AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 281: H722-H730, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H722-H730, August 2001

Calcium-mediated activation of pyruvate dehydrogenase in severely injured postischemic myocardium

Jerome Terrand, Irene Papageorgiou, Nathalie Rosenblatt-Velin, and Rene Lerch

Cardiology Center, University Hospital, CH-1211 Geneva 14, Switzerland


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Indirect evidence suggests that activity of pyruvate dehydrogenase (PDH) influences recovery of the myocardium after transient ischemia. The present study examined the relationship between postischemic injury and activity of PDH and the role of mitochondrial calcium uptake for observed changes in PDH activity. Isovolumically beating isolated rat hearts perfused with erythrocyte-enriched buffer containing glucose, palmitate, and insulin were submitted to either 20 or 35 min of no-flow ischemia. After 20 min of no-flow ischemia, hearts exhibited complete recovery of developed left ventricular pressure (DLVP). The proportion of myocardial PDH in the active state was modestly increased to 38% (compared with 13% in control hearts) without a change in glucose oxidation. In contrast, in hearts subjected to 35 min of no-flow ischemia (which exhibited poor recovery of DLVP), there was marked stimulation of glucose oxidation (+460%; P < 0.01) and pronounced increase in the active fraction of PDH to 72% (P < 0.01). Glycolytic flux was not significantly altered. Ruthenium red (6 µM) completely abolished the activation of PDH and the increase in glucose oxidation. The results indicate that variable stimulation of glucose oxidation during reperfusion is related to different degrees of activation of PDH, which depends on the severity of the ischemic injury. Activation of PDH seems to be mediated by myocardial calcium uptake.

reperfusion; substrate; metabolism; perfused heart; ruthenium red


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN RECENT YEARS evidence has accumulated that metabolism of exogenous glucose early during reperfusion favorably influences recovery of postischemic myocardium (22, 42). Identification of the subcellular mechanism(s) responsible for the beneficial effects of glucose metabolism is complicated by the multiple fates of glucose taken up by the myocardium, which include incorporation into glycogen, anaerobic glycolysis to lactate, mitochondrial oxidation, and oxidation in the pentosephosphate pathway (6). A number of observations suggest a crucial role of activation of pyruvate dehydrogenase (PDH) for the protective effect of glucose (19). PDH largely controls the rate of entry of pyruvate into mitochondrial oxidative catabolism by the tricarboxylic acid cycle. It has been proposed (39) that oxidation of glycolytically produced pyruvate avoids the production of protons during anaerobic glycolysis to lactate and thereby reduces myocyte calcium overload caused by successive transsarcolemmal H+/Na+ and Na+/Ca2+ exchange. Activity of PDH is reduced by phosphorylation, which is mediated by PDH kinase. The latter enzyme is activated by high levels of acetyl coenzyme A (acetyl-CoA) and NADH, as is observed in myocardium under conditions of high fatty acid oxidation (33), and is inhibited by dichloroacetate (35). Conversely, PDH is activated by a calcium-sensitive PDH phosphatase (25). Consistent with a protective effect of activation of PDH during reperfusion, enhancement of glucose oxidation and improvement of recovery of contractile function has been observed in postischemic hearts exposed to pyruvate (16), dichloroacetate (27), trimetazidine (43), L-carnitine (38), and ranolazine (5).

However, the rationale for implementation of an intervention aimed at activation of PDH early during reperfusion depends on the level of spontaneous activity of PDH in postischemic myocardium. In fact, reported data on carbohydrate oxidation early during reperfusion exhibit considerable variability with increased (9), unaltered (17, 20), or decreased (14, 38) glucose oxidation compared with baseline values. This suggests that activity of PDH early during reperfusion critically depends on the selected experimental conditions. Specifically, in the experiments by Lopaschuks et al. (27), in isolated working rat hearts perfused with medium containing 11 mM glucose and 1.2 mM palmitate, glucose oxidation rapidly returned to the preischemic level after 25-30 min of no-flow ischemia, which was markedly lower than in control hearts perfused with glucose as the sole substrate. This indirectly suggests persistent fatty acid-mediated inhibition of PDH during reperfusion. The concept of postischemic inhibition of PDH has been supported by 13C magnetic resonance spectroscopy (16) or direct measurement of myocardial enzyme activity in isolated perfused rat hearts subjected to 10 or 20 min of no-flow ischemia (43). In contrast to these findings, our group has observed in isolated rat hearts perfused with erythrocyte-enriched medium containing 11 mM glucose and 0.4 mM palmitate pronounced stimulation of glucose oxidation early during reperfusion after 35-40 min of no-flow ischemia (1, 9). Postischemic stimulation of myocardial glucose oxidation has also been observed in pigs (18, 26) and dogs (2, 31) after prolonged regional myocardial ischemia (>40 min). These data indirectly suggest that postischemic activation of glucose oxidation may increase with increasing severity of ischemic injury.

In the present study, we sought to determine in isolated perfused rat hearts 1) the influence of the degree of ischemic injury on myocardial activity of PDH, and 2) the potential role of myocardial mitochondrial calcium uptake in the anticipated postischemic activation of PDH.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

This study was approved by the institutional ethical committee for animal experiments and was conducted in accordance with the "Guiding Principles in the Care and Use of Animals" published by the American Physiological Society. Experiments were performed in male OFA rats (Iffa Credo; L'Arbresle, France) weighing 280-350 g. Rats were fasted for 24 h before experimentation.

