The intracellular Ca2? concentration ([Ca2?]i) isincreased during cardiac ischemia/reperfusion injury (IRI),leading to endo(sarco)plasmic reticulum (ER) stress. PersistentER stress, such as with the accumulation of [Ca2?]i,results in apoptosis. Ischemic post-conditioning (PC) canprotect cardiomyocytes from IRI by reducing the [Ca2?]i via protein kinase C (PKC). The calcium-sensing receptor(CaR), a G protein-coupled receptor, causes the productionof inositol phosphate (IP3) to increase therelease of intracellular Ca2? from the ER. This processcan be negatively regulated by PKC through the phosphorylationof Thr-888 of the CaR. This study testedthe hypothesis that PC prevents cardiomyocyte apoptosisby reducing the [Ca2?]i through an interaction of PKCwith CaR to alleviate [Ca2?]ER depletion and [Ca2?]melevation by the ER-mitochondrial associated membrane(MAM). Cardiomyocytes were post-conditioned after 3 hof ischemia by three cycles of 5 min of reperfusion and5 min of re-ischemia before 6 h of reperfusion. DuringPC, PKCe translocated to the cell membrane and interactedwith CaR. While PC led to a significant decreasein [Ca2?]i, the [Ca2?]ER was not reduced and [Ca2?]mwas not increased in the PC and GdCl3–PC groups.Furthermore, there was no evident Dwm collapse duringPC compared with ischemia/reperfusion (I/R) or PKCinhibitor groups, as evaluated by laser confocal scanningmicroscopy. The apoptotic rates detected by TUNEL andHoechst33342 were lower in PC and GdCl3–PC groupsthan those in I/R and PKC inhibitor groups. Apoptoticproteins, including m-calpain, BAP31, and caspase-12,were significantly increased in the I/R and PKC inhibitorgroups. These results suggested that PKCe interactingwith CaR protected post-conditioned cardiomyocytes fromprogrammed cell death by inhibiting disruption of themitochondria by the ER as well as preventing calciuminducedsignaling of the apoptotic pathway.

Communication between the SR (sarcoplasmic reticulum, SR) and mitochondria is important for cell survival andapoptosis. The SR supplies Ca2+ directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at closecontacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). Although it hasbeen demonstrated that CaR (calcium sensing receptor) activation is involved in intracellular calcium overload duringhypoxia/reoxygenation (H/Re), the role of CaR activation in the cardiomyocyte apoptotic pathway remains unclear. Wepostulated that CaR activation plays a role in the regulation of SR-mitochondrial inter-organelle Ca2+ signaling, causingapoptosis during H/Re. To investigate the above hypothesis, cultured cardiomyocytes were subjected to H/Re. Weexamined the distribution of IP3Rs in cardiomyocytes via immunofluorescence and Western blotting and found thattype 3 IP3Rs were located in the SR. [Ca2+]i, [Ca2+]m and [Ca2+]SR were determined using Fluo-4, x-rhod-1 and Fluo 5N,respectively, and the mitochondrial membrane potential was detected with JC-1 during reoxygenation using laserconfocal microscopy. We found that activation of CaR reduced [Ca2+]SR, increased [Ca2+]i and [Ca2+]m and decreased themitochondrial membrane potential during reoxygenation. We found that the activation of CaR caused the cleavage ofBAP31, thus generating the pro-apoptotic p20 fragment, which induced the release of cytochrome c frommitochondria and the translocation of bak/bax to mitochondria. Taken together, these results reveal that CaR activationcauses Ca2+ release from the SR into the mitochondria through IP3Rs and induces cardiomyocyte apoptosis duringhypoxia/reoxygenation.

Background: Polyamines and nitric oxide (NO) havebeen involved in the pathogenesis of cardiachypertrophy. NO can regulate cardiac ion channelsby direct actions on G-proteins and adenyl cyclase.The present study was undertaken to elucidate themolecular mechanism of interactions with polyaminesand NO in cardiac hypertrophy. Methods: Cardiaomyocytehypertrophy was induced by angiotensinII(AngII). Hypertrophy was estimated by cell-surfacearea, atrial natriuretic peptide (ANP) mRNAexpression, and the immunofluorescence of phalloidin.Pretreatment with alpha-difluoromethylornithine(DFMO) was done to deplete putrescine; KT5823pretreatment was carried out to block the nitric oxide/cGMP-dependent protein kinase type-I (NO/PKG-I)pathway. Expressions of endothelial nitric oxidesynthase (eNOS), PKG-I, c-fos and c-myc wereanalyzed by western blotting and immunofluorescence.The intracellular concentration of freecalcium ([Ca2+]i) was determined by confocal laserscanning microscopy. Results: Hypertrophy ofcardiomyocytes was induced by AngII, this caused anincrease in putrescine, spermidine and total polyaminepool in association with a decreased level of NO.Expressions of eNOS and PKG-I were down-regulated,[Ca2+]i was increased, and expressions of c-Fos andc-Myc upregulated. DFMO reversed these changesinduced by AngII. Conclusions: Downregulation ofpolyamines inhibits cardiomyocyte hypertrophy, whichis closely related to [Ca2+]i and the NO/PKG-I pathway.

