Angiotensin II in cardiac pressure-overload hypertrophy in fetal sheep.

We previously demonstrated in fetal sheep that blockade of ANG II type 1 (AT(1)) receptors did not attenuate an increase in right ventricle (RV) mass resulting from partial occlusion of the pulmonary artery (PA). We have now determined the effects of AT(2)-receptor blockade (PD-123319, 10 mg. kg(-1). day(-1) continuous iv) on the response of the fetal RV to PA banding for 7 days. Four groups of fetuses (each n = 7) were studied beginning at 126 +/- 1 days gestation (term 145 days). RV weight-to-body weight ratio (RV wt/body wt) increased (P < 0.05) in PA-banded (6.00 +/- 0.09 g/kg) and PA-banded + PD-123319 (6.19 +/- 0.27 g/kg) compared with control (5.17 +/- 0.17 g/kg) and PD-123319-infused (5.27 +/- 0.35 g/kg) fetuses (means +/- SE). Blood pressure and heart rate were similar in all groups. PD-123319 produced a decrease (P < 0.05) in AT(1) but not AT(2) mRNA levels in both fetal ventricles. To examine the effect of ANG II on fetal heart growth, twin fetal sheep were infused with either ANG II (twin received vehicle) or phenylephrine (Phe) (twin received vehicle) continuously for 7 days. Mean arterial blood pressure was 20-25 mmHg higher in ANG II and Phe fetuses compared with controls. The rate-pressure product was similar in ANG II and Phe fetuses and 40-50% greater than controls. Phe resulted in no change in RV wt/body wt or left ventricle-to-body weight ratio (LV wt/body wt) compared with controls. In contrast, ANG II produced a selective increase (27 +/- 5%, P < 0.05) in LV wt/body wt; no effect was seen on the RV. ANG II produced a decrease (P < 0.05) in LV but not RV AT(1) mRNA levels compared with controls; no effect was seen with Phe. The data demonstrate that in the ovine fetus, AT(2) receptors do not contribute to the maintenance of blood pressure or the development of pressure-overload RV hypertrophy. Elevated ANG II levels produce a selective increase in LV mass in the fetal sheep that is, in part, independent of increased systolic load.

Pediatric tele-echocardiography: evaluation of transmission modalities.

Pediatric cardiology consultation has been effectively delivered outside the tertiary care setting through the use of tele-echocardiography. This study examined the effectiveness of several tele-echocardiography connections and the satisfaction of the referring physicians using these services. Studies were transmitted via either a shared fiber-optic (DS3) connection (two sites), a dedicated fast-copper (ISDN-PRI) link, or by courier from a nearby (25-mile) or distant (170-mile) site. Time intervals between when echocardiograms were performed locally until they were received, interpreted, and reported were prospectively recorded. Referring physician satisfaction was assessed through a survey. The critical time between when a remote echocardiogram was performed and when its result was reported to the referring physician was primarily determined by the mode of transmission. The time interval between performing an echocardiogram and receiving the study was significantly longer for echocardiograms sent from the 170-mile courier site (2474 +/- 295 min) than either the 25-mile courier site (474 +/- 151 min), DS3 (374 +/- 121 min), or ISDN-PRI (129 +/- 16 min). Regardless of the method of transmission, all referring physicians felt that the service improved their ability to manage children, and they would recommend the service to their colleagues. Those using the courier service from the 25-mile away site were more concerned about the availability of a pediatric cardiologist and image quality, presumably due to the delay in response times. The time interval data provided in this study and the assessment of physician satisfaction provide important data as echocardiography laboratories implement tele-echocardiography services.

Aortic aneurysm following subclavian flap repair: diagnosis by magnetic resonance imaging.

Aortic aneurysm following subclavian flap repair of coarctation of the aorta is thought to occur infrequently. We present a case in which aneurysm of the subclavian flap was found 10 years after the original surgery. The location and extent of the aneurysm was clearly defined using cardiac magnetic resonance imaging (MRI). This report adds further support to recent recommendations to make cardiac MRI a routine imaging study for all postoperative patients from coarctation of the aorta repair.