Heart Perfusion

Rats were anesthetized with thiopental sodium (Penthotal, 60 mg/kg ip; Abbott; Chicago, IL). After thoracotomy, heparin (Liquemin, 1,000 IU; Roche; Basel, Switzerland) was injected into the inferior vena cava and the heart was quickly excised, placed in ice-cold saline, blotted, and weighed. The aorta was cannulated and retrograde perfusion was initiated at a constant flow (10 ml · min-1 · g wet wt-1) in a nonrecirculating system that included a roller pump (model IPS4, Ismatec; Glattbrugg, Switzerland). The initial perfusate was Krebs-Henseleit (KH) buffer containing (in mM) 118 NaCl, 4.0 KCl, 1.8 CaCl2, 1.4 KH2PO4, 1.2 MgSO4, 25 NaHCO3, and 11 glucose. All perfusates were equilibrated with 95% O2-5% CO2 and warmed to 37°C. The pulmonary artery was cannulated for collection of the coronary effluent. Hearts were paced at 300 beats/min throughout the experiment. A fluid-filled latex balloon was inserted into the left ventricle via a left atriotomy and was connected to a Statham P21 XL pressure transducer (Gould; Valley View, OH). At the beginning of perfusion with erythrocyte-containing medium (described in the next paragraph), filling of the balloon was adjusted to give a left ventricular (LV) systolic pressure of 80 mmHg. The balloon volume was then kept constant throughout the experiment. Isovolumic LV diastolic and systolic pressures were recorded continuously on a strip-chart recorder (type 2400S; Gould). Developed LV pressure (DLVP) was calculated as the difference between LV systolic and diastolic pressures.

After surgical preparation, perfusion was changed to an erythrocyte-enriched KH medium (EE-KH) containing 11 mM glucose, 70 mU/l insulin (Actrapid; Novo Nordisk Pharma), and 0.4 mM palmitate bound to 0.4 mM albumin. Washed human erythrocytes were added to the perfusate to yield a hematocrit of 30% allowing sufficient O2 supply to the myocardium at a physiological flow rate of 2 ml · min-1 · g wet wt-1 (9). EE-KH was passed through a 10-µm transfusion filter (MF10, Biotest; Othmarsingen, Switzerland) that was included in the perfusion system. At the end of the perfusion protocol, the heart was quickly frozen with an aluminum clamp precooled in liquid N2, powdered with a pestle in a mortar filled with liquid N2, and stored at -80°C for subsequent biochemical analysis.

Perfusion Protocols

Control experiments. Hearts of this group (n = 12) were perfused without intervention for 120 min at the control flow rate of 2 ml · min-1 · g wet wt-1 with EE-KH medium. Samples of the perfusate and the coronary effluent were withdrawn every 15 min. Radiolabeled substrates for metabolic measurements were present during the entire perfusion period. In additional hearts (n = 3), ruthenium red was infused into the perfusion system starting 55 min after the onset of perfusion with EE-KH to yield a final perfusate concentration of 6 µM. The infusion was continued for 40 min. During the last 20 min of the experiment, hearts were perfused without ruthenium red.

The commercially obtained ruthenium red batch (purity 33% wt/wt; Sigma) was purified by crystallization according to Luft (21). Purified ruthenium red was dissolved in KH buffer at a concentration of 90-120 µM, filtered (0.2 µM), analyzed for ruthenium red concentration by spectrometry, and infused into a stirring chamber just proximal to the aortic cannula at a rate that resulted in a perfusate concentration of 6 µM (1).

Effects of 20 min of ischemia. Hearts of this group (n = 12) were first equilibrated for 20 min with EE-KH; thereafter, perfusion was stopped for 20 min. Immediately after perfusion was stopped, the coronary circulation was flushed with 3 ml of erythrocyte-free perfusate to prevent intracoronary aggregation of erythrocytes. During ischemia, the heated jacket surrounding the heart was filled with warmed KH to maintain the myocardial temperature at 37°C. Hearts were then reperfused for 60 min at the control flow rate (2 ml · min-1 · g wet wt-1). Samples of the perfusate and the coronary effluent were collected after 10 and 20 min of equilibration and 5, 15, 30, 45, and 60 min after the onset of reperfusion.

Effects of 35 min of ischemia. The perfusion protocol of this group of hearts (n = 11) was identical to that of the 20-min ischemia group, with the exception that perfusion was stopped for 35 min. The duration of ischemia was selected based on previous observations that indicated pronounced stimulation of glucose oxidation (42). Although low-flow ischemia more closely simulates evolving myocardial infarction in vivo, we have previously observed (9) that the limited duration of stability of in vitro perfused hearts does not allow for extending the ischemic period long enough to reproducibly induce irreversible injury in the presence of residual perfusion.

Effects of ruthenium red after 35 min of ischemia. The same ischemia-reperfusion protocol was followed as in the hearts of the 35-min ischemia group. However, ruthenium red was infused into the perfusion system during the initial 40 min of reperfusion to yield a final concentration of 6 µM (n = 6).

Analytic Procedures

Myocardial O2 consumption. Hemoglobin content and O2 saturation of hemoglobin in both the perfusate and coronary effluent were measured with an oximeter (type IL282; Instrumentation Laboratory; Lexington, KY), and PO2 and pH were measured with a blood gas analyzer (type IL1304; Instrumentation Laboratory). Myocardial O2 consumption was calculated by multiplying the difference of the total O2 content (hemoglobin-bound and dissolved O2) between perfusate and coronary effluent by the myocardial blood flow (9).

Substrate oxidation. All labeled substrates were purchased from Amersham (Amersham Pharmacia Biotech; Zürich, Switzerland). For the measurement of substrate oxidation, either 10 µCi/l of [1-14C]lactate or 60 µCi/l of [U-14C]glucose were added to the perfusate. Oxidation of each substrate was measured by dividing myocardial release of 14CO2 (in cpm · min-1 · g wet wt-1) by the specific activity of the labeled substrate in the perfusate (in cpm/nmol). 14CO2 was measured in the perfusate and the coronary effluent as described previously (9). In brief, 1-ml samples were injected into sealed flasks containing 1 ml of 2 M NaOH. CO2 was released by addition of 3 ml of 1 M H2SO4 and trapped on small pieces of filter paper soaked with 250 µl of hyamine hydroxide (NCS; Amersham). The filter paper was then counted by liquid scintillation in a beta -spectrometer (LS 7500; Beckman Instruments; Irvine, CA) (1, 9).

