Parisa Partownavid

Phosphorylation of GSK-3β Mediates Intralipid-induced Cardioprotection against Ischemia/Reperfusion Injury

Background:Intralipid (Sigma, St. Louis, MO), a brand name for the first safe fat emulsion for human use, has been shown to be cardioprotective. However, the mechanism of this protection is not known. The authors investigated the molecular mechanism(s) of Intralipid-induced cardioprotection against ischemia/reperfusion injury, particularly the role of glycogen synthase kinase-3&bgr; (GSK-3&bgr;) and mitochondrial permeability transition pore in this protective action. Methods:In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with Intralipid (1% in ex vivo and one bolus of 20% in in vivo) or vehicle. The hemodynamic function, infarct size, threshold for the opening of mitochondrial permeability transition pore, and phosphorylation levels of protein kinase B (Akt)/extracellular signal regulating kinase (ERK)/GSK-3&bgr; were measured. Results:Administration of Intralipid at the onset of reperfusion resulted in approximately 70% reduction in infarct size in the in vivo rat model. Intralipid also significantly improved functional recovery of isolated Langendorff-perfused mouse hearts as the rate pressure product was increased from 2,999 ± 863 mmHg*beats/min in the control group to 13,676 ± 611 mmHg*beats/min (mean±SEM) and the infarct size was markedly smaller (18.3 ± 2.4% vs. 54.8 ± 2.9% in the control group, P < 0.01). The Intralipid-induced cardioprotection was fully abolished by LY294002, a specific inhibitor of PI3K, but only partially by PD98059, a specific ERK inhibitor. Intralipid also increased the phosphorylation levels of Akt/ERK1/glycogen synthase kinase-3&bgr; by eightfold, threefold, and ninefold, respectively. The opening of mitochondrial permeability transition pore was inhibited by Intralipid because calcium retention capacity was higher in the Intralipid group (274.3 ± 8.4 nM/mg vs. 168.6 ± 9.6 nM/mg in the control group). Conclusions:Postischemic treatment with Intralipid inhibits the opening of mitochondiral permeability transition pore and protects the heart through glycogen synthase kinase-3&bgr; via PI3K/Akt/ERK pathways.

Fatty-acid oxidation and calcium homeostasis are involved in the rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats.

Objectives: Lipid emulsion has been shown to be effective in resuscitating bupivacaine-induced cardiac arrest but its mechanism of action is not clear. Here we investigated whether fatty-acid oxidation is required for rescue of bupivacaine-induced cardiotoxicity by lipid emulsion in rats. We also compared the mitochondrial function and calcium threshold for triggering of mitochondrial permeability transition pore opening in bupivacaine-induced cardiac arrest before and after resuscitation with lipid emulsion. Design: Prospective, randomized animal study. Setting: University research laboratory. Subjects: Adult male Sprague-Dawley rats. Interventions: Asystole was achieved with a single dose of bupivacaine (10 mg/kg over 20 secs, intravenously) and 20% lipid emulsion infusion (5 mL/kg bolus, and 0.5 mL/kg/min maintenance), and cardiac massage started immediately. The rats in CVT-4325 (CVT) group were pretreated with a single dose of fatty-acid oxidation inhibitor CVT (0.5, 0.25, 0.125, or 0.0625 mg/kg bolus intravenously) 5mins prior to inducing asystole by bupivacaine overdose. Heart rate, ejection fraction, fractional shortening, the threshold for opening of mitochondrial permeability transition pore, oxygen consumption, and membrane potential were measured. The values are mean ± SEM. Measurements and Main Results: Administration of bupivacaine resulted in asystole. Lipid Emulsion infusion improved the cardiac function gradually as the ejection fraction was fully recovered within 5 mins (ejection fraction = 64 ± 4% and fractional shortening = 36 ± 3%, n = 6) and heart rate increased to 239 ± 9 beats/min (71% recovery, n = 6) within 10 mins. Lipid emulsion was only able to rescue rats pretreated with low dose of CVT (0.0625 mg/kg; heart rate ~ 181 ± 11 beats/min at 10 mins, recovery of 56%; ejection fraction = 50 ± 1%; fractional shortening = 26 ± 0.6% at 5 mins, n = 3), but was unable to resuscitate rats pretreated with higher doses of CVT (0.5, 0.25, or 0.125 mg/kg). The calcium-retention capacity in response to Ca2+ overload was significantly higher in cardiac mitochondria isolated from rats resuscitated with 20% lipid emulsion compared to the group that did not receive Lipid Emulsion after bupivacaine overdose (330 ± 42 nmol/mg vs. 180 ± 8.2 nmol/mg of mitochondrial protein, p < .05, n = 3 in each group). The mitochondrial oxidative rate and membrane potential were similar in the bupivacaine group before and after resuscitation with lipid emulsion infusion. Conclusions: Fatty-acid oxidation is required for successful rescue of bupivacaine-induced cardiotoxicity by lipid emulsion. This rescue action is associated with inhibition of mitochondrial permeability transition pore opening.

