Vascular dysfunction and myocardial contractility in the JCR:LA-corpulent rat

Objective: The JCR:LA-corpulent rat is a unique animal model of human vascular disease that exhibits a profound insulin resistance, vasculopathy, and cardiovascular dysfunction. We tested the hypothesis that the defects affect endothelial and smooth muscle function of the coronary microvasculature as well as cardiac contractility. Coronary, myocardial and aortic function were assessed in obese (homozygous for the cp gene, cp/cp) and lean (heterozygous or homozygous normal, 1 /?) littermates aged 7 and 18 weeks. Methods: 2 1 Coronary endothelial relaxation was examined in isolated perfused hearts by determining the effect of bradykinin (0.1–1000 nmol l ) on coronary perfusion pressure (CPP), myocardial mechanical function was evaluated in terms of left-ventricular developed pressure 2 1 (LVDevP), and aortic relaxation with the endothelium-dependent agonist, A 23187 (1–1000 nmol l ). Results: In rats aged 7 weeks, bradykinin reduced CPP from 133 6 1 mmHg to 43 6 1 mmHg ( 2 67%) in lean rats, but only to 64 6 3 mmHg ( 2 52%) in corpulent rats G ( n 5 6, P , 0.05). Similar differences were found in rats aged 18 weeks ( n 5 8). Inhibition of NO synthase with N -nitro- L -arginine 2 1 2 1 ( L -NNA; 0.2 mmol l ) impaired, and tetrahydrobiopterin (0.1 mmol l ), a NO synthase cofactor, restored relaxation in cp/cp rats. Spermine/NO equally reduced CPP in both groups ( 2 58%). Mechanical function was similar in lean and corpulent rats, aortic endothelial relaxation was attenuated by | 30% and aortic smooth muscle function was normal (7 weeks) or improved (18 weeks) in the cp/cp genotype. Conclusion: These results suggest that (i) there is a speciﬁc impairment of NO-mediated relaxation of the coronary resistance vessels in the JCR:LA-corpulent rat that is not associated with impaired baseline myocardial contractility, and (ii) exogenous tetrahydrobiopterin reversed the relaxation defects that are part of the vascular complications typical for the insulin resistance syndrome. be a viable therapeutic option to treat vascular dysfunction in insulin resistance. The evidence suggests that the dysfunction of the vascular smooth muscle cells of the cp/cp rat, while a prominent part of the disease state, may represent alterations sec-Arterioscler ondary to endothelial damage by hyperinsulinemia.


Introduction
feature of abnormal arterial vasomotion is defective endothelium-dependent relaxation in insulin resistance states The insulin resistance syndrome, a common metabolic such as hypertension and non-insulin-dependent diabetes disorder of the adult population in Western cultures, is mellitus (NIDDM). In humans, endothelial dysfunction is associated with a cluster of cardiovascular abnormalities frequently studied in terms of the blood flow response to such as hypertension, dyslipidemia and atherosclerosis [1].
local injection of endothelium-dependent agonists, whereas The relationship between the syndrome and cardiovascular vascular smooth muscle function is tested with endodisease is not completely understood, but may involve thelium-independent agents such as the classical nitroabnormalities in arterial wall function. One prominent vasodilators [2]. Such studies have shown that the leg blood flow response to intrafemoral methacholine which generates NO via activation of endothelial NO synthase [3] was considerably lower in obese insulin-resistant subjects 2. Methods or in subjects with NIDDM, compared with non-diabetic control subjects [4]. Similar differences were obtained in 2.1. Animals the forearm, even after adjustment for obesity [5]. Elevation of circulating free fatty acids to levels seen in insulin-Male rats of the JCR:LA-cp strain were bred in our resistant subjects can impair endothelial function [6] and breeding colony using standard husbandry techniques and maneuvers enforcing NO activity can improve it. There-a formal system of outbreeding [15]. The young rats can be fore, the pathogenesis of endothelial dysfunction in identified either as homozygous for the cp gene (cp / cp) NIDDM is currently believed to be a consequence of the and obese, or lean and homozygous / heterozygous normal effect of dyslipoproteinemia and of hyperoxidative stress (1 / 1, 1 / cp, a 2:1 mixture referred to as 1 / ? in this on the formation, action and disposal of NO by diverse paper) at 3 weeks of age when they are weaned. Male molecular mechanisms [7]. Clearly, endothelial dysfunc-cp / cp and 1 / ? rats aged 7 or 18 weeks were used in this tion appears to be an integral aspect of the syndrome, study (in one protocol cp / cp rats aged 37 weeks were independently of hyperglycemia. In contrast to endothelial used). The investigation conforms with the Guide for the dysfunction, vascular smooth muscle function as tested Care and Use of Laboratory Animals published by the US with the endothelium-independent agents nitroprusside or Institutes of Health (NIH Publication No. 85-23, revised nitroglycerin has been mostly normal. 1996). The insulin resistance syndrome is also an independent predictor of ischemic heart disease [8] and diabetes-related 2.2. Heart perfusion cardiomyopathy [9], but neither the coronary conductance nor microvessels can be conveniently studied in humans.
