Abstract: In earlier work, we suggested that the start of the isovolumic contraction period could be detected in arterial pressure waveforms as the start of a temporary pre-systolic pressure perturbation (AIC(start), start of the Arterially detected Isovolumic Contraction), and proposed the retrograde coronary blood volume flow in combination with a backwards traveling pressure wave as its most likely origin. In this study, we tested this hypothesis by means of a coronary artery occlusion protocol. In six Yorkshire × Landrace swine, we simultaneously occluded the left anterior descending (LAD) and left circumflex (LCx) artery for 5 s followed by a 20-s reperfusion period and repeated this sequence at least two more times. A similar procedure was used to occlude only the right coronary artery (RCA) and finally all three main coronary arteries simultaneously. None of the occlusion protocols caused a decrease in the arterial pressure perturbation in the aorta during occlusion (P > 0.20) nor an increase during reactive hyperemia (P > 0.22), despite a higher deceleration of coronary blood volume flow (P = 0.03) or increased coronary conductance (P = 0.04) during hyperemia. These results show that the pre-systolic aortic pressure perturbation does not originate from the coronary arteries.
Abstract: In view of the remarkable decrease of the relative heart weight (HW) and the relative blood volume in growing pigs, we investigated whether HW, cardiac output (CO), and stroke volume (SV) of modern growing pigs are proportional to BW, as predicted by allometric scaling laws: HW (or CO or SV) = a·BW(b), in which a and b are constants, and constant b is a multiple of 0.25 (quarter-power scaling law). Specifically, we tested the hypothesis that both HW and CO scale with BW to the power of 0.75 (HW or CO = a·BW(0.75)) and SV scales with BW to the power of 1.00 (SV = a·BW(1.0)). For this purpose, 2 groups of pigs (group 1, consisting of 157 pigs of 50 ± 1 kg; group 2, consisting of 45 pigs of 268 ± 18 kg) were surgically instrumented with a flow probe or a thermodilution dilution catheter, under open-chest anesthetized conditions to measure CO and SV, after which HW was determined. The 95% confidence intervals of power-coefficient b for HW were 0.74 to 0.80, encompassing the predicted value of 0.75, suggesting that HW increased proportionally with BW, as predicted by the allometric scaling laws. In contrast, the 95% confidence intervals of power-coefficient b for CO and SV as measured with flow probes were 0.40 to 0.56 and 0.39 to 0.61, respectively, and values obtained with the thermodilution technique were 0.34 to 0.53 and 0.40 to 0.62, respectively. Thus, the 95% confidence limits failed to encompass the predicted values of b for CO and SV of 0.75 and 1.0, respectively. In conclusion, although adult breeding sows display normal heart growth, cardiac performance appears to be disproportionately low for BW. This raises concern regarding the health status of adult breeding sows.
Abstract: 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.
Abstract: Abstract  Since the completion of the Human Genome Project and the advent of the large scaled unbiased '-omics' techniques, the field of systems biology has emerged. Systems biology aims to move away from the traditional reductionist molecular approach, which focused on understanding the role of single genes or proteins, towards a more holistic approach by studying networks and interactions between individual components of networks. From a conceptual standpoint, systems biology elicits a 'back to the future' experience for any integrative physiologist. However, many of the new techniques and modalities employed by systems biologists yield tremendous potential for integrative physiologists to expand their tool arsenal to (quantitatively) study complex biological processes, such as cardiac remodelling and heart failure, in a truly holistic fashion. We therefore advocate that systems biology should not become/stay a separate discipline with '-omics' as its playing field, but should be integrated into physiology to create 'Integrative Physiology 2.0'.
Abstract: We previously demonstrated that endothelin (ET) mediated coronary vasoconstriction wanes with increasing exercise intensity via a nitric oxide- and prostacyclin-dependent mechanism. 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 3km/h in sixteen chronically instrumented swine. In the systemic vasculature Big ET and ET induced similar changes in hemodynamic parameters at rest and during continuous exercise, indicating that continuous exercise at 3km/h does not alter ET production or receptor sensitivity in the systemic vasculature. In the coronary vasculature, administration of Big ET resulted in dose-dependent decreases in coronary blood flow (CBF) as well as coronary venous oxygen tension and saturation (cvPO(2), cvSO(2)) at rest, while the effects of Big ET were significantly reduced during exercise. In contrast, infusion of ET resulted in similar dose-dependent decreases in CBF, cvPO(2) and cvSO(2) at rest and 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, although the latter may have been masked by a baroreceptor-mediated decrease in β-adrenergic vasodilation. The decreased ET production during exercise likely contributes to metabolic coronary vasodilation.
Abstract: Coronary blood flow (CBF) is reduced and transiently reversed during systole via cardiac contraction. Cardiac contractility, coronary tone, and arterial pressure each influence systolic CBF (CBF(SYS)), particularly by modulating the retrograde component of CBF(SYS). The effect of concurrent changes in these factors on CBF(SYS) during dynamic exercise has not been examined. Using chronically instrumented swine, we hypothesized that dynamic exercise enhances retrograde CBF(SYS). Phasic CBF was examined at rest and during treadmill exercise [2-5 miles/h (mph)]. Absolute values of mean CBF over the cardiac cycle (CBF(CYCLE)) as well as mean CBF in diastole (CBF(DIAS)) and mean CBF(SYS) were increased by exercise, while relative CBF(DIAS) and CBF(SYS) expressed as percentage of mean CBF(CYCLE) were principally unchanged. Early retrograde CBF(SYS) was present at rest and increased in magnitude (-33 +/- 4 ml/min) and as a percent of CBF(CYCLE) (-0.6 +/- 0.1%) at 5 mph. This reversal was transient, comprising 3.7 +/- 0.3% of cardiac cycle duration at 5 mph. Our results also reveal that moderately intense exercise (>3 mph) induced a second CBF reversal in late systole before aortic valve closure. At 5 mph, late retrograde CBF(SYS) amounted to -53 +/- 11 ml/min (-3.1 +/- 0.7% of CBF(CYCLE)) while occupying 11.1 +/- 0.3% of cardiac cycle duration. Wave-intensity analysis revealed that the second flow reversal coincided with an enhanced aortic forward-going decompression wave (vs. rest). Therefore, our data demonstrate a predictable increase in early-systolic CBF reversal during exercise and additionally that exercise induces a late-systolic CBF reversal related to the hemodynamic effects of left ventricular relaxation that is not predictable using current models of phasic CBF.
Abstract: 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.
Abstract: 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.
