Integrated control of coronary blood flow in exercising swine by adenosine, nitric oxide, and KATP channels.

The interplay of mechanisms regulating coronary blood flow (CBF) remains incompletely understood. Previous studies in dogs indicated that CBF regulation by KATP channels, adenosine, and nitric oxide (NO) follows a nonlinear redundancy design and fully accounted for exercise-induced coronary vasodilation. Conversely, in swine, these mechanisms appear to regulate CBF in a linear additive fashion with considerable exercise-induced vasodilation remaining when all three mechanisms are inhibited. A direct comparison between these studies is hampered by the different doses and administration routes (intravenous vs. intracoronary) of drugs inhibiting these mechanisms. Here, we investigated the role of KATP channels, adenosine, and NO in CBF regulation in swine using identical drug regimen as previously employed in dogs. Instrumented swine were exercised on a motor-driven treadmill, before and after blockade of KATP channels (glibenclamide, 50 µg/kg/min ic) and combination of inhibition of NO synthase (Nω-nitro-l-arginine, NLA, 1.5 mg/kg ic) and adenosine receptors (8-phenyltheophylline, 8PT, 5 mg/kg iv) or their combination NLA + 8PT + glibenclamide. Glibenclamide and NLA + 8PT each produced coronary vasoconstriction both at rest and during exercise, whereas the combination of NLA + 8PT + glibenclamide resulted in a small further coronary vasoconstriction compared with NLA + 8PT that was, however, less than the sum of the vasoconstriction produced by NLA + 8PT and glibenclamide, each. Thus, in contrast to previous observations in the dog, 1) the coronary vasoconstrictor effect of glibenclamide was not enhanced in the presence of NLA + 8PT and 2) the exercise-induced increase in CBF was largely maintained. These findings show profound species differences in the mechanisms controlling CBF at rest and during exercise.NEW & NOTEWORTHY The present study demonstrates important species differences in the regulation of coronary blood flow by adenosine, NO, and KATP channels at rest and during exercise. In swine, these mechanisms follow a linear additive design, as opposed to dogs which follow a nonlinear redundant design. Simultaneous blockade of all three mechanisms virtually abolished exercise-induced coronary vasodilation in dogs, whereas a substantial vasodilator reserve could still be recruited during exercise in swine.

Differential impact of severe familial hypercholesterolemia on regional skeletal muscle and organ blood flows during exercise: Effects of PDE5 inhibition.

OBJECTIVE: Swine with familial hypercholesterolemia (FH) exhibit attenuated exercise-induced systemic vasodilation that is restored by phosphodiesterase 5 (PDE5) inhibition. Whether the impacts of FH and PDE5 inhibition to impair and restore exercise-induced vasodilation, respectively, results from tissue-specific or generalized effects remains unclear. Thus, we hypothesized that FH induces generalized impairment of skeletal muscle vasodilation that would be alleviated by PDE5 inhibition. METHODS: Systemic vascular responses to exercise were assessed in chronically instrumented normal and FH swine before and after PDE5 inhibition with EMD360527. Skeletal muscle and organ blood flows and conductances were determined via the microsphere technique. RESULTS: As previously reported, vs normal swine, FH swine have pronounced elevation of total cholesterol and impaired exercise-induced vasodilation that is restored by PDE5 inhibition. Blood flows to several, not all, skeletal muscle vascular beds were severely impaired by FH associated with reduced blood flow to many visceral organs. PDE5 inhibition differentially impacted skeletal muscle and organ blood flows in normal and FH swine. CONCLUSIONS: These data indicate that FH induces regional, not generalized, vasomotor dysfunction and that FH and normal swine exhibit unique tissue blood flow responses to PDE5 inhibition thereby adding to accumulating evidence of vascular bed-specific dysfunction in co-morbid conditions.

Intermittent pacing therapy favorably modulates infarct remodeling.

