RyR2-mediated Ca²⁺ Release Modulates SK Channels In Mice Cardiac Myocytes

Cardiac myocytes have the ability to produce action potential (AP) which is composed of depolarization and repolarization. During the AP, the opening and closing of different channels mediate different currents across cell membrane and are responsible for different phases of AP.The resting potential of cardiac myocyte is about-70～-90mV and depolarizes to about+15～+30mV after appropriate stimulation during depolarization. The repolarization is illustrated as the restore of membrane potential from depolarization to resting potential. Many types of currents are responsible for repolarization, including Ca2+influx, Cl-influx and especially the K+efflux induced by the activation of many types of K+channels. Different K+channels account for diffent phases of AP and small-conductance calcium-activated potassium channels (SK channels) are involved in the medium and late period of repolarization.SK channel is not a kind of voltage-gated channel but a typical Ca2+-activated channel. There are four types of SK channels according to the sensitivity of SK channels to apamin, ie., SK1, SK2, SK3and SK4. The main SK channel in cardiac myocytes is SK2channel and atrial cells showed higher expression compared with ventricular cells. It is indicated that knockdown of SK2channels induced the prolongation of AP repolarization and subsequently increased the incidence of arrhythmia including atrial fibrillation and atrial flutter. As a kind of Ca2+-activated channel, the only mechanism for activation of SK2channels is the increase in intracellular Ca2+concentration ([Ca2+]i). There are two pathways responsible for increased [Ca2+]i, including L-type voltage-gated Ca2+channels (L-VGCC)-mediated extracellular Ca2+influx and ryanodine receptors (RyRs)-mediated Ca2+release from sarcoplasmic reticulum (SR).RyR is a kind of Ca2+releasing channel located on the SR membrane. RyR1, RyR2and RyR3are three subtypes of RyRs and RyR2is the dominant one expressing in cardiac myocytes. The AP of cardiac myocyte induces Ca2+influx due to the activation of L-VGCC and subsequently results in the Ca2+releasing into the caytoplasma via RyR2, which is called Ca2+-induced Ca2+release (CICR).It is showed that inhibition of L-VGCC decreased SK2-mediated current and increased repolarization duration, suggesting the activation of SK2channels by L-VGCC and the involment of SK2current in repolarization of AP. However, it is not clear if RyR2-mediated Ca2+release is responsible for activation of SK2channels during AP. As it is indicated that RyR2-mediated Ca2+release is the main mechanism responsible for activation of SK2channels in neurons, we hypothesized that RyR2-mediated Ca2+release is a possible pathway accouting for activation of SK2channels during AP in cardiac myocytes.In our study, we investigated the effect of RyR2on AP and Ca2+transients in mice cardiac cells using whole cell patch clamp and Laser Confocal Microscope Calcium Imaging technique. The RyR2-shRNA Lentivirus vectors were constructed using siRNA technique and the effect of RyR2-shRNA on RyR2expression were identified using real-time PCR and Western blot techniques. Additionally, the effect of RyR2-shRNA on AP and Ca2+transients were also studied.Part Ⅰ Effect of RyR2on action potential in mice cardiac myocytesMethods1. Adult healthy mice atrial cells were isolated using Langendorff perfusion technique. The hearts of C57BL/6J mice were taken out after anticoagulation and anesthesia. After perfusion with enzyme and KB solution, the extracellular calcium concentration was restored step-wise.2. The whole cell patch clamp was used to recording AP in cardiac cells from mice. The patch pipettes were filled with pipette solution and the AP was induced by electrical stimulus unhder current clamp mode. The AP duration at50%and90%(APD50and APD90) were calculated respectively. Firstly, we investigated the effect of RyR2on AP and the cells were divided into3groups:control group, ryanodine group (treatment of20μM ryanodine), TG group (treatment of2μM thapsigargin for45min). Secondly, we observed the effect of SK2blockers on AP and the cells were divided into3groups:apamin group (application of500pM apamin), ryanodine+apamin group (application of apamin after AP recording in ryanodine group), TG+apamin group (application of apamin after AP recording in TG group). The difference in APD50and APD90between before and after apamin application in control group, ryanodine group and TG group were calculated as apamin-sentitive APD50and APD90.3. Confocal Microscope Calcium Imaging technique was used for recording Ca2+transients. The suspended cardiac cells were washed for three times after incubation with Fluo-3AM (5μM) at37℃for30min in the dark. The Ca2+transients were induced by field stimulation and were recorded under the excitation wave length of488nm. All the cells were divided into3groups:control group, ryanodine group and TG group.Results1. The APD50and APD90of AP were about13.7±1.9and156.4±44.7ms respectively (n=5). After applicartion of ryanodine (n=5) or TG (n=5), APD90(P<0.01) but not APD50(P>0.05) increased significantly.2. In control group, application of apamin increased APD90(P<0.001) but not APD50(P>0.05) significantly and APD-sensitive APD90was113.6±22.2ms.3. In ryanodine group, application of apamin increased APD90(P<0.05) but not APD50(P>0.05) significantly. However, APD-sensitive APD90decreased significantly compared with control group (P<0.01). 