Evaluation of cellular energetics by the pasteur effect in intact cardiomyoblasts and isolated perfused hearts.
This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (delta-lactate = stimulated [lactate] -basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 micromol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control delta-lactate was 20.9 +/- 2.31 (S.E.M.) microg/mL and markedly decreased after reperfusion (7.66 +/- 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when delta-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.
Muscari
Claudio
C
Department of Biochemistry G Moruzzi, University of Bologna, Bologna, Italy. cuore@biocfarm.unibo.it
Gamberini
Chiara
C
Bonafe'
Francesca
F
Giordano
Emanuele
E
Bianchi
Cristina
C
Lenaz
Giorgio
G
Caldarera
Claudio Marcello
CM
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000968:Antimycin A / Q000494:pharmacology*; D002460:Cell Line; D004734:Energy Metabolism / Q000187:drug effects*; D066298:In Vitro Techniques; D019344:Lactic Acid / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism*; D017202:Myocardial Ischemia / Q000378:metabolism; D015428:Myocardial Reperfusion Injury / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D010477:Perfusion; D051381:Rats; D017208:Rats, Wistar
D000900:Anti-Bacterial Agents; D019344:Lactic Acid; D000968:Antimycin A