Ablation of PLB exacerbates ischemic injury to a lesser ... - CiteSeerX

Am J Physiol Heart Circ Physiol 284: H683–H690, 2003. First published October 3, 2002; 10.1152/ajpheart.00567.2002.

Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO HEATHER R. CROSS,1 EVANGELIA G. KRANIAS,2 ELIZABETH MURPHY,3 AND CHARLES STEENBERGEN1 1 Department of Pathology, Duke University Medical Center, Durham 27710; National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709; and Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Submitted 8 July 2002; accepted in final form 30 September 2002

Cross, Heather R., Evangelia G. Kranias, Elizabeth Murphy, and Charles Steenbergen. Ablation of PLB exacerbates ischemic injury to a lesser extent in female than male mice: protective role of NO. Am J Physiol Heart Circ Physiol 284: H683–H690, 2003. First published October 3, 2002; 10.1152/ajpheart.00567.2002.—Recent studies suggest a role for phospholamban phosphorylation during ischemia and reperfusion. The role of phospholamban in ischemia was studied by subjecting hearts from male and female wild-type (MWT/FWT) and phospholamban-knockout (MKO/FKO) mice to 20 min of ischemia-40 min of reperfusion while 31P NMR spectra were acquired. ATP and pH values fell lower during ischemia, and postischemic contractility was less, in MKO and FKO versus WT hearts. After shorter ischemia (15 min), recoveries of contraction, ATP, and pH were greater in FKO than MKO hearts. To examine the role of nitric oxide (NO) synthases (NOS) in the protection in FKO versus MKO hearts, we utilized 1 ␮M L-NAME, a NOS inhibitor, or 100 ␮M S-nitroso-N-acetylpenicillamine (SNAP), an NO donor. Recoveries of function, ATP, and pH were less in L-NAMEtreated FKO than untreated FKO hearts and greater in SNAP-treated MKO than untreated MKO hearts. In conclusion, phospholamban ablation increased ischemic injury in both males and females; however, female hearts were less susceptible than male hearts. Protection in females was decreased by a NOS inhibitor and mimicked in males by an NO donor, implying that protection was NOS mediated. ischemia; energetics; sarcoplasmic reticulum

(PLB) is a sarcoplasmic reticulum (SR) protein that modifies activity of the cardiac SR Ca2⫹ATPase (SERCA2a) by reducing the affinity for Ca2⫹. In the basal, dephosphorylated state, PLB reduces SERCA2a Ca2⫹ affinity (13). However, when phosphorylated by cAMP-dependent protein kinase A (PKA) or Ca2⫹-calmodulin-dependent protein kinase, PLB dissociates from SERCA2a, leading to higher affinity of SERCA2a for Ca2⫹, higher SR Ca2⫹ load, greater SR Ca2⫹ release, and increased cardiac contractility (17– 20, 25). Consequently, PLB is the main mediator of the myocardial contractile response to catecholamines PHOSPHOLAMBAN

Address for reprint requests and other correspondence: H. R Cross, Dept. of Pathology, Box 3712, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: [email protected]). http://www.ajpheart.org

such as epinephrine and norepinephrine. Recent studies have demonstrated that PLB becomes phosphorylated on serine-16 after 20 min of ischemia by catecholamines (30), which are released endogenously from the ischemic myocardium (24). PLB is also phosphorylated on threonine-17 during early reperfusion via Ca2⫹-calmodulin-dependent protein kinase (30). It has been proposed that the ischemia-reperfusioninduced phosphorylation of PLB serves to decrease cytosolic Ca2⫹ overload by increasing sequestration of Ca2⫹ into the SR and is therefore a protective mechanism (30). However, the role of PLB in ischemic injury has not been studied. Because PLB inhibits SERCA2a activity in the unstimulated heart, removal of PLB should result in increased SERCA2a activity and thus mimic catecholamine-mediated phosphorylation of PLB. Consistent with this hypothesis, homozygous “knockout” mice with ablation of PLB (PLB-KO) exhibit increased SERCA2a activity and increased basal myocardial contractility (21). To study the role of PLB in ischemic injury, therefore, hearts from PLB-KO and wild-type mice were subjected to no-flow ischemia, and reperfusion and ischemic injury was determined by the extent of recovery of postischemic contractile function and energy metabolites. In addition, previous studies have demonstrated male/female differences in the susceptibility to myocardial ischemic injury in transgenic mice that overexpress the ␤2-adrenergic receptor (5) or Na⫹/Ca2⫹ exchanger (4) or in wild-type mice treated with the catecholamine, isoproterenol, or high extracellular Ca2⫹ (6). In all models, ischemic injury was greater in male than female hearts. Increased Ca2⫹ transport and adrenergic stimulation both lead to increased cytosolic and SR Ca2⫹ levels (6, 28). To determine whether the target of protection in females was at the level of SR Ca2⫹ homeostasis, the response to ischemia was compared in male and female hearts with ablation of PLB. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. H683

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GENDER DIFFERENCES IN PLB-KO MICE

MATERIALS AND METHODS

Animals PLB-KO mice and isogenic wild-type mice were developed as described previously (21). Twenty-three male adult homozygous PLB-KO mice (MKO) and twenty-five female adult homozygous PLB-KO mice (FKO) were used. Fifteen male and fourteen female isogenic wild-type mice (MWT and FWT, respectively) were employed as controls (21). All animals were treated in accordance with National Institutes of Health guidelines.