Metabolic adaptation of the hypertrophied heart: role of the malate/aspartate and alpha-glycerophosphate shuttles.

Activation of the malate/aspartate and alpha -glycerophosphate shuttles (the NADH shuttles) has been identified in glycolytically active newborn myocardium. The goal of this study was to determine if the NADH shuttles and their regulatory genes are activated in hypertrophied myocardium as substrate utilization shifts away from fatty acids and toward glucose and lactate. Capacity of the shuttles was determined in cardiac mitochondria isolated one week, one month, and three months following aortic banding or sham operation. Myocardial steady-state mRNA and protein levels of regulatory enzymes were also measured. Despite a significant increase in left ventricular mass and activation of the atrial natriuretic peptide gene, no change in malate/aspartate nor alpha -glycerophosphate shuttle capacity was found at any of the three time points studied. Reactivation of the genes encoding the regulatory inner mitochondrial membrane proteins was not found in the hypertrophied myocardium, though these genes were down regulated one week following aortic-banding. These results suggest that sufficient malate/aspartate and alpha -glycerophosphate shuttle capacity exists in cardiac mitochondria to accommodate increased shuttle flux as hypertrophied myocardium becomes more glycolytically active.

Balloon aortic valvuloplasty in a 1,600-gram infant.

We present a 1,600 g infant who underwent successful balloon aortic valvuloplasty from the right carotid artery approach. A simple technique to facilitate access to the left ventricle and expedite the procedure is described. Issues unique to performing balloon aortic valvuloplasty on such a small child are discussed.

Thyroid hormone regulation of the NADH shuttles in liver and cardiac mitochondria.

Thyroid hormone can potentially regulate the malate/aspartate and alpha-glycerophosphate shuttle pathways in cardiac mitochondria either directly, by altering gene expression, or indirectly, by increasing myocardial workload. The goal of the current study was to determine the influence of thyroid hormone on the NADH shuttles in cardiac and liver mitochondria. Malate/aspartate and alpha-glycerophosphate shuttle capacities were significantly increased in cardiac mitochondria from adult rats treated for 9 days with T3 compared to saline-treated controls. Liver mitochondria demonstrated a significant increase in alpha-glycerophosphate and no change in malate/aspartate shuttle capacity. T3 increased steady-state mRNA levels and activity of mitochondrial alpha-glycerophosphate dehydrogenase in both myocardium and liver. Quantitative immunoblot studies demonstrated a significant increase in aspartate-glutamate carrier levels in T3-treated myocardium suggesting a regulatory role of the aspartate/glutamate carrier in T3-treated hearts. Thyroid hormone effects on the NADH shuttles are tissue-specific. Changes in the NADH shuttles in the presence of thyroid hormone excess occur both directly at the gene level and indirectly as an adaptive response.

Optimizing utilization of pediatric echocardiography and implications for telemedicine.

Telemedicine can deliver tertiary level services to remote communities where subspecialty care is limited. Locally performed echocardiography has been initiated at several locations around Iowa. The goal of this study was to examine utilization and diagnostic yield of community-based echocardiographic services. Community physicians selected patients for remote echocardiograms (echoes), and studies were performed locally by sonographers trained in recording pediatric echoes. Echoes were sent to the pediatric echocardiography laboratory by mail or via telemedicine systems. Echoes were also ordered locally by pediatric cardiologists during outreach clinics in the same communities. Numbers of normal and abnormal echoes ordered by community physicions and pediatric cardiologists were compared by chi-square analysis. Since January 1996, community physicians ordered 378 echoes, whereas 154 echoes were ordered by pediatric cardiologists at outreach clinics. Stratifying echoes by patient age found that the percentage of normal studies in patients < 1 year of age was no different between groups (27% normal by community physicians vs 15%; chi-square 0.92; p = 0.34). The percentage of normal studies ordered by community physicians was significantly greater in patients > 1 year of age (83% normal by community physicians vs 25%; chi-square 80.2; p <0.0001). Thus, (1) community physicians effectively identified patients < 1 year of age with abnormal echoes, (2) significantly fewer echoes may be required in patients > 1 year of age if patients are first evaluated by a pediatric cardiologist, and (3) patient selection will impact cost effectiveness of remotely obtained echoes.

