Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts.

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.

Regression of left ventricular hypertrophy in human hypertension with irbesartan.

BACKGROUND: The Swedish irbesartan left ventricular hypertrophy investigation versus atenolol (SILVHIA). OBJECTIVE: Angiotensin II induces myocardial hypertrophy. We hypothesized that blockade of angiotensin II subtype 1 (AT1) receptors by the AT1-receptor antagonist irbesartan would reduce left ventricular mass (as measured by echocardiography) more than conventional treatment with a beta blocker. DESIGN AND METHODS: This double-blind study randomized 115 hypertensive men and women with left ventricular hypertrophy to receive either irbesartan 150 mg q.d. or atenolol 50 mg q.d. for 48 weeks. If diastolic blood pressure remained above 90 mmHg, doses were doubled, and additional medications (hydrochlorothiazide and felodipine) were prescribed as needed. Echocardiography was performed at weeks 0, 12, 24 and 48. RESULTS: Baseline mean blood pressure was 162/ 104 mmHg, and mean left ventricular mass index was 157 g/m2 for men and 133 g/m2 for women. Systolic and diastolic blood pressure reductions were similar in both treatment groups. Both irbesartan (P < 0.001) and atenolol (P< 0.001) progressively reduced left ventricular mass index, e.g. by 26 and 14 g/m2 (16 and 9%), respectively, at week 48, with a greater reduction in the irbesartan group (P = 0.024). The proportion of patients who attained a normalized left ventricular mass (i.e. < or = 131 g/m2 for men and < or = 100 g/m2 for women) tended to be greater with irbesartan (47 versus 32%, P = 0.108). CONCLUSIONS: Left ventricular mass was reduced more in the irbesartan group than in the atenolol group. These results suggest that blocking the action of angiotensin II at AT1-receptors may be an important mechanism, beyond that of lowering blood pressure, in the regulation of left ventricular mass and geometry in patients with hypertension.

Opening of potassium channels protects mitochondrial function from calcium overload.

Ischemic preconditioning (IPC) protects myocardium from ischemia reperfusion injury by activating mitochondrial K(ATP) channels. However, the mechanism underlying the protective effect of K(ATP) channel activation has not been elucidated. It has been suggested that activation of mitochondrial K(ATP) channels may prevent mitochondrial dysfunction associated with Ca(2+) overload during reperfusion. The purpose of this experiment was to study, in an isolated mitochondrial preparation, the effects of mitochondrial K(ATP) channel opening on mitochondrial function and to determine whether it protects mitochondria form Ca(2+) overload. Mitochondria (mito) were isolated from rat hearts by differential centrifugation (n = 5/group). Mito respiratory function was measured by polarography without (CONTROL) or with a potassium channel opener (PINACIDIL, 100 microM). Different Ca(2+) concentrations (0 to 5 x 10(-7) M) were used to simulate the effect of Ca(2+) overload; state 2, mito oxygen consumption with substrate only; state 3, oxygen consumption stimulated by ADP; state 4, oxygen consumption after cessation of ADP phosphorylation; respiratory control index (RCI: ratio of state 3 to state 4); rate of oxidative phosphorylation (ADP/Deltat); and ADP:O ratio were measured. PINACIDIL increased state 2 respiration and decreased RCI compared to CONTROL. Low Ca(2+) concentrations stimulated state 2 and state 4 respiration and decreased RCI and ADP:O ratios. High Ca(2+) concentrations increased state 2 and state 4 respiration and further decreased RCI, state 3, and ADP/Deltat. PINACIDIL improved state 3, ADP/Deltat, and RCI at high Ca(2+) concentrations compared to CONTROL. Pinacidil depolarized inner mitochondrial membrane, as evidenced by decreased RCI and increased state 2 at baseline. Depolarization may decrease Ca(2+) influx into mito, protecting mito from Ca(2+) overload, as evidenced by improved state 3 and RCI at high Ca(2+) concentrations. The myocardial protective effects resulting from activating K(ATP) channels either pharmacologically or by IPC may be the result of protecting mito from Ca(2+) overload.

Metabolic control of sodium transport in streptozotocin-induced diabetic rat hearts.

