Human red blood cell aging at 5,050-m altitude: a role during adaptation to hypoxia.

To test the hypothesis that the human red blood cell aging process participates actively in the adaptation to hypoxia, we studied some physical and biochemical hematologic variables in 10 volunteers at sea level (SL) and after 1 (1WK) or 5 wk (5WK) of exposure to 5,050-m altitude. The 2,3-diphosphoglycerate-to-hemoglobin ratio (2,3-DPG/Hb) was 0.88 +/- 0.03 (mol/mol) at SL and increased to 1.08 +/- 0.03 (P = 0.002) and 1.28 +/- 0.05 (P < 0.0001) at 1WK and 5WK, respectively. The average red blood cell density (D50), which is inversely proportional to the fraction of young red blood cells and is therefore an index of the red blood cell aging process, was 1.1053 +/- 0.0007 g/ml at SL and decreased to 1.1046 +/- 0.0008 g/ml (NS) and 1.1018 +/- 0.0008 g/ml (P < 0.001) at 1WK and 5WK, respectively. D50 was correlated with 2,3-DPG/Hb at SL (P = 0.004), only weakly at 5WK (P = 0.1), but not at all at 1WK. The arterial O2 saturation was correlated with the change of 2,3-DPG/Hb in 1WK (P = 0.02) and that of D50 in 5WK (P = 0.04). It is concluded that short-term (1WK) increase of 2,3-DPG/Hb is not associated with the erythropoietic response but is presumably due to respiratory alkalosis. By contrast, after prolonged hypoxia (5WK), erythropoiesis may provide an efficient way for increasing blood 2,3-DPG through an augmented proportion of young red blood cells.

Regulation of bioenergetics in O2-limited isolated rat hearts.

Assessing the role of O2 supply in the regulation of cardiac function in O2-limited hearts is crucial to understanding myocardial ischemic preconditioning and adaptation to hypoxia. We exposed isolated Langendorff-perfused rat hearts to either ischemia (low coronary flow) or hypoxemia (low PO2 in the perfusing medium) with matched O2 supply (10% of baseline). Myocardial contractile work and ATP turnover were greater in hypoxemic than in ischemic hearts (P < 0.05; n = 12). Thus, the energy demand was higher during hypoxemia than during ischemia, suggesting that ischemic hearts are more downregulated than hypoxemic hearts. Venous PO2 was 12 +/- 2 and 120 +/- 15 Torr (P < 0.0001) for ischemic and hypoxemic hearts, respectively, but O2 uptake was the same. Lactate release was higher during hypoxemia than during ischemia (9.7 +/- 0.9 vs. 1.4 +/- 0.2 mumol/min, respectively; P < 0.0001). Electrical stimulation (300 min-1; to increase energy demand) increased performance in ischemic (P < 0.005) but not in hypoxemic hearts without changes in venous PO2 or O2 uptake. However, venous lactate concentration and lactate release increased in ischemic (P < 0.002) but not in hypoxemic hearts, suggesting that anaerobic glycolysis provides the energy necessary to meet the increased energy demand in ischemic hearts only. We conclude that high intracellular lactate or H+ concentration during ischemia plays a major role as a downregulating factor. Downregulation disappears in hypoxemic hearts secondary to enhanced washout of lactate or H+.

Effects of energy demand in ischemic and in hypoxemic isolated rat hearts.

Aim of this study was to assess the role of O2, lactate and energy demand in the regulation of myocardial work during severe dysoxia. For this purpose, we measured function and metabolism in isolated Langendorff-perfused rat hearts exposed to either ischemia or hypoxemia (matched for the O2 supply, 10% of baseline) with/out electrical stimulation. When hearts could adjust their HR, hypoxemia demanded more energy than ischemia (p < 0.05) despite same O2 supply. Venous PO2 was 12 +/- 2 or 139 +/- 20 mmHg (p < 0.0001), respectively, but VO2 was the same. After 10 min at HR = 300 min-1, myocardial performance increased in ischemic but not in hypoxemic hearts. PvO2 and VO2 were not affected by pacing. In contrast, both venous [lactate] and lactate production rate increased, but in ischemic hearts only. We conclude that ischemic hearts were downregulated while hypoxemic hearts were not. Likely, depressed washout of lactate during ischemia could offset the effects of O2 in severely dysoxic hearts. Anaerobic glycolysis provided the energy necessary to meet increased energy demand in ischemic hearts, but could not exploit this action in hypoxemic hearts probably because in these hearts it was already working near maximum.

