The α2 Isoform of Na,K-ATPase Mediates Ouabain-induced Cardiac Inotropy in Mice

Abstract

Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific α isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the α2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive α2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the α2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice. Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be the major mechanism by which this class of drugs increases heart contractility. However, direct evidence demonstrating this is lacking. Furthermore it is unknown which specific α isoform of Na,K-ATPase is responsible for the effect of cardiac glycosides. Several studies also suggest that cardiac glycosides, such as ouabain, function by mechanisms other than inhibition of the Na,K-ATPase. To determine whether Na,K-ATPase, specifically the α2 Na,K-ATPase isozyme, mediates ouabain-induced cardiac inotropy, we developed animals expressing a ouabain-insensitive α2 isoform of the Na,K-ATPase using Cre-Lox technology and analyzed cardiac contractility after administration of ouabain. The homozygous knock-in animals were born in normal Mendelian ratio and developed normally to adulthood. Analysis of their cardiovascular function demonstrated normal heart function. Cardiac contractility analysis in isolated hearts and in intact animals demonstrated that ouabain-induced cardiac inotropy occurred in hearts from wild type but not from the targeted animals. These results clearly demonstrate that the Na,K-ATPase and specifically the α2 Na,K-ATPase isozyme mediates ouabain-induced cardiac contractility in mice. Na,K-ATPase is a transmembrane protein that transports three Na+ out of the cell and two K+ in, utilizing ATP hydrolysis as the driving force. The electrochemical gradient, established by the Na,K-ATPase, is essential for maintenance of cell-specific functions, such as muscle contraction and nerve transmission. The ion gradient also drives many plasma membrane transport processes, including Na+-coupled Ca2+ transport by the Na/Ca-exchanger and uptake of nutrients, such as glucose, in kidney and gut via Na+ cotransporters. Na,K-ATPase is composed of two subunits: the catalytic α subunit, which binds translocating cations and ATP, and the β subunit, which modulates cation affinity and is necessary for proper folding and translocation of the Na,K-ATPase to the plasma membrane (1Lingrel J.B Kuntzweiler T.A. J. Biol. Chem. 1994; 269: 19659-19662Abstract Full Text PDF PubMed Google Scholar, 2Eakle K.A. Kabalin M.A. Wang S.G. Farley R.A. J. Biol. Chem. 1994; 269: 6550-6557Abstract Full Text PDF PubMed Google Scholar, 3Eakle K.A. Lyu R.M. Farley R.A. J. Biol. Chem. 1995; 270: 13937-13947Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4McDonough A.A. Geering K Farley R.A. FASEB J. 1990; 4: 1598-1605Crossref PubMed Scopus (279) Google Scholar). There are four isoforms of the α subunit (α1, α2, α3, and α4), each exhibiting unique tissue distribution and developmental expression suggestive of differential and tissue-specific functional roles (5Shull G.E. Greeb J. Lingrel J.B Biochemistry. 1998; 25: 8125-8132Crossref Scopus (549) Google Scholar, 6Sweadner K.J. Biochim. Biophys. Acta. 1989; 988: 185-220Crossref PubMed Scopus (868) Google Scholar, 7Lingrel J.B Orlowski J. Shull M.M. Price E.M. Prog. Nucleic Acids Mol. Biol. 1990; 38: 37-89Crossref PubMed Scopus (339) Google Scholar, 8Shamraj O.I. Lingrel J.B Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12952-12956Crossref PubMed Scopus (221) Google Scholar, 9James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 10Woo A.L. James P.F. Lingrel J.B J. Biol. Chem. 2000; 275: 20693-20699Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 11He S. Shelly D.A. Moseley A.E. James P.F. James J.H. Paul R.J. Lingrel J.B Am. J. Physiol. 