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Myocardial ischemia-reperfusion (I/R) injury increases the generation of oxidized phosphatidylcholines (OxPCs), which results in cell death. However, the mechanism by which OxPCs mediate cell death and cardiac dysfunction is largely unknown. The aim of this study was to determine the mechanisms by which OxPC triggers cardiomyocyte cell death during reperfusion injury. Adult rat ventricular cardiomyocytes were treated with increasing concentrations of various purified fragmented OxPCs. Cardiomyocyte viability, bioenergetic response, and calcium transients were determined in the presence of OxPCs. Five different fragmented OxPCs resulted in a decrease in cell viability, with 1-palmitoyl-2-(5'-oxo-valeroyl)- sn -glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PONPC) having the most potent cardiotoxic effect in both a concentration and time dependent manner ( P < 0.05). POVPC and PONPC also caused a significant decrease in Ca 2+ transients and net contraction in isolated cardiomyocytes compared to vehicle treated control cells ( P < 0.05). PONPC depressed maximal respiration rate ( P < 0.01; 54%) and spare respiratory capacity ( P < 0.01; 54.5%). Notably, neither caspase 3 activation or TUNEL staining was observed in cells treated with either POVPC or PONPC. Further, cardiac myocytes treated with OxPCs were indistinguishable from vehicle-treated control cells with respect to nuclear high-mobility group box protein 1 (HMGBP1) activity. However, glutathione peroxidase 4 activity was markedly suppressed in cardiomyocytes treated with POVPC and PONPC coincident with increased ferroptosis. Importantly, cell death induced by OxPCs could be suppressed by E06 Ab, directed against OxPCs or by ferrostatin-1, which bound the sn-2 aldehyde of POVPC during I/R. The findings of the present study demonstrate that oxidation of phosphatidylcholines during I/R generate bioactive phospholipid intermediates that disrupt mitochondrial bioenergetics and calcium transients and provoke wide spread cell death through ferroptosis. Neutralization of OxPC with E06 or with ferrostatin-1 prevents cell death during reperfusion. Our study demonstrates a novel signaling pathway that operationally links generation of OxPC during cardiac I/R to ferroptosis. Interventions designed to target OxPCs may prove beneficial in mitigating ferroptosis during I/R injury in individuals with ischemic heart disease. NEW & NOTEWORTHY Oxidized phosphatidylcholines (OxPC) generated during reperfusion injury are potent inducers of cardiomyocyte death. Our studies have shown that OxPCs exert this effect through a ferroptotic process that can be attenuated. A better understanding of the OxPC cell death pathway can prove a novel strategy for prevention of cell death during myocardial reperfusion injury.

Cell death is an important component of the pathophysiology of any disease. Myocardial disease is no exception. Understanding how and why cells die, particularly in the heart where cardiomyocyte regeneration is limited at best, becomes a critical area of study. Ferroptosis is a recently described form of nonapoptotic cell death. It is an iron-mediated form of cell death that occurs because of accumulation of lipid peroxidation products. Reactive oxygen species and iron-mediated phospholipid peroxidation is a hallmark of ferroptosis. To date, ferroptosis has been shown to be involved in cell death associated with Alzheimer's disease, Huntington's disease, cancer, Parkinson's disease, and kidney degradation. Myocardial reperfusion injury is characterized by iron deposition as well as reactive oxygen species production. These conditions, therefore, favor the induction of ferroptosis. Currently there is no available treatment for reperfusion injury, which accounts for up to 50% of the final infarct size. This review will summarize the evidence that ferroptosis can induce cardiomyocyte death following reperfusion injury and the potential for this knowledge to open new therapeutic approaches for myocardial ischemia-reperfusion injury.

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.

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 Ca 2+ during ischemia or ischemia/reperfusion but did not improve Ca 2+ 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.