[Thrombolysis & arrhythmias]. / Trombolisi e aritmie.

In this study, we assessed one particular aspect of the arrhythmogenic phenomena that occur during reperfusion secondary to thrombolysis, that is the therein involved metabolic mechanisms. The employed experimental model (isolated Langendorff-perfused rat heart) allowed us to distinguish which factor involved during ischemia, low coronary flow or low oxygen tension, is primarily involved during arrhythmogenesis. This was made possible by comparing two settings characterized by the same oxygen supply, but with different coronary flows and PO2 values, i.e., ischemia and hypoxemia. As expected, the contractile dysfunction was higher during reoxygenation at the end of hypoxemia than during reperfusion at the end of ischemia (p < 0.05). However, the incidence of arrhythmias was similar in both cases. Therefore, whereas the contractile dysfunction appears to be more sensitive to coronary flow, the incidence of arrhythmias appears to be more sensitive to the total oxygen supply to the heart. This implies that the mechanisms underlying the development of contractile dysfunction and arrhythmogenesis follow different paths.

Biochemical consequences of electrical pacing in ischemic-reperfused isolated rat hearts.

It is still unclear if performance recovery in postischemic hearts is related to their tissue level of high-energy phosphates before reflow. To test the existence of this link, we monitored performance, metabolism and histological damage in isolated, crystalloid-perfused rat hearts during 20 min of low-flow ischemia (90% coronary flow reduction) and reflow. To prevent interference from different ischemia times and perfusing media compositions, the ischemic ATP level was varied by changing energy demand (electrical pacing at 330 min(-1)). Under full coronary flow conditions, work output, as well as ATP and phosphocreatine contents were the same in control, spontaneously contracting (n = 23) and paced (n = 21) hearts. During low-flow ischemia, the higher work output (p < 0.0001) in paced hearts decreased their tissue content of ATP, phosphocreatine and total adenylates and purines (p < 0.05), as opposed to maintained values in control hearts. During reflow, the recovery of mechanical performance and O2 uptake was 94 +/- 5% and 110 +/- 9% (p = NS vs. baseline) in controls, vs. 71 +/- 5% and 74 +/- 6% in paced hearts (p < 0.004 vs. baseline). The levels of ATP and total adenylates and purines remained constant in control, but were markedly depressed (p < 0.05 vs. baseline) in paced hearts. Phosphocreatine+creatine was the same in both groups. These data, together with the observed lack of creatine kinase leakage and of structural damage, indicate that myocardial recovery during reflow reflects the tissue level of ATP, phosphocreatine and total adenylates and purines during ischemia, regardless of physical cell damage.

Atenolol depresses post-ischaemic recovery in the isolated rat heart.

Metabolic events during ischaemia are probably important in determining post-ischaemic myocardial recovery. The aim of this study was to assess the effects of the beta-blocker atenolol and the high energy demand in an ischaemia-reperfusion model free of neurohormonal and vascular factors. We exposed Langendorff-perfused isolated rat hearts to low-flow ischaemia (30 min) and reflow (20 min). Three groups of hearts were used: control hearts (n =11), hearts that were perfused with 2.5 micrograms l-1atenolol (n =9), and hearts electrically paced during ischaemia to distinguish the effect of heart rate from that of the drug (n =9). The hearts were freeze-clamped at the end of reflow to determine high-energy phosphates and their metabolites. During ischaemia, the pressure-rate product was 2.3+/-0.2, 5.2+/-1.1, and 3.3+/-0.3 mmHg 10(3)min in the control, atenolol and paced hearts, respectively. In addition, the ATP turnover rate, calculated from venous (lactate), oxygen uptake and flow, was higher in atenolol (11.2+/-1.7 micromol min-1) and paced (8.1+/-0.8 micromol min-1) hearts than in control (6.2+/-0.8 micromol min-1). At the end of reflow, the pressurexrate product recovered 75.1+/-6.4% of baseline in control vs 54.1+/-9.1 and 48.8+/-4.4% in atenolol and paced hearts (P<0.05). In addition, the tissue content of ATP was higher in the control hearts (15.8+/-1. 0 micromol g(dw)(-1)) than in atenolol (10.5+/-2.6 micromol g(dw)(-1)) and paced (10.9+/-1.3 micromol g(dw)(-1)) hearts. Thus, by suppressing the protective effects of down-regulation, both atenolol and pacing apparently depress myocardial recovery in this model.