PLB ablation, to determine whether there were male/female differences in myocardial energetics or pH regulation, and to determine the energetic effects of the NO modulators. Relative changes in concentrations of phosphorus metabolites were observed during the ischemia-reperfusion protocol by acquiring consecutive 31P NMR spectra as described previously (7). The areas of the spectral peaks were expressed as a percentage of the peak areas of an initial, preischemic control spectrum from each heart. Intracellular pH was estimated from the chemical shift of the Pi peak relative to PCr using previously obtained titration curves.

Ischemia-Reperfusion Protocol

Statistics

Hearts were isolated and perfused in the Langendorff mode as described previously (7). Hearts were perfused for 30 min before being subjected to either 20 or 15 min (short ischemia; SI) of no-flow ischemia and 40 min of reperfusion. Left ventricular developed pressure (LVDP), positive and negative rate of pressure change (⫾dP/dt), and heart rate were monitored via a water-filled latex balloon inserted into the left ventricle. Before ischemia, basal end-diastolic pressure (EDP) was set between 10 and 20 cmH2O in all hearts. Recovery of contractile function was assessed by measurement of LVDP at the end of reperfusion and expressed as a percentage of preischemic LVDP.

Results are expressed as means ⫾ SE. Significance (P ⬍ 0.05) was determined by ANOVA, followed by a Fisher’s post hoc test.

Nitric Oxide Synthase Inhibitors and Nitric Oxide Donors To inhibit nitric oxide (NO) synthase (NOS), hearts from five female PLB-KO, five male wild-type, and seven female wild-type mice were perfused with the non-isoform-specific NOS inhibitor N␻-nitro-L-arginine methyl ester (L-NAME) at a final concentration of 1 ␮mol/l, beginning 5 min before ischemia and continuing until the end of reperfusion. The concentration of L-NAME used corresponded to the concentration required to inhibit NOS isoforms without inducing measurable vasoconstriction. In addition, six MKO and six FKO hearts were perfused with the NO donor dl-S-nitrosoN-acetylpenicillamine (SNAP) at a final concentration of 100 ␮mol/l, 5 min before ischemia and throughout reperfusion. NMR Spectroscopy Phosphate metabolite levels and intracellular pH were measured in all hearts to determine the energetic effects of

RESULTS

Effects of PLB Ablation Contractile function. During the preischemic period, LVDP, ⫹dP/dt, and ⫺dP/dt were higher in MKO hearts than MWT hearts (P ⬍ 0.0001; Table 1) and were higher in FKO than FWT hearts (P ⬍ 0.0001). Ablation of PLB, therefore, increased basal contractility in both male and female hearts. After 20 min of no-flow ischemia and 40 min of reperfusion, recovery of contractile function was negligible in both MKO, at ⬃1% initial LVDP, and FKO hearts, at ⬃2% initial LVDP (Fig. 1A). These values were significantly lower than MWT, at ⬃35% initial LVDP (P ⬍ 0.0001), and FWT hearts, at ⬃31% initial LVDP (P ⬍ 0.0001). Left ventricular EDP values were higher in MKO hearts, at 99 ⫾ 12 cmH2O, than in MWT hearts, at 46 ⫾ 7 cmH2O (P ⬍ 0.05), and higher in FKO, at 74 ⫾ 20 cmH2O, than in FWT, at 55 ⫾ 11 cmH2O (P ⬍ 0.05), consistent with the greater injury in PLB-KO versus wild-type hearts. Ablation of PLB, therefore, increased susceptibility to ischemic injury in both male and female hearts compared with the wildtype hearts.

Table 1. Effect of a NOS inhibitor and a NO donor on myocardial contractile function during normoxic perfusion Preinfusion Group

n

Heart rate, beats/min

MWT MKO FWT FKO MKSI FKSI FKSI ⫹ L-NAME MKSI ⫹ SNAP FKSI ⫹ SNAP

10 11 7 8 6 6 5 6 6

393 ⫾ 16 354 ⫾ 12 403 ⫾ 39 369 ⫾ 15 382 ⫾ 19 373 ⫾ 10 439 ⫾ 31 369 ⫾ 15 426 ⫾ 23

Postinfusion

LVDP, cmH2O

⫹dP/dt, cmH2O/ms

⫺dP/dt, cmH2O/ms

Heart rate, beats/min

LVDP, cmH2O

⫹dP/dt, cmH2O/ms

⫺dP/dt, cmH2O/ms

114 ⫾ 5 213 ⫾ 8*† 113 ⫾ 4 180 ⫾ 10*† 214 ⫾ 21 219 ⫾ 14 199 ⫾ 13 215 ⫾ 6 216 ⫾ 16

4.2 ⫾ 0.2 11.4 ⫾ 0.5*† 4.0 ⫾ 0.3 9.9 ⫾ 0.5*† 10.2 ⫾ 0.8 10.4 ⫾ 0.4 8.9 ⫾ 0.8 9.1 ⫾ 0.2 8.9 ⫾ 0.6

