR (+ 54 +/- 17%) and WKY hearts (+ 59 +/- 18%). However, inhibition of presynaptic alpha-2 adrenoceptors (alpha-2R) with yohimbine (1 micromol/l) resulted in a significantly larger increase in NE overflow from WKY (+ 244 +/- 42%) than from SHR hearts (+ 162 +/- 47%, P < 0.05 versus WKY), indicating impairment of presynaptic inhibitory effect of alpha-2R in SHR. Supporting this notion, mRNA concentrations of alpha-2(A), the predominant presynaptic alpha-2R subtype, were reduced in SHR compared with WKY (738 +/- 251 versus 1468 +/- 518 mRNA molecules/10 ng, n = 7, P < 0.01), as quantified by competitive reverse transcription-polymerase chain reaction derived from left stellate ganglia. The impairment of the alpha-2R mediated presynaptic negative feedback mechanism by a reduced expression of the alpha-2R subtype A may increase cardiac net secretion of NE in SHR and could therefore contribute to their hypertensive phenotype.

Initial T-cell activation required for transplant vasculopathy in retransplanted rat cardiac allografts.

A precise understanding of immunological mechanisms is needed to prevent transplant vasculopathy. The developing process of transplant vasculopathy was investigated by retransplanting rat cardiac allografts and measuring the expressions of 21 different genes inside the retransplanted allografts under nonimmunosuppressive conditions. Significant transplant vasculopathy developed if WKY hearts were grafted to LEW and retransplanted to WKY 5 days after the initial grafting, but it did not in allografts retransplanted 3 days after the initial grafting. The disease did not progress in retransplanted isografts or if nude rats were used as the initial recipients. However, the development of transplant vasculopathy was not affected by changing the second recipients to the F1 progeny of donor x recipient or to nude animals. Among the expressions of 21 different genes observed in allografts at 1, 14, 30, or 60 days after retransplantation, those of T-cell activation-related genes, such as interferon-y and Fas ligand, showed the earliest and the most dramatic difference between 3- and 5-day-retransplanted allografts whereas macrophage/monocyte activation-related genes showed little difference. Furthermore, reverse transcription-polymerase chain reaction analyses of allografts retransplanted to nude animal indicated that T cells of the initial recipient origin survive and remain activated even 60 days after retransplantation. The T-cell response occurring between 3 and 5 days after grafting was identified as the critical parameter to the disease progression. Once alloreactive T cells enter a graft, they may be able to survive a long period and promote chronic rejection.

Diminished cardiac contractile response to tetrahydropapaveroline in hypertension: role of β-adrenoceptors and intracellular Ca 2+

Tetrahydropapaveroline (THP), a condensation product of ethanol-derived acetaldehyde, potentiates cardiac function through a β-adrenergic mechanism. It is well established that β-adrenergic activity is markedly depressed in hypertension. However, little is known about the myocardial action of THP in hypertension. In this study, the effect of THP was examined using left ventricular papillary muscles and ventricular myocytes from 10-week-old normotensive (WKY) and spontaneously hypertensive (SHR) rats. The mechanical parameters evaluated include: peak tension developed (PTD), peak twitch amplitude (PTA), time-to-PTD/PTA (TPT/TPS), time-to-90% relaxation/relengthening (RT 90/TR 90), and the maximal velocities of contraction/shortening and relaxation/relengthening (±VT/±d L/d t). Intracellular Ca 2+ transients were measured as fura-2 fluorescence intensity changes (ΔFFI). THP (0.01–100 μM) produced a concentration-dependent increase in myocardial contraction on muscles and myocytes from both groups of animals. However, preparations from the SHR group were generally less responsive to THP than their normotensive counterparts. The increase in contractility by THP was associated with increases in ΔFFI and ±VT, and shortening of TPT/TPS and RT 90/TR 90. The role of β-adrenoceptor(s) in the mechanism of action of THP was explored using specific β-receptor subtype antagonists CGP 207.12A (β 1) and ICI 118,551 (β 2). In preparations from both WKY and SHR hearts, the THP-induced increase in cardiac contractility was either attenuated or blocked by CGP 207.12A and ICI 118,551. These results indicate that THP exhibits a positive action on myocardial contraction that is mediated, in part, through both β 1 and β 2 adrenergic receptors. This cardiac inotropic response, however, is markedly diminished in hypertension, which is due possibly to alterations in β-adrenergic signal transduction.

