Intracellular calcium levels are unchanged in the diabetic heart

Transcription

1 Ž. Cardovascular Research Reve Intracellular calcum levels are unchanged n the dabetc heart Jen Z. Yu a, Bran Rodrgues b, John H. McNell b,) a CardoÕascular Research Laboratory, Department of Pathology and Laboratory Medcne, Faculty of Medcne, The UnÕersty of Brtsh Columba, VancouÕer, BC V6T 1Z3, Canada b DÕson of Pharmacology and Toxcology, Faculty of Pharmaceutcal Scences, The UnÕersty of Brtsh Columba, VancouÕer, BC V6T 1Z3, Canada Receved 24 October 1996; accepted 9 January 1997 Keyords: Dabetes; Cardomyopathy; Calcum, ntracellular concentraton; SR, calcum release 1. Introducton Dabetes melltus has been assocated th cardac dsease that has been suggested to occur secondary to atheroscleross of the coronary arteres, macroangopathy and autonomc neuropathy x 1. Hoever, a cardac dsease partcular to dabetes has also been demonstrated to occur n the absence of the above factors. Ths dabetc cardomyopathy that ncludes cardomegaly, left ventrcular dysfuncton and clncally overt congestve heart falure has been strongly suggested by epdemologcal, clncal and expermental studes 2 7 x. The etology of dabetc cardomyopathy s complex and a number of factors have been suggested to be nvolved n the development of ths dsease state. These nclude: Ž. a changes n cardac metabolsm, Ž. b abnormal vascular senstvty and reactvty to varous lgands, Ž. c ncreased stffness of the ventrcular all assocated th pervascular thckenng of basement membrane and ntersttal accumulaton of glycoproten and nsoluble collagen, and Ž. d abnormaltes of varous protens hch control on movements, specfcally ntracellular calcum x 6. Streptozotocn Ž STZ. nducton of dabetes n rats s the most commonly used anmal model of dabetes. The chemcal structure of STZ comprses a glucose molecule th a hghly reactve ntrosourea sde-chan that s thought to ntate ts cytotoxc acton. The glucose moety drects ths agent to the pancreatc b-cell x 8. STZ selectvely destroys pancreatc b-cells and produces a dabetc state, the sever- ty of hch can be vared by alterng the dose of STZ. Wth tme Ž 4 6 eeks., rats so treated develop bochemcal and functonal myocardal abnormaltes hch are the result of the drug-nduced metabolc changes rather than a drect effect of the drug tself x 6. Thus, rats made dabetc th STZ exhbt depressed left ventrcular contractlty, dmnshed ventrcular complance and decreased notropc and chronotropc responses to certan drugs n solated cardac preparatons 4,9,10 x. Changes n contractle propertes of the heart have also been found n vvo n both anaesthetsed 11x and conscous 12,13x STZ-dabetc rats. Consderng that the dabetc rat does not develop atheroscleross, the n vvo and n vtro abnormaltes n contractlty and metabolsm reflect changes n the functon of the myocardal cell 14 x. Hence, smlar to the human condton, the STZ dabetc rat s a useful model to elucdate the pathogeness of dabetc cardomyopathy. In ths reve, Ž evdence s presented that basal calcum Ca x. levels n both quescent and electrcally-stmulated cardac cells from STZ dabetc rats s unchanged and may not reflect the cardac abnormaltes observed n STZ dabetes. [ 2H ] 2. Ca moblzaton n ventrcular myocytes It s ell knon that the ntegrty of the crculatory system can be nfluenced by changes n the flux of ntra- Ž x. cellular calcum Ca n heart. Ca movements are closely related to cardac electro-physologcal events, membrane ntegrty, energy metabolsm and contractle Donloaded from at Pennsylvana State Unversty on February 23, 2013 ) Correspondng author. Tel. q ; Fax q Tme for prmary reve 33 days r97r$17.00 Copyrght q 1997 Elsever Scence B.V. All rghts reserved. Ž. PII S