Hearts were perfused retrogradely (Langendorff mode) 21 21 However, ex vivo studies of the intact coronary circulation at a rate of 9.0 ml min g heart wet weight with a and isolated vessels from an insulin-resistant rat model modified Krebs-Henseleit bicarbonate buffer as previously allow assessments of coronary microvascular and conduct-described [16]. Cardiac parameters were monitored conance vessel function. These studies were undertaken in the tinuously and included heart rate, coronary perfusion JCR:LA-corpulent (JCR:LA-cp) rat, a unique animal pressure (CPP), left-ventricular developed pressure model that exhibits all aspects of the metabolic syndrome (LVDevP; difference between left-ventricular peak systolic as it occurs in humans [10]. In this model, animals pressure and end-diastolic pressure), maximal rate of rise homozygous for the autosomal recessive cp gene (cp / cp) of LVDevP (dP/dt) and left-ventricular end-diastolic presare hyperphagous and become obese and insulin-resistant, sure (LVEDP). whereas homozygous normal (1 / 1) or heterozygous (1 / cp) animals are lean and do not develop metabolic 2.3. Coronary function alterations. Animals of both genotypes are normotensive. 21 21 The cp mutation has recently been shown to create a stop Coronary flow was set at 15 ml min g resulting in a codon in the extracellular domain of the leptin receptor CPP of 130-135 mmHg. Coronary endothelium-dependent with consequent loss of receptors [11]. The male cp / cp relaxation was tested with bradykinin given as bolus animals spontaneously develop atherosclerotic disease by injections through a sideline, resulting in concentrations of 21 middle age as well as myocardial lesions, consistent with 0.1-1000 nmol l . Maximal relaxation was reached 3-4 an ischemic origin [12]. min after injection. Doses were given in cumulative We have previously characterized aortic function in manner. After the last dose, bradykinin was washed out for cp / cp rats and found evidence for a specific impairment of 20 min and CPP returned to |133 mmHg. To test the 21 endothelium-dependent relaxation which appeared to be NO-dependence of the relaxation, L-NNA (0.2 mmol l ) limited to that mediated by muscarinic receptors [13]. We was applied over 20 min and the bradykinin dose-response hypothesized that besides affecting endothelial function in curve was repeated in the continued presence of L-NNA. conductance vessels, hyperinsulinemia would impair cor-Because L-NNA constricted coronary vessels, coronary 21 onary resistance vessel and NO-mediated cardiac function. flow was decreased to |10 ml min to maintain the Therefore, we have characterized coronary microvessel baseline CPP at |130 mmHg. Finally, after washout of function and myocardial contractility in obese and lean rats L-NNA and bradykinin for 20 min, a single bolus dose of 21 using endothelium-dependent and -independent agents. spermine / NO (final concentration 0.1 mmol l ) was Additionally, we specifically tested the hypothesis that the added to the perfusion buffer to test coronary smooth endothelial dysfunction resulted from an aberrant NO muscle function. In a separate protocol, indomethacin (10 21 synthase reaction at the level of the essential cofactor, mmol l ) was used to test for a role of cyclo-oxygenase tetrahydrobiopterin (H biopterin) [14]. To this end, cor-products. The NO synthase cofactor, H biopterin (0.1 4 4 21 onary endothelium-dependent dilation was determined in mmol l ) was used in hearts from cp / cp rats aged 37 the presence of exogenous H biopterin and cofactor levels weeks to test whether an aberrant NO synthase reaction is 4 were measured in plasma. involved in the impaired relaxation response to bradykinin.