We investigated calpain activation in the heart during ischemia-reperfusion (I-R) by immunologically mapping the fragmentation patterns of calpain and selected calpain substrates. Western blots showed the intact 78 kDa large subunit of membrane-associated calpain was autolytically fragmented to 56 and 43 kDa signature immunopeptides following I-R. Under these conditions, the 78 kDa calpain large subunit from crude cytosolic fractions was markedly less fragmented, with only weakly stained autolytic peptides detected at higher molecular weights (70 and 64 kDa). Western blots also showed corresponding calpain-like degradation products (150 and 145 kDa) of membrane-associated alpha-fodrin (240 kDa) following I-R, but in crude myofibrils alpha-fodrin degradation occurred in a manner uncharacteristic of calpain. For control hearts perfused in the absence of ischemia, autolytic fragmentation of calpain and calpain-like alpha-fodrin degradation were completely absent from most subcellular fractions. The exception was sarcolemma-enriched membranes, where significant calpain autolysis and calpain-like alpha-fodrin degradation were detected. In purified sarcoplasmic reticulum membranes, RyR2 and SERCA2 proteins were also highly degraded, but for RyR2 this did not occur in a manner characteristic of calpain. When I-R-treated hearts were perfused with peptidyl calpain inhibitors (ALLN or ALLM; 25 micromol/L), calpain autolysis and calpain-like degradation of alpha-fodrin were equally attenuated by each inhibitor. However, only ALLN protected against early loss of developed pressure in hearts following I-R, with no functionally protective effect of ALLM observed. Our studies suggest calpain is preferentially activated at membranes following I-R, possibly contributing to impaired ion channel function implicated by others in I-R injury.

The Na(+)/Ca(2+) exchanger (NCX) is proposed to be an important protein in the regulation of Ca(2+) movements in the heart. This Ca(2+) regulatory action is thought to modulate contractile activity in the heart under normal physiological conditions and may contribute to the Ca(2+) overload that occurs during ischemic reperfusion challenge. To evaluate these hypotheses, adult rat cardiomyocytes were exposed to an adenovirus that codes for short hairpin RNA (shRNA) targeting NCX gene expression through RNA interference. An adenovirus transcribing a short RNA with a scrambled nucleotide sequence was compared with the NCX-shRNA nucleotide sequence and used as a control. Freshly isolated rat cardiomyocytes were infected with virus for 48 h before examination. Cardiomyocytes maintained their characteristic morphological appearance during this short time period after isolation. NCX expression was inhibited by up to approximately 60% by the shRNA treatment as determined by Western blot analysis. The depletion in NCX protein was accompanied by a significant depression of NCX activity in shRNA-treated cells. Ca(2+) homeostasis was unaltered in the shRNA-treated cells upon electrical stimulation compared with control cells. However, when cardiomyocytes were exposed to a simulated ischemic solution, NCX-depleted cells were significantly protected from the rise in cytoplasmic Ca(2+) and damage that was detected in control cells during ischemia and reperfusion. Our data support the role for NCX in ischemic injury to the heart and demonstrate the usefulness of altering gene expression with an adenoviral-delivery system of shRNA in adult cardiomyocytes.

Polyunsaturated fatty acids (PUFAs) have significant, cardioprotective effects against ischemia. Hempseed contains a high proportion of the PUFAs linoleic acid (LA) and alpha-linolenic acid (ALA), which may have opposing effects on postischemic heart performance. There are no reported data concerning the cardiovascular effects of dietary hempseed intake. A group of 40 male Sprague-Dawley rats were distributed evenly into four groups that were fed for 12 wk a normal rat chow supplemented with hempseed (5% and 10%), palm oil (1%), or a 10% partially delipidated hempseed that served as a control. Plasma ALA and gamma-linolenic acid levels were significantly elevated in the rats that were fed a 5% or 10% hempseed-supplemented diet, but in heart tissue only ALA levels were significantly elevated in the rats fed these diets compared with control. After the dietary interventions were completed, postischemic heart performance was evaluated by measuring developed tension, resting tension, the rates of tension development and relaxation, and the number of extrasystoles. Hearts from rats fed a hempseed-supplemented diet exhibited significantly better postischemic recovery of maximal contractile function and enhanced rates of tension development and relaxation during reperfusion than hearts from the other groups. These hearts, however, were not protected from the occurrence of extrasystoles, nor were the increases in resting tension altered during ischemia or reperfusion as a function of any dietary intervention. Our data demonstrate that dietary hempseed can provide significant cardioprotective effects during postischemic reperfusion. This appears to be due to its highly enriched PUFA content.