2001; 281: R917-R925Crossref PubMed Google Scholar, 12Moseley A.E. Lieske S.P. Wetzel R.K. James P.F. He S. Shelly D.A. Paul R.J. Boivin G.P. Witte D.P. Ramirez J.M. Sweadner K.J. Lingrel J.B J. Biol. Chem. 2003; 278: 5317-5324Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Cardiac glycoside sensitivity is conferred by the α subunit of the Na,K-ATPase, and this class of drugs is used to treat congestive heart failure. Inhibition of Na,K-ATPase activity by cardiac glycosides is believed to be a major mechanism by which these drugs exert their positive cardiac inotropic effect (13Akera T. Brody T.M. Pharmacol. Rev. 1977; 29: 187-220PubMed Google Scholar, 14Brody T.M. Akera T. Fed. Proc. 1977; 36: 2219-2224PubMed Google Scholar, 15Lee C.O. Am. J. Physiol. 1985; 249: C367-C378Crossref PubMed Google Scholar, 16Levi A.J. Boyett M.R. Lee C.O. Prog. Biophys. Mol. Biol. 1994; 62: 1-54Crossref PubMed Scopus (77) Google Scholar). Inhibition of the Na,K-ATPase raises intracellular Na+, which in turns increases intracellular Ca2+ concentrations. The increase in intracellular Ca2+ produces a positive inotropic response of the heart. Two isoforms, α1 and α2, are expressed in rodent heart, and three isoforms, α1, α2, and α3, are expressed in human heart. However, it is unknown which specific α isoform mediates this cardiac glycoside positive inotropic effect in either species. It has been suggested that inhibition of the high affinity α2 isoform followed by inhibition of the low affinity α1 isoform by the cardiac glycoside digoxin results in increased contractility of rat hearts (17Kang W. Weiss M. J. Pharmacol. Exp. Ther. 2002; 302: 577-583Crossref PubMed Scopus (26) Google Scholar). It has also been suggested that differences in cardiac glycoside sensitivity of Na,K-ATPase isozymes determine their therapeutic and toxic effects such that the high affinity Na,K-ATPase mediates the positive inotropic effects, whereas the low affinity Na,K-ATPase is responsible for toxic effects of cardiac glycosides (18Maixent J.M. Charlemagne D. de la Chapelle B. Lelievre L.G. J. Biol. Chem. 1987; 262: 6842-6848Abstract Full Text PDF PubMed Google Scholar). Analysis of mice expressing genetically reduced levels of α1 and α2 isoforms demonstrated differential cardiac function of these isoforms, indicating that they may differentially regulate the cardiac glycoside-induced positive inotropy (9James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). The approximate 50% reduction in α2 isoform expression resulted in an increase in contractility of the heart, whereas decreased cardiac contractility was observed in mice lacking one copy of the α1 isoform gene. As reduction of the α2 isoform mimicked the cardiac glycoside-induced positive inotropy, it was suggested that the α2 isoform might play a role in this effect, whereas inhibition of the α1 isoform may be responsible for toxic effects of cardiac glycosides. Recently it has been suggested that both α1 and α2 isoforms regulate cardiac contractility and cardiac glycoside-induced positive inotropy (19Schwartz A. Petrashevskaya N.N. Cardiovasc. Res. 2001; 51: 9-12Crossref PubMed Scopus (14) Google Scholar). However, evidence also exists implicating only the α1 isoform in regulation of cardiac contractility (20Kometiani P. Askari A. Liu J. Xie Z. Askari F.K. Am. J. Physiol. 2001; 280: H1415-H1421Google Scholar). Therefore, further studies are necessary to determine the functional role of different isoforms in the regulation of cardiac contractility and the cardiac glycoside effects. Several studies suggest that cardiac glycosides may function by mechanisms other than inhibition of Na,K-ATPase. Low concentrations of digitoxin have been shown to activate the sarcoplasmic reticulum ryanodine receptor (RyR), 1The abbreviations used are: RyRryanodine receptorESembryonic stemMAPmean arterial pressureECFextracellular fluidEOendogenous ouabain. suggesting its involvement in positive cardiac inotropy by digitoxin (21McGarry S.J. Williams A.J. Br. J. Pharmacol. 1993; 108: 1045-1050Crossref Scopus (95) Google Scholar, 22Sagawa T. Sagawa K. Kelly J.E. Tsushima R.G. Wasserstrom J.A. Am. J. Physiol. 2002; 282: H1118-H1126Crossref PubMed Scopus (62) Google Scholar). In addition to the digitoxin effect on RyR, other studies have suggested that cardiac glycosides are transported across the plasma membrane into the cell and that intracellular action is responsible for the increase in heart contractility (23Nunez-Duran H. Riboni L. Ubaldo K. Kabela E. Barcenas-Ruiz L. Am. J. Physiol. 