ryanodine and fluorexon

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.

Topics: Acetylcysteine; Adenosine Triphosphate; Animals; Calcium; Cyclosporine; Fluoresceins; Male; Membrane Potential, Mitochondrial; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocytes, Cardiac; Necrosis; Rats; Rats, Wistar; Reactive Oxygen Species; Ruthenium Compounds; Ryanodine; Thapsigargin; Tiopronin

There is solid evidence that a sudden change in mitochondrial membrane permeability (mitochondrial permeability transition, MPT) plays a critical role in reperfusion-induced myocardial necrosis. We hypothesized that sarcoplasmic reticulum (SR) Ca(2+) cycling may induce partial MPT in microdomains of close anatomic proximity between mitochondria and SR, resulting in hypercontracture and cell death. MPT (mitochondrial calcein release), cell length, and sarcolemmal rupture (Trypan blue and lactate dehydrogenase release) were measured in adult rat cardiomyocytes submitted to simulated ischemia (NaCN/2-deoxyglucose, pH 6.4) and reperfusion. On simulated reperfusion, 83 +/- 2% of myocytes developed hypercontracture. In 22 +/- 6% of cases, hypercontracture was associated with sarcolemmal disruption [Trypan blue(+)]. During simulated reperfusion there was a 25% release of cyclosporin A-sensitive mitochondrial calcein (with respect to total mitochondrial calcein content). Simultaneous blockade of SR Ca(2+) uptake and release with thapsigargin and ryanodine, respectively, significantly reduced mitochondrial calcein release, hypercontracture, and cell death during simulated reperfusion. SR Ca(2+) blockers delayed mitochondrial Ca(2+) uptake in digitonin-permeabilized cardiomyocytes but did not have any effect on isolated mitochondria. Pretreatment with colchicine to disrupt microtubule network reduced the degree of fluorescent overlap between SR and mitochondria and abolished the protective effect of SR Ca(2+) blockers on MPT, hypercontracture, and cell death during reperfusion. We conclude that SR Ca(2+) cycling during reperfusion facilitates partial mitochondrial permeabilization due to the close anatomic proximity between both organelles, favoring hypercontracture and cell death.

Topics: Animals; Calcium Signaling; Cell Death; Cells, Cultured; Colchicine; Cyclosporine; Enzyme Inhibitors; Fluoresceins; Ischemic Contracture; Male; Membrane Potential, Mitochondrial; Microtubules; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Ryanodine; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Tubulin Modulators