| Cardiac hypertrophy and failure is the common complication for such diseases as hypertension, ischemic heart disease, and heart valve disease, etc. Cardiac hypertrophy and failure often accompany with arrhythmias. Heart failure in particular is associated with a significant increase in the risk of sudden cardiac death (SCD). Despite considerable progresses have been made in the treatment of heart failure in recent years, the mortality of patients with heart failure remains high. Therefore, it is of significance to elucidate the molecular mechanism underlying the arrhythmias companying cardiac hypertrophy and failure and seek for the effective medication.The electrophysiological remodeling in cardiac hypertrophy and failure has been documented. The prolongation of action potential duration (APD) is the most consistent electrophysiological abnormality in myocytes from hypertrophied and failing hearts. However, mechanisms underling the contribution of APD prolongation to cardiac arrhythmogenesis are incompletely characterized. It is believed that amplification of transmural dispersion of repolarization (TDR) underlies reentry arrhythmias. However, the remodeling of transmural APD in experimental animal models are still not consistent although it has been shown that TDR is amplified in the heart failure.Cardiac action potential profiles depend on a delicate balance between the depolarizing and repolarizing currents. In principle, any increase in inward or decrease in outward current is capable of causing a prolongation of the cardiac action potential. Down-regulation of transient outward K+ current, Ito occurs in common, wheres ICa-L has been reported distinctively, according to experimental animal species and the progress of heart failure. Recent data obtained from many experimental models (including dog, rabbit rat and cat) of cardiac hypertrophy or failure and terminal heart failure in human, indicate that down-regulation of Ito may cause abnormalities in repolarization. Therefore, changes of Ito in myocardial hypertrophy and heart failure have been under intense investigation. However, although the mouse is being used increasingly due to the ease with which molecular genetic strategies can be applied there are highly inconsistent results on Ito in mouse cardiac hypertrophy and heart failure. In addition to Ito, IK,slow and Iss also contribute to the ventricular depolarization in mouse heart. But there are few reports about the changes of IK,slow and Iss in cardiac hypertrophy and failure. Moreover, little is known on the mechanisms underlying the changes of potassium channels in cardiac hypertrophy and failure at present.In this study, by using the patch-clamp technique, we observed characteristics of the action potential, L-type calcium current (ICa-L) and voltage-dependent K+ currents Ito, IK,slow and Iss in subepicardial and subendocardial ventricular myocardium during the devolpment of cardiac hypertrophy and failure in the mouse cardiac pressure over-loaded model. By treating animals with calcineurin inhibitor cyclosporin A (CsA), we evaluated the possible role of calcineurin pathway in the regulation of voltage-dependent potassium currents in heart failure so as to provide evidence for screening new antiarrhythmics.Part 1. Remodeling of transmural action potential during the development of mouse cardiac hypertrophy and heart failureObjective: In order to reveal the cellular mechanisms of arrhythmia, we observed the transmural action potential changes from subendocardial (Endo) and subepicardial (Epi) myocytes of left ventricular free wall during the development of mouse cardiac hypertrophy and heart failure.Methods: (1) Mouse model of cardiac pressure overload: Transverse aorta was banded by using microsurgical techniques to create cardiac pressure overload, as described by others. Briefly, Kunming male mice, 4-5 weeks old (provided by Experimental Animal Center of Hebei Province), were anesthetized with ketamine (25mg/kg, intraperitoneal injection). The mice were orally intubated and ventilated (Harvard Apparatus). The chest cavity was opened in the second intercostal space and transverse aortic banding (TAB) was performed by tying a nylon suture against a 