RESUMO
Background: Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated. Methods: 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting. Results: Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2. Conclusions: Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.
RESUMO
We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 µM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 µM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint.