⫺3.2 ⫾ 0.1 ⫺11.3 ⫾ 0.6*† ⫺3.1 ⫾ 0.3 ⫺9.3 ⫾ 0.7*† ⫺10.4 ⫾ 0.8 ⫺9.4 ⫾ 0.5 ⫺8.8 ⫾ 0.9 ⫺9.1 ⫾ 0.6 ⫺8.9 ⫾ 0.6

429 ⫾ 24 381 ⫾ 15 430 ⫾ 23

202 ⫾ 10 208 ⫾ 13 211 ⫾ 14

8.9 ⫾ 0.7 8.9 ⫾ 0.5 8.8 ⫾ 0.6

⫺8.6 ⫾ 0.6 ⫺9.1 ⫾ 0.7 ⫺9.1 ⫾ 0.6

Data are means ⫾ SE; n, number of hearts. For preinfusion values, * significant difference from MWT; † significant difference from FWT. For postinfusion values, there were no significant differences between postinfusion and preinfusion values in any groups. NO, nitric oxide; NOS, NO synthase; LVDP, left ventricular developed pressure; ⫾dP/dt, positive and negative rate of pressure development; MWT, male, wild type; MKO, male, knockout; FWT, female, wild type; FKO, female, knockout; MKSI, MKO, short ischemia; FKSI, FKO, short ischemia; L-NAME, N␻-nitro-L-arginine methyl ester; SNAP, S-nitroso-N-acetylpenicillamine. AJP-Heart Circ Physiol • VOL

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(Fig. 2B). At the end of reperfusion, PCr was lower in MKO, at ⬃14% initial PCr, than in MWT hearts, at ⬃59% initial PCr (P ⬍ 0.0001), and was also lower in FKO, at ⬃25% initial PCr, than in FWT hearts, at ⬃55% initial PCr (P ⬍ 0.0001). Intracellular pH decreased during ischemia in all hearts (Fig. 2C). At the end of 20 min of ischemia, pH was lower in MKO, at approximately pH 5.67, than in MWT hearts, at approximately pH 5.85 (P ⬍ 0.01); however, by the end of ischemia, there was no significant difference in pH between FKO, at approximately pH 5.81, and FWT hearts, at approximately pH 5.87. During reperfusion, recovery of pH was delayed in MKO and FKO hearts, being approximately pH 5.95 in MKO, compared with approximately pH 6.93 in MWT (P ⬍ 0.0001), and approximately pH 6.21 in FKO, compared with approximately pH 6.87 in FWT (P ⬍ 0.01), at 5 min of reperfusion. By the end of reperfusion, however, there were no significant differences in pH between any groups of hearts.

Fig. 1. Postischemic recovery of left ventricular developed pressure (LVDP) in male and female wild-type (MWT, n ⫽ 10; FWT, n ⫽ 7) and male and female phospholamban (PLB)-knockout (KO) (MKO, n ⫽ 11; FKO, n ⫽ 8) (A); MKO and FKO, short ischemia (MKSI, n ⫽ 6; FKSI, n ⫽ 6); N␻-nitro-L-arginine methyl ester (L-NAME)-treated FKSI (FKSI ⫹ L-NAME, n ⫽ 5) and S-nitroso-N-acetylepenacillamine (SNAP)-treated MKSI and FKSI (MKSI ⫹ SNAP, n ⫽ 6; FKSI ⫹ SNAP, n ⫽ 6) hearts (B). Data are means ⫾ SE. * Significantly different from MWT; † significantly different from FWT; ‡significantly different from MKSI; §significantly different from FKSI (P ⬍ 0.05).

Phosphate metabolite levels and intracellular pH. The ratios of the PCr/ATP peaks in the preischemic control spectra were lower in the MKO hearts, at 1.18 ⫾ 0.04, than in MWT hearts, at 1.40 ⫾ 0.05 (P ⬍ 0.05), and lower in FKO, at 1.19 ⫾ 0.07, than in FWT hearts, at 1.50 ⫾ 0.10 (P ⬍ 0.05). These findings indicate a difference in basal energetics in PLB-KO versus wild-type hearts, possibly resulting from a higher basal energy demand in the PLB-KO hearts. The lower PCr/ ATP is consistent with previous studies by Chu et al. (2), who demonstrated that PCr levels were lower in PLB-KO mice than in wild-type mice. During 20 min of ischemia, ATP levels fell lower in MKO hearts, reaching ⬃12% initial ATP, than in MWT hearts, at ⬃29% initial ATP (P ⬍ 0.001; Fig. 2A), and lower in FKO hearts, at ⬃12% initial ATP, than in FWT hearts, at ⬃28% initial ATP (P ⬍ 0.01). During reperfusion, ATP levels remained lower in MKO hearts, at ⬃12% initial ATP, than in MWT hearts, which increased to ⬃40% initial ATP by the end of reperfusion (P ⬍ 0.0001). ATP levels also remained lower in FKO hearts, at ⬃13% initial ATP, than in FWT hearts, at ⬃52% initial ATP, by the end of reperfusion (P ⬍ 0.01). PCr decreased rapidly at the onset of 20 min of ischemia and increased on reperfusion in all hearts AJP-Heart Circ Physiol • VOL

