Monoamine oxidase inhibition prevents mitochondrial dysfunction and apoptosis in myoblasts from patients with collagen VI myopathies.

Although mitochondrial dysfunction and oxidative stress have been proposed to play a crucial role in several types of muscular dystrophy (MD), whether a causal link between these two alterations exists remains an open question. We have documented that mitochondrial dysfunction through opening of the permeability transition pore plays a key role in myoblasts from patients as well as in mouse models of MD, and that oxidative stress caused by monoamine oxidases (MAO) is involved in myofiber damage. In the present study we have tested whether MAO-dependent oxidative stress is a causal determinant of mitochondrial dysfunction and apoptosis in myoblasts from patients affected by collagen VI myopathies. We find that upon incubation with hydrogen peroxide or the MAO substrate tyramine myoblasts from patients upregulate MAO-B expression and display a significant rise in reactive oxygen species (ROS) levels, with concomitant mitochondrial depolarization. MAO inhibition by pargyline significantly reduced both ROS accumulation and mitochondrial dysfunction, and normalized the increased incidence of apoptosis in myoblasts from patients. Thus, MAO-dependent oxidative stress is causally related to mitochondrial dysfunction and cell death in myoblasts from patients affected by collagen VI myopathies, and inhibition of MAO should be explored as a potential treatment for these diseases.

The contribution of reactive oxygen species and p38 mitogen-activated protein kinase to myofilament oxidation and progression of heart failure in rabbits.

BACKGROUND AND PURPOSE: The formation of reactive oxygen species (ROS) is increased in heart failure (HF). However, the causal and mechanistic relationship of ROS formation with contractile dysfunction is not clear in detail. Therefore, ROS formation, myofibrillar protein oxidation and p38 MAP kinase activation were related to contractile function in failing rabbit hearts. EXPERIMENTAL APPROACH AND KEY RESULTS: Three weeks of rapid left ventricular (LV) pacing reduced LV shortening fraction (SF, echocardiography) from 32 +/- 1% to 13 +/- 1%. ROS formation, as assessed by dihydroethidine staining, increased by 36 +/- 8% and was associated with increased tropomyosin oxidation, as reflected by dimer formation (dimer to monomer ratio increased 2.28 +/- 0.66-fold in HF vs. sham, P < 0.05). Apoptosis (TdT-mediated dUTP nick end labelling staining) increased more than 12-fold after 3 weeks of pacing when a significant increase in the phosphorylation of p38 MAP kinase and HSP27 was detected (Western blotting). Vitamins C and E abolished the increases in ROS formation and tropomyosin oxidation along with an improvement of LVSF (19 +/- 1%, P < 0.05 vs. untreated HF) and prevention of apoptosis, but without modifying p38 MAP kinase activation. Inhibition of p38 MAP kinase by SB281832 counteracted ROS formation, tropomyosin oxidation and contractile failure, without affecting apoptosis. CONCLUSIONS AND IMPLICATIONS: Thus, p38 MAP kinase activation appears to be upstream rather than downstream of ROS, which impacts on LV function through myofibrillar oxidation. p38 MAP kinase inhibition is a potential target to prevent or treat HF.

6-Aminoquinolones: photostability, cellular distribution and phototoxicity.

Three selected aminoquinolones endowed with a potent antibacterial (compounds 1 and 2) and antiviral activity (compound 3) have been evaluated for their phototoxic properties in vitro. Photostability studies of these compounds indicate that compound 3 is photostable whereas compound 1 and in particular, compound 2 are rapidly photodegraded upon UVA irradiation, yielding a toxic photoproduct. Intracellular localization of these compounds has been evaluated by means of fluorescence microscopy using tetramethylrhodamine methyl ester and acridine orange, which are specific fluorescent probes for mitochondria and lysosomes, respectively. No co-staining was observed with lysosomal stain for all the test compounds. On the contrary compound 3 was found to be specifically incorporated in mitochondria. The compounds exhibited remarkable phototoxicity in two cell culture lines: human promyelocytic leukaemia (HL-60) and human fibrosarcoma (HT-1080). The quinolone-induced photodamage was also evaluated measuring the photosensitizing cross-linking in erythrocyte ghost membranes, the strand breaks activity and oxidative damage on plasmid DNA. The results show that these derivatives are able to photoinduce crosslink of erythrocytes spectrin, whereas do not significantly photocleavage DNA directly, but single strand breaks were observed after treatment of photosensitized DNA with two base excision repair enzymes, Fpg and Endo III respectively.

