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Critical Care Medicine  |   October 2012
Intralipid, a Clinically Safe Compound, Protects the Heart Against Ischemia-Reperfusion Injury More Efficiently Than Cyclosporine-A
Author Affiliations & Notes
  • Jingyuan Li, M.D., Ph.D.
    *
  • Andrea Iorga, B.Sc.
  • Salil Sharma, Ph.D.
    *
  • Ji-Youn Youn, Ph.D.
    *
  • Rod Partow-Navid, B.Sc.
  • Soban Umar, M.D., Ph.D.
    *
  • Hua Cai, M.D., Ph.D.
    §
  • Siamak Rahman, M.D.
  • Mansoureh Eghbali, Ph.D.
    #
  • *Postdoctoral Fellow, Ph.D. Student, Master's Student, §Professor, Associate Clinical Professor and Director of Acute Pain Service, #Assistant Professor, Department of Anesthesiology, Division of Molecular Medicine, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California.
Article Information
Critical Care Medicine / Cardiovascular Anesthesia / Critical Care
Critical Care Medicine   |   October 2012
Intralipid, a Clinically Safe Compound, Protects the Heart Against Ischemia-Reperfusion Injury More Efficiently Than Cyclosporine-A
Anesthesiology 10 2012, Vol.117, 836-846. doi:10.1097/ALN.0b013e3182655e73
Anesthesiology 10 2012, Vol.117, 836-846. doi:10.1097/ALN.0b013e3182655e73
What We Already Know about This Topic
  • Previous studies have demonstrated that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury

  • This study compared the cardioprotective effect of intralipid with cyclosporine-A when administered at the onset of myocardial reperfusion

What This Article Tells Us That Is New
  • Intralipid is more effective than cyclosporine-A in reducing myocardial infarct size and improving cardiac functional recovery after ischemia-reperfusion

ACUTE myocardial infarction is responsible for the death of millions of people worldwide each year. Although early reperfusion is the only way to salvage an ischemic organ, during the crucial early moments of reperfusion, significant reversible and irreversible organ damage is initiated, a process referred to as reperfusion injury. The reperfusion injury oftentimes is even more damaging than the ischemia itself because of oxidative damage caused by free radicals and calcium overload as a result of reintroduction of blood to the tissue.1 Pharmacological postconditioning has been used to protect the heart against ischemia-reperfusion (I/R) injury. Cardioplegic arrest and cardiopulmonary bypass are also other key triggers of myocardial injury during cardiac surgery, and multiple techniques have been used to protect the heart during the surgical requirement for global or regional ischemia. Many pharmacological agents have been shown in experimental studies to have the ability to induce a polarized arrest and to better protect the heart, such as a cardio-selective β1-blocker2 and the adenosine triphosphate-sensitive potassium channel openers.3,4 However, none of pharmacological candidates have been widely accepted.5 Recently we have shown that postischemic treatment with itralipid, the first safe fat emulsion for human use, improves the cardiac functional recovery of isolated Langendorff-perfused mouse hearts by approximately 4-fold and results in 70% reduction in the myocardial infarct size.6 
The mitochondrial permeability transition pore (mPTP) is a large nonselective conductance pore located in the inner membrane of mitochondria. Although the exact molecular identity of mPTP has been questioned recently, cychlophilin-D (CypD) is still an established component of mPTP.7,8 The mPTP remains closed during ischemia, but opens during the reperfusion period.9,10 Opening of the mPTP is favored by events occurring during ischemia and reperfusion, including overproduction of reactive oxygen species and accumulation of Ca2+in the mitochondrial matrix.10 Delaying the opening of the mPTP upon reperfusion has been a potential target to reduce myocardial injury. To date, the most specific inhibitor of the mPTP is cyclosporine-A,11 which acts by directly inhibiting the peptidyl-prolyl cis-trans isomerase activity of CypD, which is a key component of the mPTP.12,13 Recently we demonstrated that intralipid inhibits the opening of the mPTP and protects the heart by recruiting the reperfusion injury salvage kinase pathway, phosphatidylinositol 3-kinase/Akt/ERK1, and leading to phosphorylation of GSK-3β.6 
Here we compared the cardioprotective effect of intralipid with cyclosporine-A when administrated at the onset of reperfusion. Our data revealed that although intralipid inhibits the mPTP opening as efficiently as cyclosporine-A, intralipid is more effective in protecting the heart against I/R injury than cyclosporine-A both in vivo  and ex vivo. 
Materials and Methods
Animals
Male Sprague-Dawley rats 250–300 g and 3-month-old wild type male mice (C57BL/6) were used. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (publication No. 85–23, revised 1996; Bethesda, Maryland). The animal protocol received institutional approval by the Animal Research Committee of University of California Los Angeles.
Left Anterior Descending Coronary Artery Occlusion and Measurement of Infarct Size
Male Sprague-Dawley rats were anesthetized with ketamine (80 mg/kg, intraperitoneally) and xylazine (8 mg/kg, intraperitoneally). The rats were intubated and ventilated with a ventilator (CWE SAR-830/P, Ardmore, PA). A catheter filled with heparinized saline was placed into the right carotid artery for the measurement of blood pressure and heart rate. Pressure and heart rate were monitored using a pressure transducer (Power Lab, ADInstruments, Colorado Springs, CO) throughout the experiment. The hearts were exposed through a left thoracotomy in the fourth intercostal space. The pericardium was opened, and a 5.0 Prolene suture was tightened around the proximal left anterior descending coronary artery. Ischemia was confirmed by ST elevation in electrocardiogram.
At the end of the experiment, 2.5 ml of 1% Evans blue dye was injected into the femoral vein, and the myocardial ischemic area at risk was identified as the region lacking blue staining. The ventricles of the hearts were sliced transversely into 2-mm thick slices. The slices were incubated in 1% triphenyltetrazolium chloride at 37°C for 15 min to identify the noninfarcted and infarcted areas. The infarcted area was displayed as the area unstained by triphenyltetrazolium chloride. Infarct size was expressed as a percentage of the area at risk.
Langendorff Preparation
Mice were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and heparin (200 U/kg) was injected to prevent blood coagulation. The heart was quickly removed and placed in ice-cold Krebs-Henseleit buffer solution (KH, in mM): glucose 11.1, NaCl 118, KCl 4.7, MgSO41.2, KH2PO41.2, NaHCO325.0, and CaCl22 at pH 7.4 bubbled with 95% O2/5% CO2at 37°C.
Experimental Protocol
In vivo  , we used the well-established method to induce I/R14;15 . The heart was subjected to 30 min of ischemia by ligating the proximal left anterior descending coronary artery, followed by 180 min of reperfusion, which was achieved by releasing the tension on the ligature. A bolus of intralipid (20%, 5 ml/kg body weight) or cyclosporine-A (10 mg/kg body weight) were applied via  the femoral vein 5 min before reperfusion (fig. 1A). These doses of intralipid and cyclosporine-A have been already used in vivo  by our group or others.6,16,17 The rats that died during 180 min reperfusion were excluded from the study (two only in the control group because of a technical problem with the ventilator).
Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo  ischemia-reperfusion rat model. (A  ) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B  ) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area  shows infarcted area, blue area  shows noninfarcted area, red  and white areas  show risk area. (C  ) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P  < 0.001 versus  CTRL, #P  < 0.05 versus  ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo 
	ischemia-reperfusion rat model. (A 
	) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B 
	) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area 
	shows infarcted area, blue area 
	shows noninfarcted area, red 
	and white areas 
	show risk area. (C 
	) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P 
	< 0.001 versus 
	CTRL, #P 
	< 0.05 versus 
	ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo  ischemia-reperfusion rat model. (A  ) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B  ) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area  shows infarcted area, blue area  shows noninfarcted area, red  and white areas  show risk area. (C  ) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P  < 0.001 versus  CTRL, #P  < 0.05 versus  ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
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Ex vivo,  we used the well-established protocol to induce I/R injury in isolated mouse hearts as shown by our group and others.6,18,19 The heart was connected to the perfusion cannula via  the aorta and perfused with KH solution. Once the equilibration was achieved, the aorta was clamped for 20 min to induce global normothermic (37°C) ischemia (the heart was immersed in the 37°C Krebs solution during ischemia), followed by reperfusion for 40 min with KH alone (CTRL group), or with additional of intralipid or cyclosporine-A. The ex vivo  hearts that during stabilization had 1) poor contractile function (less than 60 mmHg developed pressure after stabilization), 2) bradycardic (heart rate less than 140 beats/min), or 3) sign of arrhythmia were not subjected to ischemia and were excluded from the study.
To obtain the optimal doses of intralipid and cyclosporine-A in ex vivo  , different doses of intralipid (0.5, 1% and 2% intralipid) or cyclosporine-A (0.2, 0.8 and 1.5 μM) were applied at the onset of reperfusion. The lower concentration of intralipid or cyclosporine-A which resulted in higher heart functional recovery and smaller infract size was used as the optimal concentration. The ex vivo  doses of intralipid between 0.5 and 2% were based on the in vivo  dose of 5 ml/kg of 20%, following these assumptions: 1) for a given rat of 250 g, the volume of the intralipid bolus in in vivo  is 1.25 ml of intralipid (or 20%); 2) the blood volume in a rat is estimated to be between 4.3–8 times the body weight,20 thus, the maximum blood volume is about 8 × 0.250g = 20 ml; 3) the intralipid will be diluted in the blood, to 1.25 ml/(1.25 + 20) ml = 0.058 of 20%, which is equal to 1.17% or intralipid = approximately 1%. Our ex vivo  doses of cyclosporine-A between 0.2 and 1.5 μM were based on previous in vitro  studies showing that cyclosporine-A inhibits mitochondrial swelling and loss of membrane potential in a dose-dependent manner with partial inhibition at approximately 0.2 μM and full inhibition at 1 μM or more.21 Cyclosporine-A also protects against apoptosis in a dose-dependent fashion between 0.5 and 2 μM, and starts to present cell toxicity at concentrations greater than 2 μM.22 
Cardiac Functional Measurements
A catheter (1.4F SPR-671; Millar Instruments, Colorado Springs, CO) connected to a pressure transducer was directly inserted into the left ventricle (LV) to measure left ventricular systolic pressure, left ventricular end-diastolic pressure, and heart rate. The LV developed pressure (LVDP) was calculated as LVDP = left ventricular systolic pressure − left ventricular end-diastolic pressure, and rate pressure product (RPP) = heart rate · LVDP. The maximum rate of LV pressure rise (dP/dtmax) and decline (dP/dtmin) were directly calculated from the selected stable recordings.
Myocardial Necrosis
At the end of the reperfusion, the hearts were cut into four transverse slices and myocardial necrosis was assessed by measurement of the infarct size using triphenyltetrazolium chloride staining. The slices were fixed in 4% paraformaldehyde, and the area of necrosis was quantified by Photoshop (Adobe Systems Incorporated, San Jose, CA) and expressed as the percentage of total ventricular area.
Ca2+-induced Mitochondrial Permeability Transition
Preparation of Isolated Mitochondria.
Mitochondria was prepared from the hearts reperfused with KH, 1% intralipid, or 1.5 μM cyclosporine-A for 10 min. Briefly, myocardial sections were placed in isolation buffer A containing mM: 70 sucrose, 210 mannitol, 1 EDTA, and 50 Tris-HCl, pH 7.4 at 4°C. The tissue was finely minced with scissors and homogenized in the same buffer A (1 ml buffer/0.1g of tissue) using Kontes and Potter-Elvehjem tissue grinders (Fisher Scientific, Pittsburgh, PA). The homogenate was centrifuged at 1,300 g for 3 min; the supernatant was filtered through a cheesecloth and centrifuged at 10,000 g for 10 min. The mitochondrial pellet was resuspended in isolation buffer B containing mM: 70 sucrose, 210 mannitol, 0.1 EDTA, and 50 Tris-HCl, pH 7.4. Mitochondrial protein concentration was measured using the Bradford assay method.
Calcium Retention Capacity (CRC)
Ca2+accumulation during reperfusion is one of the major triggers of the opening of the mPTP. Therefore we measured calcium retention capacity (CRC) in the mitochondria isolated from the hearts subjected to I/R. The onset of the mPTP opening was assessed following in vitro  Ca2+overload as previously described.6 Free Ca2+concentration outside the mitochondria was recorded with 0.5 μM calcium green-5N (Invitrogen, Carlsbad, CA) using excitation and emission wavelengths set at 500 and 530 nm, respectively. Isolated mitochondria (500 μg of protein) were suspended in 2 ml buffer C (mM: 150 sucrose, 50 KCl, 2 KH2PO4, 5 succinic acid, and 20 Tris-HCl, pH 7.4). Samples were preincubated for 90 s in the spectrofluorometer cuvette, and 10 nmol CaCl2pulses were applied every 60 s in the spectrofluorometer. The Ca2+pulses induced a peak of extra-mitochondrial Ca2+concentration that returned to near-baseline level as Ca2+entered the mitochondrial matrix via  the Ca2+uniporter. With increasing Ca2+loading, the extra-mitochondrial Ca2+concentration started accumulating, reflecting a lower capacity for mitochondria Ca2+uptake, which was followed by a sustained Ca2+increase, indicating a massive release of the mitochondria Ca2+by the mPTP opening. The CRC was defined as the amount of Ca2+required to trigger this massive Ca2+release, which was used here as an indicator of the mPTP sensitivity to Ca2+. CRC was expressed as nmol of CaCl2per mg of mitochondrial protein.
Production of Reactive Oxygen Species in the Heart Tissue and in Isolated Cardiac Mitochondria
Reactive oxygen species (ROS) production during reperfusion triggers the opening of the mPTP. We therefore measured ROS production in the hearts subjected to 20 min ischemia followed by reperfusion for 5 min with KH, 1% intralipid, or 1.5 μM cyclosporine-A.
Dihydroethidium Staining to Measure ROS Production in the Heart Tissue Sections.
