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 opening of the mitochondrial permeability transition pore (PTP) has been suggested to play a key role in various forms of cell death, but direct evidence in intact tissues is still lacking. We found that in the rat heart, 92% of NAD ؉ glycohydrolase activity is associated with mitochondria. This activity was not modified by the addition of Triton X-100, although it was abolished by mild treatment with the protease Nagarse, a condition that did not affect the energy-linked properties of mitochondria. The addition of Ca 2؉ to isolated rat heart mitochondria resulted in a profound decrease in their NAD ؉ content, which followed mitochondrial swelling. Cyclosporin A(CsA), a PTP inhibitor, completely prevented NAD ؉ depletion but had no effect on the glycohydrolase activity. Thus, in isolated mitochondria PTP opening makes NAD ؉ available for its enzymatic hydrolysis. Perfused rat hearts subjected to global ischemia for 30 min displayed a 30% decrease in tissue NAD ؉ content, which was not modified by extending the duration of ischemia. Reperfusion resulted in a more severe reduction of both total and mitochondrial contents of NAD ؉ , which could be measured in the coronary effluent together with lactate dehydrogenase. The addition of 0.2 M CsA or of its analogue MeVal-4-Cs (which does not inhibit calcineurin) maintained higher NAD ؉ contents, especially in mitochondria, and significantly protected the heart from reperfusion damage, as shown by the reduction in lactate dehydrogenase release. Thus, upon reperfusion after prolonged ischemia, PTP opening in the heart can be documented as a CsA-sensitive release of NAD ؉ , which is then partly degraded by glycohydrolase and partly released when sarcolemmal integrity is compromised. These results demonstrate that PTP opening is a causative event in reperfusion damage of the heart. Depending on the duration and severity of myocardial ischemia, reperfusion can result in either recovery of contractile function or rapid transition toward tissue necrosis (for review see Refs. 1-3). Paradoxically, both events require coupled mitochondrial respiration (4). Indeed, cyanide (5) or 2,4-dinitrophenol (6) largely reduce the release of intracellular enzymes, the marker of cell death induced by postischemic reperfusion. However, after more than 25 years, the specific mechanisms underlying these phenomenological observations have yet to be elucidated.A large body of experimental evidence suggests that a suboptimal mitochondrial function could produce low levels of ATP, which in the presence of even a modest rise in [Ca 2ϩ ] i might cause hypercontracture in isolated cardiomyocytes (7) and sarcolemma rupture in intact hearts (8,9). Such a sequence of events could be set in motion by the opening of the mitochondrial PTP, 1 a high conductance channel located in the inner mitochondrial membrane (10). The open probability of this channel is regulated by several factors including mitochondrial membrane potential difference (⌬ m ), Ca 2ϩ , matrix pH, and CsA, a high affinity inhibitor (1...

Major factors linking mitochondrial dysfunction with myocardial injury are analyzed along with protective mechanisms elicited by endogenous processes and pharmacological treatments. In particular, a reduced rate of ATP hydrolysis and a slight increase in ROS formation appear to represent the prevailing components of self-defense mechanisms, especially in the case of ischemic preconditioning. These protective processes are activated by signaling pathways, which converge on mitochondria activating the mitochondrial K(ATP) channels and/or inhibiting the mitochondrial permeability transition pore. These pathways can also be stimulated by pharmacological treatments. Another major goal for cardioprotection is decreasing the burst in mitochondrial ROS formation that characterizes post-ischemic reperfusion. Finally, mitochondrial targets for therapeutic intervention may include the switch of substrate being utilized, because inhibition of fatty acid oxidation is associated with cardioprotective effects.

This study demonstrated that oxidative/nitrosylative changes of actin and tropomyosin are largely increased in human failing hearts. Because these changes are inversely correlated to LVEF, actin and tropomyosin oxidation are likely to contribute to the contractile impairment evident in end-stage HF.

Myofibrillar protein oxidation may represent a mechanistic link between inflammation and contractile dysfunction following coronary microembolization.