Intralipid Prevents and Rescues Fatal Pulmonary Arterial Hypertension and Right Ventricular Failure in Rats

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling leading to right ventricular (RV) hypertrophy and failure. Intralipid (ILP), a source of parenteral nutrition for patients, contains &ggr;-linolenic acid and soy-derived phytoestrogens that are protective for lungs and heart. We, therefore, investigated the therapeutic potential of ILP in preventing and rescuing monocrotaline-induced PAH and RV dysfunction. PAH was induced in male rats with monocrotaline (60 mg/kg). Rats then received daily ILP (1 mL of 20% ILP per day IP) from day 1 to day 30 for prevention protocol or from day 21 to day 30 for rescue protocol. Other monocrotaline-injected rats were left untreated to develop severe PAH by day 21 or RV failure by approximately day 30. Saline or ILP-treated rats served as controls. Significant increase in RV pressure and decrease in RV ejection fraction in the RV failure group resulted in high mortality. Therapy with ILP resulted in 100% survival and prevented PAH-induced RV failure by preserving RV pressure and RV ejection fraction and preventing RV hypertrophy and lung remodeling. In preexisting severe PAH, ILP attenuated most lung and RV abnormalities. The beneficial effects of ILP in PAH seem to result from the interplay of various factors, among which preservation and/or stimulation of angiogenesis, suppression and/or reversal of inflammation, fibrosis and hypertrophy, in both lung and RV, appear to be major contributors. In conclusion, ILP not only prevents the development of PAH and RV failure but also rescues preexisting severe PAH.

Ultrasound-guided peripheral nerve block.

With the common use of ultrasound imaging by anesthesiologists, especially for peripheral nerve blocks, this article will review basic physics of ultrasound machine, use of ultrasound in regional anesthesia, review of recent reports in the literature, and the outcome data.

Involvement of Opioid Receptors in the Lipid Rescue of Bupivacaine-Induced Cardiotoxicity.

BACKGROUND:Lipid emulsion (LE) has been successfully used for resuscitation of local anesthetic cardiotoxicity caused by bupivacaine overdose. Opioid receptors have been shown to play a key role in cardio protection. We explored whether this rescue action of LE is mediated through opioid receptors. METHODS:Asystole was induced by bupivacaine (10 mg/kg over 20 seconds, IV) in young male Sprague-Dawley rats, and resuscitation with LE (intralipid 20%; 5 mL/kg bolus and 0.5 mL/kg/min maintenance) was started immediately. The rats were pretreated 2 minutes before inducing asystole with nonselective opioid receptor antagonists such as naloxone and naloxone methiodide, as well as highly selective opioid receptor antagonists for subtype &kgr;, &dgr;, and µ or phosphate buffer solution as a control. Heart rates and ejection fractions were measured using echocardiography. RESULTS:LE rescue of bupivacaine cardiotoxicity was prevented by high-dose (1 mg/kg) naloxone but not by lower doses of naloxone (1, 5, and 10 µg/kg), by naloxone methiodide (which does not cross the blood–brain barrier), and by a selective &dgr;- and &kgr;-opioid receptor antagonists at a higher (10 mg/kg) dose. Successful LE rescue was not affected by highly selective µ-opioid receptor antagonists. &dgr;-Opioid receptor antagonist (10 mg/kg) pretreatment also resulted in reduced phosphorylation level of cardiac glycogen synthase kinase-3&bgr; in rats that were not resuscitated by LE compared with control. CONCLUSIONS:Our data highlight the involvement of peripheral &dgr;- and &kgr;-opioid receptors in the rescue action of LE.