Myocardial glycolytic flux was estimated based on myocardial release of 3H2O from [5-3H]glucose added to the perfusate. Samples of the perfusate and the coronary effluent (0.5 ml) were deproteinized with 1 ml of 0.6 M HClO4 immediately after withdrawal and were neutralized with 75 µl of 5 M K2CO3. Tritiated water was separated from [5-3H]glucose by anion-exchange chromatography on Dowex-1-borate columns (11). Dowex 1 × 4 resin (chloride form, 200-400 mesh) was converted to Dowex-1-borate by washing with 0.05 M K2B4O7. Deproteinized samples (0.2 ml) were loaded onto a 0.5-ml volume bed of resin and eluted with 0.8 ml of H2O into scintillation vials. Analysis of standard solutions containing known amounts of 3H2O and [U-14C]glucose revealed that >99% of the labeled glucose and <5% of labeled water was retained by the column.

Creatine kinase. The coronary effluent was collected during the entire postischemic reperfusion period for the determination of cumulative myocardial release of creatine kinase (CK NAC kit; bioMérieux; Marcy-l'Etoile, France) (41).

PDH activity. Myocardial PDH activity was measured in additional hearts of each perfusion protocol by the procedure described by McCormack and Denton (23). Hearts were freeze-clamped after 20, 60, or 120 min of control perfusion or after 5 and 60 min of postischemic reperfusion. Frozen samples (100-200 mg) were homogenized in buffer (100 mM potassium phosphate, 2 mM EDTA, and 1 mM dithiothreitol) containing 0.1% (wt/vol) Triton X-100 and 50 µl/ml of rat serum to stabilize the enzyme. Samples were centrifuged at 10,000 g for 2 min. The active form of PDH (PDHa) was assayed immediately by spectrophotometry (model LS 7500; Beckman Instruments; Irvine, CA). An aliquot of 100-200 µl of the supernatant was added to a cuvette containing 1.5 ml of buffer (100 mM Tris · HCl and 1 mM MgSO4 at pH 7.8), 20 µl/ml p-(p-aminophenylazo)benzenesulfonic acid (AABS), 0.2 µl/ml mercaptoethanol, 50 µl of substrate mix, and 20 µl of arylamine acetyltransferase (AAT; EC 2.3.1.5; 60-100 mU). The substrate mix contained (in mM) 78 thiamine pyrophosphate, 34 NAD+, 80 pyruvate, and 9.8 acetyl-CoA dissolved in 1 ml of H2O. The acetylation of the AABS was established spectrophotometrically at 460 nm. Total amount of PDH (PDHt) was obtained by incubating the cardiac homogenate with PDH phosphatase isolated from the pig heart and incubated with calcium (1 mM) and magnesium (20 mM) during 30 min at 30°C before centrifugation. Supernatant (50 µl) was added to the reaction cuvette described above. The activation state, i.e., percent active PDH, was calculated as (PDHa/PDHt) × 100. PDH phosphatase was prepared from freshly removed pig hearts as described by McCormack and Denton (24). The final preparation of PDH phosphatase was free of detectable PDH activity. Total protein concentration was determined spectrophotometrically at 595 nm using the Bradford method (Bio-Rad; Munich, Germany) (3).

Statistical Analysis

Results are presented as means ± SD. Data of myocardial metabolism and contractile function were analyzed by unpaired t-test for repeat measurements. Cumulative release of creatine kinase and PDH activity were analyzed by unpaired t-test. Statistical tests were conducted using StatView II software for Apple Macintosh (Abacus Concepts; Berkeley, CA). Differences were considered significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Ischemia And Reperfusion on Myocardial Contractile Function, O2 Consumption, and Release of Creatine Kinase

Figure 1 depicts LV diastolic pressure, DLVP, and myocardial O2 consumption of control hearts and hearts subjected to either 20 or 35 min of no-flow ischemia. In control hearts, contractile function was stable throughout the 120-min observation period. DLVP averaged 78 ± 2.3 mmHg after 10 min of perfusion with EE-KH and 82.5 ± 3.4 mmHg after 120 min (see Fig. 1).


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  		Fig. 1.   Time course of left ventricular (LV) diastolic pressure (A), developed LV pressure (B), and myocardial O2 consumption (C) in control hearts (open circle , n = 12), hearts subjected to 20 min of ischemia (black-lozenge , n = 12), hearts subjected to 35 min of ischemia (, n = 11), and hearts subjected to 35 min of ischemia and subsequently reperfused with ruthenium red (RR)-containing medium (black-triangle, n = 6). Isolated rat hearts were perfused in a retrograde manner with erythrocyte-enriched Krebs-Henseleit buffer containing 0.4 mM palmitate and 11 mM glucose. Ruthenium red was administered during the initial 40 min of reperfusion. Values are means ± SD. *P < 0.05 vs. control hearts; **P < 0.01 vs. control hearts; +P < 0.05 and ++P < 0.01 vs. hearts subjected to 35 min of ischemia.

