The importance of the Thr 17 residue of phospholamban as a phosphorylation site under physiological and pathological conditions

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1 Phsphrylatin Brazilian Jurnal f f Thr Medical 17 residue and Bilgical f phsphlamban Research (26) 39: ISSN 1-879X Review 563 The imprtance f the Thr 17 residue f phsphlamban as a phsphrylatin site under physilgical and pathlgical cnditins A. Mattiazzi 1, C. Mundiña-Weilenmann 1, L. Vittne 1, M. Said 1 and E.G. Kranias 2 1 Centr de Investigacines Cardivasculares, Facultad de Medicina, La Plata, Argentina 2 Department f Pharmaclgy and Cell Biphysics, University f Cincinnati Cllege f Medicine, Cincinnati, OH, USA Crrespndence A. Mattiazzi Centr de Investigacines Cardivasculares Facultad de Ciencias Médicas 6 y 12, (19) La Plata Argentina Fax: ramattia@atlas.med.unlp.edu.ar Presented at the XX Annual Meeting f the Federaçã de Sciedades de Bilgia Experimental, Águas de Lindóia, SP, Brazil, August 24-27, 24. Research supprted by PICT #8592 (FONCYT), PIP #2256 and 2257 (CONICET) and Fgarty Internatinal Research Award Grant #1-R3-TW (NIH). Received August 9, 25 Accepted January 2, 26 Abstract The sarcplasmic reticulum (SR) Ca -ATPase (SERCA2a) is under the cntrl f an SR prtein named phsphlamban (PLN). Dephsphrylated PLN inhibits SERCA2a, whereas phsphrylatin f PLN at either the Ser 16 site by PKA r the Thr 17 site by CaMKII reverses this inhibitin, thus increasing SERCA2a activity and the rate f Ca uptake by the SR. This leads t an increase in the velcity f relaxatin, SR Ca lad and mycardial cntractility. In the intact heart, ß- adrenceptr stimulatin results in phsphrylatin f PLN at bth Ser 16 and Thr 17 residues. Phsphrylatin f the Thr 17 residue requires bth stimulatin f the CaMKII signaling pathways and inhibitin f PP1, the majr phsphatase that dephsphrylates PLN. These tw prerequisites appear t be fulfilled by ß-adrenceptr stimulatin, which as a result f PKA activatin, triggers the activatin f CaMKII by increasing intracellular Ca, and inhibits PP1. Several pathlgical situatins such as ischemia-reperfusin injury r hypercapnic acidsis prvide the required cnditins fr the phsphrylatin f the Thr 17 residue f PLN, independently f the increase in PKA activity, i.e., increased intracellular Ca and acidsis-induced phsphatase inhibitin. Our results indicated that PLN was phsphrylated at Thr 17 at the nset f reflw and immediately after hypercapnia was established, and that this phsphrylatin cntributes t the mechanical recvery after bth the ischemic and acidic insults. Studies n transgenic mice with Thr 17 mutated t Ala (PLN-T17A) are cnsistent with these results. Thus, phsphrylatin f the Thr 17 residue f PLN prbably participates in a prtective mechanism that favrs Ca handling and limits intracellular Ca verlad in pathlgical situatins. Key wrds Phsphlamban Thr 17 site phsphrylatin ß-adrenergic stimulatin Acidsis Ischemia Braz J Med Bil Res 39(5) 26

2 564 A. Mattiazzi et al. I Ca SL Ca DHPR NCX RyR 2 Intrductin Figure 1. Central players f excitatin-cntractin cupling (ECC) in cardiac muscle. Scheme f the ECC mechanism in cardiac muscle. Upn deplarizatin, Ca influx thrugh L-type Ca channels r dihydrpyridine receptr (DHPR) triggers the release f mre Ca frm the sarcplasmic reticulum (SR). This Ca binds t the myfilaments (MF) t prduce cntractin. Part f the Ca is then extruded utside f the cell thrugh the Na + / Ca exchange mechanism (NCX), wrking in the frward mde, but mst f it is re-taken up by SR Ca -ATPase (SERCA2a). Phsphlamban (PLN) is a small SR prtein assciated with SERCA2a. The state f PLN phsphrylatin regulates the activity f this Ca pump. RyR 2 = ryandine receptr; SL = sarclemma. Figure 2. Phsphlamban phsphrylatin