Despite early revascularization, remodeling and dysfunction of the left ventricle (LV) after acute myocardial infarction (AMI) remain important therapeutic targets. Intermittent pacing therapy (IPT) of the LV can limit infarct size, when applied during early reperfusion. However, the effects of IPT on post-AMI LV remodeling and infarct healing are unknown. We therefore investigated the effects of IPT on global LV remodeling and infarct geometry in swine with a 3-day old AMI. For this purpose, fifteen pigs underwent 2 h ligation of the left circumflex coronary artery followed by reperfusion. An epicardial pacing lead was implanted in the peri-infarct zone. After three days, global LV remodeling and infarct geometry were assessed using magnetic resonance imaging (MRI). Animals were stratified into MI control and IPT groups. Thirty-five days post-AMI, follow-up MRI was obtained and myofibroblast content, markers of extracellular matrix (ECM) turnover and Wnt/frizzled signaling in infarct and non-infarct control tissue were studied. Results showed that IPT had no significant effect on global LV remodeling, function or infarct mass, but modulated infarct healing. In MI control pigs, infarct mass reduction was principally due to a 26.2 ± 4.4% reduction in infarct thickness (P ≤ 0.05), whereas in IPT pigs it was mainly due to a 35.7 ± 4.5% decrease in the number of infarct segments (P ≤ 0.05), with no significant change in infarct thickness. Myofibroblast content of the infarct zone was higher in IPT (10.9 ± 2.1%) compared to MI control (5.4 ± 1.6%; P ≤ 0.05). Higher myofibroblast presence did not coincide with alterations in expression of genes involved in ECM turnover or Wnt/frizzled signaling at 5 weeks follow-up. Taken together, IPT limited infarct expansion and altered infarct composition, showing that IPT influences remodeling of the infarct zone, likely by increasing regional myofibroblast content.

Altered purinergic signaling in uridine adenosine tetraphosphate-induced coronary relaxation in swine with metabolic derangement.

We previously demonstrated that uridine adenosine tetraphosphate (Up4A) induces potent and partially endothelium-dependent relaxation in the healthy porcine coronary microvasculature. We subsequently showed that Up4A-induced porcine coronary relaxation was impaired via downregulation of P1 receptors after myocardial infarction. In view of the deleterious effect of metabolic derangement on vascular function, we hypothesized that the coronary vasodilator response to Up4A is impaired in metabolic derangement, and that the involvement of purinergic receptor subtypes and endothelium-derived vasoactive factors (EDVFs) is altered. Coronary small arteries, dissected from the apex of healthy swine and swine 6 months after induction of diabetes with streptozotocin and fed a high-fat diet, were mounted on wire myographs. Up4A (10-9-10-5 M)-induced coronary relaxation was maintained in swine with metabolic derangement compared to normal swine, despite impaired endothelium-dependent relaxation to bradykinin and despite blunted P2X7 receptor and NO-mediated vasodilator influences of Up4A. Moreover, a thromboxane-mediated vasoconstrictor influence was unmasked. In contrast, an increased Up4A-mediated vasodilator influence via P2Y1 receptors was observed, while, in response to Up4A, cytochrome P450 2C9 switched from producing vasoconstrictor to vasodilator metabolites in swine with metabolic derangement. Coronary vascular expression of A2A and P2X7 receptors as well as eNOS, as assessed with real-time PCR, was reduced in swine with metabolic derangement. In conclusion, although the overall coronary vasodilator response to Up4A was maintained in swine with metabolic derangement, the involvement of purinergic receptor subtypes and EDVF was markedly altered, revealing compensatory mechanisms among signaling pathways in Up4A-mediated coronary vasomotor influence in the early phase of metabolic derangement. Future studies are warranted to investigate the effects of severe metabolic derangement on coronary responses to Up4A.

Coronary microvascular dysfunction after long-term diabetes and hypercholesterolemia.

Coronary microvascular dysfunction (CMD) has been proposed as an important component of diabetes mellitus (DM)- and hypercholesterolemia-associated coronary artery disease (CAD). Previously we observed that 2.5 mo of DM and high-fat diet (HFD) in swine blunted bradykinin (BK)-induced vasodilation and attenuated endothelin (ET)-1-mediated vasoconstriction. Here we studied the progression of CMD after 15 mo in the same animal model of CAD. Ten male swine were fed a HFD in the absence (HFD, n = 5) or presence of streptozotocin-induced DM (DM + HFD, n = 5). Responses of small (∼300-µm-diameter) coronary arteries to BK, ET-1, and the nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine were examined in vitro and compared with those of healthy (Normal) swine (n = 12). Blood glucose was elevated in DM + HFD (17.6 ± 4.5 mmol/l) compared with HFD (5.1 ± 0.4 mmol/l) and Normal (5.8 ± 0.6 mmol/l) swine, while cholesterol was markedly elevated in DM + HFD (16.8 ± 1.7 mmol/l) and HFD (18.1 ± 2.6 mmol/l) compared with Normal (2.1 ± 0.2 mmol/l) swine (all P < 0.05). Small coronary arteries showed early atherosclerotic plaques in HFD and DM + HFD swine. Surprisingly, DM + HFD and HFD swine maintained BK responsiveness compared with Normal swine due to an increase in NO availability relative to endothelium-derived hyperpolarizing factors. However, ET-1 responsiveness was greater in HFD and DM + HFD than Normal swine (both P < 0.05), resulting mainly from ETB receptor-mediated vasoconstriction. Moreover, the calculated vascular stiffness coefficient was higher in DM + HFD and HFD than Normal swine (both P < 0.05). In conclusion, 15 mo of DM + HFD, as well as HFD alone, resulted in CMD. Although the overall vasodilation to BK was unperturbed, the relative contributions of NO and endothelium-derived hyperpolarizing factor pathways were altered. Moreover, the vasoconstrictor response to ET-1 was enhanced, involving the ETB receptors. In conjunction with our previous study, these findings highlight the time dependence of the phenotype of CMD.

