Effects Of Na～+ /H~+ Exchanger And Na~+ , K~+ -ATPase On The Cardiac Contraction And Intracellular Calcium In Heart Failure Mice

Heart failure (HF) is a series of multiplicity clinical symptom, and the end stage of many cardiac disease, such as hypertension, ischemic cardiomyopathy, valvular disease and so on. The incidence and prevalence of heart failure (HF) is high and the prognosis of HF is rather poor. It is one of the most harmful problems which is increasingly severe.Cardiotonic steroid (Cs) had been used for the congestive heart failure (CHF) for more than 200 years. But the mechanism of its positive inotropic effect is still controversial. It is generally believed that Cs produce positive inotropic actions in failure heart via inhibition of Na+, K+-ATPase (NKA) activities which leads to an increase in intracellular Na+ concentration ([Na+]i), and in turn raises intracellular Ca2+ concentration ([Ca2+]i) via Na+/Ca2+ exchange, thus increasing the uptake and subsequent release of Ca2+ by the sarcoplasmic reticulum (SR).Na+/H+ exchanger (NHE) as a plasma membrane exchange glycoprotein transporter that functions in intracellular pH regulation, cell volume regulation, and cellular response to many different hormones and mitogens. Following allosteric activation by intracellular acidification, NHE exchanges extra- cellular sodium for intracellular hydrogen with stoichiometry of 1:1. Na+ influx via activation of Na+/H+ exchanger reactivates PKC in myocytes, which is one of the important signaling molecules in the development of the cardiac hypertrophic response. Recently, Baartscheer et al reported that two months of dietary treatment with the NHE-1 inhibitor cariporide caused regression of hypertrophy, heart failure and ionic and electrophysiological remodelling.Saini HK et al reported NHE play a critical role in the ouabain-induced [Ca2+]i increase in cardiomyocytes. However, inotropic effect of methyl-n- isobutyl amiloride (MIA), a NHE inhibitor, in isolated ischemia-perfusion hearts was not consistent with its effect on [Ca2+]i in quiescent or KCl- depolarized cardiomyocytes exposed to ouabain. Therefore, it is hard to explain the interacton between NKA and NHE, and effect of them on the pathological condition. Thus mice were chosen as test animals. The mouse CHF models were developed. The present study is to observe the effects of the NHE on inotropic effects and change of [Ca2+]i of normal and CHF ventricular myocytes in the presence or obsence of ouabain in order to investigate inhibition of NHE as a potentiality treatment of CHF.Objective: To detect the effects of NHE-1 on the contractile and calcium transient in the ventricular myocytes from heart failure mice through establishing CHF animal model and the effects of CHF on the expression of NKAα1 andα2 isoform and NHE-1 in protein level to identify the mechanism of the inotropic effect of NHE-1 and NKA in isolated mouse ventricular myocytes.Methods: Mice CHF model was made by constricting aortic arch. Left ventricular myocytes were enzymatically isolated. Then the contractile and calcium transient of a single myocyte from normal and CHF mice were assessed by a video-based motion edge-detection system simultaneously. At the same time, effects of the same concentration of ouabian on inotropic effects and calcium transient in CHF mouse ventricular myocytes were compared with those in normal mouse ventricular myocytes. Then the experiments were repeated when the myocytes were preincubated with 10nM ouabain. The effects of MIA with ouabain on contractility and calcium transient were recorded and the results were compared with those of MIA-only. Then the expressions of NKAα1 andα2 isoform and NHE-1 in protein level were detected with Western blot.Results:⑴The effects of ouabain on the contractility and calcium transient of NC or CHFC from mice. The extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i was normalized using the value before ouabain perfusion as 100%. In NC, OUA 0.5μM, 1μM, 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased the extent of cell shortening by (124.60±5.02)%, (133.02±5.65)%, (148.42±6.63)%, (174.14±6.29)%, (204.64±3.66)%, (228.50±10.87)%, (286.42±27.57)%, and (288.80±23.82)% (p< 0.01); The concentration of OUA above increased the ampitude of calcium transient by (119.26±4.30)%, (125.43±5.64)%, (126.10±6.77)%, (133.92±10.26)%, (139.72±6.43)%, (152.90±8.10)%, (157.38±11.53)%, and (156.39±12.24)% (p<0.01); OUA 5μM, 10μM, 50μM, 100μM, 500μM, and 1 mM increased basal level of [Ca2+]i by (107.33±2.18)%, (110.82±2.32)%, (111.10±2.81)%, (112.24±2.56)%, (115.72±4.63)%, and (116.70±3.80)% (p< 0.01), while OUA 0.5μM and, 1μM had no effect on basal level of [Ca2+]i, which are (103.64±2.26)% and (102.47±3.29)% (p>0.05), respectively; 0.1μM OUA had no effect on the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i, which are (104.59±2.35)%, (101.41±1.82)% and (99.62±2.23)% (p>0.05),respectively; and OUA 500μM and 1 mM caused the cell irregular contraction, shorter length even became to"round-up". In CHFC, OUA 50μM increased extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i by (351.71±90.6)%, (164.77±6.87) % and (117.67±7.70)% (p<0.01); At the same concentration, effect of Str was more potent in CHFC than in NC.