Elena Simplaceanu

31P NMR measurements of myocardial pH invivo

A 31P NMR magnetization transfer method for measuring myocardial pH in vivo is demonstrated in the lamb, dog and cat. The method involves measuring the difference in chemical shift between the resonances of phosphocreatine and inorganic phosphate in magnetization transfer difference spectra in which the gamma-phosphate resonance of ATP has been saturated. The method has been verified by measuring the chemical shift difference between the resonances of 2-deoxyglucose 6-phosphate and phosphocreatine following infusion of the animals with 2-deoxyglucose. The measured pH values are significantly lower than those obtained in previous studies on the heart in vivo.

ADMINISTRATION OF FRUCTOSE 1,6-DIPHOSPHATE DURING EARLY REPERFUSION SIGNIFICANTLY IMPROVES RECOVERY OF CONTRACTILE FUNCTION IN THE POSTISCHEMIC HEART☆☆☆★

OBJECTIVES Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.

Improved Protection of the Hypertrophied Left Ventricle by Histidine-Containing Cardioplegia

BACKGROUND Myocardial hypertrophy has been shown to lead to increased susceptibility to ischemia with accelerated loss of high-energy nucleotides, greater accumulation of H+ and lactate, and earlier onset of contracture. METHODS AND RESULTS To determine whether promoting anaerobic glycolysis during ischemia by buffering H+ results in improved preservation of the hypertrophied heart, we studied the effect of a histidine-containing solution (HBS) on recovery of contractile function and energetic state. Hypertrophied rabbit hearts (aortic banding at 10 days) were subjected to 40 minutes of 37 degrees C ischemia and reperfusion in an isolated Langendorff model. This group was compared with groups receiving St Thomas solution and high-potassium Krebs buffer solution (KCl). Although both phosphocreatine (PCr) and ATP were lower in hypertrophied hearts by end-ischemia compared with nonhypertrophied age-matched controls, there was significantly higher PCr, ATP, and intracellular pH in the HBS group compared with the St Thomas and KCl groups. Recovery of left ventricular developed pressure was best in the HBS group (91% of preischemic values) as was end-diastolic pressure after 30 minutes of reperfusion. Lactate production was also significantly greater in the HBS group, suggesting augmentation of anaerobic glycolysis. CONCLUSIONS We concluded that administration of histidine-containing cardioplegia promotes anaerobic glycolysis and improves recovery of high-energy phosphates and contractile function in hypertrophied myocardium.

Protein Kinase C activation in the heart: Effects on calcium and contractile proteins**

BACKGROUND Cardiac contractile function is dependent on the energetic state of the heart, intracellular calcium levels, and the interaction of the contractile proteins with both adenosine triphosphate and calcium. Protein kinase C (PKC) is a ubiquitous intracellular mediator that has been found in the heart and has been shown to phosphorylate proteins that regulate calcium homeostasis (calcium channels) and the contractile proteins themselves (troponin I and troponin T). METHODS To determine the role of PKC activation on cardiac contractile function, direct activation of PKC was achieved by the infusion of phorbol 12-myristate 13-acetate, an activating phorbol ester. The effects of PKC activation were evaluated in Langendorff-perfused rabbit hearts. Contractile function, high-energy phosphate content (phosphorous-31 nuclear magnetic resonance spectroscopy), oxygen consumption, and intracellular calcium levels (calcium fluorescent dye Rhod-2) were determined. RESULTS Activation of PKC in the heart by phorbol 12-myristate 13-acetate resulted in a significant decrease in both systolic and diastolic function while oxygen consumption and adenosine triphosphate production remained unchanged. Both baseline and peak intracellular calcium levels decreased, which may contribute to the impaired systolic function. CONCLUSIONS Activation of PKC in the heart leads to significant loss of contractile function without affecting energetics. The effect is most likely due to alteration in cytosolic calcium regulation and altered contractile sensitivity to calcium.

Changes in cerebral blood flow and distribution associated with acute increases in plasma sodium and osmolality of chronic hyponatremic rats.

The cause of the osmotic demyelination syndrome that follows too rapid correction of chronic hyponatremia (CHN) is unknown. Recently, we reported in CHN rats an association between blood-brain barrier (BBB) disruption occurring as early as 3 h into correction and subsequent demyelination. Given the changes in brain water and blood volume which occur during correction of CHN, we hypothesized that the same correction protocol that causes demyelination might alter cerebral blood flow (CBF) during correction, thereby possibly contributing to BBB disruption and demyelination. Ten CHN rats were given hypertonic sodium intraperitoneally and its effect on CBF was continuously monitored for 3 h by magnetic resonance flow imaging. Over the subsequent 3 h, plasma sodium rose from 110.8 to 127.6 mEq/liter (P < 0.001) but neither mean arterial blood pressure nor arterial CO(2) tension changed significantly. By 30 min, CBF increased by 50% in cortical and subcortical areas (P < 0.001) and remained elevated for the next 60 min. After 2 h, cortical flow was no longer elevated significantly and by 3 h it had returned to control values. Subcortical flow, however, significantly exceeded control values throughout the 3 h so that after 2 h the ratio of cortical to subcortical blood flow had fallen from 1.17 to 0.91 (P < 0.05). Although the mechanism by which increased plasma sodium and osmolality alters CBF is uncertain, the results suggest that changes in CBF may be part of a cascade of cerebrovascular disturbances including endothelial or parenchymal damage, mechanical events, metabolic disturbances, or cytokine release which eventually lead to BBB disruption and subsequent demyelination.