Compound-embedded 6 μm heart tissue sections were incubated with 10 μM dihydroethidium in Krebs-HEPES buffer (mM: 99 NaCl, 4.69 KCl, 25 NaHCO3, 1.03 KH2PO4, 5.6 D-Glucose, 20 Na-HEPES, 2.5 CaCl2, and 1.2 MgSO4) for 2 h in the dark at room temperature. The sections were then washed three times for 1.5 h in the dark with Krebs-HEPES buffer and mounted with prolong antifade reagent (Invitrogen Carlsbad, CA). Images were acquired with a confocal microscope (Olympus Fluoview, Hicksville, NY).
Electron Spin Resonance to Quantify O2·−Production in Isolated Myocardial Mitochondria.
Superoxide was detected by electron spin resonance as previously reported.23  26 The superoxide (O2·−) spin probe methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine (0.5 mM, Alexis, San Diego, CA) solution was prepared freshly in nitrogen gas-bubbled Krebs-HEPES buffer containing diethyldithiocarbamic acid (5 μM, Sigma-Aldrich, St. Louis, MO) and deferoxamine (25 μM, Sigma-Aldrich). Freshly isolated mitochondria were mixed with the O2·−-specific spin probe methoxyc, rbonyl-2,2,5,5-tetramethyl-pyrrolidine in the presence or absence of 100 U/ml of superoxide dismutases and loaded in glass capillaries for analysis of O2·−production kinetically for 10 min. The electron spin resonance settings used were as follows: center field, 3475; sweep width, 9G; static field, 3484.981; microwave frequency, 9.75 GHz; microwave power 21.02 mW; modulation frequency 86 KHz; modulation amplitude, 2.47 G; resolution in X, 512; and number of x-scans, 10. The superoxide dismutase inhibitable O2·−signals at 10 min time-point, normalized by protein concentrations, were compared among different experimental groups.
Western Blot Analysis
Intralipid-induced cardioprotection is associated with increased phosphorylation levels of Akt and GSK-3β.6 Therefore we performed Western blot analysis to examine the possible involvements of Akt and GSK-3β in cardioprotection offered by cyclosporine-A. The entire ex vivo  hearts that were reperfused with KH, 1% intralipid, or 1.5 μM cyclosporine-A for 10 min were used for making whole heart lysates, because in this model the whole heart is considered to be the area at risk. Hearts were homogenized at 4°C (mM: 150 NaCl, 50 Tris-HCl, 1 EGTA, 1 EDTA, 1 NaF, 1 PMSF, 1 Na3VO4, 1% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate, pH 7.4, supplemented with protease and phospatase inhibitor cocktails [Roche, San Francisco, CA]). The samples were centrifuged at 12,000 g for 10 min and the supernatants were collected. Protein concentration was measured and 100 μg of total protein was loaded on a 4–20% gradient Tris-HCl SDS polyacrylamide gel, electrotransferred to nitrocellulose paper, blocked with 5% nonfat dry milk in 20 mM Tris-buffered saline with 0.1% Tween and 0.5% Triton · 100, and incubated with primary antibodies. Blots were then indirectly labeled using infrared fluorophore-conjugated secondary antibodies for 1 h at room temperature, and visualized with the Odyssey Imaging System (Li-Cor, Lincoln, NE). Equal loading of protein onto each lane in the gel was confirmed with Vinculin. The proteins were first normalized to their corresponding Vinculin and then the phosphorylated proteins were normalized to their corresponding total protein levels.
Statistics
For cardiac infarct size and CRC, ROS production means were compared between groups using one-way ANOVA. For ex vivo  cardiac function, means were compared among doses and over time using two-way repeated measure ANOVA, where both dose and time are repeated factors. Pairwise mean comparisons were judged significant using the Tukey post hoc  test. For dose comparisons over time, contrasts were computed under a given ANOVA model to test for trends. A priori  sample size/power analysis was not carried out. However, our sample size was intuitively based on our previous experience and our published results.6 All statistical analyses were performed using SPSS 13.0 (SPSS Inc, Chicago, IL) or SAS 9.3 (SAS Institute, Cary NC). As all outcomes were continuous, results were summarized with means ± standard errors of the mean (SEM). All P  values are two-sided, and P  < 0.05 was considered statistically significant.
Results
Intralipid Protects the Heart against I/R Injury More Efficiently Than Cyclosporine-A in the In Vivo  Rat Model
The cardioprotective effect of intralipid was compared with cyclosporine-A in an in vivo  rat model of I/R injury. The area at risk to LV ratio was similar in all groups (62.4 ± 2.0 in CTRL [n = 7], 58.0 ± 3.0 in intralipid group [n = 7], and 59.4 ± 2.8 in cyclosporine-A group [n = 7]), indicating that all three groups were subjected to a comparable degree of ischemic risk. However, the ratio of infarct size to area at risk was significantly smaller in the intralipid group compared with cyclosporine-A (22.9 ± 2.5 in intralipid group vs.  35.2 ± 3.5 in cyclosporine-A group, P  = 0.030) (figs. 1B, C). Intralipid and cyclosporine-A both reduced the infarct size significantly compared with the CTRL group (59.8 ± 3.3 in CTRL, P  < 0.001). The differences in the infarct size were not because of the hemodynamic changes as the heart rate and mean arterial pressure were not significantly different among the three groups at baseline, during ischemia, and at reperfusion (table 1).
Table 1. Systemic Hemodynamics in In Vivo  Model of Ischemia-Reperfusion Injury
Image not available
Table 1. Systemic Hemodynamics in In Vivo  Model of Ischemia-Reperfusion Injury
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Dose Response Curves to Obtain the Optimal Dose of Postischemic Administration of Intralipid and Cyclosporine-A
Next we compared the cardioprotective effect of intralipid with cyclosporine-A in isolated Langendorff perfused hearts. Figure 2shows the RPP and infarct size as a function of intralipid (figs. 2A, B) and cyclosporine-A (figs. 2C, D) concentrations. Although 0.5% intralipid induced some degree of protection (RPP = 6,909 ± 2,055 mmHg · beats/min [n = 5] at 40 min of reperfusion, infarct size = 28.1 ± 4.4 [n = 5], the cardioprotection achieved by 1% and 2% intralipid were similar and significantly better than 0.5% (1% intralipid: RPP = 12,740 ± 675 mmHg · beats/min [n = 7] at the end of reperfusion, infarct size = 17.3 ± 2.9 [n = 7]; 2% intralipid: RPP = 12,117 ± 1,527 mmHg · beats/min [n = 5], infarct size = 19.1 ± 2.6 [n = 5]). Cyclosporine-A at 0.2 μM had no apparent cardioprotective effect (RPP = 3,968.6 ± 624 mmHg · beats/min [n = 5] at end of reperfusion, infarct size = 46.7 ± 4.6 [n = 5]). Postischemic administration of 0.8 μM cyclosporine-A significantly improved the cardiac functional recovery and reduced the infarct size compared with 0.2 μM cyclosporine-A (RPP = 9,203 ± 1,078 mmHg · beats/min [n = 5] at end of reperfusion, infarct size = 27.8 ± 1.6 [n = 5]). Increasing the concentration of cyclosporine-A from 0.8 to 1.5 μM did not result in further cardioprotection as the RPP and the infarct size for 1.5 μM were not significantly different than 0.8 μM (RPP = 8,652 ± 2,182 mmHg · beats/min [n = 5] at end of reperfusion, infarct size = 28.9 ± 2.5 [n = 5]).
Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo  ischemia-reperfusion mouse model. (A  ) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B  ) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C  ) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D  ) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P  = 0.022, 2% intralipid versus  0.5% intralipid; **P  = 0.004, 1% intralipid versus  0.5% intralipid; &&P  = 0.008, 0.8 μM cyclosporine-A versus  0.2 μM cyclosporine-A; ^^P  = 0.009, 1.5 μM cyclosporine-A versus  0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo 
	ischemia-reperfusion mouse model. (A 
	) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B 
	) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C 
	) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D 
	) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P 
	= 0.022, 2% intralipid versus 
	0.5% intralipid; **P 
	= 0.004, 1% intralipid versus 
	0.5% intralipid; &&P 
	= 0.008, 0.8 μM cyclosporine-A versus 
	0.2 μM cyclosporine-A; ^^P 
	= 0.009, 1.5 μM cyclosporine-A versus 
	0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo  ischemia-reperfusion mouse model. (A  ) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B  ) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C  ) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D  ) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P  = 0.022, 2% intralipid versus  0.5% intralipid; **P  = 0.004, 1% intralipid versus  0.5% intralipid; &&P  = 0.008, 0.8 μM cyclosporine-A versus  0.2 μM cyclosporine-A; ^^P  = 0.009, 1.5 μM cyclosporine-A versus  0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
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Intralipid Is More Effective Than Cyclosporine-A in Protecting the Heart against I/R Injury in Ex Vivo  Mouse Model
We compared the effect of intralipid and cyclosporine-A on the cardiac functional recovery and infarct size at their optimal dose of 1% and 0.8 μM, respectively. Typical examples of LVDP and dP/dt are shown in figures 3A–C. Although the baseline RPP before ischemia was similar in all groups, the functional recovery was very poor in the CTRL group; RPP was 2,077 ± 100 mmHg · beats/min (n = 7) after 10 min of reperfusion and did not change significantly throughout the 40 min reperfusion (RPP = 2,791 ± 758 mmHg · beats/min [n = 7] at 40 min of reperfusion, fig. 3D). The functional recovery in the intralipid group was much higher, as the RPP was 9,873 ± 995 mmHg · beats/min (n = 7) after 10 min of reperfusion (recovery of 63%), improved further to 11,705 ± 1021 mmHg · beats/min(n = 7) at 20 min of reperfusion (recovery of 75%), and to 12,217 ± 1123 mmHg · beats/min(n = 7) at 30 min and to 12,740 ± 675 mmHg · beats/min (n = 7) after 40 min (recovery of 81%, fig. 3D). The intralipid group also showed a much better LV dP/dtmax, LV dP/dtmin, and LVDP compared with CTRL hearts (figs. 3E, F). In the cyclosporine-A group, the RPP was 4,159 ± 739 mmHg · beats/min (n = 5) at 10 min of reperfusion (recovery of 28%) and 4,728 ± 823 mmHg · beats/min (n = 5) at 20 min of reperfusion (recovery of 32%), improved to 8,188 ± 1087 mmHg · beats/min(n = 5) at 30 min (recovery of 55%) and 9,203 ± 1,078 mmHg · beats/min at 40 min, which was significantly lower than the intralipid group (P  = 0.024, fig. 3D). The LV dP/dtmax(2,373 ± 261 in intralipid group [n = 7]vs.  1,286 ± 147 mmHg/s in cyclosporine-A group [n = 5], P  = 0.031) and LVDP (66 ± 6 in intralipid group [n = 7]vs.  39 ± 4 mmHg in cyclosporine-A group [n = 5], P  = 0.005) were also significantly lower than intralipid at the end of 40 min reperfusion (figs. 3E, F). Consistent with the cardiac function, the infarct size was also significantly smaller in the intralipid group compared with the cyclosporine-A group (17.3 ± 2.9% in intralipid [n = 7]vs  . 27.8 ± 1.6% in cyclosporine-A group [n = 5], 54.4 ± 3.8% in CTRL [n = 7], P  < 0.001 vs.  CTRL, fig. 4).
Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A  ), 1% intralipid (B  ), and 0.8 μM cyclosporine-A (C  ). Rate pressure product (D  ), dP/dtMax(E  ), and left ventricular developed pressure (F  ) as a function of time in control (filled circles  , n = 7), intralipid (open circles  , n = 7), and cyclosporine-A group (diamonds  , n = 5). *P  < 0.05 and **P  < 0.001 versus  CTRL, #P  < 0.05, ##P  < 0.01 versus  CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A 
	), 1% intralipid (B 
	), and 0.8 μM cyclosporine-A (C 
	). Rate pressure product (D 
	), dP/dtMax(E 
	), and left ventricular developed pressure (F 
	) as a function of time in control (filled circles 
	, n = 7), intralipid (open circles 
	, n = 7), and cyclosporine-A group (diamonds 
	, n = 5). *P 
	< 0.05 and **P 
	< 0.001 versus 
	CTRL, #P 
	< 0.05, ##P 
	< 0.01 versus 
	CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A  ), 1% intralipid (B  ), and 0.8 μM cyclosporine-A (C  ). Rate pressure product (D  ), dP/dtMax(E  ), and left ventricular developed pressure (F  ) as a function of time in control (filled circles  , n = 7), intralipid (open circles  , n = 7), and cyclosporine-A group (diamonds  , n = 5). *P  < 0.05 and **P  < 0.001 versus  CTRL, #P  < 0.05, ##P  < 0.01 versus  CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
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Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A  ) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area  represents the infarct zone and the red  shows the viable area. (B  ) The area of necrosis as the percentage of total ventricular area in control (black  ), intralipid (red  ), and cyclosporine-A group (blue  ). **P  < 0.001 versus  CTRL, #P  = 0.014 versus  ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A 
	) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area 
	represents the infarct zone and the red 
	shows the viable area. (B 
	) The area of necrosis as the percentage of total ventricular area in control (black 
	), intralipid (red 
	), and cyclosporine-A group (blue 
	). **P 
	< 0.001 versus 
	CTRL, #P 
	= 0.014 versus 
	ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A  ) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area  represents the infarct zone and the red  shows the viable area. (B  ) The area of necrosis as the percentage of total ventricular area in control (black  ), intralipid (red  ), and cyclosporine-A group (blue  ). **P  < 0.001 versus  CTRL, #P  = 0.014 versus  ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
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Intralipid Inhibits the Opening of mPTP as Efficiently as Cyclosporine-A after I/R
Delaying the opening of the mPTP upon reperfusion has been a potential target to reduce myocardial injury. Figures 5A, B represents a typical recording of fluorescence showing the time course of Ca2+concentration in the mitochondrial external medium. In the CTRL group, eight pulses were sufficient to trigger the opening of mPTP. Interestingly, the number of calcium pulses required for opening of the mPTP in both intralipid and cyclosporine-A groups were increased to 14 and 13, respectively (fig. 5A). These data suggest that postischemic treatment of intralipid increases the resistance of the mPTP to Ca2+overload to a comparable level with the cyclosporine-A group. In fact, the CRC was about 2-fold higher in intralipid and cyclosporine-A groups compared with CTRL (280 ± 8.2 in intralipid and 260 ± 29 in cyclosporine-A vs.  157 ± 25 nmol/mg protein in CTRL, n = 6, P  < 0.001, fig. 5C). As expected, sham hearts had much higher CRC (370 ± 20 nmol/mg protein).
Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A  , B  ) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A  ) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B  ). (C  ) Calcium retention capacity in the absence of cyclosporine-A (red bars  ) and after addition of cyclosporine-A in the cuvette (black bars  ). **P  < 0.001 versus  CTRL; ##P  < 0.001 versus  sham+ cyclosporine-A group; ∧∧P  < 0.001 versus  sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A 
	, B 
	) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A 
	) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B 
	). (C 
	) Calcium retention capacity in the absence of cyclosporine-A (red bars 
	) and after addition of cyclosporine-A in the cuvette (black bars 
	). **P 
	< 0.001 versus 
	CTRL; ##P 
	< 0.001 versus 
	sham+ cyclosporine-A group; ∧∧P 
	< 0.001 versus 
	sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A  , B  ) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A  ) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B  ). (C  ) Calcium retention capacity in the absence of cyclosporine-A (red bars  ) and after addition of cyclosporine-A in the cuvette (black bars  ). **P  < 0.001 versus  CTRL; ##P  < 0.001 versus  sham+ cyclosporine-A group; ∧∧P  < 0.001 versus  sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
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Inhibition of mPTP Opening by Intralipid is CypD-dependent
We examined the involvement of CypD in intralipid-induced inhibition of mPTP opening by comparing the CRC after addition of 1.5 μM of cyclosporine-A directly in the cuvette in CTRL, intralipid, and cyclosporine-A groups. Administration of exogenous cyclosporine-A increased the number of Ca2+pulses required to trigger the opening of mPTP in all groups, but to different extents (figs. 5B, C). In sham and CTRL groups, the addition of cyclosporine-A to the cuvette greatly increased the CRC (from 370 ± 20 to 620 ± 10 nmol/mg protein in sham and from 157 ± 25 to 347 ± 30 nmol/mg protein in CTRL, n = 6, P  < 0.001). In intralipid and cyclosporine-A groups, however, the CRC increased mildly to 445 ± 25 and 415 ± 45 nmol/mg protein, respectively, in the presence of cyclosporine-A.
Postischemic Treatment of Intralipid and Cyclosporine-A Decreases ROS Production after I/R
Next we investigated whether the delay of the mPTP opening in intralipid and cyclosporine-A groups is in part because of decreased ROS/superoxide generation. Dihydroethidium staining of heart sections revealed a significantly lower ROS production in the intralipid and cyclosporine-A groups when compared with CTRL (normalized to CTRL, 0.57 ± 0.04 in intralipid group and 0.49 ± 0.01 in cyclosporine-A, P  < 0.001 vs.  CTRL, fig. 6A, B). We then examined the superoxide production in isolated cardiac mitochondria from intralipid and cyclosporine-A. The mitochondrial superoxide production was significantly lower in the intralipid and cyclosporine-A groups compared with CTRL (normalized to CTRL: 0.34 ± 0.11 in intralipid group, 0.57 ± 0.14 in cyclosporine-A group; n = 4, P  = 0.005 intralipid group vs.  CTRL; P  = 0.041 cyclosporine-A group vs.  CTRL, fig. 6C).
Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A  ) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). (B  ) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P  < 0.001 vs.  control, n = 3). (C  ) Superoxide production in isolated mitochondria using electron spin resonance in control (black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). **P  = 0.005 intralipid versus  control; *P  = 0.041 cyclosporine-A versus  control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A 
	) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black 
	), 1% intralipid (red 
	), and 1.5 μM cyclosporine-A (blue 
	). (B 
	) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P 
	< 0.001 vs. 
	control, n = 3). (C 
	) Superoxide production in isolated mitochondria using electron spin resonance in control (black 
	), 1% intralipid (red 
	), and 1.5 μM cyclosporine-A (blue 
	). **P 
	= 0.005 intralipid versus 
	control; *P 
	= 0.041 cyclosporine-A versus 
	control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A  ) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). (B  ) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P  < 0.001 vs.  control, n = 3). (C  ) Superoxide production in isolated mitochondria using electron spin resonance in control (black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). **P  = 0.005 intralipid versus  control; *P  = 0.041 cyclosporine-A versus  control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
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Unlike Intralipid, Cyclosporine-A Does Not Induce Akt and GSK-3β Phosphorlyation
Intralipid-induced cardioprotection is associated with increased phosphorylation levels of Akt and GSK-3β.6 Here we examined the possible involvements of Akt and GSK-3β in cardioprotection offered by cyclosporine-A. Western blot analysis revealed that intralipid-induced cardioprotection was associated with an approximately 6-fold increase in phosphorylation of Akt, and an approximately 5-fold increase in phosphorylation of GSK-3β. Cyclosporine-A, however, did not affect the phosphorylation levels of Akt and GSK-3β (fig. 7), because there were no significant differences between the phosphorylation levels of these prosurvival kinases in cyclosporine-A and CTRL groups.
Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A  , B  ) Representative immunoblots of pAkt and total Akt (A  ), and pGSK-3β and total GSK-3β (C  ) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C  , D  ) Western blot analysis of pAkt protein to total Akt (B  ) and pGSK-3β to total GSK-3b (D  ) ratios in control group (black bars  ), intralipid group (white bars  ), and cyclosporine-A group (gray bars  ). **P  < 0.001 versus  control; ##P  < 0.001 versus  intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A 
	, B 
	) Representative immunoblots of pAkt and total Akt (A 
	), and pGSK-3β and total GSK-3β (C 
	) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C 
	, D 
	) Western blot analysis of pAkt protein to total Akt (B 
	) and pGSK-3β to total GSK-3b (D 
	) ratios in control group (black bars 
	), intralipid group (white bars 
	), and cyclosporine-A group (gray bars 
	). **P 
	< 0.001 versus 
	control; ##P 
	< 0.001 versus 
	intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A  , B  ) Representative immunoblots of pAkt and total Akt (A  ), and pGSK-3β and total GSK-3β (C  ) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C  , D  ) Western blot analysis of pAkt protein to total Akt (B  ) and pGSK-3β to total GSK-3b (D  ) ratios in control group (black bars  ), intralipid group (white bars  ), and cyclosporine-A group (gray bars  ). **P  < 0.001 versus  control; ##P  < 0.001 versus  intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
×
Discussion
Our previous study has shown that postischemic administration of intralipid can protect the heart against I/R injury in both the in vivo  and ex vivo  models. Here we compared the cardioprotective effect of intralipid with cyclosporine-A. Our data demonstrate that intralipid, a safe fat emulsion for human use, is more effective than cyclosporine-A in protecting the heart against ischemic I/R injury. A bolus of intralipid right before the onset of reperfusion resulted in smaller infarct size of approximately 40% compared with cyclosporine-A in the in vivo  rat model. Postischemic administration of intralipid was also more efficient than cyclosporine-A in improving the heart functional recovery and reducing the infarct size in isolated Langendorff perfused ex vivo  hearts. Intralipid is as effective as cyclosporine-A in inhibiting the mPTP opening, most likely by increasing mitochondrial resistance to Ca2+overload and reducing mitochondrial superoxide production during the first few minutes of reperfusion.
