The H₁-H₂Domain Of The α₁Isoform Of Na⁺-K⁺-ATPase Is Involved In Ouabain Toxicity In Rat Ventricular Myocytes

Cardiac glycosides(CGs) have been used for the treatment of thecongestive heart failure for more than200years. But the mechanism is stillcontroversial. It is generally believed that CGs produce the positive inotropic(therapeutic) actions in failure heart via the inhibition of Na⁺-K⁺-ATPaseactivities, which raises intracellular sodium concentration ([Na⁺]i) and asubsequent increase of intracellular Ca²⁺([Ca²⁺]i) via Na⁺/Ca²⁺exchange, thusincreasing the uptake and subsequent release of Ca²⁺by the sarcoplasmicreticulum (CICR), the consequence is a rise in [Ca²⁺]ithat produces positiveinotropic responses. The toxic (arrhythmogenic) effects of CGs occur when[Ca²⁺]iincreases to a level exceeding SR storage capacity. These resultssuggest that the toxic and therapeutic actions of CGs might in fact share acommon mechanism. Thus, there is a great need for safer therapeutic strategyto show a greater separation between inotropic efficacy and toxicity.The Na⁺-K⁺-ATPase (NKA), or sodium pump, is a member of theP-ATPase family, and widely found in various cells in humans and animals,whose role is to transports three Na⁺out of the cell and two K⁺in, utilizing anATP hydrolysis as the driving force. So far it was found that sodium pump iscomposed of α, β, γ-subunits. The α-subunit is the catalytic subunit and mainlyimplements the pump function. The smaller β-subunit spans once through themembrane to form a large extracellular structure that sits on top of the externalsurface of the α-subunit. The β-subunit is essential for trafficking and K⁺transport. The γ-subunit belongs to the FXYD family, most members of thisgroup have now been shown to be associated with the α/β-subunit of NKA andto modulate some aspect of its function. The NKA isoforms are expressed in atisssue-specific and developmental regulated manner, indicates that they mayplay particular roles in specific physiological procedures and development. Four different isoforms (α₁, α₂, α₃, and α₄) of the α-subunit have beenidentified. The α₁isoform is the most widely distributed isoform and expressedin almost all tissues where it functions as a housekeeping isozyme. Theexpressions of NKA isoforms were in a tissue-and species-specific manner,for example, in adult rat heart, α₁and α₂isoforms are mainly expressed, andthe expression of α₃isoform disappeared, and in the heart of guinea pigs onlyα₁and α₂isoforms are expressed.The α-subunit forms transmembrane domains H₁to H₁0by crossing themembrane10times, catalyzes the ion-dependent ATPase activity, and carriesthe binding sites for ATP and the specific inhibitor ouabain (OUA). The α₁isoform is the most widely distributed isoform and expressed in almost alltissues where it functions as a housekeeping isozyme. While the α₂-isoform ismainly expressed in skeletal muscle, heart, brain, adipocytes, vascular smoothmuscle, and eye, as well as a number of other tissues. The α₃-isoform is foundalmost exclusively in neurons and ovaries, but also occurs in white blood cellsand heart of some species, such as humans. The α₄-isoform is expressed insperm. It is reasonable to believe that the tissue-specific distribution of theα-isoforms indicates that each isoform exhibits a particular function associatedwith the tissue in which it is expressed.In recent years, as molecular biology technology and traditional electricphysiology techniques and application, a large amount of data obtained to theNKA structure and functional relationships. The initial efforts to localize theouabain-binding site on NKA were carried out by chemical labeling studies.Furthermore, since ouabain interacts with the exreacellular portion of NKA,the early studies concentrated on substituting amino acids in the extracellularloops of the enzyme. The rat and mouse enzymes are, for instance, a thousandtimes less sensitive than those of sheep and human. These research generatedchimeras between the ouabain-resitant rat α₁and the ouabain-sensitive sheepα₂-isozyme and found that when the border residues (Glu¹¹¹and Asn¹²²) of thefirst extracellular loop in the sheep α₁subunit were substituted with thoseamino acids (Arg¹¹¹and Asp¹²²) present in the rat α₁subunit, an ouabain-resitant enzyme was produced. These observations suggest that thefirst extracellular domain (H₁-H₂) of the NKA a subunit is