Role Of The Na～+, K～+-ATPase In The Alteration Of Myocyte Contractility Induced By Hypertension

Hypertension is a main risk factor for human with high attack rate, mutilation and mortality rate. The influence of hypertension on heart includes disfunction of contraction and diastolic, ventricular hypertrophy, cardiac failure and coronary artery disease. The change of heart function is a gradual course with the disfunction of contraction and diastole at earlier stage of hypertension and the left ventricular hypertrophy and cardiac failure with increasing systolic blood pressure (SBP) and diastolic pressure (DP). Generally, Ca2+ is a key factor for muscle contraction and diastole. The disfunction of calcium metabolism and signal transduction system are closely related to the change of contraction and diastole, myocardial hypertrophy and cardiac ventricle reconstitution. But the physiological mechanism is not knownNa+, K+-ATPase in the myocyte mainly regulates the active transport of Na+ and K+ across membrane, and also regulate Ca2+ indirectly by Na+/Ca2+ exchange. The expression of Na+, K+-ATPaseα-isoform is impacted by hypertension, the level of hormone, myocardial hypertrophy and cardiac failure. The present study, carried out mostly on the change of the activity of Na+, K+-ATPase, the inotropy of myocyte, Ca2+ and Na+, the expression ofαisoform of Na+, K+-ATPase by two different models of the hypertensive rats, is to explore the role of the Na+, K+-ATPase in the alteration of myocyte contractibility induced by hypertension.I. Effects of hypertension on the contractility and calcium transient in the rat ventricular myocytesObjective : To approach the effects and possible mechanisms of hypertension on the contractility of myocytes by detemining the systolic and diastolic function, the Na+ concentration, Ca2+ transient and the sensitivity of myocytes to Ca2+ in different phases of hypertension development .Methods : Choosing 4-month-old rats were used to prepare the hypertensive models of 1k1c rat (the rat with one kidney and the constriction of one renal artery). These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The caudilis arterial pressure were measured by the rat caudae, the index of hemodynamics on hypertensive rats were monitored by quantified system analysis, left ventricular mass index were determined by wet weight, the left ventricle myocyte were acutely isolated by enzymolysis method, and the changes of the systolic and diastolic function, the rest Na+ concentration, Ca2+ transient and the sensitivity of myocytes to Ca2+ were observed by video-motion edge-detection system. In the meantime, the distinction of above-mentioned indices of 6-, 12- and 18-month-old rats in different hypertensive models were compared. The rest Na+ concentration in myocyte were observed by video-motion edge-detection system.Results:(1)Hypertension in 1k1c rats was induced at two months after operating, i.e. at 6 months old. The achievement ratio was 73%. The SBP of 1k1c rats and SHR at 6, 12 and 18 months old were higher than those of control rats with the same age, and the SBP of SHR were higher than that of 1k1c rats with the same age(P<0.05 or 0.01). The elevating time of the SHR systolic pressure (at 6 months old) was earlier than that of 1k1c rats (at 12 months old).(2)The left ventricular mass index of 1k1c rat and SHR at 6, 12 and 18 months old were higher than those of control rats with the same age, indicating the formation of myocardial hypertrophy. Furthermore, The degree of myocardial hypertrophy in SHR were obviously greater than those in 1k1c rats (P<0.01). Nevertheless, the obvious accrescent time of myocardial hypertrophy in SHR (at 12 months old) was earlier than those in 1k1c rats (at 18 months old). It is indicated that the process and degree of myocardial hypertrophy in rats induced by hypertension is highly correlated with the elevation of systolic blood pressure.(3)In both hypertensive models, LVEDP, dp/dt and -dp/dt increased significantly in 6, 12, and 18-month old rats, compared with control rats with the same age. But dp/dt and -dp/dt in 1k1c rats at 12 months old were higher than that at 6 months old, while that at 18 months old is lower than at 12 months old. And the -dp/dt in SHR at 12 and 18 months old were lower than that at 6-month-old ones. Above trends indicate that the maximal values of compensation of the diastolic and contractile function were achieved in 12-month-old 1k1c rats and in 6-month-old SHR.