Relaxation of aortic rings
spher 100 RP-18, 5-mm particle size, Merck, Vienna, 21 Austria), eluted with 20 mmol l sodium phosphate The thoracic aorta was cleaned of connective tissue and buffer, pH 3, containing 5% (v / v) methanol, at 0.7 21 cut into rings |3-mm long with endothelium present. The ml min and detected by fluorescence spectrophotometry rings were suspended in tissue baths containing 5 ml (Hitachi F 1050; extinction 350 nm, emission 440 nm). Krebs-Henseleit solution maintained at 378C, pH 7.4 and Calibration curves were recorded with authentic tetrahy- 21 gassed with carbogen and tension was recorded isometri-drobiopterin (10-500 nmol l ). cally. Basal tension before addition of agonist was 2 g. Experimental groups consisted of 5 animals aged 7 and 8 2.7. Data analysis and calculations animals aged 18 weeks, both 1 / ? and cp / cp. Four to five aortic rings were prepared from animals aged 7, and 6-8 Coronary relaxation was calculated as reduction in CPP rings from animals aged 18 weeks. The preparations were from the maximal pressure (130-135 mmHg), and relaxastudied after 90-120 min of equilibration, with changes of tion of aortic rings as reduction in tone from the maximal bath fluid every 30 min. Tissues were precontracted with U induced tone (4-6 g) and expressed as a percentage. 21 21 46619 (100 nmol l ) and angiotensin II (5 mmol l ) Individual concentration-response curves were fitted to a which gave a maximal tension of |5 g. When the contrac-Hill-type model giving estimates of agonist potency (EC ) 50 tion had reached a stable plateau, the relaxation to the and efficacy (E ). Significance was assumed at P,0.05. mmol l ) or the ATP-sensitive K -channel opener ril- 21 21 makalim (3 nmol l -100 mmol l ). To test the relaxant 3.1. Experimental groups effect of rilmakalim, two modes of precontraction were used, i.e. U 466191angiotensin II (as in all other cases), or The body weight of cp / cp rats was significantly higher K (40 mmol l ). All experiments were performed in the than that of lean controls in all age groups: 24662 vs. 21 presence of indomethacin (10 mmol l ) to block the 18363 g (7 weeks, n56) and 57465 vs. 35767 g (18 formation of vasoactive prostanoids by cyclo-oxygenases. weeks, n58; P,0.05 in both cases). However, the corresponding heart weights were not different between groups: 0.6960.025 vs. 0.7860.018 g (7 weeks) and 1.2360.01 2.5. Assay for NO synthase activity in cell homogenates vs. 1.1860.02 g (18 weeks). Systolic arterial blood pressure was measured in animals aged 14-16 weeks and 21 Left ventricular tissue was homogenized in 50 mmol l was not different between groups (13461.5 vs. 13861.6 tetraethyl ammonium containing 1% b-mercaptoethanol, mmHg, n59). All animals showed similar heart rate, 21 10 mmol l CHAPS [((3-chol-amidopropyl)dimethylam-LVDevP and CPP irrespective of age and experimental 21 monio)-1-propanesulfonate] and 0.5 mmol l EDTA using group (Table 1). a glass homogeniser. Aortas were finely cut using a scalpel and suspended in the same buffer. After 3 cycles of 3.2. Coronary circulation freeze-thawing followed by centrifugation at 10.000 g, the protein concentration in the supernatant was determined The coronary relaxant effect of bradykinin in hearts using the Bradford assay. NO synthase activity was from rats aged 7 weeks is shown in Fig. 1. The agonist was 3 3 determined by the conversion of H-L-arginine to H-Lless effective in corpulent rats (maximal relaxation: citrulline [17].  The corresponding effects of bradykinin in hearts from reduction of CPP by bradykinin, both in control and cp / cp rats aged 18 weeks are shown in Fig. 2. Again, both the hearts (n55; not shown). maximal effect and the potency of the agonist was lower in The effect of the natural cofactor for NO synthase, tor restored agonist potency to the level observed in lean 21 21 As in the younger rats, spermine / NO (0.1 mmol l ) was rats aged 18 weeks (EC : 0.3560.05 nmol l ) and 50 similarly effective in both experimental groups (Fig. 2). significantly increased its maximal relaxant effect We also tested whether products of cyclo-oxygenase were (25562%; compare Figs. 2 and 3). involved in the impaired relaxation response in cp / cp rats. 21 However, indomethacin (10 mmol l ) had no effect on the 3.