leads t an increase in SR Ca uptake which accelerates relaxatin and enhances SR Ca lad. Mre Ca is then available fr release frm the SR, resulting in increased cntractility. Thus, phsphlamban is a majr regulatr f mycardial relaxatin and cntractility. SR = sarcplasmic reticulum. Ca 3 Na + Ca SR Ca SERCA2a PLN NCX 3 Na + Ca ATPase MF Figure 1 is a schematic illustratin f the central players in the excitatin-cntractin cupling mechanism. Upn deplarizatin, Ca enters the cell thrugh L-type Ca channels and triggers the release f mre Ca frm the sarcplasmic reticulum (SR) thrugh the activatin f the ryandine receptrs. This prcess, knwn as Ca -induced-ca release (1), amplifies and crdinates the Ca signal t prduce cntractin by interacting with myfilament prteins. The decrease in cytslic Ca t prduce relaxatin is mainly induced by SR Ca -ATPase (SERCA2a), which mediates Ca uptake int the SR, and, t a lesser extent, by the Na + /Ca exchanger, which transfers Ca t the extracellular space (2). The activity f SERCA2a is under the cntrl f a clsely assciated SR prtein named phsphlamban (PLN) (3). In the dephsphrylated frm, PLN decreases the apparent affinity f SERCA2a fr Ca (4,5). Phsphrylatin f PLN relieves this inhibitin, thus increasing SR Ca uptake. Experiments n the intact heart have shwn that ß-adrenceptr stimulatin phsphrylates PLN at the Ser 16 residue by campdependent prtein kinase (PKA) and at the Thr 17 site by Ca -calmdulin-dependent prtein kinase II (CaMKII). Phsphrylatin f these sites reverses the inhibitin f SERCA2a by PLN, thus increasing the affinity f the enzyme fr Ca and the rate f Ca uptake by the SR. This in turn leads t an increase in the velcity f relaxatin, SR Ca lad and, as a cnsequence, increased SR Ca release and mycardial cntractility (6-9) (Figure 2). The status f phsphrylatin f PLN als depends n the activity f type 1 phsphatase (PP1), the majr SRphsphatase, which specifically dephsphrylates PLN (1). Althugh, as stated abve, ß-adrenergic stimulatin phsphrylates Ser 16 and Thr 17 f PLN, the rle f the phsphrylatin f Thr 17 has been generally disregarded. The present article fcuses n the rle f the Thr 17 site f PLN during ß-adrenergic stimulatin and under different pathlgical cnditins such as acidsis and ischemia/reperfusin injury. Rle f the phsphrylatin f Thr 17 f PLN during ß-adrenergic stimulatin Althugh in vitr studies indicate that PKA and CaMKII phsphrylatins are independent f each ther (11), earlier at- Braz J Med Bil Res 39(5) 26

3 Phsphrylatin f Thr 17 residue f phsphlamban 565 tempts t phsphrylate PLN by CaMKII in the intact heart usually failed, unless cellular camp levels increased (6,7,12). These findings suggested an interactin between the PKA and CaMKII pathways fr PLN phsphrylatin. The availability f transgenic mdels expressing wild-type PLN (PLN- WT), the Ser 16 Ala mutant PLN (PLN- S16A) r the Thr 17 Ala mutant PLN (PLN- T17A) in the cardiac cmpartment f PLN knck-ut mice, and f phsphrylatin sitespecific antibdies t PLN, which precisely discriminate between the Ser 16 and the Thr 17 phsphrylatin sites, in cmbinatin with the quantificatin f 32 P incrpratin int PLN, helped t clarify the relative rle f Ser 16 and Thr 17 phsphrylatin (9,13,14). Experiments in transgenic mice expressing either PLN-WT r the PLN-S16A, indicated that the phsphrylatin f Ser 16 f PLN is a prerequisite fr the phsphrylatin f Thr 17 (13). Other experiments cnducted with PLN-T17A hearts further indicated that the Ser 16 site can be phsphrylated independently f Thr 17 in viv and that phsphrylatin f Ser 16 was sufficient t evke the maximal effect f ß-adrenergic stimulatin (14). Figure 3A, shws the increase in ttal PLN phsphrylatin prduced by increasing isprterenl cncentratin in the cardiac perfusate. When