Severe familial hypercholesterolemia impairs the regulation of coronary blood flow and oxygen supply during exercise.

Accelerated development of coronary atherosclerosis is a defining characteristic of familial hypercholesterolemia (FH). However, the recent data highlight a significant cardiovascular risk prior to the development of critical coronary stenosis. We, therefore, examined the hypothesis that FH produces coronary microvascular dysfunction and impairs coronary vascular control at rest and during exercise in a swine model of FH. Coronary vascular responses to drug infusions and exercise were examined in chronically instrumented control and FH swine. FH swine exhibited ~tenfold elevation of plasma cholesterol and diffuse coronary atherosclerosis (20-60 % plaque burden). Similar to our recent findings in the systemic vasculature in FH swine, coronary smooth muscle nitric oxide sensitivity was increased in vivo and in vitro with maintained endothelium-dependent vasodilation in vivo in FH. At rest and during exercise, FH swine exhibited increased myocardial O2 extraction resulting in reduced coronary venous SO2 and PO2 versus control. During exercise in FH swine, the transmural distribution of coronary blood flow was unchanged; however, a shift toward anaerobic cardiac metabolism was revealed by increased coronary arteriovenous H(+) concentration gradient. This shift was associated with a worsening of cardiac efficiency (relationship between cardiac work and O2 consumption) in FH during exercise owing, in part, to a generalized reduction in stroke volume which was associated with increased left atrial pressure in FH. Our data highlight a critical role for coronary microvascular dysfunction as a contributor to impaired myocardial O2 balance, cardiac ischemia, and impaired cardiac function prior to the development of critical coronary stenosis in FH.

Reduced contribution of endothelin to the regulation of systemic and pulmonary vascular tone in severe familial hypercholesterolaemia.

Vascular dysfunction has been associated with familial hypercholesterolaemia (FH), a severe form of hyperlipidaemia. We recently demonstrated that swine with FH exhibit reduced exercise-induced systemic, but not pulmonary, vasodilatation involving reduced nitric oxide (NO) bioavailability. Since NO normally limits endothelin (ET) action, we examined the hypothesis that reduced systemic vasodilatation during exercise in FH swine results from increased ET-mediated vasoconstriction. Systemic and pulmonary vascular responses to exercise were examined in chronically instrumented normal and FH swine in the absence and presence of the ETA/B receptor antagonist tezosentan. Intrinsic reactivity to ET was further assessed in skeletal muscle arterioles. FH swine exhibited ∼9-fold elevation in total plasma cholesterol versus normal swine. Similar to our recent findings, systemic, not pulmonary, vasodilatation during exercise was reduced in FH swine. Blockade of ET receptors caused marked systemic vasodilatation at rest and during exercise in normal swine that was significantly reduced in FH swine. The reduced role of ET in FH swine in vivo was not the result of decreased arteriolar ET responsiveness, as responsiveness was increased in isolated arterioles. Smooth muscle ET receptor protein content was unaltered by FH. However, circulating plasma ET levels were reduced in FH swine. ET receptor antagonism caused pulmonary vasodilatation at rest and during exercise in normal, but not FH, swine. Therefore, contrary to our hypothesis, FH swine exhibit a generalised reduction in the role of ET in regulating vascular tone in vivo probably resulting from reduced ET production. This may represent a unique vascular consequence of severe familial hypercholesterolaemia.