Ontogeny of malate-aspartate shuttle capacity and gene expression in cardiac mitochondria.

Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by approximately 50% during the first 5 wk of life (from 921 +/- 48 to 531 +/- 53 nmol.min-1.mg protein-1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.

Angiotensin AT1 receptor blockade fails to attenuate pressure-overload cardiac hypertrophy in fetal sheep.

We examined the hypothesis that endogenous angiotensin II and angiotensin type 1 (AT1) receptors participate in the development of fetal right ventricular hypertrophy by studying the effects of AT1 receptor blockade on cardiac growth in fetal sheep subjected to constrictive banding of the pulmonary artery (PA). Seven pairs of twin fetuses were studied beginning at 126 +/- 1 days gestation (term = 145 days). One twin was given losartan (10 mg kg(-1) x day(-1) i.v.) for 7 consecutive days after PA banding, and the other twin served as a saline-treated, PA-banded control. Four additional pairs of twins served as sham-operated controls. Fetal heart rate (HR) and mean arterial blood pressure (MABP) were similar in the two groups of PA-banded animals before treatment and remained unchanged in the PA-banded control group. Losartan resulted in a significant decrease (P < 0.05) in MABP between days 0 and 7, whereas HR was not affected. Total body weight of the losartan-treated animals was significantly less (P < 0.05) than twin PA-banded controls and nonbanded fetuses. Right ventricle weight-to-body weight ratios were similar in saline (2.29 +/- 0.34 g/kg) and losartan-treated (2.11 +/- 0.15 g/kg) PA-banded animals and significantly greater than that in nonbanded fetuses (1.52 +/- 0.07 g/kg). Similar differences were seen in the right ventricle weight-to-left ventricle weight ratios. Right and left ventricle AT1 receptor mRNA and protein expression were also similar among the three groups, as were AT2 receptor mRNA levels. These data suggest that endogenous angiotensin II does not contribute to the development of pressure overload-induced right ventricular hypertrophy during fetal life and that expression of angiotensin receptors is not altered by increased afterload in the ovine fetus.

Developmental regulation of the alpha-glycerophosphate shuttle in porcine myocardium.

The alpha-glycerophosphate (alpha-GP) shuttle has been shown to play a role in reducing equivalent transfer in neonatal cardiac mitochondria. In adult heart mitochondria, alpha-GP shuttle activity is not detectable. The goals of the current study were to define the time course of the age-dependent decline in alpha-GP shuttle capacity and to identify the enzymatic step(s) of the alpha-GP shuttle which are regulated during development. Intact mitochondria were isolated from porcine hearts of various ages and assayed for alpha-GP shuttle capacity. By 5 weeks of age, alpha-GP shuttle capacity had decreased by nearly 39%. The cytosolic step of the shuttle, catalysed by cytosolic alpha-glycerophosphate dehydrogenase (c alpha-GPDH), demonstrated a significant increase between 0-2-day-old animals and adults. Partial cDNA clones of porcine c alpha-GPDH and mitochondrial alpha-glycerophosphate dehydrogenase (m alpha-GPDH) were prepared and used to quantitate expression of these genes. Using mRNA isolated from neonatal and adult porcine myocardium, expression of the c alpha-GPDH was unchanged, while expression of the m alpha-GPDH gene was present in neonatal but absent in adult myocardium. These results demonstrate a rapid postnatal decline in myocardial alpha-GP shuttle capacity which appears to be regulated by a decline in m alpha-GPDH gene expression.

