Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Abstract

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.

Fig 1

Inhibition of MPTP, MCU as well as SR Ca²⁺ uptake or release routes significantly attenuated spontaneous Ca²⁺ oscillations during cardiomyocyte reperfusion. (A–B) Single cell recordings of cytosolic fura-2 ratio (340/380 nm) during reperfusion under control conditions or under treatment with ryanodine. Dashed line represents pre-ischemic cytosolic fura-2 ratio at the beginning of ischemia. Arrow indicates the time of the fura-2 loss due to necrosis. (C) Maximal amplitude of cytosolic Ca²⁺ oscillation defined between 5th and 10th min. of reperfusion in control cells or in cells treated during reperfusion with ryanodine (5 μmol/l), thapsigargine (100 nmol/l), Ru360 (1 μmol/l) or cyclosporine A (0.5 μmol/l). (D) Maximal oscillation frequency of cytosolic Ca²⁺ (min.⁻¹) defined between 5th and 10th min. of reperfusion. Similar treatments as in (C). Data are mean ± S.E.M., n= 52–60 cells from five different preparations. *P < 0.05 versus control.

Fig 2

Time course of the mitochondrial calcein fluorescence (in percentage to normoxic level) during 80 min. of ischemia and 20 min. of reperfusion in control cells (•) or in cells treated during reperfusion with 5 μmol/l ryanodine (♦), 100 nmol/l thapsigargine (◊), 1 μmol/l Ru360 (▪) or 0.5 μmol/l cyclosporine A (□). Data are mean ± S.E.M., n= 44–48 cells from five different preparations. *P < 0.05 versus control. Note that in control cells a dramatic loss of the calcein fluorescence was found between 5th and 10th min. of reperfusion indicating MPTP opening.

Fig 3

Time course of ΔΨ expressed as JC-1 fluorescence ratio (590/530 nm) during 80 min. of ischemia and 20 min. of reperfusion in control cells (•) or in cells treated during reperfusion with 5 μmol/l ryanodine (♦), 100 nmol/l thapsigargine (◊), 1 μmol/l Ru360 (▪) or 0.5 μmol/l cyclosporine A (□). Data are mean ± S.E.M., n= 44–50 cells from five different preparations. *P < 0.05 versus control. Values at the beginning of reperfusion were set to 100%. The decline of the JC-1 ratio represents mitochondrial membrane depolarization.

Fig 4

Time course of the mitochondrial calcium concentration indicated by fura-2 fluorescence ratio (340/380 nm) in the presence of MnCl₂ (to bleach the fluorescence of cytosolic fura-2) during 80 min. of ischemia and 20 min. of reperfusion in control cells (•) or in cells treated during reperfusion with 5 μmol/l ryanodine (♦), 100 nmol/l thapsigargine (◊), 1 μmol/l Ru360 (▪) or 0.5 μmol/l cyclosporine A (□). Data are mean ± S.E.M., n= 48–55 cells from five different preparations. The rise of the fura-2 ratio represents the increase of the mitochondrial Ca²⁺ concentration.

Fig 5

Time course of cellular Mg²⁺-concentration measured by mag-fura-2 fluorescence ratio (340/380 nm) during 80 min. of ischemia and 20 min. of reperfusion in control cells (•) or in cells treated during reperfusion with 5 μmol/l ryanodine (♦), 100 nmol/l thapsigargine (◊), 1 μmol/l Ru360 (▪) or 0.5 μmol/l cyclosporine A (□). Data are mean ± S.E.M., n= 44–48 cells from five different preparations. *P < 0.05 versus control. Values at the beginning of reperfusion were set to 100%. The rise of the mag-fura-2 ratio represents the increase of the intracellular Mg²⁺ concentration.

Fig 6

Time course of mitochondrial ROS measured as MitoSox fluorescence during 80 min. of ischemia and 20 min. of reperfusion in control cells (•) or in cells treated during reperfusion with 5 μmol/l ryanodine (♦), 100 nmol/l thapsigargine (◊), 1 μmol/l Ru360 (▪), 0.5 μmol/l cyclosporine A (□), 100 μmol/l 2-MPG (▴) or 500 μmol/l NAC (▵). Data are mean ± S.E.M., n= 44–48 cells from five different preparations. *P < 0.05 versus control. Values at the beginning of reperfusion were set to 100%.

Fig 7

Inhibition of MPTP, MCU and SR Ca²⁺ uptake or release routes significantly reduced reperfusion-induced cardiomyocyte hypercontracture and necrosis. (A) Length of cardiac myocytes after 20 min. of reperfusion (in percentage to end-ischemic length) under control conditions or in the presence of 5 μmol/l ryanodine, 100 nmol/l thapsigargine, 1 μmol/l Ru360, 0.5 μmol/l cyclosporine A, 100 μmol/l 2-MPG or 500 μmol/l NAC. Data are mean ± S.E.M., n= 50–55 cells from five different preparations. *P < 0.05 versus control. (B) Cell necrosis after 30 min. of reperfusion defined by PI staining and expressed as a percentage of the total cell count. Data are mean ± S.E.M., n= 200–300 cells from five different preparations. *P < 0.05 versus control.