. 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.

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.

Gap-junctions are specialized regions of intercellular contacts allowing electrical impulse propagation among adjacent cardiomyocytes. Connexin43 (Cx43) is the predominant gap-junction protein in the working ventricular myocardium and its reduced expression has been extensively implicated in the genesis of conduction abnormalities and re-entry arrhythmia of chronically hypertrophied hearts. In contrast, data on the role played by this protein during cardiac remodeling and early phases of developing hypertrophy are lacking. Therefore, in the present study, we investigated this issue using an experimental model of pig left ventricle (LV) volume overloading consisting in the creation of an aorto-cava fistula. At scheduled times (6, 24, 48, 96, 168 h, and 2, 3 months after surgery) echocardiographic and haemodynamic measurements were performed and myocardial biopsies were taken for the morphological and biochemical analyses. When faced with the increased load, pig myocardium underwent an initial period (from 6 up to 48 h) of remarkable tissue remodeling consisting in the occurrence of cardiomyocyte damage and apoptosis. After that time, the tissue developed a hypertrophic response that was associated with early dynamic changes (up-regulation) in Cx43 protein expression, as demonstrated by Western blot and confocal immunofluorescence analyses. However, an initial transient increase of this protein was also found after 6 h from surgery. With the progression of LV hypertrophy (from 168 hr up to 3 months), a reduction in the myocardial Cx43 expression was, instead, observed. The increased expression of Cx43 protein during acute hypertrophic response was associated with a corresponding increase in the levels of its specific mRNA, as detected by RT-PCR. We concluded that up-regulation of Cx43 gap-junction protein could represent an immediate compensatory response to support the new working conditions in the early stages of ventricular overloading.

We previously reported that acylphosphatase, a cytosolic enzyme present in skeletal and heart muscle, actively hydrolyzes the phosphoenzyme (EP) of cardiac sarcoplasmic reticulum (SR) Ca2+-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.

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.

To identify early adaptive processes of cardiac remodeling (CR) in response to volume overload, we investigated the molecular events that may link intracellular Ca2+ homeostasis alterations and cardiomyocyte apoptosis. In swine heart subjected to aorto-cava shunt for 6, 12, 24, 48 and 96 h sarcoplasmic reticulum (SR) Ca2+ pump activity was reduced until 48 h (−30%), but a recovery of control values was found at 96 h. The decrease in SR Ca2+-ATPase (SERCA2a) expression at 48 h, was more marked (−60%) and not relieved by a subsequent recovery, while phospholamban (PLB) concentration and phosphorylation were unchanged at all the considered times. Conversely, acylphosphatase activity and expression significantly increased from 48 to 96 h (+40%). Bcl-2 expression increased significantly from 6 to 24 h, but at 48 h, returned to control values. At 48 h, microscopic observations showed that overloaded myocardium underwent substantial damage and apoptotic cell death in concomitance with an enhanced Fas/Fas-L expression. At 96 h, apoptosis appeared attenuated, while Fas/Fas-L expression was still higher than control values and cardiomyocyte hypertrophy became to develop. These data suggest that in our experimental model, acylphosphatase could be involved in the recovery of SERCA2a activity, while cardiomyocyte apoptosis might be triggered by a decline in Bcl-2 expression and a concomitant activation of Fas.