Ca influx t cardiac cells was virtually ablished by the presence f lw extracellular Ca (Ca ) and the Ca channel blcker nifedipine t prevent CaMKII activatin, ttal PLN phsphrylatin decreased at the highest isprterenl cncentratins (1, 3, and 3 nm), but did nt significantly change at the lwest levels f ß-adrenceptr stimulatin (.3 and 3 nm). These results suggested that there was n cntributin f the CaMKII pathways t the ttal phsphrylatin f PLN at the lwest isprterenl cncentratins. Immundetectin f site-specific phsphrylated PLN revealed an isprterenl cncentratin-dependent increase in bth Ser 16 and Thr 17 residues f PLN at nrmal (Ca ) % f maximum 32 A P-PLN B C [Ca] 1.35 mm [Ca] 7 µm + nifedipine Is (nm) % f maximum [Ca] 1.35 mm 12 [Ca] 7 µm + nifedipine 1 8 PSer 16 PSer 16 PThr PThr Is (nm) % f maximum Is (nm) Figure 3. Isprterenl (Is) cncentratin-dependent increase in 32 P incrpratin int phsphlamban (PLN) and in the phsphrylatin f Ser 16 and Thr 17 residues at tw different levels f extracellular Ca. A, Rat hearts were perfused with 32 P and then with different isprterenl cncentratins at tw different levels f Ca. The virtual suppressin f Ca supply t the heart (7 µm (Ca) +.4 µm nifedipine) significantly decreased 32 P incrpratin int PLN ( 32 P-PLN), at 1, 3 and 3 nm Is, but failed t affect it at.3 and 3 nm Is. Data are reprted as percent maximal 32 P incrpratin int PLN. B and C, Densitmetric analysis f the signal f immunblts f sarcplasmic reticulum membrane vesicles islated frm rat hearts perfused with different Is cncentratins at tw (Ca ), and prbed with anti-pser 16 -PLN and anti-pthr 17 -PLN. At 1.35 mm (Ca), Is increased the phsphrylatin f bth Ser 16 and Thr 17 (B). Phsphrylatin f Thr 17 was shifted t the right relative t the increase in the phsphrylatin f Ser 16. The decrease in (Ca ), as shwn in panel A, did nt significantly affect the phsphrylatin f Ser 16 but inhibited the increase in the phsphrylatin f Thr 17 at all Is cncentratins (C). Data are reprted as percent f the maximal signal achieved in each experimental series. P <.5 cmpared t n drug values at nrmal (Ca ) (B) and at lw (Ca ) (C) (Student t-test fr unpaired samples). Mdified frm Ref. 15, with permissin. Braz J Med Bil Res 39(5) 26

4 566 A. Mattiazzi et al. (Figure 3B). Hwever, when isprterenl was perfused in the presence f lw (Ca ), Ser 16 was the nly site t shw a cncentratin-dependent increase in phsphrylatin (Figure 3C). At each isprterenl cncentratin, Ser 16 was phsphrylated t a similar extent, independently f the phsphrylatin f Thr 17. In rder t crrelate the changes bserved at the specific phsphrylatin sites f PLN with the mechanical activity f the heart during ß-adrenergic stimulatin, Ca supply t the cell was decreased s as t cancel the psitive intrpic effect f each isprterenl cncentratin. Figure 4 shws that this maneuver decreased the phsphrylatin f Thr 17 in assciatin with a significant reductin f the relaxant effect f the ß-agnist at 1 and 3 nm isprterenl (9,15). Hwever, at 3 nm isprterenl, decreasing (Ca ) had n significant effect n Thr 17 r Ser 16 phsphrylatin, nr did it mdify the relaxant effect f isprterenl, supprting earlier experiments in which ttal PLN phsphrylatin was evaluated with 32 P (15,16). The lack f a cntributin by Thr 17 t the ttal PLN phsphrylatin at the lwest isprterenl cncentratins might be attributed t a mdest PKA activity, unable t cause an increase in intracellular Ca, sufficient t activate CaMKII, as well as a significant phsphatase inhibitin necessary t detect the phsphrylatin f the Thr 17 residue (15), as it will be discussed belw. +dp/dt (% cntrl) PThr -PLN (% 3 nm Is) 17 A C dp/dt Cntrl [Ca ] [Ca ] Is (nm) PThr 17 - PLN Cntrl [Ca ] [Ca ] B 16 PLN (% 3 nm Is) PSer PSer -PLN t 1/2 