Reduction of myocardial ischaemia-reperfusion injury by inactivating oxidized phospholipids.

Aims: Myocardial ischaemia followed by reperfusion (IR) causes an oxidative burst resulting in cellular dysfunction. Little is known about the impact of oxidative stress on cardiomyocyte lipids and their role in cardiac cell death. Our goal was to identify oxidized phosphatidylcholine-containing phospholipids (OxPL) generated during IR, and to determine their impact on cell viability and myocardial infarct size. Methods and results: OxPL were quantitated in isolated rat cardiomyocytes using mass spectrophotometry following 24 h of IR. Cardiomyocyte cell death was quantitated following exogenously added OxPL and in the absence or presence of E06, a 'natural' murine monoclonal antibody that binds to the PC headgroup of OxPL. The impact of OxPL on mitochondria in cardiomyocytes was also determined using cell fractionation and Bnip expression. Transgenic Ldlr-/- mice, overexpressing a single-chain variable fragment of E06 (Ldlr-/--E06-scFv-Tg) were used to assess the effect of inactivating endogenously generated OxPL in vivo on myocardial infarct size. Following IR in vitro, isolated rat cardiomyocytes showed a significant increase in the specific OxPLs PONPC, POVPC, PAzPC, and PGPC (P < 0.05 to P < 0.001 for all). Exogenously added OxPLs resulted in significant death of rat cardiomyocytes, an effect inhibited by E06 (percent cell death with added POVPC was 22.6 ± 4.14% and with PONPC was 25.3 ± 3.4% compared to 8.0 ± 1.6% and 6.4 ± 1.0%, respectively, with the addition of E06, P < 0.05 for both). IR increased mitochondrial content of OxPL in rat cardiomyocytes and also increased expression of Bcl-2 death protein 3 (Bnip3), which was inhibited in presence of E06. Notably cardiomyocytes with Bnip3 knock-down were protected against cytotoxic effects of OxPL. In mice exposed to myocardial IR in vivo, compared to Ldlr-/- mice, Ldlr-/--E06-scFv-Tg mice had significantly smaller myocardial infarct size normalized to area at risk (72.4 ± 21.9% vs. 47.7 ± 17.6%, P = 0.023). Conclusions: OxPL are generated within cardiomyocytes during IR and have detrimental effects on cardiomyocyte viability. Inactivation of OxPL in vivo results in a reduction of infarct size.

Alpha linolenic acid decreases apoptosis and oxidized phospholipids in cardiomyocytes during ischemia/reperfusion.

The omega-3 fatty acid, alpha linolenic acid (ALA) found in plant-derived foods induces significant cardiovascular benefits when ingested. ALA may be cardioprotective during ischemia; however, the mechanism(s) responsible for this effect is unknown. Isolated adult rat cardiomyocytes were exposed to medium containing ALA for 24 h and then exposed to non-ischemic (control), simulated ischemia (ISCH), or simulated ischemia/reperfusion (IR) conditions. Cardiomyocyte phospholipids were extracted and analyzed by an HPLC/electrospray ionization tandem mass spectrometry system. Pre-treatment of cells with ALA resulted in a significant incorporation of ALA within cardiomyocyte phosphatidylcholine. Cell death, DNA fragmentation and caspase-3 activity increased during ischemia and ischemia/reperfusion. Two pro-apoptotic oxidized phosphatidylcholine (OxPC) species, 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC), and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC) were significantly increased during both ischemia and ischemia/reperfusion. Pre-treatment of the cells with ALA resulted in a significant reduction in cell death during ischemia and ischemia/reperfusion challenge. Apoptosis was also inhibited during ischemia and ischemia/reperfusion as shown by reduced DNA fragmentation and decreased caspase activation. ALA pre-treatment significantly decreased the production of POVPC and PGPC during ischemia and ischemia/reperfusion. ALA pre-treatment also significantly increased in resting Ca2+ during ischemia or ischemia/reperfusion but did not improve Ca2+ transients. ALA protects the cardiomyocyte from apoptotic cell death during simulated ISCH and IR by inhibiting the production of specific pro-apoptotic OxPC species. OxPCs represent a viable interventional target to protect the heart during ischemic challenge.

Identification of Oxidized Phosphatidylinositols Present in OxLDL and Human Atherosclerotic Plaque.