27-gauge needle to produce an aortic narrowing 0.4 mm in diameter when the needle was removed. The procedure resulted in a reproducible transverse aortic banding of 65-70%. Age-matched mice were subjected to a sham operation in which the aortic arch was visualized but not banded. Mice were then maintained for 13 weeks after operation. Hemodynamic variables in some TAB or sham-operated mice were measured by placing a catheter in the isolated right carotid artery, which was advanced as far as the aorta and left ventricle. Cardiac mass index was also assessed and compared to the sham-operated animals at the different time after operation. (2) Single myocyte dissociation: Single myocytes were enzymatically isolated from left ventricle. In briefly, after retrograde perfusion with Tyrode's solution, hearts was perfused with Tyrode's solution containing collagenase for 25-35 minutes. The septum, apex, and the base of heart were then removed. Subendocardium and subepicardium (0.5mm, respectively)were carefully dissected from the left ventricular free wall and placed in separate plates. After trituration, both Endo and Epi myocytes were filtered through a 200 mesh sieve and stored in KB fluid at room temperature (23°C to 25°C) for 2 h. (3) Action potential recordings: Action potentials (APs) were recorded by using the perforated patch technique. The cardiac action potentials were induced by wave-width of 10 ms, 1Hz, 100% ~ 120% threshold currents stimulation. Action potential durations including APD50 and APD90 at 50% and 90% repolarization respectively were calculated. The cells with resting membrane potential below -65mV were discarded. Cell capacitance was calculated from uncompensated capacity current transient elicited by a 20-mV hyperpolarizing voltage step from a holding potential of -40 mV.Results: (1) A mouse pressure over-loaded cardiac hypertrophy and failure model was established by aorta banding. The results showed that hearts in first 7 week from banded mice were manifested the progressive increase in heart mass index and contractile function, which we defined the stage of compensatory hypertrophy. The following was the stage of heart failure, characterized by declining contraction. (2) The cell capacitances from left ventricular Endo and Epi myocytes in Sham mice were very similar, that is, the cell sizes of two areas were no difference. Capacitances of Endo myocytes began to increase (P<0.01) at 2 weeks after operation, compared to age-matched sham-operated animals, with the increase by 25.5%,29.1%,34.9% and 54.4% at 2, 5, 9, 13 weeks respectively, Similarly, Epi myocytes showed a similar increase as Endo myocytes (P>0.05). (3) There were a marked difference in AP profiles and APD between Endo and Epi cells. APD from Endo myocytes was significantly longer than that from Epi cells. Compared to age-matched sham-operated group, APD50 of Endo cell was no change in 2w Band mice. Whereas it was significantly prolonged by 27% , 82% and 406% in 5w, 9w and 13w Band mice respectively. APD90 in compensatory hypertrophy hearts (Band 2w, 5w) was not significantly different from that in sham hearts (P<0.05), but in failing hearts it was significantly prolonged, with an increment of 96% and 364% at 9w and 13w Band, respectively (P<0.01). Different from Endo myocytes, APD90 of Epi myocytes in 2w Band mice was significantly longer than that in Sham mice and did not progressively prolonged during the development of hypertrophy to heart failure. The increment of APD90 was 116%, 192%, 174% and 154% at 2, 5, 9, 13w, respectively (P<0.01) and for APD50 was 58%, 84% and 67% at 5w, 9w and 13w, respectively(P<0.01). (4) Resting potentials were about -70 mV in different periods, and there are no significantly difference between Sham and Band group. AP overshoot value in Endo is greater than that in Epi in both sham and Band group (P<0.01). Compared to sham 2w, only Endo AP overshoot value was greater than that in 2w Band mice.Conclusions: (1) It was in the early phase (Band 2w) that action potential started to be prolonged in hypertrophied heart. Because the prolongation of APD from Epi myocytes was more remarkable than that from Endo myocytes, transmural repolarization gradient of APD