4. In TG group, application of apamin increased APD90(P<0.05) but not APD50(P>0.05) significantly. However, APD-sensitive APD90decreased significantly compared with control group (P<0.01).5. APD90(P<0.05) but not APD50(P>0.05) in ryanodine or TG group decreased significantly compared with apamin group. Both APD50and APD90in ryanodine+apamin group or TG+apamin group were not altered significanrly compared with apamin group (P>0.05).6. The amplitude of Ca2+transient induced by local field stimulation was about1.13±0.29(n=8), which was significantly inhibited by application of ryanodine (n=8) or TG(n=10)(P<0.01).Summary1. Inhibition of SK2channels prolonged repolarization duration of AP significantly, indicating the involvement of SK2channels in the repolarization of AP within cardiomyocytes.2. Blocking of RyR2-mediated Ca2+release by application of ryanodine or TG statistically increased AP repolarization, demonstrating the regulatory effect of RyR2-mediated Ca2+release on SK2channel.3. Addition of ryanodine or TG decreased the amplitude of Ca2+transients induced significantly.ConclusionsDuring AP period, RyR2-mediated Ca2+release from SR is a possible mechanism for the SK2channel activation in mouse cardiomyocyte. Part II Effect of RyR2-siRNA on action potential in mice cardiac myocytesMethods1. Neonatal mice hearts were dissected from the chest and were subsequently subjected to digestion with an enzyme solution including collagenase and trypsin. Puried cardiomyocytes were abtained by differential velocity adherent technique.2. The construction of RyR2-shRNA Lentivirus vector:Four siRNAs targeting different nucleotide sites of mouse RyR2mRNA were designed in accordance to siRNA design principles. According to the targeting sequences, four pairs of oligonucleotides coding shRNAs were synthesized. Both sense and antisense oligomers of RyR2shRNAs were annealed and inserted into psiHIV-U6-EGFP vector after treatment of T4DNA ligase and digestion by BamH I, EcoR I to eonstruct the recombinant lentiviral vectors of RyR2-shRNA. The recombinant lentiviral vectors were identified by sequencing and were named after psiHIV-U6-shRNAl, psiHIV-U6-shRNA2, psiHIV-U6-shRNA3and psiHIV-U6-shRNA4, respectively. The recombinant lentivirus vectors were packaged and amplified in293T cells in accordance to the manufacturer’s protocol of Lenti-PacTM HIV Expression Packaging Kit to obtain Lenti-siRNA-RyR2-1, Lenti-siRNA-RyR2-2, Lenti-siRNA-RyR2-3, Lenti-siRNA-RyR2-4and Lenti-scramble siRNA (negative control). Cardiac myocytes isolated from neonatal mice were infected with recombinant lentivirus vectors of Lenti-siRyR2-1, Lenti-siRyR2-2, Lenti-siRyR2-3, Lenti-siRyR2-4and Lenti-scramble siRNA, respectively.48h after infection, the green fluorescent protein was examined using fluorescence microscopy. Flow cytometry technique was used to test infection efficiency.3. The identification of RyR2-shRNA Lentivirus vector:Expression of RyR2mRNA in cardiac myovytes isolated from neonatal mice was assessed by real-time PCR with GAPDH as an internal standard.4. Adult healthy mice atrial cells were isolated using Langendorff perfusion technique. The hearts of C57BL/6J mice were taken out after anticoagulation and anesthesia. After perfusion with enzyme including and KB solution, the extracellular calcium concentration was restored step-wise.5. The isolated cardiomyocytes from adult mice were cultured with FBS-free DMEM/F-12for48h and an appropriate titer of gene-carrying Lenti-siRyR2-2+Lenti-siRyR2-4and Lenti-scramble siRNA was added to the culture medium.6. Western blot technique was used to test the expression of RyR2. Protein samples were extracted from cultured adult cardiac myocytes. Protein sample were separated and then transferred to polyvinylidene difluoride (PVDF) membranes. Following transfer, the membrane was blocked and subsequently incubated with anti-RyR2antibody and then, was incubated with secondary antibody. The bands were detected by chemiluminescence and quantification of the signals was performed.7. The whole cell patch clamp was used to recording AP in cardiac cells from mice. The patch