| Since K2P channels were found in the middle-1990s, it attracted considerable research interest. TREK-1, as one member of K2P family, has become the focus of attention in recent years because of their own unique channel characteristics. There was extensive evidence that TREK-1was highly expressed in some vital organs such as brain and heart, so it can be speculated that TREK-1plays a very important role in the maintenance of normal physiological function. With the development of a variety of biological technology, the function of TREK-1in the central nervous system and cardiovascular system has been revealed gradually. But because no specific blocker of TREK-1was available, its role in cardiomyocytes and astrocytes still remained unclear. Hypertrophy also presented substrates for lethal ventricular arrhythmias, resulting in sudden arrhythmic deaths that account for about one third of deaths in cardiac hypertrophy. Accumulated experimental data suggested that the most consistent electrical change which has been described in association with the chronic stage of cardiac hypertrophy is prolongation of action potential duration (APD).Considering TREK-1was a stretch-activated potassium channel and meanwhile a background potassium channel, its role in regulating the cardiac action potential was anticipating. Astrocytes are non-excitable cells of the central nervous system and provide many important functions that are critical for the normal activity of neurons. TREK-1was sensitive to some pathological conditions in astrocytes such as cell swelling, a rise in free fatty acid level and intracellular acidification, its role in astrocytes also remained unknown. Pathological changes of TREK-1in cardiomyocytes and electrophysiological identification of TREK-1in astrocytes were uncertain. So in this study we focused on the expressional and functional studies in normal or pathological conditions of caidiomyocytes or astrocytes, and provided theoretical basis for further clarifying the concise function of TREK-1and seachering for new drug targets.Part I:Changes of protein expression and current of TREK-1in endocardial myocytes during left ventricular hypertrophy1. Hypertrophy in rat induced by isoproterenol Left ventricular hypertrophy was evoked by repeated subcutaneous administration of isoproterenol at a dosage of5mg/kg for7days. The results showed that compared to normal rats, the heart weight, heart weight/body weight and left ventricular wall thickness of hypertrophic rats were increased by44.9%,45.2%and34.9%, respectively. It meant isoproterenol-induced hypertrophy was successful.2. Changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophyWestern blot was used to observe changes of TREK-1protein expression in endocardial myocytes during left ventricular hypertrophy. The result showed compared to control group, TREK-1protein expression was enhanced by26.6%in hypertrophy group. It meant hypertrophy significantly up-regulated TREK-1protein expression in endocardial myocytes.3Identification of cardiac TREK-1currents3.1Outward single channel currents activated by stretch (negative pressure) in cardiomyocytesStretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.064±0.01could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.3.2Outward single channel currents induced by intracellular acidification in cardiomyocytesObserve the effects of intracellular acidification on cardiac outward single channel currents and CHO-TREK-1. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes but flittering openings could be detected in CHO-TREK-1cells. When the pH was6.8and6.3, both in Cardiomyocytes and CHO-TREK-1cells visible opening could be seen.PH6.8and6.3solution elicited an outward single channel in cardiomyocytes with an open probability of0.037±0.007and0.124±0.009, respectively. pH7.3,6.8and6.3solution elicited an outward single channel in CHO-TREK-1cells with open probabilities of0.068±0.002,0.133±0.017and0.225±0.022, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.3.3Outward single channel currents induced by arachidonic acid in cardiomyocytesObserve the effects of arachidonic acid on cardiac outward single channel currents. The results showed in inside-out recordings10μM arachidonic acid could obviously induce an outward single channel current in cardiomyocytes while evident openings also could be detected in CHO-TREK-1cells without any treatment. It suggested arachidonic acid could activated an activated an outward current in cardiomyocytes.3.4Conductance of cardiac TREK-like channelsSingle channel conductances of two cell types calculated according to the fitted current amplitude of the above-recorded currents were123±7pS (CHO-TREK-1) and113±17pS (cardiomyocyte) with no statistical significance. The conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was close to the previously reported conductance of TREK-1. It meant the conductance of our recorded currents induced by arachidonic acid in cardiomyocyts was consistent with the conductance of CHO-TREK-1.According to the above results, the outward channels we recorded in cardiomyocyes could be activated by stretch, intracellular acidification and AA and had a similar conductance to CHO-TREK-1, so