Cntrl [Ca ] [Ca ] -2 [Ca ] Is (nm) Is (nm) 1/2 ms) t ( D Is (nm) Cntrl [Ca ] Figure 4. Effects f decreasing (Ca ) n mechanical parameters and phsphlamban (PLN) phsphrylatin sites at different isprterenl (Is) cncentratins. A, Ca supply t the heart was decreased s as t ablish the Is-induced psitive intrpic effect at the different cncentratins. (Ca) was.25 mm at 3 and 1 nm Is and.5 mm at 3 nm Is. Cntractility was evaluated by the maximal rate f rise in pressure (+dp/dt). Phsphrylatin f Ser 16 f PLN (PSer 16 -PLN), induced by the different cncentratins f Is, was nt affected by the decrease in (Ca ) (B); hwever, this decrease, reduced the phsphrylatin f Thr 17 (C) as well as the isprterenl-induced relaxant effect (decrease in half relaxatin time, t 1/2 ) (D), at 3 and 1 nm Is. At 3 nm Is the decrease in (Ca ) did nt affect either the Is-induced phsphrylatin f Thr 17 r the relaxant effect. +dp/dt is expressed as percentage f cntrl values. t 1/2 is expressed as difference frm cntrl values ( ms). Site-specific phsphrylatin f PLN is expressed as percent f the signal btained with 3 nm Is at 1.35 mm (Ca). P <.5 cmpared t the crrespnding Is cncentratin at cntrl (Ca ) (Student t-test fr unpaired bservatins). Mdified frm Ref. 15, with permissin. Braz J Med Bil Res 39(5) 26

5 Phsphrylatin f Thr 17 residue f phsphlamban 567 Taken tgether, these findings demnstrate: a) the additive nature f the PKA and CaMKII pathways f PLN phsphrylatin, in agreement with in vitr results (11); b) that bth sites equally cntribute t the ttal PLN phsphrylatin at the highest levels f ß-adrenergic stimulatin; c) that at lw isprterenl cncentratins ( 3 nm), the increase in PLN phsphrylatin and the relaxant effect f isprterenl appear t be exclusively determined by the increase in the phsphrylatin f the Ser 16 residue. Phsphrylatin f Thr 17 f PLN in the absence f ß-adrenergic stimulatin The cnclusin that CaMKII-dependent PLN phsphrylatin can nly ccur in the intact heart in the presence f ß-adrenergic stimulatin, i.e., when the camp levels within the cell and PKA activity increase (7,12), was in sharp cntrast with the independence f bth pathways f PLN phsphrylatin described in in vitr systems (11). Figure 5 shws that the increase in cntractility (intracellular Ca ) prduced by increasing extracellular Ca, in the presence f the phsphatase inhibitr, kadaic acid, evked a significant increase in Thr 17 phsphrylatin assciated with a relaxant effect, in the absence f any significant increase in camp levels r in the phsphrylatin f Ser 16 f PLN (9). These results indicate that the Thr 17 residue can be phsphrylated independently f Ser 16 phsphrylatin, as was described in vitr and further emphasize that phsphatases are as imprtant as kinases in determining the level f phsphrylatin f any prtein. Accrdingly, phsphrylatin f the Thr 17 residue in the intact heart culd be detected in tw situatins: a) in the presence f high extracellular Ca (t activate CaMKII) and f kadaic acid (t inhibit PP1), and b) at high intracellular camp, which, by activatin f PKA, accunts fr bth effects, i.e., the increase in intracellular Ca and the inhibitin f PP1. In the cntext f the experiments decribed abve, it is imprtant t discuss the apparent cntradictin between sme f the results f the experiments n transgenic mice (13,14) and f phsphrylatin experiments cnducted n animals with an intact PLN (7-9,15). The failure t find PLN phsphrylatin (i.e., Thr 17 phsphrylatin) in transgenic mice in which the Ser 16 site was mutated t Ala (13) - which wuld apparently cntradict the results f the phsphrylatin experiments shwing that Thr 17 can be phsphrylated independently f Ser 16 phsphrylatin (9,22) - must be attributed t the fact that the lack f phsphrylatin f the Ser 16 site