Fig. 2. Myocardial levels of ATP (A) and PCr (B) and intracellular pH (C) during ischemia and reperfusion. Points are means ⫾ SE.

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In summary, ablation of PLB resulted in a greater loss of ATP during ischemia in both male and female hearts, indicating greater ischemic energy demand. During reperfusion, recoveries of ATP and PCr were lower and recovery of intracellular pH was delayed in MKO and FKO hearts compared with wild-type hearts, indicating greater injury. Male/Female Differences During Short Ischemia and Effects of L-NAME and SNAP Contractile function. Because 20 min of ischemia resulted in negligible recovery of contractile function in both MKO and FKO hearts, a second group of PLB-KO hearts (MKSI and FKSI) was subjected to a shorter ischemic insult of 15-min duration to allow recovery of function to be compared. After 15 min of no-flow ischemia and 40 min of reperfusion, recovery of contractile function was greater in FKSI hearts, at ⬃22% initial LVDP, than in MKSI hearts, at ⬃1% initial LVDP (P ⬍ 0.001; Fig. 1B). Left ventricular EDP values were higher in MKSI hearts, at 119 ⫾ 4 cmH2O, than in FKSI hearts, at 43 ⫾ 12 cmH2O (P ⬍ 0.05), consistent with the greater injury in MKSI versus FKSI hearts. Therefore, female hearts were less susceptible to the effects of PLB ablation, with respect to ischemic injury, than male hearts. To determine the role of NO in the observed female cardioprotection, FKSI hearts were pretreated with 1 ␮mol/l of the non-isoform-specific NOS inhibitor, LNAME, beginning 5 min before ischemia, and MKSI and FKSI hearts were perfused with 100 ␮mol/l SNAP, an NO donor, also beginning 5 min before ischemia. During normoxic perfusion, neither L-NAME nor SNAP had effects on heart rate, LVDP, ⫹dP/dt, or ⫺dP/dt in any group (Table 1). Coronary flow was higher in MKO, at 2.8 ⫾ 0.2 ml/min, and FKO, at 2.5 ⫾ 0.1 ml/min, than in MWT, at 1.9 ⫾ 0.1 ml/min, and FWT hearts, at 1.6 ⫾ 0.1 ml/min (P ⬍ 0.05). Coronary flow was unaltered by perfusion with L-NAME, at 2.7 ⫾ 0.1 ml/min preinfusion and 2.5 ⫾ 0.1 ml/min postinfusion in FKSI hearts. There was similarly no effect of 1 ␮mol/l L-NAME on contractile function and coronary flow in MWT and FWT hearts (Table 2). There was no significant effect of SNAP on coronary flow at 2.9 ⫾ 0.1 ml/min preinfusion and 3.0 ⫾ 0.1 ml/min postinfusion in MKSI hearts and 2.6 ⫾ 0.1 ml/min preinfusion and 2.9 ⫾ 0.2 ml/min postinfusion in FKSI hearts. After 15 min of ischemia and 40 min of reperfusion, recovery of contractile function was lower in FKSI ⫹ L-NAME hearts, at ⬃1% initial LVDP, than in untreated FKSI hearts (P ⬍ 0.001; Fig. 1B) and as low as

