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+.

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Correlation between myocardial contractile force and cytosolic inorganic phosphate during early ischemia

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.272.3.h1333 ◽

1997 ◽

Vol 272 (3) ◽

pp. H1333-H1341 ◽

Cited By ~ 3

Author(s):

M. X. He ◽

S. Wang ◽

H. F. Downey

Keyword(s):

Inorganic Phosphate ◽

Contractile Force ◽

Left Ventricular ◽

Resonance Spectroscopy ◽

Rat Hearts ◽

Initial Control ◽

Myocardial Contractile Force ◽

Myocardial Contractile ◽

Developed Pressure

To test the role of inorganic phosphate (Pi) in downregulation of myocardial contractile force at the onset of ischemia, Pi of rat hearts was determined with 31P nuclear magnetic resonance spectroscopy. Forty cycles of brief hypoperfusion (30% of baseline flow for 33 s) were used to achieve a time resolution of 0.512 s for comparing dynamic changes in Pi and contractile force. Initial control values of left ventricular developed pressure (LVP), heart rate, and oxygen consumption were 136 +/- 11 mmHg, 236 +/- 4 beats/min, and 95 +/- 3 microl O2 x min(-1) x g(-1); these values were unchanged at the end of the experiment. During the first 10 s of hypoperfusion, Pi increased at a rate (percentage of the total observed change) faster than the decrease in LVP; Pi and LVP then changed at the same rate during the remainder of the hypoperfusion. ADP did not change in advance of LVP. Intracellular pH did not change. The results indicate that Pi plays an important role in initiating the downregulation of myocardial contractile force at the onset of ischemia. Perfusion pressure also declined faster than LVP at the onset of ischemia, indicating potential importance of vascular collapse in contractile downregulation during early ischemia.

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Dynamics of myocardial adaptation to low-flow ischemia and hypoxemia

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1996.271.6.h2300 ◽

1996 ◽

Vol 271 (6) ◽

pp. H2300-H2305

Author(s):

G. Merati ◽

S. Allibardi ◽

L. D. Monti ◽

J. W. de Jong ◽

M. Samaja

Keyword(s):

Low Flow ◽

Control Performance ◽

Perfusion Medium ◽

Atp Production ◽

Lactate Release ◽

Rat Hearts ◽

O2 Supply ◽

Developed Pressure ◽

Myocardial Adaptation ◽

Isolated Rat

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.

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Downregulation of ventricular contractile function during early ischemia is flow but not pressure dependent

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1998.275.5.h1520 ◽

1998 ◽

Vol 275 (5) ◽

pp. H1520-H1523 ◽

Cited By ~ 1

Author(s):

Miao-Xiang He ◽

H. Fred Downey

Keyword(s):

Coronary Artery ◽

Perfusion Pressure ◽

Contractile Function ◽

Coronary Perfusion Pressure ◽

Coronary Perfusion ◽

Rat Hearts ◽

Myocardial Contractile Force ◽

Myocardial Contractile ◽

Artery Obstruction

The mechanism responsible for the abrupt fall in myocardial contractile function following coronary artery obstruction is unknown. The “vascular collapse theory” hypothesizes that the fall in coronary perfusion pressure after coronary artery obstruction is responsible for contractile failure during early ischemia. To test the role of vascular collapse in downregulating myocardial contractile force at the onset of ischemia, coronary flow of isolated rat hearts was abruptly decreased by 50, 70, 85, and 100% of baseline, and subsequent changes in coronary perfusion pressure and ventricular function were recorded at 0.5-s intervals. At 1.5 s after flow reductions ranging from 50 to 100%, decreases in contractile function did not differ, although perfusion pressure varied significantly from 45 ± 1 to 20 ± 2 mmHg. When function fell to 50% of baseline, perfusion pressures ranged from 35 ± 0.5 to 2.5 ± 1 mmHg for flow reductions ranging from 50 to 100%. Identical contractile function at widely differing coronary perfusion pressures is incompatible with the vascular collapse theory.

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Oxygen conformance of cellular respiration in hepatocytes

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.265.4.l395 ◽

1993 ◽

Vol 265 (4) ◽

pp. L395-L402 ◽

Cited By ~ 15

Author(s):

P. T. Schumacker ◽

N. Chandel ◽

A. G. Agusti

Keyword(s):