2 92 ( ) J.Z. Yu et al.rcardoõascular Research functon. Mantanng the ntracellular free Ca concentraton requres a fne balance n the co-ordnated functon of many organelles. Intracellular organelles hch ncorporate Ca from and return Ca to the cytosol play a central role n determnng Ca x n the cell 15 x. In a mammalan ventrcular cell, Ca moves across the sarcolemma Ž SL., sarcoplasmc retculum Ž SR., and mtochondral membranes th the help of L-type Ca channels, Na Ca exchangers, Ca -ATPase, and Ca bndng stes thn the SL, Ca release channels and Ca - ATPase thn the SR and Na Ca exchangers and Ca - bndng protens thn the mtochondral membranes. It s assumed that the cytosolc concentraton Ca x and spa- tal dstrbuton of the Ca x transents are unform thn x the cell 16. Durng cardac exctaton, extracellular Ca moves nto the cells through actvated Ca channels and x reverse Na Ca exchange 17,18. Ths Ca current Ž I Ca. may n turn control SR Ca release va the SR Ca release channelrryanodne receptor 19x and contracton ensues. For relaxaton to occur, Ca must be removed from the cytoplasm, ether by transport nto the SR Žby SR. Ž Ca -ATPase or out of the cell by sarcolemmal Ca - ATPase or Na Ca exchange.. These transport systems are thus n competton for cytoplasmc Ca and act n concert, both to regulate the ntracellular Ca concentra- ton and to modulate Ca -dependent events 15,20 x. 3. Intracellular Ca 2H n the dabetc heart Evdence for abnormal myocardal cell functon n dabetes melltus, nfluenced by metabolc changes, has appeared n recent years. Expermental studes at the cellular level have provded data for several possble explanatons for ths cardac dysfuncton. Among these, an abnormal ntracellular Ca homeostass and trans-sarcolemmal re5,21 x. For ceptor sgnallng defects have been suggested example, durng chronc dabetes ntracellular Ca homeostass n the heart s altered, possbly as a result of an mparment n SR hch acts as a Ca store and takes up and releases Ca on a beat-to-beat bass 22 x. Addtonally, SL Ca pump and Na Ca exchange actvty, together th the efflux of Ca through the SL are de x pressed n the dabetc heart 23,24. Mtochondral Ca abnormaltes have also been reported n the chroncally dabetc rat heart x 5. Among the receptor defects, cardac b-adrenoceptor functon s altered n dabetc anmals. These alteratons nclude a depressed b-receptor number, a reduced response to b-adrenergc agonsts, and a sgnfcant alteraton n the response and senstvty to perfusate Ca stmulaton 10,25 27 x. These results collectvely support the hypothess that dabetes melltus leads to an alteraton n Ca movements n the heart. In more recent years, ntracellular Ca moblzaton and contractlty n the dabetc heart have been studed usng solated cardomyocytes that have the responses of an ntact heart but are not affected by non-myocardal tssues 28 x. As sngle cells, they are sutable for measurng contractlty, on actvtes and Ca moblzaton, and an ncreasng number of reports have documented ntracellular Ca transents and on channel and transporter actvtes n parallel th sngle cell contracton n dabetc heart cells x. Utlzaton of fluorescent dyes n solated myocytes allos for the analyss of Ca x, and the study of the mechansm by hch stmulaton causes x Ca changes. Moreover, the restoraton of Ca x fol- long stmulaton-nduced elevaton can be observed, especally th rapdly desenstzng agonsts that produce a bref ncrease n Ca n the cytosol x. Usng these methods, t has been repeatedly reported that cardomy29,31,40 x. Cell shortenng and veloctes of contracton Ž qdlrdt. and relaxaton Ž ydlrdt. ere sgnfcantly loer n da- ocyte contracton s mpared n dabetc hearts betc myocytes than controls, hereas tme to peak short34 x. These observa- enng as prolonged n dabetc cells tons agree th the results from ntact hearts and tssue preparatons of dabetc rats hch sho decreased peak ventrcular pressure development and rates of ventrcular pressure development and declne Ž "dprdt.. The data on basal levels of Ca x n dabetc myocytes are more nconsstent. Early studes have reported an n crease n restng Ca x, an effect that appeared to support the hypothess of Ca overload n dabetc rat hearts 30,41 x. Hoever, the anmals used n the above studes ere clamed to be a model of non-nsuln-dependent dabetes, and dfferent from STZ-dabetc rats, often referred to as a model of poorly controlled nsuln-dependent dabetes. Subsequently, Noda