under cnditins f ß-stimulatin precludes the increase in intracellular Ca necessary t phsphrylate the Thr 17 residue. Mrever, the experiments shwing that in transgenic mice in which the Thr 17 site was mutated t Ala, Ser 16 phsphrylatin was sufficient t mediate the maximal respnse t isprterenl (14), d nt necessarily cntradict the results f the phsphrylatin experiments which indicate that, when bth % 3 nm Is t ( ms) A 1/ B PSer -PL N 17 PThr -PLN Ca (mm ) + OA Figure 5. Effects f increasing (Ca ) and inhibitin f phsphatases n the phsphrylatin f Thr 17 f phsphlamban (PLN) and mechanical relaxatin. A, The increase in (Ca ) in the presence f kadaic acid (OA) increased the phsphrylatin f Thr 17 site f PLN. B, The increased phsphrylatin f the Thr 17 site was assciated with a decrease in half relaxatin time (t 1/2 ). P <.5 cmpared t.25 mm Ca + OA. (Student t-test fr paired (mechanical) r unpaired (bichemical) data). Mdified frm Ref. 9, with permissin. Braz J Med Bil Res 39(5) 26

6 568 A. Mattiazzi et al. A +dp/dt (%) B Tau ( ms) sites are present, they bth cntribute t the ttal phsphrylatin f PLN and t the mechanical effect f ß-adrenceptr stimulatin (7,9,15). Rle f the phsphrylatin f Thr 17 f PLN in pathlgical cnditins Acidsis Intracellular acidsis is assciated with a decrease in the ability f the heart t gener- +dp/dt 17 PThr -PLN Time f acidsis (min) 17 PThr -PLN Figure 6. Effect f acidsis n left ventricular functin and n the phsphrylatin f Thr 17 f phsphlamban (PLN). Hypercapnic acidsis prduced an initial (3 s) decrease in cntractility (maximal rate f rise in pressure, +dp/dt) (A) and an antirelaxant effect (prlngatin f the time cnstant f left ventricular develped pressure decay, Tau) (B), fllwed by mechanical recvery, mst f which ccurred during the first 3 min f acidsis. Phsphrylatin f the Thr 17 site f PLN increased simultaneusly with the early recvery f cntractility and relaxatin (A and B) and then returned t basal levels. Phsphrylatin f Ser 16 remained at basal levels thrughut the acidsis perid (nt shwn). Phsphrylatin f Thr 17 is reprted as indicated in Figure 4C. +dp/dt is reprted as percent f basal values. Tau is reprted as ms f basal values. P <.5 cmpared t basal values (Student t-test fr paired (mechanical) r unpaired (bichemical) data). Mdified frm Ref. 2, with permissin. Tau Time f acidsis (min) % 3 nm Is % 3 nm Is ate tensin (17). Acidsis prduces a rapid decrease in the cntractin f cardiac muscle mainly due t a decrease in myfilament Ca respnsiveness (18) and an impairment f relaxatin, which ccurs in spite f the decrease in the respnsiveness f the cntractile prteins and appears t be mainly prduced by a direct inhibitin f SERCA2a (19). This initial impairment f cntractility and relaxatin is fllwed by a spntaneus mechanical recvery. The mechanisms underlying this recvery are prly understd. Figure 6 shws that phsphrylatin f the Thr 17 site f PLN transiently increases at the nset f acidsis and is assciated with a large part f the cntractile and relaxatin recveries, mst f which ccurred within the first 3 min f acidsis. This phsphrylatin wuld prvide a mechanism t vercme the direct depressant effect f acidsis n SERCA2a (19). Whereas Ser 16 was nt phsphrylated during the perid f acidsis, the Thr 17 site became dephsphrylated after 5 min f acidsis (2), which wuld indicate that the phsphrylatin f these sites des nt cntribute t the cntractile and relaxatin recveries bserved after 5 min f acidsis r that the phsphrylatin f the Thr 17 residue has memry, triggering a mechanism that persists thrughut the perid f acidsis. The increase in the phsphrylatin f the Thr 17 site at the beginning f acidsis might be attributed t the increase in intracellular Ca that has been shwn t ccur during acidsis (19), and t the acidsis-induced phsphatase inhibitin (21). These tw phenmena prvide the necessary cnditins t increase the phsphrylatin f the Thr 17 site (22). Dephsphrylatin f the Thr 17 residue wuld ccur after the recvery f phsphatase activity due t the return f intracellular ph t values clse t cntrl (23). Cnsistent with the imprtant rle f Thr 17 phsphrylatin in the mechanical recvery frm acidsis, experiments by DeSantiag et al. (24) shwed absence f mechanical recvery in my- Braz J Med Bil Res 39(5) 26