was blunted. (2) The major change in heart failure stage is that APD from Endo myocytes was much more prolonged than from Epi myocyes and therefore, transmural repolarization dispersion was greatly increased. The results suggest that different cellular mechanism contributes to arrhythmogenesis in hypertrophied and failing hearts.Part 2 Transmural L-type calcium current during the development of mouse cardiac hypertrophy and heart failureObjective: To observe the changes of ICa-L density and channel kinetics in Endo and Epi myocytes of left ventricular free wall in mouse hypertrophied and failure hearts.Methods: The changes of ICa-L density and the channel kinetic properties were recorded from isolated Endo and Epi myocytes of mouse left ventricular free wall by whole-cell patch-clamp current recordings at 5w ( in hypertrophy phase without dysfunction of contraction) and 13w (decompensate heart failure phase) after operation.Results: (1) there was no significant difference in ICa-L density between Endo and Epi myocytes in Sham mice. (2) ICa-L density in Endo myocytes was significantly decreased in hypertrophied and failing hearts compared with age-matched sham-operated mice. From +10 mV to +40mV of four test potentials the density of ICa-L from Endo myocytes in hypertrophied hearts was respectively decreased 43%, 43.5%, 43.9% and 43.6% (P<0.01), and in heart failure was respectively decreased 20.2%, 21.4%, 21.6% and 25.7% (P<0.01), but the activation voltage, the maximal activation voltage and reversal potential of ICa-L were not significantly altered. The others L-type channel kinetic properties including activation, instant inactivation constant, steady state inactivation and recovery from steady state inactivation were not significantly altered. Whereas we didn't observed any significant difference in ICa-L density in Epi myocytes either from hypertrophied or failure hearts.Conclusions: (1) L-type calcium current may not contribute to the physiological transmural electrical heterogeneity in mouse hearts. (2) The density of ICa-L was decreased in Endo myocytes, but not in Epi myocytes in the hypertrophied hearts. The results suggested that such non-synchronous changes of ICa-L in Endo and Epi myocytes could contribute to AP transmural repolarization gradient disappear. (3) The density of ICa-L was decreased in Endo myocytes, but not in Epi myocytes in the failing hearts. The result suggests that the decreased density of ICa-L in Endo myocytes may be an adaptive response to the prolongation of action potential due to delayed depolarization and may undertake an effect of reducing the transmural dispersion of repolarization in the heart failure. Taken together, down regulation of change of ICa-L in Endo myocytes has different effect on the transmural repolarization gradient in hypertrophied or failure hearts.Part 3. Remodeling of transmural voltage-dependent potassium currents during the development of mouse cardiac hypertrophy and heart failureObjective: In order to reveal the ion currents underlying AP transmural heterogeneity change, we observed the changes of voltage-dependent potassium currents including Ito, IK,slow and Iss and channel kinetics in subendocardial (Endo) and subepicardial (Epi) myocytes of left ventricular free wall in hypertrophied and failure hearts.Methods: Voltage-dependent potassium currents including Ito, IK,slow and Iss and channel kinetics were recorded from isolated Endo and Epi myocytes of left ventricular free wall by whole cell patch-clamp at 5w and 13w after mouse aorta banded. The decay phases of the currents evoked by long (4.5 s) depolarizing voltage steps from -60 mV to +60 mV from a holding potential of -80 mV were fitted by the sum of two exponentials, and then three current components Ito, IK,slow, Iss and their inactivation time constant (τ1andτ2) were distinguished.Results: (1) Ito: There was a significant transmual gradient of Ito density in all of sham-operated mice. Ito current density was significant smaller in Endo than in Epi myocytes. Compared to the age-matched sham-operated mice, Ito current density in 5w Band mice was significantly decreased(P<0.01), with a reduction of 52.5% in Endo and a reduction of 41% in Epi myocytes