F. Di Lisa

Myocardial recovery during post-ischaemic reperfusion: Effects of nifedipine, calcium and magnesium

We studied the effects of various interventions introduced at the time of post-ischaemic reperfusion on mechanical activity, tissue and mitochondrial calcium, mitochondrial function and tissue ATP and CP of isolated perfused rabbit hearts. These interventions were: nifedipine, low calcium (0.75 mM, 0.15 mM and 0.05 mM), high magnesium (15 mM) and high magnesium (15 mM) with low calcium (0.75 mM). Ischemia was induced by abolishing coronary flow for 60 min, followed by 30 min of reperfusion. The results indicate that nifedipine when given during reperfusion had no protective effect, whilst it was beneficial when administrated before ischaemia. Lowering calcium content of the perfusate during reperfusion may be advantageous, depending on the degree of calcium reduction. Reperfusion with high magnesium reduced the mitochondrial calcium overload and maintained the mitochondrial ATP-producing capacity but failed to modify the increase of tissue calcium and of diastolic pressure. Lowering calcium content in the presence of high magnesium resulted in better protection. These data suggest that the conditions of reperfusion may influence the capacity of myocardial recovery.

Influence of L-carnitine administration on maximal physical exercise

SummaryThe effects of L-carnitine administration on maximal exercise capacity were studied in a double-blind, cross-over trial on ten moderately trained young men. A quantity of 2 g of L-carnitine or a placebo were administered orally in random order to these subjects 1 h before they began exercise on a cycle ergometer. Exercise intensity was increased by 50-W increments every 3 min until they became exhausted. After 72-h recovery, the same exercise regime was repeated but this time the subjects, who had previously received L-carnitine, were now given the placebo and vice versa. The results showed that at the maximal exercise intensity, treatment with L-carnitine significantly increased both maximal oxygen uptake, and power output. Moreover, at similar exercise intensities in the L-carnitine trial oxygen uptake, carbon dioxide production, pulmonary ventilation and plasma lactate were reduced. It is concluded that under these experimental conditions pretreatment with L-carnitine favoured aerobic processes resulting in a more efficient performance. Possible mechanisms producing this effect are discussed.

Uptake of spermine by rat liver mitochondria and its influence on the transport of phosphate

Spermine, a polyamine present in the mammalian cells at rather high concentration, has, among other actions, a remarkable stabilizing effect on mitochondria, functions which have generally been attributed to the capability of this and other polyamines to bind to membrane anionic sites. In the present paper evidence is provided that at physiological concentrations spermine may also be transported into rat liver mitochondrial matrix space, provided that mitochondria are energized and inorganic phosphate is simultaneously transported. The close dependence of spermine transport is also demonstrated by the concurrent efflux of spermine and inorganic phosphate when mitochondria preloaded with the two ionic species are deenergized either with uncouplers or respiratory chain inhibitors. Furthermore, Mersalyl, the known inhibitor of phosphate transport, prevents both spermine uptake and release. Mg2+ inhibits the transport of spermine conceivably by competing for the some binding sites on the mitochondrial membrane. The physiological significance of these results is discussed.

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.

Isovalerylcarnitine is a specific activator of the high calcium requiring calpain forms

Isovalerylcarnitine, a product of the catabolism of L-leucine, is a potent activator of rat calpains isolated from erythrocytes, kidney, liver, skeletal and heart muscle. Only calpains II, but not calpains I, are activated by IVC, with the only exception of rat erythrocyte calpain I, the only species present in these cells which has a Ca2+ requirement higher than that of most calpain I isoenzymes. Activation by IVC involves a dual effect: 1) a ten fold increase in the affinity of calpain for Ca2+, and 2) an increase in the Vmax 1.3-1.6 fold above the values observed with the native enzymes at saturating [Ca2+] as well as with the autolyzed fully active calpain form at 5 microM Ca2+. The increased affinity for calcium results in an increased rate of autoproteolysis of calpain II. Activation by IVC is additive to that promoted by interaction (or association) to phospholipids vesicles. Together these results suggest that IVC may operate as a selective activator of calpain both in the cytosol and at the membrane level; in the latter case in synergism with the activation induced by association of the proteinase to the cell membrane.

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.

Protective and restorative action of Mn2+ on membrane potential of rat liver mitochondria de-energized by Ca2+ and phosphate cycling