[The effects due to reduced coronary inflow and hypoxemia during myocardial ischemia]. / Effetti dovuti al ridotto afflusso coronarico e all'ipossemia durante l'ischemia del miocardio.

The events associated to myocardial ischemia result from 2 overlapping phenomena due to reduced blood flow (ischemia) and reduced O2 supply (hypoxemia). To distinguish these effects, 2 groups of isolated rat hearts were perfused through the aorta (Langendorff's method) with Krebs-Henseleit buffer, and were exposed for 20 min to hypoxemia or ischemia, matched in terms of the O2 supply (10% of baseline), with continuous monitoring of cardiac contractility, O2 uptake and lactate production. The developed pressure and the O2 uptake were similar in hypoxemic and ischemic hearts; heart rate, end-diastolic pressure and lactate production rate were higher in hypoxemia than in ischemia; the recovery from hypoxemia was less than that from ischemia, despite the same O2 supplies; treatment with superoxide dismutase and catalase, scavengers of the O2 derived free radicals, during hypoxemia, allowed hypoxemic hearts to recover as ischemic hearts. Therefore, the main determinant of the reperfusion injury is to be attributed to the low O2 supply rather than to the low coronary flow; part of the injury is due to free radicals; a substantial portion is mediated by the energy demand during the stress which was higher in hypoxemia than in ischemia.

Dynamics of myocardial adaptation to low-flow ischemia and hypoxemia.

We investigated whether one or more factors control performance in O2-limited hearts. For this purpose, we measured the dynamics of myocardial adaptation to reduced O2 supply with a specially designed setup, analyzing early changes after reduction in either flow of the perfusion medium or its PO2. For 10 min, 38 isolated rat hearts underwent low-flow ischemia or hypoxemia, matched for O2 supply. Early during ischemia, developed pressure declined at a rate of 311 +/- 25 mmHg/s; lactate release increased and then leveled off to 3.4 +/- 0.7 mumol/min within 2 min. During hypoxemia, pressure dropped initially, as observed during ischemia. However, it then increased before slowly decreasing. Lactate release during hypoxemia peaked at 13.0 +/- 2.3 mumol/min after 2 min, leveling off to 3.5 +/- 1.3 mumol/min. Glycogen decreased by 52 and 81% in ischemic and hypoxemic hearts, respectively (P < 0.05). Reexposure to ischemia or hypoxemia induced comparable changes in both groups. We conclude that, at the beginning of ischemia, a single factor does limit myocardial performance. This variable, which remains undisturbed for 10 min, is presumably O2 availability. In contrast, approximately 20 s after induction of hypoxemia, glycolytic ATP production can partially override low O2 availability by providing most of the energy needed. During repeated restriction of O2 supply, O2 availability alone limits performance during both ischemia and hypoxemia.

Swim training improves myocardial resistance to ischemia in rats.

As the relationship between training and ischemic heart disease is not yet unraveled, we test the hypothesis that, in a model free from environmental, behavioural, and neuro-hormonal factors, endurance training improves myocardial resistance to ischemia. As carbohydrate metabolism is relevant for myocardial resistance to ischemia, we also test whether hyperglycemia blunts the protective effect of training. Eight-week old rats were randomly assigned to four groups (n = 6-8): sedentary or trained (3-week swim program, up to 2 h/day), and normal or high-carbohydrate diet (50 g/l sucrose in drinking water). Excised hearts were perfused isovolumically (flow = 15 ml/min) with Krebs-Henseleit (2 mM free Ca++, 11 mM glucose, pH 7.38 +/- 0.02, PO2 = 670 +/- 6 mmHg, PCO2 = 43 +/- 1 mmHg, mean +/- SE), exposed to 60 min low-flow (1.5 ml/min) ischemia, and then reperfused for 30 min (15 ml/min). In normally fed rats training increased the stroke volume index (97.5 +/- 13.0 vs. 72.6 +/- 6.2 microl, P = 0.05), depressed diastolic contracture (+2.3 +/- 2.0 vs. +24.2 +/- 6.7 mmHg, P = 0.02), improved the recovery of developed pressure x heart rate (33.8 +/- 2.3 vs. 24.1 +/- 3.3 mmHg/min/1000, P = 0.05), and decreased arrhythmias (P = 0.05). In high-carbohydrate-fed rats training induced myocardial hypertrophy (1.95 +/- 0.08 vs. 1.67 +/- 0.03 g, P = 0.02) and decreased arrhythmias but did not affect stroke volume, developed pressure x heart rate, and diastolic contracture. Thus endurance training improves myocardial resistance to ischemia but a high-carbohydrate diet partially blunts this protection. The occurrence of an inducible alteration able to modulate myocardial tolerance to ischemia may give clues to extend our knowledge of ischemic preconditioning.