at +60 mV test potential. In Endo myocytes, Ito current density was further down regulated in 13w Band mice, with a decrease of 67.3% (P<0.01) However, there were no significantly difference in Ito current density between 5w and 13w Band (P >0.05). (2) IK,slow: There were no significant difference of IK, slow between Endo and Epi in Sham-operated group(P>0.05). Compared to sham-operated group, IK,slow current density at +60mV test potential in 5w Band was decreased by 40% (P<0.01) in Endo myocytes, whereas no change in Epi myocytes (P>0.05). It was further down regulated in 13w Band mice, with a decrease of 61.7% (P<0.01). Also, it was decreased by 32% in Epi cells (P<0.01). Obviously, there was more down regulation of IK,slow in Endo than in Epi myocytes in 13w Band mice. (3) Iss: Iss current density in Sham group was mild greater in Epi than in Endo myocytes(P>0.05). Compared to age-matched sham mice, Iss current density in Band mice was decreased in a parallel manner in Endo and Epi cells, with the reduction 42%, 43% in 5w Band and 61%, 57% in 13w Band, respectively. (4) Instant inactivation constant (τ): It was noticed that inactivation time constant of Ito (τ1) both in Endo and Epi myocytes was singnificant longer in 5w and 13w Band mice compared to Sham mice.τ2 (inactivation time constant of IK, slow in 13w Band was remarkable shorter either in Endo or in Epi cell. (5) Steady-state inactivation: Steady state inactivation curve of Ito was shifted to left, i.e. hyperpolarizing direction shift in Endo myocytes in 13w Band mice. In Epi myocytes, inactivation curves of Ito in 5w and 13w Band mice were also shifted to left. Inactivation curves of IK,slow in Endo and Epi myocytes were shifted to hyperpolarizing direction in 13w Band mice.Conclusions: (1) The study showed a distinct distribution of Ito density in Endo and Epi myocytes under physiological conditions. Lower Ito density in Endo than in Epi primarily contributes to AP transmural heterogeneity. There were no significantly difference in IK,slow and Iss density between Endo and Epi myocytes. (2) In myocardial hypertrophy stage, there were significant down-regulations of voltage-dependent K+ currents, which caused the prolongation of action potential. Both Ito and Iss were simultaneously reduced in Endo and Epi myocytes, while IK,slow was down-regulated only in Endo cells. Obviously, in addition to the down regulation of K+ currents, the down regulation of ICa-L in Endo cells also contributes to the remodeling of AP transmural gradient. (3) In heart failure stage the prominent change was the asynchronous down-regulation of Ito and IK,slow, that is, the current densities were much more decreased in Endo than in Epi myocytes. Remarkable down regulation of Ito and IK,slow may result in the amplification of TDR. The result suggests that down-regulations of voltage-gated K+ currents and L-type Ca2+ current contribute to the remodeling of AP transmural heterogeneity during the development of myocardial hypertrophy and heart failure.Part 4. The effect of calcineurin signaling pathway on down-regulation of voltage-dependent K+ currents in decompensated failing heartsObjective: To evaluate the role of calcineurin signaling pathway in down-regulation of voltage-dependent K+ currents in decompensated failing hearts by treating aorta-banded mice with calcineurin inhibitor cyclosporin A (CsA). Methods: Sham or aorta banded (Band) mice were randomized to receive CsA (25mg/kg, injected subcutaneously twice daily, North China Pharmaceutical Group Corporation) or vehicle (Veh) for 2 weeks. Treatments were started from 9 weeks after operation, which represented heart failure phases. At the end of treatment, i.e. 11 weeks after operation, the densities and kinetics properties of voltage-dependent K+ currents and cell capacitance from Endo and Epi myocytes were determined by using whole-cell patch-clamp technique in CsA or Veh treatment mice, which including following groups: Band+CsA, Band+Veh, Sham+CsA and Sham+Veh. Meantime, cardiac mass index and cardiac histopathology were assessed. Ito, IK,slow, Iss and their inactivation time constant (τ1andτ2) were distinguished as mentioned above.Results: (1) There were no significant