Vincent J. de Beer

Sunitinib-Induced Systemic Vasoconstriction in Swine Is Endothelin Mediated and Does Not Involve Nitric Oxide or Oxidative Stress

Angiogenesis inhibition with agents targeting tyrosine kinases of vascular endothelial growth factor receptors is an established anticancer treatment, but is, unfortunately, frequently accompanied by systemic hypertension and cardiac toxicity. Whether vascular endothelial growth factor receptor antagonism also has adverse effects on the pulmonary and coronary circulations is presently unknown. In chronically instrumented awake swine, the effects of the vascular endothelial growth factor receptor antagonist sunitinib on the systemic, pulmonary, and coronary circulation were studied. One week after sunitinib (50 mg PO daily), mean aortic blood pressure (MABP) had increased from 83±5 mm Hg at baseline to 97±6 mm Hg (P<0.05) because of a 57±20% increase in systemic vascular resistance as cardiac output decreased. In contrast, sunitinib had no discernible effects on pulmonary and coronary hemodynamics or cardiac function. We subsequently investigated the mechanisms underlying the sunitinib-induced systemic hypertension. Intravenous administration of NO synthase inhibitor NG-nitro-L-arginine increased MABP by 24±1 mm Hg under baseline conditions, whereas it increased MABP even further after sunitinib administration (32±3 mm Hg; P<0.05). Reactive oxygen species scavenging with a mixture of antioxidants lowered MABP by 13±2 mm Hg before but only by 5±2 mm Hg (P<0.05) after sunitinib administration. However, intravenous administration of the dual endothelin A/endothelin B receptor blocker tezosentan, which did not lower MABP at baseline, completely reversed MABP to presunitinib values. These findings indicate that sunitinib produces vasoconstriction selectively in the systemic vascular bed, without affecting pulmonary or coronary circulations. The sunitinib-mediated systemic hypertension is principally attributed to an increased vasoconstrictor influence of endothelin, with no apparent contributions of a loss of NO bioavailability or increased oxidative stress.

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 (≈200-&mgr;m 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.

Prevention of Myofilament Dysfunction by β-Blocker Therapy in Postinfarct Remodeling

Background—Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether &bgr;-blocker therapy could restore myofilament contractility. Methods and Results—In pigs with a MI induced by ligation of the left circumflex coronary artery, &bgr;-blocker therapy (bisoprolol, MI+&bgr;) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (Fmax) was lower, whereas Ca2+ sensitivity was higher in untreated MI compared with sham (both P<0.05). The difference in Ca2+ sensitivity was abolished by treatment of cells with the &bgr;-adrenergic kinase, protein kinase A. &bgr;-blocker therapy partially reversed Fmax and Ca2+ sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca2+ sensitivity in MI+&bgr; compared with untreated myocardium, the protein kinase A induced reduction in Ca2+ sensitivity was largest in cardiomyocytes from myocardium treated with &bgr;-blockers. Phosphorylation of &bgr;-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. &bgr;-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions—&bgr;-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of &bgr;-blockade on global left ventricular function after MI.

Both β1- and β2-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.

Enhanced myofilament responsiveness upon β-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 β-adrenergic receptor (β-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 β-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.

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 β-adrenergic or endothelial NO-mediated coronary vasodilator influences, and are opposed by an increased vasodilator influence through opening of KATP channels. Unexpectedly, we observed that despite increased circulating levels of noradrenaline, angiotensin II and endothelin-1, α-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.

Transmural Heterogeneity of Myofilament Function and Sarcomeric Protein Phosphorylation in Remodeled Myocardium of Pigs with a Recent Myocardial Infarction

Aim: Transmural differences in sarcomeric protein composition and function across the left ventricular (LV) wall have been reported. We studied in pigs sarcomeric function and protein phosphorylation in subepicardial (EPI) and subendocardial (ENDO) layers of remote LV myocardium after myocardial infarction (MI), induced by left circumflex coronary artery ligation. Methods: EPI and ENDO samples were taken 3 weeks after sham surgery (n = 12) or induction of MI (n = 12) at baseline (BL) and during β-adrenergic receptor (βAR) stimulation with dobutamine. Isometric force was measured in single cardiomyocytes at various [Ca2+] and 2.2 μm sarcomere length. Results: In sham hearts, no significant transmural differences were observed in myofilament function or protein phosphorylation. Myofilament Ca2+-sensitivity was significantly higher in both EPI and ENDO of MI compared to sham hearts. Maximal force was significantly reduced in MI compared to sham, but solely in ENDO cells. A higher passive force was observed in MI hearts, but only in EPI cells. The proportion of stiff N2B isoform was higher in EPI than in ENDO in both sham and MI hearts, and a trend toward increased N2B-proportion appeared in MI EPI, but not MI Endo. Analysis of myofilament protein phosphorylation did not reveal significant transmural differences in phosphorylation of myosin binding protein C, desmin, troponin T, troponin I (cTnI), and myosin light chain 2 (MLC-2) both at BL and during βAR stimulation with dobutamine infusion. A significant increase in MLC-2 phosphorylation was observed during dobutamine only in sham. In addition, the increase in cTnI phosphorylation upon dobutamine was twofold lower in MI than in sham. Conclusion: Myofilament dysfunction is present in both EPI and ENDO in post-MI remodeled myocardium, but shows a high degree of qualitative heterogeneity across the LV wall. These heterogeneous transmural changes in sarcomeric properties likely contribute differently to systolic vs. diastolic global LV dysfunction after MI.

Nitroso-redox balance in control of coronary vasomotor tone

Reactive oxygen species (ROS) are essential in vascular homeostasis but may contribute to vascular dysfunction when excessively produced. Superoxide anion (O(2)(·-)) can directly affect vascular tone by reacting with K(+) channels and indirectly by reacting with nitric oxide (NO), thereby scavenging NO and causing nitroso-redox imbalance. After myocardial infarction (MI), oxidative stress increases, favoring the imbalance and resulting in coronary vasoconstriction. Consequently, we hypothesized that ROS scavenging results in coronary vasodilation, particularly after MI, and is enhanced after inhibition of NO production. Chronically instrumented swine were studied at rest and during exercise before and after scavenging of ROS with N-(2-mercaptoproprionyl)-glycine (MPG, 20 mg/kg iv) in the presence or absence of prior inhibition of endothelial NO synthase (eNOS) with N(ω)-nitro-L-arginine (L-NNA, 20 mg/kg iv). In normal swine, MPG resulted in coronary vasodilation as evidenced by an increased coronary venous O(2) tension, and trends toward increased coronary venous O(2) saturation and decreased myocardial O(2) extraction. These effects were not altered by prior inhibition of eNOS. In MI swine, MPG showed a significant vasodilator effect, which surprisingly was abolished by prior inhibition of eNOS. Moreover, eNOS dimer/monomer ratio was decreased after MI, reflecting eNOS uncoupling. In conclusion, ROS exert a small coronary vasoconstrictor influence in normal swine, which does not involve scavenging of NO. This vasoconstrictor influence of ROS is slightly enhanced after MI. Since inhibition of eNOS abolished rather than augmented the vasoconstrictor influence of ROS in swine with MI, while eNOS dimer/monomer ratio was decreased, our data imply that uncoupled eNOS may be a significant source of O(2)(·-) after MI.

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.