⑵The effects of MIA on the contractility and calcium transient of NC or CHFC from mice. In NC, after 5 min perfusion MIA 0.1 and 1μM decreased the extent of cell shortening by (78.82±5.44)% and (46.93±7.36)% (p<0.01); and decreased the ampitude of calcium transient by (73.41±8.73)% and (49.59±4.01)% (p<0.01); but had no effect on basal level of [Ca2+]i (p>0.05). After 15 min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (142.91±12.64)% and (204.36±27.22)% (p<0.01), increased the ampitude of calcium transient by (117.51±2.68)% and (132.51±4.30)% (p<0.01), and increased basal level of [Ca2+]i by (115.22±1.90)% and (126.93±3.97)% (p<0.01). When the positive inotropic effect reach the maximal value, MIA caused the shorter length even became to"round-up". In CHFC, however, after 5min perfusion MIA 0.1 and 1μM increased the extent of cell shortening by (117.01±1.11)% and (130.08±3.61)% (p<0.01), increased the ampitude of calcium transient by (104.64±2.18)% (p>0.05) and (113.38±2.61)% (p<0.01), and had no effect on basal level of [Ca2+]i, (101.13±4.90)% and (104.21±2.49)% (p>0.05). In CHFC, after 15min perfusion MIA 0.1 and 1μM had no effect on the extents of cell shortening, which were (104.55±3.89)% and (101.83±2.62)%, the ampitudes of calcium transient, which were (100.11±4.59)% and (97.27±3.72)%, and basal levels of [Ca2+]i, which were (103.63±6.91)% and (99.01±4.82)% (p>0.05), respectively.⑶The effects of MIA on the contractility and calcium transient of NC or CHFC from mice in the present of low concentration of ouabain. In NC, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA decreased the extent of cell shortening form (46.93±7.36)% to (78.49±9.19)% (p<0.01) in 5 min perfusion, increased the extent of cell shortening form (204.36±27.22)% to (135.42±10.74)% (p<0.01) in 15 min perfusion; and decreased the ampitude of calcium transient from (49.59±4.01)% to (78.51±10.83)% (p<0.01) in 5 min perfusion, increased the ampitude of calcium transient from (132.51±4.30)% to (108.64±2.68)% (p<0.01) in 15 min perfsion; and had no effect on basal level of [Ca2+]i in 5 min perfusion, while increased basal level of [Ca2+]i from (126.93±3.97)% (p<0.01) to (103.11±2.07)% (p>0.05), which had no significant difference compared with those before perfusion. In CHFC, however, after preincubation with 10 nM OUA for 10 min, 10 nM OUA+1μM MIA was perfused. Compared with the effect of MIA in NC, OUA+MIA increased the extent of cell shortening form (130.08±3.61)% to (183.14±23.77)% (p<0.01) in 5 min perfusion, and from significant difference to increased the extent of cell shortening by (180.56±18.31)% in 15 min perfusion; and increased the ampitude of calcium transient from (113.38±2.61)% to (155.37±4.89)% (p<0.01) in 5 min perfusion, and from no significant difference (97.27±3.72)% (p>0.05) to increased the ampitude of calcium transient by (158.48±8.62)% (p<0.01) in 15 min perfusion; OUA+ MIA increased basal level of [Ca2+]i from no significant difference (104.21± 2.49)% and (99.01±4.82)% (p>0.05) to increased by (115.19±1.98)% and (114.37±2.05)% (p<0.01), respectively, after 5 min and 15 min perfusion.⑷The expression of NKAα1 andα2 isoform and NHE-1 in protein level in ventricular myocytes from mice with CHF. Compared with NC, the protein level of NKAα1 andα2 isoform and NHE-1 were decreased in CHFC (p<0.01).Conclusions: Ouabain increases the extent of cell shortening, ampitude of calcium transient and basal level of [Ca2+]i. The reason why effects of OUA is more potent in CHFC than in NC may be related to that the more protein level of NKAα1 andα2 isoform are decreased in CHFC than in NC. MIA enhances the contractility and calcium transient followed by a weaken effect; whlie MIA moderately increased the inotropy and calcium transient of CHFC. Low concentration of OUA can partially abolish MIA-induced the changes in NC; however, MIA-induced changes in inotropic effect and [Ca2+]i were augmented by OUA in CHFC. It is prompted that there is a colsed relationship between NHE and NKA on the process of regulating cadiocytes, and the regulation is dependent on the condition of cadiocytes. In NC, NHE and NKA showed synergistic action, while in CHFC, they showed antagonistic effect. In addition, our study also suggest that research and development of the inhibitor of NHE-1 have the prospect of as adjunctive therapy with Cs to treat CHF.