Virginia M. Stiffel

Effects of Long-Term Hypoxia and Development on Cardiac Contractile Proteins in Fetal and Adult Sheep

Objective: We studied the effect of long-term, high-altitude hypoxia on cardiac myosin, actin, and troponin T (TnT) isoforms and Ca2+ and Mg2+-activated myofibrillar adenosine triphosphatase (ATPase) activities in fetal and adult sheep. Methods: We exposed pregnant (beginning at day 30 of gestation) and nonpregnant sheep to high altitude (3820 m) for 110 days. Myosin, actin, and TnT isoforms were analyzed by Western analysis. In purified myofibrillar preparations, Ca2+ and Mg2+-ATPase activities were measured by the appearance of inorganic phosphate after the addition of NaATP and various concentrations of either calcium or magnesium to the reaction mixture. Results: We found no change in myosin, actin, or TnT isoform composition after exposure to long-term hypoxia in either fetal or adult sheep. However, Mg2+-activated myofibrillar ATPase activity decreased significantly in the right ventricle of both fetus and adult after hypoxic exposure. There was also a significant maturational increase in both Ca2+ and Mg2+-ATPase activity in control animals. Conclusion: The decrease in Mg2+-activated myofibrillar ATPase activity might affect the decrease in cardiac contractility previously noted in the right ventricle of fetal sheep after exposure to long-term hypoxia. Likewise, the increase in Ca2+ and Mg2+-activated ATPase activities from the fetus to adult could partially explain the previously found maturational increase in cardiac contractility.

Effects of Long-Term High-Altitude Hypoxia on Ioslated Fetal Ovine Coronary Arteries

Objective: To examine the effects of long-term high-altitude hypoxia on the contractile properties of isolated fetal coronary arteries. Methods: Maximal contractile responses (Tmax) to 90 mmol/L KCl and the thromboxane A2 mimetic U46619 were measured in proximal (PLCx) and distal left circumflex (DLCx), left anterior descending (LAD), and right coronary arterial (RCA) rings from high-altitude and control fetuses. Paired studies were conducted with and without nitric oxide synthase (NOS) inhibitors, Nω-nitro-L-arginine and Nω-nitro-L-arginine ester. Results: In high-altitude fetuses, 90 mmol/L KCl Tmax responses in both intact and NOS-blocked rings decreased by ∼62% in PLCx, ∼59% in DLCx, ∼57% in LAD, and ∼47% in RCA (n = 9-18/group; P < .05). High-altitude vessels also exhibited decreased sensitivity to U46619. NOS blockade potentiated Tmax to U46619 in the high-altitude RCA segments and augmented Tmax to U46619 in high-altitude RCA compared with its treated control counterpart (P < .05). Conclusion: These results suggest that nitric oxide influences the pharmacologic responsiveness of the RCA to U46619. Furthermore, long-term high-altitude hypoxia significantly alters the contractile capabilities of fetal coronary arteries. These observations may partiall explain the maintained redistribution of cardiac output to the fetal heart during exposure to long-term high-altitude hypoxia.

Ca2+ Sensitivity of Fetal Coronary Arteries Exposed to Long-Term, High-Altitude Hypoxia

Objective: To determine the contribution of decreased calcium responsiveness of fetal coronary arteries to decreased contractile responses to potassium and the thromboxane A2 analogue U46619 in these arteries after exposure to chronic hypoxemia. Methods: Concentration-response curves to Ca2+ in β-escin-permeabilized left circumflex (LCx), left anterior descending (LAD), and right coronary artery (RCA) rings from high-altitude (HA) and control (CON) fetuses were measured. In a second set of β-escin-permeabilized coronary artery rings, the effect of U46619 on Ca2+ sensitivity was tested. Results: Maximum Ca2+-activated force (Tmax) was decreased in HA LCx (CON 0.091 ± 0.010 versus HA 0.057 ± 0.006 g/cm2; P < .05) and HA LAD (CON 0.065 ± 0.012 versus HA 0.031 ± 0.007 g/cm2; P < .05). No significant difference was observed in the RCA. There was no change in the pD2 (-log EC50) values between CON and HA coronary rings. The Ca2+ sensitizing effect of U46619 on submaximal Ca2+-activated force was lower only in the HA LCx (CON 0.044 ± 0.010 versus HA 0.023 ± 0.006 g/cm2 at 10-5 mol/L; P < .05). Conclusion: These results indicate that maximum tension development in response to Ca2+ was decreased in the HA LCx and LAD but not the RCA; however, Ca2+ sensitivity of the contractile apparatus was unaltered in all of them. Decreased Ca2+ responsiveness may partially explain the decreased contractile capability of fetal LCx and LAD during long-term, high-altitude intrauterine hypoxemia.

