Activation of a calcium-sensing receptor (Ca-SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca-SR has been identified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca-SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca-SR were identified in rat heart tissues usingRT-PCR that was further confirmed by sequence analysis. Ca-SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect any specific bands after preadsorption with standard Ca-SR antigens. An immunohistochemistry study revealed the presence of Ca-SR in rat cardiac as well as other tissues. An increase in extracellular calcium or gadolinium induced a concentration-dependent sustained increase in [Ca2+]i in isolated ventricular myocytes from adult rats. Spermine (1–10 mM) also increased [Ca2+]i. Pre-treatment of cardiac myocytes with thapsigargin or U73122 abolished the extracellular calcium, gadolinium or spermine-induced increase in [Ca2+]i. The blockade of Na+/Ca2+ exchanger or voltagedependent calcium channels did not alter the extracellular calcium-induced increase in [Ca2+]i. Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5-triphosphate (IP3) levels. Our results demonstrated that Ca-SR was expressed in cardiac tissue and cardiomyocytes and its function was regulated by extracellular calcium and spermine.

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.

Polyamines (putrescine, spermidine, and spermine)play an essential role in cell growth, differentiation, andapoptosis. Protein kinase C (PKC) stimulates polyaminebiosynthesis through the induction of ornithine decarboxylase(ODC), a rate-limiting enzyme in polyaminebiosynthesis. Activation of PKC mediates ischemic preconditioningto reduce necrosis and apoptosis in intacthearts and in isolated culture cardiomyocytes. In this study,we examined whether the ODC/polyamine system isinvolved in the ischemic preconditioning signaling pathwayand whether this system interacts with PKC inpreconditioning-induced cardioprotection. Hearts werepreconditioned with three cycles of 5-min ischemia and5-min reflow, which caused an increase of ODC expressionand spermidine, spermine, and total polyamine pool levels.a-Difluoromethylornithine (DFMO) and ethylglyoxal bis(guanylhydrazone) (EGBG) inhibited the key enzymesinvolved in polyamine biosynthesis, and abolished thepreconditioning-induced reduction in infarct size andimprovement in postischemic heart contractility function.They also increased cell apoptosis extent and aggravatedmyocardium ultrastructure damage. Inhibition also attenuatedthe preconditioning-induced translocation and activationof the PKC-d,-e isoforms from the cytosol to theparticulate. Conversely, activation of PKC by phorbol 12-myristate 13-acetate (PMA) upregulated the ODC/polyaminesystem, whereas the PKC inhibitor chelerythrine(Che) downregulated the ODC/polyamine system. Thesefindings suggest that upregulation of the polyamine synthesismetabolism occurs in response to preconditioningand mediates preconditioning-induced cardioprotection.The ODC/polyamine system and PKC signals may "crosstalk" in preconditioned hearts such that inhibiting onepathway leads to a reduction in the activity of the otherpathway and vice versa.

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.

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.