The present study was undertaken to comprehensively characterize low-flow ischemia and reperfusion in single adult cardiomyocytes and to determine whether it is important to control contractile activity. The ischemia-mimetic solution was hypoxic, acidic (pH 6.0), and deficient in glucose but contained elevated KCl. Cardiomyocytes were stimulated to contract throughout ischemia and during reperfusion with control perfusate. After the ischemia-reperfusion insult, cells exhibited poor recovery of active cell shortening, a decrease in passive cell length, increased frequency of necrosis, lower ATP content, and evidence of the generation of oxygen-derived free radicals within the cells. Intracellular lactate concentration increased, pH decreased, and Ca(2+) transients were depressed during the ischemic insult, but the latter two parameters recovered partially on reperfusion. Basal intracellular Ca(2+) concentration was elevated during ischemia and early into reperfusion. Recovery was attenuated in cells that were electrically stimulated to contract throughout ischemia. The duration of ischemia, stimulation frequency, and composition of the ischemia-mimetic solution were important variables. The inclusion of 10 mM lactate in the ischemia-mimetic solution significantly aggravated all the parameters examined above. Our data demonstrate that 1) an ischemia-mimetic solution administered to single, isolated adult cardiomyocytes can reproduce many of the responses observed in whole hearts, 2) caution should be used in adding lactate to an ischemic solution, and 3) it is important to stimulate contractile activity throughout ischemia to reproduce the effects of ischemia in whole hearts.

Ischemic heart disease is a significant problem in the diabetic population. Animal models of diabetes show a paradoxical resistance to ischemic challenge. The present treatise will discuss the mechanics involved and the central role that Na+-H+ exchanger plays in this response to ischemic-reperfusion injury.

Previous work has demonstrated that drugs which inhibit Na+ entry through voltage-sensitive Na+ channels, or via Na(+)-H+ exchange protect the heart from ischemic reperfusion damage. The purpose of our study was to determine whether these drugs in combination will have an additive protective effect in Langendorff-perfused hearts. During reperfusion following 30 min of ischemia, developed tension and resting tension were 24 +/- 3 and 162 +/- 5%, respectively, of pre-ischemic values in non-treated ischemic hearts. The administration of HOE-642 to inhibit Na+/H+ exchange increased active developed tension (DT) to 58 +/- 2% of pre-ischemic levels and decreased resting tension (RT) to 111 +/- 3% of pre-ischemic levels. The administration of tetrodotoxin (TTX) to block the Na+ channel increased DT to 56 +/- 3% of the pre-ischemic level and reduced the RT to 126 +/- 12% of the pre-ischemic level. Together, HOE-642 and TTX increased recovery of DT to 63 +/- 2% of pre-ischemic levels and improved RT to 116 +/- 4% of pre-ischemic levels after 30 min of reperfusion. All drug treatment protocols significantly lowered the creatine phosphokinase activity measured in the coronary effluent in comparison to that observed in the non-treated hearts. These data demonstrate that inhibition of Na+ entry through either Na(+)-H+ exchange or the Na+ channel protects the heart from ischemic injury, but there is no additional benefit of blocking both routes of Na+ entry simultaneously. This suggests that a threshold level of Na+i may be a critical factor in ischemic cardioprotection.

Recent data from a number of independent laboratories have implicated ionic disturbances within the myocardium as being a critical contributory factor in injury to the heart during ischemia/reperfusion. The present treatise discusses the data supporting a role for Ca2+, Na+ and K+ in ischemic/reperfusion injury. The mechanism whereby intracellular ionic homeostasis becomes altered is the focus of the review. In particular, the evidence in support of an involvement of abnormal ion movements through Na+/H+ exchange, Na+ channels and the ATP-sensitive K+ channel in ischemic/reperfusion injury is advanced.

Previous work has associated cardiac dysfunction and damage after ischemia/reperfusion with metabolic alterations in the heart or alterations in the myocardial ionic homeostasis. Unfortunately, neither mechanism on its own has been able to conclusively explain the pathology. Instead, recent data suggest that the two mechanisms may be interrelated. The low intracellular pH during ischemia (due to the accumulation of metabolic by-products) may stimulate the Na(+)-H+ exchange pathway during reperfusion to remove H+ from the cell in exchange for will lead to accelerated Ca2+ entry via Na+. The subsequent accumulation of Na+ in the cell Na(+)-Ca2+ exchange, which can ultimately result in intracellular Ca2+ overload, contractile dysfunction and damage. This hypothesis is supported by the known biochemical characteristics of the cardiac Na(+)-H+ exchanger. Pharmacological studies also support this hypothesis as a mechanism involved in ischemic/reperfusion damage. Dimethylamiloride, a blocker of Na(+)-H+ exchange, has provided significant protection against ischemic/reperfusion injury to the heart. A series of studies have indicated that the mechanism through which dimethylamiloride acts is via inhibition of the Na(+)-H+ exchange pathway. The data, therefore, are consistent with an important interaction between metabolism and ionic alterations, which includes a central role for Na(+)-H+ exchange in ischemic/reperfusion damage to the heart.

