| Objective:In this study,we investigate the "membrane clock" and "calcium clock" in injured sinus node cells(SNC)of neonatal rats induced by hypoxia/reoxygenation(H/R),and discuss the regulation of AC-cAMP-PKA signaling,further reveal the pathogenesis and possible treatment of sinus syndrome.The First Part Study on "Membrane Clock" and "Calcium Clock" in injuried sinus node cells of neonatal rats induced by hypoxia/reoxygenation(H/R)Methods:1.The sinus node cells were isolated by “enzymolysis with differential attachment” method.To identificate sinus node cells,patch clamp technique was used to record the action potential(AP)and Immunofluorescence experiments were conducted to detection of HCN4 expression.2.The sinus node cells were assigned into control group,H/R group(hypoxia/reoxygenation treatment).Patch-clamp recordings were performed to measure the AP and the I-V curve 、steady-state activation(SSA)and steady-state activation(SSI)of If and ICa,L.3.The expression of HCN4 and Cav1.3 was analyzed by immunofluorescence staining.4.To measure intracellular calcium concentration,cells were loaded with fluo-4,fluorescence was detected with a laser-scanning confocal microscope.Results:1.Isolated cells were divided into two types: spindle and triangle.The spontaneous action potential can be recorded on the spontaneously beating spindle-shaped cells and the HCN4 channel protein only located in the membrane of spindle cell.2.The spontaneous beating rates in H/R group were significantly reduced from(142.63±8.75)beats/min to(72.52±5.61)beats/min compared with the control group.The maximum diastolic potential(MDP)in the H/R group decreased from(-58.45 ± 0.5)m V to(-42.20 ± 0.7)m V,the diastolic depolarization(DDR)decreased from(105.45 ± 16.69)m V/s to(37.55 ± 5.49)m V/s,and the action potential amplitude(APA)decreased from(72.25 ± 2.72)m V to(54.39 ± 3.12)m V in the H/R group,all of that wit statistic differences(P <0.01).3.The peak current density of If in the control group was(-213.97 ± 22.77)p A/ p F,and was significantly decreased to(-106.83 ± 3.89)p A / p F in the H/R group.The steady-state activation curve in H/R group shifted left,V1/2 was shifted from(-142.61 ± 2.32)m V to(-149.38 ± 1.67)m V,(P <0.01)compared with the control group.Apparent difference was not abserved in the slope factor k between H/R group and H/R+FSK group.4、The peak current density of ICa,L was significantly decreased by about 56%in H/R group compared to the control group.The SSA of ICa,L in the H/R group shifted right,the V1/2 was shifted from(-18.94±0.22)m V to(-17.11±0.19)m V,(P<0.01).The slope factor k in the control group was(4.87±0.16)m V,and was(6.16±0.22)m V in the H/R group,with a statistic difference(P <0.01).Compared with the control group,the steady-state inactivation curve in H/R group was significantly shifted left,the V1/2 shifted from(-41.58±1.13)m V to(-47.55±0.95)m V,and the slope factor k increased from(6.88±0.98)m V to(8.21±1.10)m V,all of that with statistic differences(P <0.01).5.HCN4 and Cav1.3 channel proteins were mainly located in the cell membrane,the fluorescence intensities of both HCN4 and Cav1.3 were significantly decreased in H/R group compared to the control group.6.The mean fluorescence intensities of Ca2+ was stronger in H/R group than that in control groupConclusions:1.Sinus node cell has a spindle shape,with spontaneous beating.2.Hypoxia / reoxygenation can cause changes in the "membrane clock" and "calcium clock" of the sinoatrial node cells of the neonatal rat,leading to a decrease in maximum diastolic potential,a decrease in diastolic depolarization rate,slow the diastolic depolarization and reduce the magnitude of the action potential,resulting in sinus node dysfunction.The Second Part The study on the effect of AC-cAMP-PKA signaling pathway in injuried sinus node cells of neonatal rats induced by hypoxia/reoxygenation(H/R)Methods:The sinoatrial node cells were assigned into H/R group and H/R+Forskolin group.Patch-clamp recordings were performed to record AP 、 If and ICa,L.The concentration of intracellular calcium concentration was detected with a laser-scanning confocal microscope.Results:1.The spontaneous beating rates in H/R group were(72.52 ± 5.61)beats /min,and increased to(148.86 ± 8.30)beats/min after 10 μmol/L forskolin was added(P<0.01).Compared with H/R group,the MDP increased from(-42.20 ± 0.70)m V to(-45.3 ± 0.62)m V,the DDR increased from(37.55 ± 5.49)m V/s to(97.02±12.16)m V,and the APA increased from(54.39 ± 3.12)m V to(71.7 ± 2.16)m V after 10μmol/L forskolin was added,all of that with statistic differences(P <0.01).2.The peak current density of If in H/R was(-106.83 ± 3.89)p A/p F,and was increased to(-138.36 ± 5.27)p A/p F after adding 10 μmol / L Forskolin(P <0.01).Compared with the H/R group,the SSA curve in the H/R + FSK group shifted right after addition of 10 μmol/L forskolin,and the V1/2 was shifted from(-149.38 ± 1.67)m V to(-138.36 ± 5.27)m V(P <0.01).There was no significant difference in the slope factor k between two group.3.The peak current density of ICa,L in the H/R group was(-4.26±0.98)p A/p F,increased significantly to(-8.66±1.74)p A/p F after 10 μmol/L forskolin was added(P <0.01).Compared with H/R group,the SSA curve was not significantly altered by adding 10 μmol /L Forskolin.The V1/2 between the H/R group and the H/R+FSK group had no statistic difference.The slop factor k was increased from(4.87±0.16)m V to(5.49±0.41)m V(P< 0.01).Compared with the H/R group,the SSI curves of the H/R + FSK group shifted right after adding 10 μmol /L forskolin,the V1/2 was shifted from(-47.55±0.95)m V to(-42.47±1.68)m V(P< 0.01).The slop factor k was significant increased from(4.56±0.45)m V to(8.21±1.10)m V(P< 0.01).4.The mean fluorescence intensity of Ca2+ was weaken after the 10 μmol /L forskolin was added.Conclusions:Hypoxia / reoxygenation may decrease the phosphorylation of the AC-cAMP-PKA signaling pathway in neonatal rat sinoatrial node cells,thereby affecting the opening of the current channel and the gating dynamics mechanism of "membrane clock" as well as causing intracellular calcium disorders.Which leading to a decrease in maximum diastolic potential,a decrease in diastolic depolarization rate,slow the diastolic depolarization and reduce the magnitude of the action potential,resulting in sinus node dysfunction.AC-cAMP-PKA signaling pathway can be activated by Forskolin to improve function of the injured sinus node by hypoxia/reoxygenation. |
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