in untreated MKSI hearts. This is in contrast to MWT and FWT hearts, in which treatment with 1 ␮mol/l L-NAME before 20 min of ischemia had no effect on postischemic functional recovery (Table 3). Left ventricular EDP values were higher in FKSI ⫹ L-NAME hearts at 83 ⫾ 11 cmH2O than untreated FKSI hearts at 43 ⫾ 12 cmH2O (P ⬍ 0.05), consistent with the greater injury in the L-NAME-treated FKSI hearts. Therefore, the protection from ischemic injury observed in FKO, compared with MKO hearts, was abolished by pretreatment with L-NAME. These results imply that the protection in FKO hearts is mediated by NOS. After 15 min of ischemia and 40 min of reperfusion, recovery of contractile function was higher in MKSI ⫹ SNAP hearts, at ⬃17% initial LVDP, than in untreated MKSI hearts (P ⬍ 0.01; Fig. 1B) and as high as in untreated FKSI hearts. There was no difference in postischemic functional recovery between FKSI ⫹ SNAP hearts (at ⬃24% initial LVDP) and untreated FKSI hearts. Left ventricular EDP values were lower in MKSI ⫹ SNAP hearts at 68 ⫾ 13 cmH2O than in untreated MKSI hearts at 119 ⫾ 4 cmH2O (P ⬍ 0.05) consistent with the lower injury in the SNAP-treated MKSI hearts. Therefore, perfusion of MKO hearts with an NO donor increased recovery from ischemia to the same level as observed with FKO hearts, consistent with a role for NO in the protection observed in FKO hearts. Phosphate metabolite levels and intracellular pH. There were no differences in ATP levels between MKSI and FKSI hearts during 15 min of ischemia (Fig. 3A). However, during reperfusion, ATP was higher in FKSI, reaching ⬃38% initial ATP, than MKSI hearts, at ⬃6% initial ATP at the end of reperfusion (P ⬍ 0.01). Neither L-NAME nor SNAP had an effect on ATP levels during ischemia in any hearts (Fig. 3A). However, ATP levels were lower in FKSI ⫹ L-NAME hearts at ⬃7% initial ATP at the end of reperfusion than in untreated FKSI hearts (P ⬍ 0.01) and as low as untreated MKSI hearts. ATP levels recovered to a greater extent in MKSI ⫹ SNAP hearts, reaching ⬃36% initial ATP by the end of reperfusion, than in untreated MKSI hearts (P ⬍ 0.05). There were no differences in ATP levels between FKSI ⫹ SNAP and FKSI hearts during reperfusion. At the end of 15 min of ischemia, there were no significant differences in PCr levels between MKSI and FKSI hearts (Fig. 3B). However, at the end of reperfusion, PCr was higher in FKSI, reaching ⬃44% initial PCr, than MKSI hearts, at ⬃20% initial PCr (P ⬍

Table 2. Effect of L-NAME on myocardial contractile function and coronary flow during normoxic perfusion Preinfusion

Postinfusion

Group

n

Coronary flow, ml/min

LVDP, cmH2O

⫹dP/dt, cmH2O/ms

⫺dP/dt, cmH2O/ms

Coronary flow, ml/min

LVDP, cmH2O

⫹dP/dt, cmH2O/ms

⫺dP/dt, cmH2O/ms

MWT ⫹ L-NAME FWT ⫹ L-NAME

5 7

1.6 ⫾ 0.2 1.8 ⫾ 0.2

112 ⫾ 1 117 ⫾ 2

3.9 ⫾ 0.1 3.6 ⫾ 0.2

⫺3.0 ⫾ 0.1 ⫺2.6 ⫾ 0.2

1.9 ⫾ 0.1 1.6 ⫾ 0.1

116 ⫾ 4 117 ⫾ 3

3.7 ⫾ 0.1 3.3 ⫾ 0.1

⫺2.8 ⫾ 0.1 ⫺2.5 ⫾ 0.1

Data are means ⫾ SE; n, no. of hearts. There were no significant differences between postinfusion and preinfusion values in either group. AJP-Heart Circ Physiol • VOL

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Table 3. Effect of L-NAME during 20 min of ischemia-40 min of reperfusion: phosphate metabolites and postischemic contractile recovery Ischemia

Reperfusion

Group

n

ATP, %initial

PCr, %initial

pH

ATP, %initial

PCr, %initial

Recovery, %LVDP

MWT FWT MWT ⫹ L-NAME FWT ⫹ L-NAME

10 7 5 7

29 ⫾ 4 28 ⫾ 5 39 ⫾ 7 30 ⫾ 6

9⫾5 4⫾1 4⫾1 3⫾5

5.85 ⫾ 0.04 5.87 ⫾ 0.07 5.84 ⫾ 0.04 5.83 ⫾ 0.05

40 ⫾ 5 52 ⫾ 8 42 ⫾ 6 45 ⫾ 6

59 ⫾ 8 55 ⫾ 8 60 ⫾ 5 61 ⫾ 9

39 ⫾ 4 34 ⫾ 8 35 ⫾ 6 31 ⫾ 4

Data are means ⫾ SE; n, no. of hearts. There were no significant differences in any values between treated and untreated groups.

0.01). Neither L-NAME nor SNAP had an effect on PCr levels during ischemia in any hearts (Fig. 3B). PCr levels were lower in FKSI ⫹ L-NAME hearts, at ⬃16% initial PCr at the end of reperfusion, than in untreated FKSI hearts (P ⬍ 0.01) and as low as untreated MKSI hearts. PCr levels recovered to a greater extent in MKSI ⫹ SNAP hearts, reaching ⬃46% initial PCr by