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.

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.

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 induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co(2+) 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 with. Cyclosporin A-inhibitable release of cytochrome c and 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.

Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart.

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 microm 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.

Mitochondrial contribution in the progression of cardiac ischemic injury.

The multifaceted relationship between mitochondria and the rest of the cell is reviewed in the context of myocardial ischemia. Paradoxically, mitochondria can exacerbate the ischemic damage, especially at the onset of reperfusion. Indeed, the recovery of oxidative phosphorylation in the presence of an excessive energy demand is likely to represent a crucial factor in the ensuing irreversible damage of cardiomyocytes. A major role in the progression towards cell death might be attributed to the opening of the permeability transition pore, which besides abolishing mitochondrial ATP production might amplify the damage by causing NAD+ release. This damaging role is balanced by the contribution of mitochondria in self-defense mechanisms operating in the ischemic cardiomyocytes. The mitochondrial ATP-sensitive K+ channel and a slight increase in the production of reactive oxygen species appear to mediate the attempt of the heart to maintain its viability under conditions of acute and chronic ischemia. The significance of the various processes is discussed along with the critical evaluation of both the difficulties in studying mitochondria in situ and the possible sources of errors or misinterpretations.

Mitochondria and cell death. Mechanistic aspects and methodological issues.

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.

Chloromethyltetramethylrosamine (Mitotracker Orange) induces the mitochondrial permeability transition and inhibits respiratory complex I. Implications for the mechanism of cytochrome c release.

We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker Orange) that is increasingly used to monitor the mitochondrial membrane potential (Deltapsi(m)) in situ. CMTMRos binds to isolated mitochondria and undergoes a large fluorescence quenching. Most of the binding is energy-independent and can be substantially reduced by sulfhydryl reagents. A smaller fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spontaneously reverts to the same level obtained by treating mitochondria with the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. We show that this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRos, with concomitant depolarization, swelling, and release of cytochrome c. After staining cells with CMTMRos, depolarization of mitochondria in situ with protonophores is accompanied by changes of CMTMRos fluorescence that range between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochondria only results from treatment with thiol reagents, suggesting that CMTMRos binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Deltapsi(m). Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Deltapsi(m) in situ and demands a reassessment of the conclusion that cytochrome c release can occur without membrane depolarization and/or onset of the permeability transition.

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 cytochrome c 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.

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.

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 deltapsi(m) 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.

The role of mitochondria in the salvage and the injury of the ischemic myocardium.

The relationships between mitochondrial derangements and cell necrosis are exemplified by the changes in the function and metabolism of mitochondria that occur in the ischemic heart. From a mitochondrial point of view, the evolution of ischemic damage can be divided into three phases. The first is associated with the onset of ischemia, and changes mitochondria from ATP producers into powerful ATP utilizers. During this phase, the inverse operation of F0F1 ATPase maintains the mitochondrial membrane potential by using the ATP made available by glycolysis. The second phase can be identified from the functional and structural alterations of mitochondria caused by prolongation of ischemia, such as decreased utilization of NAD-linked substrates, release of cytochrome c and involvement of mitochondrial channels. These events indicate that the relationship between ischemic damage and mitochondria is not limited to the failure in ATP production. Finally, the third phase links mitochondria to the destiny of the myocytes upon post-ischemic reperfusion. Indeed, depending on the duration and the severity of ischemia, not only is mitochondrial function necessary for cell recovery, but it can also exacerbate cell injury.

Imaging the mitochondrial permeability transition pore in intact cells.

The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co-loaded with calcein-AM and CoCl2, resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells.

Troponin T cross-linking in human apoptotic cardiomyocytes.