pipettes were filled with pipette solution and the AP was induced by electrical stimulus unhder current clamp mode. The AP duration at50%and90%(APD50and APD90) were calculated. Firstly, we investigated the effect of RyR2on AP and the cells were divided into3groups:blank control group, negative control group (transfected with Lenti-scramble siRNA), siRyR2group (transfected with Lenti-siRyR2-2+Lenti-siRyR2-4). Secondly, we observed the effect of SK2blockers on AP and the cells were divided into3groups:apamin group (application of500pM apamin after AP recording in blank control group), Lenti-scramble siRNA+apamin group (application of apamin after AP recording in negative control group), Lenti-siRyR2-2+Lenti-siRyR2-4+apamin group (application of apamin after AP recording in Lenti-siRyR2-2group). The difference in APD50and APD90between before and after apamin in blank control group, negative control group and siRyR2group were calculated as apamin-sentitive APD50and APD90.8. Confocal Microscope Calcium Imaging technique was used for recording Ca2+transients. The suspended cardiac cells were washed for three times after incubation with Fluo-3AM (5μM) at37℃for30min in the dark, the Ca2+transients were induced by field stimulation and recorded under the excitation wave of488nm. All the cells were divided into3groups:blank control group, negative control group (transfection with Lenti-scramble siRNA) and siRyR2group (transfection with Lenti-siRyR2-2+Lenti-siRyR2-4).Results1. Four lentiviral expressive vectors of shRNA targeting RyR2were successfully constructed.2. The lentivirus particles were successfully packaged in293T cells and the viral titers of Lenti-siRyR2-1, Lenti-siRyR2-2, Lenti-siRyR2-3, Lenti-siRyR2-4and Lenti-scramble siRNA were1.2×107IU/ml、1.1×107IU/ml、1.3×107IU/ml1.4×107IU/ml and1.1×107IU/ml respectively.3. Flow cytometry results showed that the infection efficiency of combinant lentivirus vectors were79.35%、82.47%、81.62%、87.91%and86.18%, respectively.4. Real-time PCR results showed that RyR2mRNA expression levels in cardiac myocytes isolated from neonatal mice were significantly lower in Lenti-siRyR2-1, Lenti-siRyR2-2, Lenti-siRyR2-3and Lenti-siRyR2-4groups compared-with blank control and negative control groups (P<0.01). There was no difference in RyR2mRNA expression level between blank control and negative control groups (P>0.05).5. In cardiac myocytes of adult mice, western blot results showed that the expression of RyR2protein in siRyR2group decreased significantly compared with blank control and negative control groups (P<0.05). There was no difference in RyR2mRNA expression level between blank blank control and negative control groups (P>0.05).6. The APD50and APD90of AP were about13.7±1.9and156.4±44.7ms respectively (n=5). After transfection with Lenti-scramble siRNA (n=5), both APD50and APD90were not altered (P>0.05). After transfection with Lenti-siRyR2-2+Lenti-siRyR2-4(n=5), APD90(P<0.01) but not APD50(P>0.05) increased significantly.7. In blank control group, application of apamin increased APD90(P<0.001) but not APD50(P>0.05) significantly and apamin-sensitive APD90was113.6±22.2ms.8. In negative control group, application of apamin increased APD90(P<0.001) but not APD50(P>0.05) significantly and apamin-sensitive APD90was not altered significantly compared with blank control group (P>0.05).9. In siRyR2group, application of apamin increased APD90(P<0.05) but not APD50(P>0.05) significantly. However, apamin-sensitive APD90decreased significantly compared with blank control group (P<0.01).10.APD90(P<0.05) but not APD50(P>0.05) in siRyR2group decreased significantly compared with apamin group. Both APD50and APD90in siRyR2+apamin group was not altered significanrly compared with apamin group (P>0.05).11. The amplitude of Ca2+transient induced by local field stimulation was about1.13±0.29(n=8), which was significantly inhibited by transfection with Lenti-siRyR2-2(P<0.01, n=5) but not Lenti-scramble siRNA (P>0.05, n=5).Summary1. We designed and constructed Lenti-siRyR-1/2/3/4which were specific to RyR2. Real-time PCR results showed that all Lenti-siRyR-1/2/3/4could slience RyR2effectively and specifically and Lenti-siRyR2-2and Lenti-siRyR2-4showed the best effects.2. Cardiomyocytes transfected with Lenti-siRyR2-2and Lenti-siRyR2-4developed prolonged AP repolarization duration and decrased Ca2+transients, indicating the involvement of SK2channels in the repolarization of AP within cardiomyocytes.Conclusions1. During AP period, knockdown of RyR2showed a significant. prolongation of AP and lower the peak amplitude of calcium transient. Data suggested that the RyRs-mediated Ca2+release from SR is a possible mechanism for SK channels activation in the mouse cardiac myocytes.2. Lentivirus-mediated shRNA specific to RyR2gene was constructed successfully.