OBJECTIVE:1,25-Dihydroxyvitamin D3 [1,25(OH)2D3], the hormonal active form of vitamin D3, could represent a potentially therapeutic agent in autoimmune diseases. Cyclosporin A (CsA) shows immunoregulatory properties, which, in many respects, seem to be similar to those of 1,25(OH)2D3. Our aim was to investigate the possible synergistic effect exerted by CsA in combination with 1,25(OH)2D3 or its nonhypercalcemic analogues, EB 1089 and KH 1060, on the proliferative response of T lymphocytes obtained from active ulcerative colitis patients. METHODS:The T lymphocyte-enriched population was treated with phytohemagglutinin and CsA (doses from 1 ng to 1000 ng/ml) alone or in association with 1,25(OH)2D3 or EB 1089 or KH 1060 (0.1, 1, 10 nM final concentration). Cell proliferation was determined by [3H]thymidine incorporation and analyzed on day 5 of culture. RESULTS:After incubation with CsA, T lymphocyte proliferation was significantly inhibited in comparison with the vehicle-treated cultures. However, T lymphocytes from ulcerative colitis patients were significantly more sensitive to CsA than those from healthy controls. The inhibition in T lymphocyte proliferation, after treatment of the cultures with CsA associated with either 1,25(OH)2D3 or EB 1089 or KH 1060, was synergistic at well-defined concentrations. CONCLUSIONS:Taking into account the lowest CsA dose (1 ng/ml), the highest synergistic inhibition in the proliferation of T lymphocytes prepared from ulcerative colitis patients was found combining CsA and 10 nM of 1,25(OH)2D3 or 10 nM of EB 1089 or KH 1060 at the three concentrations. The results obtained, associating the lowest CsA dose and the lowest KH 1060 concentration, may suggest an alternative therapeutic approach in these patients, reducing the dose, and consequently the toxicity, of CsA.

The response of C2C12 myoblasts to 1 nM 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 100 nM retinoids (9-cis retinoic acid, all-trans retinoic acid) and to combination treatments, after 72 h incubation, was studied. The incubation with 1,25(OH)2D3 was ineffective on either cell proliferation or [3H]thymidine incorporation (expressed as DPM per cell) or protein content per cell. On the contrary, all the other treatments inhibited cell proliferation, this inhibition being synergistic when the vitamin D derivatives were combined with 9-cis or all-trans retinoic acid, and increased [3H]thymidine incorporation and protein content per cell. The levels of the VDR protein remarkably increased in comparison with control cells, except for the incubation with 9-cis retinoic acid. This increase was particularly accentuated in C2C12 cells treated with KH 1060 and 9-cis retinoic acid in combination. These results, taken together, suggest a role for vitamin D derivatives and retinoids on C2C12 cells.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)2D3 or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)2D3 or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)2D3 or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)2D3 or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)2D3 or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)2D3 or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)2D3 or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.

This study examines the effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on proliferation of SH-SY5Y human neuroblastoma cells and demonstrates, for the first time, the presence of vitamin D receptor (VDR) in this cell line. Cell number showed a significant decrease, when the cells were incubated with 1 or 10 nM 1,25(OH)2D3 for 24, 48, 72, 96 and 144 h, while 100 nM 1,25(OH)2D3 was ineffective after 24 and 96 h incubation. The highest inhibition (about 35%) was observed after 72 h treatment with the hormone at the three concentrations used. Protein content per cell increased, in comparison with controls, after treatment of SH-SY5Y neuroblastoma cells with 1,25(OH)2D3, at the three concentrations, up to 96 h incubation. 1, 10 or 100 nM 1,25(OH)2D3 positively affected [3H]thymidine incorporation after treatment of the cells for 48 and 72 h, while, after 24 h, only 10 nM 1,25(OH)2D3 exerted a stimulatory action. To study the expression of the VDR gene, Northern blot analysis was performed. Subconfluent SH-SY5Y neuroblastoma cells were treated for 24 h with medium containing 10 nM 1,25(OH)2D3 or vehicle alone. A main transcript of an approximate size of 4.5 kb, present either in controls or in cells incubated with the hormone, was revealed. A limited increase in VDR mRNA levels was observed in the cells treated with 1,25(OH)2D3, fetal bovine serum or forskolin. Only slight differences in morphology were perceived between SH-SY5Y cultures maintained with or without 10 nM 1,25(OH)2D3 for 7 days.

This study examines the effect of 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], serum or forskolin on the proliferation of IMR‐90 fetal lung fibroblasts and demonstrates, for the first time, the presence of 1,25(OH)2D3 receptor (VDR) in this cell line. In quiescent, subconfluent cultures neither the treatment with 100 nM 1,25(OH)2D3 nor that with 50 μM forskolin influenced proliferation, while a significant increase was observed after incubation of the cells with 10% fetal bovine serum. Either cell number, determined on growing IMR‐90 human fibroblasts after 48 or 72 h incubation with 100 nM 1,25(OH)2D3 or [3H]thymidine incorporation (24, 48 or 72 h incubation) significantly decreased, while protein content per cell increased. Northern blot analysis revealed the expression of the VDR gene, the VDR mRNA bands being prominent in 1,25(OH)2D3, serum or forskolin treated fibroblasts. VDR mRNA levels slightly decreased, when growing fibroblasts were exposed to 100 nM 1,25(OH)2D3 for 48 or 72 h.