Abstract: Protocols for the extraction of nuclear proteins have been developed for cultured cells and fresh tissue, but sometimes only frozen tissue is available. We have optimized the homogenization procedure and subsequent fractionation protocol for the preparation of nuclear protein extracts from frozen porcine left ventricular (LV) tissue. This method gave a highly reproducible protein yield (6.5 ± 0.7% of total protein; mean±SE, n = 9) and a 6-fold enrichment of the nuclear marker protein B23. The nuclear protein extracts were essentially devoid of cytosolic, myofilament, and histone proteins. Compared to nuclear extracts from fresh LV tissue, some loss of nuclear proteins to the cytosolic fraction was observed. Using this method, we studied the distribution of tyrosine-phosphorylated signal transducer and activator of transcription 3 (PY-STAT3) in LV tissue of animals treated with the β-agonist dobutamine. Upon treatment, PY-STAT3 increased 30.2 ± 8.5-fold in total homogenates, but only 6.9 ± 2.1-fold (n = 4, P = 0.03) in nuclear protein extracts. Of all PY-STAT3 formed, only a minor fraction appeared in the nuclear fraction. This simple and reproducible protocol yielded nuclear protein extracts that were highly enriched in nuclear proteins with almost complete removal of cytosolic and myofilament proteins. This nuclear protein extraction protocol is therefore well-suited for nuclear proteome analysis of frozen heart tissue collected in biobanks.
Abstract: In view of the remarkable decrease of the relative heart weight and the relative blood volume in growing pigs, we investigated whether cardiac output (CO) and stroke volume (SV) of modern growing pigs are proportional to body mass (M), as predicted by allometric scaling laws: CO (or SV) = a.M(b), in which b is a multitude of 0.25 (quarter power scaling law). Specifically, we tested the hypothesis that CO scales with M to the power of 0.75 (CO = a.M(0.75)) and SV scales with M to the power of 1.00 (SV = a.M(1.0)) and investigated whether these relations persisted during increased cardiac stress. For this purpose, 2 groups of pigs (group 1 of 57 +/- 3 kg in Lelystad, and group 2 of 28 +/- 1 kg in Rotterdam) were chronically instrumented with a flow probe to measure CO and SV; instrumented pigs were studied at rest and during strenuous exercise (at approximately 85% of maximum heart rate). Analysis of both groups of pigs (analyzed separately or combined) under resting conditions demonstrated that the 95% confidence intervals of power-coefficient b for CO encompassed 0.75 and for SV encompassed 1.0. During exercise, similar results were obtained, except for SV in group 2, in which the 95% confidence limits remained below 1.0, which may have been due to the relatively small range of BW in group 2. These observations indicate that CO and SV of growing pigs with M less than 75 kg are still proportional to M, even during strenuous exercise, and that CO and SV scale with M according to the quarter power scaling laws. In conclusion, the concerns about disproportional growth and development of modern growing pigs with BW up to 75 kg were not confirmed by the present study.
Abstract: 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 beta-blocker therapy could restore myofilament contractility.
Abstract: 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.
Abstract: Several studies have indicated an interaction between the renin-angiotensin (ANG II) system and endothelin (ET) in the regulation of vascular tone. Previously, we have shown that both ET and ANG II exert a vasoconstrictor influence on the coronary resistance vessels of awake normal swine. Here, we investigated whether the interaction between ANG II and ET exists in the control of coronary resistance vessel tone at rest and during exercise using single and combined blockade of angiotensin type 1 (AT(1)) and ET(A)/ET(B) receptors. Since both circulating ANG II and ET levels are increased after myocardial infarction (MI), we investigated if the interaction between these systems is altered after MI. In awake healthy swine, coronary vasodilation in response to ET(A)/ET(B) receptor blockade in the presence of AT(1) blockade was similar to vasodilation produced by ET(A)/ET(B) blockade under control conditions. In awake swine with a 2- to 3-wk-old MI, coronary vasodilator responses to individual AT(1) and ET(A)/ET(B) receptor blockade were virtually abolished, despite similar coronary arteriolar AT(1) and ET(A) receptor expression compared with normal swine. Unexpectedly, in the presence of AT(1) blockade (which had no effect on circulating ET levels), ET(A)/ET(B) receptor blockade elicited a coronary vasodilator response. These findings suggest that in normal healthy swine the two vasoconstrictor systems contribute to coronary resistance vessel control in a linear additive manner, i.e., with negligible cross-talk. In contrast, in the remodeled myocardium, cross-talk between ANG II and ET emerges, resulting in nonlinear redundant control of coronary resistance vessel tone.
Abstract: Sarcomeric dysfunction plays a central role in reduced cardiac pump function in heart failure. This review focuses on the alterations in sarcomeric proteins in diseased myocardium that range from altered isoform expression to post-translational protein changes such as proteolysis and phosphorylation. Recent studies in animal models of heart failure and human failing myocardium converge and indicate that sarcomeric dysfunction, including altered maximum force development, Ca(2+) sensitivity, and increased passive stiffness, largely originates from altered protein phosphorylation, caused by neurohumoral-induced alterations in the kinase-phosphatase balance inside the cardiomyocytes. Novel therapies, which specifically target phosphorylation sites within sarcomeric proteins or the kinases and phosphatases involved, might improve cardiac function in heart failure.
Abstract: 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.
Abstract: In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.
Abstract: 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.
Abstract: Secondary pulmonary hypertension after myocardial infarction (MI) has been associated with endothelial dysfunction and activation of the endothelin (ET) system. Here, we investigated whether an increased ET-mediated pulmonary vasoconstrictor influence contributes to pulmonary hypertension after MI, and whether this increased ET vasoconstriction is caused by impaired nitric oxide (NO) and prostanoid production. For this purpose, chronically instrumented swine with and without MI ran on a treadmill at 0-4 km h(-1). Mixed ET(A)/ET(B) receptor blockade (tezosentan) was performed in the absence and presence of single or combined inhibition of endothelial NO synthase (eNOS, with N(omega)-nitro-l-arginine) and cyclo-oxygenase (COX, with indometacin). In normal swine, mixed ET(A)/ET(B) blockade decreased pulmonary vascular resistance, but only during exercise. In MI swine, an increased ET-mediated vasoconstrictor influence was observed in the pulmonary circulation both at rest and during exercise. Inhibition of COX resulted in pulmonary vasoconstriction at rest in MI, but not in normal swine; this vasoconstriction in MI swine was normalized by ET(A)/ET(B) receptor blockade. Inhibition of eNOS enhanced the vasodilator response to ET(A)/ET(B) blockade, indicating that NO blunts the pulmonary vasoconstrictor influence of ET. However, this vasodilator response was enhanced to a similar degree in MI and normal swine. In summary, swine with a recent MI are characterized by an exaggerated pulmonary vasoconstrictor influence of ET. This increased ET-mediated pulmonary vasoconstrictor influence is not caused by a loss of NO bioavailability, and is blunted by an increased prostanoid-mediated vasodilatation. In conclusion, an increased ET-mediated vasoconstriction, which does not appear to be the result of loss of endothelial vasodilators, contributes to pulmonary hypertension after MI.