Metabolic Activity ◽

Energy Demand ◽

Lactic Acid Production ◽

Rat Hepatocytes ◽

Cellular Respiration ◽

O2 Uptake ◽

Rapid Reduction ◽

Isolated Rat Hepatocytes ◽

Cell Densities ◽

O2 Supply

Cellular respiratory rates are normally determined by metabolic activity, but become rate limited by O2 availability if the cell O2 tension (PO2) falls below a critical value (typically 1–10 Torr). An ability to reduce metabolic activity and energy demand in response to a falling O2 availability might confer an increased resistance to a diminished O2 supply. Isolated rat hepatocytes were studied in primary culture under controlled O2 tensions. Cells were obtained by collagenase digestion and seeded into nutritive media in control and experimental spinner flasks at identical cell densities. Cells subjected to rapid reduction in PO2 (100⇢0 Torr over < 40 min) exhibited undiminished O2 uptake until PO2 fell below 10 Torr. By contrast, when cell PO2 was reduced over several hours, significant decreases in O2 uptake became evident at O2 tensions as high as 70 Torr. These decreases were associated with a reduction in ATP concentration and an increase in NAD(P)H, compared with rapidly deoxygenated cells at the same PO2. No loss in cell viability was detected after 24 h at reduced PO2. The decrease in respiratory rate was associated with an increased rate of lactic acid production relative to normoxic controls. Restoration of normoxia was associated with an immediate return of O2 uptake to control levels. These results demonstrate that hepatocytes are capable of reversibly decreasing metabolic activity and O2 demand during sustained moderate reductions in PO2, via a mechanism that appears to involve an inhibition of mitochondrial function other than O2 supply limitation. This response may alter cellular susceptibility to physiological stresses including hypoxia.

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Direct evidence for a role of intramitochondrial Ca2+ in the regulation of oxidative phosphorylation in the stimulated rat heart. Studies using31P n.m.r. and ruthenium red

Biochemical Journal ◽

10.1042/bj2620293 ◽

1989 ◽

Vol 262 (1) ◽

pp. 293-301 ◽

Cited By ~ 63

Author(s):

J F Unitt ◽

J G McCormack ◽

D Reid ◽

L K MacLachlan ◽

P J England

Keyword(s):

Oxidative Phosphorylation ◽

Direct Evidence ◽

Respiratory Control ◽

Ruthenium Red ◽

Phosphorylation Potential ◽

Mitochondrial Inner Membrane ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Inotropic Stimulation

1. The concentrations of free ATP, phosphocreatine (PCr), Pi, H+ and ADP (calculated) were monitored in perfused rat hearts by 31P n.m.r. before and during positive inotropic stimulation. Data were accumulated in 20 s blocks. 2. Administration of 0.1 microM-(-)-isoprenaline resulted in no significant changes in ATP, transient decreases in PCr, and transient increases in ADP and Pi. However, the concentrations of all of these metabolites returned to pre-stimulated values within 1 min, whereas cardiac work and O2 uptake remained elevated. 3. In contrast, in hearts perfused continuously with Ruthenium Red (2.5 micrograms/ml), a potent inhibitor of mitochondrial Ca2+ uptake, administration of isoprenaline caused significant decreases in ATP, and also much larger and more prolonged changes in the concentrations of ADP, PCr and Pi. In this instance values did not fully return to pre-stimulated concentrations. Administration of Ruthenium Red alone to unstimulated hearts had minor effects. 4. It is proposed that, in the absence of Ruthenium Red, the transmission of changes in cytoplasmic Ca2+ across the mitochondrial inner membrane is able to maintain the phosphorylation potential of the heart during positive inotropic stimulation, through activation of the Ca2+-sensitive intramitochondrial dehydrogenases (pyruvate, NAD+-isocitrate and 2-oxoglutarate dehydrogenases) leading to enhanced NADH production. 5. This mechanism is unavailable in the presence of Ruthenium Red, and oxidative phosphorylation must be stimulated primarily by a fall in phosphorylation potential, in accordance with the classical concept of respiratory control. However, the full oxidative response of the heart to stimulation may not be achievable under such circumstances.

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Role of dual-site phospholamban phosphorylation in the stunned heart: insights from phospholamban site-specific mutants

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00209.2003 ◽

2003 ◽

Vol 285 (3) ◽

pp. H1198-H1205 ◽

Cited By ~ 33

Author(s):

M. Said ◽

L. Vittone ◽

C. Mundiña-Weilenmann ◽

P. Ferrero ◽

E. G. Kranias ◽

...

Keyword(s):

Ischemia Reperfusion ◽

Phosphorylation Sites ◽

Adrenergic Stimulation ◽

Rat Hearts ◽

Adrenergic Blocker ◽

Perfused Rat Hearts ◽

A Site ◽

Contractile Recovery ◽

Mechanical Recovery

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on β-adrenergic stimulation and an ischemia and reperfusion-induced Thr17 phosphorylation that was dependent on Ca2+ influx. To elucidate the relationship between these two PLB phosphorylation sites and postischemic mechanical recovery, rat hearts were submitted to ischemia-reperfusion in the absence and presence of the CaMKII inhibitor KN-93 (1 μM) or the β-adrenergic blocker dl-propranolol (1 μM). KN-93 diminished the reperfusion-induced Thr17 phosphorylation and depressed the recovery of contraction and relaxation after ischemia. dl-Propranolol decreased the ischemia-induced Ser16 phosphorylation but failed to modify the contractile recovery. To obtain further insights into the functional role of the two PLB phosphorylation sites in postischemic mechanical recovery, transgenic mice expressing wild-type PLB (PLB-WT) or PLB mutants in which either Thr17 or Ser16 were replaced by Ala (PLB-T17A and PLB-S16A, respectively) into the PLB-null background were used. Both PLB mutants showed a lower contractile recovery than PLB-WT. However, this recovery was significantly impaired all along reperfusion in PLB-T17A, whereas it was depressed only at the beginning of reperfusion in PLB-S16A. Moreover, the recovery of relaxation was delayed in PLB-T17A, whereas it did not change in PLB-S16A, compared with PLB-WT. These findings indicate that, although both PLB phosphorylation sites are involved in the mechanical recovery after ischemia, Thr17 appears to play a major role.

