Effects Of Oua On Calcium Transients And Contraction In Rat Cardiocytes

The Na+/K+-ATPase (NKA), or sodium pump, is widely found in various cells in humans and animals, whose role is to transport three Na+ out of the cell and two K+ in, utilizing ATP hydrolysis as the driving force. So far found that sodium pump was composed ofα,β,γsubunits. Where theαsubunit is referred to as the catalytic subunit, that contains binding sites for Na+, K+, ATP and cardiac glycosides (CGs), mainly implement pump function. Four different isoforms (α1,α2,α3, andα4) of theαsubunit have been described. Previous studies have shown that DHO (dihydroouabain, a CGs) increase diastolic [Ca2+] and the contraction, without altering the calcium transient.There are also reports OUA (ouabain, a CGs) can increase the calcium transient and the myocardial contractile force. Our previous study suggests Str (strophanthidin, a CGs) can increase the extent of contraction and calcium transients in a concentration-dependent manner.Previous study reported that rat vertricular myocytes express two functionally distinct NKA: theα2-isoform has a high affinity for CGs and theα1-isoform has a low affinity. A concentration of 0.3μM OUA blocked 94% of theα2-isoform, but less than 1% of theα1-isoform. And 0.3 mM OUA blocked 90%α1-isoform. However, the regulation of diastolic [Ca2+] and calcium transients by which isoform has not been reported.Our previous studies have shown that Str has more potent contraction at the same concentration in heart failure guinea pig vertricular myocytes than in normal one. The heart failure model was made by constricting descent aorta. But the role of cardiac glycosides play in other type's heart failure has not been reported.A number of studies have suggested that 10-7-10-3 M inhibit NKA, the lower concentrations of CGs show positive inotropic effect and the higher concentrations induce Ca2+ overload and cardiac arrhythmia in the isolated heart or single myocytes. And 10-10-10-8 M OUA can stimulate cardiac NKA and show positive inotropic effect in the isolated heart. However, the positive inotropic effect cannot be detected in single myocytes.Isolated adult rat cardiac ventricular myocytes have been a useful model for contraction research, but they have to require high quality. They must have not only the sustained excitability, but also stable contraction. In the past, lots of research explored the objective conditions to improve the method of the cardiocyte isolation, including digestive enzymes, the composition, pH, and temperature of perfusion solution, and so on. However, because of subjective, the determination of digestion could not judge properly by the traditional Langendorff perfusion. The nature and quality of the cardiocytes was various. And the efficiency was reduced in contraction research.The goal of the present study was to improve the traditional Langendorff perfusion. And then we synchronously detected diastolic [Ca2+], calcium transients and the shortening in vertricular myocytes from normal and heart failure rat. The heart failure rat was induced by adriamycin (ADR). The different concentrations of OUA were used to inhibit Na+/K+-ATPaseα1 andα2 isoform, respectively, to investigate their isoform-specific regulation on diastolic [Ca2+] and calcium transients. By using low concentrations OUA to measure sensitivity in rats different hierarchical left ventricular myocytes. This study will aid us to further develop the mechanism of isolated cardiocytes exposed to nanomolar cardiac glycosidesPart 1 An improved method of rat cardiocyte isolationObjective: To develop an improved method of Langendorff perfusion for isolating rat cardiac myocytes that is easy to determine the termination of digestion.Methods: Hearts were excised quickly from anesthetized SD rats and were perfused with normal Tyrode solution. After the perfusate was free of blood, the normal Tyrode solution was changed to Tyrode solution contained the following: 0.6% Collagenase B, 0.6% BSA and 30μM Ca2+ at 37℃. Single cardiac myocytes were enzymatically isolated by traditional and improved Langendorff perfusion devices. The isolated myocardial cell was measured by video-based motion edge-detection system (IonOptix, USA).Results: One group of heart was digested by traditional Langendorff perfusion for 13~16 min, in which the termination of digestion could not be judged properly and the nature and quality of the isolated cardiocytes were not stable. The cardiocytes could not keep their survival and viability when exposed to Tyrode Solution contained 1.8 mM Ca2+. Another group was digested by improved Langendorff perfusion. More than 80% survival cardiac ventricle myocytes could be obtained by improved Langendorff perfusion. Moreover, about 50% cardiocytes could retain rod-shaped and be used to contraction research when exposed to Tyrode solution with 1.8 mM Ca2+. The contraction of cardiocyte was stabile within 1000 s.Conclusion: Cardiocytes isolated by improved Langendorff perfusion