1988; 255: C479-C485Crossref PubMed Google Scholar, 24Isberg G. Cardiac Glycoside Receptors and Positive Inotropy. Steinkopf, Darmstadt, Germany1984: 56-71Crossref Scopus (21) Google Scholar). Furthermore non-Na,K-ATPase receptors for ouabain have been suggested, dissociating its involvement in positive cardiac inotropy (25Fujino M. Fujino S. Satoh K. Nakai T. Kado T. Adv. Exp. Med. Biol. 1992; 311: 449-451Crossref PubMed Google Scholar). The possibility that cardiac glycoside receptors other than Na,K-ATPase exist raises the question whether inhibition of Na,K-ATPase is the only mechanism for cardiac glycoside-induced cardiac positive inotropy. ryanodine receptor embryonic stem mean arterial pressure extracellular fluid endogenous ouabain. To determine whether the Na,K-ATPase, and specifically the α2 isoform of Na,K-ATPase, mediates the cardiac glycoside-induced positive inotropy we developed animals expressing the α2 isoform with reduced cardiac glycoside affinity (α2R/R) and analyzed cardiac contractility after administration of ouabain. If an increase in cardiac contractility is not observed in targeted animals in the presence of cardiac glycosides, this that the Na,K-ATPase and specifically the α2 Na,K-ATPase isozyme is ouabain-induced cardiac positive inotropy. However, ouabain an increase in heart contractility in the other receptor be this analyzed the cardiac response to ouabain administration using both isolated heart and intact animals. results clearly demonstrate that the increase in heart contractility by low concentrations of cardiac glycosides is the α2 Na,K-ATPase and not other receptors as ouabain-induced cardiac positive inotropy was not observed in the animals. of the Cardiac α2 Na,K-ATPase and were into the α2 isoform by two in using as shown in The was used as for and was from an of the α2 isoform gene. Furthermore two were to a The the was into the The was into a of The was of the The presence of the in the was by both analysis and The cell was with the by and was by The was from whereas the was from of the α2 isoform gene. The presence of the and in was analyzed in targeted by The was that the of a in the presence of the as analysis of the demonstrated the of this with the The was by using and and the were with targeted were to the with the the were and one was in with was from the with and analyzed by using the and The presence of the and the presence of the in was analyzed in targeted by as mice were via of the cell and were to mice for two to a knock-in mice were by was by analysis across the in of the The presence of the in homozygous mice was by analysis of using and tissue were used for each tissue were and on with two of a in on the of were further and was by protein were used for and were as Lingrel J.B J. Biol. Chem. Full Text PDF PubMed Google for each were from animals and on with two in of and were for The was and for using an the was in and was by protein analysis was via (9James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). In protein were for in and and by in The were to and in in for The were in α1 of α2 K. and which the of α isoforms with was with the by using the protein for each analysis was by the with In were in for followed by their in for was used as was as Analysis of by Na,K-ATPase in from and was as (9James P.F. Grupp I.L. Grupp G. Woo A.L. Askew G.R. Croyle M.L. Walsh R.A. Lingrel J.B Mol. Cell. 1999; 3: 555-563Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). of membrane were for in and either ouabain. the membrane were and with of using a cell The was by for each was by using an of ouabain and was from using ouabain. of from in kidney from both and in heart and muscle from targeted animals resulted in Analysis of in and mice were with an of of and of in was into the from the and to a low pressure for of mean arterial high was into the and into the to cardiac The was for of ouabain. arterial pressure and pressure from the and the were analyzed using a to a for each for heart and pressure were from the pressure and were for each from the of each and for each and ouabain were from the of the pressure were also using a heart as I.L. A. J. Grupp G. Am. J. Physiol. 