Iintralipid Is More Effective Than Cyclosporine-A in Protecting the Heart against I/R Injury
We report that intralipid was more effective than cyclosporine-A in protecting the heart against I/R injury because the infarct size was significantly smaller and the heart functional recovery indices were all significantly better. Although the optimal concentration of cyclosporine-A, if applied before ischemia, has been shown to be 0.2 μM,27,28 here we found that cyclosporine-A at this concentration does not induce cardioprotection if applied at the onset of reperfusion. Interestingly, administration of 1% intralipid at the onset of reperfusion rapidly restored heart function, as 40–60% recovery was observed in the RPP and LVDP parameters within 10 min of reperfusion. On the other hand, the cardiac functional recovery in the cyclosporine-A group during the first 10 min of reperfusion was only 20–30%. Because the first few minutes of reperfusion are critical in myocardial protection, intralipid could be an ideal safe pharmacological agent to rapidly restore the heart function, resulting in smaller infarct size.
Our data demonstrate that intralipid is a very powerful postischemic pharmacological agent reducing infarct size (both in vivo  and ex vivo  ) and improving cardiac contractility.6 The role of intralipid in preconditioning, however, seems to be controversial. In the ex vivo  model of I/R injury, intralipid failed to protect the myocardium from contractile dysfunction when administered 10 min before the onset of ischemia and throughout the reperfusion phase.29 Administration of intralipid before ischemia in an in vivo  model of myocardial I/R injury was also not able to reduce the infarct size.30,31 However, in another work from the same group, intralipid was shown to reduce the infarct size in the ex vivo  model of I/R in rats if administered before ischemia.32 Further studies are required to clarify the role of intralipid in preconditioning.
Intralipid Is as Effective as Cyclosporine-A in Inhibiting mPTP Opening by Increasing Mitochondrial Resistance to Ca2+Overload and Reducing Mitochondrial Superoxide Production
The opening of the mPTP during reperfusion has been implicated in cell death.33,34 Ca2+accumulation and overproduction of ROS during reperfusion are the two major triggers of the opening of the mPTP. It has been demonstrated that ischemic preconditioning and postconditioning induce cardioprotection by increasing mitochondrial resistance to Ca2+overload.35 Mitochondria are also the major source of ROS generation through their respiratory chain and are also the target organelle of oxidative damage.35 Decreasing ROS generation during reperfusion has been considered to induce cardioprotection against I/R injury.36 Recently we showed that intralipid inhibits the opening of the mPTP.6 Here our data demonstrate that intralipid is as efficient as cyclosporine-A in increasing the mitochondrial calcium uptake for the opening of the mPTP compared with CTRL (fig. 5). We propose that intralipid enhances the homeostasis of cardiomyocytes in the same manner as cyclosporine-A to better regulate calcium overload and therefore increase the threshold for opening of the mPTP. Intralipid exerts its action in a CypD-dependent manner as cyclosporine-A, which results in attenuation of the interaction between CypD with mPTP and therefore increases the mitochondrial CRC.
We also found that ROS generation in the heart tissue, as well as the production of superoxide in cardiac mitochondria during the first 5 min of reperfusion, was significantly reduced by postischemic administration of intralipid. In fact, intralipid was as effective as cyclosporine-A, if not better, in reducing the production of superoxide in mitochondria. Since it is well accepted that overproduction of ROS in the mitochondria is one of the triggers of the opening of the mPTP, reduced superoxide production in the mitochondria by intralipid could delay the opening of mPTP. Cyclosporine-A is currently the most specific inhibitor of the mPTP and has been demonstrated in many species including pigs, as well as in clinical trials, to be cardioprotective.37,38 However, cyclosporine-A is known to have undesirable side effects. Intralipid, which has been in clinical use for more than four decades with no known side effects, is as effective as cyclosporine-A in inhibiting the mPTP opening, most likely by increasing mitochondrial resistance to Ca2+overload and reducing mitochondrial superoxide production during the first few minutes of reperfusion.
Unlike Intralipid, Cyclosporine-A-induced Cardioprotection Is Not Mediated via  Akt/GSK-3β
Phosphatidylinositol 3-kinase-protein kinase B (PKB)/Akt pathway plays an important role in reperfusion injury.39  41 GSK-3β phosphorylation has emerged as an end-effector step where multiple protective signaling pathways converge.39  42 We have recently shown that postischemic administration of intralipid increases the phosphorylation levels of Akt and GSK. Here we report that cyclosporine-A-induced cardioprotection is not mediated through Akt/GSK, as cyclosporine-A had no effect on the phosphorylation levels of these kinases. The cyclosporine-A-induced protection of mesenchymal stem cells against apoptosis has also been reported to be independent of Akt/ERK, because cyclosporine-A did not promote the phosphorylation of these prosurvival proteins.43 The lack of involvement of the Akt/GSK pathway in the cyclosporine- -induced cardioprotection could be one of the reasons behind the slow action of cyclosporine-A in improving the postischemic heart function.
Limitations
To date, cyclosporine-A11 is known to be the most specific inhibitor of the mPTP. It exerts its action by directly inhibiting the peptidyl-prolyl cis-trans isomerase activity of CypD, which is a key component of the mPTP.12,13 Here we report that intralipid inhibits the opening of the mPTP as efficiently as cyclosporine-A. Our previous study demonstrated that intralipid increases the phosphorylation of GSK-3β, leading to inhibition of the mPTP opening.6,18,19 However, it is not clear if intralipid directly inhibits the mPTP in a similar manner as cyclosporine-A. Further experiments using genetically modified mice of mPTP component are required to demonstrate a possible direct action of intralipid on the mPTP.
Our data demonstrate that cyclosporine-A-induced cardioprotection is most likely not mediated via  the Akt/GSK-3β pathway. However, we did not examine the involvement of the protein kinase G nor the survivor activating factor enhancement pathway in cyclosporine-A-induced cardioprotection. Because the activation of the reperfusion injury salvage kinases pathway does not seem to be crucial for postconditioning in pigs,44 it remains to be seen whether reperfusion injury salvage kinases, protein kinase G, or the survivor activating factor enhancement pathways play a role in cyclosporine-A- or intralipid-induced cardioprotection in larger animals.