Hearts subjected to 20 min of no-flow ischemia exhibited only a slight increase of LV end-diastolic pressure at the end of the ischemic period (8.0 ± 0.9 vs. 0.1 ± 0.2 mmHg in control hearts; P = not significant), which returned to baseline during reperfusion. DLVP recovered rapidly after the onset of reperfusion and averaged 83% of the corresponding value of control hearts after 5 min and 99% after 60 min. Myocardial O2 consumption returned to baseline within 5 min after the onset of reperfusion and remained constant thereafter. Myocardial release of creatine kinase was low during reperfusion after 20 min of no-flow ischemia and did not significantly differ from control hearts (see Fig. 2).


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  		Fig. 2.   Cumulative myocardial release of creatine kinase (CK) during the last 60 min of perfusion of control hearts or during 60 min of reperfusion of hearts subjected to 20 or 35 min of ischemia without or with ruthenium red. Values are means ± SD. **P < 0.01 vs. control hearts; +P < 0.05 vs. hearts subjected to 35 min of ischemia.

Hearts subjected to 35 min of no-flow ischemia developed marked diastolic contracture later than 20 min after the onset of ischemia with LV diastolic pressure averaging 52 ± 2.3 mmHg at the end of the ischemic period (P < 0.01 vs. control hearts). LV diastolic pressure did not decrease during the 60-min reperfusion period. Recovery of DLVP was incomplete, averaging after 60 min of reperfusion 37% of the value measured in the control group (P < 0.01). Despite poor recovery of contractile function, myocardial O2 consumption rapidly returned to baseline and 5 min after the onset of reperfusion averaged 98% of the value measured in the control group (P = not significant). Myocardial O2 consumption subsequently gradually decreased to 59.8% of control after 60 min (P < 0.01). Cumulative myocardial release of creatine kinase during reperfusion was increased sixfold compared with control hearts (P < 0.01; see Fig. 2).

Glucose Oxidation, Glycolytic Rate, and Lactate Oxidation During Postischemic Reperfusion

During reperfusion after 20 min of no-flow ischemia, glucose oxidation did not differ from values measured in control hearts (see Fig. 3). In contrast, in hearts subjected to 35 min of no-flow ischemia without intervention, glucose oxidation was increased sevenfold compared with control hearts at 15 min after the onset of reperfusion (see Fig. 3). Glucose oxidation subsequently gradually returned toward baseline but was still slightly elevated after 60 min.


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  		Fig. 3.   Time course of myocardial oxidation of glucose in control hearts (open circle , n = 6), hearts subjected to 20 min of no-flow ischemia (black-lozenge , n = 7), and hearts subjected to 35 min of no-flow ischemia and reperfused without (, n = 6) and with (black-triangle, n = 6) ruthenium red. Hearts were perfused as described for Fig. 1. Myocardial oxidation of glucose was measured based on myocardial release of 14CO2 from [U-14C]glucose. Values are means ± SD. *P < 0.05 and **P < 0.01 vs. control hearts; +P < 0.05 vs. hearts subjected to 35 min of no-flow ischemia.

In contrast to glucose oxidation, the glycolytic rate did not significantly differ from that of control hearts after both 20 min and 35 min of ischemia (see Fig. 4).


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  		Fig. 4.   Glycolytic flux measured after 60 min of control perfusion (control) or 15 min of reperfusion after 20 or 35 min of no-flow ischemia without or with RR. Hearts were perfused as described for Fig. 1. Glycolytic flux was measured based on myocardial release of 3H2O from [5-3H]glucose. Differences were statistically not significant. Values are means ± SD.

Because oxidation of lactate more closely reflects activity of PDH than glucose oxidation, myocardial oxidation of [1-14C]lactate was measured in additional experiments. Similar to oxidation of glucose, myocardial oxidation of lactate was not altered in hearts reperfused after 20 min of no-flow ischemia but was markedly increased during reperfusion in hearts subjected to 35 min of no-flow ischemia (see Fig. 5).


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  		Fig. 5.   Time course of myocardial oxidation of lactate in control hearts (open circle , n = 5) and hearts subjected to 20 min of ischemia (black-lozenge , n = 5) or 35 min of ischemia (, n = 5). Hearts were perfused as described for Fig. 1. Myocardial oxidation of lactate was measured based on release of 14CO2 from [1-14C]lactate. Values are means ± SD. *P < 0.05 and **P < 0.01 vs. control hearts.

Myocardial Activity of PDH

Total PDH activity after enzymatic dephosphorylation was comparable among the control group, the 20-min ischemia group, and the 35-min ischemia group (4.9 ± 1.7, 5.2 ± 1.1, and 4.0 ± 0.8 nmol · min-1 · mg protein-1, respectively; values measured after 60 min control perfusion or 5 min after the onset of reperfusion). In control hearts, the proportion of active PDH complex averaged 13.4 ± 6.3% at the end of 20 min of equilibration with EE-KH and remained stable throughout the perfusion protocol (18.0 ± 11.0% after 120 min of perfusion).

Five minutes after the onset of reperfusion after 20 min of no-flow ischemia, the active fraction of the PDH was modestly but significantly increased to 38.0 ± 12.3% (P < 0.05; Fig. 6A). Activation of PDH was entirely reversible after 60 min of reperfusion (see Fig. 6B).


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  		Fig. 6.   Effect of transient ischemia on activation of PDH after 5 min (A) and 60 min of reperfusion (B). Values of control hearts were obtained after 60 min (A) and 120 min (B) of perfusion at control flow. Bars on the far right display the values obtained in hearts subjected to 35 min of no-flow ischemia and reperfusion with medium containing RR. Values are means ± SD. *P < 0.05 and **P < 0.01 vs. control hearts.

In hearts subjected to 35 min of no-flow ischemia there was a sixfold increase in the active PDH fraction 5 min after the onset of reperfusion to 72.8 ± 15.2% (P < 0.01; Fig. 6A). PDH activity decreased during the reperfusion period but still averaged 52.0 ± 18.6% after 60 min (P < 0.01; Fig. 6B).

