The Regulation Of Calcineurin Inhibitor On Transmural L-type Calcium Current In A Pressure-overloaded Mouse Model With Hypertrophy And Failure

Cardiac hypertrophy and heart failure often accompany with arrhythmias. Especially, heart failure is associated with a significant increase in the risk of sudden cardiac death (SCD), which becomes a main reason of the death caused by cardiovascular disease. The latest data show that the incidence of SCD in China averages 41.84 cases in 100,000 peoples, and to estimate by the 1.3 billion populations, the quantity of SCD is up to 54.4 million in 1 year and can rank first in all countries. Since 1998, Molkentin et al have put forward that the Ca2+i/CaM/Calcineurin/NFAT signaling transduction pathway plays an important role in cardiac hypertrophy and heart failure. More and more experiments have found that Calcineurin signaling pathway also contributes to the pathological electrical remodeling.Previous experiments of our group showed that in cardiac hypertrophy stage, transmural dispersion of repolarization (TDR) of APD was disappeared because the prolongation of APD from subepicardial (Epi) myocytes was more remarkable than that from subendocardial (Endo) myocytes. The major change in heart failure stage was that APD from Endo myocytes was much more prolonged than from Epi myocyes and therefore, TDR greatly increased. By applying Calcineurin inhibitors cyclosporin A (CsA) in heart failure stage, the significant increase of voltage-dependent K+ currents including Ito, IK,slow and Iss was more predominant in Endo myocytes than in Epi myocytes, and thus attenuated the amplification of transmural repolarization dispersion (see Shi chenxia PhD thesis). Little is known whether Calcineurin signaling pathway also contributes to the reduction of TDR in cardiac hypertrophy stage. Cardiac hypertrophy and heart failure is a dynamic process of development, we hypothesized that the activation, function and the electrophysiological characteristics of Calcineurin signaling pathway are different on different models of hypertrophy, even on different stages of the same models.In this study, by treating aorta-banded mice in cardiac hypertrophy and heart failure stage with Calcineurin inhibitor cyclosporin A (CsA), the cardiac hemodynamics parameters were measured and the transmural AP and ICa-L density from Endo and Epi myocytes of left ventricular free wall were also determined by using whole-cell patch-clamp technique, in order to evaluate whether the same role of Calcineurin signaling pathway plays on the heart structure, function and electrophysiological characteristics in the development of cardiac hypertrophy and heart failurePart1. The regulation of Calcineurin inhibitor on hemodynamics in a pressure-overloaded mouse model with hypertrophy and failureObjective: In order to evaluate whether the same role of Calcineurin signaling pathway plays in structure, function and the physiological characteristic of cardiac hypertrophy and heart failure sage. We applied with Calcineurin inhibitor cyclosporin A (CsA) in a pressure-overloaded mouse model with hypertrophy and failure stage.Methods: (1) Mouse model of cardiac pressure overload: Kunming male mice, 4-5 weeks old (provided by Experimental Animal Center of Hebei Province) were anesthetized ketamine (25mg/kg, intraperitoneal injection). The chest cavity was opened and transverse aortic banding (Band) was partly ligature. The procedure resulted in a transverse aortic banding of 65-70%.(2)Experimental groups: Treatments were started from 5w or 11w after operation, which represented cardiac hypertrophy and heart failure phases. Sham or aorta banded (Band) mice in 3w and 9w mice after operation were randomized to receive CsA (25mg/kg, injected subcutaneously twice daily) or vehicle (Veh) for 2 weeks. The mice were divided into following eight groups: Sham5w+Veh, Sham5w+CsA, Band5w+Veh, Band5w+CsA, Sham11w+Veh, Sham11w+CsA, Band11w+Veh and Band11w+CsA. The animial body weight, food consumption, cardiac mass index, lung mass index and hemodynamic parameters such as SBP, LVSP, LVEDP, dp/dt max and dp/dt min were detected.Results: (1) The mouse in 5w Sham and Band group were in a good condition, and no death was happened. The body weight and food consumption were no significant differences in all groups of 5 weeks, whereas they significant reduced in Band11w+Veh and Band11w+CsA mcie compared to sham groups(P<0.01). (2) Compared to age-matched Sham+Veh mice, the cardiac weight, cardiac mass index, lung weight and lung mass index in Band5w+Veh and Band11w+Veh mice were significant increased(P<0.01)and they were no difference in Sham5w+CsA and Sham11w+CsA mice. The parameters of Band5w+CsA mice were significantly decreased compared to Band5w+Veh group (P<0.01), but not reached to the level in Sham5w+Veh group. There were no differences in Band11w+CsA and Band11w+Veh in all parameters (P>0.05). (3)The heart rates were no significant differences in all groups. Compared to age-matched Sham+Veh mice, the parameters including SBP, LVSP, LVEDP, dp/dt max and dp/dt min were significant increased in Band5w+Veh mice (P<0.01), whereas they also increased except that dp/dt max and dp/dt min were lower in Band11w+Veh mice (P<0.05). The parameters of Band5w+CsA mice were significantly decreased