Clinical Perspectives
Acute myocardial infarction is still a major cause of mortality, despite advances in the management of patients. Although prompt reperfusion is critical in salvaging the ischemic heart, it also has the potential to induce reperfusion injury. Currently, there is no effective postischemic pharmacological agent for protecting the heart against the detrimental effects of lethal myocardial reperfusion injury. Delaying the opening of the mPTP upon reperfusion has been a potential target to reduce myocardial injury. Cyclosporine-A, which is one of the most potent inhibitors of mPTP opening, is emerging as a new postconditioning cardioprotective agent and it has been used recently in small clinical trials both in Europe and the United States. Chronic treatment with cyclosporine-A, however, has been associated with a number of potentially serious adverse drug reactions such as high blood pressure, potassium retention, and possibly hyperkalemia, kidney and liver dysfunction, and an increased vulnerability to opportunistic fungal and viral infections. Therefore, because of cyclosporine-A's undesirable side effects, it is questionable whether cyclosporine-A is an ideal pharmacological postconditioning agent. Intralipid, on the other hand, is a clinically safe lipid emulsion that is generally well tolerated and is widely used in different clinical settings for parenteral nutrition for almost four decades. Our results demonstrate that not only is intralipid as potent as cyclosporine-A in inhibiting mPTP opening, but is in fact more effective than cyclosporine-A in reducing the infarct size. Therefore, intralipid could be a better alternative and an ideal safe pharmacological postconditioning agent for targeting the critical first few minutes of reperfusion, to rapidly restore heart function and therefore reduce infarct size. Although our in vivo  dose of intralipid at 5 ml/kg is within the range that has been recommended by American Society of Regional Anesthesia and Pain Medicine for rescuing bupivacaine cardiotoxicity in patients, the exact dose of intralipid still needs to be determined in clinical trials for reperfusion injury. Our findings raise the intriguing concept that intralipid could serve as a novel therapeutic agent for acute myocardial infarction patients, which certainly warrants further investigation in the human heart.
The authors thank Jeffrey Gornbein, Ph.D., Senior Statistician, Department of Biomathematics at University of California Los Angeles, Los Angeles, California, for statistical assistance.
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Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo  ischemia-reperfusion rat model. (A  ) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B  ) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area  shows infarcted area, blue area  shows noninfarcted area, red  and white areas  show risk area. (C  ) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P  < 0.001 versus  CTRL, #P  < 0.05 versus  ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo 
	ischemia-reperfusion rat model. (A 
	) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B 
	) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area 
	shows infarcted area, blue area 
	shows noninfarcted area, red 
	and white areas 
	show risk area. (C 
	) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P 
	< 0.001 versus 
	CTRL, #P 
	< 0.05 versus 
	ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
Fig. 1. Intralipid reduces the infarct size more efficiently than cyclosporine-A in the in vivo  ischemia-reperfusion rat model. (A  ) The left coronary artery was occluded for 30 min followed by 3 h of reperfusion. One single IV bolus of 20% intralipid (5 ml/kg body weight, intralipid group), cyclosporine-A (10 mg/kg body weight, cyclosporine-A group), and phosphate-buffered saline (control group) was administered 5 min before reperfusion. (B  ) Representative triphenyltetrazolium chloride-stained heart slices from control, intralipid, and cyclosporine-A group. The white area  shows infarcted area, blue area  shows noninfarcted area, red  and white areas  show risk area. (C  ) Percentage of area at risk divided by left ventricle (AAR/LV), infarct size divided by area at risk (IS/AAR), and infarct size divided by left ventricle in control (IS/LV) (n = 7), intralipid group (n = 7), and cyclosporine-A group (n = 7). **P  < 0.001 versus  CTRL, #P  < 0.05 versus  ILP. AAR = area at risk, CsA = cyclosporine-A; CTRL = control; ILP = intralipid; IS = infarct size; LV = left ventricle.
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Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo  ischemia-reperfusion mouse model. (A  ) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B  ) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C  ) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D  ) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P  = 0.022, 2% intralipid versus  0.5% intralipid; **P  = 0.004, 1% intralipid versus  0.5% intralipid; &&P  = 0.008, 0.8 μM cyclosporine-A versus  0.2 μM cyclosporine-A; ^^P  = 0.009, 1.5 μM cyclosporine-A versus  0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo 
	ischemia-reperfusion mouse model. (A 
	) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B 
	) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C 
	) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D 
	) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P 
	= 0.022, 2% intralipid versus 
	0.5% intralipid; **P 
	= 0.004, 1% intralipid versus 
	0.5% intralipid; &&P 
	= 0.008, 0.8 μM cyclosporine-A versus 
	0.2 μM cyclosporine-A; ^^P 
	= 0.009, 1.5 μM cyclosporine-A versus 
	0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
Fig. 2. Dose response of intralipid and cyclosporine-A in ex vivo  ischemia-reperfusion mouse model. (A  ) Rate pressure product as a function of time in 0.5%, 1%, and 2% intralipid (n = 7). (B  ) The area of necrosis as the percentage of total ventricular area in 0.5%, 1%, and 2% intralipid group. (C  ) Rate pressure product as a function of time in 0.2 μM, 0.8 μM, and 1.5 μM cyclosporine-A (n = 5). (D  ) The area of necrosis as the percentage of total ventricular area in control group, 0.5%, 1%, and 2% intralipid group. *P  = 0.022, 2% intralipid versus  0.5% intralipid; **P  = 0.004, 1% intralipid versus  0.5% intralipid; &&P  = 0.008, 0.8 μM cyclosporine-A versus  0.2 μM cyclosporine-A; ^^P  = 0.009, 1.5 μM cyclosporine-A versus  0.2 μM cyclosporine-A. CsA = cyclosporine-A; ILP = intralipid; RPP = rate pressure product.
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Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A  ), 1% intralipid (B  ), and 0.8 μM cyclosporine-A (C  ). Rate pressure product (D  ), dP/dtMax(E  ), and left ventricular developed pressure (F  ) as a function of time in control (filled circles  , n = 7), intralipid (open circles  , n = 7), and cyclosporine-A group (diamonds  , n = 5). *P  < 0.05 and **P  < 0.001 versus  CTRL, #P  < 0.05, ##P  < 0.01 versus  CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A 
	), 1% intralipid (B 
	), and 0.8 μM cyclosporine-A (C 
	). Rate pressure product (D 
	), dP/dtMax(E 
	), and left ventricular developed pressure (F 
	) as a function of time in control (filled circles 
	, n = 7), intralipid (open circles 
	, n = 7), and cyclosporine-A group (diamonds 
	, n = 5). *P 
	< 0.05 and **P 
	< 0.001 versus 
	CTRL, #P 
	< 0.05, ##P 
	< 0.01 versus 
	CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
Fig. 3. Administration of intralipid at reperfusion improves heart functional recovery against reperfusion injury more efficiently than cyclosporine-A. Representatives of the left ventricular developed pressure and left ventricle pressure rise (dP/dtmax) and decline (dP/dtmin) as a function of time in control group (A  ), 1% intralipid (B  ), and 0.8 μM cyclosporine-A (C  ). Rate pressure product (D  ), dP/dtMax(E  ), and left ventricular developed pressure (F  ) as a function of time in control (filled circles  , n = 7), intralipid (open circles  , n = 7), and cyclosporine-A group (diamonds  , n = 5). *P  < 0.05 and **P  < 0.001 versus  CTRL, #P  < 0.05, ##P  < 0.01 versus  CsA. CsA = cyclosporine-A; ILP = intralipid; KH = Krebs-Henseleit buffer solution; LVDP = left ventricular developed pressure; RPP = rate pressure product.
