Vasodilator reactive oxygen species ameliorate perturbed myocardial oxygen delivery in exercising swine with multiple comorbidities.

Multiple common cardiovascular comorbidities produce coronary microvascular dysfunction. We previously observed in swine that a combination of diabetes mellitus (DM), high fat diet (HFD) and chronic kidney disease (CKD) induced systemic inflammation, increased oxidative stress and produced coronary endothelial dysfunction, altering control of coronary microvascular tone via loss of NO bioavailability, which was associated with an increase in circulating endothelin (ET). In the present study, we tested the hypotheses that (1) ROS scavenging and (2) ETA+B-receptor blockade improve myocardial oxygen delivery in the same female swine model. Healthy female swine on normal pig chow served as controls (Normal). Five months after induction of DM (streptozotocin, 3 × 50 mg kg-1 i.v.), hypercholesterolemia (HFD) and CKD (renal embolization), swine were chronically instrumented and studied at rest and during exercise. Sustained hyperglycemia, hypercholesterolemia and renal dysfunction were accompanied by systemic inflammation and oxidative stress. In vivo ROS scavenging (TEMPOL + MPG) reduced myocardial oxygen delivery in DM + HFD + CKD swine, suggestive of a vasodilator influence of endogenous ROS, while it had no effect in Normal swine. In vitro wire myography revealed a vasodilator role for hydrogen peroxide (H2O2) in isolated small coronary artery segments from DM + HFD + CKD, but not Normal swine. Increased catalase activity and ceramide production in left ventricular myocardial tissue of DM + HFD + CKD swine further suggest that increased H2O2 acts as vasodilator ROS in the coronary microvasculature. Despite elevated ET-1 plasma levels in DM + HFD + CKD swine, ETA+B blockade did not affect myocardial oxygen delivery in Normal or DM + HFD + CKD swine. In conclusion, loss of NO bioavailability due to 5 months exposure to multiple comorbidities is partially compensated by increased H2O2-mediated coronary vasodilation.

Cardiovascular performance of adult breeding sows fails to obey allometric scaling laws.

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.

Does cardiovascular performance of modern fattening pigs obey allometric scaling laws?

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.

Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.

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.

Regulation of coronary blood flow. Effect of coronary artery stenosis.

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.

Nitric oxide limits coronary vasoconstriction by a shear stress-dependent mechanism.

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.

Stenosis differentially affects subendocardial and subepicardial arterioles in vivo.

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.

Adenosine preconditions against endothelin-induced constriction of coronary arterioles.

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.

Functional characteristics of the coronary microcirculation.

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.

Prolonged diastolic time fraction protects myocardial perfusion when coronary blood flow is reduced.

BACKGROUND: 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. METHODS AND RESULTS: In anesthetized, open-chest dogs, diastolic time fraction (DTF) increased significantly when coronary flow was reduced by lowering perfusion pressure from 100 to 70, 55, and 40 mm Hg. On average, DTF increased from 0.47+/-0.04 to 0.55+/-0.03 after a pressure step from 100 to 40 mm Hg in control, from 0.42+/-0.04 to 0.47+/-0.04 after administration of adenosine, and from 0.46+/-0.07 to 0.55+/-0.06 after L-NMMA (mean+/-SD, 6 dogs for control and adenosine, 4 dogs for L-NMMA, all P<0.05). Flow normalized to its value at full dilation and pressure of 90 mm Hg (375+/-25 mL/min) increased during the period of reduced pressure at 40 mm Hg; control, from 0.005+/-63 (2 seconds after pressure step) to 0.09+/-0.06 (15 seconds after pressure step); with adenosine, from 0.19+/-0.06 to 0. 22+/-0.06; and with L-NMMA, from 0.013+/-0.007 to 0.12+/-0.02 (all P<0.05). The increase in DTF at low pressure may be explained by a decrease in interstitial volume at low pressure, which either decreases the preload of the myocytes or reduces the buffer capacity for ions determining repolarization, thereby causing an earlier onset of relaxation. CONCLUSIONS: Because the largest increase in DTF occurs at pressures below the autoregulatory range when blood flow to the subendocardium is closely related to DTF, modulation of DTF by coronary blood flow can provide an important regulatory mechanism to match supply and demand of the myocardium when vasodilatory reserve is exhausted.

Chronic cardiac denervation affects the speed of coronary vascular regulation.

OBJECTIVE: We tested the hypothesis that the rate of adaptation of coronary metabolic vasodilatation and autoregulation is modulated by the cardiac nerves. METHODS: Anaesthetised dogs (seven innervated (control) and seven with denervated hearts) were subjected to controlled pressure perfusion of the left main coronary artery. Heart rate was controlled by pacing. RESULTS: The steady state autoregulation curves and metabolic regulation curves were similar in the two groups. A sudden increase or decrease in heart rate was associated with a faster response (22% shorter half-times) in the innervated than the denervated dogs (P < 0.001). A sudden increase or decrease in coronary arterial perfusion pressure was associated with a slower response (24% longer half-times) in the innervated than the denervated hearts (P < 0.005). CONCLUSIONS: We conclude that the speed of response to metabolic and perfusion pressure changes is partly mediated by cardio-cardiac reflexes. Reflex coronary vasodilatation appears to reinforce the metabolic vasodilatation of a heart rate increase and oppose the vasoconstriction in response to increased perfusion pressure.

Impairment of contraction increases sensitivity of epicardial lymph pressure for left ventricular pressure.

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.

Changes in myocardial fluid filtration are reflected in epicardial lymph pressure.

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.

Synthetic peptides representing T-cell epitopes act as carriers in pneumococcal polysaccharide conjugate vaccines.

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.