Abstract: Nitric oxide (NO) plays a key role in regulating vascular tone. Mice overexpressing endothelial NO synthase [eNOS-transgenic (Tg)] have a 20% lower systemic vascular resistance (SVR) than wild-type (WT) mice. However, because eNOS enzyme activity is 10 times higher in tissue homogenates from eNOS-Tg mice, this in vivo effect is relatively small. We hypothesized that the effect of eNOS overexpression is attenuated by alterations in NO signaling and/or altered contribution of other vasoregulatory pathways. In isoflurane-anesthetized open-chest mice, eNOS inhibition produced a significantly greater increase in SVR in eNOS-Tg mice compared with WT mice, consistent with increased NO synthesis. Vasodilation to sodium nitroprusside (SNP) was reduced, whereas the vasodilator responses to phosphodiesterase-5 blockade and 8-bromo-cGMP (8-Br-cGMP) were maintained in eNOS-Tg compared with WT mice, indicating blunted responsiveness of guanylyl cyclase to NO, which was supported by reduced guanylyl cyclase activity. There was no evidence of eNOS uncoupling, because scavenging of reactive oxygen species (ROS) produced even less vasodilation in eNOS-Tg mice, whereas after eNOS inhibition the vasodilator response to ROS scavenging was similar in WT and eNOS-Tg mice. Interestingly, inhibition of other modulators of vascular tone [including cyclooxygenase, cytochrome P-450 2C9, endothelin, adenosine, and Ca-activated K(+) channels] did not significantly affect SVR in either eNOS-Tg or WT mice, whereas the marked vasoconstrictor responses to ATP-sensitive K(+) and voltage-dependent K(+) channel blockade were similar in WT and eNOS-Tg mice. In conclusion, the vasodilator effects of eNOS overexpression are attenuated by a blunted NO responsiveness, likely at the level of guanylyl cyclase, without evidence of eNOS uncoupling or adaptations in other vasoregulatory pathways.
Abstract: Coronary blood flow is tightly coupled to myocardial oxygen consumption to maintain a consistently high level of myocardial oxygen extraction over a wide range of physical activity. This tight coupling has been proposed to depend on periarteriolar oxygen tension, signals released from cardiomyocytes (adenosine acting on K(ATP) channels) and the endothelium (prostanoids(,) nitric oxide, endothelin) as well as neurohumoral influences (catecholamines, endothelin), but the contribution of each of these regulatory pathways, and their interactions, to exercise hyperaemia in the human heart are still incompletely understood. Thus, in the human heart, nitric oxide, prostanoids, adenosine and K(ATP) channels each contribute to resting tone, but evidence for a critical contribution to exercise hyperaemia is lacking. In dogs K(ATP) channel activation together with adenosine and nitric oxide contribute to exercise hyperaemia in a non-linear redundant fashion. In contrast, in swine nitric oxide, adenosine and K(ATP) channels contribute to resting coronary resistance vessel tone control in a linear additive manner, but are not mandatory for exercise hyperaemia in the heart. Rather, exercise hyperaemia in swine appears to involve K(Ca) channel opening that is mediated, at least in part, by exercise-induced beta-adrenergic activation, possibly in conjunction with exercise-induced blunting of an endothelin-mediated vasoconstrictor influence. In view of these remarkable species differences in coronary vasomotor control during exercise, future studies are required to determine whether exercise hyperaemia in humans follows a canine or porcine control design.
Abstract: Phosphorylation of cardiac myofilament proteins represents one of the main post-translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time- (or dose-) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2-DE and Pro-Q® Diamond stained gradient gels using minor amounts (˜0.5 mg dry weight) of human and pig cardiac tissue.
Abstract: We previously observed that pulmonary hypertension secondary to myocardial infarction (MI) in swine is characterized by elevated plasma endothelin (ET) levels and pulmonary vascular resistance (PVR). Consequently, we tested the hypothesis that an increased ET-mediated vasoconstrictor influence contributes to secondary pulmonary hypertension after MI and investigated the involvement of ET(A) and ET(B) receptor subtypes. Chronically instrumented swine with (MI swine; n = 25) or without (normal swine; n = 19) MI were studied at rest and during treadmill exercise (up to 4 km h(-1)), in the absence and presence of the ET(A) antagonist EMD 122946 or the mixed ET(A)/ET(B) antagonist tezosentan. In normal swine, exercise caused a small decrease in PVR. ET(A) blockade had no effect on PVR at rest or during exercise. Conversely, ET(A)/ET(B) blockade decreased PVR but only during exercise (at 4 km h(-1), from 3.0 +/- 0.1 to 2.3 +/- 0.1 mmHg min l(-1); P <or= 0.05). MI increased pulmonary arterial pressure and PVR both at rest and during exercise (both P <or= 0.05). The increased pulmonary arterial pressure correlated with the increased plasma ET levels in resting MI swine (r = 0.71; P <or= 0.01). Furthermore, the pulmonary vasoconstrictor response to ET-1 infusion was enhanced after MI (P <or= 0.05). ET(A)/ET(B) blockade decreased PVR in MI swine from 3.6 +/- 0.3 to 3.1 +/- 0.5 mmHg min l(-1) at rest and from 3.4 +/- 0.3 to 2.4 +/- 0.2 mmHg min l(-1) during exercise at 4 km h(-1) (both P <or= 0.05). This increased response to mixed ET(A)/ET(B) blockade in MI compared to normal swine appeared to be the result of an increased ET(A)-mediated vasoconstriction, as ET(A) blockade decreased PVR in MI swine from 3.4 +/- 0.4 to 2.8 +/- 0.2 mmHg min l(-1) at rest and from 3.1 +/- 0.3 to 2.6 +/- 0.2 mmHg min l(-1) at 4 km h(-1) (both P <or= 0.05). In conclusion, increased plasma ET levels together with increased pulmonary resistance vessel responsiveness to ET result in an exaggerated pulmonary vasoconstrictor influence of ET in swine with a recent MI. This vasoconstrictor influence is the result of an emergent tonic ET(A)-mediated vasoconstriction in addition to the exercise-induced ET(B)-mediated vasoconstriction that is already present in normal swine.
Abstract: Coronary blood flow is controlled via several vasoactive mediators that exert their effect on coronary resistance vessel tone through activation of K(+) channels in vascular smooth muscle. Because Ca(2+)-activated K(+) (K(Ca)(+)) channels are the predominant K(+) channels in the coronary vasculature, we hypothesized that K(Ca)(+) channel activation contributes to exercise-induced coronary vasodilation. In view of previous observations that ATP-sensitive K(+) (K(ATP)(+)) channels contribute, in particular, to resting coronary resistance vessel tone, we additionally investigated the integrated control of coronary tone by K(Ca)(+) and K(ATP)(+) channels. For this purpose, the effect of K(Ca)(+) blockade with tetraethylammonium (TEA, 20 mg/kg iv) on coronary vasomotor tone was assessed in the absence and presence of K(ATP)(+) channel blockade with glibenclamide (3 mg/kg iv) in chronically instrumented swine at rest and during treadmill exercise. During exercise, myocardial O(2) delivery increased commensurately with the increase in myocardial O(2) consumption, so that myocardial O(2) extraction and coronary venous Po(2) (Pcv(O(2))) were maintained constant. TEA (in a dose that had no effect on K(ATP)(+) channels) had a small effect on the myocardial O(2) balance at rest and blunted the exercise-induced increase in myocardial O(2) delivery, resulting in a progressive decrease of Pcv(O(2)) with increasing exercise intensity. Conversely, at rest glibenclamide caused a marked decrease in Pcv(O(2)) that waned at higher exercise levels. Combined K(Ca)(+) and K(ATP)(+) channel blockade resulted in coronary vasoconstriction at rest that was similar to that caused by glibenclamide alone and that was maintained during exercise, suggesting that K(Ca)(+) and K(ATP)(+) channels act in a linear additive fashion. In conclusion, K(Ca)(+) channel activation contributes to the metabolic coronary vasodilation that occurs during exercise. Furthermore, in swine K(Ca)(+) and K(ATP)(+) channels contribute to coronary resistance vessel control in a linear additive fashion.