et al. 31,42x reported a reduced basal Ca x n the dabetc heart and suggested a relatonshp beteen depressed basal Ca x and reduced cardac contractlty. Hoever, ther measurements ere made at an extracellular Ca concentraton of 0.5 mm that s not physologcal for rats. In our laboratory, a large amount of data has been collected that demonstrates no dfferences n basal Ca x levels beteen dabetc and control cardomyocytes 32,34,40 x. Hence, under normal condtons for solated cardomyocytes, basal Ca x Ž both restng and electrcally stmulated. does not necessarly reflect the depressed contracton n the dabetc heart and Ca x transents n response to dfferent physo- logcalrpharmacologcal stmulants may be better parameters to represent cardac dysfuncton. [ 2H ] 4. Ca moblzaton n the dabetc heart 4.1. Sarcolemmal Ca 2 q channels and Ca 2 q and K q currents n dabetc cardomyocytes Ca channel actvty n sarcolemmal membrane of dabetc hearts has been extensvely studed. Unfortunately, the data are controversal. Increased PH Ža dhydropyrdne dervatve. bndng stes n dabetc car- Donloaded from at Pennsylvana State Unversty on February 23, 2013

3 ( ) J.Z. Yu et al.rcardoõascular Research dac SL membrane 43 x, and decreased ntrendpne bndng stes th ncreased affnty ere observed n dabetc x cardac membranes 44. Net nflux of Ca as reported to be sgnfcantly reduced n chronc Ž 4 8 eeks. dabetc x rat myocardum 45. L-type Ca current as not sgnf46 x. There are data ndcatng a prolonga- cantly changed ton of the acton potentals n dabetc rat papllary mus47 x, and n solated dabetc myocytes 29x perhaps cles due to enhanced Ca current 47,48 x. Prolonged acton potental duraton n 30-eek dabetc myocardum has also been reported n rat ventrcular muscle and ventrcular myocytes 49 x. Whole cell clamp n cardomyocytes revealed a consderable reducton of the transent outard current Ž I. to, but the nard rectfer potassum current Ž I. as found to be unaltered 49 x K. The dabetes-related suppresson of Ito th accelerated nactvaton explans the decreased repolarzaton rate of the acton potental x 49, and may act to force more Ca to enter the myoplasm. Thus, the Ca transent probably has an ncreased ampltude, and lasts longer. Due to ths, the contractle response s prolonged Ž sometmes potentated. n papllary muscles x 4, and n solated myocytes 29 x. Under x such condtons, Ca may be ncreased hle the Ca content of the SR s probably decreased. Ths stuaton can explan hy the total Ca content of solated myocytes s smaller, and the effect of some notropc agents s dmn29 x. The transent outard potassum current densty shed as sgnfcantly reduced by dabetes hereas the voltage dependence of the nactvaton and the tme dependence of recovery ere not modfed 46 x. Furthermore, the lengthenng of the acton potental nduced by dabetes results manly from a decrease of the transmembrane Ca -nde46 x. Insuln restored the pendent potassum permeablty densty of the 4-amnopyrdne-senstve early transent component of I hch decreased n dabetes 50 x to. The prolongaton of APD suppresses the extruson of Ca va Ž electrogenc. Na Ca exchange, and facltates the sus- taned ncrease of the net Ca -nflux 48 x. The lack of effect of dabetes on I 49 x k, the man component of the restng conductance of cardomyocytes, ould account for the lack of change n restng potental of myocytes from dabetc rats as compared to controls. Decreased Ito n dabetc cells ll manly delay the early phase of repolarzaton of the acton potental. Ito and the Ca current Ž I. Ca actvate n the same range of potentals. One consequence of a depressed Ito durng dabetes ould be a longer Ca nflux through transmembrane Ca channels. Ths could explan the greater reducton of APD nduced by Ca blockers n ventrcular myocardum of dabetc rats 48 x. Delayed rectfer current Ž I. K s nvolved n the sloer phase of acton potental repolarzaton; decrease of ths current n dabetc myocytes may contrbute to the lengthenng of the late repolarzaton phase of the acton potental 46 x. In a recent report, rate-dependent attenuaton of IK