Response of normal and reperfused livers to glucagon stimulation : NMR detection of blood flow and high-energy phosphates

The effects of glucagon on blood flow and high-energy phosphates in control and in rat livers damaged by ischemia were studied using in vivo nuclear magnetic resonance (NMR) spectroscopy. Normal livers and livers which had been made ischemic for 20, 40, and 60 min followed by 60 min of reperfusion were studied. Ischemia led to a loss in adenosine triphosphate (ATP) within 30 min. Reperfusion after 20 min of ischemia led to complete recovery of ATP. 60 min of reperfusion after 40 or 60 min of ischemia led to only a 76% and 48% recovery of ATP, respectively. Glucagon, at doses up to 2.5 mg/kg body weight, caused no changes in the inorganic phosphate (P(i)) to ATP ratio in normal livers as measured by 31P-NMR spectroscopy. In livers which had been made ischemic for 20, 40, or 60 min, glucagon caused an increase in the P(i)/ATP ratio of 18%, 40%, and 40%, respectively. 19F-NMR detection of the washout of trifluoromethane from liver was used to measure blood flow. Glucagon-stimulated flow in the normal liver in a dose-dependent manner, with 2.5 mg glucagon/kg body weight leading to a 95% increase in flow. Ischemia for 20, 40, and 60 min followed by 60 min of reperfusion led to hepatic blood flows which were 63%, 68%, and 58% lower than control liver. In reperfused livers, blood flow after glucagon-stimulation was reduced to 56%, 43%, and 48% of control glucagon-stimulated flow after 20, 40, and 60 min of ischemia. These results indicate that ischemia followed by reperfusion leads to decreases in hepatic blood flow prior to alterations in ATP and the response of the liver to glucagon is altered in the reperfused liver.

Testing causal mechanisms of nonsyndromic craniosynostosis using path analysis of cranial contents in rabbits with uncorrected craniosynostosis.

Objective: Various causal mechanisms of familial nonsyndromic craniosynostosis have been presented. One hypothesis suggests that overproduction of bone at the suture is the primary origin of craniosynostosis, which affects brain and cranial growth secondarily through altered intracranial pressure (Primary Suture Fusion Model). Other hypotheses suggest that decreased cranial base growth or abnormal brain growth are the primary cause of craniosynostosis (Cranial Base, Brain Parenchyma Models, respectively). This study was designed to investigate which model best describes neurocranial changes associated with craniosynostosis in a rabbit model through multivariate path analysis. Design: Serial magnetic resonance imaging scans and intracranial pressure measurements were obtained at 10, 25, and 42 days of age from 18 rabbits: six controls, six with delayed-onset synostosis, and six with early-onset synostosis. Five variables were collected from each rabbit: calvarial thickness at the affected suture, cranial base length, brain volume, cerebrospinal fluid volume, and intracranial pressure. This data set was used to test causal pathway relationships generated by the proposed models. Goodness of fit was measured by experimental group for each model. Results: Primary Suture Fusion Model best explained the variables in both delayed-onset and early-onset synostotic rabbits (Goodness of fit = 93%, 97%, respectively). Cranial Base Model (Goodness of fit = 94%) best explained the data in control rabbits. Conclusion: Results suggest that the primary site of craniosynostosis in craniosynostotic rabbits is most likely the synostosed suture. Other cranial vault anomalies are most likely secondary compensatory changes. Results of the present study may provide insight regarding the causal pathway of craniosynostosis.

CYCLOSPORINE AND LIVER REGENERATION STUDIED BY IN VIVO 31P NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

The changes in fructose-1-phosphate (F-1-P), intracellular pH, and ATP content of the liver after a fructose challenge were investigated noninvasivelyin vivo using phosphorus-31 nuclear magnetic resonance spectroscopy of dog liver four days after a portacaval shunt (PCS) with or without portal venous infusion of cyclosporin (CsA). The F-1-P metabolism was slower in PCS dogs (N=2) as compared to either the normal (N=2) or PCS+CsA-treated dogs (N=3) (P<0.05). The intracellular pH temporarily decreased from 7.3±0.05 to 7.0±0.05 during the fructose challenge. The regenerative indexes were increased in the PCS+CsA group (P<0.01). These data obtainedin vivo using31P-NMR spectroscopy in the liver following a portacaval shunt, suggest that : (1) the energy status of the liver and the metabolic response to fructose are reduced in PCS compared to normal animals and (2) CsA treatment enhances the regenerative response of the liver and prevents the reduction in hepatic function associated with portacaval shunting.