Pulmonary vasoconstrictor influence of endothelin in exercising swine depends critically on phosphodiesterase 5 activity.

Both phosphodiesterase 5 (PDE5) inhibition and endothelin (ET) receptor blockade have been shown to induce pulmonary vasodilation. However, little is known about the effect of combined blockade of these two vasoconstrictor pathways. Since nitric oxide (NO) exerts its pulmonary vasodilator influence via production of cyclic guanosine monophosphate (cGMP) as well as through inhibition of ET, we hypothesized that interaction between the respective signaling pathways precludes an additive vasodilator effect. We tested this hypothesis in chronically instrumented swine exercising on a treadmill by comparing the vasodilator effect of the PDE5 inhibitor EMD360527, the ETA/ETB antagonist tezosentan, and combined EMD360527 and tezosentan. In the systemic circulation, vasodilation by tezosentan and EMD360527 was additive, both at rest and during exercise, resulting in a 17 ± 2% drop in blood pressure. In the pulmonary circulation, both EMD360527 and tezosentan produced vasodilation. However, tezosentan produced no additional pulmonary vasodilation in the presence of EMD360527, either at rest or during exercise. Moreover, in isolated preconstricted porcine pulmonary small arteries (∼300 µm) EMD360527 (1 nM-10 µM) induced dose-dependent vasodilation, whereas tezosentan (1 nM-10 µM) failed to elicit vasodilation irrespective of the presence of EMD360527. However, both PDE5 inhibition and 8Br-cGMP, but not 8Br-cAMP, blunted pulmonary small artery contraction to ET and its precursor Big ET in vitro. In conclusion, in healthy swine, either at rest or during exercise, PDE5 inhibition and the associated increase in cGMP produce pulmonary vasodilation that is mediated in part through inhibition of the ET pathway, thereby precluding an additional vasodilator effect of ETA/ETB receptor blockade in the presence of PDE5 inhibition.

Phosphodiesterase-5 activity exerts a coronary vasoconstrictor influence in awake swine that is mediated in part via an increase in endothelin production.

Nitric oxide (NO)-induced coronary vasodilation is mediated through production of cyclic guanosine monophosphate (cGMP) and through inhibition of the endothelin-1 (ET) system. We previously demonstrated that phosphodiesterase-5 (PDE5)-mediated cGMP breakdown and ET each exert a vasoconstrictor influence on coronary resistance vessels. However, little is known about the integrated control of coronary resistance vessel tone by these two vasoconstrictor mechanisms. In the present study, we investigated the contribution of PDE5 and ET to the regulation of coronary resistance vessel tone in swine both in vivo, at rest and during graded treadmill exercise, and in vitro. ETA/ETB receptor blockade with tezosentan (3 mg/kg iv) and PDE5 inhibition with EMD360527 (300 µg·min(-1)·kg(-1) iv) each produced coronary vasodilation at rest and during exercise as well as in preconstricted isolated coronary small arteries. In contrast, tezosentan failed to produce further coronary vasodilation in the presence of EMD360527, both in vivo and in vitro. Importantly, EMD360527 (3 µM) and cGMP analog 8-Br-cGMP (100 µM) had no significant effects on ET-induced contractions of isolated porcine coronary small arteries, suggesting unperturbed ET receptor responsiveness. In contrast, PDE5 inhibition and cGMP blunted the contractions produced by the ET precursor Big ET, but only in vessels with intact endothelium, suggesting that PDE5 inhibition limited ET production in the endothelium of small coronary arteries. In conclusion, PDE5 activity exerts a vasoconstrictor influence on coronary resistance vessels that is mediated, in part, via an increase in endothelial ET production.

Cytochrome P-450 2C9 exerts a vasoconstrictor influence on coronary resistance vessels in swine at rest and during exercise.

