We report here that mPTP inhibition limited apoptosis associated with ACGR more efficiently than Fas inactivation. This protective effect may be related to a direct effect on cardiomyocyte apoptosis and/or to the rerouting of the classical acute graft rejection pathway involving CD8+ T lymphocytes.
Classical activation of graft damage during acute rejection
Cardiomyocyte injury following acute rejection involves T-cell-mediated cytotoxicity (1). At day 5, we observed a rapid infiltration of lymphocytes, plasmocytes and macrophages into allogenic transplants, with surrounding edema, fibrosis and myocyte damage. Studies have determined that cytotoxicity can be mediated through several major molecular pathways, mainly the granule exocytosis or perforin-granzyme pathway, and the Fas (Apo-1, CD95)/Fas ligand (FasL, CD95L)-mediated apoptosis pathway (6,16,17). In our model, expression of perforin, granzyme B and TNFα was clearly upregulated in allogenic grafts, and concomitant with an infiltration of CD8+ T cells.
Activation of the Fas/FasL system can trigger apoptosis in various cell types, including cardiomyocytes (4). Expression of FasL, essentially undetectable in syngenic groups, was strongly upregulated in allografts and correlated with a significantly increased cytochrome c release and caspase-3 activity, a pivotal effector caspase in the apoptotic program of cell death (18). In order to investigate the role of Fas in cardiomyocyte injury during acute graft rejection, we used lpr mice that lack a functional Fas receptor (19). Lpr allografts did not develop different myocardial damage when compared to wild-type allografts, in agreement with Djamali et al. who demonstrated that lpr allografts are often rejected in a near-normal time course (20,21). The increase of caspase-3 activation in these Fas-deficient transplants remained lesser than in wild-type grafts, confirming a nonnegligible role of the Fas/FasL pathway in cardiomyocyte apoptosis activation in this experimental model (22,23). Yet, the fact that NIM811 equally reduced caspase-3 activity and cytochrome c release in allogenic and Fas-deficient grafts indirectly suggests that Fas-induced apoptosis may possibly act via a cross-talk with mitochondria through caspase-8 activation.
Alternative pathway for apoptosis activation in myocardial transplant
Mitochondrial permeability transition is recognized as a key event in cardiomyocyte death after ischemia-reperfusion (24–26). We previously demonstrated that mPTP is also involved in apoptosis during acute graft rejection (3). Opening of the mPTP results in the collapse of the membrane potential and efflux of cytochrome c and downstream effector caspases activation that may lead to either apoptosis or necrosis (27).
Acute graft rejection causes accumulation of cytosolic Ca2+ through activation of IP3 pathway (28). Accumulated cytosolic Ca2+ enters the mitochondria through the Ca2+ uniporter, and high matrix Ca2+ concentrations provide an ideal condition for mPTP opening (29). We demonstrated here that mitochondria isolated from allogenic grafts display a significant reduction in their resistance to Ca2+ overload. Involvement of mPTP opening in cardiomyocyte death was strongly suggested by the fact that the specific inhibitor NIM811 clearly inhibited Ca2+-induced mPTP opening and significantly reduced cytochrome c release and caspase-3 activity. We then first interpreted that inhibition of mPTP opening in cardiomyocytes limited cardiac apoptosis during acute rejection.
We were, however, surprised to observe that NIM811 dramatically modified the expression of FasL, perforin, granzyme B and TNFα, that represent major CTL effector molecules. NIM811 is known as a nonimmunosuppressive derivative ([Me-Ile4]-cyclosporine) of CsA. Several reports indicate that this CsA analogue does not inhibit the calcium/calmodulin-dependent protein phosphatase, calcineurin, and does not inhibit NFAT migration into the nucleus (30). Our results are in agreement with those of Rosenwirth et al., who demonstrated that NIM811 does not alter IL-2 up-regulation into allogenic transplant and consequently, does not alter CD4+ proliferation (31). Indeed, allogenic grafts that received NIM811 did not exhibit an inhibition of acute rejection. Despite this, we observed that infiltration of CD8+ T cells into allogenic grafts was significantly reduced by NIM811. Expression of the CTL effector molecules (perforin, granzyme, FasL and TNFα) was consequently markedly reduced by NIM811, and in vitro dose-response of NIM811 on T-cells proliferation was in agreement with in vivo results. In other words, the specific mPTP inhibitor NIM811 inhibited CD8+ (but not CD4+) T-cells proliferation and activation, and the protective effect of NIM811 might not be solely due to inhibition of mPTP in cardiomyocytes.
What is the link between mitochondrial permeability transition and CD8+ T-cell activation? One may first hypothesize that cardiomyocyte apoptosis may stimulate CD8+ T lymphocyte infiltration in the rejecting graft. Faouzi et al. reported that caspase-3 activation promotes inflammatory cells attraction through chemokines induction in mice liver (32). Using a murine model of renal ischemia-reperfusion injury, Daemen et al. demonstrated that caspase inactivators not only limited apoptosis but also prevented inflammation and organ injury (33). NIM811 might therefore attenuate T-cell infiltration during acute graft rejection via a reduction of cardiomyocyte apoptosis secondary to the inhibition of mPTP opening and subsequent cytochrome c release and caspase3 activation.
Second, Ca2+, that is essential for regulating a host of distinct processes involving enzyme control, exocytosis, gene regulation, cell growth and proliferation and apoptosis, may play an important role. Lichtman et al. demonstrated that calcium is required for the stimulation of lymphocytes (34). Calcium enters the mitochondria as a result of a respiration-dependent mitochondrial membrane potential. Mitochondria are Ca2+-excitable organelles involved in the transduction of cell Ca2+ signals, and seem to play a major part in the regulation of regulation of Ca2+-release-activated Ca2+ current (ICRAC) (11,35). Calcium release from the mitochondria may also occur via opening of the mitochondrial transition pore, under either a low-conductance or a high-conductance state (11,36,37). In light of our present results, and in agreement with Hoth et al. (38), we can hypothesize that mPTP opening may contribute to increase, or maintain at a high level, cytosolic Ca2+ concentration, and thereby favor CD8+ T-cell activation.
The present data confirm that cardiomyocyte apoptosis is associated with acute graft rejection. Although cardiomyocyte apoptosis may be induced by different pathways, mitochondria appear to be a predominant player. Inhibition of mitochondrial permeability transition may, in the case of ACGR, act on two different targets: first, CD8+ T lymphocyte infiltration via either reduced local apoptosis or limited calcium release in the lymphocyte cytosol; second, cardiac apoptosis via mPTP inhibition within jeopardized cardiomyocytes.