Effects of stretch on transient outward potassium and inward rectifier potassium current in cultured neonatal rat atrial myocytes

AIM：To investigate the effects of mechanical stretch on transient outward potassium current (Ito), inward rectifier potassium current (IK1) and action potential duration(APD) of cultured neonatal rat atrial myocytes. METHODS：Neonatal rat atrial myocytes were isolated and cultured on silicone sheeting with or without stretch for 24 h. The silicone membrane area was increased by 12% in stretched group. The cells without stretch served as control. Ito, IK1 and APD were recorded by the technique of whole-cell patch clamp. RESULTS：Compared with control group, Ito density in stretched myocytes was significantly reduced ［(16±04) pA/pF vs (121±29) pA/pF, P<001］, whereas IK1 density was increased ［(-108±08) pA/pF vs (-88±09) pA/pF, P<001］. The APDs at 50% and 90% levels of repolarization (APD50 and APD90) in the stretched cells were obviously decreased than those in non-stretched cells ［(105±14) ms vs (155±24) ms, (300±28) ms vs (563±36) ms, P<001］. CONCLUSION：Stretch stimulation leads to the reduction of Ito density, the increase in IK1 density and the shortness of APD in cultured rat atrial neonatal myocytes, which may contribute to atrial electrical remodeling induced by pressure overload.     

Effect of experimental acute necrotizing pancreatitis on sodium and L-type calcium current in rat cardiomyocytes

AIM: To study the effect of experimental acute necrotizing pancreatitis (ANP) on sodium and L-type calcium current in rat cardiomyocytes. METHODS: I_Na and I_Ca-L were recorded using whole cell patch-clamp techniques from left ventricular myocytes in ANP model established by retrograde injection of 3.5% sodium taurocholate 2.5 mL/kg into pancreatic duct. RESULTS: Peak I_Na current density (at -30 mV) was significantly reduced in ANP [(12.45±2.26)pA/pF,n=16] compared with sham [(25.32±3.31)pA/pF,n=14], P<0.01; I_Ca-L current density (at +10 mV) was also significantly reduced in ANP [(3.63±0.65)pA/pF,n=16] compared with sham [(5.46±1.03)pA/pF,n=12], P<0.05. CONCLUSIONS: There were changes in both I_Na and I_Ca-L in cardiomyocytes of ANP. These changes may underlie the altered excitability and abnormally short transmembrane action potentials and repolarization of cardiomyocytes, thus contributing to arrhymias in ANP.     

Cardiac IKs as repolarization reserve plays a role in gender difference of diabetic QT prolongation

AIM: To explore the basic ionic mechanisms underlying long-QT syndrome by observing the changes of slowly activating outward rectifying potassium current (I_Ks) and its proteins in abnormal QT prolongation in different genders of diabetic rabbits.METHODS: A single injection of pre-warmed (37 ℃) alloxan (140 mg/kg) was used to establish a rabbit model of insulin-dependent diabetes mellitus. Eight weeks after alloxan injection, the levels of blood glucose in the rabbits were monitored and standard lead II electrocardiogram was recorded. The myocardial cells were isolated from the ventricle of the rabbits via enzymatic digestion. Whole-cell patch clamp technique was performed to study the action potential duration (APD) and I_Ks. The changes of both KvLQT1 and mink proteins were detected by Western blotting analysis.RESULTS: The QT interval and APD were prolonged apparently both in male and female diabetic rabbits. The increased APD/QT-I of the male diabetic rabbits is more remarkable than that of the female. The I_Ks step current density of the male diabetic rabbits was decreased at test potentials ranging from+40 mV to+70 mV compared with that of the control animals (P<0.05), which was lowered from (3.08±0.67) pA/pF (n=17) to (1.27±0.20) pA/pF (n=16) at+70 mV. However, the I_Ks step current density of the female diabetic rabbits was increased at test potentials ranging from 0 mV to+70 mV compared with that of control group (P<0.05), which was increased from (1.56±0.20) pA/pF (n=13) to (3.65±0.50) pA/PF (n=14) at+70 mV. The expression of KvLQT1 and mink in male diabetic group decreased by 21.6% and 18.5%, respectively. However, the expression of KvLQT1 and mink in female diabetic group were increased by 42.3% and 20.5%, respectively.CONCLUSION: The I_Ks may be a protective factor in the early period of diabetic development in female rabbits. As a repolarization reserve, cardiac I_Ks is likely to restrict the effects of excessively slowing repolarization.     