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Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A  ) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area  represents the infarct zone and the red  shows the viable area. (B  ) The area of necrosis as the percentage of total ventricular area in control (black  ), intralipid (red  ), and cyclosporine-A group (blue  ). **P  < 0.001 versus  CTRL, #P  = 0.014 versus  ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A 
	) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area 
	represents the infarct zone and the red 
	shows the viable area. (B 
	) The area of necrosis as the percentage of total ventricular area in control (black 
	), intralipid (red 
	), and cyclosporine-A group (blue 
	). **P 
	< 0.001 versus 
	CTRL, #P 
	= 0.014 versus 
	ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
Fig. 4. The infarct size is significantly smaller in intralipid than cyclosporine-A group. (A  ) Four slices of the same heart after 2,3,5-triphenyltetrazolium chloride (TTC) staining in control group (n = 7), 1% intralipid group (n = 7), and 0.8 μM cyclosporine-A group (n = 5). The white area  represents the infarct zone and the red  shows the viable area. (B  ) The area of necrosis as the percentage of total ventricular area in control (black  ), intralipid (red  ), and cyclosporine-A group (blue  ). **P  < 0.001 versus  CTRL, #P  = 0.014 versus  ILP. CsA = cyclosporine-A; CTRL = control; ILP = intralipid.
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Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A  , B  ) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A  ) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B  ). (C  ) Calcium retention capacity in the absence of cyclosporine-A (red bars  ) and after addition of cyclosporine-A in the cuvette (black bars  ). **P  < 0.001 versus  CTRL; ##P  < 0.001 versus  sham+ cyclosporine-A group; ∧∧P  < 0.001 versus  sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A 
	, B 
	) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A 
	) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B 
	). (C 
	) Calcium retention capacity in the absence of cyclosporine-A (red bars 
	) and after addition of cyclosporine-A in the cuvette (black bars 
	). **P 
	< 0.001 versus 
	CTRL; ##P 
	< 0.001 versus 
	sham+ cyclosporine-A group; ∧∧P 
	< 0.001 versus 
	sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
Fig. 5. Postischemic treatment of intralipid increases mitochondrial calcium retention capacity (CRC) after ischemia-reperfusion injury as cyclosporine-A in a cychlophilin-D-dependent manner. (A  , B  ) Typical recordings of the mitochondrial permeability transition pore opening in isolated mitochondria from control group, 1% intralipid, and 1.5 μM cyclosporine-A groups subjected to 20 min of global ischemia followed by 10 min of reperfusion as well as sham hearts before (A  ) and after addition of 1.5 μM cyclosporine-A directly in the cuvette (B  ). (C  ) Calcium retention capacity in the absence of cyclosporine-A (red bars  ) and after addition of cyclosporine-A in the cuvette (black bars  ). **P  < 0.001 versus  CTRL; ##P  < 0.001 versus  sham+ cyclosporine-A group; ∧∧P  < 0.001 versus  sham (n = 6). CsA = cyclosporine-A; CTRL = control; ILP = intralipid; mPTP = mitochondrial permeability transition pore.
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Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A  ) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). (B  ) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P  < 0.001 vs.  control, n = 3). (C  ) Superoxide production in isolated mitochondria using electron spin resonance in control (black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). **P  = 0.005 intralipid versus  control; *P  = 0.041 cyclosporine-A versus  control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A 
	) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black 
	), 1% intralipid (red 
	), and 1.5 μM cyclosporine-A (blue 
	). (B 
	) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P 
	< 0.001 vs. 
	control, n = 3). (C 
	) Superoxide production in isolated mitochondria using electron spin resonance in control (black 
	), 1% intralipid (red 
	), and 1.5 μM cyclosporine-A (blue 
	). **P 
	= 0.005 intralipid versus 
	control; *P 
	= 0.041 cyclosporine-A versus 
	control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
Fig. 6. Postischemic treatment of intralipid and cyclosporine-A decreases cardiac reactive oxygen species generation as well as mitochondrial superoxide production. (A  ) Representative dihidroethidium staining of transverse heart sections subjected to 20 min of global ischemia followed by 5 min reperfusion with phosphate-buffered saline (control group, black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). (B  ) Fluorescence quantification of dihydroethidium staining: average intensity represents area · fluorescence intensity and is normalized to control (**P  < 0.001 vs.  control, n = 3). (C  ) Superoxide production in isolated mitochondria using electron spin resonance in control (black  ), 1% intralipid (red  ), and 1.5 μM cyclosporine-A (blue  ). **P  = 0.005 intralipid versus  control; *P  = 0.041 cyclosporine-A versus  control (n = 4). CsA = cyclosporine-A; CTRL = control; DHE = dihydroethidium; ILP = intralipid.
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Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A  , B  ) Representative immunoblots of pAkt and total Akt (A  ), and pGSK-3β and total GSK-3β (C  ) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C  , D  ) Western blot analysis of pAkt protein to total Akt (B  ) and pGSK-3β to total GSK-3b (D  ) ratios in control group (black bars  ), intralipid group (white bars  ), and cyclosporine-A group (gray bars  ). **P  < 0.001 versus  control; ##P  < 0.001 versus  intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A 
	, B 
	) Representative immunoblots of pAkt and total Akt (A 
	), and pGSK-3β and total GSK-3β (C 
	) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C 
	, D 
	) Western blot analysis of pAkt protein to total Akt (B 
	) and pGSK-3β to total GSK-3b (D 
	) ratios in control group (black bars 
	), intralipid group (white bars 
	), and cyclosporine-A group (gray bars 
	). **P 
	< 0.001 versus 
	control; ##P 
	< 0.001 versus 
	intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
Fig. 7. The lack of involvement of protein kinase B (Akt)/glycogen synthase kinase (GSK) pathways in cyclosporine-A-induced protection. (A  , B  ) Representative immunoblots of pAkt and total Akt (A  ), and pGSK-3β and total GSK-3β (C  ) in heart homogenates subjected to ischemia-reperfusion injury from control group, 1% intralipid, or 1.5 μM cyclosporine-A. (C  , D  ) Western blot analysis of pAkt protein to total Akt (B  ) and pGSK-3β to total GSK-3b (D  ) ratios in control group (black bars  ), intralipid group (white bars  ), and cyclosporine-A group (gray bars  ). **P  < 0.001 versus  control; ##P  < 0.001 versus  intralipid (n = 4–6/group). Akt = protein kinase B; CsA = cyclosporine-A; CTRL = control; GSK = glycogen synthase kinase; ILP = intralipid.
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Table 1. Systemic Hemodynamics in In Vivo  Model of Ischemia-Reperfusion Injury
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Table 1. Systemic Hemodynamics in In Vivo  Model of Ischemia-Reperfusion Injury
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