Diabetic and control cardiomyocytes encapsulated in agarose beads and superfused with modified medium 199 were studied with 23Na- and 31P-NMR. Baseline intracellular Na+ was higher in diabetic (0.076 +/- 0.01 micromoles/mg protein) than in control (0.04 +/- 0.01 micromoles/mg protein) (p < 0.05). Baseline betaATP and phosphocreatine (PCr) (peak area divided by the peak area of the standard, methylene diphosphonate) were lower in diabetic than in control, e.g., betaATP control, 0.70 +/- 0.07; betaATP diabetic, 0. 49 +/- 0.04 (p < 0.027); PCr control, 1.20 +/- 0.13; PCr diabetic, 0. 83 +/- 0.11 (p < 0.03). This suggests that diabetic cardiomyocytes have depressed bioenergetic function, which may contribute to abnormal Na,K-ATPase function, and thus, an increase in intracellular Na+. In the experiments presented herein, three interventions (2-deoxyglucose, dinitrophenol, or ouabain infusions) were used to determine whether, and the extent to which, energy deficits or abnormalities in Na,K-ATPase function contribute to the increase in intracellular Na+. In diabetic cardiomyocytes, 2-deoxyglucose and ouabain had minimal effect on intracellular Na+, suggesting baseline depression of, or resetting of both glycolytic and Na,K-ATPase function, whereas in control both agents caused significant increases in intracellular Na+after 63 min exposure: 2-deoxyglucose control, 32.9 +/- 8.1%; 2-deoxyglucose diabetic, -4.6 +/- 6% (p < 0.05); ouabain control, 50.5 +/- 8.8%; ouabain diabetic, 21.2 +/- 9.2% (p < 0.05). In both animal models, dinitrophenol was associated with large increases in intracellular Na+: control, 119.0 +/- 26.9%; diabetic, 138.2 +/- 12.6%. Except for the dinitrophenol intervention, where betaATP and PCr decreased to levels below 31P-NMR detection, the energetic metabolites were not lowered to levels that would compromise sarcolemmal function (Na,K-ATPase) in either control or diabetic cardiomyocytes. In conclusion, in diabetic cardiomyocytes, even though abnormal glycolytic and Na, K-ATPase function was associated with increases in intracellular Na+, these increases were not directly related to global energy deficit.

Mitochondrial oxidative phosphorylation in heart from stressed cardiomyopathic hamsters.

Stress alone is generally not sufficient to produce serious disease, but stress imposed upon pre-existing disease can contribute to disease progression. To explore this phenomenon, cold-immobilization stress was imposed on young 12.5 month, necrotic phase with small vessel coronary spasm) and older (5 month, quiescent phase, between necrosis and heart failure) cardiomyopathic hamsters. Our hypothesis was that changes in mitochondrial energy processes are involved in stress induced pathology. Polarographic and high performance liquid chromatography (HPLC) techniques were used to measure mitochondrial respiration and oxidative phosphorylation and concentrations of phosphocreatine and adenylates, respectively, in hearts from young and old cardiomyopathic hamsters (stressed and unstressed). No significant differences were found between the young (2.5 month) and old (5 month) age groups in unstressed and stressed healthy hamsters and between young (2.5 month) and old (5 month) unstressed cardiomyopathic hamsters with respect to different parameters of mitochondrial oxidative phosphorylation and with respect to concentration of bioenergetic metabolites, except that ADP concentration was higher in older cardiomyopathic hamsters. Application of stress uncovered differences between young and old cardiomyopathic hamsters: respiration control index was lower and State 4 respiration was higher in young compared to old cardiomyopathic hamsters; whereas the total concentration of ATP was decreased to the same level in both cardiomyopathic groups when compared to control. Mitochondrial oxidative phosphorylation in young cardiomyopathic hamsters was more sensitive to Ca2+, as evidenced by partial uncoupling of respiration and oxidative phosphorylation, than in older cardiomyopathic hamsters and controls. In conclusion, young cardiomyopathic hamsters, i.e. in the necrotic phase of disease, were more susceptible to stress induced changes in mitochondrial oxidative phosphorylation than older cardiomyopathic hamsters and controls.

Irbesartan reduces QT dispersion in hypertensive individuals.