Characteristics of pacemaker current in aged rat atrial myocytes

AIM: To investigate the characteristics of pacemker current (I_f) of atrium myocytes in aged rats. METHODS: Aged SD rats (22-24 months) were used in the study. The single atrium myocytes were isolated. I_f current was recorded by the technique of whole-cell patch clamp. RESULTS: The densities of I_f increased markedly in aged rat atrium myocytes compared with that in the cells of young rats. At the test potential of-150 mV, the average amplitude of I_f was (382±23)pA and the current density was (3.2±0.4)pA/pF, while the average amplitude of I_f in control group was (86.9±8.4)pA and the current density was (0.9±0.1)pA/pF,P<0.01. I_f current was augmented at negative hyperpolarzing test potential. Time constants of activation in aged and control cells were (230.2±14.4)ms and (670.5±23.6)ms, respectively. Furthermore, steady state curves in the elder cells was shifted to positive potentials and the voltages for half maximal activation were (-87.2±2.3)mV (aged cells) and (-104.4±6.3)mV (control cells). In contrast, the slope of activation curves and reversed potential of I_f were slightly different between aged cells and control cells. CONCLUSION: The amplitude and densities of I_f in atrium cells of aged rats were larger than that of control rats.     

Effect of hypercholesterolemia on the ionic currents in cardiac ventricular myocytes of rats

AIM: To determine whether chronic hypercholesterolemia affects ionic currents on cardiac ventricular myocytes of rats. METHODS: Whole-cell patch-clamp technique was used to record the ionic currents in single cardiac myocytes isolated from normal cholesterolemia and hypercholesterolemia rats. RESULTS: In the hypercholesterol group (group Ⅱ), serum total-cholesterol level was significantly higher than that of normal group (group Ⅰ) ［(3.10±0.62)mmol·L-1 vs (1.18±0.37)mmol·L-1, P<0.01, n=20］. The serum triglyceride content of group II was remarkably higher than that of group Ⅰ ［(1.51±0.30)mmol·L-1 vs (0.43±0.15)mmol·L-1, P<0.01, n=20］. In ventricular myocytes of rats, 50% repolarization of action potential duration (APD50) prolonged from (70.86±8.12)ms (group Ⅰ) to (116.16±6.90)ms (group Ⅱ) (n=10 in each group, P<0.01); APD90 prolonged from (95.10±7.27)ms (group Ⅰ) to (144.04±7.39)ms (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of -120 mV, Ik1 increased from (-16.98±4.54) pA/pF(group Ⅰ) to (-19.92±4.08) pA/pF (group Ⅱ) (n=12 in each group, P<0.05); at the test potential of 0 mV, ICa-L decreased from (-8.56±1.29) pA/pF (group Ⅰ) to (-5.24±0.90) pA/pF (group Ⅱ) (n=10 in each group, P<0.01); at the test potential of +60 mV, Ito decreased from (13.20±1.97) pA/pF (group Ⅰ) to (10.30±1.97) pA/pF (group Ⅱ) (n=8 in each group, P<0.05). CONCLUSION: Hypercholesterolemia affects the ionic currents on cardiomyocytes of rats greatly, which may be the ionic mechanism of cardiac toxicity induced by hypercholesterolemia.     

Effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells from rats exposed to chronic hypoxia

AIM: To investigate the effect of cGMP on voltage-gated potassium channel in pulmonary artery smooth muscle cells (PASMCs) from rats exposed to chronic hypoxia. METHODS: (1) Wistar rats were randomly divided into control group (group A) and chronic hypoxia group (group B). Then group B received hypoxia 8 hours per day for 4 consecutive weeks. (2) Single PASMC was obtained via acute enzyme separation method. (3) Conventional whole-cell patch clamp technique was used to record resting membrane potential (Em) and ion currents of voltage-gated potassium channel. The changes of ion currents of voltage-gated potassium channel before and after applying cGMP (1 mmol/L), an agonist of protein kinase G (PKG), and cGMP plus H-8 (1 mmol/L), an inhibitor of PKG were compared between two groups. RESULTS: The Em of group B were significantly lower than that of group A. The ion currents of voltage-gated potassium channel in group A and group B were all significantly inhibited by cGMP [control group: from (118.0±5.0) pA/pF to (89.9±16.5) pA/pF, n=6, P＜0.05;chronic hypoxia group: from (81.0±5.0) pA/pF to (56.8±9.1) pA/pF, n=6, P＜0.05]and these effects were reversed by H-8 [control group: from (119.2±10.3) pA/pF to (117.8±9.1) pA/pF, n=6, P＞0.05;chronic hypoxia group: from (96.8±6.2)　pA/pF to (98.0±2.2)　pA/pF, n=6, P＞0.05]. CONCLUSIONS: The currents of voltage-gated potassium channel was inhibited by chronic hypoxic. The inhibitory effect of cGMP on currents of voltage-gated potassium channel in PASMCs from both normal and chronic hypoxic rats may be probably through the phosphorylation of voltage-gated potassium channel.     