Phosphorylation of GSK-3b is Required for Intralipid to Protect the Heart Against Ischemia/Reperfusion Injury

Recently we found that administration of Intralipid (ILP) during reperfusion significantly improves post-ischemic cardiac function and reduces the myocardial infarct size by ∼70% , both in the isolated mouse heart and in-vivo rat heart. Here we investigated whether ILP-induced cardioprotection is mediated through the inhibition of GSK-3b. Wild type (WT) C57BL/6 male mice and GSK-3b Knockin (KI) were used. The isolated hearts were subjected to 20 min of global normothermic ischemia followed by reperfusion with 1% ILP (40 min for heart function and infarct size and 10 min for calcium retention capacity (CRC) experiments). The left ventricular (LV) systolic pressure, LV end-diastolic pressure (LVDP), heart rate, maximum velocity of contraction (dP/dt max) and maximum velocity of relaxation (dP/dt min) were recorded. Myocardial necrosis was assessed using TTC staining. Mitochondria were isolated to measure CRC by calculating the number of pulses required to trigger the opening of the mitochondrial transition permeability pore as a result of calcium overload. Before ischemia, the baseline RPP, LVDP, dP/dtmax and dP/dtmin in GSK-3b KI mice were similar to WT. However, the functional recovery during reperfusion was very poor in GSK-3b KI mice. At the end of 40 min of reperfusion, the RPP was 1990±499 in GSK-3b KI mice vs. 15405±1011mmHg∗beats/min in WT, the LV dP/dtmax was 239.7±17.8 in GSK-3b KI vs. 2703±145 mmHg/s in WT and the LV dP/dtmin was 219±14 in GSK-3b KI vs. 1683±66 mmHg/s in WT. The infarct size was significantly larger compared to WT (45.3±10.3 vs. 16.7±2.33% in WT, P<0.001). Postischemic administration of ILP in GSK-3b KI mice demonstrated lower CRC than WT (1.3±0.1 vs. 2.7±0.06 µM/mg-mitochondrial protein in WT). In conclusions, these data demonstrate that phosphoryltaion of GSK-3b is required for the cardioprotective action of ILP.

CVT Inhibits the Intralipid Rescue of Bupivacaine-Induced Cardiotoxicity in a Dose-Dependant Manner

Intralipid (ILP) is effective in resuscitating Bupivacaine-induced cardiac arrest, but its mechanism of action is not clear. Here we investigated whether protective action of ILP is mediated through fatty acid oxidation pathway using CVT-4325, a fatty acid oxidation inhibitor. Male Sprague-Dawley rats (300-350 g) were anesthetized (ketamine (80mg/kg) and xylazine (8mg/kg, i.p.)) and then ventilated. In control (CTRL, n=8), asystole was achieved with a single dose of Bupivacaine (10mg/kg over 20 seconds, i.v.) and then resuscitation was started immediately using ILP (5ml/kg bolus, and 0.5ml/kg/min maintenance) together with cardiac massage. In CVT group (n=12), the protocol was identical to CTRL, except that rats were pre-treated with different doses of CVT (0.5, 0.25, 0.125 and 0.0625mg/kg bolus i.v.) for 5 min. The heart rate (HR), ejection fraction (EF) and fractional shortening (FS) were measured by echocardiography. As expected, in CTRL group, administration of Bupivacaine resulted in asystole and ILP improved HR and cardiac function gradually within 10 min; HR increased from 73±3beats/min at 1 min to 180±23beats/min at 5min, and further to 243±20beats/min at 10min. The left ventricular systolic function fully recovered in all rats within 5min of ILP treatment (EF=70±3%, FS=40±3%). In CVT pretreated group, however, there was no recovery of cardiac function with ILP at CVT doses of 0.5, 0.25 and 0.125mg/kg within 10min of ILP therapy. ILP was only able to rescue Bupivacaine-induced cardiotoxicity at lowest dose of 0.0625 mg/kg CVT as cardiac function improved gradually within 10min (HR from 65 at 1min to ∼170min at 10 min; EF=∼55%, FS=∼29% at 10min and QRS from 33 ms at 1 min to 27 ms at 10 min). In conclusion CVT-4325 prevents intralipid rescue of Bupivacaine-induced cardiotoxicity in a dose dependant manner.