Angiotensin type 1 receptor antagonists have direct effects on the autonomic nervous system and myocardium. Because of this, we hypothesized that irbesartan would reduce QT dispersion to a greater degree than amlodipine, a highly selective vasodilator. To test this, we gathered electrocardiographic (ECG) data from a multinational, multicenter, randomized, double-blind parallel group study that compared the antihypertensive efficacy of irbesartan and amlodipine in elderly subjects with mild to moderate hypertension. Subjects were treated for 6 months with either drug. Hydrochlorothiazide and atenolol were added after 12 weeks if blood pressure (BP) remained uncontrolled. ECGs were obtained before randomization and at 6 months. A total of 188 subjects (118 with baseline ECGs) were randomized. We analyzed 104 subjects who had complete ECGs at baseline and after 6 months of treatment. Baseline characteristics between treatments were similar, apart from a slight imbalance in diastolic BP (irbesartan [n=53] versus amlodipine [n=51], 99.2 [SD 3. 6] versus 100.8 [3.8] mm Hg; P=0.03). There were no significant differences in BP normalization (diastolic BP <90 mm Hg) between treatments at 6 months (irbesartan versus amlodipine, 80% versus 88%; P=0.378). We found a significant reduction in QT indexes in the irbesartan group (QTc dispersion mean, -11.4 [34.5] milliseconds, P=0.02; QTc max, -12.8 [35.5] milliseconds, P=0.01), and QTc dispersion did not correlate with the change in BP. The reduction in QT indexes with amlodipine (QTc dispersion, -9.7 [35.4] milliseconds, P=0.06; QTc max, -8.6 [33.2] milliseconds, P=0.07) did not quite reach statistical significance, but there was a correlation between the change in QT indexes and changes in systolic BP. In conclusion, irbesartan improved QT dispersion, and this effect may be important in preventing sudden cardiac death in at-risk hypertensive subjects.

Adenosine improves cardiomyocyte respiratory efficiency.

The role of adenosine on the regulation of mitochondrial function has been studied. In order to evaluate this the following experiments were done in isolated rat cardiomyocites and mitochondria using polarographic techniques. Cardiomyocyte oxygen consumption (MVO2) and mitochondrial respiratory function (State 3 and State 4, respiratory control index, and ADP/O ratio) were evaluated after exposure to adenosine. Cardiomyocyte MVO2 was significantly lower in cells previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) than in cells not exposed to adenosine (control). Addition of dipyridamole (10 microM) or 8-(p-Sulfophenyl) theophylline (50 microM) to cardiomyocytes before adenosine incubation prevented the adenosine-induced changes in MVO2. Mitochondria obtained from isolated perfused beating heart previously perfused with adenosine (10 microM, 30 min heart perfusion) also resulted in significant increases in ADP/O and respiratory control index compared to matching control. Mitochondria isolated from cardiomyocytes previously exposed to adenosine (10 microM, 15 min or 30 min cell incubation) resulted in a significant increase in mitochondrial ADP/O ratio compared to control. Adenosine-induced decrease in cardiomyocyte MVO2 may be related to an increase in efficiency of mitochondrial oxidative phosphorylation, and more economical use of oxygen, which is necessary for survival under ischemic stress.

Metabolic abnormalities and differential responses to stress associated with hamster cardiomyopathy.

Metabolic differences between cardiomyopathic hamsters (CMHs), as they progress through various physiologic phases before reaching end-stage heart failure (HF), and healthy hamsters (HHs) are often difficult to demonstrate. We suggest that metabolic differences, magnified by application of chronic stress (S: cold immobilization 2 hr/day for 5 days) followed by acute stress (AS: 55 min global ischemia /30 min reperfusion), can be used to characterize different stages in this cardiomyopathic process. High performance liquid chromatography (HPLC) and 31P NMR methods were used to monitor the effects of acute stress applied to nonstressed (NS) and previously stressed CMHs (NS-2.5-month NS-5-month; S-2.5-month, S-5-month) and HHs (NS-HH, S-HH). Cardiac tissue extracts from nonstressed and stressed hamsters were analyzed for ATP and PCr at baseline and after completion of ischemia/reperfusion (AS) using HPLC. In nonstressed hamsters, ATP and PCr were 12% lower in CMHs (both NS-2.5- and NS-5-month) than in NS-HHs. After exposure to stress, ATP was 26% lower in CMHs (S-2.5- and S-5-month) compared to S-HHs, whereas there were minimal differences in PCr between the groups. 31P NMR monitoring of metabolism in the perfused beating heart during application of acute stress produced similar changes (%) in ATP and PCr in all groups (NS and S), whereas Pi increase was less in NS-5-month (118%) compared to NS-2.5-month (179%) and NS-HHs (306.8%), P < 0.05; and in S-5-month (148%) compared to S-2.5-month (216%) and S-HHs (222%). The changes in myocardial pH were inversely related to changes in Pi: NS-5-month (-13.5%); NS-2.5-month (-9.7%); NS-HH (-17.7%). pH changes in stressed cardiomyopathic hamsters were similar to those of S-HHs. The postischemic recovery of ATP and Pi return closer to baseline values in cardiomyopathic hamsters (both NS and S) compared to healthy hamsters. The data suggest that cardiomyopathic hamsters have baseline metabolic abnormalities, and their responses to chronic cold immobilization stress, acute ischemia, and chronic cold immobilization stress plus acute ischemia are different from those in HHs. These responses may help to characterize specific stages of disease.