the end of reperfusion, than in untreated MKSI hearts (P ⬍ 0.01). There were no differences in PCr levels between FKSI ⫹ SNAP and FKSI hearts during reperfusion. There was no difference in intracellular pH at the end of 15 min of ischemia between MKSI and FKSI hearts (Fig. 3C). During reperfusion, recovery of pH was delayed in MKSI hearts, being approximately pH 6.04 at 5 min of reperfusion compared with approximately pH 6.88 in FKSI hearts (P ⬍ 0.001). Recovery of pH was delayed in FKSI ⫹ L-NAME hearts, being approximately pH 5.98 at 5 min of reperfusion, compared with untreated FKSI hearts (P ⬍ 0.001; Fig. 3C). In contrast, recovery of pH was faster in MKSI ⫹ SNAP hearts, being approximately pH 6.81 at 5 min of reperfusion, than untreated MKSI hearts (P ⬍ 0.01). There were no differences in pH recovery between FKSI ⫹ SNAP and FKSI hearts. By the end of reperfusion, there were no significant differences in pH between any groups of hearts. To summarize, during the shorter 15-min ischemic insult, there were still no differences in energy metabolites and intracellular pH between MKO or FKO hearts. However, during reperfusion, male/female differences became apparent; recoveries of ATP, PCr, and intracellular pH were greater in FKO than MKO hearts. Pretreatment of FKO hearts with L-NAME lowered postischemic recoveries of energy metabolites and pH to similar levels as those observed in untreated MKO hearts, whereas pretreatment of MKO hearts with SNAP increased postischemic recoveries of energy metabolites and pH. These results provide further support for the hypothesis that protection in females is mediated by NOS. DISCUSSION

Effects of PLB Ablation on Contractility and Ischemic Injury in Male and Female Hearts

Fig. 3. Myocardial levels of ATP (A) and PCr (B) and intracellular pH (C) during ischemia and reperfusion. Points are means ⫾ SE. AJP-Heart Circ Physiol • VOL

In the present study, perfused hearts from both MKO and FKO mice exhibited an approximate 70% increase in peak LVDP, an approximate 150% increase in ⫹dP/dt, and an approximate 250% increase rate of relaxation ⫺dP/dt, compared with wild-type hearts, consistent with previous studies (21) and also consistent with the role of PLB in inhibiting SERCA2a activity. Interestingly, the rate of relaxation was increased in PLB-KO hearts to a greater extent than the

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rate of contraction, consistent with the primary role of SERCA2a in restoring cytosolic Ca2⫹ levels and mediating the relaxation phase of the cardiac cycle (1). Recent studies demonstrate that cardiac PLB becomes phosphorylated during ischemia via endogenous catecholamines and during reperfusion via Ca2⫹-calmodulin-dependent protein kinase (30). Because phosphorylation and ablation of PLB both result in increased SERCA2a activity, we studied the role of PLB in ischemic injury by subjecting hearts from PLB-KO and wild-type mice to no-flow ischemia and reperfusion. Ischemic injury was increased in both MKO and FKO hearts, as indicated by negligible postischemic recovery of contractile function, lower postischemic recoveries of ATP and PCr, and slower recovery of intracellular pH than in MWT and FWT hearts. During ischemia, ATP levels fell lower in MKO and FKO hearts than MWT and FWT hearts, reflecting greater ischemic energy utilization. Because H⫹ are produced by ATP hydrolysis, the greater net ATP utilization in the MKO and FKO hearts was consistent with the lower pH observed in these hearts. Ablation of PLB, therefore, resulted in increased ischemic energy demand and increased susceptibility to ischemic injury in male and female hearts compared with wild-type hearts. As discussed earlier, it has been proposed that the ischemia-reperfusion-induced phosphorylation of PLB and consequent decrease in PLB inhibition of SERCA2a is a protective mechanism (30). However, our present findings indicate that removal of PLB is detrimental. Although permanent PLB ablation is not directly comparable to transient removal of PLB activity via phosphorylation, our results highlight the possibility of a maladaptive role of the reported ischemic phosphorylation of PLB. Our results imply conversely that the presence of PLB is beneficial to the ischemic-reperfused heart. The mechanism of exacerbation of ischemia-reperfusion injury via PLB ablation is unknown. We assume during our discussion of male/female differences below that increased SR Ca2⫹ plays a major role in the effects of PLB ablation. However, there are a number of energetic alterations in PLB-KO versus wild-type hearts, as reviewed by Kiriazis and Kranias (14), which may play a direct role, such as increased oxygen consumption, decreased PCr levels, or an increase in the active fraction of mitochondrial pyruvate dehydrogenase, reflecting increased ATP synthesis. Male/Female Differences in Ischemic Injury in PLB-KO Mouse Hearts Previous studies demonstrated protection from myocardial ischemic injury in female transgenic mice that overexpress the ␤2-adrenergic receptor (5) or Na⫹/Ca2⫹ exchanger (4) or in wild-type mice treated with the catecholamine, isoproterenol, or high extracellular Ca2⫹(6). Because increased Ca2⫹ transport and adrenergic stimulation both lead to increased cytosolic and SR Ca2⫹ levels, we determined whether the target of protection in females was at the level of SR Ca2⫹ AJP-Heart Circ Physiol • VOL