Effects of long-term high-altitude hypoxia and troponin I phosphorylation on cardiac myofilament calcium responses in Fetal and nonpregnant sheep

Objective: We studied the effects of long-term high-altitude hypoxia and protein kinase A (PKA) phosphorylation on calcium (Ca2+) responses of skinned cardiac papillary muscles from fetal and adult sheep. Methods: Fetal and nonpregnant adult sheep were exposed to high-altitude (3820 m), long-term (approximately 110 days) hypoxia. Papillary muscles were isolated and mounted in well-oxygenated, temperature-controlled baths. After the papillary muscles were stimulated electrically to establish the diastolic tension that produced the maximum active contraction, the electrical stimulation was stopped, and the muscles were skinned with 1% vol/vol Triton-X-100. In protocol 1, the skinned muscles were exposed to activating solutions containing different calcium concentrations (pCa; from pCa 8.0 to pCa 4.0), which were prepared by varying the Ca-EGTA/EGTA ratio, and the steady-state tension was measured at each pCa. In protocol 2, the skinned muscles were contracted with activating solution containing a pCa of 5.0. After equilibration, the solution in some baths was changed to activating solution at the same pCa of 5.0 but also containing the catalytic subunit of PKA. The other baths were exchanged with activating solution at a pCa of 5.0 containing PKA. We then measured the degree of tension reduction caused by PKA until tension reached a new steady state. Results: In the long-term hypoxic fetal heart, the maximum tension response of right, but not left, ventricular skinned papillary muscle to Ca2+ was significanlty less than that in control muscles. In the long-term hypoxic adult heart, the left ventricle, but not the right ventricle, displayed an increased maximum tension response to Ca2+ compared with control. Phosphorylation of troponin I (TnI) with PKA reduced active tension in both fetal ventricles of the long-term hypoxic group more than in hearts from control fetuses. In the adult, phosphorylation with PKA resulted in a larger decrease in tension in the left venitricle and a smaller decrease in tension in the right ventricle in the long-term hypoxic group, although the differences were small. Conclusion: In the long-term hypoxic fetal right ventricle, the decreased maximum tension response to Ca2+ is consistent with the decrease in myofibrillar magnesium-activated adenosine triphophatase activity observed previously. The larger decrease in tension after PKA phosphroylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.

Effects of Long-Term High-Altitude Hypoxia on Myocardial Protein Kinase A Activity and Troponin I Isoforms in Fetal and Nonpregnant Sheep:

Objective: In fetal sheep, we found that the augmentation of cardiac contractility by β-adrenergic receptor (β-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after β-AR stimulation was higher in long-term hypoxic fetal sheep than in normoxic ones. Therefore, we studied the potential role of changes in myocardial protein kinase A (PKA) activity and troponin I (TnI) isoforms in fetal nonpregnant sheep exposed to approximately 112 days of hypoxia at high altitude (3820 m). Methods: Resting and maximally stimulated (by cAMP) PKA activity was measured by phosphorylation of the artificial peptide, Kemptide. Specificity was confirmed by inhibition with PKI, a specific PKA inhibitor. For TnI isoforms, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to resolve the proteins. We used monoclonal anti-cardiac TnI antibody (clone C5), which also cross-reacted with slow skeletal muscle TnI, to identify TnI isoforms. Results: For the fetal hearts, resting PKA activity was significantly higher in the high-altitude group than the control group, but total PKA activity was not different between the normoxic and hypoxic groups. In the adult hearts, no significant difference was observed in either resting or total PKA activity between normoxic and hypoxic groups. For both the fetal and adult sheep, the predominant TnI was the cardiac isoform, and hypoxic exposure produced no change in the TnI isoform composition. Conclusion: Neither a reduction in PKA activity nor a change in TnI isoforms could explain the reduction in beta-receptor augmentation of cardiac contractility in fetal sheep exposed to long-term hypoxia.