Improvement of myocardial mitochondrial function after hemodynamic support with left ventricular assist devices in patients with heart failure.

OBJECTIVES: Mitochondrial abnormalities have been described in cardiac tissue of patients with heart failure. These changes may result from chronic hypoxia. Our goal was to determine whether mitochondrial functional capacity can be improved in patients with heart failure by means of long-term left ventricular assist device therapy, which improves myocardial oxygen supply by decreasing myocardial work. METHODS: Mitochondria were isolated from myocardial tissue obtained from 13 patients with heart failure without a left ventricular assist device (HF group) and seven patients with heart failure treated with a left ventricular assist device (LVAD-HF group). Mitochondrial respiratory rates (State 2, State 3, and State 4) were measured by means of polarographic techniques with reduced nicotinamide adenine dinucleotide-dependent (pyruvate/malate, alpha-ketoglutarate, glutamate) and -independent (succinate) substrates. The respiratory control index of Chance (State 3/State 4) and Lardy (State 3/State 2) and phosphorus to oxygen ratios were determined. RESULTS: The respiratory control index of Chance was higher in LVAD-HF than in HF when using NADH-dependent substrates pyruvate/malate and alpha-ketoglutarate (pyruvate/malate HF: 4.9 +/- 1.0; LVAD-HF: 6.5 +/- 1.5; alpha-ketoglutarate HF: 8.5 +/- 2.4; LVAD-HF: 11.8 +/- 2.9; both p = 0.04). Similarly, the respiratory control index of Lardy was greater in the LVAD-HF than the HF group when alpha-ketoglutarate and glutamate were used as substrates (alpha-ketoglutarate HF: 7.8 +/- 1.7; LVAD-HF: 9.9 +/- 1.5; glutamate HF: 7.6 +/- 2.2; LVAD-HF: 10.7 +/- 2.1; both p = 0.04). The phosphorus to oxygen ratio was comparable for both groups using all substrates. No change in mitochondrial respiration was observed after left ventricular assist device therapy with the NADH-independent substrate, succinate. CONCLUSION: Cardiomyocyte mitochondrial function is improved by long-term therapy with a left ventricular assist device. This improvement suggests that cardiomyocyte metabolic dysfunction in heart failure may be reversed with left ventricular assist device support.

A randomised, double-blind comparison of the angiotensin II receptor antagonist, irbesartan, with the full dose range of enalapril for the treatment of mild-to-moderate hypertension.

OBJECTIVE: To compare the anti-hypertensive efficacy, safety, and tolerability of irbesartan with those of the full dose range of enalapril in patients with mild-to-moderate hypertension. DESIGN AND METHODS: A total of 200 patients were randomised to irbesartan 75 mg or enalapril 10 mg (once daily). Doses were doubled at Weeks 4 and/or 8 if seated diastolic blood pressure (DBP) was > or = 90 mm Hg. Trough blood pressure was measured after completion of a 4- to 5-week placebo lead-in period and again after 2, 4, 8, and 12 weeks of treatment. MAIN OUTCOME MEASURES: Efficacy was evaluated by determining the change from baseline in trough seated blood pressure and the proportion of patients normalised (seated DBP <90 mm Hg) at Week 12. Safety and tolerability were assessed by adverse events reported by physicians, by patients in response to a specific-symptoms questionnaire, by open-ended questioning of patients by physicians, and by clinical laboratory evaluations. RESULTS: Both treatments significantly lowered blood pressure with no significant difference in efficacy between treatment groups. At Week 12, the percentage of patients titrated to either enalapril 40 mg or irbesartan 300 mg was 24% and 28%, respectively. The frequency of overall adverse events was similar in both groups. The incidence of cough in the enalapril and irbesartan groups was 17% and 10%, respectively. In contrast to other AII receptor antagonists, there was no change in uric acid concentrations with irbesartan. CONCLUSIONS: Irbesartan was as effective as the full dose range of enalapril. Irbesartan also demonstrated an excellent tolerability profile.

