Roberta A. Gottlieb

Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death

Mitochondria play a critical role in mediating both apoptotic and necrotic cell death. The mitochondrial permeability transition (mPT) leads to mitochondrial swelling, outer membrane rupture and the release of apoptotic mediators. The mPT pore is thought to consist of the adenine nucleotide translocator, a voltage-dependent anion channel, and cyclophilin D (the Ppif gene product), a prolyl isomerase located within the mitochondrial matrix. Here we generated mice lacking Ppif and mice overexpressing cyclophilin D in the heart. Ppif null mice are protected from ischaemia/reperfusion-induced cell death in vivo, whereas cyclophilin D-overexpressing mice show mitochondrial swelling and spontaneous cell death. Mitochondria isolated from the livers, hearts and brains of Ppif null mice are resistant to mitochondrial swelling and permeability transition in vitro. Moreover, primary hepatocytes and fibroblasts isolated from Ppif null mice are largely protected from Ca2+-overload and oxidative stress-induced cell death. However, Bcl-2 family member-induced cell death does not depend on cyclophilin D, and Ppif null fibroblasts are not protected from staurosporine or tumour-necrosis factor-α-induced death. Thus, cyclophilin D and the mitochondrial permeability transition are required for mediating Ca2+- and oxidative damage-induced cell death, but not Bcl-2 family member-regulated death.

Enhancing Macroautophagy Protects against Ischemia/Reperfusion Injury in Cardiac Myocytes

Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-xL were protective against I/R injury, and expression of a Beclin1 Bcl-2/-xL binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3ΔTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3ΔTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

New paradigm for lymphocyte granule-mediated cytotoxicity: Target cells bind and internalize granzyme B, but an endosomolytic agent is necessary for cytosolic delivery and subsequent apoptosis

Lymphocyte granule-mediated apoptosis is postulated to entail the formation of membrane pores by perforin. Then soluble granzyme reaches the cytosol either through these pores or by reparative pinocytosis. We demonstrate here that Jurkat cells bind and internalize granzyme B via high affinity binding sites without toxic consequence. Apoptosis occurs, however, if sublytic perforin is added to targets washed free of soluble granzyme B. We suggest that granule-mediated apoptosis mimics viral strategies for cellular entry. Accordingly, co-internalization of granzyme B with adenovirus, a virus that escapes endosomes to reach the cytosol, also induced apoptosis. Poly(ADP-ribose) polymerase cleavage and processing of CPP32, ICE-LAP3, and Mch2 were detected at 30 min, while cytosolic acidification and DNA fragmentation occurred at 60 min. Annexin V binding and membrane permeabilization arose at 4 h. The concurrent activation of the Ced-3 proteases differed from the rate at which each cysteine protease is cleaved in vitro by granzyme B. Thus, granzyme B may not directly process these proteases in whole cells but rather may function by activating a more proximal enzyme. These results indicate that adenovirus-mediated delivery of granzyme B is suitable for elucidating biochemical events that accompany granule-mediated apoptosis.

Calpain and Mitochondria in Ischemia/Reperfusion Injury

Studies of ischemia/reperfusion (I/R) injury and preconditioning have shown that ion homeostasis, particularly calcium homeostasis, is critical to limiting tissue damage. However, the relationship between ion homeostasis and specific cell death pathways has not been investigated in the context of I/R. Previously we reported that calpain cleaved Bid in the absence of detectable caspase activation (1). In this study, we have shown that an inhibitor of the sodium/hydrogen exchanger prevented calpain activation after I/R. Calpain inhibitors prevented cleavage of Bid as well as the downstream indices of cell death, including DNA strand breaks, creatine kinase (CK) release, and infarction measured by triphenyl tetrazolium chloride (TTC) staining. In contrast, the broad spectrum caspase inhibitor IDN6734 was not protective in this model. To ascertain whether mitochondrial dysfunction downstream of these events was a required step, we utilized a peptide corresponding to residues 4–23 of Bcl-xL conjugated to the protein transduction domain of HIV TAT (TAT-BH4), which has been shown to protect mitochondria against Ca2+-induced ΔΨm loss (2). TAT-BH4 attenuated CK release and loss of TTC staining, demonstrating the role of mitochondria and a pro-apoptotic Bcl-2 family member in the process leading to cell death. We propose the following pathway. (i) Reperfusion results in sodium influx followed by calcium accumulation. (ii) This leads to calpain activation, which in turn leads to Bid cleavage. (iii) Bid targets the mitochondria, causing dysfunction and release of pro-apoptotic factors, resulting in DNA fragmentation and death of the cell. Ischemia/reperfusion initiates a cell death pathway that is independent of caspases but requires calpain and mitochondrial dysfunction.