Myocardial function
The effect of bradykinin on LVDevP is shown in Fig. 4. identical results were obtained in rats aged 18 weeks: LVDevP was reduced from 8061 mmHg (baseline) to 21 6161 mmHg (at 1 mmol l bradykinin) in the absence of L-NNA, and from 6162 mmHg (baseline) to 4762 mmHg in the presence of L-NNA. Indomethacin had no effect on the bradykinin-induced reduction of LVDevP in any group. Comparable results as for LVDevP were obtained for the second contractility parameter, dP/dt (not shown).

NO synthase activity
NOS activity was determined in terms of formation of 3 [ H]citrulline in aorta and left ventricle. In 1 / ? tissue, the rates of formation were 8.461.1 and 3.460.5 21 21 pmol mg min ; in cp / cp tissues they were 10.660.5 sensitive HPLC method. Similar levels were found in lean and obese rats aged 18 weeks (40.362.84 and 41.963.20 21 Baseline LVDevP was 80-81 mmHg in both experimental nmol l ; P.0.05, n58). groups and was reduced to 6462 mmHg by L-NNA (P, 0.05). Bradykinin weakly, but significantly, reduced contractility in concentration-dependent fashion in lean and 3.6. Aorta corpulent rats, both in the absence and presence of L-NNA. A positive inotropic effect was never observed. Virtually Aortic ring relaxation was studied after submaximal stable contraction using the organ bath setup. The calcium ionophore A 23187 relaxed tissues from cp / cp and 1 / ? animals with similar potency (no difference in EC 50 values), but the maximum relaxation response was less (P,0.05) in the obese genotype of both age groups ( Table  2). L-NNA reduced relaxation to #20% and abolished the difference in E between genotypes (N.S. cp / cp vs. max 1 /?). Fig. 5 shows the relaxation of aortic rings in response to the endothelium-independent agent spermine / NO. In both age groups, the agonist was similarly effective in relaxing rings from lean and corpulent rats (N.S. for EC and 50 maximal relaxation), but the tissues from rats aged 18 weeks showed a greater relaxation (E |60%) than those max from the younger rats (E |37%; P,0.05; Table 2). max Finally, to determine whether hyperpolarization-induced relaxation was affected in cp / cp rats, the relaxant potency 1 of rilmakalim, a K channel agonist was tested in aortic rings from rats aged 18 weeks following contraction with 1 Fig. 4. Effect of bradykinin on left-ventricular developed pressure U 466191angiotensin II or K depolarization (Fig. 6).
(LVDevP) in hearts from lean (1 / ?) and obese (cp / cp) rats aged 7 After either precontraction, the agonist was similarly weeks. Hearts were perfused with agonist, followed by infusion of effective in both groups (251% and 250% relaxation, larization than receptor-mediated contraction (

Discussion
hyperinsulinemia is present at 4 weeks and develops rapidly, and at 12 weeks of age there is no insulin-The JCR:LA-corpulent rat is a unique genetic animal mediated glucose uptake or turnover. The profound strain that closely resembles the features of humans with peripheral insulin resistance in cp / cp rats leads to the early stage type II diabetes characterized by insulin resistance and hyperinsulinemia. Animals of the cp / cp strain become detectably obese at 3 weeks of age, mild 1 Fig. 6. Aortic relaxation response to the K channel opener rilmakalim in Fig. 5. Aortic relaxation response to the endothelium-independent vasodi-rings from lean (1 / ?) and obese (cp / cp) rats aged 18 weeks. Tissues  Table 2.
Values are the mean6S.E.M. of the number of tissues given in Table 2. diversion of glucose to triglyceride synthesis and marked NO, followed by production of hydrogen peroxide and / or VLDL hyperlipidemia [10]. Our major finding was a peroxynitrite [14,25]. These biochemical investigations specific impairment of NO-mediated relaxation of the support the concept that an uncoupled NO synthase is a coronary resistance vessels that is completely reversed by source of reactive oxygen metabolites that may play an exogenous H biopterin. Furthermore, we have shown that important role in the endothelial dysfunction and oxidative 4 endothelium-dependent but not endothelium-independent vascular injury described in several diseases, including aortic relaxation was reduced, and myocardial contractility diabetic endothelial dysfunction [26,27]. The substantially was unaffected, in these relatively young obese animals.