Zacopride attenuates pressure overload-induced ventricular remodeling in rats

AIM:To investigate the protective effect of zacopride (ZAC) on the pressure-overload left ventricular remodeling in the rats induced by coarctation of abdominal aorta. METHODS:Male Sprague-Dawley (SD) rats with pressure overload were induced by the coarctation of abdominal aorta. The model rats were intraperitoneally administered with ZAC, chloroquine (Chlor), and zacopride+chlorquine (ZAC+Chlor). The study duration was 8 weeks. The cardiac structure and function were assessed by echocardiography. The heart weight/body weight (HW/BW) ratio and the left ventricular weight/body weight (LVW/BW) ratio were calculated. The changes of structure and shape in myocardial tissue were observed with HE staining. The ultrastructure of the myocytes was observed under transmission electron microscope. The inward rectifier potassium channel (I_K1) protein expression was determined by Western blot. The mRNA expression of Kir2.1 was detected by RT-PCR. RESULTS:Compared with vehicle group, ZAC improved cardiac function, as indicated by the decreased left ventricular end-diastolic dimension (LVEDD) and left ventricular end systolic dimension (LVESD) (P<0.05), and the increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (P<0.01). The HW/BW and LVW/BW ratios were significantly decreased, and the cross-sectional area of the cardiomyocytes was significantly less in ZAC group than that in vehicle group (P<0.01). The ultrastructure of the myocytes was significantly improved. Chlor blocked the protective effect of zacopride on the pressure-overload left ventricular remodeling. The protein level of I_K1 and mRNA expression of Kir2.1 in the cardiac tissues in ZAC group were significantly increased compared with vehicle group (P<0.01). CONCLUSION:I_K1 agonist ZAC significantly attenuates pressure overload-induced ventricular remodeling in rats.     

Effects of piperine on abnormalities of inward rectifier potassium current and ultra rapid delayed rectifier potassium current induced by hydrogen peroxide in rabbit atrial myocytes

AIM: To study the protective effect of piperine on abnormalityies of inward rectifier potassium current (I_K1) and ultra rapid delayed rectifier potassium current (I_KUr) induced by hydrogen peroxide (H₂O₂) in single rabbit atrial myocytes. METHODS: The technique of whole-cell patch-clamp was used to study the effect of H₂O₂ at concentration of 50 μmol/L on I_K1 and I_KUr in single rabbit atrial myocytes. The protective effect of pretreatment with piperine (7 μmol/L) was also observed. RESULTS: The piperine at concentration of 7 μmol/L had no significant effect on I_K1 and I_KUr and their channel dynamics. In the presence of H₂O₂ at concentration of 50 μmol/L, the peak currents of I_K1 and I_KUr reduced significantly (P<0.05).The steady-state activation curve of I_KUr was shifted right, the steady-state inactivation curve of I_KUr was shifted left, and the recovery from inactivation of I_KUr was shifted downward. The I_KUr showed frequency-dependent characteristics. Piperine at concentration of 7 μmol/L significantly alleviated the inhibitory effect of H₂O₂ on I_K1 and I_KUr (P<0.01). In addition, piperine protected against the changes of I_KUr dynamics induced by H₂O₂. CONCLUSION: Piperine alleviates the abnormalities of I_K1 and I_KUr induced by oxidative stress in atrial myocytes.     