A significant endothelium-dependent vasodilation persists after inhibition of nitric oxide synthase (NOS) and cyclooxygenase (COX) in the coronary vasculature, which has been linked to the activation of cytochrome P-450 (CYP) epoxygenases expressed in endothelial cells and subsequent generation of vasodilator epoxyeicosatrienoic acids. Here, we investigated the contribution of CYP 2C9 metabolites to regulation of porcine coronary vasomotor tone in vivo and in vitro. Twenty-six swine were chronically instrumented. Inhibition of CYP 2C9 with sulfaphenazole (5 mg/kg iv) alone had no effect on bradykinin-induced endothelium-dependent coronary vasodilation in vivo but slightly attenuated bradykinin-induced vasodilation in the presence of combined NOS/COX blockade with N(ω)-nitro-L-arginine (20 mg/kg iv) and indomethacin (10 mg/kg iv). Sulfaphenazole had minimal effects on coronary resistance vessel tone at rest or during exercise. Surprisingly, in the presence of combined NOS/COX blockade, a significant coronary vasodilator response to sulfaphenzole became apparent, both at rest and during exercise. Subsequently, we investigated in isolated porcine coronary small arteries (∼250 µm) the possible involvement of reactive oxygen species (ROS) in the paradoxical vasoconstrictor influence of CYP 2C9 activity. The vasodilation by bradykinin in vitro in the presence of NOS/COX blockade was markedly potentiated by sulfaphenazole under control conditions but not in the presence of the ROS scavenger N-(2-mercaptoproprionyl)-glycine. In conclusion, CYP 2C9 can produce both vasoconstrictor and vasodilator metabolites. Production of these metabolites is enhanced by combined NOS/COX blockade and is critically dependent on the experimental conditions. Thus production of vasoconstrictors slightly outweighed the production of vasodilators at rest and during exercise. Pharmacological stimulation with bradykinin resulted in vasodilator CYP 2C9 metabolite production when administered in vivo, whereas vasoconstrictor CYP 2C9 metabolites, most likely ROS, were dominant when administered in vitro.

Enhanced myofilament responsiveness upon ß-adrenergic stimulation in post-infarct remodeled myocardium.

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed ß-adrenergic receptor (ß-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F(max)) and higher Ca(2+)-sensitivity in MI compared to sham. F(max) did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca(2+)-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of ß-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca(2+)-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.

Prostanoids suppress the coronary vasoconstrictor influence of endothelin after myocardial infarction.

Myocardial infarction (MI) is associated with endothelial dysfunction resulting in an imbalance in endothelium-derived vasodilators and vasoconstrictors. We have previously shown that despite increased endothelin (ET) plasma levels, the coronary vasoconstrictor effect of endogenous ET is abolished after MI. In normal swine, nitric oxide (NO) and prostanoids modulate the vasoconstrictor effect of ET. In light of the interaction among NO, prostanoids, and ET combined with endothelial dysfunction present after MI, we investigated this interaction in control of coronary vasomotor tone in the remote noninfarcted myocardium after MI. Studies were performed in chronically instrumented swine (18 normal swine; 13 swine with MI) at rest and during treadmill exercise. Furthermore, endothelial nitric oxide synthase (eNOS) and cyclooxygenase protein levels were measured in the anterior (noninfarcted) wall of six normal and six swine with MI. eNOS inhibition with N(ω)-nitro-L-arginine (L-NNA) and cyclooxygenase inhibition with indomethacin each resulted in coronary vasoconstriction at rest and during exercise, as evidenced by a decrease in coronary venous oxygen levels. The effect of l-NNA was slightly decreased in swine with MI, although eNOS expression was not altered. Conversely, in accordance with the unaltered expression of cyclooxygenase-1 after MI, the effect of indomethacin was similar in normal and MI swine. L-NNA enhanced the vasodilator effect of the ET(A/B) receptor blocker tezosentan but exclusively during exercise in both normal and MI swine. Interestingly, this effect of L-NNA was blunted in MI compared with normal swine. In contrast, whereas indomethacin increased the vasodilator effect of tezosentan only during exercise in normal swine, indomethacin unmasked a coronary vasodilator effect of tezosentan in MI swine both at rest and during exercise. In conclusion, the present study shows that endothelial control of the coronary vasculature is altered in post-MI remodeled myocardium. Thus the overall vasodilator influences of NO as well as its inhibition of the vasoconstrictor influence of ET on the coronary resistance vessels were reduced after MI. In contrast, while the overall prostanoid vasodilator influence was maintained, its inhibition of ET vasoconstrictor influences was enhanced in post-MI remote myocardium.

Exercise limits the production of endothelin in the coronary vasculature.