s more pronounced n cells from dabetc rats 51 x Na Ca exchange actõty n SL of dabetc myocardum Depressed Na Ca exchange actvty thout change n affnty to Ca has been prevously reported n dabetc rats 24 x. These changes may be due to compostonal modfcatons n the SL membrane, possbly as a result of dabetes-nduced hyperlpdema x 7. In ths regard, unpublshed observatons from our laboratory have ndcated that the Na Ca exchange n hypertensve-dabetc rats demonstrates a more sgnfcant ncrease n the affnty for Ca. Interestngly, the hypertensve-dabetc rat model s knon for ts severe hyperlpdema 7,52 x SR Ca 2 q -ATPase n dabetc cardomyocytes It has been ell documented that SR functon n dabetc hearts s depressed. The early report of SR dysfuncton n the dabetc rat heart came from solated cardac membrane vescles hereby the uptake of 45 Ca nto SR Ž vescles a functon of SR Ca -ATPase. as reported to x be reduced 22. More recently, Ca transents have been measured to elucdate SR functon. In ths regard, a depressed rapd-coolng contracture Ž; 18C, an ndrect mea- sure of SR releasable Ca. as reported n papllary muscles 53x and solated cardomyocytes, accompaned by a reducton n ntracellular Ca transents 34 x. Caffene can also nduce Ca release from the SR, and the resultng contracture can be used as an ndrect measure of SR Ca avalable for release. Caffene-nduced Ca transents and contracture are both depressed n dabetc my32,34 x. ocytes Rapd-coolng and caffene contractures The Ca content of the SR that s avalable for release s clearly an mportant determnant of the contractle state. To approaches have been used to study SR Ca n cardomyocytes: rapd applcaton of Ž. a cold soluton Ž. Ž. RCC, ; 18C or b caffene to nduce SR Ca release. The resultng contracture can be used as an ndex of SR Ca avalable for release. The advantage of usng the above approaches s that they can be done on lne th contracted lvng cells 15 x. Rapd coolng of cardac muscle results n a contracture that can be attrbuted to the quck release of SR Ca to the cytoplasm. The ampltude of the contracture s ndcatve of the amount of Ca avalable for release from SR at the tme of coolng. Durng the coolng process, the probablty of the SR Ca release channel openng n54 x, the acton potental duraton s ncreased 55 x, peak Ca current decreases, myoflament Ca senstv- creases ty s reduced 56 x, and Na Ca exchange actvty s attenu x ated 56. The duraton of Ca x elevaton durng an RCC n unloaded condtons prolongs the actve state and may allo the myocyte to shorten progressvely. Due to nhb- Ž ton of membrane Ca transport Ca pump and Na Ca Donloaded from at Pennsylvana State Unversty on February 23, 2013

4 94 ( ) J.Z. Yu et al.rcardoõascular Research exchange., cell shortenng declnes sloly and the rse n Ca x durng an RCC s less transent than that observed durng caffene-nduced contractures. As Ca x s much hgher durng an RCC than durng a normal ttch, t appears that rapd coolng can release all of the avalable x SR Ca 15, hle only a fracton of the SR Ca avalable for release s dscharged durng a normal ttch. Caffene ncreases SR Ca channel openng and hence promotes a Ca leak nto the myoplasm. Ths process effectvely prevents the SR from accumulatng Ca.Ca pumped nto the SR s mmedately rentroduced nto the myoplasm va the open SR channels, and can then be removed from the cell by Na Ca exchange. Hence, caffene can nhbt SR Ca uptake thout drectly effectng SR Ca pump. One caveat s that caffene can cause myoflament senstzaton and phosphodesterase nhbton that can ncrease camp and camp-dependent proten knase and both these effects can complcate nterpretaton of the results 56 x. Moreover, Indo-1 fluorescence s strongly quenched by caffene. Hoever, ths occurs n a avelength-ndependent manner so that the fluorescence rato used to estmate Ca x s unaffected Caffene-nduced calcum transents n quescent myocytes The SR content of Ca can also be assessed by measurng Ca x response to caffene th the fluores- cence Ca -ndcator, fura-2. Bref exposure to caffene nduces a transent nard