1993; Google Scholar). In wild type and targeted mice were and the hearts were and in a The was and the heart was via its on a for of the with occurred from the heart to a pressure of which was via an was the of the the into the The was the of the the of the the The and The was and the was to The pressure and its and were via a pressure and and were also on a and cardiac were with mean pressure and was with a was via a were with Ca2+ Ca2+ and functional were the different Ca2+ concentrations. The effects of the cardiac glycoside ouabain on the functional of the hearts were concentrations to of ouabain, to a were and into the via into the the heart. The hearts were to each effect was a of The of ouabain to the heart was on and cardiac analysis of for and with was and of was of a Cardiac α2 Na,K-ATPase ouabain sensitivity of the α2 isoform was reduced by and in the extracellular of this isoform The and are in the low affinity rodent α1 isoforms and were shown to cardiac glycoside of the rodent α1 has demonstrated that these in the α1 isoform reduced its ouabain affinity by its activity E.M. Lingrel J.B Biochemistry. 1988; PubMed Scopus Google Scholar, E.M. D.A. Lingrel J.B J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). In a was as a for of the targeted The ouabain-insensitive α2 isoform mice were by the as in The of two cell The the into one of the wild type by results in cell a targeted the and the and The which the from the targeted by of two the in and one in the targeted the cell two targeted cell and were by analysis the of their The which was in cell to the wild type The to the targeted and results from the the of the targeted The was in cell and and it was that and were also not of resulted in of the in one cell As of the resulted in of the both targeted and wild type are as by analysis of the The of the is in than in the indicating that it is composed of two one to the wild type and the other to the targeted cell a and the and in not animals were via of cell and were to to animals. was used to wild type and homozygous (α2R/R) that were born in normal Mendelian The of the animals was by the of their which the wild type as a and the targeted as a The animals the as shown by the of their The the α2 of the Na,K-ATPase with and and of the and in the α2 isoform reduced its ouabain affinity in the mice as demonstrated by As was in kidney from each to expression of only the low affinity α1 in wild type heart and muscle was to the high affinity α2 was in from the of the mice. demonstrated a reduction in the ouabain affinity of the α2 isoform the and The in tissue from the animals was not to expression of high affinity isoform of the Na,K-ATPase. However, it was reduced by which with the of the α2 isoform expressed in James and J. B. also analyzed the protein expression of the α2 isoform in from mice to determine whether the its expression and tissue analysis of tissue from the and mice normal protein expression and tissue distribution of the α2 isoform the and The α2 isoform of Na,K-ATPase was expressed in heart, and muscle in both wild type and targeted animals. It is to out that an of the α2 isoform was observed in heart and muscle from both and resulted from protein in these The expression and tissue distribution of the α1 and isoforms in mice were analyzed to determine whether mechanisms in expression of these two α isoforms resulted from of the α2 isoform gene. analysis normal protein expression and tissue distribution of both the α1 and the isoforms in mice The α1 isoform was expressed in heart, and and protein expression levels in these were the two The isoform its expression in targeted and its protein expression was from that in wild type Furthermore Na,K-ATPase protein as by that the α isoforms, normal in of animals with wild type There was in expression in each indicating protein not reduced ouabain affinity of the α2 isoform but not its normal protein expression tissue distribution in the mice. In normal expression and tissue distribution of both α1 and isoforms were not in the mice. The presence of one of the α2 isoform not its normal the α2 isoform the and has ouabain affinity to the α1 Na,K-ATPase activity be two Therefore, the activity of the α2 isoform the not be in from mice. However, Na,K-ATPase activity was the two in determine whether the and in the α2 Na,K-ATPase