Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor

Abstract

Ischemic postconditioning (IPC) and ATP sensitive potassium channel (KATP) agonists (e.g. pinacidil and diazoxide) postconditioning are effective methods to defeat myocardial ischemia-reperfusion (I/R) injury, but their specific mechanisms of reducing I/R injury are not fully understood. We observed an intracellular free calcium ([Ca²⁺]_i) overload in Anoxia/reoxygenation (A/R) cardiomyocytes, which can be reversed by KATP agonists diazoxide or pinacidil. The calcium-sensing receptor (CaSR) regulates intracellular calcium homeostasis. CaSR was reported to be involved in the I/R-induced apoptosis in rat cardiomyocytes. We therefore hypothesize that IPC and pinacidil postconditioning (PPC) reduce calcium overload in I/R cardiomyocytes by the down-regulation of CaSR. A/R model was established with adult rat caridomyocyte. mRNA and protein expression of CaSR were detected, IPC, PPC and KATP's effects on [Ca²⁺]_i concentration was assayed too. IPC and PPC ameliorated A/R insult induced [Ca²⁺]_i overload in cardiomyocytes. In addition, they down-regulated the mRNA and protein level of CaSR as we expected. CaSR agonist spermine and KATP blocker glibenclamide offset IPC's effects on CaSR expression and [Ca²⁺]_i modulation. Our data indicate that CaSR down-regulation contributes to the mitigation of calcium overload in A/R cardiomyocytes, which may partially represents IPC and KATP's myocardial protective mechanism under I/R circumstances.

Keywords: ATP sensitive potassium channel (KATP); Calcium-sensing receptor (CaSR); Ischemia-reperfusion (I/R) injury; Postconditioning.

Figure 1. Illustration of A/R protocols.

After 20 h culture in normoxic incubator, cardiomyocytes were randomly distributed to different groups. Cardiomyocytes of Control were continuously cultured in a normoxic incubator for 105 min. Medium of other groups was replaced with N₂ bubbled (95% N₂, 5% CO₂) M199 for the first 45 min, then replaced with O₂ bubbled M199. IPC group underwent three cycles of reoxygenation/anoxia (5 min: 5 min) before 30 min normal culture. Different concentration of pinacidil was added into the M199 and incubated with cardiomyocytes for 5 min at the beginning of reoxygenation in PPC groups. Glibenclamide + IPC group and spermine + IPC group were treated with 5 min glibenclamide (Gli) or spermine (Sper), respectively before IPC treatments (3 reoxygenation/anoxia (5 min: 5 min)). A/R + glibenclamide and A/R + spermine group were treated with 5 min glibenclamide or spermine, respectively at the end of anoxia periods before reoxygenation. Each protocol took 105 min in total.

Figure 2. [Ca²⁺]_i and cell viability detection in acutely isolated rat cardiomyocytes after A/R, IPC, PPC, KATP blocker or CaSR agonist treatment.

(A–B) The morphology of acutely isolated adult rat cardiomyocytes. The ventricular myocytes were rod-shaped, with clear cross striations. (C–L) The effect of different treatment on the [Ca²⁺]_i level in adult rat cardiomyocytes. At the end point of reoxygenation, cells of Control (C), A/R (D), IPC (E), PPC (10 μM) (F), PPC (30 μM) (G), PPC (100 μM) (H), glibenclamide + IPC (I), spermine + IPC (J), A/R + glibenclamide (K) and A/R + spermine (L) group were incubated in 10 μM Fluo-3-AM for 60 min at 37 °C and detected with a confocal microscope. (M) The [Ca²⁺]_i fluorescence intensity comparison. [Ca²⁺]_i in A/R group increased dramatically compared with the Control group (P < 0.01). IPC and 30 or 100 μM pinacidil reduced the [Ca²⁺]_i intensity. After glibenclamide administration, fluorescence intensity increased to the A/R group level. CaSR agonist spermine offset IPC’s effect on [Ca²⁺]_i level too. *, P < 0.01 compared with Control, IPC and PPC group. #, P < 0.01 compared with glibenclamide + IPC, spermine + IPC, A/R + glibenclamide or A/R + spermine group. ★, P < 0.01 compared with PPC (10 μM) or PPC (100 μM) group. Data are mean ± SD, n = 25 cells for each group. (N) CCK-8 assay showed that A/R insult significantly decreased the cell viability while IPC and 30 μM PPC reversed the decrease. Glibenclamide or spermine use can offset IPC’s effect on cell viability. *, P < 0.01 compared with Control, IPC and PPC group. #, P < 0.01 compared with glibenclamide + IPC, spermine + IPC, A/R + glibenclamide or A/R + spermine group. Data are mean ± SD, n = 6.

Figure 3. CaSR mRNA expression.

At the end point of reoxygenation, cells of eight groups were subjected to RT-PCR to detect CaSR expression at the mRNA level. CaSR mRNA showed apparently increase in A/R group compared with the Control group. IPC and 30 μM pinacidil reduced this trend profoundly. After glibenclamide administration, CaSR expression increased nearly to the A/R group level. CaSR agonist spermine offset IPC’s effect on CaSR mRNA level too. Data are expressed as mean ± SD. n = 6 for each group. *, P < 0.01 compared with Control, IPC and PPC group. #, P < 0.01 compared with glibenclamide + IPC, spermine + IPC, A/R + glibenclamide or A/R + spermine group.

Figure 4. CaSR protein expression.

At the end point of reoxygenation, cells were subjected to Western blot to detect CaSR ptotein expression change after different treatments. CaSR protein level increased dramatically after A/R insult compared with the Control group (P < 0.01). IPC and 30 μM pinacidil supressed CaSR over-expression after I/R injury. After the administration of glibenclamide or spermine, much of IPC’s suppression effects on CaSR expression were diminished. Data are mean ± SD. Three replicates for each group. *, P < 0.01 compared with Control, IPC and PPC group. #, P < 0.01 compared with glibenclamide + IPC, spermine + IPC, A/R + glibenclamide or A/R + spermine group.