Abstract: The renin-angiotensin system plays an important role in cardiovascular homeostasis by contributing to the regulation of blood volume, blood pressure, and vascular tone. Because AT(1) receptors have been described in the coronary microcirculation, we investigated whether ANG II contributes to the regulation of coronary vascular tone and whether its contribution is altered during exercise. Since the renin-angiotensin system is activated after myocardial infarction, resulting in an increase in circulating ANG II, we also investigated whether the contribution of ANG II to the regulation of vasomotor tone is altered after infarction. Twenty-six chronically instrumented swine were studied at rest and while running on a treadmill at 1-4 km/h. In 13 swine, myocardial infarction was induced by ligation of the left circumflex coronary artery. Blockade of AT(1) receptors (irbesartan, 1 mg/kg iv) had no effect on myocardial O(2) consumption but resulted in an increase in coronary venous O(2) tension and saturation both at rest and during exercise, reflecting coronary vasodilation. Despite increased plasma levels of ANG II after infarction and maintained coronary arteriolar AT(1) receptor levels, the vasodilation evoked by irbesartan was significantly reduced both at rest and during exercise. In conclusion, despite elevated plasma levels, the vasoconstrictor influence of ANG II on the coronary circulation in vivo is reduced after myocardial infarction. This reduction in ANG II-induced coronary vasoconstriction may serve to maintain perfusion of the remodeled myocardium.
Abstract: Withdrawal of the endothelin (ET)-mediated vasoconstrictor influence contributes to metabolic coronary vasodilation during exercise. Because production of nitric oxide (NO) and prostanoids increases with increasing shear stress and because NO and prostanoids are able to modify the release of ET, we hypothesized that the withdrawal of ET-mediated coronary vasoconstriction during exercise is mediated through NO and/or prostanoids. To test this hypothesis, 19 chronically instrumented swine were studied at rest and while running on a treadmill up to 85-90% of maximal heart rate. Blockade of ET(A)/ET(B) receptors with tezosentan resulted in an increase in coronary venous O(2) levels (i.e., in coronary vasodilation) at rest, which waned at increasing levels of exercise intensity. Inhibition of either NO synthase [N(omega)-nitro-l-arginine (l-NNA)] or cyclooxygenase (indomethacin) did not affect the response to tezosentan under resting conditions but unmasked a vasodilator response to tezosentan during exercise. The vasodilator response to tezosentan during exercise increased progressively after combined administration of l-NNA and indomethacin. These findings suggest that NO and prostanoids act synergistically to inhibit the vasoconstrictor influence of ET on the coronary circulation during exercise, thereby facilitating the exercise-induced vasodilation of coronary resistance vessels.
Abstract: Experimental evidence has emerged that myocardial ischemic preconditioning can prime the mitochondria into a "stress-resistant state", so that cell death is reduced following prolonged severe ischemia and reperfusion. Using a swine model of chronically ischemic myocardium, we tested the hypothesis that mitochondria within the ischemic territory have also acquired a protective phenotype. Eleven swine underwent a left thoracotomy with placement of an external constrictor around the proximal left anterior descending (LAD) artery. By 10 weeks, a severe stenosis of the LAD artery was documented by quantitative coronary angiography (92 +/- 2%). Animals were sacrificed and myocardium was extracted from the LAD and remote regions. Mitochondria were isolated from subendocardium and subepicardium from LAD and remote regions and state 2 (substrate alone) and state 3 (+ADP) respiration were assessed with a Clark electrode. Within the LAD subendocardium, the respiratory control index was 2.68 +/- 0.17 and was lower than the remote subendocardium (3.64 +/- 0.08; P < 0.05). When exposed to 20 min anoxia with reoxygenation, the LAD region demonstrated a more preserved state 3 respiration compared with the remote region (99 +/- 14 versus 65 +/- 9 nmol O2/mg, respectively; P < 0.05). In parallel mitochondrial experiments, chemiluminescence was detected with the probe coelenterazine and superoxide generation in the LAD region in the presence of antimycin A was 574 +/- 108 RLU/30 s/microg and was nearly 50% lower than the remote region (979 +/- 175 RLU/30 s/microg; P < 0.05). Within the mitochondria, the expression of uncoupling protein (UCP) 2 by western gels was 20% higher in the LAD region compared with the remote region (P < 0.05) with no differences noted in UCP-3. In this swine model of chronic myocardial ischemia, isolated mitochondria from the ischemic tissue demonstrate preserved state 3 respiration following anoxia/reoxygenation, consistent with a stress-resistant state. This state is characterized by a mild degree of uncoupling under basal conditions and decreased superoxide generation. Uncoupling protein 2 expression is enhanced in the mitochondria, providing a potential mechanism for these favorable mitochondrial adaptations.
Abstract: Alpha-adrenergic vasoconstriction in the coronary circulation is mediated through alpha-adrenoceptors on cardiac myocytes and subsequent release of endothelin, a very potent, long-lasting vasoconstrictor. Recent studies found that adult cardiac myocytes do not express the preproendothelin gene. Thus we hypothesized that alpha-adrenoceptor stimulation on the cardiac myocytes results in the production of an endothelin-releasing factor, which stimulates the coronary vasculature to produce endothelin. We tested this hypothesis by using an in vitro model in which isolated adult rat cardiac myocytes can be stimulated with an alpha-adrenoceptor agonist (phenylephrine). Their bathing fluid is then transferred to isolated coronary arterioles, and vasoactive responses are measured. To identify the source of endothelin, the endothelin-converting enzyme inhibitor phosphoramidon was added to either the myocytes or the isolated arterioles. Phenylephrine enhanced the vasoconstrictor properties of the myocyte bathing fluid. Administration of phosphoramidon (in either the presence or the absence of phenylephrine) to the myocytes had no effect on the vasoactive properties of the bathing fluid. In contrast, administration of phosphoramidon to the isolated arteriole before administration of the bathing fluid converted vasoconstriction to vasodilation, similar to the effect of the endothelin A receptor antagonist JKC-301, indicating that the endothelin is indeed produced by the coronary vasculature. Administration of the angiotensin type 1 receptor antagonist losartan to the vessel bath enhanced vasodilation to the bathing fluid of the phenylephrine-treated but not control myocytes. In conclusion, during alpha-adrenergic activation cardiac myocytes release a factor, probably angiotensin II, that stimulates the vascular production of endothelin. Although the physiological implications of this mechanism are not obvious, this may represent a protective mechanism that integrates neuronal vasoconstrictor mechanisms with myocardial metabolism, which minimizes periods of both coronary underperfusion and overperfusion.