homeostasis by comparing the response to ischemia in male versus female hearts with ablation of PLB. Because 20 min of ischemia resulted in negligible recovery of contractile function in both MKO and FKO hearts, a second group of MKO and FKO hearts were subjected to a shorter ischemic insult of 15-min duration to allow injury to be compared. During the shorter ischemic insult there were still no differences in energy metabolites and intracellular pH between MKO and FKO hearts. However, on reperfusion, male/female differences became apparent, recoveries of contractile function, ATP, PCr, and intracellular pH being greater in FKO than MKO hearts. Therefore, female hearts were less susceptible to the effects of PLB ablation, with respect to ischemia-reperfusion injury, than male hearts. These results support a role for adrenergic targets downstream of PLB, such as SR Ca2⫹ homeostasis, in sex-specific injury. Interestingly, recent studies have revealed that females are protected from cardiac failure resulting from overexpression of a mutant PLB, which acts as a super inhibitor of SERCA2a (9). Females, therefore, appear to be protected from the consequences of both increased or decreased SERCA2a inhibition by PLB, indicating that SR Ca2⫹ homeostasis may be more tightly regulated in female than male hearts. Role of NOS in Protection From Ischemic Injury in Female PLB-KO Mouse Hearts The observed protection in FKO hearts is consistent with clinical findings indicating that females are protected from cardiovascular injury (8, 10, 12, 26). The protection observed clinically is mediated via estrogen. Alterations in atherosclerosis and vascular function contribute to the protection; however, direct protective effects of estrogen on the heart have also been implicated by isolated tissue and organ studies (16, 22). Estrogen increases expression of several NOS isoforms in the myocardium (23), and a number of studies have found NO produced via inducible NOS, endothelial NOS, or NO donors to be cardioprotective (11, 27, 31). Because NO is known to affect SR Ca2⫹ homeostasis (29, 32), we studied the role of NO and of the estrogen effector, NOS, in the observed protection in FKO hearts. To determine the role of NOS in the observed female cardioprotection, FKO mouse hearts were pretreated with 1 ␮mol/l of the non-isoform-specific NOS inhibitor, L-NAME, 5 min before ischemia and throughout reperfusion. The concentration of L-NAME used corresponded to the concentration required to inhibit NOS isoforms without inducing measurable vasoconstriction; we observed no effect of 1 ␮mol/l L-NAME on contractile function and coronary flow in any hearts during normoxic perfusion. There was no effect of pretreatment with L-NAME on ATP, PCr, or intracellular pH during ischemia in FKO hearts. However, the postischemic recoveries of contractile function, ATP, and PCr were less and the recovery of intracellular pH was delayed in the L-NAME-treated FKO hearts compared

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with untreated FKO hearts, indicating greater injury. Notably, postischemic recoveries of contractile function and energy metabolites were as low in the L-NAMEtreated FKO hearts as in untreated MKO hearts. Therefore, the protection from ischemic injury observed in female, compared with male, PLB-KO hearts was abolished by pretreatment with L-NAME. These results imply that the protection in FKO hearts is mediated by NOS. To determine whether NO could provide protection against ischemic injury in males, we studied the ischemic response of MKO and FKO hearts pretreated with 100 ␮mol/l of the NO donor, SNAP, 5 min before ischemia and throughout reperfusion. There was no effect of pretreatment with SNAP on ATP, PCr, or intracellular pH during ischemia in MKO or FKO hearts. However, the postischemic recoveries of contractile function, ATP, and PCr were greater, and the recovery of intracellular pH was faster, in the SNAP-treated MKO hearts compared with untreated MKO hearts, indicating less injury. Notably, postischemic recoveries of contractile function and energy metabolites were as high in the SNAP-treated MKO hearts as in untreated FKO hearts. Therefore, the protection from ischemic injury observed in FKO hearts was mimicked in MKO hearts by pretreatment with an NO donor. These results are consistent with a role for NO in the protection observed in FKO hearts. In summary, by comparing the ischemic response of hearts from MKO and FKO mice to that of wild-type mice, we demonstrated that ablation of PLB results in increased ischemic energy demand and increased injury. Because PLB becomes phosphorylated during ischemia and reperfusion and phosphorylation and ablation of PLB have the similar effects on SERCA2a activity, these results, although not definitive, may imply that phosphorylation of PLB has a pathophysiological role in ischemic injury. Interestingly, because PLB is decreased and SERCA2a activity is increased in hyperthyroid hearts (15), our findings indicate that these alterations could contribute to the increased susceptibility to ischemic injury observed in hyperthyroid hearts (3). A comparison of the responses of MKO and FKO hearts to shorter ischemia indicated that female hearts were less susceptible to the effects of PLB ablation, with respect to ischemia-reperfusion injury, than males. These results support a role for SR Ca2⫹ homeostasis in sex-specific injury. In addition, pretreatment of FKO mouse hearts with the nonspecific NOS inhibitor L-NAME, and examination of the ischemic response, revealed that the protection in female, compared with male, PLB-KO mouse hearts was mediated via NOS. Further support for the role of NO in the protection observed in PLB-KO females was provided by pretreatment of hearts with the NO donor SNAP; we demonstrated that the protection from ischemic injury observed in FKO hearts was mimicked in MKO hearts by SNAP. Taken together, these results imply that females may be protected from cardiovascular AJP-Heart Circ Physiol • VOL