We previously demonstrated that endothelin (ET)-mediated coronary vasoconstriction wanes with increasing exercise intensity via a nitric oxide- and prostacyclin-dependent mechanism (Ref. 23). Therefore, we hypothesized that the waning of ET coronary vasoconstriction during exercise is the result of decreased production of ET and/or decreased ET receptor sensitivity. We investigated coronary ET receptor sensitivity using intravenous infusion of ET and coronary ET production using intravenous infusion of the ET precursor Big ET, at rest and during continuous treadmill exercise at 3 km/h in 16 chronically instrumented swine. In the systemic vasculature, Big ET and ET induced similar changes in hemodynamic parameters at rest and during continuous exercise at 3 km/h, indicating that exercise does not alter ET production or receptor sensitivity in the systemic vasculature. In the coronary vasculature, infusion of ET resulted in similar dose-dependent decreases in coronary blood flow and coronary venous oxygen tension and saturation at rest and during exercise. In contrast, administration of Big ET resulted in dose-dependent decreases in coronary blood flow, as well as coronary venous oxygen tension and saturation at rest. These effects of Big ET were significantly reduced during exercise. Altogether, our data indicate that continuous exercise at 3 km/h attenuates ET-mediated coronary vasoconstriction through reduced production of ET from Big ET rather than through reduced ET sensitivity of the coronary vasculature. The decreased ET production during exercise likely contributes to metabolic coronary vasodilation.

Left ventricular remodeling in swine after myocardial infarction: a transcriptional genomics approach.

Despite the apparent appropriateness of left ventricular (LV) remodeling following myocardial infarction (MI), it poses an independent risk factor for development of heart failure. There is a paucity of studies into the molecular mechanisms of LV remodeling in large animal species. We took an unbiased molecular approach to identify candidate transcription factors (TFs) mediating the genetic reprogramming involved in post-MI LV remodeling in swine. Left ventricular tissue was collected from remote, non-infarcted myocardium, 3 weeks after MI-induction or sham-surgery. Microarray analysis identified 285 upregulated and 278 downregulated genes (FDR < 0.05). Of these differentially expressed genes, the promoter regions of the human homologs were searched for common TF binding sites (TFBS). Eighteen TFBS were overrepresented >two-fold (p < 0.01) in upregulated and 13 in downregulated genes. Left ventricular nuclear protein extracts were assayed for DNA-binding activity by protein/DNA array. Out of 345 DNA probes, 30 showed signal intensity changes >two-fold. Five TFs were identified in both TFBS and protein/DNA array analyses, which showed matching changes for COUP-TFII and glucocorticoid receptor (GR) only. Treatment of swine with the GR antagonist mifepristone after MI reduced the post-MI increase in LV mass, but LV dilation remained unaffected. Thus, using an unbiased approach to study post-MI LV remodeling in a physiologically relevant large animal model, we identified COUP-TFII and GR as potential key mediators of post-MI remodeling.

Both beta1- and beta2-adrenoceptors contribute to feedforward coronary resistance vessel dilation during exercise.

During exercise, beta-feedforward coronary vasodilation has been shown to contribute to the matching of myocardial oxygen supply with the demand of the myocardium. Since both beta(1)- and beta(2)-adrenoceptors are present in the coronary microvasculature, we investigated the relative contribution of these subtypes to beta-feedforward coronary vasodilation during exercise as well as to infusion of the beta(1)-agonist norepinephrine and the beta(1)- and beta(2)-agonist isoproterenol. Chronically instrumented swine were studied at rest and during graded treadmill exercise (1-5 km/h) under control conditions and after beta(1)-blockade with metoprolol (0.5 mg/kg iv) and beta(1)/beta(2)-blockade with propranolol (0.5 mg/kg iv). The selectivity and degree of beta-blockade of metoprolol and propranolol were confirmed using isoproterenol infusion (0.05-0.4 microg. kg(-1).min(-1)) under resting conditions. Isoproterenol-induced coronary vasodilation was mediated through the beta(2)-adrenoceptor, whereas norepinephrine-induced coronary vasodilation was principally mediated through the beta(1)-adrenoceptor. Exercise resulted in a significant increase in left ventricular norepinephrine release and epinephrine uptake. beta(1)-Adrenoceptor blockade with metoprolol had very little effect under resting conditions. However, during exercise, metoprolol attenuated the increase in myocardial oxygen supply in excess of the reduction in myocardial oxygen demand, as evidenced by a progressive decrease in coronary venous Po(2). Consequently, metoprolol caused a clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous Po(2). Additional beta(2)-adrenoceptor blockade with propranolol further inhibited myocardial oxygen supply during exercise, resulting in a further clockwise rotation of the relationship between myocardial oxygen consumption and coronary venous Po(2). In conclusion, both beta(1)- and beta(2)-adrenoceptors contribute to the beta-feedforward coronary resistance vessel dilation during exercise.