Abstract: Previous studies demonstrated a decreased flow reserve in the hypertrophied myocardium early after myocardial infarction (MI). Previously, we reported that exacerbation of hemodynamic abnormalities and neurohumoral activation during exercise caused slight impairment of myocardial O(2) supply in swine with a recent MI. We hypothesized that increased metabolic coronary vasodilation [via ATP-sensitive K(+) (K(ATP)(+)) channels and adenosine] may have partially compensated for the increased extravascular compressive forces and increased vasoconstrictor neurohormones, thereby preventing a more severe impairment of myocardial O(2) balance. Chronically instrumented swine were exercised on a treadmill up to 85% of maximum heart rate. Under resting conditions, adenosine receptor blockade [8-phenyltheophylline (8-PT), 5 mg/kg i.v.] and K(ATP)(+) channel blockade (glibenclamide, 3 mg/kg i.v.) produced similar decreases in myocardial O(2) supply in normal and MI swine. However, while glibenclamide's effect waned in normal swine during exercise (P < 0.05), it was maintained in MI swine. 8-PT's effect was maintained during exercise and was not different between normal and MI swine. Finally, in normal swine combined treatment with 8-PT and glibenclamide produced a vasoconstrictor response that equaled the sum of the responses to blockade of the individual pathways. In contrast, in MI swine the vasoconstrictor response to 8-PT and glibenclamide was similar to that produced by glibenclamide alone. In conclusion, despite significant hemodynamic abnormalities in swine with a recent MI, myocardial O(2) supply and O(2) consumption in remodeled myocardium are still closely matched during exercise. This close matching is supported by increased K(ATP)(+) channel-mediated coronary vasodilation. Although the net vasodilator influence of adenosine was unchanged in remodeled myocardium, it became exclusively dependent on K(ATP)(+) channel opening.
Abstract: Severe congestive heart failure is associated with autonomic imbalance consisting of an increased sympathetic and decreased parasympathetic activity. In the present study, we investigated the influence of alterations in autonomic balance on cardiovascular function in 11 swine with left ventricular (LV) dysfunction produced by a 2- to 3-week-old myocardial infarction (MI).
Abstract: The endothelium releases substances that affect both vascular and cardiac myocytes. However, under conditions of augmented metabolic demands and cardiac work, signals from the cardiac myocytes may be critical for the endothelium to fulfill its secretory and regulatory function in the vascular bed. Therefore, we hypothesized that cardiac myocytes produce substances that alter the resting membrane potential of endothelial cells and thus vascular tone. Isolated rat cardiac myocytes were electrically stimulated at the rate of 0 and 400 beats/min (Po2 = 150 mmHg), and supernatants were collected from each group (Sup-0; control) and (Sup-400) and used within 6 mo. These supernatants were applied to human coronary endothelial cells that were subsequently analyzed by using the whole cell and cell-attached patch-clamp modes. Sup-0 had no effect on the whole cell current and the zero-current potential. The Sup-0 from myocytes treated with aprotinin, an inhibitor of kallikrein and serine protease, reduced whole cell current between -120 and -60 mV. Sup-400 depolarized endothelial cells from the resting membrane potential of -45 to -5 mV (P < 0.05), increased the magnitude of an inward current, and activated an outward current. Moreover, Sup-400 cells assayed in cell-attached patches increased single channel amplitude and the probability of a channel being in the open state. These effects were reversed by the Sup-400 from aprotinin-treated cells. We conclude that under certain metabolic conditions, isolated cardiac myocytes produce and release vasoactive substances that alter the function of K+ channels in vascular endothelial cells. Thus cardiac myocytes seem to communicate metabolic information to the endothelium, which could potentially influence vascular tone.
Abstract: We have previously shown that vasodilators and vasoconstrictors that are produced by the vascular endothelium, including nitric oxide (NO), prostanoids and endothelin (ET), contribute to the regulation of systemic and pulmonary vascular tone in swine, in particular during treadmill exercise. Since NO and prostanoids can modulate the release of ET, and vice versa, we investigated the integrated endothelial control of pulmonary vascular resistance in exercising swine. Specifically, we tested the hypothesis that increased NO and prostanoid production during exercise limits the vasoconstrictor influence of ET, so that loss of these vasodilators results in exaggerated ET-mediated vasoconstriction during exercise. Fifteen instrumented swine were exercised on a treadmill at 0-5 km h(-1) before and during ET(A)/ET(B) receptor blockade (tezosentan, 3 mg kg(-1) I.V.) in the presence and absence of inhibition of NO synthase (N(omega)-nitro-L-arginine, 20 mg kg(-1) I.V.) and/or cyclo-oxygenase (indometacin, 10 mg kg(-1) I.V.). In the systemic circulation, ET receptor blockade decreased vascular resistance at rest, which waned with increasing exercise intensity. Prior inhibition of either NO or prostanoid production augmented the vasodilator effect of ET receptor blockade, and these effects were additive. In contrast, in the pulmonary bed, ET receptor blockade had no effect under resting conditions, but decreased pulmonary vascular resistance during exercise. Prior inhibition of NO synthase enhanced the pulmonary vasodilator effect of ET receptor blockade, particularly during exercise, whereas inhibition of prostanoids had no effect, even after prior NO synthase inhibition. In conclusion, endogenous endothelin limits pulmonary vasodilatation in response to treadmill exercise. This vasoconstrictor influence is blunted by NO but not by prostanoids.
Abstract: Coronary blood flow is tightly coupled to myocardial oxygen consumption to maintain a consistently high level of myocardial oxygen extraction. This tight coupling has been proposed to depend on periarteriolar oxygen tension, signals released from cardiomyocytes (adenosine acting on K+(ATP) channels), and/or the endothelium (prostanoids, nitric oxide, endothelin [ET]) and autonomic influences (catecholamines), but the contribution of each of these regulatory pathways and their interactions are still incompletely understood. Until recently, experimental studies into the regulation of coronary blood flow during exercise were principally performed in the dog. We have performed several studies on the regulation of vasomotor tone in coronary resistance vessels in chronically instrumented exercising swine. These studies have shown that the coronary resistance vessels in swine lack significant alpha-adrenergic control, but that these vessels are subject to beta-adrenergic feed-forward control during exercise, which is aided by a parasympathetic withdrawal. In addition, withdrawal of an ET-mediated vasoconstrictor influence also contributes to exercise-induced coronary vasodilation. Coronary blood flow regulation by endothelial and metabolic vasodilator pathways contributes to resting vasomotor tone regulation but does not appear to contribute to the exercise-induced coronary vasodilation. Furthermore, blockade of one vasodilator pathway is not compensated by an increased contribution of the other vasodilator mechanisms, suggesting that porcine coronary vasomotor control by endothelial and metabolic factors occurs in a linear additive rather than a nonlinear synergistic fashion.