probably part ofthe ouabain-binding site of the enzyme. The generally accepted cellularreceptor for OUA and other CGs is the α-subunit of membrane-bound NKA,and site-directed mutagenesis has shown that OUA affinity is controlled byresidues in extracellular loop H₁-H₂, leading to the logical conclusion that thisis an important region of NKA. In addition to the H₁-H₂region, subsequentstudies dimonstrated that other parts of the α-subunit transmembrane domain,such as H3-H4and H₅-H₆, might be involved in OUA binding, indicating thatplay an important role in OUA binding.Previous study reported that rat vertricular myocytes express twofunctionally distinct NKA: the α₂-isoform has a high affinity for CGs and theα₁-isoform has a low affinity. A concentration of0.3μM OUA blocked94%of the α₂-isoform, but less than1%of the α₁-isoform. And0.3mM OUAblocked90%α₁-isoform. However, the regulation of diastolic [Ca²⁺] andcalcium transients by which isoform has not been reported. Su et al indicatedthat the positive inotropic effects and irregular contraction produced bystrophanthidin (Str) at higher concentrations result from NKA inhibition, andthe positive inotropic effects of Str at lower concentrations are not related toNKA inhibition. Ventricular myocytes round up into a contracture state oncethe peak level of [Ca²⁺]iis achieved in the presence of10μM Str, but there isno change in the presence of100nM Str. Gao et al have demonstrated thatCGs1to10nmol/L stimulated NKA activities, and pointed out that thisexciting role relevant to α₂-isoform. Our studies also found that0.1to10nmol/L DHO increased NKA activity. However, the therapeutic concentrations(1¹0nmol/L, low concentrations) of CGs in patients with the heart failure aremuch lower than those shown to inhibit the NKA in vitro. Thus, the inhibitiontheory of NKA seems to be inadequate in explaining the inotropic action ofCGs at therapeutic levels. The relationship between the inotropic actions andNKA activities in low concentrations of CGs need further to be confirmed.Therefore, it may be of importance for the development of new drugs that are able to discriminate between the 'inotropic' α₂and the 'toxic' α₁isoform ofNKA. More recently, various reports have suggested that differences in CGssensitivity of NKA isozymes determine their therapeutic and toxic effects suchthat the high affinity NKA mediates the positive inotropic effects, whereas thelow affinity NKA is responsible for toxic effects of CGs. Our work provides aclue for us to screen specific drugs that act on α₂receptors to treat heartfailure.For at least200years digitalis glycosides have been widely used for thetreatment of cardiac insufficiency. In spite of several advances in the treatmentof cardiovascular disorders, digoxin has remained one of the most commonlyprescribed drugs in the clinical practice. The foreign researchers suggest thatthe H₁-H₂domain of the NKA α₁isoform is a critical determinant of enzymeactivity, and an antibody directed against the loop is known to enhance Ca²⁺flux and myocyte inotropic action. They also reported that the H₁-H₂domainof the α₁isoform is capable of regulating enzyme function in two distinctways, by inhibiting or enhancing NKA activity. These findings motivated usto use polyclonal antibodies as an important tool to look for reduced thetoxical α₁or specific role in α₂and base to look for more safe role is musclestrength drug? In view of the above research, this experiment is divided intothree parts:Part1Preparation, purification and identification of polyclonalantibody of anti NKA α₁and α₂H₁-H₂domainAim: To use sodium pump α₁and α₂isoforms H₁-H₂amino acidsequence as an antigen to immunize New Zealand rabbits and preparepolyclonal antibody through the preparation of immune animal serumtechnique. The indirect enzyme-linked immunosorbent assay (ELISA),Western blot and immunohistochemistry method was used to characterizepolyclonal antibody, which lay the foundation for the study of mechanism ofaction of the sodium pump in the cell and the relationship with the positiveinotropy.Methods:(1) Acquisition and purification of the antibodies: We producted sodium pump α₁and α₂isoforms H₁-H₂amino acid sequence bychemical process, and immunize New Zealand rabbits as an antigen andprepare polyclonal antibody through the preparation of immune animal serumtechnique. The positive antisera were applied on the Hi Trap protein A HPcolumn, which was an affinity chromatography column for purification of IgGfrom serum. Titers of the antibodies were determined by indirect ELISA.