7 Phsphrylatin f Thr 17 residue f phsphlamban 569 cytes lacking PLN. Finally, a mre prminent rle f the phsphrylatin f PLN residues might be expected in viv. Systemic acidsis is knwn t increase sympathetic nerve activity (25). This increased sympathetic tne may prduce a mre persistent phsphrylatin f bth the Thr 17 and Ser 16 residues f PLN, with a further cntributin t mechanical recvery. Stunning Ischemic heart diseases still cnstitute the leading cause f mrtality and mrbidity in industrialized cuntries. Mycardial stunning is recgnized as the reversible decrease in mycardial cntractility that fllws a brief ischemic episde, clinically manifested as sluggish recvery f the pump functin PSer 16 -PLN PThr 17 -PLN A PI Isch R1min R5min R3min Is B PI Isch R1min R5min R3min Is PSer 16 -PLN (% 3 nm Is) LVDP (%) C Cntrl Ischemia Reperfusin Cntrl Ischemia Reperfusin PI Cntrl Ischemia Reperfusin P <.5 PI PThr 17 -PLN (% 3 nm Is) t1/2 ( ms) D PI Cntrl Ischemia Reperfusin P <.5 PI Figure 7. Time curse f phsphlamban (PLN) Ser 16 and Thr 17 phsphrylatin and f left ventricular functin during ischemia and reperfusin. Representative immunblts (A and B, abve) and verall results f densitmetric analysis f the signals f inmunblts (A and B, belw) f sarcplasmic reticulum membrane vesicles islated frm hearts freeze-clamped after 2 min f glbal ischemia (Isch) and at 1, 5, and 3 min during reperfusin (R1- R3 min). The blts were prbed with anti PSer 16 -PLN (A) and anti PThr 17 -PLN (B). Phsphrylatin f PLN sites induced by 3 nm isprterenl (Is) in nn-ischemic hearts is shwn in the blts fr reference. Ser 16 phsphrylatin significantly increased after 2 min f ischemia and Thr 17 phsphrylatin transiently increased at 2-5 min f ischemia and at 1 min f reperfusin. Left ventricular develped pressure (LVDP) (C) decreased t virtually nndetectable levels after cessatin f flw and reperfusin resulted in a partial recvery f cntractility. In spite f the impairment in cntractility, t 1/2 (D) recvered immediately after ischemia and then significantly decreased belw pre-ischemic values (PI), indicating an acceleratin f ventricular relaxatin. Pints represent the mean ± SEM f data frm 51 hearts freeze-clamped at 2 min f ischemia and 1, 2, 3, 5, 15, and 3 min f reperfusin. LVDP is reprted as percent PI and t 1/2 as ms f PI. Site-specific PLN phsphrylatin is reprted as shwn in previus figures. P <.5 cmpared t PI (Student t-test fr paired (mechanical) r unpaired (bichemical) data). Mdified frm Ref. 35, with permissin. Braz J Med Bil Res 39(5) 26

8 57 A. Mattiazzi et al. Figure 8. Functinal rle f Thr 17 phsphrylatin in the ischemiareperfused muse heart. Time curse f left ventricular develped pressure (LVDP; A) and Tau (B) during ischemia and reperfusin in wild-type phsphlamban (PLN-WT) and PLN with a Thr 17 t Ala mutatin (PLN- T17A) muse hearts. Mutatin f Thr 17 t Ala is assciated with diminished cntractin and relaxatin recveries after the ischemic insult cmpared t PLN- WT muse hearts. P <.5 cmpared t PLN-WT (Student t-test fr unpaired bservatins). Mdified frm Ref. 36, with permissin. after revascularizatin (26,27). Althugh the phenmenn f mycardial stunning was described ver 25 years ag (28), the mechanisms respnsible fr the delayed recvery f