can be used in these studies of detecting contraction and relaxation. This method of cardiac myocyte isolation is economical and easy to control. The beginners are able to acquire the technological method over a short-term practice.Part 2 Effects of OUA on calcium transients and contraction in rat cardiocytesObjective: To detect the effects of OUA on the contractility, diastolic [Ca2+] and calcium transients of cardiac myocytes from normal and failure rat hearts to investigate the regulation of Na+/K+-ATPaseα1 andα2 isoform on them and the contractile sensitivity in different hierarchical left ventricular myocytes. Methods: Rat heart failure model was made by intraperitoneal injecton of adriamycin. Left ventricular myocytes were isolated as described in part 1 and then loaded with Fluo-4. The effects of different concentration OUA on the contractility, diastolic [Ca2+] and calcium transients were assessed by the video-based motioned detection system (IonOptix, USA). Isolated cardiac myocytes from the endocardium, mid-myocardium and epicardium of left ventricular were exposed to 1 nM OUA and detected the contractility by the video-based motioned detection system (IonOptix, USA).Results:1The injection of adriamycin could induce the heart failure in rats Rats showed the symptoms of HF after intraperitoneal injecting adriamycin: Psyche depressed, activity and taking food reduced, breathing accelerated, body weight obviously lightened, the heart rate, left ventricular systole pressure (LVSP) and±dp/dtmax significantly reduced, left ventricular end-diastolic pressure (LVEDP) significantly increased. The morphological changes of heart from HF rats under 400 times light microscope showed widened cardiocyte interstice, irregular myocytes arrange, nuclear deformity, and partial cell necrosis.2 Effects of OUA on calcium transient and shortening in normal rat cardiocytes10-7~10-3 M OUA were able to increase the extent of cardiocyte shortening and calcium transients in a concentration-dependent manner. 3×10-7 M OUA significantly increased the extent of shortening (58.00±27.86, P<0.05) and calcium transient (27.38±14.47, P<0.05), 3×10-4 M OUA increased by 217.34±63.80% (P<0.01) and 70.95±15.81% (P<0.05). Although 10-3 M OUA concentration can further enhance the extent of shortening and calcium transients, there were no significant difference between extents induced by 3×10-4 and 10-3 M OUA, and the cardiocytes be induced Ca2+ overload and cardiac arrhythmia suggesting that 3×10-4 M OUA was the minimum concentration that produced the maximum shortening and calcium transients. 10-5~10-3 M OUA could not only increase the extent of calcium transient, but also diastolic [Ca2+], while a lower concentrations of OUA had no effect on diastolic [Ca2+].10μM nimodipine significantly inhibited the shortening, diastolic [Ca2+] and calcium transient in rat cardiocytes. 3×10-4 M OUA was able to reverse the negative inotropic effect of nimodipine. However, the 3×10-7 M OUA had no any effect on the action of nimodipine.3 Effects of OUA on Ca2+ transient and shortening in heart failure rat cardiocytesThe cardiocytes from adriamycin-induced heart failure rats had lower sensitivity to the OUA. 3×10-7 M OUA that inreased the extent of shortening and calcium transients in normal myocytes had no effect on HF myocytes. Although 3×10-4 M OUA significantly increased the contractility in HF myocytes, the magnitude was significantly less than that in the normal myocytes. What's more, the extents of diastolic [Ca2+] and calcium transients increased by 3×10-4 M OUA in HF myocytes were also lower than those in the normal myocytes.4 Nanomolar OUA enhanced the positive inotropic effect of endocardium cells1 nM OUA could enhance the positive inotropic effect of left ventricular myocytes. The increased amplitude in contraction was 21.32±9.05% (P<0.05, n=4). However, this positive response rate for the positive inotropic effect was very low at nM concentration. Further experiment found that the positive response rate induced myocyte shortening at nM OUA concentration was depended on location of myocyte in left ventricular myocardium, which respective were 4/6 in the endocardial cells, 1/4 in the mid-myocardial cells, and 0/4 in the epicardial cells, suggesting that the endocardium cells were more sensitivity to nanomolar OUA.Conclusion: 10-7~10-3 M OUA are able to increase the extent of cardiocytes shortening and Ca2+ transients in a concentration-dependent manner; 10-5~10-3 M OUA not only increased the extent of calcium transient, but also diastolic [Ca2+]. The Na+/K+-ATPaseα2-isoform involves in the increases of contractility and calcium transients, but did not affect diastolic [Ca2+]. The Na+/K+-ATPaseα1-isoform can not only enhance contractility and calcium transients, but also increase diastolic [Ca2+]. Adriamycin-induced heart failure can lower the sensitivity of rat cardiocytes to the OUA. Nanomolar OUA can selectively enhanced the positive inotropic effect of endocardial cells.