Fabio Di Lisa

Transient and Long-Lasting Openings of the Mitochondrial Permeability Transition Pore Can Be Monitored Directly in Intact Cells by Changes in Mitochondrial Calcein Fluorescence

The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

The mitochondrial permeability transition from in vitro artifact to disease target

The mitochondrial permeability transition pore is a high conductance channel whose opening leads to an increase of mitochondrial inner membrane permeability to solutes with molecular masses up to ≈ 1500 Da. In this review we trace the rise of the permeability transition pore from the status of in vitro artifact to that of effector mechanism of cell death. We then cover recent results based on genetic inactivation of putative permeability transition pore components, and discuss their meaning for our understanding of pore structure. Finally, we discuss evidence indicating that the permeability transition pore plays a role in pathophysiology, with specific emphasis on in vivo models of disease.

Postconditioning and protection from reperfusion injury: where do we stand?Position Paper from the Working Group of Cellular Biology of the Heart of the European Society of Cardiology

Ischaemic postconditioning (brief periods of ischaemia alternating with brief periods of reflow applied at the onset of reperfusion following sustained ischaemia) effectively reduces myocardial infarct size in all species tested so far, including humans. Ischaemic postconditioning is a simple and safe manoeuvre, but because reperfusion injury is initiated within minutes of reflow, postconditioning must be applied at the onset of reperfusion. The mechanisms of protection by postconditioning include: formation and release of several autacoids and cytokines; maintained acidosis during early reperfusion; activation of protein kinases; preservation of mitochondrial function, most strikingly the attenuation of opening of the mitochondrial permeability transition pore (MPTP). Exogenous recruitment of some of the identified signalling steps can induce cardioprotection when applied at the time of reperfusion in animal experiments, but more recently cardioprotection was also observed in a proof-of-concept clinical trial. Indeed, studies in patients with an acute myocardial infarction showed a reduction of infarct size and improved left ventricular function when they underwent ischaemic postconditioning or pharmacological inhibition of MPTP opening during interventional reperfusion. Further animal studies and large-scale human studies are needed to determine whether patients with different co-morbidities and co-medications respond equally to protection by postconditioning. Also, our understanding of the underlying mechanisms must be improved to develop new therapeutic strategies to be applied at reperfusion with the ultimate aim of limiting the burden of ischaemic heart disease and potentially providing protection for other organs at risk of reperfusion injury, such as brain and kidney.

A mitochondrial perspective on cell death

The role of mitochondria as crucial participants in cell death programs is well established, yet the mechanisms responsible for the release of mitochondrial activators and the role of BCL2 family proteins in this process remain controversial. Here, we point out the limitations of current approaches used to monitor the physiological responses of mitochondria during cell death, the implications arising from modern views of mitochondrial structure, and briefly assess two proposed mechanisms for the release of mitochondrial proteins during apoptosis.

The Mitochondrial Permeability Transition, Release of Cytochrome c and Cell Death CORRELATION WITH THE DURATION OF PORE OPENINGS IN SITU

We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Treatment with arachidonic acid or A23187 induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co2+ quenching technique (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725–734). Yet depolarization, as assessed from the changes of mitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapid and extensive with arachidonic acid and slow and partial withA23187. Cyclosporin A-inhibitable release of cytochrome cand cell death correlate with the changes of TMRM fluorescence but not with those of calcein fluorescence. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. Modulation of the open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.

Beat‐to‐beat oscillations of mitochondrial [Ca2+] in cardiac cells

The Ca2+‐sensitive photoprotein aequorin and the new green fluorescent protein‐based fluorescent Ca2+ indicators ‘ratiometric‐pericam’ were selectively expressed in the mitochondria, cytosol and/or nucleus of spontaneously beating ventricular myocytes from neonatal rats. This combined strategy reveals that mitochondrial [Ca2+] oscillates rapidly and in synchrony with cytosolic and nuclear [Ca2+]. The Ca2+ oscillations were reduced in frequency and/or amplitude by verapamil and carbachol and were enhanced by isoproterenol and elevation of extracellular [Ca2+]. An increased frequency and/or amplitude of cytosolic Ca2+ spikes was rapidly mirrored by similar changes in mitochondrial Ca2+ spikes and more slowly by elevations of the interspike Ca2+ levels. The present data unequivocally demonstrate that in cardiac cells mitochondrial [Ca2+] oscillates synchronously with cytosolic [Ca2+] and that mitochondrial Ca2+ handling rapidly adapts to inotropic or chronotropic inputs.