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injury via an NO-mediated mechanism involving maintenance of SR Ca2⫹ homeostasis. The authors thank Karen B. Young for mouse genotyping and Dr. Robert E. London for use of NMR facilities. We thank the National Heart, Lung, and Blood Institute for the following grants: HL-37952 (to C. Steenbergen) and HL-26057, HL52318, P40RR12358, and HL-64018 (to E. G. Kranias). REFERENCES 1. Balke CW, Egan TM, and Wier WG. Processes that remove calcium from the cytoplasm during excitation-contraction coupling in intact rat heart cells. J Physiol 474: 447–462, 1994. 2. Chu G, Luo W, Slack JP, Tilgmann C, Sweet WE, Spindler M, Saupe KW, Boivin GP, Moravec CS, Matlib MA, Grupp IL, Ingwall JS, and Kranias EG. Compensatory mechanisms associated with the hyperdynamic function of phospholambandeficient mouse hearts. Circ Res 79: 1064–1076, 1996. 3. Clarke K, Sunn N, and Willis RJ. 31P NMR spectroscopy of hyperthyroid rat hearts: effect of graded global ischemia. J Mol Cell Cardiol 21: 1315–1325, 1989. 4. Cross HR, Lu L, Steenbergen C, Philipson KD, and Murphy E. Overexpression of the cardiac Na⫹/Ca2⫹ exchanger increases susceptibility to ischemia/reperfusion injury in male, but not female, transgenic mice. Circ Res 83: 1215–1223, 1998. 5. Cross HR, Murphy E, Koch WJ, and Steenbergen C. Male and female mice overexpressing the beta(2)-adrenergic receptor exhibit differences in ischemia/reperfusion injury: role of nitric oxide. Cardiovasc Res 53: 662–671, 2002. 6. Cross HR, Murphy E, and Steenbergen C. Ca2⫹ loading and adrenergic stimulation reveal male/female differences in susceptibility to ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 283: H481–H489, 2002. 7. Cross HR, Steenbergen C, Lefkowitz RJ, Koch WJ, and Murphy E. Overexpression of the ␤2-adrenergic receptor and a ␤ARK1 inhibitor both increase contractility but have differential effects on susceptibility to ischemic injury. Circ Res 85: 1077– 1084, 1999. 8. Godsland IF, Wynn V, Crook D, and Miller NE. Sex, plasma lipoproteins and atherosclerosis: prevailing assumptions and outstanding questions. Am Heart J 114: 1467–1471, 1987. 9. Haghighi K, Schmidt AG, Hoit BD, Brittsan AG, Yatani A, Lester JW, Zhai J, Kimura Y, Dorn II GW, MacLennan DH, and Kranias EG. Superinhibition of sarcoplasmic reticulum function by phospholamban induces cardiac contractile failure. J Biol Chem 276: 24145–24152, 2001. 10. Heckbert SR, Weiss NS, Koepsell TD, Lemaitre RN, Smith NL, Siscovick DS, Lin D, and Psaty BM. Duration of estrogen replacement therapy in relation to the risk of incident myocardial infarction in postmenopausal women. Arch Intern Med 157: 1330–1336, 1997. 11. Hoshida S, Yamashita N, Igarashi J, Nishida M, Hori M, Kamda T, Kuzuya T, and Tada M. Nitric oxide synthase protects the heart against ischemia-reperfusion injury in rabbits. J Pharmacol Exp Ther 274: 413–418, 1995. 12. Isles CG, Hole DH, Hawthorne VM, and Lever AF. Relation between coronary risk and coronary mortality in women of the Renfrew and Paisley survey: comparison with men. Lancet 339: 702–706, 1992. 13. Kim HW, Steenaart NAE, Ferguson DG, and Kranias EG. Functional restitution of the cardiac sarcoplasmic reticulum Ca2⫹ ATPase with phospholamban in phospholipid vesicles. J Biol Chem 265: 1702–1709, 1990. 14. Kiriazis H and Kranias EG. Genetically engineered models with alterations in cardiac membrane calcium-handling proteins. Annu Rev Physiol 62: 321–351, 2000. 15. Kiss E, Jakab G, Kranias EG, and Edes I. Thyroid hormoneinduced alterations in phospholamban protein expression. Circ Res 75: 245–251, 1994. 16. Kolodgie FD, Farb A, and Litovsky SH. Myocardial protection of contractile function after global ischemia by physiologic estrogen in the ovariectomized rat. J Mol Cell Cardiol 29: 2403– 2414 1997.

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