current hch reflects the electrogenc extruson of Ca across the membrane by the Na Ca exchange 57 x. Wth ths method, t as demon strated that the peak Ca x transent n response to caf- fene as sgnfcantly decreased n dabetes, suggestng a reducton n Ca storage n the SR 34 x. Insuln treatment prevented ths effect 34x and ths as probably related to the fact that nsuln treatment of STZ dabetc rats reverses the depresson of Ca-ATPase actvty and Ca uptake by the SR 22 x. In dabetc hearts, there has been some debate as to hether Ca overload or Ca underload occurs, and hether the decrease n cardac contractlty n the dabetc rat s accompaned by reduced or excessve loadng of the Ca nto SR. Our results, documentng the declne n SR Ca store and release, as assessed by RCC and caffene nduced Ca x transents 34 x, agree th the results of Bouchard and Bose 53x ho reported a reducton n SR Ca stores and decreased fractonal release of Ca durng stmulaton of papllary muscles from STZ-treated rats. Thus, the marked reducton of developed tenson n dabetc tssues s suggested to be a consequence of depleted SR Ca stores, rather than a result of chronc SR Ca overloadng. In ths regard, the reduced Ca uptake x by the SR 22 could dmnsh Ca stores and hence mpar Ca release, th a consequent reducton n cardac contracton Mtochondral functon n dabetc hearts The current ve of Ca accumulaton by heart mtochondra s that t may act as a snk for Ca under pathologcal condtons. Therefore, mtochondra may act as a potent and mportant bufferng component of Ca n the heart. A generalzed depresson of mtochondral functon exsts n hearts from dabetc rats. Mtochondral Ca-ATPase actvty and Ca transport capacty are sgnfcantly mpared n hearts from chroncally dabetc rats x 58. Although the alteraton n Ca accumulaton by mtochondra may have lttle effect on cardac functon on a beat-to-beat bass, any stmulus to the heart that results n elevated Ca x may not be adequately buffered n da- betc hearts. Drect measurement of mtochondral calcum concentraton n dabetc cardomyocytes has not been made, although a technque for such a measurement s avalable and has been appled n other pathologcal mod59,60 x. In a report els such as anoxa and reoxygenaton usng solated cardomyocytes, mtochondral uptake of 45 Ca, transmembrane potental gradent across the nner membrane of mtochondra, and cell respraton ere sgnfcantly decreased n dabetc myocytes compared to control 61 x. These changes ere restored to normal by nsuln treatment 61 x Myoflaments and Ca 2 q senstõty n dabetc cardomyocytes To knds of Ca -dependent alteratons may affect contractlty of the dabetc heart: Ž. a changng the aval- ablty of Ca to the myoflaments, and Ž. b modfyng the responsveness of the myoflaments to actvaton by ntracellular Ca. The avalablty of ntracellular Ca s regulated by the SL and SR, and Ca responsveness s controlled by the myoflaments and the regulatory tro62 x. There has been no consstent fndng regardng the ponn tropomyosn complex senstvty of the myoflament to Ca. The senstvty of solated myofbrls to Ca x has been reported to be unchanged 63x or ncreased 64x durng dabetes. The ncreased senstvty has been suggested to contrbute to the slo tme-course of relaxaton. More recently, to studes have suggested that the senstvty of myoflaments to Ca as decreased durng dabetes 65,66 x. Although these experments ere performed under dfferent condtons, the data appear to suggest that altered ntracellular Ca transents are not the only cause of cardac dysfuncton durng dabetes. It has been reported that the loer actvty of myosn ATPase and abnormal myosn soenzyme dstrbuton could be responsble for the depressed contractle functon n x dabetc myocardum 67. Addtonally, Ca -senstvty of sknned dabetc myocytes s sgnfcantly enhanced th unchanged maxmal actvated tenson 64 x. In con- Donloaded from at Pennsylvana State Unversty on February 23, 2013