Apoptosis in Myocardial Ischemia‐Reperfusion

Abstract: The signal transduction pathways by which ischemia‐reperfusion leads to apoptosis may involve the JNK pathway, ceramide generation, and inhibition of protective PKC pathways. The biochemical events associated with apoptosis include mitochondrial inactivation, cytochrome c dislocation, caspase activation, and cytoplasmic acidification. Through the concerted efforts of multiple classes of enzymes, apoptosis is accomplished, resulting in the death of a cell in which potentially transforming oncogenes have been degraded and inflammatory contents are contained within the plasma membrane until the fragments can be ingested by phagocytes. This non‐inflammatory mode of cell death permits tissue remodeling with minimal scar formation, and so is preferable to necrotic cell death. The distinction between apoptosis and necrosis, which implies different mechanisms of cell death, is blurred in the case of a pathologic insult such as ischemia‐reperfusion. It is suggested that it is more useful to view cell death in the context of whether or not it can be prevented

Mitochondria: execution central

Mitochondria play an essential function in eukaryotic life and death. They also play a central role in apoptosis regulation, reflected by the convergence of Bcl‐2 family members on the mitochondrial outer membrane, and the presence of ‘death factors’ in the intermembrane space. Mitochondrial structure and function must be taken into consideration when evaluating mechanisms for cytochrome c release. The core machinery for caspase activation is conserved from Caenorhabditis elegans to man, and we consider parallels in the role of mitochondria in this process.

Bcl-2 and the Outer Mitochondrial Membrane in the Inactivation of Cytochrome c during Fas-mediated Apoptosis

Fas-driven apoptosis in Jurkat cells results in the inactivation of cytochrome c with cessation of oxygen consumption. Overexpression of Bcl-2 was found to protect against acidification and apoptosis mediated by Fas ligation in these cells. Bcl-2 is present in the outer mitochondrial membrane, but the molecular mechanism by which it protects cells is unknown. Because Bcl-2 projects into the mitochondrial intermembrane space and cytochrome cis located in the intermembrane space, we considered the possibility that Bcl-2 might protect cytochrome c from inactivation during Fas-mediated apoptosis. The present study shows that 1) in Jurkat cells, cytochrome c inactivation during Fas-driven apoptosis requires the permeabilization of the outer mitochondrial membrane; and 2) the post-mitochondrial fraction from CEM cells that overexpress Bcl-2 both prevents and reverses cytochrome cinactivation.

Regulation of the activity of caspases by L-carnitine and palmitoylcarnitine

L‐Carnitine facilitates the transport of fatty acids into the mitochondrial matrix where they are used for energy production. Recent studies have shown that L‐carnitine is capable of protecting the heart against ischemia/reperfusion injury and has beneficial effects against Alzheimers disease and AIDS. The mechanism of action, however, is not yet understood. In the present study, we found that in Jurkat cells, L‐carnitine inhibited apoptosis induced by Fas ligation. In addition, 5 mM carnitine potently inhibited the activity of recombinant caspases 3, 7 and 8, whereas its long‐chain fatty acid derivative palmitoylcarnitine stimulated the activity of all the caspases. Palmitoylcarnitine reversed the inhibition mediated by carnitine. Levels of carnitine and palmitoyl‐CoA decreased significantly during Fas‐mediated apoptosis, while palmitoylcarnitine formation increased. These alterations may be due to inactivation of β‐oxidation or to an increase in the activity of the enzyme that converts carnitine to palmitoylcarnitine, carnitine palmitoyltransferase I (CPT I). In support of the latter possibility, fibroblasts deficient in CPT I activity were relatively resistant to staurosporine‐induced apoptosis. These observations suggest that caspase activity may be regulated in part by the balance of carnitine and palmitoylcarnitine.

TAT protein transduction into isolated perfused hearts: TAT-apoptosis repressor with caspase recruitment domain is cardioprotective.

Background—Linkage of the 11–amino-acid transduction domain of HIV TAT to a heterologous protein allows the protein to be transduced readily into cells. Methods and Results—In this study, we inserted the apoptosis repressor with caspase recruitment domain (ARC) or &bgr;-galactosidase (&bgr;-gal) cDNA into the pTAT-hemagglutinin bacterial expression vector to produce genetic in-frame TAT-ARC or TAT-&bgr;-gal fusion proteins for use in cell culture and in Langendorff perfusion of adult rat hearts. TAT-&bgr;-gal and TAT-ARC were conjugated with Texas Red and could be detected in >95% of cells. TAT-ARC was able to protect H9c2 cells against cell death mediated by hydrogen peroxide, as measured by protection against the loss of mitochondrial membrane potential and preservation of nuclear morphology. Isolated adult hearts were perfused with recombinant TAT-&bgr;-gal or TAT-ARC (20 nmol/L) for 15 minutes and then subjected to 30 minutes of global no-flow ischemia, followed by 2 hours of reperfusion. Protein transduction was assessed by Western blotting of cell lysates and cytosolic and mitochondrial fractions and by fluorescence microscopy of Texas Red–conjugated TAT proteins. TAT-&bgr;-gal and TAT-ARC readily transduced into perfused hearts and were homogeneously distributed. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining, and creatine kinase release was measured. Transduction of TAT-ARC was cardioprotective when administered before global ischemia and reperfusion. Conclusions—Our results demonstrate that TAT-linked fusion protein transduction into the myocardium is feasible and that transduction of TAT-ARC is protective in cell culture and in the perfused heart.