Abstract: Left ventricular dysfunction in swine with a recent myocardial infarction (MI) is associated with neurohumoral activation, including increased catecholamines and endothelin (ET). Although the increase in ET may serve to maintain blood pressure and, hence, perfusion of essential organs such as the heart and brain, it could also compromise myocardial perfusion by evoking coronary vasoconstriction. In the present study, we tested the hypothesis that endogenous ET contributes to perturbations in myocardial O2 balance during exercise in remodeled myocardium of swine with a recent MI. For this purpose, 26 chronically instrumented swine (10 with and 16 without MI) were studied at rest and while running on a treadmill at 1-4 km/h. After MI, plasma ET increased from 3.2 +/- 0.4 to 4.9 +/- 0.3 pM (P < 0.05). In normal swine, blockade of ETA (by EMD-122946) or ETA-ETB (by tezosentan) receptors resulted in an increase in coronary venous PO2, i.e., coronary vasodilation at rest, which decreased during exercise. In contrast, neither ETA nor ETA-ETB receptor blockade resulted in coronary vasodilation in swine with MI. Coronary vasoconstriction to intravenous ET-1 infusion in awake resting swine was blunted after MI. To investigate whether factors released by cardiac myocytes contributed to decreased vascular responsiveness to ET, we performed ET-1 dose-response curves in isolated coronary arterioles (70-200 microm). Vasoconstriction to ET-1 in isolated arterioles from MI swine was enhanced. In conclusion, the vasoconstrictor influence of endogenous as well as exogenous ET on coronary circulation in vivo is reduced. Because the response of isolated coronary arterioles to ET is increased after MI, the reduced vasoconstrictor influence in vivo suggests modulation of ET receptor sensitivity by cardiac myocytes, which may serve to maintain adequate myocardial perfusion.
Abstract: The heart constitutively expresses heme oxygenase (HO)-2, which catabolizes heme-containing proteins to produce biliverdin and carbon monoxide (CO). The heart also contains many possible substrates for HO-2 such as heme groups of myoglobin and cytochrome P-450s, which potentially could be metabolized into CO. As a result of observations that CO activates guanylyl cyclase and induces vascular relaxation and that HO appears to confer protection from ischemic injury, we hypothesized that the HO-CO pathway is involved in ischemic vasodilation in the coronary microcirculation. Responses of epicardial coronary arterioles to ischemia (perfusion pressure approximately 40 mmHg; flow velocity decreased by approximately 50%; dL/dt reduced by approximately 60%) were measured using stroboscopic fluorescence microangiography in 34 open-chest anesthetized dogs. Ischemia caused vasodilation of coronary arterioles by 36 +/- 6%. Administration of N(G)-monomethyl-L-arginine (L-NMMA, 3 micromol.kg(-1).min(-1) intracoronary), indomethacin (10 mg/kg iv), and K(+) (60 mM, epicardial suffusion) to prevent the actions of nitric oxide, prostaglandins, and hyperpolarizing factors, respectively, partially inhibited dilation during ischemia (36 +/- 6 vs. 15 +/- 4%; P < 0.05). The residual vasodilation during ischemia after antagonist administration was inhibited by tin mesoporphyrin IX (SnMP, 10 mg/kg iv), which is an inhibitor of HO (15 +/- 4 vs. 7 +/- 2%; P < 0.05 vs. before SnMP). The guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (10(-5) M, epicardial suffusion) also inhibited vasodilation during ischemia in the presence of L-NMMA with indomethacin and KCl. Moreover, administration of heme-L-arginate, which is a substrate for HO, produced dilation after ischemia but not after control conditions. We conclude that during myocardial ischemia, HO-2 activation can produce cGMP-mediated vasodilation presumably via the production of CO. This vasodilatory pathway appears to play a backup role and is activated only when other mechanisms of vasodilation during ischemia are exhausted.
Abstract: Prostacyclin and nitric oxide (NO) are produced by the endothelium in response to physical forces such as shear stress. Consequently, both NO and prostacyclin may increase during exercise and contribute to metabolic vasodilation. Conversely, NO has been hypothesized to inhibit prostacyclin production. We therefore investigated the effect of cyclooxygenase (COX) inhibition on exercise-induced vasodilation of the porcine systemic, pulmonary, and coronary beds before and after inhibition of NO production. Swine were studied at rest and during treadmill exercise at 1-5 km/h, before and after COX inhibition with indomethacin (10 mg/kg iv), and in the absence and presence of NO synthase inhibition with N(omega)-nitro-l-arginine (l-NNA; 20 mg/kg iv). COX inhibition produced systemic vasoconstriction at rest, which waned during exercise. The systemic vasoconstriction by COX inhibition was enhanced after l-NNA, particularly at rest. In the coronary circulation, COX inhibition also resulted in vasoconstriction at rest and during exercise. However, vasoconstriction was not modified by pretreatment with l-NNA. In contrast, COX inhibition had no effect on the pulmonary circulation, either at rest or during exercise. Moreover, a prostanoid influence in the pulmonary circulation could not be detected after l-NNA. In conclusion, endogenous prostanoids contribute importantly to systemic and coronary tone in awake swine at rest but are not mandatory for exercise-induced vasodilation in these beds. Endogenous prostanoids are not mandatory for the regulation of pulmonary resistance vessel tone. Finally, NO blunts the contribution of prostanoids to vascular tone regulation in the systemic but not in the coronary and pulmonary beds.
Abstract: Myocardial infarction (MI) initiates cardiac remodeling, depresses pump function, and predisposes to heart failure. This study was designed to identify early alterations in Ca2+ handling and myofilament proteins, which may contribute to contractile dysfunction and reduced beta-adrenergic responsiveness in postinfarct remodeled myocardium. Protein composition and contractile function of skinned cardiomyocytes were studied in remote, noninfarcted left ventricular (LV) subendocardium from pigs 3 weeks after MI caused by permanent left circumflex artery (LCx) ligation and in sham-operated pigs. LCx ligation induced a 19% increase in LV weight, a 69% increase in LV end-diastolic area, and a decrease in ejection fraction from 54+/-5% to 35+/-4% (all P<0.05), whereas cardiac responsiveness to exercise-induced increases in circulating noradrenaline levels was blunted. Endogenous protein kinase A (PKA) was significantly reduced in remote myocardium of MI animals, and a negative correlation (R=0.62; P<0.05) was found between cAMP levels and LV weight-to-body weight ratio. Furthermore, SERCA2a expression was 23% lower after MI compared with sham. Maximal isometric force generated by isolated skinned myocytes was significantly lower after MI than in sham (15.4+/-1.5 versus 19.2+/-0.9 kN/m2; P<0.05), which might be attributable to a small degree of troponin I (TnI) degradation observed in remodeled postinfarct myocardium. An increase in Ca2+ sensitivity of force (pCa50) was observed after MI compared with sham (DeltapCa50=0.17), which was abolished by incubating myocytes with exogenous PKA, indicating that the increased Ca2+ sensitivity resulted from reduced TnI phosphorylation. In conclusion, remodeling of noninfarcted pig myocardium is associated with decreased SERCA2a and myofilament function, which may contribute to depressed LV function. The full text of this article is available online at http://circres.ahajournals.org.
Abstract: The consistently high level of myocardial oxygen extraction requires tight control of coronary blood flow, because an increase in myocardial oxygen demand, as occurs during exercise, cannot be obtained by a further increase in oxygen extraction. Consequently, adequate control of coronary vascular resistance is critical. Coronary resistance vessel tone is the result of a myriad of vasodilator and vasoconstrictor influences, which are exerted by the myocardium, endothelium and neurohumoral status. Unraveling of the integrative mechanisms controlling metabolic vasodilation has been difficult, more than likely due to the redundancy design of vasomotor control. In contrast to the traditional view that myocardial ischemia produced by a coronary artery stenosis causes maximal microvascular dilation, more recent studies have shown that the coronary microvessels retain some degree of vasodilator reserve during ischemia and remain responsive to vasoconstrictor stimuli. These observations raise the question of whether pharmacologic vasodilators acting at the microvascular level might be therapeutically useful. The critical property of effective vasodilator therapy requires selective dilation of small arteries, while avoiding coronary steal by not interfering with metabolic vasoregulation at the level of the arterioles.