Abstract Inorganic phosphate and Ca 2+ in the incubation medium induce a parallel efflux of endogenous Mg 2+ and adenine nucleotides from rat liver mithochondria [1]. At first no release of accumulated Ca 2+ occurs during Mg 2+ efflux; however as soon as 50% of endogenous Mg 2+ has been lost, Ca 2+ begins to escape as well. It was also shown that addition of Mg 2+ to deenergized mitochondria restores the original membrane potential and confers to mitochondria the full capacity to reaccumulate the Ca 2+ lost [2]. Here, we report that the membrane potential of liver mitochondria incubated in the presence of Ca 2+ and phosphate is preserved by Mn 2+ and restored by the same cation plus ATP or ADP when collapsed. In the typical experiment reported in Fig. 1 Δψ of liver mitochondria, incubated in the presence of Ca 2+ and Pi was fully preserved by 50 μM Mn 2+ . Furthermore, when Mn 2+ was added to mitochondria deenergized by the action of external Ca 2+ and phosphate, Mn 2+ restored the original Δψ provided that ADP was also added. Concordantly, as shown in Fig. 2, Mn 2+ prevented the parallel efflux of endogenous Mg 2+ and adenine nucleotides induced by the flux of Ca 2+ and phosphate. This action of Mn 2+ is very similar to that of Mg 2+ with three major differences: (1) Mn 2+ is active at much lower concentrations; Mg 2+ exhibits the same action [2] of 50 μ M Mn 2+ when added in concentrations above 1 m M. (2) Unlike Mg 2+ , Mn 2+ restores collapsed Δψ only when added together with ATP or ADP. (3) Mg 2+ are unable to restore collapsed Δψ if Mn 2+ are previously added. These results showed that Mg 2+ can be replaced by Mn 2+ in some of their roles in mitochondria and provide preliminary evidence that Mg 2+ and Mn 2+ compete for the same binding sites located on the inner mitochondrial membrane. This assumption explains the inability of Mg 2+ to act as once Mn 2+ has been added to the mitochondrial suspension.

Mitochondria in Myocardial Ischemia

Due to the abundance of mitochondria in cardiac myocytes and the strict dependence of myocardial function on oxidative phosphorylation, it is hardly surprising that tight links have been described between mitochondrial dysfunction and cardiovascular diseases, especially in the relevant case of structural and functional alterations induced by ischemia and reperfusion. The elucidation of the underlying mechanisms have also indicated relevant targets for novel and focused therapeutic approaches, as was recently validated in patients undergoing myocardial ischemia. This article analyzes the processes by which mitochondria contribute to the loss of cardiomyocyte viability, especially during post-ischemic reperfusion, due to a deadly mixture of reduced adenosine triphosphate synthesis, elevated [Ca 2+ ], and increased formation of reactive oxygen species. These elements synergize in promoting opening of the mitochondrial permeability transition pore (PTP) that eventually hampers the maintenance of cardiomyocyte viability. Interventions and compounds antagonizing PTP opening have been demonstrated to afford significant cardioprotection against ischemia/reperfusion injury. Similarly, endogenous self-defenses triggered by conditioning stimuli appear to depend on inhibition of PTP opening through signaling mechanisms that have not yet been elucidated conclusively.

P.1.8 Genetic ablation of p66Shc rescues functional and morphological abnormalities in collagen VI dystrophic mice

Several studies documented the key role of abnormal production of reactive oxygen species (ROS) in the pathophysiology of muscular dystrophies (MDs) that are contributed also by mitochondrial dysfunction. The sources of ROS, however, are still controversial as well as their major molecular targets. This study investigated whether ROS produced in mitochondria by p66 Shc contributes to MD pathogenesis. p66 Shc is a growth factor adapter that is phosphorylated upon oxidative stress. In this form, a fraction of p66shc localizes to mitochondria, where it binds to cytochrome c and acts as an oxidoreductase, generating ROS and leading to organelle dysfunction and cell death. We provide clear evidence that inactivation of the gene encoding for p66 Shc reduced ROS accumulation along with a beneficial effect on the dystrophic phenotype of Col6a1 −/− mice, a model of Bethlem myopathy and Ullrich congenital MD. Based upon our previous observations on oxidative damage of myofibrillar proteins in heart failure and MDs, we hypothesized that p66 Shc -dependent ROS formation might impair contractile function in dystrophic muscles. Indeed, oxidation of myofibrillar proteins, as probed by formation of disulphide cross-bridges in tropomyosin, was detected in Col6a1 −/− mice. Notably, genetic ablation of p66 Shc significantly reduced myofiber apoptosis and ameliorated muscle strength in Col6a1 −/− mice. These findings demonstrate a novel and determinant role of p66 Shc in MDs, adding evidence of the pivotal role of mitochondria and suggesting p66 Shc as a novel pharmacological target for the therapy of human ColVI myopathies.

771 The opening of the permeability transition pore is responsible for mitochondrial dysfunction and cell death caused by N‐methyl‐N'‐nitro‐N‐nitrosoguanidine (MNNG)

a.u. for control, p<0.01, n=6). However, this increase was abolished in the presence of MPG (96±2 a.u., p<0.01). Co-treatment of ceramide with cyclooxygenase inhibitors, 10μM NS 398 or 1μM nimesulide also abolished the cytoprotection afforded by ceramide (cell viability: NS 398 + Cer 66±2% and nimesulide + Cer 66±1%; p<0.01 vs Cer; n=4) and partially decreased the amount of ROS produced by ceramide. Inhibition of other potential ROS-producing systems with NOS inhibitor (L-NAME, 5mM), xanthine oxidase inhibitor (allopurinol, 0.1mM) or NADPH oxidase inhibitor (Cadmium chloride, 1mM) failed to abolish ceramide cytoprotection. In conclusion, these data strongly suggest that ROS production mediated, in part, by cyclooxygenase would play a major role in the cytoprotective effect of ceramide.