Myocardial metabolism and function in acutely ischemic and hypoxemic isolated rat hearts.

We tested the hypothesis that residual oxygen supply during acute low-flow ischaemia or hypoxemia is a major regulator of myocardial performance, metabolism and recovery. Rat hearts were exposed for 20 min to either ischemia (coronary flow reduced to 10% of baseline), hypoxemia (oxygen content reduced to 10% baseline) or a "mixed" condition (combined ischaemia and hypoxemia). The oxygen supply (coronary flow x oxygen content) was matched in all groups (n = 16 per group). Hypoxemic hearts had the highest performance (systolic and developed pressures, +/- dP/dtmax and oxygen uptake) and content of IMP and AMP. Ischaemic hearts had the highest content of ATP, phosphocreatine, adenine nucleotides and purines. As flow and/or oxygenation were restored, post-ischemic hearts showed better functional and metabolic recovery than post-hypoxemic ones. "Mixed" hearts were more similar to hypoxemic ones during oxygen shortage but to ischemic ones during recovery. We conclude that as oxygenation is critically limiting, coronary flow is relatively more important than oxygen supply in determining myocardial function, metabolism and recovery, most likely secondary to changes in the metabolism of diffusible substances.

High-energy phosphates metabolism and recovery in reperfused ischaemic hearts.

BACKGROUND: The aim of this study was to assess how coronary flow, oxygen supply and energy demand affect myocardial ATP, phosphocreatine and their metabolites during oxygen shortage and recovery. METHODS: Isolated rat hearts were exposed for 20 min to either low-flow ischaemia or hypoxaemia at the same oxygen supply, followed by return to baseline conditions (20 min). Seventy-three hearts were divided into four groups: ischaemic or hypoxaemic, spontaneously beating or paced to increase energy demand. RESULTS: During O2 shortage, myocardial performance was less in ischaemic, spontaneously beating hearts (SpIs), than in the other groups (14 +/- 1% of baseline vs. 25-48%). Consequently, the tissue levels of ATP, total adenylates and phosphocreatine were maintained in SpIs, in contrast to marked decreases in the other groups. Upon reflow, the recovery of performance and of myocardial ATP was 94 +/- 5% in SpIs (P = NS vs. baseline) compared with 64-85% (P < 0.05 vs. baseline) in the other groups. The degree of recovery was positively related to the ischaemic contents of ATP (P = 0.03) and adenylates (P = 0.001), but not to that of phosphocreatine (P = NS). CONCLUSION: The maintenance of the ATP pool under low oxygen supply conditions is essential for good recovery. The most important factors that determine the ATP pool size are the energy demand, which increases the formation of diffusible ATP catabolites, and the coronary flow, which removes these catabolites, rather than the oxygen supply per se.

Triglycerides impair postischemic recovery in isolated hearts: roles of endothelin-1 and trimetazidine.