Impairment of Diazoxide-Induced Formation of Reactive Oxygen Species and Loss of Cardioprotection in Connexin 43 Deficient Mice

Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43+/−) mice. Because connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/− mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 &mgr;mol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/− cardiomyocytes. In contrast, diazoxide (200 &mgr;mol/L) increased ROS formation by 43±10% versus vehicle in WT, but only by 18±4% in Cx43+/− cardiomyoctes (P<0.05). Two hour–simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7±1% versus 39±2%, Cx43+/−: 8±1% versus 40±3%, P<0.01). Although menadione protected WT and Cx43+/− cardiomyocytes, diazoxide increased viability (17±2%, P<0.01) in WT, but not in Cx43+/− (9±1%). Menadione (37 &mgr;g/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/− mice (24±4% versus 24±5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35±4% versus 55±3% of area at risk, P<0.01), but not in Cx43+/− mice (56±2% versus 54±3%). Cardiomyocytes of Cx43+/− mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

Mitochondrial function as a determinant of recovery or death in cell response to injury

Many pathological conditions can be the cause or the consequence of mitochondrial dysfunction. For instance anoxia, which is initiated by a critical reduction of oxygen availability for mitochondrial oxidations, is followed by a wide variety of mitochondrial alterations. A crucial role in the evolution of cell injury is to be attributed to the direction of operation of the F0F1 ATPase, which may turn mitochondria into the major consumers of cellular ATP in the futile attempt to restore the proton electrochemical gradient. On the other hand, functional mitochondria can paradoxically accelerate or exacerbate cell damage. This concept is particularly relevant for the ischemic myocardium. Indeed, inhibition of the respiratory chain or addition of uncouplers of oxidative phosphorylation can both limit the extent of enzyme release in the intact heart and prevent the onset of irreversible morphological changes in isolated myocytes. From studies on different tissues in a variety of pathological conditions a general consensus emerges on the role of intracellular Ca2+ overload as a pivotal link between cellular alterations and mitochondrial dysfunction. Oxidative phosphorylation is reduced by a massive mitochondrial uptake of Ca2+, resulting in a vicious cycle whereby the reduced ATP availability is followed by a failure of the mechanisms which extrude Ca2+ from the sarcoplasm. In addition, the rise in [Ca2+]i could promote opening of the cyclosporin-sensitive mitochondrial permeability transition pore, leading to a sudden ΔΨm dissipation. Here, we review the changes in intracellular and intramitochondrial ionic homeostasis occurring during ischemia and reperfusion. In particular, we evaluate the potential contribution of the permeability transition pore to cellular damage and discuss the mechanisms which can determine the cellular fate from a mitochondrial point of view.(Mol Cell Biochem 184: 379–391, 1998)

Commitment to apoptosis by GD3 ganglioside depends on opening of the mitochondrial permeability transition pore.

We have studied the effects of GD3 ganglioside on mitochondrial function in isolated mitochondria and intact cells. In isolated mitochondria, GD3 ganglioside induces complex changes of respiration that depend on the substrate being oxidized. However, these effects are secondary to opening of the cyclosporin A-sensitive permeability transition pore and to the ensuing swelling and cytochromec depletion rather than to an interaction with the respiratory chain complexes. By using a novel in situ assay based on the fluorescence changes of mitochondrially entrapped calcein (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725–734), we unequivocally show that GD3 ganglioside also induces the mitochondrial permeability transition in intact cells and that this event precedes apoptosis. The mitochondrial effects of GD3 ganglioside are selective, in that they cannot be mimicked by either GD1a or GM3 gangliosides, and they are fully sensitive to cyclosporin A, which inhibits both the mitochondrial permeability transition in situ and the onset of apoptosis induced by GD3 ganglioside. These results provide compelling evidence that opening of the permeability transition pore is causally related to apoptosis.

Pathophysiological relevance of mitochondria in NAD+ metabolism

Pyridine nucleotides are mostly stored within mitochondria where they are involved in different functions ranging from energy metabolism to cellular signaling. Here we discuss the mechanisms of mitochondrial NAD+ metabolism and release that may contribute to the crucial roles played by these organelles as triggers or amplifiers of physiological and pathological events.