differences in animal appearances between Band+CsA and Band+Veh mice, also in Sham+CsA and Sham+Veh groups. The mortality was 8% in Band+CsA group, which was similar to that in Band+Veh mice. There was no mortality in sham-operated mice. The cardiac mass index of Band+CsA mice was significantly decreased compared to Band+Veh group (P<0.01), but not reached to the level in Sham+Veh group. Compared to Sham+Veh group, cell capacitances in Band+Veh mice were greatly increased both in Endo and Epi myocytes (P<0.01), with the increase 63% and 67% (P<0.01) respectively. The cell capacitances of Band+CsA mice were significantly decreased compared to Band+Veh group (P<0.01), but not reversed to the level in Sham+Veh group (P<0.05). The cell sizes of Endo or Epi myocytes were no difference in all of the four groups (P>0.05). (2) Cardiac histopathology: Cardiac myocytes from Sham+Veh and Sham+CsA had well-arranged appearance, with similar sizes and clear transverse lines. There was little collagen between the cardiomyocytes. However, cardiomyocytes in Band+Veh mice were lightly dyed and had blurry-transverse lines, with bigger size and disorganized appearance. There were a lot of denatured cells with dark-dyed and blurry nucleus. Compared to Band+Veh group, myocardium in Band+CsA was with a more clear structure, less edema and less denaturing. (3) Ito: Compared to the Sham+Veh mice, Ito current density in Band+Veh mice was significantly decreased (P< 0.01), with a reduction of 66% in Endo and a reduction of 44% in Epi myocytes at +60 mV test potential. CsA treatment up-regulated Ito density both in Endo and Epi myocytes in banded mice (P<0.01, P<0.05), with a increase of 84.7% and 25.6% respectively, but not reversed to the level in Sham+Veh group(P<0.01). (4) IK,slow : Compared to Sham+Veh group, IK,slow current density at +60mV test potential in Band+Veh was decreased by 51.5% and 29.3%(P<0.01) in Endo and Epi myocytes, and IK,slow current density in Band+Veh mice was singnificantly smaller in Endo than in Epi myocytes. Compared to Band+Veh mice, IK,slow current density in Band+CsA mice were significantly increased either in Endo or in Epi myocytes, with an increase of 87.5% and 37.9% respectively, and completely restored to the level Sham+Veh group (P>0.05). Similar to Sham+Veh group, there was no difference in IK,slow density between Endo and Epi myocytes in Band+CsA mice. (5) Iss: Compared to Sham+Veh mice, Iss current density in Sham+Veh mice was decreased in a parallel manner in Endo and Epi cells, with the same reduction of 56%. After CsA treatment, Iss current density was partially up-regulated both in Endo and Epi myocytes in banded mice (P<0.01), with an increase of 100% and 26.7% respectively. CsA could completely revised the increase of Iss current density in Endo, but not up to the level in Epi myocytes of Sham+Veh group (P<0.01). (6) Instantanous inactivation constant (τ): It was noticed that inactivation time constant of Ito (τ1) both in Endo and Epi myocytes was singnificant longer in Band+Veh mice compared to Sham+Veh mice.τ1 was no sigificiant differences in Endo or Epi myocytes between Band+CsA and Band+Veh groups (P>0.05).τ2 (inactivation time constant of IK,slow) in Band+Veh group was remarkable shorter either in Endo or in Epi cell. Compared to Band+Veh mice,τ2 in Band+CsA mice were significantly longer either in Endo or in Epi myocytes, with an increase of 22.7% and 47.3% respectively, and completely restored to the level Sham+Veh group (P>0.05). (7) Steady-state inactivation: Compared to Sham+Veh mice, steady state inactivation curve of Ito was shifted to left, i.e. hyperpolarizing direction shift either in Endo and Epi myocytes in Band+Veh mice. CsA completely antagonized the shift of steady state inactivation curve and shifted the curve from left to right in Endo myocytes, but had no impact on the curve in Epi myocytes. It was noticeable that CsA completely reversed the shift of steady state inactivation curve of IK,slow in Endo and Epi myocytes and resulted in a curve, which was similar to that in Sham+Veh mice. (8) Compared to Sham+Veh mice, APD of Endo cell was significantly prolonged by 438.6% and 410.5% in APD50 and APD90. CsA treatment in banded