Reducing equivalent shuttles in developing porcine myocardium: enhanced capacity in the newborn heart.

The metabolic demands of the newborn heart are met primarily by glucose and lactate. Mitochondria are impermeable to the NADH produced by these cytosolic reactions. The malate/aspartate and alpha-glycerophosphate (alpha-GP) shuttles provide two pathways to transport reducing equivalents into mitochondria. The goals of this study were to compare the capacity of these shuttles in newborn and adult cardiac mitochondria and to measure the maximal activity of the mitochondrial enzymes involved in these shuttles. Shuttle and enzyme capacities were measured in isolated mitochondria from the left and right ventricular free wall of 0-3-d-old and adult pig hearts. Malate/aspartate shuttle capacity was nearly three times greater in the newborn left ventricle compared with adult (newborn, 616 +/- 24; adult, 232 +/- 28 nmol/min/mg; mean +/- SEM; n = 8; p < 0.00001). The capacity of the malate/aspartate shuttle of the right ventricular free wall was greater than the left in the adult heart. Despite a decrease in malate/aspartate shuttle capacity, maximal activity of mitochondrial matrix enzymes involved in this pathway were increased in adult mitochondria. alpha-GP shuttle activity was absent in adult myocardium. Newborn left ventricular myocardium had significant alpha-GP shuttle activity (44 +/- 4 nmol/min/mg) due to enhanced flavin-linked mitochondrial alpha-GP dehydrogenase activity compared with adult. Interventricular differences in the alpha-GP shuttle capacity were not found in newborn or adult hearts. These findings suggest a mechanism for the substrate preference of neonatal myocardium.

Water-macromolecular proton magnetization transfer in infarcted myocardium: a method to enhance magnetic resonance image contrast.

Water proton nuclear magnetic resonance relaxation times and magnetization transfer (MT) parameters of rat hearts were studied 24 h or 4 weeks after ligation of the left coronary artery or sham operation. Compared with sham-operated controls, measured relaxation times (T1sat and T2) of both acute and chronic myocardial infarction increased. The MT effect significantly decreased in the infarcted myocardium. The changes in relaxation times and MT effect were significantly greater in chronic infarcts compared with acute infarcts. Improvements in calculated image contrast between normal and infarcted tissue were supported by images of ex vivo hearts with chronic infarction. Image contrast was increased at short echo times in the presence of macromolecular proton pool irradiation. Exploiting changes in tissue MT following myocardial infarction to enhance contrast between normal and infarcted tissue should allow improved identification and characterization of infarcted myocardium.

Measurement of kinetic perfusion parameters of gadoteridol in intact myocardium: effects of ischemia/reperfusion and coronary vasodilation.

Quantitation of myocardial perfusion is feasible using contrast enhanced magnetic resonance imaging. A method to quantitate myocardial blood flow is provided by the Kety model modified to account for a diffusable tracer such as gadoteridol. In the present study, perfusion parameters of the modified Kety model (partition coefficient and extraction efficiency) were determined for gadoteridol in intact myocardium using a constant flow, isolated, perfused heart model. Perfusion conditions included hearts with normal perfusion, hearts made globally ischemic for 20 min then perfused normally, and hearts whose coronary flow was more than doubled with 9 microM adenosine. T1 relaxation times were rapidly measured at 0.5 T following step increases in perfusate gadoteridol concentration and at steady state. Both the partition coefficient and extraction efficiency were found to be significantly increased in ischemic/reperfused hearts compared to normal. While flow rates in adenosine hearts were too high for accurate extraction efficiency determination using this technique, the partition coefficient was no different between adenosine and normally perfused hearts. The method described in this article allowed the kinetic parameters of the modified Kety model to be determined in intact heart using NMR relaxation time measurements as the basis of the calculation.

Effect of substrate on mitochondrial NADH, cytosolic redox state, and phosphorylated compounds in isolated hearts.