Abstract: In dogs, only combined blockade of vasodilator pathways [via adenosine receptors, nitric oxide synthase (NOS) and ATP-sensitive K+ (KATP) channels] results in impairment of metabolic vasodilation, which suggests a redundancy design of coronary flow regulation. Conversely, in swine and humans, blocking KATP channels, adenosine receptors, or NOS each impairs coronary blood flow (CBF) at rest and during exercise. Consequently, we hypothesized that these vasodilators act in parallel rather than in redundancy to regulate CBF in swine. Swine exercised on a treadmill (0-5 km/h), during control and after blockade of KATP channels (with glibenclamide), adenosine receptors [with 8-phenyltheophylline (8-PT)], and/or NOS [with Nomega-nitro-l-arginine (l-NNA)]. l-NNA, 8-PT, and glibenclamide each reduced myocardial O2 delivery and coronary venous O2 tension. These effects of l-NNA, 8-PT, and glibenclamide were not modified by simultaneous blockade of the other vasodilators. Combined blockade of KATP channels and adenosine receptors with or without NOS inhibition was associated with increased H+ production and impaired myocardial function. However, despite an increase in O2 extraction to >90% during administration of l-NNA + 8-PT + glibenclamide, vasodilator reserve could still be recruited during exercise. Thus in awake swine, loss of KATP channels, adenosine, or NO is not compensated for by increased participation of the other two vasodilator mechanisms. These findings suggest a parallel rather than a redundancy design of CBF regulation in the porcine circulation.
Abstract: Exercise-induced vasodilation is thought to be mediated through various vasodilator substances, but blunting the influence of vasoconstrictors such as ET may also play a role. However, the role of ET and its receptors in the regulation of systemic, pulmonary and coronary vascular resistance is incompletely understood. The aim of this study was to identify the contribution of ET-1 through the ET(A) and ET(B) receptors to the regulation of tone in the systemic, coronary and pulmonary beds at rest and during exercise.
Abstract: Left ventricular (LV) dysfunction caused by myocardial infarction (MI) is accompanied by endothelial dysfunction, most notably a loss of nitric oxide (NO) availability. We tested the hypothesis that endothelial dysfunction contributes to impaired tissue perfusion during increased metabolic demands as produced by exercise, and we determined the contribution of NO to regulation of regional systemic, pulmonary, and coronary vasomotor tone in exercising swine with LV dysfunction produced by a 2- to 3-wk-old MI. LV dysfunction resulted in blunted systemic and coronary vasodilator responses to ATP, whereas the responses to nitroprusside were maintained. Exercise resulted in blunted systemic and pulmonary vasodilator responses in MI that resembled the vasodilator responses in normal (N) swine following blockade of NO synthase with N(omega)-nitro-L-arginine (L-NNA, 20 mg/kg iv). However, L-NNA resulted in similar decreases in systemic (43 +/- 3% in N swine and 49 +/- 4% in MI swine), pulmonary (45 +/- 5% in N swine and 49 +/- 4% in MI swine), and coronary (28 +/- 4% in N and 35 +/- 3% in MI) vascular conductances in N and MI swine under resting conditions; similar effects were observed during treadmill exercise. Selective inhibition of inducible NO synthase with aminoguanidine (20 mg/kg iv) had no effect on vascular tone in MI. These findings indicate that while agonist-induced vasodilation is already blunted early after myocardial infarction, the contribution of endothelial NO synthase-derived NO to regulation of vascular tone under basal conditions and during exercise is maintained.
Abstract: Coronary tone is determined by a balance between endogenously produced endothelin and metabolic dilators. We hypothesized that coronary vasodilation during augmented metabolism is the net result of decreased endothelin production and increased production of vasodilators. Isolated rat myocytes were stimulated at 0, 200, and 400 beats/min to modify metabolism. Supernatant from these preparations was added to isolated coronary arterioles with and without blocking vasoactive pathways (adenosine, bradykinin, and endothelin). Chronically instrumented swine were studied while resting and running on a treadmill before and after endothelin type A (ET(A)) receptor blockade. The vasodilatory properties of the supernatant increased with increased stimulation frequencies. Combined blockade of adenosine and bradykinin receptors abolished vasodilation in response to supernatant of stimulated myocytes. ET(A) blockade increased vasodilation to supernatant of unstimulated myocytes but did not affect dilation to supernatant of myocytes stimulated at 400 beats/min. In vivo, ET(A) blockade resulted in coronary vasodilation at rest, which waned during exercise. Thus endothelin has a tonic constrictor influence through the ET(A) receptor at low myocardial metabolic demand but its influence decreased during increased metabolism.
Abstract: The presence of a coronary stenosis results primarily in subendocardial ischemia. Apart from the decrease in coronary perfusion pressure, a stenosis also decreases coronary flow pulsations. Applying a coronary perfusion system, we compared the autoregulatory response of subendocardial (n = 10) and subepicardial (n = 12) arterioles (<120 microm) after stepwise decreases in coronary arterial pressure from 100 to 70, 50, and 30 mmHg in vivo in dogs (n = 9). Pressure steps were performed with and without stenosis on the perfusion line. Maximal arteriolar diameter during the cardiac cycle was determined and normalized to its value at 100 mmHg. The initial decrease in diameter during reductions in pressure was significantly larger at the subendocardium. Diameters of subendocardial and subepicardial arterioles were similar 10--15 s after the decrease in pressure without stenosis. However, stenosis decreased the dilatory response of the subendocardial arterioles significantly. This decreased dilatory response was also evidenced by a lower coronary inflow at similar average pressure in the presence of a stenosis. Inhibition of nitric oxide production with N(G)-monomethyl-L-arginine abrogated the effect of the stenosis on flow. We conclude that the decrease in pressure caused by a stenosis in vivo results in a larger decrease in diameter of the subendocardial arterioles than in the subepicardial arterioles, and furthermore stenosis selectively decreases the dilatory response of subendocardial arterioles. These two findings expand our understanding of subendocardial vulnerability to ischemia.
Abstract: Increases in shear stress promote coronary vasodilation by stimulating the production of nitric oxide (NO). Whether shear stress-induced NO production also limits vasoconstriction in the coronary microcirculation in vivo is unknown. Accordingly, we measured microvascular diameter and flow velocity in the beating heart along with estimated blood viscosity to calculate shear stress during vasoconstriction with endothelin or vasopressin. Measurements were repeated in the presence of NG-monomethyl-L-arginine (L-NMMA) to inhibit NO production and BQ-788 to block NO-linked endothelin type B receptors. BQ-788 did not augment steady-state constriction to endothelin, suggesting that NO production via activation of this receptor is inconsequential. L-NMMA potentiated constriction to both agonists, particularly in small arteries (inner diameter >120 microm). Shear stresses in small arteries were elevated during constriction and further elevated during constriction after L-NMMA. These observations suggest that NO production limits vasoconstriction in the coronary microcirculation and that the principal stimulus for this governance is elevated shear stress. The degree of shear stress moderation of constriction is heterogeneously distributed, with small arteries displaying a higher degree of shear stress regulation than arterioles. These results provide the strongest evidence to date that shear stress-mediated production of NO exerts a "braking" influence on constriction in the coronary microcirculation.