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with 31P-NMR spectroscopy.

An NMR method to study on-line mitochondrial function was developed. Mitochondria were maintained in a stable physiologic state in agarose beads that were continuously superfused with oxygenated buffer at 28 degrees C. Oxidative function of both heart and liver mitochondria was evaluated with 31P NMR at 9.4 T using pyruvate plus malate as substrate. This method allows clear resolution of adenosine triphosphate-gamma (ATPgamma) and adenosine diphosphate-beta (ADPbeta) phosphate signals, whereas alpha signals of ATP and ADP overlap. ATP production by mitochondria was documented to be very sensitive to different interventions (hypoxia, ischemia, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP)) and depended on the ADP concentration in superfusion medium. These data demonstrate that the new application of NMR to study mitochondrial function can discriminate, on-line, between several physiologic and biochemical processes in intact physiologically stable mitochondria.

Temporal relationships between eating behavior and liver adenine nucleotides in rats treated with 2,5-AM.

Administration of the fructose analog 2,5-anhydro-D-mannitol (2,5-AM) elicits eating behavior in rats by its action in the liver. To evaluate whether the decrease in liver ATP levels produced by injection of 2,5-AM plays a role in the eating response, we examined the relationship between changes in eating behavior and liver adenine nucleotide levels over time in rats given 2,5-AM. Liver ATP concentrations decreased within 15 min after injection of 2,5-AM (300 mg/kg ip), remained low for up to 90 min postinjection, and returned to control (saline injection) levels by 4 h after treatment. Rats fed ad libitum initiated eating between 15 and 45 min after 2,5-AM treatment, after liver ATP levels had declined. Rats given food 1 h after 2,5-AM treatment increased food intake, but if access to food was delayed for 4 h after 2,5-AM injection the eating response was attenuated or absent. Whereas liver AMP and ADP levels were also altered by injection of 2,5-AM, changes in food intake did not consistently track changes in these nucleotides. The results support the hypothesis that the eating response to 2,5-AM is triggered by a decrease in liver ATP level.

Comparison of the angiotensin II receptor antagonist irbesartan with atenolol for treatment of hypertension.

In this multicenter, double-blind study, the antihypertensive efficacy and safety of irbesartan were compared with those of atenolol in patients with mild-to-moderate hypertension. Following a 4- to 5-week placebo lead-in period, 231 patients with seated diastolic blood pressure (SeDBP) 95-110 mmHg were randomized to irbesartan 75 mg or atenolol 50 mg once daily for 24 weeks. Doses were doubled at Week 6 for SeDBP > or = 90 mmHg. At Week 12, or anytime thereafter, doses were doubled for SeDBP > or = 90 mmHg if not done at Week 6, and hydrochlorothiazide and then nifedipine were added. Efficacy was determined by change from baseline in blood pressure and by therapeutic response rates. Safety was assessed by monitoring adverse events (AEs). Both treatments significantly lowered blood pressure from baseline. There were no significant differences between treatment groups with respect to blood pressure changes or therapeutic response. Atenolol significantly reduced seated heart rate compared with irbesartan at Week 12. The incidences of serious AEs and discontinuations due to AEs were approximately twice as high in the atenolol group compared with the irbesartan group. Thus, in comparison to atenolol, irbesartan < or = 150 mg provided at least equivalent blood pressure control while demonstrating an excellent safety and tolerability profile.

The effects of irbesartan added to hydrochlorothiazide for the treatment of hypertension in patients non-responsive to hydrochlorothiazide alone.