(2)Accroding to Beatty's method, the affinity constants of the polyclonalantibodies were measured with indirect ELISA.(3) The specificity of antibodywas confirmed by Westernblot.(4) Polyclonal antibodies are successfullydetected on the membrane surface of cardiac myocytes by immunofluorescentstaining methods.Results:(1) The results of titers, concentrations and purification:Immunize New Zealand rabbits third time and prepare polyclonal antibodythrough the preparation of immune animal serum technique. After purificationwith protein A HP column, concentrations of antibodies against α₁(SSA78)and α₂(WJS) were0.949mg/ml and1.03mg/ml. Titers of the antibodiesagainst α₁and α₂reached1:240000by indirect ELISA.(2) Accroding toWestern blotting method, SSA78could specifically recognize the Hl-H₂domain of NKA α₁isoform and detect the NKA α₁isoform collected from ratcardiac myocytes, as the same as the commercial antibody. WJS couldspecifically recognize the Hl-H₂domain of NKA α₂isoform and detect theNKA α₂isoform collected from rat cardiac myocytes, as the same as thecommercial antibody. These results suggest that SSA78and WJS havecharacter of α₁and α₂antibodies of NKA.(3) SDS-PAGE analysis showed thatmost purified antibodies were separated into2bands, the molecularmasses ofwhich are about110kD.(4) All results showed that polyclonal antibodies aresuccessfully detected on the membrane surface of cardiac myocytes byimmunofluorescent staining methods, and revealed that the interactions of theantibodies with their antigenic sites on the surface of rat myocytes wereabolished not by1mM or1μM OUA, but by peptide blockers PB78and RE2.Conclusion: The above results suggest that SSA78and WJS specifically bind with the Hl-H₂region of α₁and α₂isoforms of NKA on the surface of ratmyocytes, and interact with their respective antigenic sites on the H₁-H₂domain of NKA α₁or α₂isoforms.Part2Effects of SSA78and WJS on Na/K pump current, the riseof [Ca²⁺]iand contraction of the cardiomyocytes induced by OUAAim: To further study the antibody on activity of NKA, the rise of [Ca²⁺]iand contraction of the cardiomyocytes induced by ouabain (OUA).Methods: On enzymatic isolation of rats ventricular myocytes,whole-cell patch-clamp technique was performed to record the Na+/K+pumpcurrent (Ip), in order to observe the antibodies SSA78and WJS onOUA-induced NKA activity. The [Ca²⁺]iwas detected with confocal laserscanning system and the contraction of a single myocyte was assessed by avideo-based motion edge-detection systemResults:1When perfusion of10^-6mol·L^-1or10^-3mol·L^-1OUA shows arepresentative inward shift of the holding current.10^-6mol·L^-1OUA was usedto assay high OUA affinity pump current (Iph) of the α₂isoform and10^-3mol·L^-1OUA was applied to assay low OUA affinity pump current (Ipl).2Preincubation of myocytes with SSA78inhibited Ipl elicited by10^-3mol·L^-1OUA, preincubation of myocytes with WJS inhibited Iph elicited by10^-6mol·L^-1OUA.310^-6mol·L^-1and10^-3mol·L^-1OUA elevated [Ca²⁺]iin thecardiomyocytes in a concentration and time-dependent manner. Thepretreatment of SSA78significantly attenuated the elevation of [Ca²⁺]iinduced by10^-3mol·L^-1not10^-6mol·L^-1OUA, which were151.6±4.9%at5min and166.7±10.9%at15min, they were lower than those in thecardiomyocytes without the pretreatments of SSA78or WJS (249.6±18.3%and281.1±11.4%, p <0.05). Pretreatment of WJS significantly decreased therise of [Ca²⁺]1iinduced by10^-6mol·L^-1OUA not10^-3mol·L-OUA, which were152.6±6.7%at5min and156.0±7.8%at15min. They were lower thanthose in the cardiomyocytes without the pretreatments of SSA78or WJS (189.2±14.0%and184.6±8.9%, p <0.05).4Our result showed that SSA78and WJS themselves did not havesignificant positive inotropic effects.5The results measured indicate that the extents of cell shorteninginduced by10^-6mol·L^-1OUA were increased by142.9±8.3%at5min and141.4±6.6%at15min, respectively; the extents of cell shortening induced by10^-3mol·L^-1OUA was obviously increased by284.6±12.4%at5min,whereas at15min after continual perfusion of10^-3mol·L^-1OUA, the extentsof cell shortening not only did not increase but also decreased by101.8±3.3%, suggesting that the contractile function of the myocytes has beenimpaired by high concentration