| Background: The nonezymatic reaction between protein, lipids,ribonucleotide and aldoses produced unstable compound known as a Schiff base. This compound can undergo rearrangement to form the more stable Amadori-type products-AGEs. Recently studies suggested that AGEs may play a key role in the pathogenesis of cardiovascular diseases such as atherosclerosis, hypertension, diabetic crdiomyopathy and may participate in the left ventricle diastolic dysfunction in patients with type 1 diabetes mellitus. Our previous studies found that AGEs could significantly increase the number of apoptosis body/nucleus on CMs in a time- and dose- dependent manner. Antibody of RAGE and inhibitor of P38MAPK-SB203580 could inhibit AGEs induced apoptosis in CMs. Meanwhile we found AGEs could significantly increase Ang Ⅱ autocrining in CMs. Losartan could inhibit AGEs induced apoptosis in CMs. On the other hand, we found exogenous AGEs could result in dcrease of left ventricle diastolic function in CRF rat model. The effects and the pathway of advanced glycation end products on cytosolic free calcium concentration in CMs and whether the changes was involved in the CMs injury byAGEs are unknown. It will provide a new target for clinical therapy to investigate the effects and mechanisms of AGEs on cytosolic free calcium in CMs.Ojective: In this study we investigated the effects of advanced glycation end products on cytosolic free calcium concentration in cultured neonatal rat cardiac myocytes. We also studied the interfering action of verapamil (inhibitor of L-type calcium channel), procaine (inhibitor of RyRs), heparin (inhibitor of IP3R), Thapsigargin (inhibitor of SERCA2), antibody of RAGE, SB203580 (inhibitor of P38MAPK) and Losartan (inhibitor of ATI) on CMs cytosolic free calcium concentration's changes induced by AGEs. Meanwhile we measured the expressions of SERCA2, RyR and phospholamban in CMs treated with AGEs. We examined the effects of Angllon cytosolic free calcium concentration in CMs preconditioning with AGEs to elucidated whether AGEs enhanced the effects of Ang II on CMs.Methods: CMs cultured by trypsin digestion method for 3-5days in vitro wereincubated with Ca2+-sensitive fluorescent indicator Fluo-3/AM with light screening at 37 °C with 5%CO2 for 60 minutes. Changes of fluorescence singal of free calcium caused by AGEs(50ug/ml, lOOug/ml , 200ug/ml )and HSA ((50ug/ml, lOOug/ml , 200ug/ml )were measured under the LSCM. In the studies of the interfering action of verapamil, procaine, heparin, Thapsigargin, antibody of RAGE, SB203580 and Losartan, CMs were preconditioned with these drugs at different doses as designed for 2 hours, then process of fluorescence labeling and LSCM same as above. The expressions of SERCA2, RyRs2 and phospholamban were measured by western blotting. CMs had been pretreated with AGEs and HSA for 12 and 24 hours, then labeled with Fluo-3/AM. Changes of fluorescence singal of free calcium caused by Ang II were measured under the LSCM.Results:1.Compared with the control group, AGEs could rapidly increased fluorescence value of CMs. The mean fluorescence peak value and mean fluorescence elevated amplitude of CMs increased to 1.72, 2.2, 2.7 times and 2.96,4.09 ,4.52 times in a dose-dependent manner at 50ug/ml, lOOug/ml, 200ug/ml respectively (p<0.05). Five minutes after AGEs administration, increased fluorescence could not recovery.2. Interfering action of some drugs.(1).Antibody of RAGE pretreatment could inhibit fluorescence increasing in CMs caused by AGEs compared with control group and Gamma globulin group. Antibody of RAGE groups displayed decreased fluorescence after AGEs administration.(2).Verapamil, an inhibitor of L-type calcium channel, could not inhibit AGEs induced increasing of fluorescence in CMs.(3). An inhibitor of RyRs-procaine could not inhibit AGEs induced increasing of fluorescence in CMs.(4). Heparin could competitive inhibit IP3R. Pretreated with heparin could partly inhibit AGEs's effect, the mean fluorescence peak value and mean fluorescence elevated amplitude of CMs decreased in heparin group, and the recovery time of fluorescence was significantly shorten by heparin.(5).Thapsigargin, an inhibitor of SERCA2, could completely inhibit AGEs induced increasing of fluorescence in CMs. Thapsigargin groups displayed decreased fluorescence after AGEs administration.3. SB203580 and Losartan all could inhibit AGEs induced increasing of fluorescence in CMs. In addition SB203580 could shorten the recovery time of fluorescence.4. Ang II could increased the fluorescence of CMs, preconditioning with AGEs for 12h could enhance the effect of Ang II, and lengthen recovery time of fluoresce-ence, preconditioning with AGEs for 24h only lengthen recovery time of fluorescence, had no effect on mean fluorescence elevated amplitude.5. The expression of SERCA2 and RyR in CMs with AGEs treatment for 12, 24 and 48 hours had no significant difference compare with control group. AGEs decreased the expression of phospholamban in CMs in a dose-dependent manner compare with control group, this effect abolished at 48h.Conclusions:l.AGEs could increased the cytosolic free calcium concentration in cultured neonatal rat cardiac myocytes in a dose-dependent manner.This effect was mediated by RAGE in cell membrane. Antibody of RAGE could inhibit this effect.2. Predominant source of increased cytosolic free calcium caused by AGEs was calcium released from sarcoplasmic reticulum through IP3R. Heparin and Thapsigargin could inhibit this effect, verapamil and procaine could not inhibit this effect.3. P38MAPK and Angll were involved in the effect of AGEs. SB203580 and Losartan all could inhibit increasing of cytosolic free calcium concentration in CMs caused by AGEs.4. Preconditioning with AGEs could enhance the effect of Ang II on cytosolic free calcium concentration in CMs,and lengthen recovery time of calcium.5. AGEs had no effect on the expression of SERCA2 and RyRs in CMs, but could decrease the expression of phospholamban in CMs in a dose-dependent manner.
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