Intracellular calcium overload of guinea pig cardiomyocytes is accompanied by troponin T cross-linking, which is revealed by changes in immunoreactivity of anti-troponin T antibodies. We presently investigated whether the same process is detectable in the human heart. Immunohistochemistry shows myofibrillar staining with BN-59 anti-troponin T antibody with rare cardiomyocytes in samples obtained at surgery, whereas approximately 50% of myocytes are labeled in heart samples taken at autopsy within 3 hours of death, and every cardiomyocyte is stained after exposure of biopsy sections to 10 mmol/L calcium. Western blot analysis shows reactive polypeptides of approximately 70 and 85 to 90 kd in addition to troponin T in both treated and autopsy heart sections. Neither reactivity in immunohistochemistry nor additional reactive polypeptides in Western blot are detectable when calpain or transglutaminase is inhibited during exposure of sections to high calcium. Troponin T crosslinking occurs also in isolated myofibrils, which show staining with BN-59 at either sarcomeric A or I bands. Labeling with TdT-mediated dUTP nick and labeling (TUNEL) to demonstrate apoptosis reveals DNA fragmentation in BN-59-positive myocytes. Thus, troponin T cross-linking occurs in human cardiac myocytes concomitantly with apoptosis and autopsy autolysis, suggesting that similar cytosolic alterations can be produced by different types of myocyte death.

Effects of prolonged L-carnitine administration on delayed muscle pain and CK release after eccentric effort.

Eccentric muscle effort is known to induce delayed muscle soreness (DOMS) and muscle damage which are not responsive to medical treatment with the most common analgesic agents. The aim of the study was to investigate the effects of oral L-carnitine supplementation on pain (VAS scale), tenderness (pain thresholds) and CK release induced by a 20-min eccentric effort of the quadriceps muscle. A single-blind study was carried out on 6 untrained subjects (mean age: 26 +/- 3.8 yrs; mean height: 173 +/- 4.6 cm; mean body weight, 68.3 +/- 4.5 kg) over 7 weeks during which each subject: a) was given 3 g/day of placebo for 3 weeks and, after a week's interval, 3 g/day of L-carnitine for 3 weeks: b) performed 2 step tests on the first day of the 3rd and 7th week inverting the order of the exercising limb. In a separate set of experiments carried out 8 months later, the possible effects of training on pain parameters and CK levels were also investigated in the same subjects who performed 2 step tests at a 4-weeks' interval, without medication. L-carnitine significantly reduced pain, tenderness and CK release after the effort with respect to placebo. In contrast, no significant difference was found in the parameters measured between the two tests performed without medication. It is concluded that L-carnitine has a protective effect against pain and damage from eccentric effort. This effect is mainly attributed to the vasodilatation property of the compound, which both improves energetic metabolism of the hypoxic/damaged muscle and enhances wash-out of algogenic metabolites.

Binding of cytosolic proteins to myofibrils in ischemic rat hearts.

Myofibrillar proteins (MPs) were extracted from isolated and perfused rat hearts subjected to different periods of ischemia to investigate the occurrence of protein degradation and/or the association of cytosolic proteins with the myofibrillar pellet. A 23-kD band was detected by SDS-PAGE of MPs after 5 minutes of ischemia, with its density gradually increasing to a plateau after 20 minutes. Longer periods of ischemia were associated with the appearance of a 39-kD band. Irrespective of the duration of ischemia, both these bands persisted during reperfusion. A partial proteolytic degradation of troponin T (TnT) and troponin I (TnI) has been claimed to be responsible for the generation of these peptides. However, the N-terminal sequence of the 39-kD band was identical to that of GAPDH, whereas Edman sequencing after pepsin digestion showed that the 23 kD is alpha B-crystallin. The binding of the two cytosolic proteins to myofibrils was confirmed by immunofluorescence analysis on cryosections of ischemic hearts. In vitro studies showed that acidosis was sufficient to induce the binding of alpha B-crystallin, whereas the inhibition of ATP depletion prevented the binding of GAPDH. Thiol oxidation is unlikely to promote GAPDH binding, since perfusion with iodoacetate under aerobic conditions or treatment of homogenates with N-ethylmaleimide or diamide failed to induce GAPDH association with the myofibrils. These changes of the myofibrillar proteins could be considered as intracellular markers of the evolution of the ischemic damage. In addition, the binding of the 23-kD peptide might be involved in alterations of contractility.