The response of IMR-90 human fetal lung fibroblasts at high population doubling level (PDL > 42) to 1,25-dihydroxyvitamin D3[1,25(OH)2D3] was investigated to clarify whether some metabolic and molecular parameters of senescent cells are affected by the hormone treatment. Pyruvate kinase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity significantly increased after treatment of confluent-phase cells with 10 nM 1,25(OH)2D3 for 24 h. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3 to affect the enzyme activities, while estradiol-17 beta and progesterone produced a slight increase in glucose-6-phosphate dehydrogenase and lactate dehydrogenase levels, respectively. 1,25(OH)2D3 also affected fibroblast proliferation, protein content/cell and DNA synthesis. The cell number significantly decreased after a 48 h incubation with 1,25(OH)2D3 at various concentrations (0.01-1 nM) when compared with control fibroblasts, while an increase in the protein content/cell was demonstrated. The same experiment, carried out by protracting the incubation with the hormone for 72 h, showed a similar trend, but 10 nM 1,25(OH)2D3 was also able to inhibit cell proliferation and to stimulate protein synthesis. The incorporation of [3H]thymidine into DNA increased after the treatment of high PDL fibroblasts with 0.01-1 nM of hormone for 48 h in comparison with controls.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a role in regulating some aspects of metabolism in IMR-90 normal human fetal lung fibroblasts. Among the enzymes studied, only pyruvate kinase showed a significant increase after treatment of confluent-phase cells with 1,25(OH)2D3 at various concentrations (0.1-100 nM range) for 24 h. A parallel increase in lactate output was observed. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3, estradiol-17 beta and progesterone to affect pyruvate kinase activity. The determination of the time course of [3H]-2-deoxy-D-glucose transport indicated that the hormone did not influence the transmembrane transport system of D-glucose. The addition of the inhibitors cycloheximide and actinomycin D to the culture medium abolished, at least in part, the 1,25(OH)2D3 stimulation of pyruvate kinase activity, suggesting the probable dependence of the hormone effect on cellular RNA and protein synthesis. 1,25(OH)2D3 also affected fibroblast growth and DNA synthesis. Cell number significantly decreased after 2-5 days treatment with 10 nM hormone in comparison with control fibroblasts, and also the incorporation of [3H]thymidine into DNA decreased after treatment of the cells with 1 and 10 nM hormone for 48 h. In conclusion, these data demonstrate that 1,25(OH)2D3 stimulates pyruvate kinase activity in confluent-phase IMR-90 human fibroblasts by a mechanism probably dependent on de novo protein synthesis, and also affects cell growth and DNA synthesis in sub-confluent-phase cells.

We previously demonstrated that feeding rats the Steenbock and Black rickets-inducing diet produces remarkable changes in the metabolic pattern of intestinal mucosa, kidney, liver, cerebral cortex and heart. We have now determined the levels of calcium, phosphorus and citrate in cerebral cortex and the activity of some enzymes in synaptosomes and cerebral cortex mitochondria of three rat groups: control (Group A), fed a vitamin D-deficient diet (Group B), fed a vitamin D-deficient diet and treated with 1,25-dihydroxyvitamin D3 (Group C). While calcium content increased in Groups B and C, phosphorus concentration increased only in Group C and citrate in Group B in comparison with control. The increase in acetylcholinesterase and citrate synthase registered in Group B was restored to control values by 1,25-dihydroxyvitamin D3 treatment, while, neither the decrease in cytochrome c oxidase, nor the increase in glucose-6-phosphate dehydrogenase, acid phosphatase and NADP+(-)isocitrate dehydrogenase observed in Group B were corrected by 1,25-dihydroxyvitamin D3 supply. Acyl phosphatase showed a remarkable increase in consequence of 1,25-dihydroxyvitamin D3 administration.