Changes of transmural repolarization heterogeneity and ion currents in rabbits with left ventricular hypertrophy

AIM: To investigate the changes of transmural repolarization heterogeneity and ion currents in rabbits with left ventricular hypertrophy. METHODS: Ventricular hypertrophy was induced by a partial constriction of the abdominal aorta in rabbits. Myocytes were isolated by a two steps enzymological method. The sub-endocardial (Endo) and sub-epicardium (Epi) tissues were separated from other region (midmyocardium, Mid) with a razor. Whole cell patch clamp technique was used to record the action potential and ion currents. RESULTS: The action potentials duration at 90% repolarization (APD₉₀) of Epi, Mid and Endo were all prolonged significantly in hypertrophy group compared to control group. This prolongation of APD₉₀ was more pronounced in Mid (26.0%±2.7%) than that in Epi (14.0%±1.6%) and Endo (10.0%±1.1%). The transmural repolarization heterogeneity was increased significantly in the hypertrophy group. The I_Ks and I_to density in Epi, Mid and Endo was decreased significantly in hypertrophy group compared to those in control group. This decrease in I_Ks and I_to density was more pronounced in Mid than in Epi and Endo. No significantly difference of I_Ca,L and I_Kr density between hypertrophy group and control group in three layers was observed. The I_K1 density decreased significantly in hypertrophy group compared to control group, but the extent of the decrease had no differences among the three layers. CONCLUSIONS: The transmural repolarization heterogeneity increases significantly in rabbit hypertrophied ventricle. The decrease in transmural heterogeneity of I_to and I_Ks is the main causes.     

Characteristics of transient outward potassium current in repolarization 1 phase from the canine right ventricular mid-myocardial cells

AIM: To examine the electrophysiological characteristics of transient outward potassium current(Ito₁) in repolarization 1 phase from the canine right ventricular M cells. METHODS: By use of whole cell patch-clamp technique, we quantitatively researched the ionic intensity, density of Ito₁ and the notch magnitude of action potential in repolarization 1 phase. RESULTS: (1) The activating process of Ito₁ of canine right ventricular M cells presented evident voltage-dependency. Under the condition of 37℃, 5 000 ms, 0 mV and +70 mV, the average peak Ito₁ intensity of right ventricular M cell were (690±380) pA and (3 130±1 910) pA, respectively (P＜0.01). (2) The Ito₁ intensity of canine right ventricular M cell possessed obvious frequent dependency. Under the condition of 37℃,+70 mV, 500 ms and 5 000 ms, the average peak Ito₁ intensity were (1 690±830) pA,(3 130±1 910) pA, respectively(P＜0.01), corresponding to the increase of action potential "spike-dome" magnitude in repolarization 1 phase. CONCLUSION: Potent Ito₁ as well as the "spike-dome"-like action potential figure mediated by Ito₁ is one of the prominent electrophysiological characteristics of the canine right ventricular M cells.     

Blunted response of L-type calcium current to simulated ischemia and reperfusion in ventricular myocytes from diabetic rabbits

AIM: To evaluate the effects of simulated acute ischemia and reperfusion on L-type calcium current (I_{Ca, L}) in ventricular myocytes from diabetic and non-diabetic rabbits.METHODS: Using whole-cell patch clamp techniques, I_{Ca, L} was measured in left ventricular myocytes isolated from 6-week alloxan-induced diabetic rabbits and age-matched control ones at baseline, 5 min of simulated ischemia, and 5 min of reperfusion.RESULTS: There were no significant differences on baseline maximum I_{Ca, L} densities between diabetic and control ventricular myocytes. In control cells (n=11), maximal I_{Ca, L} densities of baseline, ischemia and reperfusion were (-8.36±1.63)pA/pF, (-5.90±1.75)pA/pF and (-4.22±1.02)pA/pF, respectively. The I_{Ca, L} of ischemia was less than that of baseline (P<0.01), while the I_{Ca, L} of reperfusion was less than those of baseline (P<0.01) and ischemia (P<0.05). In diabetic cells (n=9), the I_{Ca, L} of baseline, ischemia and reperfusion were (-7.55±1.62)pA/pF, (-6.05±1.58)pA/pF and (-5.12±1.13)pA/pF, respectively. Only I_{Ca, L} of reperfusion was less than that of baseline (P<0.01), while I_{Ca, L} of ischemia was not significantly different from that of baseline (P>0.05) or reperfusion (P>0.05).CONCLUSION: I_{Ca, L} in diabetic ventricular myocytes represents blunted response to acute ischemic injury, being decreased more slowly than that in control cells. Post-ischemic reperfusion is still a potent inhibitor against I_{Ca, L} in both diabetic and non-diabetic cells. This study may be indicative of the mechanism about ischemia-reperfusion injury to diabetic myocardium and the therapy for diabetic patients with ischemic heart disease.     