5 ( ) J.Z. Yu et al.rcardoõascular Research trast, ncreased expresson n cardac b-myosn heavy chan and changes n troponn T expresson may contrbute to the decrease n Ca -senstvty of myoflaments at ph 7.0, and decreased maxmum tenson-generatng ablty at ph x 6.6 n dabetes 66. A decreased Ca -senstvty of sometrc tenson n sknned cardomyocytes from dabetc rats suggests that decreased cardac output n the hole heart can occur ndependently of alteratons n Ca -handlng by the SR or SL. Durng n vvo acdotc condtons such as schema and hypoxa, a substantal decrease n ventrcular pressure may occur, n part due to changes n x myoflament proten expresson 66. The apparent Ca - senstvty as greatly dmnshed at a sarcomere length of 1.9 mm but as not affected at a longer length Ž 2.4 mm.. Another possblty s that dabetc Ca -senstvty changes n the myocardum are coupled th troponn T alteratons 65 x. [ 2 q ] 4.6. Ca responses to b-adrenergc stmulaton Dabetc rat hearts are characterzed by dmnshed re10,68 x. Ths has been sponses to b-adrenergc stmulaton suggested to be due to a reducton n b-adrenergc receptor 25 x. As electrcally stmulated dabetc cardomyocytes demonstrate a depressed maxmum Ca x response to soproterenol and 8-bromo-cAMP thout a change n senstvty 31,40 x, t suggests that n addton to alteratons n b-adrenoceptor functon there are postreceptor defects n the dabetc myocardum. The response of Ca x to soproterenol can be blocked by thapsgargn, suggestng that the b-agonst-nduced Ca x changes are medated, n part, by SR Ca -ATPase 40 x. Insuln treatment of dabetc rats reversed the depressed response of Ca x to soproterenol 40 x. [ 2 q ] 4.7. Ca responses to dfferent pharmacologcal agents As stated earler, the peak Ca x transent nduced by caffene may be used as an ndrect ndex of SR releasable Ca. The maxmum Ca x ncrease n response to ouaban s reduced n dabetc cells hle the senstvty of dabetc myocytes to ouaban as enhanced 32 x. KCl-n duced Ca x ncrease as enhanced n dabetc cells accompaned by a decreased caffene and dchlorobenza32 x. Ntrendpne block- ml blockade of the KCl effects ade effects ere smlar n dabetc and control cells x 30,32. The maxmum responses of Ca x to exogenous ATP as ncreased n dabetc cells 32 x Isoproterenol and 8-bromo-cAMP In addton to the depressed response to soproterenol, dabetc myocytes also demonstrate an attenuated Ca x response to 8-bromo-cAMP at 10 y5 M and hgher concen32 x. Ths suggests that at steps dstal to the b- traton adrenoceptor and adenylate cyclase, dabetc myocytes exhbt a defcency. Hence, there may be an alteraton beteen camp and Ca x ncrease such as PKA actva- ton and the phosphorylaton of protens. The phosphorylaton process n the SR of dabetc myocytes s as yet unclear Ouaban The response to ouaban n papllary muscle and left atra from hearts of dabetc rats as reported to be markedly depressed 69,70 x. It as also reported that the maxmum number of hgh- and lo-affnty ouaban bndng stes n membrane preparatons obtaned from chroncally dabetc rats as sgnfcantly reduced to 60 and 49% of controls, respectvely. These results suggest that the decreased notropc response of ouaban n the ntact cardac tssue obtaned from dabetc rats may be related to a decreased number of ouaban bndng stes 71 x. The altered Kd could be due to an alteraton n the ouaban bndng stes or an altered composton of the membrane n dabetes. The actvty of Na-K ATPase s dramatcally reduced n the dabetc heart 72 x. As ths enzyme nd rectly regulates Ca x levels through ts modulaton of Na q x, the dabetes-lnked decrease n Na-K ATPase ac- tvty ould be expected to medate a net ncrease n both q x Na and Ca x. Myocardum from dabetc rats s susceptble to Ca loadng by ouaban ncubaton Žas measured by afterdepolarzatons. 