Effects of Ruthenium Red

To determine the potential involvement of mitochondrial calcium uptake in the activation of PDH after 35 min of no-flow ischemia, ruthenium red (6 µM) was added to the perfusate to block the mitochondrial Ca2+ uniporter at the inner mitochondrial membrane. In nonischemic control hearts (n = 3), ruthenium red exerted a pronounced negative inotropic effect with a reduction in DLVP after 30 min of exposure by 45% (P < 0.01). Myocardial O2 consumption was reduced by 61% from 3.3 ± 0.2 to 1.2 ± 0.7 nmol · min-1 · g wet wt-1 (P < 0.01), and oxidation of glucose was reduced by 62% from 18.2 ± 6.0 to 6.9 ± 4.4 nmol · min-1 · g wet wt-1 (P < 0.05).

Consistent with previous observations from our laboratory (1), ruthenium red markedly reduced diastolic contracture and improved recovery of contractile function during reperfusion in hearts subjected to 35 min of no-flow ischemia (see Fig. 1). Specifically, LV diastolic pressure steadily decreased during ruthenium red infusion to 12.2 ± 8.0 mmHg (P < 0.05 vs. control hearts) at the end of the 40-min infusion and only slightly increased after cessation of ruthenium red infusion to 20.6 ± 11.4 mmHg (vs. 60.2 ± 9.7 mmHg in untreated postischemic hearts; P < 0.01).

DLVP recovered completely during ruthenium red infusion and remained high after cessation of ruthenium red administration (84.1 ± 9.5 mmHg at the end of the observation period; P = not significant vs. control hearts). Finally, cumulative myocardial release of creatine kinase was reduced by ruthenium red and did not differ significantly from control hearts.

Myocardial O2 consumption was slightly lower early during reperfusion in the presence of ruthenium red compared with untreated postischemic hearts despite higher contractile function, which indicates enhancement of metabolic efficiency (see Fig. 1). Ruthenium red did not alter glycolytic flux (see Fig. 4). However, glucose oxidation was markedly reduced during reperfusion by ruthenium red compared with untreated hearts subjected to 35 min of no-flow ischemia (by 55.7% after 15 min of reperfusion; Fig. 3). By 30 min of reperfusion, glucose oxidation in the ruthenium red-treated hearts did not differ significantly from nonischemic control hearts.

Ruthenium red completely abolished postischemic activation of PDH in hearts subjected to 35 min of no-flow ischemia (see Fig. 6). Relative PDH activity closely matched the corresponding values in nonischemic control hearts after 5 min (14.3 ± 6.0 vs. 13.4 ± 5.8%; P = not significant) and 60 min (14.0 ± 11.4 vs. 18.6 ± 11.0%; P = not significant) of reperfusion.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

There is increasing evidence that activity of PDH early during reperfusion may critically influence recovery of postischemic myocardium (27). In this study, we measured in isolated rat hearts perfused with fatty acid-containing medium the effect of increasing degrees of ischemic injury on myocardial glucose oxidation and activity of PDH during reperfusion. The main findings are the following: 1) PDH is largely inhibited in hearts isolated from fasted rats and perfused with 11 mM glucose and 0.4 mM palmitate; 2) PDH is activated after transient ischemia, whereby the degree and duration of activation increases with increasing severity of the ischemic insult; and 3) pronounced activation of PDH in advanced ischemic injury seems to be related to mitochondrial calcium uptake.

Characteristics of the Model

To identify activation of PDH during postischemic reperfusion, experimental conditions were selected to maintain low baseline activation during perfusion without intervention. This was achieved by fasting the rats for 24 h before experimentation and inclusion of a physiological concentration of palmitate (0.4 mM) in the perfusate. Accordingly, the proportion of active PDH was low (13.4 and 18.0% after 20 min and 120 min of perfusion, respectively) in control hearts perfused for 120 min without intervention. We have previously observed in this model (29) that fasting results in high fatty acid oxidation rates and low glucose oxidation. The concomitant increase of myocardial acetyl-CoA content in the latter experiments (29) most likely contributed to inhibition of PDH by stimulation of PDH kinase-mediated phosphorylation.

Twenty minutes of no-flow ischemia resulted in entirely reversible postischemic injury on the basis of complete recovery of both contractile function and oxidative metabolism and the absence of excess creatine kinase release compared with control hearts. In contrast, 35 min of no-flow ischemia most likely resulted in a mixture of reversible and irreversible injury indicated by marked diastolic contracture, incomplete recovery of DLVP, and enhancement of release of creatine kinase. It is likely that myocardial injury was aggravated early during reperfusion because ventricular contracture and enzyme release were markedly reduced by ruthenium red, which was administered exclusively during the initial 40 min of reperfusion.

Consistent with previous observations (9, 30), reperfusion after 35 min of ischemia resulted in complete recovery of myocardial O2 consumption early after restoration of perfusion despite persistent depression of contractile function, which suggests reduced efficiency of oxidative metabolism in terms of contractile function (9, 30). Because efficiency of oxidative metabolism was normalized by ruthenium red [an inhibitor of the mitochondrial calcium uniporter (4)], contraction-independent stimulation of the respiratory chain seems to be related to increased calcium concentration in the mitochondrial matrix (37). At least two possible mechanisms of stimulation of oxidative metabolism by mitochondrial calcium uptake may be considered: 1) dissipation of respiratory energy by calcium-induced mitochondrial permeability pore transition (8, 40) may enhance metabolism; and 2) oxidative metabolism may be accelerated by activation of calcium-sensitive dehydrogenases in the tricarboxylic acid cycle, including isocitrate dehydrogenase and oxoglutarate dehydrogenase (24, 32).