The Effect of Long-term Hypoxia on Tension and Intracellular Calcium Responses Following Stimulation of the Thromboxane A2 Receptor in the Left Anterior Descending Coronary Artery of Fetal Sheep:

Objective: The purpose of this study was to investigate the mechanisms of tension and intracellular calcium regulation following stimulation with the thromboxane A2 receptor agonist U46619 in the left anterior descending coronary artery of fetal sheep exposed to long-term hypoxia. We hypothesized that there would be a reduction in intracellular calcium responses in long-term hypoxic left anterior descending coronary artery accompanied by an increase in calcium sensitivity of the contractile mechanism. Methods: Pregnant sheep were kept at altitude (3820 m) from day 30 of gestation until day 140. Fetal hearts from long-term hypoxic and from a control, normoxic group were obtained and the left anterior descending coronary artery of the fetus was dissected, cleaned, and mounted in a bath (Jasco) in which tension and intracellular calcium [Ca2+]i, using Fura-2, could be measured simultaneously following stimulation of the thromboxane A2 receptor with U46619. The role of intracellular calcium and the Rho kinase and protein kinase C pathways in the tension responses were investigated by maintaining intracellular calcium constant or by using the Rho kinase blocker, Y27632, or the protein kinase C blocker, GF109203-X. Results: There was no difference in the tension dose-response to U46619 between the normoxic fetal and hypoxic fetal left anterior descending, although [Ca2+]i was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca2+] i was maintained constant at baseline levels, U46619 produced the same tension dose-response in both normoxic fetal and hypoxic fetal left anterior descending as when [Ca2+]i was allowed to rise. The tension response was abolished in both groups when the Rho kinase inhibitor, Y27632, was given either during or before stimulation with U46619. The protein kinase C blocker, GF109203-X, had no effect on the tension response in either group. Conclusions: Long-term hypoxia did not alter the tension response to thromboxane A2 receptor stimulation in fetal left anterior descending. The contractions in response to U46619 were produced apparently completely by changes in calcium sensitivity through the Rho kinase pathway.

Effects of long-term, high-altitude hypoxia on tension and intracellular calcium responses in coronary arteries of fetal and adult sheep.

Objectives: We have previously shown that after exposure to long-term hypoxia, fetal coronary flow is maintained at control levels despite a 25% reduction in cardiac output. We also demonstrated that coronary vascular rings isolated from the long-term hypoxic fetuses and studied in well-oxygenated bath system displayed significantly reduced depolarization-induced contraction strength in response to KCl. To study the mechanism of reduced fetal coronary vascular responses to KCl-induced contractions following exposure to long-term hypoxia, we measured tension and intracellular calcium simultaneously, as well as L-type Ca2+ channel density and sensitivity. Methods: Pregnant ewes were housed at altitude (3820 m) for approximately 110 days. At 138 to 141 days of gestation, long-term hypoxic and control animals were killed and fetal and adult left anterior descending coronary artery (LAD) was isolated and studied in a well-oxygenated bath system. Tension and intracellular calcium ([Ca2+]i) were measured simultaneously in response to increasing concentrations of KCl and, in addition, the sensitivity to the calcium channel blocker nifedipine was measured at a half maximal concentration of KCl. We also measured L-type Ca2+ channel density with (+)-[3H]PN200-110. Results: L-type Ca2+ channel density was decreased by ∼31% in the long-term hypoxic fetal, but not adult, LAD. Tension in the long-term hypoxic fetal and adult LAD was significantly lower at all concentrations of KCl. [Ca2+]i was lower at rest in both fetal and adult LAD from long-term hypoxic animals and increased to lower levels at all concentrations of KCl. The ratio of tension to [Ca2+]i was also lower at all concentrations of KCl. Sensitivity to nifedipine was unchanged. Conclusions: The reduced L-type Ca2+ channel density and the reduced [Ca2+]i response to KCl, as well as the reduced tension response to [Ca2+]i, could potentially be involved in the reduction in depolarization-induced contractions in LAD from long-term hypoxic fetuses. In hypoxic adults, reduced [Ca2+]i and reduced tension response to [Ca2+]i may be involved in the lower tension response to KCl-induced contractions.