There is growing evidence that hypertriglyceridemia exacerbates ischemic injury. We tested the hypothesis that triglycerides impair myocardial recovery from low-flow ischemia in an ex vivo model and that such an effect is related to endothelin-1. Hyperglycemic (glucose concentration = 12 mmol/l) and hyperinsulinemic (insulin concentration = 1.2 micromol/l) isolated rat hearts were perfused with Krebs-Henseleit buffer (PO(2) = 670 mmHg, pH 7.4, 37 degrees C) added with increasing triglycerides (0, 1,000, 2,000, and 4,000 mg/dl, n = 6-9 rats/group). Hearts were exposed to 60 min of low-flow ischemia (10% of basal coronary flow), followed by 30 min of reperfusion. We found that increasing triglycerides impaired both the diastolic (P < 0.005) and systolic (P < 0.02) recovery. The release of endothelin-1 during reperfusion increased linearly with triglyceride concentration (P = 0.0009). Elevated triglycerides also increased the release of nitrite and nitrate (NO(x)), the end products of nitric oxide, up to 6 micromol/min. Trimetazidine (1 micromol) further increased NO(x) release, blunted endothelin-1 release, and protected myocardial function during recovery. We conclude that high triglyceride levels impair myocardial recovery after low-flow ischemia in association with endothelin-1 release. The endothelium-mediated effect of triglycerides on both contractile recovery and endothelin-1 release is prevented by 1 microM trimetazidine.

Differential depression of myocardial function and metabolism by lactate and H+.

The effects of both high blood H+ concentration ([H+]) and high blood lactate concentration ([lactate]) under ischemia-reperfusion conditions are receiving attention, but little is known about their effects in nonischemic hearts. Isolated rat hearts were Langendorff perfused at constant flow with media at two pH values (7.4 and 7.0) and two [lactate] (0 and 20 mM) in various sequences (n = 6/group). Coronary flow and arterial O2 content were kept constant at levels that allowed hearts to function without O2 supply limitation. We measured contractility, O2 uptake, diastolic pressure, and at the end of the protocol, tissue [lactate] and pH. Perfusion with high [lactate] raised tissue [lactate] from 5.5 +/- 0.1 to 17.5 +/- 2.6 micromol/heart (P < 0.0001), whereas decreasing the pH of the medium decreased tissue pH from 6.94 +/- 0.02 to 6.81 +/- 0.06 (P = 0.002). Heart rate was not affected by high [lactate] but was reversibly depressed by high [H+] (P = 0.004). Developed pressure declined by 20% in response to high [lactate], high [H+], and high [lactate] + high [H+] (P = 0.002). After the high-[lactate] challenge was withdrawn, pressure continued to decline. In contrast, withdrawing the high [H+] challenge allowed partial recovery. The behavior of diastolic pressure mirrored that of developed pressure. Although unaffected by high [lactate], the O2 uptake was reversibly depressed by high [H+]. This suggests higher O2 cost per contraction in the presence of high [lactate]. We conclude that for similar acute contractility depression, high [lactate] induces irreversible damage, likely at some point in the pathway of O2 utilization. In contrast, the effect of high [H+] appears reversible. These differential behaviors may have implications for heart function during heavy exercise and ischemia-reperfusion events.

Effects of trimetazidine on metabolic and functional recovery of postischemic rat hearts.

The objective of this study was to test the hypothesis that the beneficial effect of trimetazidine during reflow of ischemic hearts is mediated by energy sparing and ATP pool preservation during ischemia. Isolated rat hearts (controls and rats treated with 10(-6) M trimetazidine, n = 17 per group) underwent the following protocol: baseline perfusion at normal coronary flow (20 minutes), low-flow ischemia at 10% flow (60 minutes), and reflow (20 minutes). We measured contractile function, O2 uptake, lactate release, venous pH and PCO2, and the tissue content of high-energy phosphates and their metabolites. During baseline, trimetazidine induced higher venous pH and lower PCO2 without influencing performance and metabolism. During low-flow ischemia, trimetazidine reduced myocardial performance (P = 0.04) and ATP turnover (P = 0.02). During reflow, trimetazidine improved performance (91 +/- 6% versus. 55 +/- 6% of baseline), prevented the development of diastolic contracture and coronary resistance, and reduced myocardial depletion of adenine nucleotides and purines. ATP turnover during low-flow ischemia was inversely related to recovery of the rate-pressure product (P = 0.002), end-diastolic pressure (P = 0.007), and perfusion pressure (P = 0.05). We conclude that trimetazidine-induced protection of ischemic-reperfused hearts is also mediated by energy sparing during ischemia, which presumably preserves the ATP pool during reflow.