Effects of simvastatin on transient outward potassium current in ventricular myocytes of normocholesterolemic rabbits suffering from ischemia and reperfusion

AIM: To investigate the effects of simvastatin on transient outward potassium current (Ito) in left ventricular myocytes of rabbit heart undergoing ischemia-reperfusion, so as to explore the cellular (ionic) mechanism of statin treatment for antiarrhythmia. METHODS: Forty-five rabbits were randomly divided into three groups: ischemic-reperfusion group (I-R), simvastatin intervention group (statin) and sham-operation control group (sham). Anesthetized rabbits were subjected to 30 min ischemia by ligation of the left anterior descending coronary artery and 60 min reperfusion after oral administration of simvastatin at dose of 5 mg·kg-1·d-1(statin group) or placebo (I-R group) for 3 d. Single ventricular myocytes were isolated enzymatically from the epicardial zone of the infracted region derived from the hearts in I-R, statin group and the same anatomy region in sham. Whole cell patch clamp technique was used to record Ito. Simultaneously, the level of serum cholesterol was examined. RESULTS: No significant difference in serum cholesterol concentration among three groups was observed. The Ito current density (at +60 mV) was significantly decreased in I-R ［(9.49 ±1.91) pA/pF, n=11］ compared with sham ［(17.41± 3.13) pA/pF, n=15, P＜0.01］ and statin ［(15.24 ± 2.41) pA/pF, n=11, P＜0.01］, although there was slight reduction in statin group compared with sham (P＜0.05). CONCLUSION: Ischemia-reperfusion induces significant down-regulation of Ito, which may underlie the altered electrical activity and prolong abnormal transmembrane action potential duration of the surviving ventricular myocytes. Pretreatment with simvastatin attenuates these changes without lowering the serum cholesterol level, suggesting that simvastatin may reverse this electrical remodeling, thus contributing to the ionic mechanism of statin treatment for antiarrhythmia.     

Electrical heterogeneity of transient outward current in thyroxine induced ventricular myocytes of cardiomyopathy rat

AIM: To study the electrical heterogeneity of transient outward potassium current (Ito) in left and right ventricular myocytes of cardiomyopathy rat. METHODS: The rats were peritoneally injected with L-thyroxine 0.5 mg/kg for 10 d to establish the model of ventricular hypertrophy. The right and left ventricular parts of the heart were separated and the ventricular myocytes were prepared by step digestion using enzyme solution. Ito was recorded by using whole cell patch clamp technique. The change of the electrical heterogeneity was determined. RESULTS: The electrical heterogeneity of Ito existed in the normal myocytes of left and right ventricles. In the myocytes of left and right ventricles isolated from the cardiomyopathy rats, the electrical heterogeneity was enhanced obviously and showed statistical difference. At +40 mV depolarizing test potential, the current density of Ito in the myocytes of right ventricle was increased from (9.23±0.84) pA/pF to (11.19±1.73) pA/pF, while the current density of Ito in the myocytes of left ventricle was decreased from (6.99±1.14) pA/pF to (4.95 ±1.84) pA/pF and the dispersion was increased. The V1/2 of right ventricle steady inactivation was increased significantly ［from (-68.85±1.37) mV to (-49.86±0.69) mV］. The time constant τ of de-inactivation changed significantly ［τleft=(79.16±7.04) ms,τright=(53.19±3.72) ms］. CONCLUSION: Enhanced electrical heterogeneity of Ito in the left and right ventricular myocytes of cardiomyopathy rat may represent one of the important ionic mechanisms for some arrhythmia caused by myocardial hypertrophy.     