The effect of metabolic substrates on the relation among cytosolic redox state (NADHc/NAD+) mitochondrial NADH (NADHm), and [ATP]/([ADP] x [Pi]) was studied in isolated working rabbit hearts. Substrates were varied from 5.6 mM glucose alone to glucose in combination with 10 mM lactate and/or 10 mM pyruvate while afterload and preload were held constant. Changes in NADHm were determined from epicardial NADH fluorescence. The ratio of glycerol 3-phosphate (G-3-P) to dihydroxyacetone phosphate (DHAP), determined from tissue extracts, was used as an index of cytosolic redox. Myocardial 31P metabolites were measured using nuclear magnetic resonance spectroscopy. The addition of pyruvate to the perfusion medium caused increases in myocardial oxygen consumption (MVo2), NADHm fluorescence, phosphocreatine (PCr), and [ATP]/([ADP] x [Pi]) and a decrease in NADHc/NADc+ (decreased G-3-P/DHAP). Although the addition of lactate to the perfusion medium caused an increase in NADHm similar to pyruvate, MVo2 and PCr did not change significantly, [ATP]/([ADP] x [Pi]) increased less than with pyruvate, and there was an increase in NADHc/NADc+. The findings suggest that variations in the cytosolic redox state do not necessarily result in obligatory changes in either the mitochondrial redox state or in the [ATP]/([ADP] x [Pi]). This implies that under the conditions of this study an equilibrium is not maintained between [ATP]/([ADP] x [Pi]) and NADHc/NADc+. Furthermore, similar levels of NADHm can be associated with different values for [ATP]/([ADP] x [Pi]) and MVo2, depending on the substrates available to the heart.

Mitochondrial F1-ATPase activity of canine myocardium: effects of hypoxia and stimulation.

Recent studies have suggested that modifications in mitochondrial F1-adenosinetriphosphatase (ATPase) activity may play an important role in the regulation of myocardial oxidative phosphorylation. The goal of the present study was to develop and characterize an assay of F1-ATPase activity that could be performed repeatedly on an intact heart under various physiological states. With the use of submitochondrial particles prepared from biopsy samples of canine myocardium, we found reproducible F1-ATPase activity when normalized to the activity of the intramitochondrial enzyme citrate synthase. The oligomycin-sensitive component of the ATPase activity was found to be mainly F1-ATPase. F1-ATPase activity of normal myocardium increased by incubation in high salt-pH buffer, suggesting baseline inhibition. Five minutes after global ischemia, F1-ATPase activity decreased to 60% of baseline. Hypoxia for 10 min resulted in no significant change in F1-ATPase activity. With phenylephrine infusion, myocardial oxygen consumption more than doubled, whereas F1-ATPase activity increased by approximately 30%. Both returned to baseline levels after discontinuation of the drug. With the use of an assay developed to measure F1-ATPase activity of intact myocardium, changes of the enzyme activity were found during both ischemia and at increased work loads. These data suggest that alterations of F1-ATPase activity may contribute to the regulation of myocardial oxidative phosphorylation.

Reduction in myocardial high energy phosphate spin lattice relaxation times using manganese.

Paramagnetic contrast agents are being widely used in proton magnetic resonance imaging. The present study demonstrates the potential usefulness of paramagnetic contrast agents in 31P NMR spectroscopy. Using the Langendorff perfused rat heart, manganese chloride (Mn2+) at a concentration of 50 microM/L was added to the perfusate for 10 or 30 min. 31P NMR T1 relaxation times were subsequently measured at 121.5 MHz. After the 10 min exposure to Mn2+, 31P NMR T1 measurements of phosphocreatine and ATP were reduced by ca 25 and 50%, respectively, with no significant change in linewidths. A small additional decrease in T1 relaxation times after infusion of Mn2+ for 30 min was not significantly different from the values at 10 min. Potential uses of Mn2+ in 31P NMR spectroscopy include improving the S/N ratio of spectra and assessment of calcium ion channel activity.

Selenophosphate synthetase. Enzyme properties and catalytic reaction.