(4)Dep v and Peak of rat myocytes from both models were higher than those from control rats with the same age. Ret v of rat myocytes from 1k1c rats exceed that of control rats with the same age, but Ret v of SHR is lower than that of rat myocytes from control rats with the same age. This effect indicates that in the earlier period of hypertension, the diastolic and contractile function of myocytes in 1k1c rats all enhanced. But in SHR rats, only contractile function enhanced, and the diastolic function started to decrease at 6 months old. The above results were same as those observed in hemodynamic research.(5)DCa of rat myocytes from two models at 6-months old were not obviously different from those from control rats, but was lower at 12 and18 months old than that of control group at the same age and that of corresponding model group at 6 months old. These results indicated that 6-month-old 1k1c rat and SHR occupy the compensation stage and the main change is diastolic Ca2+. ?Ca2+ in 1k1c rats at 6-month old have no obvious discrepancy with the same age control group, those of SHR were higher than those in control rats with the same age, those of both 1k1c rat and SHR in 12 and18 month old exceeded those in 6-months group and in control group with the same age. These results indicated that ?Ca2+in 1k1c rats and SHR increased with in the development of hypertensive course, which was likely to be one of the main compensative modes of myocardial contractility.Judged from above results, it is obviously that Ca2+ transient in rat myocytes from two models at 6-months old was not significantly different from the control group, the myocardial contractility of myocytes in 1k1c rat was higher than that in control rats with the same age and lower than that in SHR. Both of Ca2+ transient in myocytes from 1k1c rat and SHR were higher than that in control rats with the same age, but myocardial contractility decreased gradually.(6)Resting Na+ concentration in rat myocytes of two models at 6-months old was not obviously different from the control group(p>0.05), those at 12 and 18-months old were higher than those in control rats with the same age and at 6-month-old model group (P<0.05 or 0.01). The resting Na+ concentration in myocytes of control rats had no change from 6-month old to 18-month old, but the [Na+]i in rat myocytes from two models increased gradually with the age (P<0.01). It was indicated that the level of [Na+]i was increased by hypertension.(7)The contractile amplitude-fluorescence intensity curve in 6-months old 1k1c rats shift left comparing with that in control rats with the same age, which indicated that the sensitivity of myocyte to Ca2+ enhanced. The sensitivity in both 12 and 18-month-old rats decreased. The sensitivity in 6-month-old rats was significantly lower than that in control rats with the same age, and decreased with the prolongation of hypertension duration. The depressed sensitivity of myocyte to Ca2+ induced by hypertension could be one of causes in gradual decline of myocyte contractility by the increasing ofΔCa2+ in 12 and 18-month-old 1k1c rats and SHR.Conclusion: The development of hypertension is a gradual course. The degree of SBP elevation and myocardial hypertrophy increased gradually with temporal prolongation. The degrees of them in 1k1c rats were significantly lower than those in SHR. The amplitudes of Ca2+ transient did not change in 1k1c rats and SHR at two 6-month-old, but enhanced at 12 and 18-month old. The myocardial hypertrophy and sensitivity of myocte to Ca2+ in 1k1c rats all increased at 6-months old but decreased at 12-months old, while both in SHR all started to decrease at 6 months old.II. Effects of cardiac glycosides on the contractility and Ca2+ transient of myocytes from hypertensive ratsObjective:To explore the relationship between the Na+-K+-ATPase and hypertension and the role of the Na+-K+-ATPase in the process of hypertension development by observing