Integrated control of pulmonary vascular tone by endothelin and angiotensin II in exercising swine depends on gender.

The lungs are now recognized as an active metabolic organ that is a major determinant of the plasma concentrations of the vasoconstrictors endothelin (ET) and ANG II. Several studies have suggested a complex interaction between ET and ANG II in the systemic and coronary vascular beds that is different at rest and during exercise. To date, the interaction between these vasoconstrictor peptides has barely been investigated in relation to the pulmonary vascular bed. Consequently, we investigated the integrated control of pulmonary vasomotor tone by ET and ANG II in 24 chronically instrumented swine (15 female and 9 male) at rest and during graded treadmill exercise. In the systemic circulation, ANG II type 1 (AT(1)) receptor blockade with irbesartan and mixed ET(A)/ET(B) blockade with tezosentan each produced vasodilation. The systemic vasodilator effect of ET(A)/ET(B) blockade was enhanced after AT(1) blockade in female swine, whereas a trend toward an increase was observed in male swine. In the pulmonary circulation, AT(1) receptor blockade had no effect on pulmonary vascular tone in male swine, whereas it resulted in an unexpected increase in pulmonary vasomotor tone in female swine. ET(A)/ET(B) receptor blockade did not result in a decrease in pulmonary vasomotor tone at rest but produced a decrease in vasomotor tone during exercise in both genders. This pulmonary vasodilation by ET(A)/ET(B) receptor blockade was enhanced after prior AT(1) blockade in female swine but not in male swine. In conclusion, in both the systemic and pulmonary circulation of female swine, ANG II inhibits the vasoconstrictor influence of ET. This interaction is gender specific. The observation that plasma ET levels were not altered by AT(1) blockade in either gender suggests that the interaction between these vasoconstrictors occurs locally in the vasculature.

Functional and structural adaptations of coronary microvessels distal to a chronic coronary artery stenosis.

Distal to a chronic coronary artery stenosis, structural remodeling of the microvasculature occurs. The microvascular functional changes distal to the stenosis have not been studied in detail. We tested the hypothesis that microvascular structural remodeling is accompanied by altered regulation of coronary vasomotor tone with increased responsiveness to endothelin-1. Vasomotor tone was studied in coronary microvessels from healthy control swine and from swine 3 to 4 months after implantation of an occluder that causes a progressive coronary narrowing, resulting in regional left ventricular dysfunction and blunted myocardial vasodilator reserve. Arterioles (approximately 200-microm passive inner diameter at 60 mm Hg) were isolated from regions perfused by the stenotic left anterior descending and normal left circumflex coronary arteries and studied in vitro. Passive pressure-diameter curves demonstrated reduced distensibility of subendocardial left anterior descending compared with subendocardial left circumflex or control arterioles, suggestive of structural remodeling. Myogenic responses were blunted in subendocardial left anterior descending compared with left circumflex arterioles, reflecting altered smooth muscle function. However, vasodilator responses to nitroprusside and bradykinin were not different in the endocardium, suggesting preserved endothelium and smooth muscle responsiveness. Finally, vasoconstrictor responses to endothelin-1 were enhanced in left anterior descending arterioles compared with left circumflex or control arterioles. Regional myocardial vascular conductance responses to bradykinin and endothelin in vivo confirmed the in vitro observations. In conclusion, inward remodeling of coronary microvessels distal to a stenosis is accompanied by exaggerated vasoconstrictor responses to endothelin-1. These structural and functional alterations may aggravate flow abnormalities distal to a chronic coronary artery stenosis.

Cellular, mitochondrial and molecular alterations associate with early left ventricular diastolic dysfunction in a porcine model of diabetic metabolic derangement.

