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 beta-adrenergic receptor (beta-AR) stimulation was reduced after exposure to long-term hypoxia. However, cyclic adenosine monophosphate (cAMP) production after beta-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 and 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. CONCLUSIONS: 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.

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 no 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 significantly 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 ventricle 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 triphosphatase activity observed previously. The larger decrease in tension after PKA phosphorylation of TnI in the long-term hypoxic fetal left ventricle indicates a larger reduction in Ca2+ binding to troponin C.

Effect of fetal hypoxia on heart susceptibility to ischemia and reperfusion injury in the adult rat.

OBJECTIVE: Epidemiologic studies showed an association between adverse intrauterine environment and ischemic heart disease in the adult. We tested the hypothesis that prenatal hypoxia increased the susceptibility of adult heart to ischemia-reperfusion (I-R) injury. METHODS: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% oxygen from day 15 to 21) groups. Hearts of 6-month-old male progeny were studied using Langendorff preparation and were subjected to two protocols of I-R: 10 minutes of ischemia and 3 hours of reperfusion (I-R(10)) or 25 minutes of ischemia and 3 hours of reperfusion (I-R(25)). RESULTS: Prenatal hypoxia did not change basal left ventricular (LV) function. I-R(10) produced myocardial stunning and a transient decrease in LV function in control hearts but caused myocardial infarction and a persistent decrease in postischemic recovery of LV function in hypoxic hearts. I-R(25) caused myocardial infarction in both control and hypoxic hearts, which was significantly higher in hypoxic hearts. The postischemic recovery of LV function was significantly reduced in hypoxic hearts. I-R(25)-induced activation of caspase-3 and apoptosis in the left ventricle were significantly higher in hypoxic than control hearts. There was a significant decrease in LV heat shock protein 70 and endothelial nitric oxide synthase levels in hypoxic hearts. Prenatal hypoxia did not change beta(1)-adrenoreceptor levels but significantly increased beta(2)-adrenoreceptor in the left ventricle. In addition, it increased G(s)alpha but decreased G(i)alpha. CONCLUSIONS: Prenatal chronic hypoxia increases the susceptibility of adult heart to I-R injury. Several possible mechanisms may be involved, including an increase in beta(2)-adrenoreceptor and the G(s)alpha/G(i)alpha ratio, and a decrease in heat shock protein 70 and endothelial nitric oxide synthase in the left ventricle.

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 Ca(2+)- and Mg(2+)-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, Ca(2+)(-) and Mg(2+)-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, Mg(2+)-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 Ca(2+)- and Mg(2+)-ATPase activity in control animals. CONCLUSION: The decrease in Mg(2+)-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 Ca(2+)- and Mg(2+)-activated ATPase activities from the fetus to adult could partially explain the previously found maturational increase in cardiac contractility.

Fetal cardiac and cerebrovascular acclimatization responses to high altitude, long-term hypoxia.

In response to high altitude long-term hypoxemia, the heart of fetal sheep shows a decrease in cardiac output that is secondary to a decrease in myocardial cell contractile function. The intracellular mechanisms responsible for these reductions might include reduced myofibrillar Mg(2+)-activated ATPase. There is also a decrease in beta(1)-adrenergic receptor stimulated augmentation of myocardial contraction. An overproduction of cAMP by beta(1)-adrenergic receptor stimulation, resulting in overphosphorylation of troponin I, may reduce calcium binding by troponin C. Fetal coronary arteries have a reduced contractile response to K(+) depolarization and a reduced sensitivity to a thromboxane A(2) receptor agonist-stimulated contraction. Cerebral arteries of adult sheep (but not the fetus) show decreased responses to both K(+)-depolarization and norepinephrine-induced contraction. Nonetheless, cerebral arteries in the long-term hypoxic fetus demonstrated a number of significant changes from control. For the cerebral arteries in general, high altitude hypoxia is associated with augmented or upregulation of presynaptic functions. In contrast, postsynaptic functions tend to be significantly depressed or downregulated. The results emphasize the role of high altitude, long-term hypoxemia in modulating adrenergic- and serotonergic-mediated signal transduction in the cerebral vasculature. They specifically highlight the significant differences in acclimatization responses between the fetus and adult.

The effect of long-term hypoxia on tension and intracellular calcium responses following stimulation of the thromboxane A(2) 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 A(2) 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 [Ca(2+)](i), using Fura-2, could be measured simultaneously following stimulation of the thromboxane A(2) 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 [Ca(2+)](i) was lower in the hypoxic fetal than normoxic fetal at the highest doses. When [Ca(2+)]( 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 [Ca(2+)](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 A(2) 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 approximately 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.

Prenatal cocaine exposure increases heart susceptibility to ischaemia-reperfusion injury in adult male but not female rats.

The present study tested the hypothesis that prenatal cocaine exposure differentially regulates heart susceptibility to ischaemia-reperfusion (I/R) injury in adult offspring male and female rats. Pregnant rats were administered intraperitoneally either saline or cocaine (15 mg kg(-1)) twice daily from day 15 to day 21 of gestational age. There were no differences in maternal weight gain and birth weight between the two groups. Hearts were isolated from 2-month-old male and female offspring and were subjected to I/R (25 min/60 min) in a Langendorff preparation. Preischaemic values of left ventricular (LV) function were the same between the saline control and cocaine-treated hearts for both male and female rats. Prenatal cocaine exposure significantly increased I/R-induced myocardial apoptosis and infarct size, and significantly attenuated the postischaemic recovery of LV function in adult male offspring. In contrast, cocaine did not affect I/R-induced injury and postischaemic recovery of LV function in the female hearts. There was a significant decrease in PKCepsilon and phospho-PKCepsilon levels in LV in the male, but not female, offspring exposed to cocaine before birth. These results suggest that prenatal cocaine exposure causes a sex-specific increase in heart susceptibility to I/R injury in adult male offspring, and the decreased PKCepsilon gene expression in the male heart may play an important role.