Role of Nitric Oxide in the Regulation of Coronary Vascular Tone in Hearts From Hypertensive Rats

In arterial hypertension, coronary flow reserve, expressed by the difference between autoregulated and maximal coronary flow, is frequently impaired. Previous experimental and clinical investigations using acetylcholine as a stimulus for the production of endothelium-derived relaxing factor suggested that an impaired endothelium-dependent vasodilation, presumably caused by a decreased formation of nitric oxide (NO), may account for this microvascular dysfunction. However, so far no study has been performed that quantifies the formation of NO within the coronary circulation of hypertensive hearts to assess its role in setting coronary vascular tone in the hypertensive heart. We therefore quantified NO formation within the coronary circulation of constant flow-perfused, isolated hearts from spontaneously hypertensive rats (SHR, 16th to 26th week), as a model for hypertensive heart disease, and from the normotensive control strain (Wistar-Kyoto, WKY) using the oxyhemoglobin technique. Coronary perfusion pressure and vascular resistance were almost 30% higher in SHR compared with WKY hearts. Intracoronarily applied NO decreased coronary vascular resistance by maximally 45% of resting values in a concentration-dependent manner in both groups. The bradykinin-induced decrease in coronary vascular resistance and the parallel increase in NO release were comparable in SHR and WKY hearts and fell within the vasodilator range of exogenously applied NO. Moreover, basal release of NO normalized to heart wet weight was 50% higher in SHR compared with WKY hearts. Rates of basal NO release were correlated inversely with changes in coronary perfusion pressure and vascular resistance in both groups (r = -.85 and -.84, respectively, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased superoxide dismutase activity and increased superoxide anion production in cardiac hypertrophy of spontaneously hypertensive rats.

In order to investigate SHR hypertrophied heart myocardial vulnerability, superoxide dismutase, a potent scavenger of superoxide anion, was examined in myocardium of SHR and WKY in relation to aging, as well as under doxorubicin (DOX)-induced cardiomyopathy. Superoxide anion production due to doxorubicin administration was also examined histochemically. Superoxide dismutase (SOD), either as Mn-SOD in mitochondria or as CuZn-SOD in cytoplasm, was found to be lower in aged SHR hypertrophied heart than in age-matched WKY heart. Under DOX-induced cardiomyopathy, SOD activity was significantly lower in SHR hypertrophied heart than either control SHR or treated WKY myocardium. Superoxide anion generation, examined morphologically as formazan deposits, was much more intense in SHR myocardium and some degenerative changes were found such formazan-containing cells. These results clearly indicate that the myocardial vulnerability of SHR hypertrophied heart might be a result of a lowered free radical scavenge ability.

Nuclear magnetic resonance measurement of intracellular sodium in the perfused normotensive and spontaneously hypertensive rat heart.

We have used 23Na nuclear magnetic resonance spectroscopy to examine the relationship between intracellular sodium and cardiac muscle alterations in genetic hypertension. In the spontaneously hypertensive rat (SHR) compared with the normotensive Wistar-Kyoto rat (WKY) (aged 15 to 19 weeks), mean systolic blood pressures (measured using the tail-cuff method) were significantly (P < .05) different (WKY: 118 +/- 8 mm Hg, n = 5; SHR: 185 +/- 9 mm Hg, n = 5). Heart weights were also increased significantly (P < .05) in SHR (grams dry heart to kilograms body weight ratio was 0.73 +/- 0.04, n = 5, for SHR and 0.55 +/- 0.02, n = 5, for WKY). Intracellular sodium levels, measured using shift-reagent-aided and triple quantum filtered (TQF) nuclear magnetic resonance techniques, were significantly increased (P < .05) in the isolated Langendorff perfused hypertensive rat hearts (17.3 +/- 3.6 mmol/L, n = 5) compared with normotensive rat hearts (8.4 +/- 2.3 mmol/L, n = 5). These data demonstrate increased sodium in cardiac muscle in essential hypertension. We also investigated the effect of pacing on cardiac TQF 23Na nuclear magnetic resonance and found an increase in TQF Na+ content in both WKY and SHR hearts on stepped up pacing. These results support the existence of sodium pump lag in the rat heart perfused at physiologic Ca2+ concentration and suggest that the hypertensive rat heart has adapted to compensate for increased basal intracellular Na+ and maintain a normal response to increased heart rate. Our data appear to suggest an ionic contribution to the cardiac hypertrophy of genetic hypertension in the rat.