Alteration of transient outward potassium current in ventricular myocytes from 1-week and 2-month infarcted rabbit hearts

AIM: To study the current density of transient outward potassium current (I_to) in cells from the epicardial zone of the 1-week and 2-month infarcted rabbit heart. METHODS: Rabbits were infarcted by ligation of the left anterior descending coronary artery, 1 week as well as 2 months later, the single ventricular myocytes were isolated enzymatically from the infracted area of 1-week infracted rabbit heart (PMI-1 week) and 2-month infracted heart (PMI-2 months), region remote from the infracted zone of 2-month infracted heart (REM-2 months) and free wall of left ventricule from noninfarcted heart (CON). I_to was recorded using whole cell patch-clamp techniques. RESULTS: Membrane capacitance of myocytes in REM-2 months group was signifitantly larger than that in CON. I_tocurrent density (at +60 mV) was significantly reduced in PMI-1 week and PMI-2 months compared with CON , P＜0.01. Nevertheless, there was an significant increase in PMI-2M compared with PMI-1W, P＜0.05. I_to current density was also significantly decreased in REM-2 months compared with CON (P＜0.05), while there was an significant increase in REM-2 months compared with PMI-2 months, P＜0.05. CONCLUSION: The results indicated that the reduction and change of I_to in infarcted rabbit heart were heterogenous. These changes may underlie the abnormally long transmembrane action potentials of these arrhymogenic surviving ventricular fibers of the infarcted heart, thus contributing to reentrant arrhythmias in the infarcted heart. By 2 months after myocardial infarction, the depressed I_to of infracted area had returned to nearly normal, suggesting the presence of reverse remodeling.     

Effects of cardiac remodeling on atrial arrhythmogenesis in aged rabbit left atrium

AIM: To investigate the effects of myocardial remodeling of aged left atrium (LA) on atrial arrhythmogenesis in rabbits.METHODS: The male New Zealand rabbits were divided into young LA and aged LA groups. By observing the changes of monophasic action potential (MAP) and burst-pacing in LA of the rabbits in vivo, the main cardioelectrophysiological parameters such as resting membrane potential (RMP), action potential amplitude (APA), ma-ximum rise veloctiy of action potential (dv/dt_max), plateau potential and action potential duration at 30%, 50% and 90% (APD₃₀, APD₅₀ and APD₉₀), as well as the inducibility and duration of atrial arrhythmias were recorded. L-type calcium current (I_Ca,L) was analyzed via whole-cell patch-clamp technique in enzymatically dissociated single rabbit LA myocytes. The myocardial collagen content was quantified after Masson staining, and the ultrastructure of the LA cells was observed under scanning electron microscope. The expression of Cav1.2 in LA tissue of the 2 groups was detected by Western blot.RESULTS: Compared with young LA group, dv/dt_max and plateau potential were significantly decreased, APD₃₀ and APD₅₀ were shortened, and APD₉₀ was notably prolongated in aged LA group (P<0.01). The inducibility or duration of atrial arrhythmias was severely increased or prolongated in aged LA group (P<0.01). With voltage clamp model, the density of I_Ca,L in aged LA group was significantly decreased, and current-voltage curve was notably moved upward compared with young LA group. When the clamp potential was +20 mV, the density of I_Ca,L was notably modified from (11.72±1.39) pA/pF in young LA group to (6.08±0.98) pA/pF in aged LA group (P<0.01). Compared with young LA group, the protein level of collagen was significantly increased (P<0.01), and the arrange of atrial myocytes was irregular in LA of rabbits in aged LA group. The atrial myocytes of the LA wall in aged LA group exhibited abnormal ultrastructural alterations, such as karyopyknosis, irregular and swelling mitochondria with the presence of vacuoles, and mild and severe sarcomere degeneration. Compared with those in LA tissues of young rabbits, the expression levels of Cav1.2 in the LA tissues of aged rabbits were severely reduced (P<0.01), and had a significant positive correlation with the reduction of I_Ca,L (r=0.83, P<0.01).CONCLUSION: The pathophysiological characteristics of aged LA are significantly altered, and might contribute to vulnerability and susceptibility of occurrence of atrial fibillation in aged rabbits. The mechanisms might completely attribute to the notable reduction of I_Ca,L, abnormal alterations of ultrastructures and obvious decrease in the expression of Cav1.2 in the aged LA of aged rabbits.     