Abstract: Myocardial hypoperfusion is accompanied by concomitant increases in adenosine and endothelin-1 (ET-1) production, but the vasodilatory effect of adenosine prevails over that of ET-1. Therefore, we hypothesized that adenosine-induced or ischemic preconditioning reduces the vasoconstrictive effect of ET-1. Coronary arteriolar diameter in vivo was measured using fluorescence microangiography in anesthetized open-thorax dogs. ET-1 (5 ng. kg(-1). min(-1) administered intracoronary, n = 10) induced progressive constriction over 45 min [25 +/- 6% (SE)]. The constriction was blocked by preconditioning with adenosine (25 microgram. kg(-1). min(-1) administered intracoronary) for 20 min and 10 min of washout (n = 10) or attenuated by ischemic preconditioning (four 5-min periods of ischemia, 9 +/- 5% at 45 min). To investigate the receptor involved in this process, coronary arterioles (50-150 micrometer) were isolated and pressurized at 60 mmHg in vitro. The ET-1 dose-response curve (1 pM-5 nM) was rightward shifted after preconditioning with adenosine (1 microM) for 20 min and 10 min of washout (n = 11). Blockade of A(2) receptors [8-(3-chlorostyryl)caffeine, 1 microM, n = 9] but not A(1) receptors (8-cyclopentyl-1,3-dipropylxanthine, 100 nM, n = 7) prevented this shift. These results suggest that adenosine confers a vascular preconditioning effect, mediated via the A(2) receptor, against endothelin-induced constriction. This effect may offer a new protective function of adenosine in preventing excessive coronary constriction.
Abstract: Because coronary blood flow is impeded during systole, the duration of diastole is an important determinant of myocardial perfusion. The aim of this study was to show that coronary flow modulates the duration of diastole at constant heart rate.
Abstract: For over 50 years, it has been recognized that coronary blood flow is precisely matched to cardiac metabolism. The interactions which govern this matching remain unknown. In the current review, 3 specific aspects of coronary flow regulation will be discussed: Specialization of function in different microvascular domains, influence of cardiac region on microvascular function and the interactions of vasoactive agents in control of coronary blood flow. Each level of the coronary microcirculation is affected by different physical and chemical forces within the heart. These forces place special demands on these vessels and are in turn met by specialized vasodilator responses, including metabolic and flow-mediated vasodilation. Perfusion of the heart is also profoundly affected by the region perfused. The endocardium is affected by forces, notably cardiac contraction, in a different manner than the epicardium. Thus, the microcirculation has specialized to meet these demands. Finally, the factors determining microvascular tone appear to be coordinated such that the loss of any individual dilator, such as nitric oxide, can be compensated for by the increased contribution of another, such as adenosine. This interplay may serve to protect the heart from ischemia during the early phases of coronary vascular disease when individual dilators may be impaired.
Abstract: We tested the hypothesis that the rate of adaptation of coronary metabolic vasodilatation and autoregulation is modulated by the cardiac nerves.
Abstract: In the present study, cardiac contraction was regionally impaired to investigate the relationship between contractility [maximum first time derivative of left ventricular pressure (dPLV/dtmax)] and PLV on epicardial lymph pressure (Plymph) generation. Measurements were performed in open-chest anesthetized dogs under control conditions and while local contraction was abolished by intracoronary administration of lidocaine. Lidocaine significantly lowered dPLV/dtmax and PLV pulse to 77 +/- 9 (SD; n = 5) and 82 +/- 5% of control, respectively, whereas Plymph pulse increased to 186 +/- 101%. The relative increase of maximum Plymph to PLV related inversely to the change in dPLV/dtmax after lidocaine administration. Additional data were obtained when PLV was transiently increased by constriction of the descending aorta. The ratio of pulse Plymph to PLV during aortic clamping increased after lidocaine administration, from 0.063 +/- 0.03 to 0.15 +/- 0.09. The results suggest that transmission of PLV to the cardiac lymphatic vasculature is enhanced when regional contraction is impaired. These findings imply that during normal, unimpaired contraction lymph vessels are shielded from high systolic PLV by the myocardium itself.
Abstract: The effect of increased fluid filtration on stopped-flow epicardial lymph pressure (P(lymph)), used as an indicator of myocardial interstitial volume, was investigated in the anesthetized open-chest dog. Histamine infusion resulted in an increased systolic peak in the P(lymph) signal together with an increase in diastolic P(lymph) in four of five animals. During reactive hyperemia, systolic and diastolic P(lymph) increased to 127 +/- 8 and 121 +/- 6.7% (mean +/- SE, n = 6) of control, respectively. Peak P(lymph) was approximately 15 s later than peak coronary blood flow and venous pressure (P(ven)). When P(ven) was transiently elevated to 367 +/- 72 (systolic) and 247 +/- 45% (diastolic) of control, P(lymph) increased to 132 +/- 12 and 120 +/- 5.5% of control. The time of response was similar for P(ven) and P(lymph) (t50 approximately 2 S). The increased systolic and diastolic P(lymph) can be explained by an increase in interstitial and lymph filling. It is concluded that changes in myocardial fluid filtration are reflected in epicardial P(lymph). Furthermore, it seems that cardiac contraction constitutes an important defense mechanism against the formation of myocardial edema.
Abstract: Improvement of antibody responses to polysaccharides through their linkage to proteins is thought to be mediated by protein-specific T helper (Th) cells. To investigate whether the carrier protein of a conjugate could be substituted by a Th epitope, Streptococcus pneumoniae type 17F polysaccharide (PS) was bromoacetylated and coupled to different peptides via their carboxy-terminal cysteines. Two peptides, one from the mycobacterial 65-kDa heat shock protein (hsp65) and the other from influenza virus hemagglutinin, are well-known Th epitopes. Two other peptides were selected from the pneumolysin sequence by Th epitope prediction methods; one of them was synthesized with cysteine either at the carboxy or the amino terminus. Three conjugates consistently elicited in mice anti-PS immunoglobulin M (IgM) and IgG responses that were not observed upon immunization with derivatized PS without peptide. The same conjugates induced no anti-PS antibody responses in athymic (nu/nu) mice, whereas clear responses were elicited in euthymic (nu/+) controls, demonstrating the thymus-dependent character of these conjugates. Only the three conjugates inducing anti-PS responses were capable of eliciting antipeptide antibodies. One of the immunogenic conjugates was studied in more detail. It induced significant protection and an anti-PS IgG response comprising all subclasses. On the basis of these results and proliferation studies with peptide and conjugate-primed cells, it is concluded that linkage of Th epitopes to PS in the right orientation enhances its immunogenicity in a thymus-dependent manner. Future possibilities for using peptides as carriers for inducing antibody responses to poorly immunogenic saccharide antigens are discussed.