Mechanisms of Prevention of Pulmonary Hypertension-Induced Right-Ventricular Failure by Intralipid

Intralipid (ILP) has been used for treatment of local anesthetic-induced cardiac-arrest and as source of parenteral nutrition. Some of its constituents are precursors of pulmonary vasodilator prostaglandins. Pulmonary hypertension (PH) is associated with pulmonary vascular remodeling leading to right-ventricular (RV) hypertrophy and failure. Recently we found that ILP prevents PH in rats. Here we investigated the mechanisms involved in this prevention. Rats were treated with monocrotaline(60 mg/kg) to induce PH, and then either received daily ILP (1 mL of 20% ILP/day) for 30days or left untreated to develop RV failure (RVF). Saline treated rats served as control. Serial echocardiography was performed to monitor cardiopulmonary hemodynamics., RV pressure (RVP) was recorded by direct cardiac catheterization right before sacrifice. Immunohistochemistry, confocal-microscopy, Western Blot analysis and RT-PCR were performed. RVF group developed PH that led to RVF later with significantly increased RVP (70±5mmHg,n=10) and depressed RV ejection fraction (RVEF=34±2%). Severe structural changes in RV and lung were observed in RVF group including RV-hypertrophy, increased lung weight, pulmonary medial hypertrophy, fibrosis, apoptosis (upregulation of Caspase-3 (∼10fold)), inflammation (increased expression of IL-6) and suppression of angiogenesis (decreased VEGF and capillary-density). Furthermore, RVF was associated with downregulation of phospho-eNOS(∼4fold), Caveolin-1(∼6fold) adenosine-A2B receptor and estrogen receptor-β in lungs. In the RV, adenosine-A2A receptor and estrogen receptor-b were downregulated in RVF. ILP therapy prevented PH-induced RVF by restoring RVP(30±2mmHg,n=8) and RVEF(63±1.5%) and attenuating RV hypertrophy and lung remodeling. ILP suppressed inflammation and fibrosis along-with improving angiogenesis and restoring phospho-eNOS, Caveolin-1 in lungs and Caspase-3, adenosine-A2 and estrogen receptor-β expression in lungs and RV. In conclusion ILP prevented PH and RV-failure by preserving cardiopulmonary structure and function and enhancing perfusion via adenosine receptor, estrogen receptor-b and eNOS-mediated mechanisms.

Intralipid Protects Cardiac Function of Late Pregnant Mice against Ischemia/Reperfusion Injury

Female mouse hearts show better functional recovery after ischemia/reperfusion (I/R) injury compared with males. However, the vulnerability of isolated late pregnant (LP) hearts to I/R injury is unknown. Here we investigated the susceptibility of isolated mouse hearts in LP and postpartum (PP) to I/R injury. Isolated hearts (Langendorff) from female mice in diestrus stage (NP), LP, one day PP (PP1) and 7 day PP (PP7) were subjected to 20 minutes of global normothermic (37°C) ischemia followed by 40 minutes of reperfusion. The heart function was recorded throughout the experiments and infarct size was assessed by triphenyltetrazolium staining at the end of reperfusion. Although the function was similar in all 4 groups before ischemia, the functional recovery of LP hearts at the end of reperfusion was significantly lower compared to NP hearts; the rate pressure product (RPP) was reduced from 12926±1479mmHg∗beats/min in NP to 1614±438mmHg∗beats/min in LP mice. Interestingly, the RPP recovered partially in PP1 to 4716±584mmHg∗beats/min and almost fully back to NP levels one week PP. Consistent with the functional recovery findings, the infarct size was markedly larger in LP (59.7±5.2%) compared with NP (15.2±0.8%). The infarct size was restored partially in PP1 and fully back in PP7. Recently we have observed that Intralipid can protect the male mouse heart against I/R injury. To test whether Intralipid can improve the heart function in LP mice, 1% Intralipid was applied to isolated LP hearts at the onset of reperfusion. Intralipid treatment significantly improved the cardiac function of LP mice (RPP=11565±1599mmHg∗beats/min) and reduced the infarct size (17±1.1%) to similar values as in NP. In conclusion, isolated LP hearts have high vulnerability to I/R injury and postischemic treatment with Intralipid can protect the heart against I/R injury.