OUA. However, in the presence of1:1000SSA78, the extent of cell shortening induced by10^-3mol·L^-1OUA at15minwas increased by269.7±24.5%, which was similar to278.8±11.0%at5minafter perfusion of OUA, but was significantly higher than101.8±3.3%at15min after continual perfusion of10^-3mol·L^-1OUA without SSA78preincubation (p <0.01). While in the presence of1:1000WJS, the extent ofcell shortening induced by10^-6mol·L^-1OUA was104.0±2.8%at5min and117.3±6.9%at15min after OUA perfusion (p <0.05), but no effect on theextent of cell shortening after10^-3mol·L^-1OUA perfusion (p>0.05).6Treatment with10^-3mol·L^-1OUA caused arrhythmic contractionswithin8.3±0.8min. In the presence of1:1000and1:100SSA78, the meanonset times of10^-3mol·L^-1OUA-induced arrhythmic contraction weresignificantly prolonged to11.8±0.8min and11.9±1.0min, respectively (p <0.05). When OUA concentration was reduced to0.1mol·L^-1, the mean onsettime of arrhythmic contraction was significantly prolonged to10.5±1.1min(p <0.05vs.1mol·L^-1OUA). Whereas in the presence of1:1000SSA78,arrhythmic contraction was not observed until30min after perfusion of0.1mol·L^-1OUA.Conclusion: Our results provided the direct experimental evidence thatthe H₁-H₂domain of the NKA α₁isoform can participate in OUA-induced[Ca²⁺]ioverload and arrhythmic contraction, the H₁-H₂domain of NKA α₂can participate in OUA-induced positive inotropic effects, and antibody SSA78may have a protective effect in cardiomyocytes contraction. These resultsindicate that the therapeutic effect of OUA is mediated by α₂isoform of NKA,while the toxicity effect of OUA is mediated by α₁isoform of NKA.Part3Effects of SSA78and WJS on OUA induced mitochondrialdysfunctionAim: To study the effect of antibody on OUA-induced damage tomyocardial mitochondria in rat.Methods: The mitochondrial Ca²⁺concentration ([Ca²⁺]m) andmitochondrial membrane potential (ΔΨm) was measured by laser scanningcofocal microscope. Rhodamine123as the probe of fluorescencespectrophotometer measured the fluorescence intensity to reflect the change inthe Δ Ψm; Rhod-AM as the probe of fluorescence spectrophotometermeasured the fluorescence intensity to reflect the change in the [Ca²⁺]m.Results:1.10^-3mol·L^-1OUA can effectively induce ΔΨmreduction andmitochondrial Ca²⁺overload, which was45.0±4.4%of baseline (p <0.01)and302.4±65.3%of baseline (p <0.01).2. Preincubation of cells with1:1000SSA78significantly restored theΔΨmreduced by10^-3mol·L^-1OUA in a concentration-dependent manner andattenuated the decrease of Rh^-123fluorescence density induced by10^-3mol·L^-1OUA, which could partly be restored to62.0±5.0%by SSA78(p <0.05).3. Preincubation of cells with1:1000SSA78dramatically decreased theincrease of [Ca²⁺]minduced by high concentration of OUA, which declined to118.8±20.1%of baseline (p <0.05).4. Preincubation of cells with WJS did not significantly change themitochondrial dysfunction.Conclusion: High concentration of OUA can effectively induceΔΨmreduction and mitochondrial Ca²⁺overload. NKA α₁antibody SSA78maydecrease the damage to mitochondria induced by OUA,and has protective effect on OUA-induced cardiotoxicity. NKA α₂antibody WJS had noapparent effect on mitochondrial dysfunction, and then suggested that thecardiac toxicity of OUA mediated by α₁isoform of NKA.SUMMARY1. To use sodium pump α₁and α₂isoforms H₁-H₂amino acid sequenceas an antigen to immunize New Zealand rabbits and prepare polyclonalantibody through the preparation of immune animal serum polyclonalantibody technique, the indirect ELISA, Western blot andimmunohistochemistry method was used to characterize polyclonal antibody.2. The cardiac mechanical dysfunction induced by toxic doses of OUAmay be associated with [Ca²⁺]moverload and decrease ofΔΨmresulting fromCa²⁺-overload toxicity, reduced NKA activity and caused arrhythmiccontraction. These data indicated that SSA78, could effectively resistancethese toxicity, which suggest that the low OUA affinity α₁isoform of NKAmediates the cardiac toxicity of OUA in rat ventricular myocytes.3. Antibody against α₂isoform of NKA (WJS) did not affect arrhythmiccontraction and mitochondrial dysfunction induced by OUA. Preincubation ofmyocytes with WJS abolished Iph and attenuated the elevation of positiveinotropic effects induced by low concentration of OUA. These results indicatethat the therapeutic effect of OUA is mediated by high OUA affnity pumbcurrent.