Degree of Activation of PDH During Reperfusion

The results of the present study indicate that in myocardium exhibiting low baseline activity of PDH, the multienzyme complex is activated early during postischemic reperfusion. However, the extent and duration of activation critically depend on the degree of the preceding ischemic insult: the active fraction of PDH was increased to 38.0% after 20 min of no-flow ischemia and to 72.8% after 35 min of no-flow ischemia. Activation of PDH was completely reversible after 60 min of reperfusion after 20 min of ischemia, whereas PDH activity remained elevated throughout the entire reperfusion period after 35 min of ischemia. Thus far, only a few studies have directly measured PDH activity in reperfused myocardium by tissue analysis. Consistent with the results of the present study, Pepe and colleagues (34) have recently observed a threefold increase of the active fraction of PDH in isolated rat hearts reperfused for 5 min after 15 min low-flow ischemia with medium containing 11 mM glucose and 0.2 mM octanoate. To our knowledge, PDH activity has not been measured during reperfusion after prolonged ischemia leading to stimulation of glucose oxidation.

In contrast to the present study, Kobayashi and Neely (13) have observed a reduction of PDH activity early during reperfusion. However, in their study, hearts were perfused with medium containing glucose as the sole substate, which resulted in a high baseline activation of PDH (exceeding 70% of total activity). Five minutes after the onset of reperfusion after 10 min of low-flow ischemia, the active fraction of PDH had dropped to ~60%, which is still higher than the value measured in the present study after 20 min of no-flow ischemia.

To examine the metabolic consequences of observed activation of PDH in this study, oxidation of either glucose or lactate (which more directly reflects activity of PDH) has been determined. Although PDH was slightly and transiently activated 5 min after the onset of reperfusion after 20 min of ischemia, neither glucose nor lactate oxidation was concomitantly increased. The reason for the dissociation between PDH activity and carbohydrate oxidation in reversibly injured postischemic myocardium is not apparent from the data of this study. However, it needs to be emphasized that substrate supply and accumulation of metabolic products may modulate flux through the PDH reaction independently of enzyme activity. In the present study, limited supply of pyruvate is unlikely to limit flux throughout PDH, because glycolytic flux was similar in hearts subjected to 20 or 35 min of ischemia. It is possible that the accumulation of acetyl-CoA and NADH that originated from fatty acid oxidation may have lowered flux throughout the PDH reaction despite the higher intrinsic activity of the enzyme. In fact, Kudo and colleagues (15) have shown in rat hearts that fatty acid oxidation is stimulated early during reperfusion.

Pronounced activation of PDH during reperfusion after 35 min of no-flow ischemia was associated with a marked increase of oxidation of both glucose and lactate. Enhancement of myocardial glucose oxidation during reperfusion has been observed previously after prolonged severe ischemia in isolated hearts (9, 26, 42) and animals in vivo (31, 36). On the other hand, in working rat hearts reperfused after 25 to 30 min of no-flow ischemia with medium containing 11 mM glucose and 1.2 mM palmitate, glucose oxidation did not exceed preischemic levels, which is compatible with absent or less-pronounced activation of PDH (19, 20, 39). This interpretation is indirectly supported by the marked stimulation of glucose oxidation in response to pharmacological activation of PDH by dichloroacetate (27). The apparent absence of spontaneous stimulation of PDH during reperfusion in the latter studies may be related either to the lower severity of ischemic injury or the high fatty acid concentration in the perfusate.

Indirect Evidence for a Role of Mitochondrial Calcium Uptake in Postischemic Activation of PDH

Activation of PDH after 35 min of no-flow ischemia was completely abolished by the addition of 6 µM ruthenium red to the perfusate at the moment of reperfusion. Because ruthenium red blocks the mitochondrial calcium uniporter (4), the results suggest that mitochondrial calcium uptake mediates postischemic activation of PDH by stimulation of PDH phosphatase. However, it needs to be emphasized that ruthenium red is not selective for blocking the mitochondrial calcium uniporter (12); in normal myocardium, this agent also inhibits sarcolemmal calcium binding and reduces release of calcium from the sarcoplasmic reticulum, which explains the negative inotropic effect observed in control hearts of this study (10). Furthermore, recent evidence suggests that ruthenium red may directly interact with enzymes including smooth muscle myosin light chain phosphatase (44). Although the precise mechanism of prevention of activation of PDH by ruthenium red is not known, indirect evidence suggests that reduction of mitochondrial calcium overload is involved. Pepe and co-workers (34) recently measured the effects of ruthenium red on mitochondrial calcium content by atomic absorption spectrometry. The authors observed an increase of calcium content in mitochondria isolated from perfused rat hearts submitted to 15 min of low-flow ischemia and a subsequent 5-min period of reperfusion. In these experiments, PDH was activated to a comparable extent as in the 20-min experiments of the present study. Increase of both mitochondrial calcium content and PDH activity were completely prevented by 3.4 µM ruthenium red. Therefore, mitochondrial calcium uptake is likely to be involved in activation of PDH even after comparatively mild injury.

Glucose Oxidation and Myocardial Injury

A number of observations indicate that pronounced mitochondrial calcium overload is involved in the transition from reversible to irreversible myocardial injury early during reperfusion (7, 28). Accordingly, irreversible injury and activation of PDH after 35 min of no-flow ischemia are likely to be epiphenomena of mitochondrial calcium overload. Therefore, activation of glucose oxidation is not necessarily implicated in the mechanisms that underlie irreversible myocardial injury. On the contrary, there is increasing evidence that activation of PDH improves myocardial recovery (16, 27, 43). Consistent with this interpretation, we previously observed in rat hearts using the same perfusion protocol that withdrawal of glucose during reperfusion markedly aggravated myocardial contracture and enzyme release (42). Furthermore, LV end-diastolic pressure was inversely related to glucose oxidation during reperfusion but not to the glycolytic rate (42). This observation indirectly suggests that activation of PDH during reperfusion in severely injured myocardium is likely to counteract irreversible injury.