mice partially antagonized the prolongation of APD50 and APD90 in Endo myocytes (P<0.01), with a shortening of 65.8% and 59.5%. Compared to Sham+Veh mice, APD of Epi cell was significantly prolonged by 69.4% and139.6% in APD50 and APD90. CsA treatment partially reversed the prolongation of APD50 in Epi myocytes from banded mice, with a decease of 21.3% compared to Band+Veh mice, but had no impact on APD90 (P>0.05). (9) In sham-operated mice, there was no effect of CsA on general conditions, cardiac mass index, cell capacitances, the density and function of voltage-dependent K+ currents and APD.Conclusions: (1) Calcineurin inhibitor CsA completely reversed the down-regulation of IK,slow in heart failure, suggesting that calcineurin signaling pathway primarily contributes to the down-regulation of IK,slow in the pathological condition. (2) Inhibition of calcineurin pathway partially antagonized the down-regulation of Ito, indicating that in addition to calcineurin signaling pathway, there are other signaling pathways causing the down-regulation of Ito in decompensated heart failure. (3) Antagonizing the remodeling of voltage-dependent K+ currents including Ito, IK,slow and Iss by the inhibition of calcineurin pathway was more predominant in Endo myocytes than in Epi myocytes, and thus attenuated the amplification of transmural repolarization dispersion. The result gives us a hint that interruption of calcineurin signaling pathway may be a potential new therapeutic target against arrhythmias and sudden death in heart failure.CONCLUSIONS1 Different cellular mechanism contributes to arrhythmogenesis in hypertrophied and failing mouse hearts.2 L-type calcium current may not contribute to the physiological transmural electrical heterogeneity in mouse hearts. Down regulation of change of ICa-L in Endo myocytes has different effect on the transmural repolarization gradient in hypertrophied or failure hearts: Such non-synchronous changes of ICa-L in Endo and Epi myocytes could contribute to AP transmural repolarization gradient disappear; the decreased density of ICa-L in Endo myocytes may be an adaptive response to the prolongation of action potential due to delayed depolarization and may undertake an effect of reducing the transmural dispersion of repolarization in the heart failure.3 In myocardial hypertrophy stage, there were significant down-regulations of voltage-dependent K+ currents, which caused the prolongation of action potential. Both Ito and Iss were simultaneously reduced in Endo and Epi myocytes, while IK,slow was down-regulated only in Endo cells. Obviously, in addition to the down regulation of K+ currents, the down regulation of ICa-L in Endo cells also contributes to the remodeling of AP transmural gradient. In heart failure stage the prominent change was the asynchronous down-regulation of Ito and IK,slow, that is, the current densities were much more decreased in Endo than in Epi myocytes. Remarkable down regulation of Ito and IK,slow may result in the amplification of TDR. The result suggests that down-regulations of voltage-gated K+ currents and L-type Ca2+ current contribute to the remodeling of AP transmural heterogeneity during the development of myocardial hypertrophy and heart failure.4 Calcineurin inhibitor CsA completely reversed the down-regulation of IK,slow in mouse heart failure, suggesting that calcineurin signaling pathway primarily contributes to the down-regulation of IK,slow in the pathological condition. Inhibition of calcineurin pathway partially antagonized the down-regulation of Ito, indicating that in addition to calcineurin signaling pathway, there are other signaling pathways causing the down-regulation of Ito in decompensated heart failure. Antagonizing the remodeling of voltage-dependent K+ currents including Ito, IK,slow and Iss by the inhibition of calcineurin pathway was more predominant in Endo myocytes than in Epi myocytes, and thus attenuated the amplification of transmural repolarization dispersion. The result gives us a hint that interruption of calcineurin signaling pathway may be a potential new therapeutic target against arrhythmias and sudden death in heart failure.
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