Oxidized low-density lipoprotein (OxLDL) plays an important role in initiation and progression of atherosclerosis. Proatherogenic effects of OxLDL have been attributed to bioactive phospholipids generated during LDL oxidation. It is unknown what effect oxidation has on the phosphatidylinositol (PtdIns) molecules in LDL, even though PtdIns is 6% of the total LDL phospholipid pool. We sought to identify and quantitate oxidized phosphatidylinositol (OxPtdIns) species in OxLDL and human atherosclerotic plaque. Bovine liver PtdIns was subjected to non-enzymatic and lipoxygenase-catalyzed oxidation. Reversed-phase liquid chromatography with negative ESI-MS identified and confirmed compounds by fragmentation pattern analysis from which an OxPtdIns library was generated. Twenty-three OxPtdIns molecules were identified in copper-oxidized human LDL at 0, 6, 12, 24, 30, and 48 h, and in human atherosclerotic plaque. In OxLDL, OxPtdIns species containing aldehydes and carboxylates comprised 17.3 ± 0.1 and 0.9 ± 0.2%, respectively, of total OxPtdIns in OxLDL at 48 h. Hydroperoxides and isoprostanes at 24 h (68.5 ± 0.2 and 22.8 ± 0.2%) were significantly greater than 12 h (P < 0.01) without additional changes thereafter. Hydroxides decreased with increased oxidation achieving a minimum at 24 h (5.2 ± 0.3%). Human atherosclerotic plaques contained OxPtdIns species including aldehydes, carboxylates, hydroxides, hydroperoxides and isoprostanes, comprising 18.6 ± 4.7, 1.5 ± 0.7, 16.5 ± 7.4, 33.3 ± 1.1 and 30.2 ± 3.3% of total OxPtdIns compounds. This is the first identification of OxPtdIns molecules in human OxLDL and atherosclerotic plaque. With these novel molecules identified we can now investigate their potential role in atherosclerosis.

A whole blood-based perfusate provides superior preservation of myocardial function during ex vivo heart perfusion.

BACKGROUND: Ex vivo heart perfusion (EVHP) provides the opportunity to resuscitate unused donor organs and facilitates assessments of myocardial function that are required to demonstrate organ viability before transplantation. We sought to evaluate the effect of different oxygen carriers on the preservation of myocardial function during EVHP. METHODS: Twenty-seven pig hearts were perfused ex vivo in a normothermic beating state for 6 hours and transitioned into working mode for assessments after 1 (T1), 3 (T3), and 5 (T5) hours. Hearts were allocated to 4 groups according to the perfusate composition. Red blood cell concentrate (RBC, n = 6), whole blood (RBC+Plasma, n = 6), an acellular hemoglobin-based oxygen carrier (HBOC, n = 8), or HBOC plus plasma (HBOC+Plasma, n = 7) were added to STEEN Solution (XVIVO Perfusion, Goteborg, Sweden) to achieve a perfusate hemoglobin concentration of 40 g/liter. RESULTS: The perfusate composition affected the preservation of systolic (T5 dP/dtmax: RBC+Plasma = 903 ± 99, RBC = 771 ± 77, HBOC+Plasma = 691 ± 82, HBOC = 563 ± 52 mm Hg/sec; p = 0.047) and diastolic (T5 dP/dtmin: RBC+Plasma = -574 ± 48, RBC = -492 ± 63, HBOC+Plasma = -326 ± 32, HBOC = -268 ± 22 mm Hg/sec; p < 0.001) function, and the development of myocardial edema (weight gain: RBC+Plasma = 6.6 ± 0.9, RBC = 6.6 ± 1.2, HBOC+Plasma = 9.8 ± 1.7, HBOC = 16.3 ± 1.9 g/hour; p < 0.001) during EVHP. RBC+Plasma hearts exhibited less histologic evidence of myocyte damage (injury score: RBC+Plasma = 0.0 ± 0.0, RBC = 0.8 ± 0.3, HBOC+Plasma = 2.6 ± 0.2, HBOC = 1.75 ± 0.4; p < 0.001) and less troponin-I release (troponin-I fold-change T1-T5: RBC+Plasma = 7.0 ± 1.7, RBC = 13.1 ± 1.6, HBOC+Plasma = 20.5 ± 1.1, HBOC = 16.7 ± 5.8; p < 0.001). Oxidative stress was minimized by the addition of plasma to RBC and HBOC hearts (oxidized phosphatidylcholine compound fold-change T1-T5: RBC+Plasma = 1.83 ± 0.20 vs RBC = 2.31 ± 0.20, p < 0.001; HBOC+Plasma = 1.23 ± 0.17 vs HBOC = 2.80 ± 0.28, p < 0.001). CONCLUSIONS: A whole blood-based perfusate (RBC+Plasma) minimizes injury and provides superior preservation of myocardial function during EVHP. The beneficial effect of plasma on the preservation of myocardial function requires further investigation.