During reperfusion with pH 7.2 media after 55 min of ischemia, the recovery of developed tension (DT) and the maximal resting tension (RT) of the isolated right ventricular wall of rats were 58.8 +/- 6.5 and 201 +/- 20%, respectively, of the preischemic values. DT and RT in the ventricular wall perfused with pH 7.9 media for 6 min during reperfusion were 40 +/- 5.9 and 285 +/- 13%, respectively, of the preischemic values (P less than .05 vs. pH 7.2 group). The Na(+)-H+ exchange inhibitor, 5-(N,N-dimethyl)amiloride (DMA), effectively antagonized the detrimental effect of pH 7.9 media. A pH 6.5 media inhibited DT recovery and the rise in RT in the first 6 min of reperfusion. Subsequent reperfusion with pH 7.2 media resulted in cardiac dysfunction similar to that observed when reperfused at pH 7.2 only. Cellular Na+ and Ca++ were significantly elevated after 6 min of reperfusion at pH 7.2. Na+ and Ca++ levels were increased further if reperfusion was carried out at pH 7.9. Inclusion of 20 microM DMA during reperfusion at 7.9 significantly reduced cellular Na+ and Ca++. Creatine phosphokinase activity in the coronary effluent rose significantly during reperfusion at pH 7.2 and this was exacerbated if the reperfusion pH was 7.9. DMA treatment during reperfusion could significantly inhibit this elevation. The data lend further support for an important role of Na(+)-H+ exchange in the development of ischemia-reperfusion injury.

During reperfusion in the isolated right ventricular wall of the rat after 60 min of ischemia, developed tension and resting tension were 35 +/- 4 and 221 +/- 12%, respectively, of preischemic values. Including 35 microM ouabain in the perfusate before and after ischemia resulted in more severe cardiac dysfunction during reperfusion than in drug-untreated hearts. Introduction of the Na(+)-H+ exchange inhibitor, 5-(N,N-dimethyl)-amiloride (DMA), could effectively protect the right ventricular wall against ischemia-reperfusion dysfunction in the presence or absence of ouabain. The ion content in the right ventricular wall was measured with atomic absorbance spectrophotometry. Before ischemia, Na+,Ca++ and K+ content were 53.4 +/- 6.4, 2.70 +/- 0.22 and 262 +/- 7.7 mumol/g of dry weight tissue, respectively. After 60 min of ischemia and 6 min of reperfusion, Na+,Ca++ and K+ content were 73.4 +/- 7.2, 3.79 +/- 0.31 and 180 +/- 15 mumol/g of dry weight tissue, respectively (P less than .05). Introduction of 20 microM DMA normalized ion content in the muscles which was consistent with the contractile function recovery during reperfusion. The data suggest that a rise in intracellular Na+ in the early stage of reperfusion represents a crucial or primary step for the development of cardiac contractile dysfunction. DMA, which protects against severe reperfusion-induced cardiac contractile dysfunction, appears to act via a normalization of tissue sodium levels. This action is consistent with its proposed role as a blocker of transsarcolemmal Na(+)-H+ exchange.

An Na(+)-H+ exchange inhibitor, 5-(N,N-dimethyl)amiloride (DMA), was used to probe the possible role of Na(+)-H+ exchange in ischemia-reperfusion injury in coronary perfused isolated rat right ventricular wall. In DMA-untreated hearts, 60 min of ischemia resulted in a significant rise in testing tension (RT: 174 +/- 8% of preischemic level). Thirty minutes of reperfusion further increased RT (273 +/- 12%) and induced a poor recovery in developed tension (DT: 28 +/- 4%). Both the rate of tension development and relaxation (+dT/dt and -dT/dt) recovered to a similar degree. When 1, 5, or 20 microM DMA was included in the perfusate (3 min before ischemia and in the first 3 min of reperfusion), the maximal postischemic RT of the heart was reduced to 204 +/- 21, 166 +/- 15, and 139 +/- 45% of the preischemic levels (P less than 0.05), respectively, and DT was 39 +/- 3, 63 +/- 10, and 79 +/- 8% of the preischemic levels (P less than 0.05), respectively. Similar qualitative recovery of +/- dT/dt was observed. Recovery was similar if DMA was present only during reperfusion. DMA treatment also significantly protected against creatine phosphokinase release during reperfusion. The results demonstrate that DMA can significantly protect the heart during the initial stages of reperfusion. The data suggest that Na(+)-H+ exchange may play an important role in the development of cardiac dysfunction and damage during the first minutes of reperfusion.