Inhibitory effect of zacopride on ouabain-induced arrhythmias in adult rats

AIM: To investigate the effect of zacopride, an inward rectifier potassium channel agonist, on ouabain-induced arrhythmias in adult rats, and to explore the underlying electrophysiological mechanism.METHODS: Using ouabain to establish in vitro and in vivo arrhythmic rat models, the effects of zacopride on ouabain-induced arrhythmias were observed. The technique of whole-cell patch clamp was used to observe the effects of zacopride on inward rectifier potassium current (I_K1), resting membrane potential (RMP) and delayed afterdepolarizations (DADs) in single rat ventricular myocyte. RESULTS: Zacopride at 1 μmol/L significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain in rat hearts in vitro (P<0.05). In anesthetized rats, zacopride at 15 μg/kg significantly reduced total number of premature ventricular beats, and the duration and incidence of ventricular tachycardia and ventricular fibrillation induced by ouabain (P<0.05). I_K1 was significantly inhibited by ouabain (P<0.05), which was partially and even completely reversed by zacopride at 0.1~10 μmol/L. RMP value was significantly reduced by ouabain (P<0.05), and then increased to different levels after treatment with zacopride (0.1~10 μmol/L). Zacopride at 1 μmol/L showed its maximal effect and RMP was restored to normal level. Moreover, zacopride at 1 μmol/L markedly suppressed ouabain-induced DADs in single rat ventricular myocyte. The incidence of DADs decreased from 91.67% to 12.50% after zacopride was applied (P<0.05), and this effect was abolished by 1 μmol/L BaCl₂. CONCLUSION: Inward rectifier potassium channel agonist zacopride significantly inhibits ouabain-induced ventricular arrhythmias in adult rats. The mechanism is related to increased RMP level and inhibition of DADs by activation of I_K1 channel.     

Effect of zacopride on angiotensin Ⅱ-induced myocardial fibrosis

AIM:To investigate the effect of inwardly rectifying potassium channel (I_K1) agonist zacopride (Zac) on angiotensin Ⅱ (Ang Ⅱ)-induced viability and apoptosis of cardiac fibroblasts (CFb) and to explore the underlying anti-fibrosis mechanism.METHODS:The ventricular fibroblasts from neonatal SD rats were isolated and cultured by tissue digestion and differential adherence methods. The model of rat cardiac fibroblast activation induced by angiotensin Ⅱ was established. The CFb were randomly divided into control group, Ang Ⅱ model group, Ang Ⅱ+Zac group, Ang Ⅱ+Zac+BaCl₂ group, AngⅡ+Zac+chloroquine group and Ang Ⅱ+captopril group. CCK-8 assay was used to detect the effect of Zac on the viability of CFb. The amount of collagen I and collagen Ⅲ secreted by CFb was determined by ELISA. The apoptosis of the CFb was analyzed by flow cytometry. The protein expression of Kir2.1 was determined by Western blot. RESULTS:Compared with the control group, the viability and collagen synthesis of the CFb were significantly increased, along with decreased Kir2.1 expression (P<0.05). Compared with the Ang Ⅱ model group, Zac treatment inhibited the viability and collagen synthesis of the CFb, induced apoptosis and up-regulated Kir2.1 expression (P<0.05). I_K1 blockers BaCl₂ and chloroquine reversed the effect of Zac. CONCLUSION:By enhancing I_K1 (Kir2.1) expression, Zac attenuates Ang Ⅱ-induced ventricular fibrosis, in response to the inhibition of cell viability and induction of apoptosis.     

Effect of platelet activating factor on the action potential and potassium currents in guinea-pig ventricular myocytes

AIM: To investigate the effects of platelet activating factor (PAF) on the action potential and potassium currents in guinea-pig ventricular myocytes. METHODS: By using whole-cell patch clamp technique, the effects of PAF on APD₉₀, I_K1 and I_K were investigated in enzymatically dispersed single guinea-pig ventricular myocytes. RESULTS: With 5 mmol/L ATP in the pipette electrode, 1 μmol/L PAF increased APD₉₀ from (225.8±23.3) ms to (352.8±29.8) ms (n=5, P<0.05), decreased I_K1 and I_K tail currents from (-6.1±1.3) nA to (-5.6±1.1) nA (n=5, P<0.05) at -120 mV and from (173.5±16.7) pA to (152.1±11.5) pA (P<0.05, n=4) at +30 mV, respectively. In contract, without ATP in the pipette electrode, 1 μmol/L PAF shortened APD₉₀ from (153.0±24.6) ms to (88.2±19.4) ms (n=5, P<0.01). Incubation of myocytes with 1 μmol/L glibenclamide, a blocker of I_KATP restored prolongation of APD induced by PAF. CONCLUSION: In guinea-pig ventricular myocytes, with 5 mmol/L ATP in the pipette, PAF prolonged APD partly due to the inhibition of I_K and I_K1, while with 0 mmol/L ATP in the pipette, PAF induced an activation of I_KATP, hence a decrease in APD was observed. Therefore, PAF might amplify the heterogeneity between ischemia and normal cardiac myocytes during ischemic reperfusion, which might play a vital role in the pathogenesis of the arrhythmias induced by ischemia/reperfusion.     