    ACKNOWLEDGEMENTS

The authors thank Dr. Françoise Assimacopoulos-Jeanneret for helpful advice regarding the PDH determination.


    FOOTNOTES

This study was supported by the Swiss National Science Foundation Grants 3200-04561.95 and 32-56779-99.

Address for reprint requests and other correspondence: R. Lerch, Cardiology Center, Univ. Hospital, CH-1211 Geneva 14, Switzerland (E-mail: rene.lerch{at}hcuge.ch).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 November 2000; accepted in final form 20 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Benzi, RH, and Lerch R. Dissociation between contractile function and oxidative metabolism in postischemic myocardium. Attenuation by ruthenium red administered during reperfusion. Circ Res 71: 567-576, 1992[Abstract/Free Full Text].

2.   Bergmann, SR, Weinheimer CJ, Brown MA, and Perez JE. Enhancement of regional myocardial efficiency and persistence of perfusion, oxidation, and functional reserve with paired pacing of stunned myocardium. Circulation 89: 2290-2296, 1994[Abstract/Free Full Text].

3.   Bradford, MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[Web of Science][Medline].

4.   Broekeimer, KM, Kresbsbach RJ, and Pfeiffer DR. Inhibition of the mitochondrial Ca2+ uniporter by pure and impure ruthenium red. Mol Cell Biochem 139: 33-40, 1994[Web of Science][Medline].

5.   Clarke, B, Wyatt KM, and McCormack JG. Ranolazine increases pyruvate dehydrogenase in perfused normoxic rat hearts: evidence for an indirect mechanism. J Mol Cell Cardiol 28: 341-350, 1996[Web of Science][Medline].

6.   Depre, C, Vanoverschelde L, and Taegmeyer H. Glucose for the heart. Circulation 99: 578-588, 1999[Free Full Text].

7.   Di Lisa, F, and Bernardi P. Mitochondrial function as a determinant of recovery or death in cell response to injury. Mol Cell Biochem 184: 379-391, 1998[Web of Science][Medline].

8.   Ferrari, R, Pedersini P, Bongrazio M, Gaia G, Bernocchi P, Di Lisa F, and Visioli O. Mitochondrial energy production and cation control in myocardial ischemia and reperfusion. Basic Res Cardiol 88: 495-512, 1993[Web of Science][Medline].

9.   Görge, G, Chatelain P, Schaper J, and Lerch R. Effect of increasing degrees of ischemic injury on myocardial oxidative metabolism early after reperfusion in isolated rat hearts. Circ Res 68: 1681-1692, 1991[Abstract/Free Full Text].

10.   Gupta, MP, Dixon IMC, Zhao D, and Dhalla NS. Influence of ruthenium red on rat heart subcellular calcium transport. Can J Cardiol 5: 55-63, 1989[Web of Science][Medline].

11.   Hammerstedt, RH. The use of Dowex-1-borate to separate 3HOH from 2-3H-glucose. Anal Biochem 56: 292-293, 1973[Web of Science][Medline].

12.   Kargacin, GJ, Ali Z, and Kargacin ME. Ruthenium red reduces the Ca2+ sensitivity of Ca2+ uptake into cardiac sarcoplasmic reticulum. Pflügers Arch 436: 338-342, 1998[Web of Science][Medline].

13.   Kobayashi, K, and Neely JR. Effects of ischemia and reperfusion on pyruvate dehydrogenase activity in isolated rat hearts. J Mol Cell Cardiol 6: 359-367, 1983.

14.   Kofoed, KF, Schoeder H, Knight RJ, and Buxton DB. Glucose metabolism in reperfused myocardium measured by [2-18F] 2-fluorodeoxyglucose and PET. Cardiovasc Res 45: 321-329, 2000[Abstract/Free Full Text].

15.   Kudo, N, Gillespie JG, Kung L, Witters LA, Schulz R, Clanachan AS, and Lopaschuk GD. Characterization of 5' AMP-activated protein kinase activity in the heart and its role in inhibiting acetyl-CoA carboxylase during reperfusion. Biochim Biophys Acta 1301: 67-75, 1996[Medline].

16.   Lewandowski, ED, Lawrence T, and White BS. Pyruvate dehydrogenase influences postichemic heart function. Circulation 91: 2071-2079, 1995[Abstract/Free Full Text].

17.   Liedtke, AJ, DeMaison L, Eggleston AM, Cohen LM, and Nellis SH. Changes in substrate metabolism and effects of excess fatty acids in reperfused myocardium. Circ Res 62: 535-542, 1988[Abstract/Free Full Text].

18.   Liedtke, AJ, Renstroem B, and Nellis SH. Correlation between [5-3H]glucose and [U-14C]deoxyglucose as markers of glycolysis in reperfused myocardium. Circ Res 71: 689-700, 1992[Abstract/Free Full Text].

19.   Lopaschuk, GD, and Saddik M. The relative contribution of glucose and fatty acid to ATP production in hearts reperfused following ischemia. Mol Cell Biochem 116: 111-116, 1992[Web of Science][Medline].

20.   Lopapschuk, GD, Spafford MA, Davies NJ, and Wall SR. Glucose and palmitate oxidation in isolated working rat hearts reperfused after a period of transient global ischemia. Circ Res 66: 546-553, 1990[Abstract/Free Full Text].

21.   Luft, JH. Ruthenium red and violet. Chemestry, purification, methods of use for electron microscopy and mechanism of action. Anat Rec 171: 347-368, 1971[Medline].