reduced relaxation observed in the coronary microvascula-We examined the contractility of the hearts in terms of ture of cp / cp rats in the present study, and its alleviation LVDevP and dP/ dt measurements and found matching by H biopterin, is strong evidence for a dysfunctional 4 values in both groups of hearts. Endogenous NO exerted a endothelial NO synthase enzyme in the obese, insulinstrong positive inotropic effect as evident from the reduc-resistant state. However, in view of the complexity of the tion of LVDevP in the presence of L-NNA (227%) NO synthase reaction, the mechanism responsible for the whereas NO generated by bradykinin depressed myocar-dysfunction is more difficult to discern. A reduced formadial function. Because experiments were done in the tion of intracellular H biopterin due to impaired activity of 4 presence of indomethacin, endothelial mediators generated the key enzyme, cyclohydrolase, hastened degradation or a by stimulated cyclo-oxygenase activity can be excluded.
reduced activity of the cofactor could all play a role. We These findings are reminiscent of recent evidence support-speculated that a reduced formation might translate to ing a biphasic effect of NO / cGMP on myocardial pump reduced plasma cofactor levels in cp / cp rats, but this was function [20]. The complete lack of a positive inotropic not the case. Because similar plasma levels of H biopterin 4 effect of bradykinin at low concentrations was unexpected do not necessarily indicate similar intracellular cofactor and not investigated further. It appears that the striking levels, the simplest explanation of our data assumes a myocardial lesions in cp / cp rats that we observed previ-reduced intracellular activity of H biopterin. Alternatively, 4 ously in cp / cp rats aged 39 weeks [12] are without availability of the substrate L-arginine may be limiting NO consequence to normoxic left-ventricular function in ani-synthase activity. However, under normal conditions, both mals up to 18 weeks of age, whereas older animals exhibit the concentration of L-arginine in blood and the intracelluan increased sensitivity to ischemic myocardial injury [21]. lar L-arginine concentration are far greater than the K of m In streptozotocin-diabetic rats, basal ventricular perform-NO synthase for L-arginine, suggesting that substrate ance and the responsiveness to b-adrenergic stimulation availability is unlikely to be a limiting factor (see Discus-G was suppressed, and inhibition of NO synthase with N -sion in [28]). We found similar rates of citrulline formation nitro-L-arginine methyl ester restored hemodynamic func-in two different tissues (myocardium and aorta) of lean and tion to the level of non-diabetic hearts [22]. Cardiodepres-corpulent rats, indicating that equal amounts of NO sion was attributed to products of inducible NO synthase in synthase enzyme were expressed in both groups. Finally, this model, whereas, apparently, no such induction occurs still other factors essential to the NO synthase reaction in the cp / cp rat heart in which dysfunction is related to including NADPH may be limiting or dysfunctional in the hyperinsulinemia and hypertriglyceridemia [10,15]. present model. The intact coronary circulation of cp / cp rats showed a We have tested previously whether increased degrastrikingly impaired relaxation response to bradykinin that dation of NO in obese rats might result from increased was fully expressed at 7 weeks of age. The impairment formation of reactive oxygen species. Probucol treatment was clearly due to a reduced activity of the NO / cGMP did not decrease intimal lesions, although it was cardiopathway because L-NNA inhibited the agonist's effect to protective [12]. When exposed to copper oxidation stress in the same extent in both experimental groups, whereas the vitro, acetylcholine-mediated relaxation is less impaired in cyclo-oxygenase pathway was not involved, as is evident mesenteric rings from obese cp / cp animals than from the from the lack of effect of indomethacin. The impairment is lean littermates (O'Brien et al., unpubl. observations). consistent with the 2-fold elevation of activity, and 3-fold Thus, there is no evidence that reactive oxygen species are elevation of content, of phosphodiesterase-3A in the aorta responsible for the abnormal behavior of the vessels from of cp / cp rats [23]. An important component of the the cp / cp genotype. underlying pathophysiological mechanism was revealed The responses of conductance vessels to vasodilators when hearts from cp / cp rats were perfused with have varied in different experimental models of diabetes, H biopterin which promptly resulted in restoration of and only limited studies have been performed in human 4 bradykinin-induced coronary relaxation. H biopterin is an subjects. In a previous investigation in male cp / cp rats 4 allosteric activator of NO synthase that is needed to direct aged 6 months, aortic rings from cp / cp rats, precontracted the electron flow to L-arginine, thus coupling NADPH with norepinephrine, responded more weakly to acetyloxidation to NO synthesis [24]. In the presence of sub-choline than control vessels, whereas A 23187 and sodium optimal levels of H biopterin, the NO synthase reaction nitrite were similarly active [13]. We have also found that 4 results in the generation of superoxide anions rather than both aorta and mesenteric resistance vessels from cp / cp rats have an enhanced contractile response to both norepi-