compared to Band5w+Veh group (P<0.05 or P<0.01), but not reached to the level in Sham5w+Veh group. There were no differences in Band11w+CsA and Band11w+Veh in all parameters (P>0.05).Conclusions: Calcineurin signaling pathway activated is a very important way induced myocardial hypertrophy. Heart function in myocardial hypertrophy can be improved by inhibiting the way.Part2. The regulation of Calcineurin inhibitor on transmural action potential in a pressure-overloaded mouse model with hypertrophyObjective: Inhibited Calcineurin by using the immunosuppressant CsA, in order to observe the changes of the transmural AP from subendocardial (Endo) and subepicardial (Epi) myocytes of left ventricular free wall.Methods: (1) Sham or Band mice in 3w mice after operation were randomized to receive CsA (25mg/kg, injected subcutaneously twice daily) or vehicle (Veh) for 2 weeks. The mice were divided into following groups: Sham5w+Veh, Sham5w+CsA, Band5w+Veh, Band5w+CsA. (2) Myocytes of Endo and Epi myocytes were enzymatically isolated from left ventricular free wall. APs of single myocyte were recorded by using the perforated patch technique. Action potential durations including APD50 and APD90 were calculated.Results: (1) There were a significant difference in AP profiles and APD between Endo and Epi myocytes of left ventricular free wall in Sham+Veh and Sham+CsA mice. The APD of Endo myocytes was significant prolonged than that of Epi myocytes. The APD50 and APD90 of Sham+CsA was not significant difference compared with Sham+Veh(P>0.05). (2) Compared to Sham5w+Veh, the APD50 and APD90 of Epi myocytes were significant prolonged by 23% and 185%, and APD50 in Endo myocytes was prolonged by 30%, but APD90 was not changed in Band5w+Veh mice. Thus, action potential heterogeneity was decreased. (3)Compared with Band5w+Veh, the APD50 in Epi myocytes was not changed and the APD90 was shortened by 24% in Band5w+CsA mice, though it didn't recover to the level of Sham5w+Veh mice. The APD50 and APD90 of Endo myocytes in Band5w+CsA mice were not changed. That is, although it didn't recover to normal level, transmural repolarization gradient of APD was increased after treated with CsA.Conclusions: The activation of calcineurin signaling pathway is the main reason to reduce transmural dispersion of repolarization (TDR) of APD in cardiac hypertrophy. It contributes to restore the normal TDR of APD in cardiac hypertrophy by inhibiting calcineurin signaling pathway. Part3. The regulation of Calcineurin inhibitor on transmural L-type calcium current in a pressure-overloaded mouse model with hypertrophy and failureObjective: To observe the transmural changes of L-type calcium current from subendocardial (Endo) and subepicardial (Epi) myocytes of left ventricular free wall by applying inhibitor cyclosporin A (CsA).Methods: The changes of ICa-L density and the cell capacitance 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. All mice were divided into eight groups with the same method in first part of the paper.Results: (1) Compared to age-matched Sham+Veh mice, there was no difference in the cell capacitance of myocytes from Sham5w+CsA and Sham11w+CsA mice, and the cell capacitance of Endo or Epi myocytes significantly increased by 46.7%, 42.9% respectively in Band5w + Veh mice and 61.1% , 80.4% respectively in Band11w+Veh mice(P <0.01). After given with CsA in the same period of Band mice, the cell capacitance of Endo or Epi myocytes significantly decreased by 17.7%, 18.0% respectively in Band5w +CsA mice and14.1%, 16.7% respectively in Band11w+ CsA mice compared to age-matched BAND+Veh mice (P <0.05). (2) There was no significant difference in ICa-L density between Endo and Epi myocytes in Sham mice. ICa-L density in Endo myocytes was significantly decreased in Band+Veh hearts compared with age-matched sham-operated mice. After given with CsA in the same period of Band mice, from +20 mV to +40mV of test potentials the density of ICa-L from Endo myocytes was respectively decreased 25.1%, 31.8% and 36.5% in Ban5w+ mice (P<0.01), and from +10 mV to +40mV of test potentials the density of ICa-L from Endo myocytes was respectively decreased 19.1%, 23.2%, 25.3% and 32.3% in Band11w+Veh mice (P<0.01 or P<0.05). The activation voltage, the maximal activation voltage and reversal potential of ICa-L in all groups were not significantly altered. Whereas that we didn't observe any significant difference of ICa-L density from Epi myocytes in all groups.Conclusions: That the decrease of L-type calcium current density and APD shorting in Endo myocytes is one of reason in disappearance of TDR by inhibiting Calcineurin signaling pathway in heart faulure. 1 Calcineurin signaling pathway activated is a very important way induced myocardial hypertrophy. Heart function in myocardial hypertrophy can be improved by inhibiting the way.2 The activation of calcineurin signaling pathway is the main reason to reduce transmural dispersion of repolarization (TDR) of APD in cardiac hypertrophy. It contributes to restore the normal TDR of APD in cardiac hypertrophy by inhibiting calcineurin signaling pathway.3 That the decrease of L-type calcium current density and APD shorting in Endo myocytes is one of reason in disappearance of TDR by inhibiting Calcineurin signaling pathway in heart faulure.