73 x. These data suggest a declne n the reserve capacty of the sarcolemmal Na q pump n the dabetc heart. The decrease n Na-K ATPase could enhance the senstvty to dgtals-lke compounds by reducng the reserve capacty of the Na-pump and hence, the extent of dgtals-nduced pump nhbton requred before the onset of toxcty Žn the case of sngle myocyte, hypercontracton.. A reducton n reserve capacty may loer the tolerance to ouaban by decreasng the number of pump stes that the glycosde ould have to nhbt before elctng a marked Na q x accumulaton and the resultng toxc effects beleved to be medated by Ca -overload. Cardac arrhythmas are a frequent and serous complcaton of the clncal use of dgtals glycosdes, and t s possble that the tolerance to these cardotoxc effects and the margn of safety for cardotonc sterods s reduced n dabetc patents KCl and ATP The nflux of Ca x s a prerequste of the KCl-n- duced Ca x transent because EGTA abolshed the tran- sent. The Ca x nflux s trggered by membrane depo- larzaton and thus actvaton of L-type Ca -channels and reversed mode operaton of Na Ca exchange. Ca nflux va Ca -channel and Na Ca exchange may drectly contrbute to the Ca x ncrease and, more mportantly, nduce Ca release from SR. In accordance th our results, an enhanced Ca x response to KCl Ž 30 mm. n a myocyte suspenson from dabetc rats has been docu x mented 30. Increased L-type Ca -channel actvty deter 3 HxPN bndng stes n cardac mned by mem- Donloaded from at Pennsylvana State Unversty on February 23, 2013

6 96 ( ) J.Z. Yu et al.rcardoõascular Research brane has been reported n the dabetc heart 43 x. Interest 3 HxPN bndng to control cardac ngly, the membrane as dose-dependently nhbted by verapaml, but ths as not the case n dabetc cardac membranes x 43. Ths suggests that L-type Ca -channels are quanttatvely and qualtatvely altered n dabetes, and may be related to the ntrendpne effects found n ths study. The effects of ATP n modulatng Ca -channel and ntra- cellular Ca store are very smlar to KCl 74 x. They are both senstve to BAY K 8644, nfedpne, and EGTA and both are partally nhbted by ryanodne and caffene. These smlartes suggest that the ATP mechansm s smlar to the KCl effects of membrane depolarzaton 74x and drect actvaton of Ca -channels. 5. Summary The qualty control ndces of myocyte solaton Žvabl- ty, yeld, survval tme, cell response, etc.. suggest that the adult rat myocyte model s stable and useful n Ca x measurements and functonal studes at the cellular level. Moreover, dabetc cardomyocytes are a valuable model for studyng cellular functons of the dabetc heart as they retan most of the features of cardac dysfuncton of ntact rat. Data from our studes ndcate that the basal Ca x n both quescent and electrcally-stmulated cells s not x changed. Thus, restng levels of Ca and basal Ca x transents may not reflect the abnormaltes observed n dabetes untl the system s challenged by certan stmul. Ca x responses to soproterenol are depressed n both restng and stmulated dabetc cells. Ths suggests an alteraton n the b-adrenergc pathay, possbly related to the b-adrenoceptor defcency reported n the dabetc heart. SR Ca-ATPase s also nvolved n the soproterenol-nduced Ca x changes. Moreover, the decreased maxmum re- sponse to 8-bromo-cAMP provdes evdence of a post-receptor alteraton n the pathay. Dabetc myocytes are more senstve to ouaban, hereas the maxmum response to ouaban as depressed. Ths may be the result of depressed Na-K ATPase and ncreased Na q x. In dabetc myocytes, rapd coolng contractures and caffene contractures are depressed, hereas caffene-nduced Ca transents are decreased. Ryanodne bndng suggests a decreased number of hgh-affnty bndng stes n the SR of dabetc myocytes. Addtonally, there are ndcatons that SR releasable calcum s reduced and that the major functons of SR, notably uptake, release and storage, may be depressed n dabetc myocytes. Fnally, L-type Ca -channels are quanttatvely and qualtatvely altered n dabetes. Insuln treatment normalzes most of the dabetes-nduced changes n cardomyocytes, suggestng that metabolc alteratons due to nsuln defcency play an mportant role n dabetc cardomyopathy. Results from several studes sho that n dabetes the functon of major organelles hch handle Ca x n myocytes s depressed, hch n turn causes the alteraton of Ca x moblzaton n myocytes. Dfferent second messenger systems nvolved n E-C couplng may also be altered due to the metabolc mparments. 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