the effects of strophanthidin (Str), a specific blocker of Na+,K+-ATPase, on the contractility and Ca2+ transient of myocyte, the Na+ concentration in myocytes, and the sensitivity of myocytes to Ca2+ at different stages of hypertension development in 1k1c rats and SHR.Methods: Choosing 4-month-old rats were used to prepare hypertensive models of 1k1c rat. These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The left ventricle myocyte were acutely isolated by enzymolysis method, and the effects of Str on the contractility and Ca2+ transient of myocyte, the Na+ concentration in myocytes, and the sensitivity of myocytes to Ca2+ at different stages of hypertension development in 1k1c rats and SHR were observed by video-motion edge-detection system.Results:(1)Str 1, 10, 50 and 100μmol?L-1 increased concentration-dependently the amplitude of myocyte contraction in all rats, either normal control rats, 1k1c rat, or SHR, at 6, 12 and 18 months old (P<0.05). The augmented amplitudes of myocyte contraction by Str in 1k1c rats and SHR at 6 months old were obviously higher than those in control rats with the same age. Meanwhile, the enlarged effects of Str on the amplitude of myocyte contraction in 1k1c rats were higher than those in SHR. With the growth of rats, the augmented amplitudes of myocyte contraction by different concentrations of Str did not altered in the control rats, but were decreased concentration-dependently in 1k1c rat and SHR, which indicated that the sensitivity of myocytes to Str was obviously decreased with the development of hypertension.(2)Perfused with 1mM Str, resting Na+ concentration in rat myocytes from two hypertensive models were not obviously different from the control rats at 6-months old (p>0.05), but higher at 12 and 18-months old than those in the corresponding control rats and two hypertensive model rats at 6-month-old (P<0.01), and reached to the maximal value respectively at 12-months old. The resting Na+ concentration at 12 and 18-months old were higher than that of corresponding SHR, and step up gradually with development of hypertension.(3)Str 1,10,50 and 100μmol?L-1 concentration-dependently increased the amplitudes of myocyte Ca2+ transientin in all groups at 6, 12 and 18 months old (P < 0.05). The augmented amplitude of 6, 12 and 18-month-old 1k1c rats and SHR were obviously higher than that of control group, but there were no difference between 1k1c rat and SHR. With the growth of rats, different str concentration had the same effect on the control group, but decreased concentration-dependently on 1k1c rat and SHR.(4)In 6-month-old rats, the contraction amplitude-fluorescence intensity curve of all groups didn't change, only that of 1k1c rat shifted right slightly, but there was no obvious difference.In 12-month-old rats, the contraction amplitude-fluorescence intensity curves of both in 1k1c rats and SHR shifted left, while that of control group didn't change, which indicated that Str could enhance the sensitivity of myocyte to Ca2+ in 1k1c rat and SHR.In 18-month-old rats, the contraction amplitude-fluorescence intensity curves of both 1k1c rat and SHR shifted left, while that of control group didn't change, which indicated that Str could enhance the sensitivity of myocyte to Ca2+ in 1k1c rat and SHR.Conclusion: Str which is a specific Na+,K+-ATPase blocker not only enhances the contractility and Ca2+ transient of myocytes from hypertensive rats, but also increases the sensitivity of myocyte to Ca2+ and the Na+ concentration in myocytes. Furthermore, the effects of Str enhancing the contractility and Ca2+ transient are decreased in age-dependent manner, which indicate the sensitivity of myocytes to Str obviously decreased following the develpment of hypertension.III The effects of hypertension on the activity of Na pump and expressions ofαisoform in rat myoctyteObjectives:To detect the activity of Na+, K+-ATPase and the expressions of its threeαisoforms in hypertensive rats and study the role and underlying mechanism of Na+, K+-ATPase in the alteration of rat myocyte contractility induced by hypertension.Methods:Choosing 4-month-old rats were used to prepare hypertensive models of 1k1c rat. These rats were used in the research