No abstract

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD(+) and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD(+) and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion.

We investigated the effect of 3-aminobenzamide (3-AB), an inhibitor of the nuclear enzyme poly(ADP-ribose) polymerase (PARP), against early ischemia/reperfusion (IR) injury in heart transplantation. In our experimental model, rat heart subjected to heterotopic transplantation, low temperature global ischemia (2 h) was followed by an in vivo reperfusion (60 min). In these conditions, and in the absence of 3-AB treatment, clear signs of oxidative stress, such as lipid peroxidation, increase in protein carbonyls and DNA strand breaks, were evident; PARP was markedly activated in concomitance with a significant NAD+ and ATP depletion. The results of microscopic observations (nuclear clearings, plasma membrane discontinuity), and the observed rise in the serum levels of heart damage markers, suggested the development of necrotic processes while, conversely, no typical sign of apoptosis was evident. Compared to the effects observed in untreated IR heart, the administration of 3-AB (10 mg/kg to the donor and to the recipient animal), but not that of its inactive analogue 3-aminobenzoic acid, significantly modified the above parameters: the levels of oxidative stress markers were significantly reduced; PARP activation was markedly inhibited and this matched a significant rise in NAD+ and ATP levels. PARP inhibition also caused a reduced release of the cardiospecific damage markers and attenuated morphological cardiomyocyte alterations, save that, in this condition, we noted the appearance of typical apoptotic markers: activation of caspase-3, oligonucleosomal DNA fragmentation, ISEL positive nuclei. Possible mechanisms for these effects are discussed, in any case the present results indicate that PARP inhibition has an overall beneficial effect against myocardial reperfusion injury, mainly due to prevention of energy depletion. In this context, the signs of apoptosis observed under 3-AB treatment might be ascribed to the maintenance of sufficient intracellular energy levels. These latter allow irreversible damages triggered during the ischemic phase to proceed towards apoptosis instead of towards necrosis, as it appears to happen when the energetic pools are depleted by high PARP activity.

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated the role of oxidative stress in the initial phases of cardiac remodeling induced in an animal model by volume overload. As plausible candidates for a connection between oxidative stress and cardiomyocyte apoptosis or hypertrophy, we explored the behaviour of two MAPKs, specifically JNK and ERK. At 48 h of overload, the greatest increase in oxidative stress coincided with a peak of cardiomyocyte apoptosis. This was possibly induced through the mitochondrial metabolism, as evidenced by the release of cytochrome c and a significant increase in the active forms of caspase-9 and -3, but not caspase-8. Oxidative stress markers significantly decreased at 96 h of overload, combined with a marked attenuation of apoptosis and the appearance of hypertrophy. The highest levels of JNK and the lowest levels of ERK phosphorylation were observed at 48 h of overload. Conversely, a sharp increase in ERK phosphorylation was detected at 96 h of overload coinciding with the hypertrophic response. Together these results show that oxidative stress is an early and transient event in myocardial volume overload. They suggest that oxidative stress mediates amplitude dependent apoptotic and hypertrophic responses in cardiomyocytes through the selective activation of, respectively, JNK and ERK.

We previously reported that acylphosphatase, a cytosolic enzyme present in skeletal and heart muscle, actively hydrolyzes the phosphoenzyme (EP) of cardiac sarcoplasmic reticulum (SR) Ca 2þ -ATPase (SERCA2a), inducing an increased activity of this pump. We hypothesized that acylphosphatase-induced stimulation of SERCA2a, in addition to enhanced EP hydrolysis, may be due to a displacement of phospholamban (PLN), removing its inhibitory effect. To verify this hypothesis co-immunoprecipitation experiments were performed by adding recombinant muscle acylphosphatase to solubilized heart SR vesicles, used as a source of SERCA2a and PLN. With anti-acylphosphatase antibodies only SERCA2a was co-immunoprecipitated in an amount which increased in parallel to the concentrations of our enzyme. Conversely, using anti-SERCA2a antibody, both PLN and acylphosphatase were co-immunoprecipitated with SERCA2a, and the PLN amount in the precipitate decreased with increasing acylphosphatase concentrations. SERCA2a and PLN were co-immunoprecipitated by anti-phospholamban antibodies, but while the amount of precipitated phospholamban increased in the presence of acylphosphatase, the level of SERCA2a decreased. These preliminary results strengthen the supposed displacement of phospholamban by acylphosphatase. . The cardiac sarcoplasmic reticulum (SR) Ca 2þ -ATPase (SERCA2a) is regulated by its interactions with the intrinsic membrane protein phospholamban. Phospholamban (PLN) inhibits the activity of SERCA2a, but its phosphorylation by protein kinase A or Ca 2þ -calmodulin kinase reverses the inhibitory interaction, presumably through dissociation of phosphorylated phospholamban from SERCA2a [3]. An additional regulatory effect on SERCA2a could be exerted by acylphosphatase (EC 3.6.1.7), a cytosolic 11 kDa enzyme well represented in cardiac muscle that catalyzes the hydrolysis of acylphosphates [4][5][6]. In previous studies we have shown that acylphosphatase actively hydrolyzes the EP intermediate of this transport system, an effect that results in an enhanced activity of the SR Ca 2þ pump [7,8]. We have also demonstrated that acylphosphatase, in addition to its hydrolytic activity on EP, stimulated SERCA2a activity through another mechanism, probably due to its conformational properties. In fact, using an acylphosphatase mutant, Asn41Ser, devoid of catalytic activity but unchanged as for the three-dimensional structure [9], the stimulatory effect on SERCA2a was retained, but disappeared when the conformational structure of the mutant was altered by thermal denaturation. To explain these results we supposed that acylphosphatase could interact with SERCA2a taking the place of unphosphorylated PLN whose inhibitory effect could be removed [8]. In fact, NMR studies showed that acylphosphatase contains a structural motif where 12 residues (from 55 to 66) form an amphipathic a-helix with a prevalence of basic groups [10] that resembles that of the PLN cytoplasmic 1A domain, essential for the association with SERCA2a [11].