Calcium-induced Permeability Transition Pore Opening Research Articles

The Obesity-Related Peptide Leptin Sensitizes Cardiac Mitochondria to Calcium-Induced Permeability Transition Pore Opening and Apoptosis

The obesity-related 16 kDa peptide leptin is synthesized primarily in white adipocytes although its production has been reported in other tissues including the heart. There is emerging evidence that leptin may contribute to cardiac pathology especially that related to myocardial remodelling and heart failure. In view of the importance of mitochondria to these processes, the goal of the present study is to determine the effect of leptin on mitochondria permeability transition pore opening and the potential consequence in terms of development of apoptosis. Experiments were performed using neonatal rat ventricular myocytes exposed to 3.1 nM (50 ng/ml) leptin for 24 hours. Mitochondrial transition pore opening was analyzed as the capacity of mitochondria to retain the dye calcein-AM in presence of 200 µM CaCl2. Leptin significantly increased pore opening although the effect was markedly more pronounced in digitonin-permeabilized myocytes in the presence of calcium with both effects prevented by the transition pore inhibitor sanglifehrin A. These effects were associated with increased apoptosis as evidenced by increased TUNEL staining and caspase 3 activity, both of which were prevented by the transition pore inhibitor sanglifehrin A. Leptin enhanced Stat3 activation whereas a Stat 3 inhibitor peptide prevented leptin-induced mitochondrial transition pore opening as well as the hypertrophic and pro-apoptotic effects of the peptide. Inhibition of the RhoA/ROCK pathway prevented the hypertrophic response to leptin but had no effect on increased pore opening following leptin administration. We conclude that leptin can enhance calcium-mediated, Stat3-dependent pro-apoptotic effects as a result of increased mitochondrial transition pore opening and independently of its hypertrophic actions. Leptin may therefore contribute to mitochondrial dysfunction and the development of apoptosis in the diseased myocardium particularly under conditions of excessive intracellular calcium accumulation.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

In brain mitochondria, calcium and cell death-associated permeability transition are controlled by possibly associated proteins, 2',3'-CNPase, Centaurin-alpha1 and peripheral benzodiazepine receptor, and their substrates/ligands

Mitochondria play a central role in calcium homeostasis and cellular calcium signaling. During cellular calcium overload, mitochondria take up cytosolic calcium, which, in turn, induces opening of the permeability transition pore (PTP), disruption of mitochondrial membrane potential and cell death. PTP is a protein complex changing according to the needs of the cell and responding to different external and internal stimuli. The identity of the PTP is still unresolved. 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and p42(IP4) (centaurin-alpha1) have been shown to be associated with rat brain mitochondria (RBM), but the exact role of these proteins in mitochondria is still obscure. Localization of p42(IP4) and CNP within the inner membrane and contact sites indicates further functions for these proteins. We found interaction of p42(IP4) with CNP by pull-down binding assay and by immunoprecipitation. Since PTP opening is important in mitochondrial events leading to programmed cell death, we studied whether p42(IP4) and CNP are involved in calcium-induced calcium release and consequently PTP. Simultaneous measurements of the respiratory rate, trans-membrane potential and calcium transport in the mitochondrial suspension were performed. We also developed the method of isolation of functionally active mitochondria from several cell types. We determined the calciumcapacity and lag-phase for PTP opening in mitochondria isolated from p42(IP4)-transfected and from control neuroblastoma cells. Overexpression of p42(IP4) led to promotion of calcium-induced PTP opening. The enzymatic activity of CNP was reduced under PTP opening, whereas the level of CNP detected in RBM before and after PTP opening were unchanged. Involvement of CNP in PTP operation was confirmed in further experiments using mitochondria isolated from CNP-knock-down oligodendrocytes (OLN93 cells). In mitochondria isolated from OLN93 cells transfected with CNP-targeting siRNA, CNP reduction was correlated with facilitation of calcium-induced PTP opening. The CNP substrates, 2',3'-cAMP and 2',3'-cNADP, induced PTP opening in RBM. The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive functions. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold calcium load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the calcium-induced calcium efflux, and AIF release from mitochondria. In summary, our results provide evidence that PBR, CNP and p42(IP4) are involved in regulation of calcium-induced PTP opening, important stage of initiation of programmed cell death.

Cadmium directly induced the opening of membrane permeability pore of mitochondria which possibly involved in cadmium-triggered apoptosis.

The mitochondrial damage induced by cadmium has been well established, but its mechanism and its relationship with cadmium-induced apoptosis are elusive until now. Our research showed that cadmium could directly lead to the dysfunction of isolated mitochondria from mouse liver, including the inhibition of respiration, the opening of permeability transition pore (PTP), the loss of transmembrane potential, and the release of cytochrome c. These mitochondrial changes were completely suppressed by Bcl-xL and Ruthenium Red (RR). Bongkrekic acid (BK), an inhibitor of the PTP opening directly via adenine nucleotide translocator (ANT), also completely inhibited the PTP opening and loss of transmembrane potential. However, cyclosporin A (CsA), another inhibitor of the PTP opening indirectly via ANT, had not any inhibitory effect. When cadmium being pre-incubated with proteins containing abundant thiol groups, its effect was partially reversed. These results revealed that mitochondria pathway may involve in cadmium-induced apoptosis, and cadmium caused the PTP opening possibly through its binding to thiol groups of ANT. Furthermore, the mechanism of the PTP opening induced by cadmium was probably distinct from that of the calcium-induced PTP opening.