it could be proved that the current we recorded in cardiomyocytes was native TREK-1.4. Effects of L-NBP on the open probability of TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytesInside-out recordings were used to observe the inhibitive effects of L-NBP on TREK-1. In inside-out recording, obvious channel openings could be recorded without any treatment in CHO-TREK-1cells, while after application of10μM L-NBP for5min, the channel activity could be apparently inhibited but could not be reversed totally. In normal and hypertrophic cardiomyocytes,0.18chloroform could induce burst TREK-1openings, while after application of10μM L-NBP for5min, the openings were significantly decreased but could not be inhibited completely. The inhibition rate of10μM L-NBP on TREK-1in CHO-TREK-1cells, normal and hypertrophic cardiomyocytes were48.5±8%,54.3±3%and55.5±4%, no statistical significance existed among the three groups. These results indicated that chloroform could easily elicit TREK-1in cardiomyocytes and10μM L-NBP had significant inhibitory effects on TREK-1in three cell types.5. Effects of L-NBP on outward currents in normal and hypertrophic cardiomyocytesWhole cell recordings were used to observe the effects of TREK-1blocker L-BNP on outward currents in normal and hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of10μM L-NBP for5min could reduce the outward current from10.21±3.02pA/pF to8.99±2.40pA/pF in normal cardiomyocytes and from7.43±1.72pA/pF to5.73±1.75pA/pF in hypertrophic cardiomyocytes.10μM L-NBP-inhibited outward current in hypertrophic cardiomyocytes (1.70±0.4pA/pF) was significantly larger than in normal cardiomyocytes (1.22±0.36pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.6. Effects of chloroform on outward currents in normal and hypertrophic cardiomyocytesWhole cell recordings were used to observe the effects of TREK-1selective agonist chloroform on outward currents in normal and hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current was considered as an index to evaluate the difference of TREK-1between normal and hypertrophic cardiomyocytes. On the base of blocking other major potassium channels, application of0.18mM chloroform for5min could enhance the outward current from6.47±2.13pA/pF to8.89±2.38pA/pF in normal cardiomyocytes and from6.04±1.57pA/pF to9.47±2.16pA/pF in hypertrophic cardiomyocytes.0.18mM chloroform-activated outward current in hypertrophic cardiomyocytes (3.43±0.7pA/pF) was obviously larger than in normal cardiomyocytes (2.42±0.5pA/pF). The results suggested the background current of TREK-1in hypertrophic cardiomyocytes was larger than in normal cardiomyocytes.The above results meant that there was an increasing tendency of TREK-1in hypertrophic cardiomyocytes compared to normal cardiomyocytes, which was consistent with our western blot data.Part II:Electrophysiological Identification of a native TREK-like channel in rat brain astrocytes1. TREK-like outward channels activated by arachidonic acid and chloroformObserve the effects of arachidonic acid and chloroform on outward single channel currents in astrocytes. In inside-out recordings,10μM arachidonic acid and0.2mM chloroform could obviously induce outward openings in cultured astrocytes. The open probabilities of10μM arachidonic acid-activated and0.2mM chloroform-activated outward channels were0.173±0.01and0.255±0.02. The calculated single channel conductance of the recorded channels was107±16pS, which was close to previous literature. These results indicated that arachidonic acid and chloroform could activate outward channel openings with a similar conductance to TREK-1in astrocytes.2Outward single channel currents activated by stretch (negative pressure) in astrocytesStretch was elicited via suction of a syringe connected to a manometer. The results showed in inside-out recordings little outward current could be recorded when no stretch was applied to the excised patches. When a-30cmH2O negative pressure was applied, obvious outward openings with an open probability of0.142±0.02could be detected. After the negative pressure was removed, the openings were almost reversed. It meant stretch could activate an outward single channel in cardiomyocytes.3.2Outward single channel currents induced by intracellular acidification in astrocytesObserve the effects of intracellular acidification on outward single channel currents in cultured astrocytes. The results showed in inside-out recordings when the pH of the external solution was7.3, no obvious openings could be recorded in cardiomyocytes. When the pH was6.8and6.3, visible opening could be seen. Outward channel openings could be increased pH-dependently. PH6.8and6.3solutions elicited an outward single channel in astrocytes with open probabilities of0.064±0.01and0.176±0.01, respectively. These results indicated that intracellular acidification could activate an outward current in cardiomyocytes.According to the above results, the outward channels we recorded in astrocytes could be activated by stretch, intracellular acidification, AA and chloroform and had a similar conductance to TREK-1, so it could be proved that the current we recorded in astrocytes was native TREK-1. |
PDF Full Text Request