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

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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Probing the link between citrate and malonyl-CoA in perfused rat hearts

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00244.2002 ◽

2002 ◽

Vol 283 (4) ◽

pp. H1379-H1386 ◽

Cited By ~ 17

Author(s):

Myriame Poirier ◽

Geneviève Vincent ◽

Aneta E. Reszko ◽

Bertrand Bouchard ◽

Joanne K. Kelleher ◽

...

Keyword(s):

Carbon Sources ◽

Acid Oxidation ◽

Atp Citrate Lyase ◽

Acetyl Coa ◽

Rat Hearts ◽

Citrate Lyase ◽

Malonyl Coa ◽

Perfused Rat Hearts ◽

Citrate Release

Little is known about the sources of cytosolic acetyl-CoA used for the synthesis of malonyl-CoA, a key regulator of fatty acid oxidation in the heart. We tested the hypothesis that citrate provides acetyl-CoA for malonyl-CoA synthesis after its mitochondrial efflux and cleavage by cytosolic ATP-citrate lyase. We expanded on a previous study where we characterized citrate release from perfused rat hearts (Vincent G, Comte B, Poirier M, and Des Rosiers C. Citrate release by perfused rat hearts: a window on mitochondrial cataplerosis. Am J Physiol Endocrinol Metab 278: E846–E856, 2000). In the present study, we show that citrate release rates, ranging from 6 to 22 nmol/min, can support a net increase in malonyl-CoA concentrations induced by changes in substrate supply, at most 0.7 nmol/min. In experiments with [U-13C](lactate + pyruvate) and [1-13C]oleate, we show that the acetyl moiety of malonyl-CoA is derived from both pyruvate and long-chain fatty acids. This 13C-labeling of malonyl-CoA occurred without any changes in its concentration. Hydroxycitrate, an inhibitor of ATP-citrate lyase, prevents increases in malonyl-CoA concentrations and decreases its labeling from [U-13C](lactate + pyruvate). Our data support at least a partial role of citrate in the transfer from the mitochondria to cytosol of acetyl units for malonyl-CoA synthesis. In addition, they provide a dynamic picture of malonyl-CoA metabolism: even when the malonyl-CoA concentration remains constant, there appears to be a constant need to supply acetyl-CoA from various carbon sources, both carbohydrates and lipids, for malonyl-CoA synthesis.

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Mathematical analysis of isotope labeling in the citric acid cycle with applications to 13C NMR studies in perfused rat hearts.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)43987-1 ◽

1983 ◽

Vol 258 (22) ◽

pp. 13785-13794 ◽

Cited By ~ 8

Author(s):

E M Chance ◽

S H Seeholzer ◽

K Kobayashi ◽

J R Williamson

Keyword(s):

Citric Acid ◽

13C Nmr ◽

Mathematical Analysis ◽

Citric Acid Cycle ◽

Isotope Labeling ◽

Nmr Studies ◽

Acid Cycle ◽

Rat Hearts ◽

Perfused Rat Hearts

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13C-NMR spectroscopic evaluation of the citric acid cycle flux in conditions of high aspartate transaminase activity in glucose-perfused rat hearts

Biochimie ◽

10.1016/s0300-9084(99)80006-6 ◽

1998 ◽

Vol 80 (12) ◽

pp. 1013-1024 ◽

Cited By ~ 1

Author(s):

Son Tran-Dinh ◽

Jacqueline A. Hoerter ◽

Philippe Mateo ◽

Franck Gyppaz ◽

Martine Herve

Keyword(s):

Citric Acid ◽

13C Nmr ◽

Citric Acid Cycle ◽

Aspartate Transaminase ◽

Transaminase Activity ◽

Acid Cycle ◽

Rat Hearts ◽

Perfused Rat Hearts

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Probing the Origin of Acetyl-CoA and Oxaloacetate Entering the Citric Acid Cycle from the13C Labeling of Citrate Released by Perfused Rat Hearts

Journal of Biological Chemistry ◽

10.1074/jbc.272.42.26117 ◽

1997 ◽

Vol 272 (42) ◽

pp. 26117-26124 ◽

Cited By ~ 43

Author(s):

Blandine Comte ◽

Geneviève Vincent ◽

Bertrand Bouchard ◽

Christine Des Rosiers

Keyword(s):

Citric Acid ◽

Citric Acid Cycle ◽

Acetyl Coa ◽

Acid Cycle ◽

Rat Hearts ◽

Perfused Rat Hearts

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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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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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