cntractile functin are nt cmpletely clear (29). Different types f evidence btained in rdents and humans indicate that the ultimate cause f the alteratin f mycardial cntractility is a decrease in myfilament Ca respnsiveness, since the Ca transient is nt altered, althugh cntractility is decreased (29). On the ther hand, experimental evidence indicates that the functin f the SR is altered bth in reversible and irreversible ischemiareperfusin injury (3-34). In particular, in the case f mycardial stunning, a decrease in the activity f SERCA2a and/r in the rate f Ca reuptake by the SR has been described in several species (31-34). Thus, an bvius questin is: why des the intracellular Ca transient remain unaltered in species in which SR functin is depressed? A pssible explanatin fr this apparent cntradictin is that cmpensatry mechanisms can vercme the depressed SERCA2a activity. One pssible cmpensatry mechanism is the phsphrylatin f PLN. T test this hypthesis, we studied the time curse f phsphrylatin f PLN residues at different times during ischemia/reperfusin (35). Figure 7 shws the time curse f left ventricular develped pressure (LVDP), half relaxatin time (t 1/2 ) and the phsphrylatin f Ser 16 and Thr 17 residues f PLN during ischemia and reperfusin. LVDP decreased t virtually undetectable levels after cessatin f LVDP (%) A flw. Reperfusin resulted in a partial recvery f LVDP. In spite f the reductin f cntractility, t 1/2 recvered immediately after ischemia and then significantly decreased belw pre-ischemic values, indicating an acceleratin f ventricular relaxatin. Phsphrylatin f Ser 16 was significantly increased at the end f ischemia, but returned t basal values during the reperfusin perid. Phsphrylatin f the Thr 17 residue f PLN transiently increased bth at the beginning f ischemia (2-5 min) and at the beginning f reperfusin (1 min), remaining at pre-ischemic values fr the rest f the reperfusin perid. The decrease in Thr 17 phsphrylatin by CaMKII inhibitin, prlnged t 1/2, which indicates that the phsphrylatin f Thr 17, when present, attenuates the impaired relaxatin that ccurs at the beginning f reperfusin (36). Figure 8 shws the time curse f cntractile and relaxatin parameters during ischemia and reperfusin in PLN-WT and PLN-T17A mutant hearts. The recvery f LVDP was significantly lwer in PLN-T17A mutant hearts than in PLN-WT hearts thrughut the reperfusin perid, whereas the time cnstant f pressure decay, Tau, was significantly prlnged at the beginning f reperfusin and then recvered t values clse t cntrl. Additinal experiments als shwed that the cntractile recvery was depressed in PLN-S16A mutant hearts cmpared t the heart f PLN- WT mice. Hwever, this depressin nly ccurred at the beginning f reperfusin (36). These results indicate that phsphrylatin f Cntrl Ischemia Reperfusin Cntrl Ischemia Reperfusin -1 PLN-WT PLN-T17A Tau ( ms) B PLN-WT PLN-T17A Braz J Med Bil Res 39(5) 26

9 Phsphrylatin f Thr 17 residue f phsphlamban 571 Thr 17, althugh transient, may play a significant rle in the critical phase f initial reperfusin, favring Ca re-uptake by the SR. This wuld tend t cmpensate fr the depressin f SERCA2a and wuld amelirate Ca verlad, typical f the beginning f reflw (29). Mrever, these experiments indicate that activatin f SERCA2a by any ther means, at this crucial mment f reperfusin, may cntribute significantly t Ca handling. In agreement with this view, recent experiments suggested that the cardiprtectin prduced by cgmp-mediated stimuli in rexygenated cells is assciated with an increase in the phsphrylatin f Ser 16 f PLN (37). Cnclusins Taken tgether, these findings suggest that the Thr 17 site f PLN has an imprtant rle, nt nly under physilgical cnditins, such as ß-adrenceptr stimulatin, but