Congenital absence of nitric oxide synthase 3 potentiates cardiac dysfunction and reduces survival in doxorubicin- and trastuzumab-mediated cardiomyopathy.

BACKGROUND: Doxorubicin (DOX) and trastuzumab (TRZ) are highly effective chemotherapeutic agents in the breast cancer setting, limited by their cardiotoxic side effects. Among the potential mechanisms for this drug-induced cardiomyopathy, increased production of oxidative stress (OS) through a nitric oxide synthase 3 (NOS3)-dependent pathway has gained recent attention. The objective of the study was to determine the role of NOS3 and OS in a clinically relevant female murine model of DOX- and TRZ-induced heart failure. METHODS: A total of 120 female mice (60 wild-type [WT] and 60 NOS3 knockout [NOS3(-/-)]) were treated with either 0.9% saline, DOX, TRZ, or DOX with TRZ (DOX+TRZ). Serial echocardiography was performed for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. RESULTS: In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75 ± 3% at baseline to 46 ± 2% at day 10 (P < 0.05). In the NOS3(-/-) group, LVEF decreased from 72 ± 3% at baseline to 35 ± 2% at day 10 (P < 0.05). LVEF was significantly lower in NOS3(-/-) female mice receiving DOX+TRZ than WT mice at day 10 (P < 0.05). Compared with WT, NOS3(-/-) female mice also demonstrated increased mortality after treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis demonstrated increased myofibrillar degradation and loss of cell integrity in NOS3(-/-) female mice treated with DOX+TRZ. There was increased generation of oxidized phosphatidylcholine, a marker of OS, in NOS3(-/-) female mice receiving DOX+TRZ compared with control mice. CONCLUSIONS: Congenital absence of NOS3 potentiates the cardiotoxic side effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

A cardioprotective preservation strategy employing ex vivo heart perfusion facilitates successful transplant of donor hearts after cardiocirculatory death.

BACKGROUND: Ex vivo heart perfusion (EVHP) has been proposed as a means to facilitate the resuscitation of donor hearts after cardiocirculatory death (DCD) and increase the donor pool. However, the current approach to clinical EVHP may exacerbate myocardial injury and impair function after transplant. Therefore, we sought to determine if a cardioprotective EVHP strategy that eliminates myocardial exposure to hypothermic hyperkalemia cardioplegia and minimizes cold ischemia could facilitate successful DCD heart transplantation. METHODS: Anesthetized pigs sustained a hypoxic cardiac arrest and a 15-minute warm ischemic standoff period. Strategy 1 hearts (S1, n = 9) underwent initial reperfusion with a cold hyperkalemic cardioplegia, normothermic EVHP, and transplantation after a cold hyperkalemic cardioplegic arrest (current EVHP strategy). Strategy 2 hearts (S2, n = 8) underwent initial reperfusion with a tepid adenosine-lidocaine cardioplegia, normothermic EVHP, and transplantation with continuous myocardial perfusion (cardioprotective EVHP strategy). RESULTS: At completion of EVHP, S2 hearts exhibited less weight gain (9.7 ± 6.7 [S2] vs 21.2 ± 6.7 [S1] g/hour, p = 0.008) and less troponin-I release into the coronary sinus effluent (4.2 ± 1.3 [S2] vs 6.3 ± 1.5 [S1] ng/ml; p = 0.014). Mass spectrometry analysis of oxidized pleural in post-transplant myocardium revealed less oxidative stress in S2 hearts. At 30 minutes after wean from cardiopulmonary bypass, post-transplant systolic (pre-load recruitable stroke work: 33.5 ± 1.3 [S2] vs 19.7 ± 10.9 [S1], p = 0.043) and diastolic (isovolumic relaxation constant: 42.9 ± 6.7 [S2] vs 65.2 ± 21.1 [S1], p = 0.020) function were superior in S2 hearts. CONCLUSION: In this experimental model of DCD, an EVHP strategy using initial reperfusion with a tepid adenosine-lidocaine cardioplegia and continuous myocardial perfusion minimizes myocardial injury and improves short-term post-transplant function compared with the current EVHP strategy using cold hyperkalemic cardioplegia before organ procurement and transplantation.