Asics Expression And Distribution On Cardiac Tissues And Special Electrophysiological Properties

PartⅠASICs mRNA and Protein Expression on Cardiomyocytes and Heart TissueAim:In order to explore the expressions of ASICs in heart tissue and cardiomyocytes.Methods: ASICs expressions in heart sections and cardiomyocytes from SD rats were detected by RT-PCR,western blotting and immunofluorescent methods.Results: ASICs mRNA and proteins were expressed on cardiomyocytes and heart tissue.1. ASIC1, ASIC2a and ASIC3 mRNA were proved to be existed on heart tissues by RT-PCR. The base number is 140bp, 139bp, and 107bp respectively.2. ASIC1,ASIC2a and ASIC3 proteins were expression in rat heart tissues and cardiomyocytes by Western Blotting. Proteins on heart tissues were more than those in cardiomyocytes. Similar to nervous tissues, locations of all ASIC proteins were from 55 to 72 KDa, and molecular weight of ASIC1 proteins was a little heavier than ASIC2a and ASIC3.3. ASIC1, ASIC2a and ASIC3 Proteins were proved to distributing on heart tissue and cultured cardiomyocytes by immunofluorescence. The intensity of three subunits were almost the same, however, the distribitions were different. ASIC1 proteins predominantly expressed in nucleuses, and little in endochylema and membrane; Most ASIC2a proteins expressed in nucleuses, as well as in endochylema and membrane; on the contrary, ASIC3 proteins were major in endochylema and membrane, a little of them in nucleuses. Statistical data showed the intensity of membrane and cytoplasm: ASIC1 32.01±5.98 (n=8); ASIC2a 21.83±4.83 (n=10,); and 12.43±3.46 (n=11).Conclusion: 1. ASIC1, ASIC2a and ASIC3 mRNA were proved existing on heart tissue by RT-PCR.2. ASIC1, ASIC2a and ASIC3 Proteins were proved existing on heart tissues and cultured cardiomyocytes by Western blotting and immunofluorescence. The distrubitions of three subunits were different.PartⅡAcid-Evoked Currents in Cultured CardiomyocytesAim: To investigate the electrophysiological properties of ASICs in cultured cardiomyocytes.Methods: Acid-evoked currents from cultured cardiomyocytes were recorded by using the whole-cell patch clamps.Results:1. ASICs-like currents were recorded from cultured cardiomyocytes.(1)ASICs-like currents reversiblely inhibited by 100μM amiloride. There were almost all kinds of ASICs currents in cultured cardiomyocytes, including ASIC1a,ASIC1b,ASIC2a and ASIC3 currents,and even heterologous currents.(2)ASICs-like currents were evoked by a rapid lowering of pH from 7.4 to 7.0. The current densities were pH-dependent, and the curve of currents density-pH was like obverse"S". Current evoked by pH 6.0 and 5.0 changed sharply. The current densities were: pH7.0, 8.51±4.37 pA/pF (n=8), pH6.0, 17.03±5.02 pA/pF (n=11), pH5.0, 50.20±6.94 pA/pF (n=7) and pH4.0, 53.73±9.87 pA/pF (n=7), respectively. pH50 was 5.73±0.18, similar to ASIC1b (about 5.9).2. TRPV1-like currents were also recorded from cultured cardiomyocytes.TRPV1 currents of cultured cardiomyocytes were reversiblely inhibited by 20μM CPZ. Currents were pH-dependent and the current density in pH6.0 was 9.52±4.89 pA/pF (n=5). Conclusions:(1) There were two types of acid-evoked currents in cultured cardiomyocytes. ASICs-like currents of cultured cardiomyocytes were pH-dependent, and reversiblely inhibited by 100μM amiloride. TRPV1-like currents of cultured cardiomyocytes were pH-dependent, and reversiblely inhibited by 20μM CPZ.(2) For ASICs-like currents of cultured cardiomyocytes, pH50 was 5.73±0.18, similar to ASIC1b (about 5.9), but there were almost all kinds of ASICs currents similar to nervous system.PartⅢAcid-Evoked Currents in Acute Isolated CardiomyocytesAim: To further investigate the electrophysiological properties of ASICs in acute isolated cardiomyocytes, and compare the difference of ASIC currents between two kinds of cells.Methods: Acid-evoked currents from acute isolated cardiomyocytes were recorded by using the whole-cell patch clamps.Results:1. ASICs-like currents were recorded from acute isolated cardiomyocytes. There were three types of ASICs-like currents and the the current density was 9.22±5.19 pA/pF (n=5) in pH6.0. ASICs-like currents were reversiblely inhibited by 100μM amiloride.2. TRPV1 currents were also recorded from acute isolated cardiomyocytes. Current density was 1.69±0.26 pA/pF (n=3) in pH6.0。The currents were reversiblely inhibited by 20μM CPZConclusions: There were two types of acid-evoked currents in acute isolated cardiomyocytes. ASICs-like currents of acute isolated cardiomyocytes could be reversiblely inhibited by 100μM amiloride. TRPV1-like currents of acute isolated cardiomyocytes could be reversiblely inhibited by 20μM CPZ.Summary:1. ASIC1, ASIC2a and ASIC3 mRNA and proteins were proved existing on heart tissues and cardiac myocytes, the ditributions were different.2. ASIC current densities of cultured cardiomyocytes were pH-dependent, and could be reversiblely inhibited by 100μM amiloride. TRPV1-like currents of cultured cardiomyocytes were pH-dependent, and reversiblely inhibited by 20μM CPZ. 75.34% of all cells tested induced acid-evoked currents, 43.83% of which were ASICs-like currents and 34.25% were TRPV1-like currents. In some cells, two types of acid-evoked currents could be co-existed.3. ASICs-like currents of acute isolated cardiomyocytes were mostly reversiblely by 100μM amiloride. TRPV1-like currents of acute isolated cardiomyocytes were reversiblely inhibited by 20μM CPZ. 84.62% of all cells tested induced acid-evoked currents, 61.54% of which were ASICs-like currents and 23.08% were TRPV1-like currents. In some cells, these two types of acid-evoked currents could be co-existed.