22.   Mallet, RT, Hartmann DA, and Bünger R. Glucose requirement for postischemic recovery of perfused working heart. Eur J Biochem 188: 481-493, 1990[Web of Science][Medline].

23.   McCormack, JG, and Denton RM. Influence of calcium ions on mammalian intramitochondrial dehydrogenases. Methods Enzymol 174: 95-118, 1989[Web of Science][Medline].

24.   McCormack, JG, and Denton RM. A comparative study of the regulation of Ca2+ of the activities of the 2-oxoglutarate dehydrogenase complex and NAD+-isocitrate dehydrogenase from a variety of sources. Biochem J 196: 619-624, 1981[Web of Science][Medline].

25.   McCormack, JG, Halestrap AP, and Denton RM. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev 70: 391-425, 1990[Free Full Text].

26.   McGowan, FX, Jr, Lee FA, Chen V, and Downing SE. Oxidative metabolism and mechanical function in reperfused neonatal pig heart. J Mol Cell Cardiol 24: 831-840, 1992[Web of Science][Medline].

27.   McVeight, JJ, and Lopapschuk GD. Dichloroacetate stimulation of glucose oxidation improves recovery of ischemic rat hearts. Am J Physiol Heart Circ Physiol 259: H1079-H1085, 1990[Abstract/Free Full Text].

28.   Miyata, H, Lakatta EG, Stern MD, and Silverman HS. Relation of mitochondrial and cytosolic free calcium to cardiac myocyte recovery after exposure to anoxia. Circ Res 71: 605-613, 1992[Abstract/Free Full Text].

29.   Montessuit, C, Papageorgiou I, Tardy I, and Lerch R. Effect of nutritional state on substrate metabolism and contractile function in postischemic rat myocardium. Am J Physiol Heart Circ Physiol 271: H2060-H2070, 1996[Abstract/Free Full Text].

30.   Montessuit, C, Papageorgiou I, Tardy-Cantalupi I, Rosenblatt-Velin N, and Lerch R. Postischemic recovery of heart metabolism and function: role of mitochondrial fatty acid transfer. J Appl Physiol 89: 111-119, 2000[Abstract/Free Full Text].

31.   Myears, DW, Sobel BE, and Bergmann SR. Substrate use in ischemic and reperfused canine myocardium: quantitative considerations. Am J Physiol Heart Circ Physiol 253: H107-H114, 1987[Abstract/Free Full Text].

32.   Nichols, BJ, Rigoulet M, and Denton RM. Comparison of the effect of Ca2+, adenine nucleotides, and pH on the kinetic properties of mitochondrial NAD+-isocitrate dehydrogenase and oxoglutarate dehydrogenase from yeast Saccharomyces cerevisiae and rat heart. Biochem J 303: 461-465, 1994.

33.   Patel, TB, and Olson MS. Regulation of pyruvate dehydrogenase complex in ischemic rat heart. Am J Physiol Heart Circ Physiol 246: H858-H864, 1984.

34.   Pepe, S, Tsuchiya N, Lakatta EG, and Hansford R. PUFA and aging modulate cardiac mitochondrial membrane lipid composition and Ca2+ activation of PDH. Am J Physiol Heart Circ Physiol 276: H149-H158, 1999[Abstract/Free Full Text].

35.   Randle, PJ. Fuel selection in animals. Biochem Soc Trans 14: 799-806, 1986[Web of Science][Medline].

36.   Renstrom, B, Nellis SH, and Liedtke AJ. Metabolic oxidation of glucose during early myocardial reperfusion. Circ Res 65: 1094-1101, 1989[Abstract/Free Full Text].

37.   Robb-Gaspers, LD, Burnett P, Rutter GA, Denton RM, Rizzuto R, and Thomas AP. Integrating cytosolic calcium signals into mitochondrial metabolic responses. EMBO J 17: 4987-5000, 1998[Web of Science][Medline].

38.   Schoeder, H, Knight RJ, Kofoed KF, Schelbert HR, and Buxton DB. Regulation of pyruvate dehydrogenase activity and glucose metabolism in post-ischaemic myocardium. Biochim Biophys Acta 1406: 62-72, 1998[Medline].

39.   Stanley, WC, Lopaschuk GD, Hall JL, and McCormack JG. Regulation of myocardial carbohydrate metabolism under normal and ischaemic conditions. Potential for pharmacological interventions. Cardiovasc Res 33: 243-257, 1997[Free Full Text].

40.   Steenbergen, C, Murphy E, Watts JA, and London RE. Correlation between cytosolic free calcium, contracture, ATP, and irreversible ischemic injury in perfused rat heart. Circ Res 66: 135-146, 1990[Abstract/Free Full Text].

41.   Szasz, G, Gruber W, and Bernt E. Creatine kinase in serum. I. Determination of optimum reaction conditions. Clin Chem 22: 650-656, 1976[Abstract/Free Full Text].

42.   Tamm, C, Benzi RH, Papageorgiou I, Tardy I, and Lerch R. Substrate competition in postischemic myocardium: effect of substrate availability during reperfusion on metabolic and contractile recovery in isolated rat hearts. Circ Res 75: 1103-1112, 1994[Abstract/Free Full Text].

43.   Veith, K, Moisin L, and Hue L. Trimetazidine effects on the damage to mitochondrial functions caused by ischemia and reperfusion. Am J Cardiol 76: 25B-30B, 1995[Medline].

44.   Yamada, A, Ohya S, Hirano M, Watanabe M, Walsh MP, and Imaizumi Y. Ca2+ sensitization of smooth muscle contractility induced by ruthenium red. Am J Physiol Cell Physiol 276: C566-C575, 1999[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 281(2):H722-H730
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