als in the mechanical recvery under sme pathlgical cnditins such as acidsis and stunning. An interesting pint is that, althugh the phsphrylatin experiments reveal that the phsphrylatin f Thr 17 is transient and ccurs nly at the beginning f bth acidsis and reperfusin, the presence f this residue and/r f an intact PLN seems t be necessary fr mechanical recvery during the reperfusin r acidsis perid. References 1. Fabiat A & Fabiat F (1997). Calcium release frm the sarcplasmic reticulum. Circulatin Research, 4: Bers DM (21). Excitatin-Cntractin Cupling and Cardiac Cntractile Frce. 2nd edn. Kluwer Academic Publishers, Drdrecht, Netherlands. 3. Tada M, Kirchberger MA & Katz AM (1975). Phsphrylatin f a 22,-daltn cmpnent f the cardiac sarcplasmic reticulum by adensine 3',5'-mnphsphate-dependent prtein kinase. Jurnal f Bilgical Chemistry, 25: James P, Inui M, Tada M et al. (1989). Nature and site f phsphlamban regulatin f the Ca² + pump f sarcplasmic reticulum. Nature, 342: Kim HW, Steenaart NA, Fergusn DG et al. (199). Functinal recnstitutin f the cardiac sarcplasmic reticulum Ca -ATPase with phsphlamban in phsphlipid vesicles. Jurnal f Bilgical Chemistry, 265: Lindemann JP, Jnes LR, Hathaway DR et al. (1983). ß-Adrenergic stimulatin f phsphlamban phsphrylatin and Ca -ATPase activity in guinea pig ventricles. Jurnal f Bilgical Chemistry, 258: Vittne L, Mundiña C, Chiappe de Cinglani G et al. (199). camp and calcium dependent mechanisms f phsphlamban phsphrylatin in intact hearts. American Jurnal f Physilgy, 258: H318-H Naplitan R, Vittne L, Mundiña-Weilenmann C et al. (1992). Phsphrylatin f phsphlamban in the intact heart. A study n the physilgical rle f the Ca-calmdulin-dependent prtein kinase system. Jurnal f Mlecular and Cellular Cardilgy, 24: Mundiña-Weilenmann C, Vittne L, Ortale M et al. (1996). Immundetectin f phsphrylatin sites gives new insights int the mechanisms underlying phsphlamban phsphrylatin in the intact heart. Jurnal f Bilgical Chemistry, 271: MacDugall LK, Jnes LR & Chen P (1991). Identificatin f the majr prtein phsphatases in mammalian cardiac muscle which dephsphrylate phsphlamban. Eurpean Jurnal f Bichemistry, 196: Bilezikjian LM, Kranias EG, Ptter JD et al. (1981). Studies n phsphrylatin f canine cardiac sarcplasmic reticulum by calmdulin-dependent prtein kinase. Circulatin Research, 49: Lindemann JP & Watanabe AM (1985). Phsphrylatin f phsphlamban in intact mycardium. Rle f Ca -calmdulin-dependent mechanisms. Jurnal f Bilgical Chemistry, 26: Lu W, Chu G, Sat Y et al. (1998). Transgenic appraches t define the functinal rle f dual site phsphlamban phsphrylatin. Jurnal f Bilgical Chemistry, 273: Chu G, Lester JW, Yung KB et al. (2). A single site (Ser 16 ) phsphrylatin in phsphlamban is sufficient in mediating its maximal cardiac respnses t ß-agnists. Jurnal f Bilgical Chemistry, 275: Said M, Mundiña-Weilenmann C, Vittne L et al. (22). The relative relevance f phsphrylatin f the Thr 17 residue f phsphlamban is different at different levels f ß-adrenergic stimulatin. Pflügers Archiv, 444: Mundiña de Weilenmann C, Vittne L, de Cinglani G et al. (1987). Dissciatin between cntractin and relaxatin: the pssible rle f phsphlamban phsphrylatin. Basic Research in Cardilgy, 82: Gaskell WH (188). On the tnicity f the heart and bld vessels. Jurnal f Physilgy, 3: Fabiat A & Fabiat F (1978). Myfilament-generated tensin scillatins during partial calcium activatin and activatin dependence f the sarcmere length-tensin relatin f skinned cardiac cells. Jurnal f General Physilgy, 72: Hulme JT & Orchard CH (1998). Effect f acidsis n Ca uptake and release by sarcplasmic reticulum f intact rat ventricular my- Braz J Med Bil Res 39(5) 26

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