AIM: To evaluate the antihypertensive efficacy and safety of adding irbesartan to hydrochloride (HCTZ) in patients not adequately controlled by HCTZ alone. PATIENTS AND METHODS: In this multicenter study, after a single-blind, placebo lead-in period, hypertensive patients received single-blind HCTZ 25 mg once daily. After 4 weeks, 238 patients with seated diastolic blood pressure of 93-110 mmHg continued on HCTZ 25 mg once daily and were randomized to double-blind irbesartan 75 mg once daily or matching placebo for 12 weeks. At week 6, the dosage of irbesartan or placebo was doubled for seated diastolic blood pressure > or = 90 mmHg. RESULTS: At weeks 2, 6, and 12, irbesartan/HCTZ resulted in significantly greater (P<0.01) reductions from baseline in trough seated diastolic and systolic blood pressure compared with placebo/HCTZ. At week 12, the mean reductions in trough seated diastolic and systolic blood pressure were 7.2 mmHg (95%, C.I., 5.1-9.3 mmHg) and 11.1 mmHg (95% C.I., 7.9-14.3 mmHg) greater, respectively, with irbesartan/HCTZ compared with placebo/HCTZ. At week 12, significantly (P < 0.01) more patients were normalized (trough seated diastolic blood pressure < 90 mmHg) with irbesartan/HCTZ (67%) compared with placebo/HCTZ (29%). The frequency of adverse events, serious adverse events, and discontinuations attributed to adverse events was similar in both groups, and there were no clinically relevant changes in serum creatinine, potassium, or any other laboratory parameter. CONCLUSION: Irbesartan was effective and well tolerated when added to a background of HCTZ 25 mg in patients whose blood pressure was not adequately controlled by HCTZ alone.

Multiparameter monitoring and analysis of in vivo ischemic and hypoxic heart.

We describe a unique in vivo technique which addresses the multifactorial function of the heart, i.e., simultaneous measurement of myocardial ion transport (two mini-electrode systems to measure K+e and Ca2+e), energy metabolism (NADH fluorescence to measure NADH redox state), and coronary flow (laser-Doppler perfusion) using a multiprobe assembly (MPA) which contains transducers for all measurements. The MPA (which is 6 mm in diameter) was applied to the external surface of the heart in an open chest dog model. To test MPA function, myocardial ischemia was produced by application of a balloon occluder to the left anterior descending coronary (LAD) artery, and hypoxia was produced by changing the inspired O2-N2 ratio until the PaO2 was 20-30 torr. The MPA simultaneously monitored changes in ion flux, heart metabolism, and tissue perfusion during pathophysiological intervention.

Metabolic regulation of in vivo myocardial contractile function: multiparameter analysis.

To gain insight into the mechanisms of myocardial regulation as it relates to the interaction of mechanical and metabolic function and perfusion, intact animal models were instrumented for routine physiological measurements of mechanical function and for measurements of metabolism (31P NMR, NADH fluorescence (redox state)) and perfusion (2H NMR and Laser doppler techniques). These techniques were applied to canine and cat models of volume and/or pressure loading, hypoxia, ischemia and cardiomyopathic states. Data generated using these techniques indicate that myocardial bioenergetic function is quite stable under most loading conditions as long as the heart is not ischemic. In addition, these data indicate that there is no universal regulator and that different biochemical regulators appear to mediate stable function under different physiological and pathophysiological conditions: for example; during hypoxia, NADH redox state appears to play a regulatory role; and in pressure loading, ADP, phosphorylation potential and free energy of ATP hydrolysis as well as NADH redox state appear to be regulatory.

Intracellular sodium in cardiomyocytes using 23Na nuclear magnetic resonance.

Intracellular sodium content in superfused isolated rat cardiomyocytes was measured using 23Na nuclear magnetic resonance. The shift reagent dysprosium tripolyphosphate was added to the buffer to distinguish between NMR signals from the intracellular region and the extracellular buffer. The NMR visibility of the intracellular sodium signal was experimentally determined by measuring the changes induced in the sodium NMR signals by application of ischemia as an intervention. Intracellular volume was accounted for by determining the change in the sodium signal upon adding cells (in beads) to the buffer solution at the beginning of each experiment and by killing the cells (in beads) with Triton X-100 at the end of each experiment. The visibility of intracellular sodium (relative to extracellular) was 0.47 +/- 0.12 (mean +/- S.D., n = 12). The average intracellular sodium concentration using this visibility is 29 +/- 4.5 mM (n = 12). This value is much higher than results obtained by some investigators using NMR techniques and by others using different standard methods, with the exception of those methods which evaluate the total intracellular sodium (atomic absorption spectroscopy and X-ray microanalysis). We conclude that total Nai is higher than generally reported, using other accepted techniques such as ion-specific electrodes, and that 23Na-NMR analysis can be used to accurately determine Nai in intact cells.