isozyme the and cardiovascular of the we analyzed the heart of contraction of contraction and of contraction and after administration of a β There were differences in heart pressure and pressure of the and the mice and after β These demonstrate that expression of the ouabain-insensitive α2 isoform not normal in mice. Analysis of cardiac function in intact animals demonstrated that was also in not and in the and the mice and after β that animals have normal heart function. Low of of the effect of ouabain on contractility in isolated hearts was analyzed to determine whether reduced ouabain affinity of the α2 isoform ouabain-induced cardiac positive inotropy. hearts from wild type and mice were with concentrations of ouabain, and the of heart contraction and were and The increase in cardiac function of wild type as demonstrated by the increase in of contraction and was observed ouabain and increased with concentrations of ouabain. positive cardiac inotropic effect of ouabain was to that shown Grupp I.L. A. Grupp G. Prog. Biol. Res. 1988; Google Scholar, I.L. Lee C.O. Lee A. J. Physiol. 1985; PubMed Scopus Google Scholar). observed only a response of cardiac contractility to concentrations of ouabain as a is concentrations of ouabain than In to wild cardiac contractility in hearts not increase with concentrations of ouabain of ouabain that increased cardiac function in wild type hearts not of contraction of of also analyzed the in and of wild type and hearts from of the ouabain and increased heart contractility in wild type and decreased cardiac contractility in the different effects of ouabain on hearts from the two clearly demonstrate that the increase in heart contractility by low of ouabain is the α2 Na,K-ATPase Low of in the but in the contractility as a function of ouabain was also analyzed in intact animals. of the and of contraction and and of contraction and of ouabain were As an of the concentrations of ouabain in heart we the of ouabain in extracellular fluid The ouabain concentrations in are with to concentrations in of intact animals. extracellular fluid to of ouabain of and of to and ouabain, that and of ouabain were the which was were increased not and in wild type mice. The increase was ouabain of and ouabain of increase in not and was observed in the targeted mice. also analyzed the in and in each from two analysis of each not into the effect of ouabain as be differences in animals analysis of the in contractility ouabain for each analysis of ouabain effects on cardiac contractility in intact animals. in and in and mice from ouabain of a increase in contractility in wild which was different from the observed in targeted animals. These demonstrate that low of ouabain increase the cardiac contractility by the α2 Na,K-ATPase isozyme in hearts of intact animals. It is to that inhibition of the low sensitivity α2 isoform in mice was responsible for this but not increase in contractility ouabain of As the for the low affinity α isoforms is ouabain, inhibition of the low affinity α2 isoform in mice occurred ouabain of and resulted in the increase in cardiac contractility. further the of the α2 isoform in ouabain-induced cardiac positive inotropy. In addition to the α2 inhibition of the α1 isoform to a increase in cardiac contractility in mice. inhibition of the high affinity isoform in have also resulted in increased nerve activity and increased cardiac contractility. It is to also out that of of of ouabain, which to an ouabain of ouabain, to cardiac in wild type animals but not in mice not this of ouabain the α2 isoform be and only inhibition of the α1 isoform have the of the α2 isoform in regulation of normal cardiac contractility. The mechanism of ouabain-induced cardiac positive inotropy has been and studies suggest that the Na,K-ATPase mediates ouabain-induced cardiac positive inotropy, direct evidence is lacking. In it is unknown whether this effect in is the α1 α2 isoforms, which are both expressed in In the presence of the high affinity isoform in of the heart it for this isoform to be in the ouabain As mechanisms of cardiac glycoside action non-Na,K-ATPase receptors have also been suggested (21McGarry S.J. Williams A.J. Br. J. Pharmacol. 