Selenophosphate synthetase, the product of the selD gene, produces the biologically active selenium donor compound, monoselenophosphate, from ATP and selenide. Isolation of the enzyme and characterization of some of its physical and catalytic properties are described. Magnesium ion and a monovalent cation, K+, NH4+, or Rb+, are required for catalytic activity. Polyphosphates and other common nucleotide triphosphates do not replace ATP as substrate. The stoichiometry of the catalytic reaction (Reaction 1) was established using 31P NMR, anaerobic molecular sieve chromatography, and radiochemical labeling procedures. ATP+selenide+H2O-->selenophosphate+Pi+AMP. In the absence of selenide, ATP is converted completely to AMP and orthophosphate upon prolonged incubation with elevated levels of enzyme. AMP is a competitive inhibitor of ATP, Ki = 170 microM, whereas selenophosphate and orthophosphate are weak inhibitors indicating a multistep reaction. Attempts to obtain direct evidence for a postulated enzyme-pyrophosphate intermediate using several experimental approaches are described. No exchange of [14C]AMP with ATP could be detected after the enzyme was freed of traces of contaminating adenylate kinase by chromatography on phenyl-Sepharose.

Myocardial energy metabolism and morphology in a canine model of sepsis.

The mechanism responsible for sepsis-induced myocardial depression is not known. To determine if sepsis-induced myocardial depression is caused by inadequate free energy available for work, we studied myocardial energy metabolism in a canine model of sepsis. Escherichia coli-infected (n = 18) or sterile (n = 16) fibrin clots were implanted intraperitoneally into beagles. Myocardial function and structure was assessed using radionuclide ventriculograms, echocardiograms, and light and electron microscopy. The adequacy of energy metabolism was evaluated by comparing catecholamine-induced work increases [myocardial O2 consumption (MVO2) and rate pressure product (RPP)] with a simultaneously obtained estimate of intracellular free energy [phosphocreatine-to-adenosine triphosphate ratio (PCr:ATP)] determined by 31P-magnetic resonance spectroscopy. When compared with control animals, septic animals had a decrease in left ventricular ejection fraction (EF, P < 0.0001) on day 1 and fractional shortening (FS, P < 0.0003) on day 2 after clot implantation. On day 2, neither septic nor control animals had statistically significant decreases in PCr:ATP, despite catecholamine-induced increases in MVO2 and RPP (mean maximal increases in septic animals 135 +/- 31 and 51 +/- 10%, respectively). Light and electron microscopic findings showed that hearts of septic animals, compared with control animals, had a greater degree of morphological abnormalities. Thus, in a canine model of sepsis with alterations in myocyte ultrastructure and documented myocardial depression (decreased EF and FS), intracellular free energy levels (PCr:ATP) were maintained despite catecholamine-induced increases in myocardial work (increased MVO2 and RPP), suggesting high-energy synthetic capabilities are not limiting cardiac function.

Monoselenophosphate: synthesis, characterization, and identity with the prokaryotic biological selenium donor, compound SePX.

A labile, selenium donor compound required for synthesis of selenium-dependent enzymes and seleno-tRNAs is formed from ATP and selenide by the SELD enzyme. This compound, tentatively identified as a selenophosphate [Veres, Z., Tsai, L., Scholz, T. D., Politino, M., Balaban, R. S., & Stadtman, T. C. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 2975-2979], is indistinguishable from chemically prepared monoselenophosphate by 31P NMR spectroscopy and ion pairing HPLC. Furthermore, addition of chemically prepared monoselenophosphate caused a dose-dependent decrease in the amount of 75Se incorporated into tRNAs from 75SePX generated in situ by SELD enzyme. A procedure is described for the chemical synthesis of monoselenophosphate in which the readily prepared (MeO)3PSe is converted in quantitative yield to (TMSO)3PSe followed by complete cleavage of the latter to monoselenophosphate in oxygen-free aqueous buffer. The chemical properties of chemically synthesized monoselenophosphate are described.