Fas-independent mitochondrial damage triggers cardiomyocyte death after ischemia-reperfusion

Abstract

The Fas/Fas ligand and mitochondria pathways have been involved in cell death in several cell types. We combined the genetic inactivation of the Fas receptor (lpr mice), on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of these pathways is predominantly activated during prolonged ischemia-reperfusion. Anesthetized C57BL/6JICO (control) and C57BL/6-lpr mice were pretreated with either saline or cyclosporin A (CsA; 40 mg/kg, 3 times a day), an inhibitor of the mPTP, and underwent 25 min of ischemia and 24 h of reperfusion. After 24 h of reperfusion, hearts were harvested: infarct size was assessed by 2,3,5-triphenyltetrazolium chloride staining, myocardial apoptosis by caspase 3 activity, and mitochondrial permeability transition by Ca2+-induced mPTP opening using a potentiometric approach. Infarct size was comparable in untreated control and lpr mice, ranging from 77 +/- 5% to 83 +/- 3% of the area at risk. CsA significantly reduced infarct size in control and lpr hearts. Control and lpr hearts exhibited comparable increase in caspase 3 activity that averaged 57 +/- 18 and 49 +/- 5 pmol x min(-1) x mg(-1), respectively. CsA treatment significantly reduced caspase 3 activity in control and lpr hearts. The Ca2+ overload required to open the mPTP was decreased to a similar extent in lpr and controls. CsA significantly attenuated Ca2+-induced mPTP opening in both groups. Our results suggest that the Fas pathway likely plays a minor role, whereas mitochondria are preferentially involved in mice cardiomyocyte death after a lethal ischemia-reperfusion injury.

Increased mitochondrial calcium coexists with decreased reperfusion injury in postconditioned (but not preconditioned) hearts

“…An earlier time point was not chosen to avoid large variability in mitochondrial calcium concentration among experiments, partly due to increased instability in calcium homeostasis in the early minutes of reperfusion. Previous reports from our laboratory, as well as unpublished data, show that CRC changes are detectable in the early minutes following reflow and remain present at least for several hours thereafter (2,4,23).…”

The SR/ER-mitochondria calcium crosstalk is regulated by GSK3β during reperfusion injury

“…C57BL/6 J (male, 8-10 weeks old) were anesthetized by intraperitoneal mixture of 70 mg/kg pentobarbital sodium and 0.1 mg/kg buprenorphine as described previously. 7,[38][39][40] The animals were orally intubated, ventilated and body temperature was maintained at 37°C. A left thoracotomy was performed in the fourth intercostal space.…”

“…Myocardial infarct size was then determined by triphenyltetrazolium staining as described previously. 7,[38][39][40] Statistical analysis. Data are presented as mean ± S.E.M., unless otherwise specified.…”

Effective Blockage of Both the Extrinsic and Intrinsic Pathways of Apoptosis in Mice by TAT-crmA

“…Expression of Fas and FasL are increased in hypoxic myocytes, and activation of Fas has been shown to induce apoptosis in cardiac myocytes (21,22). However, mice with a functionally relevant mutation in the extracellular portion of Fas (lpr mice) or Fas ligand (gld mice) exhibited similar infarction sizes compared with wild-type mice (23,24). In contrast, vanadyl sulfate treatment caused decreased expression of FasL, and this was correlated with reduced caspase-3 activation and reduced myocardial infarction in vivo (25).…”