1993; 108: 1045-1050Crossref Scopus (95) Google Scholar, 22Sagawa T. Sagawa K. Kelly J.E. Tsushima R.G. Wasserstrom J.A. Am. J. Physiol. 2002; 282: H1118-H1126Crossref PubMed Scopus (62) Google Scholar, H. Riboni L. Ubaldo K. Kabela E. Barcenas-Ruiz L. Am. J. Physiol. 1988; 255: C479-C485Crossref PubMed Google Scholar, 24Isberg G. Cardiac Glycoside Receptors and Positive Inotropy. Steinkopf, Darmstadt, Germany1984: 56-71Crossref Scopus (21) Google Scholar, M. Fujino S. Satoh K. Nakai T. Kado T. Adv. Exp. Med. Biol. 1992; 311: 449-451Crossref PubMed Google it is to the functional role of Na,K-ATPase in the cardiac glycoside-induced positive inotropy. we have analyzed the role of Na,K-ATPase in the ouabain-induced cardiac inotropy and which specific α isoform mediates this mice expressing the α2 Na,K-ATPase isoform with reduced cardiac glycoside sensitivity and analyzed the contractility low of ouabain in both isolated hearts and intact animals. clearly demonstrate that the ouabain-induced positive cardiac inotropic effect is the α2 Na,K-ATPase. direct evidence that this effect is inhibition of the Na,K-ATPase and specifically the α2 Na,K-ATPase inhibition of the high affinity isoform by ouabain may have occurred in of the it a role in the ouabain-induced cardiac positive inotropy in intact animals. not whether the inhibition of the isoform by ouabain in a role in heart contractility as ouabain not across the In analysis in intact animals that inhibition of the α1 isoform of Na,K-ATPase have effect in ouabain-induced cardiac positive inotropy. The of ouabain increased cardiac contractility in wild type animals and effect in mice. If the α1 isoform a major role in ouabain-induced positive inotropy, a increase in cardiac contractility have been observed in mice. is in to was suggested (19Schwartz A. Petrashevskaya N.N. Cardiovasc. Res. 2001; 51: 9-12Crossref PubMed Scopus (14) Google Scholar, P. Askari A. Liu J. Xie Z. Askari F.K. Am. J. Physiol. 2001; 280: H1415-H1421Google Scholar). receptors have been in the positive inotropic effect of ouabain. Low concentrations of digitoxin have been shown to activate in sarcoplasmic reticulum T. Sagawa K. Kelly J.E. Tsushima R.G. Wasserstrom J.A. Am. J. Physiol. 2002; 282: H1118-H1126Crossref PubMed Scopus (62) Google Scholar, H. Riboni L. Ubaldo K. Kabela E. Barcenas-Ruiz L. Am. J. Physiol. 1988; 255: C479-C485Crossref PubMed Google Scholar). Furthermore it has been suggested that ouabain low concentrations is transported into the cell in intracellular of G. Cardiac Glycoside Receptors and Positive Inotropy. Steinkopf, Darmstadt, Germany1984: 56-71Crossref Scopus (21) Google Scholar, M. Fujino S. Satoh K. Nakai T. Kado T. Adv. Exp. Med. Biol. 1992; 311: 449-451Crossref PubMed Google Scholar). out the possibility that intracellular receptors studies clearly that α2 Na,K-ATPase isozyme is the cardiac glycoside receptor which the increase in heart contractility by low of ouabain is of low of cardiac glycosides is as a of the α2 Na,K-ATPase isozyme Inhibition of α2 Na,K-ATPase the Na/Ca-exchanger of the Na+ gradient a of its and of in intracellular Ca2+ in of and the Ca2+ from sarcoplasmic Therefore, of is a effect of the α2 isoform the of Ca2+ to the necessary in cardiac muscle It is to that animals a for the role of endogenous ouabain endogenous have been and isolated from human plasma J.M. J.H. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). These endogenous cardiac glycosides have been in a W. J. 2002; 269: PubMed Scopus Google Scholar). However, their role is As we have demonstrated that the ouabain-induced increase in heart contractility is the α2 Na,K-ATPase, it is to suggest that endogenous ouabain cardiac contractility it be the α2 Na,K-ATPase. As the animals have normal normal and normal cardiac function with their wild type it is that regulation of α2 Na,K-ATPase is not necessary for and normal function is not as levels of are low in animals. However, an increase in has been in such as congestive heart and W. J. 2002; 269: PubMed Scopus Google Scholar). Therefore, it is of to the animals to have high levels of As may also the it be to animals in which this isoform is also to cardiac glycosides. M. L. and for for also for and E. Shull for with