Dopamine receptors exist in many tissues, including rat cardiac tissue. However, the physiological importance ofdopamine receptors in the homeostatic regulation of cardiac function is unclear. In this study, a model of ischaemia/reperfusion was established by culturing primary neonatal rat cardiomyocytes in ischaemia-mimetic solution for 2 hr, followedby incubation in normal culture medium for 24 hr. Lactate dehydrogenase activity, superoxide dismutase activity andmalondialdehyde content were determined colorimetrically with a spectrophotometer. Apoptotic cell death was assayed by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, terminal deoxynucleotidyl transferase-mediateddUTP-biotin nick end labelling staining and flow cytometry, and morphological alterations were observed with transmissionelectron microscopy. The intracellular free calcium concentration ([Ca2+]i) was measured by confocal laser scanningmicroscopy. Finally, the expression of dopamine receptor 1 (DR1), caspase-3, -8 and -9, Fas, Fas ligand and Bcl-2 and therelease of cytochromecwere analysed by Western blot. The results showed that DR1 expression was increased markedlyduring ischaemia/reperfusion. Treatment with 10M SKF-38393 (DR1 agonist) significantly increased lactate dehydrogenaseactivity, decreased superoxide dismutase activity and increased malondialdehyde content in the culture medium. The DR1agonist promoted the release of cytochromec, accumulation of [Ca2+]i, and apoptosis induced by ischaemia/reperfusion.Furthermore, SKF-38393 up-regulated the expression of caspase-3, -8 and -9, Fas and Fas ligand, and down-regulatedBcl-2 expression. In contrast, 10M SCH-23390 (DR1 antagonist) had no significant effects on the above indicators. Inconclusion, DR1 activation is involved in the apoptosis of cultured neonatal rat cardiomyocytes in simulated ischaemia/reperfusion through the mitochondrial and death receptor pathways.

Polyamines (putrescine, spermidine and spermine) are essential for cell growth and differentiation. Nitric oxideexhibits antihypertrophic functions and inhibits cardiac remodelling. However, the metabolism of polyamines and thepotential interactions with nitric oxide in cardiac hypertrophy remain unclear. We randomly divided Wistar rats into fourtreatment groups: controls, isoproterenol (ISO), ISO and -arginine, and -arginine. Isoproterenol (5mg/kg/day, subcutaneously)and/or?-arginine (800 mg/kg/day, intraperitoneally) was administered once daily for 7 days. The expression ofatrial natriuretic peptide mRNA was determined by reverse transcription-polymerase chain reaction, and fibrogenesis ofheart was assessed by Van Gieson staining. Polyamines were measured with high-performance liquid chromatography, andplasma nitric oxide content and lactate dehydrogenase (LDH) activity were determined with a spectrophotometer. Theexpression levels of ornithine decarboxylase, spermidine/spermine N1-acetyltransferase (SSAT), endothelial nitric oxidesynthase (eNOS) and inducible nitric oxide synthase (iNOS) were analysed by Western blot. Heart-to-body weight ratio,left ventricle-to-body weight ratio, atrial natriuretic peptide mRNA expression, collagen fibres and LDH activity wereelevated, both ornithine decarboxylase and SSAT proteins were up-regulated, and total polyamines were increased in thegroup treated with ISO. Additionally, the expression of iNOS was up-regulated, eNOS was down-regulated, and nitricoxide levels were low. Notably, cotreatment with -arginine reversed most of these changes except for SSAT expression,which was further up-regulated. We propose that increased polyamines and decreased nitric oxide are involved in cardiachypertrophy induced by ISO and suggest that -arginine pre-treatment can attenuate cardiac hypertrophy through theregulation of key enzymes of the polyamine and nitric oxide pathways.