The prevalence of diabetic metabolic derangement (DMetD) has increased dramatically over the last decades. Although there is increasing evidence that DMetD is associated with cardiac dysfunction, the early DMetD-induced myocardial alterations remain incompletely understood. Here, we studied early DMetD-related cardiac changes in a clinically relevant large animal model. DMetD was established in adult male Göttingen miniswine by streptozotocin injections and a high-fat, high-sugar diet, while control animals remained on normal pig chow. Five months later left ventricular (LV) function was assessed by echocardiography and hemodynamic measurements, followed by comprehensive biochemical, molecular and histological analyses. Robust DMetD developed, evidenced by hyperglycemia, hypercholesterolemia and hypertriglyceridemia. DMetD resulted in altered LV nitroso-redox balance, increased superoxide production-principally due to endothelial nitric oxide synthase (eNOS) uncoupling-reduced nitric oxide (NO) production, alterations in myocardial gene-expression-particularly genes related to glucose and fatty acid metabolism-and mitochondrial dysfunction. These abnormalities were accompanied by increased passive force of isolated cardiomyocytes, and impaired LV diastolic function, evidenced by reduced LV peak untwist velocity and increased E/e'. However, LV weight, volume, collagen content, and cardiomyocyte cross-sectional area were unchanged at this stage of DMetD. In conclusion, DMetD, in a clinically relevant large-animal model results in myocardial oxidative stress, eNOS uncoupling and reduced NO production, together with an altered metabolic gene expression profile and mitochondrial dysfunction. These molecular alterations are associated with stiffening of the cardiomyocytes and early diastolic dysfunction before any structural cardiac remodeling occurs. Therapies should be directed to ameliorate these early DMetD-induced myocardial changes to prevent the development of overt cardiac failure.

Control of pulmonary vascular tone during exercise in health and pulmonary hypertension.

Despite the importance of the pulmonary circulation as a determinant of exercise capacity in health and disease, studies into the regulation of pulmonary vascular tone in the healthy lung during exercise are scarce. This review describes the current knowledge of the role of various endogenous vasoactive mechanisms in the control of pulmonary vascular tone at rest and during exercise. Recent studies demonstrate an important role for endothelial factors (NO and endothelin) and neurohumoral factors (noradrenaline, acetylcholine). Moreover, there is evidence that natriuretic peptides, reactive oxygen species and phosphodiesterase activity can influence resting pulmonary vascular tone, but their role in the control of pulmonary vascular tone during exercise remains to be determined. K-channels are purported end-effectors in control of pulmonary vascular tone. However, K(ATP) channels do not contribute to regulation of pulmonary vascular tone, while the role of K(V) and K(Ca) channels at rest and during exercise remains to be determined. Pulmonary hypertension is associated with alterations in pulmonary vascular function and structure, resulting in blunted pulmonary vasodilatation during exercise and impaired exercise capacity. Although there is a paucity of studies pertaining to the regulation of pulmonary vascular tone during exercise in idiopathic pulmonary hypertension, the few studies that have been performed in models of pulmonary hypertension secondary to left ventricular dysfunction suggest altered control of pulmonary vascular tone during exercise. Since the increased pulmonary vascular tone during exercise limits exercise capacity, future studies are needed to investigate the vasomotor mechanisms that are responsible for the blunted exercise-induced pulmonary vasodilatation in pulmonary hypertension.

Alterations in vasomotor control of coronary resistance vessels in remodelled myocardium of swine with a recent myocardial infarction.

The mechanism underlying the progressive deterioration of left ventricular (LV) dysfunction after myocardial infarction (MI) towards overt heart failure remains incompletely understood, but may involve impairments in coronary blood flow regulation within remodelled myocardium leading to intermittent myocardial ischemia. Blood flow to the remodelled myocardium is hampered as the coronary vasculature does not grow commensurate with the increase in LV mass and because extravascular compression of the coronary vasculature is increased. In addition to these factors, an increase in coronary vasomotor tone, secondary to neurohumoral activation and endothelial dysfunction, could also contribute to the impaired myocardial oxygen supply. Consequently, we explored, in a series of studies, the alterations in regulation of coronary resistance vessel tone in remodelled myocardium of swine with a 2 to 3-week-old MI. These studies indicate that myocardial oxygen balance is perturbed in remodelled myocardium, thereby forcing the myocardium to increase its oxygen extraction. These perturbations do not appear to be the result of blunted beta-adrenergic or endothelial NO-mediated coronary vasodilator influences, and are opposed by an increased vasodilator influence through opening of K(ATP) channels. Unexpectedly, we observed that despite increased circulating levels of noradrenaline, angiotensin II and endothelin-1, alpha-adrenergic tone remained negligible, while the coronary vasoconstrictor influences of endogenous endothelin and angiotensin II were virtually abolished. We conclude that, early after MI, perturbations in myocardial oxygen balance are observed in remodelled myocardium. However, adaptive alterations in coronary resistance vessel control, consisting of increased vasodilator influences in conjunction with blunted vasoconstrictor influences, act to minimize the impairments of myocardial oxygen balance.