Effects of docosahexaenoic acid on large-conductance calcium-activated potassium channels in rat pulmonary artery smooth muscle cells

AIM：To investigate the effects of docosahexaenoic acid (DHA) on large-conductance calcium-activated potassium channels (BKCa) in rat pulmonary artery smooth muscle cells (PASMCs)．METHODS：BKCa currents in individual PASMCs were recorded by patch-clamp technique in whole-cell configuration．Calcium sparks in PASMCs caused by DHA were recorded by confocal microscopy. RESULTS：DHA activated BKCa . BKCa current densities were (30.5±6.5)pA/pF,(59.4±5.8)pA/pF, (87.2±4.3)pA/pF and (117.3±7.1) pA/pF (P<0.01) with the addition of DHA at concentrations of 0, 0.1, 1 and 10 μmol/L, respectively. Hypoxia inhibited BKCa currents in PASMCs, but this inhibition was reversed by DHA (10 μmol/L). DHA (10 μmol/L) induced an increase in ［Ca2+］i with a maximal increase rate of (71.9±4.1)%. CONCLUSION：DHA activates BKCa in rat PASMCs, leading to the vasodilation of pulmonary arteries.     

Effects of apelin-13 on transient sodium currents in rat ischemic ventricular myocardial cells

AIM：To investigate the effects of apelin on ventricular arrythmias and cardiac functions in rat Langendorff perfusion-simulated myocardial ischemia model by observing the changes of transient sodium currents (INa) in normal cells and the simulated ischemic cells in rat left ventricle. METHODS：Ventricular cells were enzymatically isolated by the Langendorff perfusion system. INa was recorded by the technique of whole-cell patch-clamp. Some elements in the extracellular fluid were changed to simulate the normal or ischemic status. Forty Wistar rats were divided into 4 groups：normal group, ischemic group, normal with apelin group and ischemic with apelin group. The effect of apelin-13 on INa was observed. The method of rat Langendorff perfusion was used to simulate the ischemic heart model. The ventricular arrhythmia scores and heart functional parameters were compared. The expression level of sodium channel protein,type V,alpha subunit (SCN5A) in ventricular ischemic cells was measured by Western blotting. RESULTS： Apelin-13 increased INa amplitude in both normal myocardial cells ［(-86±13） pA/pF］ and ischemic myocardial cells ［(-52±15） pA/pF］. The results of current-voltage curve analysis indicated that apelin-13 did not change the conduction velocity of INa in the 4 groups ［(3.2±0.2） pS/pF, （3.1±0.3） pS/pF,（2.9±0.1）pS/pF and （2.8±0.4） pS/pF,respectively, P>0.05］. The membrane potentials at 50% maximal activation in the 4 groups were （-21.9±0.6） mV, （-28.7±0.3） mV, （-30.5±0.7） mV and （-36.8±0.2） mV, respectively, and the slope of activation curves was 5.6±0.3, 5.1±0.4, 4.3±0.3 and 4.9±0.6 (P>0.05), respectively. No difference of ventricular arrhythmia scores between normal group and normal with apelin group, as well as between ischemic group and ischemic with apelin group was observed. LVEDP in normal with apelin group was lower than that in normal group.The dp/dtmax and dp/dtmin in normal with apelin group were higher than those in normal group. Apelin improved cardiac function parameters in the ischemic hearts. The expression of SCN5A was not affected by apelin (28.8±3.6, 29.4±4.1, 30.1±2.9 and 31.3±3.8,respectively,P>0.05). CONCLUSION：Apelin-13 changes the gating properties of sodium channel, enhances the peak INa and facilitates the opening of sodium channel without inducing ventricular arrhythmias. Apelin-13 has a positive inotropic effect on both normal and ischemic hearts.