E abdominal cavity was exposed, hearts were quickly JW-74 site isolated and rinsed with ice-cold Ca2+ free buffer as we have reported before [7]. Oxygenated KHB was perfused through Mouse heart perfusion system (TME Technology, Chengdu, CHINA) in the experiment [22].GPR30 and Chronic CardioprotectionFigure 4. Function determination of the single cardiac cell. Myocytes of the six groups incubated in veh medium, shortening amplitude, TTP and R90 are shown as figure (A), (B) and (C) respectively. Data shown are mean6 S.E.M. n = 10 hearts. *p,0.05 order AN-3199 versus Sham, #p,0.05 versus OVX+ISO and p,0.05 versus OVX. OVX+ISO 
and OVX+ISO+G-1 groups were incubated in veh, CGP or ICI medium. Shortening amplitude, TTP and R90 are shown as figure (D), (E) and (F) respectively. Data shown are mean6S.E.M. n = 10 hearts. #p,0.05 versus OVX+ISO of each subgroup. doi:10.1371/journal.pone.0048185.gGPR30 and Chronic CardioprotectionFigure 5. The expression of beta-AR. The expression of b-actin was detected as an internal standard. The relative arbitrary unit for sham group was assigned as (A). Figure (B) and (C) showed the expression of b1-AR and b2-AR. Each value represents the mean6S.E.M. n = 10 hearts in each group, *P,0.05 versus Sham, #P,0.05 versus OVX+ISO, p,0.05 versus OVX. doi:10.1371/journal.pone.0048185.gDetermination of Cardiac FunctionLinked aortic side of the isolated heart to perfusion device, left ventricular pressure was recorded using a Biopac system (BIOPAC) via a pressure sensor Millar transducer instrument (1.4F, Millar). Hearts were equilibrated for 30 minutes with KHB. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), and the rate in rise and fall of ventricular pressure (+dp/dtmax, -dp/dtmax) were recorded as left ventricular functional parameters. The left ventricular developed pressure (LVDP) were calculated as LVDP = LVSPLVEDP; rate pressure product (RPP) was calculated as RPP = HR6LVDP.Masson’s Trichrome StainRat hearts were perfused with KHB for 20 minutes to remove the residual blood, and then fixed with 10 formalin before embedding in paraffin. All hearts were embedded in a cross section orientation, and all slices were cross sections 
of the heart. All 1081537 slices were taken from the midpoint of the left ventricles. Fourmicrometer slices were deparaffinized and rehydrated. Then the instruction of masson’s trichrome stain kit was followed. Microscope (IX 71, OLYMPUS, Japan) was used to get the pictures. The normal cardiac myocytes were stained red, and fibrotic areas were stained green.GPR30 and Chronic CardioprotectionIsolation and Culture of Myocardial CellsVentricular myocytes were isolated from the hearts as we described before [7,22]. Cardiac ventricular myocytes were isolated from the hearts then the cells were suspended in dulbecco’s minimal essential medium (DMEM) at a density of 36105 cells per well, finally took them to carbon dioxide incubator (Heraeus, Germany), parameters were set as below: 5 CO2, 37uC, cultured for 1 hour to stabilize the cells. The rat cardiomyocytes were incubated with vehicle (veh), CGP20712A (CGP, 300 nM), or ICI118551 (ICI, 55 nM), for 2 h to study the impact of different b-AR antagonists on shortening amplitude in rat myocytes.myocytes shortened upon electrical stimulation, an indicative of peak ventricular; contractility time-to-peak contraction (TTP), the duration of myocyte shortening, an indicative of systolic duration; and time-to-90 relaxation (R.E abdominal cavity was exposed, hearts were quickly isolated and rinsed with ice-cold Ca2+ free buffer as we have reported before [7]. Oxygenated KHB was perfused through Mouse heart perfusion system (TME Technology, Chengdu, CHINA) in the experiment [22].GPR30 and Chronic CardioprotectionFigure 4. Function determination of the single cardiac cell. Myocytes of the six groups incubated in veh medium, shortening amplitude, TTP and R90 are shown as figure (A), (B) and (C) respectively. Data shown are mean6 S.E.M. n = 10 hearts. *p,0.05 versus Sham, #p,0.05 versus OVX+ISO and p,0.05 versus OVX. OVX+ISO and OVX+ISO+G-1 groups were incubated in veh, CGP or ICI medium. Shortening amplitude, TTP and R90 are shown as figure (D), (E) and (F) respectively. Data shown are mean6S.E.M. n = 10 hearts. #p,0.05 versus OVX+ISO of each subgroup. doi:10.1371/journal.pone.0048185.gGPR30 and Chronic CardioprotectionFigure 5. The expression of beta-AR. The expression of b-actin was detected as an internal standard. The relative arbitrary unit for sham group was assigned as (A). Figure (B) and (C) showed the expression of b1-AR and b2-AR. Each value represents the mean6S.E.M. n = 10 hearts in each group, *P,0.05 versus Sham, #P,0.05 versus OVX+ISO, p,0.05 versus OVX. doi:10.1371/journal.pone.0048185.gDetermination of Cardiac FunctionLinked aortic side of the isolated heart to perfusion device, left ventricular pressure was recorded using a Biopac system (BIOPAC) via a pressure sensor Millar transducer instrument (1.4F, Millar). Hearts were equilibrated for 30 minutes with KHB. Heart rate (HR), left ventricular systolic pressure (LVSP), left ventricular enddiastolic pressure (LVEDP), and the rate in rise and fall of ventricular pressure (+dp/dtmax, -dp/dtmax) were recorded as left ventricular functional parameters. The left ventricular developed pressure (LVDP) were calculated as LVDP = LVSPLVEDP; rate pressure product (RPP) was calculated as RPP = HR6LVDP.Masson’s Trichrome StainRat hearts were perfused with KHB for 20 minutes to remove the residual blood, and then fixed with 10 formalin before embedding in paraffin. All hearts were embedded in a cross section orientation, and all slices were cross sections of the heart. All 1081537 slices were taken from the midpoint of the left ventricles. Fourmicrometer slices were deparaffinized and rehydrated. Then the instruction of masson’s trichrome stain kit was followed. Microscope (IX 71, OLYMPUS, Japan) was used to get the pictures. The normal cardiac myocytes were stained red, and fibrotic areas were stained green.GPR30 and Chronic CardioprotectionIsolation and Culture of Myocardial CellsVentricular myocytes were isolated from the hearts as we described before [7,22]. Cardiac ventricular myocytes were isolated from the hearts then the cells were suspended in dulbecco’s minimal essential medium (DMEM) at a density of 36105 cells per well, finally took them to carbon dioxide incubator (Heraeus, Germany), parameters were set as below: 5 CO2, 37uC, cultured for 1 hour to stabilize the cells. The rat cardiomyocytes were incubated with vehicle (veh), CGP20712A (CGP, 300 nM), or ICI118551 (ICI, 55 nM), for 2 h to study the impact of different b-AR antagonists on shortening amplitude in rat myocytes.myocytes shortened upon electrical stimulation, an indicative of peak ventricular; contractility time-to-peak contraction (TTP), the duration of myocyte shortening, an indicative of systolic duration; and time-to-90 relaxation (R.