at 6 months old (2 months after operating), 12 months old and 18 months old. The SHR and control rats were chosen by corresponding age. The left ventricle myocyte were acutely isolated by enzymolysis method, Na+, K+-ATPase activity was measured through spectrophotometry method,The expression and distribution of the Na+, K+-ATPaseαisofroms were determined with Western blot.Results:(1) At 6 months old, the activity of Na+,K+-ATPase in mycoytes from 1k1c hypertensive rats was significantly higher (P<0.05) than those from SHR and control rats. At 12 and 18 months old, the activities of Na+,K+-ATPase in mycoytes from both 1k1c and SHR rats were significantly lower (P<0.05 or 0.01) than that from control rats. In control rats, there is no significant difference in activity of Na+,K+-ATPase among 6, 12, and 18 months old. While in 1k1c and SHR rats, the activity of Na+,K+-ATPase at 12 and 18 months old is significantly lower than those at 6 months old (P<0.01). The above results indicates that there changes of Na+,K+-ATPase function are highly correlated with development of hypertension.(2) At 6 months old, the expressions ofα1,α2 andα3 isoforms of Na+,K+ -ATPase in 1k1c rats did not change significantly (p>0.05), while those in SHR rats increased significantly comparing with the corresponding period control rats (P<0.05 or 0.01).At 12 months old, the expressions ofα1 andα2 isoforms of Na+,K+-ATPase in 1k1c rats did not change significantly (p>0.05), only that ofα3 isoforms increased (P<0.01), while those ofα1 andα3 isoforms in SHR rats increased significantly comparing with the corresponding period control group (P<0.05 or 0.01).At 18 months old, expressions ofα1 isoforms of Na+,K+-ATPase in 1k1c rats decreased, that ofα2 isoforms did not change significantly (p>0.05), and that ofα3 isoforms increased (P<0.01), while those ofα1 andα2 isoforms in SHR rats decreased significantly andα3 isoforms increased comparing with the corresponding period control group (P<0.05 or 0.01).Going up from six months to 18 months, expressions ofα1,α2 andα3 isoforms of control group did not change significantly (p>0.05). The expression ofα3 isoforms in 1k1c rats at 18-month old decreased (P<0.01),α2 isoforms had no change (p>0.05)andα3 isoforms increased age-dependently(P<0.01). The expression ofα1 andα2isoforms of SHR decreased and that ofα3isoforms increased age-dependently(P<0.01). The above result implied that the development of hypertension is related with the decreasingα1 andα2 isoforms and increasingα3 isoforms.Conclusion:With development of hypertension, the activity of Na+, K+-ATPase increases but subsequently decreases. In the earlier period of hypertension, the expressions ofα1,α2 andα3 isoforms all enhance. In 12-month-old SHR and 18-month-old 1k1c rats, the expressions ofα1 andα2 isoform all decrease and the expression ofα3 isoform increases gradually. These results imply that Na+,K+-ATPase play an important role in the development of hypertension. Summary1.The development of hypertension is a gradual course. The elevated degrees of SBP and myocardial hypertrophy and the amplitude of Ca2+ transient are gradually increased, but the contractility of myocyte and sensitivity of myocyte to Ca2+ are gradually decreased, with the development of hypertension in SHR and 1k1c rats, indicating that both amplitude of Ca2+ transient and the sensitivity of myocyte to Ca2+ are responsible to the contractility of myocyte.2.With the development of hypertension, the elevated amplitude of myocyte contraction induced by strophanthidin, a specific Na+,K+-ATPase blocker, is decreased in ventricular myocytes from SHR and 1k1c rats in an age-dependent manner, but does not change in those from normal control rats. These results indicate that the development of hypertension may decrease the sensitivity of myocytes to strophanthidin by lowering the affinity of Na+, K+-ATPase to strophanthidin.3.With development of hypertension, the activity of Na+, K+-ATPase increases but subsequently decreases, which is consistent with the expressions ofα1 andα2 isoforms. But the expression ofα3 isoform increases gradually. These results imply that Na+,K+-ATPase play an important role in the development of hypertension.