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Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2000.278.5.e846 ◽

2000 ◽

Vol 278 (5) ◽

pp. E846-E856 ◽

Cited By ~ 30

Author(s):

Geneviève Vincent ◽

Blandine Comte ◽

Myriame Poirier ◽

Christine Des Rosiers

Keyword(s):

Citric Acid Cycle ◽

Citrate Synthase ◽

Specific Inhibitor ◽

Release Rates ◽

Fuel Selection ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Citrate Release ◽

Labeling Pattern

Cytosolic citrate is proposed to play a crucial role in substrate fuel selection in the heart. However, little is known about factors regulating the transfer of citrate from the mitochondria, where it is synthesized, to the cytosol. Further to our observation that rat hearts perfused under normoxia release citrate whose 13C labeling pattern reflects that of mitochondrial citrate (B. Comte, G. Vincent, B. Bouchard, and C. Des Rosiers. J. Biol. Chem. 272: 26117–26124, 1997), we report here data indicating that this citrate release is a specific process reflecting the mitochondrial efflux of citrate, a process referred to as cataplerosis. Indeed, measured rates of citrate release, which vary between 2 and 21 nmol/min, are modulated by the nature and concentration of exogenous substrates feeding acetyl-CoA (fatty acid) and oxaloacetate (lactate plus pyruvate) for the mitochondrial citrate synthase reaction. Such release rates that represent at most 2% of the citric acid cycle flux are in agreement with the activity of the mitochondrial tricarboxylate transporter whose participation is also substantiated by 1) parallel variations in citrate release rates and tissue levels of citrate plus malate, the antiporter, and 2) a lowering of the citrate release rate by 1,2,3-benzenetricarboxylic acid, a specific inhibitor of the transporter. Taken together, the results from the present study indicate that citrate cataplerosis is modulated by substrate supply, in agreement with the role of cytosolic citrate in fuel partitioning, and occurs, at least in part, through the mitochondrial tricarboxylate transporter.

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Presynaptic Effects of Moxonidine in Isolated Buffer Perfused Rat Hearts: Role of Imidazoline-1 Receptors and α2-Adrenoceptors

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.102.041657 ◽

2002 ◽

Vol 303 (3) ◽

pp. 1163-1170 ◽

Cited By ~ 12

Author(s):

Ulrich Schäfer ◽

Christof Burgdorf ◽

Astrid Engelhardt ◽

Thomas Kurz ◽

Gert Richardt

Keyword(s):

Α2 Adrenoceptors ◽

Rat Hearts ◽

Perfused Rat Hearts

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Src tyrosine kinase is the trigger but not the mediator of ischemic preconditioning

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.281.3.h1066 ◽

2001 ◽

Vol 281 (3) ◽

pp. H1066-H1074 ◽

Cited By ~ 34

Author(s):

Reiji Hattori ◽

Hajime Otani ◽

Takamichi Uchiyama ◽

Hiroji Imamura ◽

Jianhua Cui ◽

...

Keyword(s):

Tyrosine Kinase ◽

Ischemic Preconditioning ◽

Tyrosine Kinases ◽

Src Kinase ◽

Src Tyrosine Kinase ◽

Kinase Activation ◽

Rat Hearts ◽

Perfused Rat Hearts ◽

Src Activation

The signal cascade that triggers and mediates ischemic preconditioning (IPC) remains unclear. The present study investigated the role of the Src family of tyrosine kinases in IPC. Isolated and buffer-perfused rat hearts underwent IPC with three cycles of 5-min ischemia and 5-min reperfusion, followed by 30-min ischemia and 120-min reperfusion. The Src tyrosine kinase family-selective inhibitor PP1 was administered between 45 and 30 min before ischemia (early PP1 treatment) or for 15 min before IPC [early PP1-preconditioning (PC) treatment]. PP1 was also administered for 5 min before the sustained ischemia (late PP1 treatment) or after IPC (late PP1-PC treatment). Src kinase was activated after 30 min of ischemia in both the membrane and cytosolic fractions. Src kinase was also activated by IPC but was attenuated after the sustained ischemia. Early and late PP1 treatment inhibited Src activation after the sustained ischemia and reduced infarct size. Early PP1-PC inhibited Src activation after IPC but not after the sustained ischemia and blocked cardioprotection afforded by IPC. Late PP1-PC treatment abrogated IPC-induced activation of Src and protein kinase C (PKC)-ε in the membrane but not in the cytosolic fraction. This treatment modality abrogated Src activation after the sustained ischemia and failed to block cardioprotection afforded by IPC. These results suggest that Src kinase activation mediates ischemic injury but triggers IPC in the position either upstream of or parallel to membrane-associated PKC-ε.

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