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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.

Cardiovascular disease remains the leading cause of mortality and morbidity worldwide. The inclusion of functional foods and natural health products in the diet are gaining increasing recognition as integral components of lifestyle changes in the fight against cardiovascular disease. Several preclinical and clinical studies have shown the beneficial cardiovascular effects of dietary supplementation with flaxseed. The cardiovascular effects of dietary flaxseed have included an antihypertensive action, antiatherogenic effects, a lowering of cholesterol, an anti-inflammatory action, and an inhibition of arrhythmias. Its enrichment in the ω-3 fatty acid α-linolenic acid and the antioxidant lignan secoisolariciresinol diglucoside as well as its high fiber content have been implicated primarily in these beneficial cardiovascular actions. Although not as well recognized, flaxseed is also composed of other potential bioactive compounds such as proteins, cyclolinopeptides, and cyanogenic glycosides, which may also produce biological actions. These compounds could also be responsible for the cardiovascular effects of flaxseed. This article will not only summarize the cardiovascular effects of dietary supplementation with flaxseed but also review its bioactive compounds in terms of their properties, biological effects, and proposed mechanisms of action. It will also discuss promising research directions for the future to identify additional health-related benefits of dietary flaxseed.

Oxylipins are a group of fatty acid metabolites generated via oxygenation of polyunsaturated fatty acids and are involved in processes such as inflammation, immunity, pain, vascular tone, and coagulation. As a result, oxylipins have been implicated in many conditions characterized by these processes, including cardiovascular disease and aging. The best characterized oxylipins in relation to cardiovascular disease are derived from the ω-6 fatty acid arachidonic acid. These oxylipins generally increase inflammation, hypertension, and platelet aggregation, although not universally. Similarly, oxylipins derived from the ω-6 fatty acid linoleic acid generally have more adverse than beneficial cardiovascular effects. Alternatively, most oxylipins derived from 20- and 22-carbon ω-3 fatty acids have anti-inflammatory, antiaggregatory, and vasodilatory effects that help explain the cardioprotective effects of these fatty acids. Much less is known regarding the oxylipins derived from the 18-carbon ω-3 fatty acid α-linolenic acid, but clinical trials with flaxseed supplementation have indicated that these oxylipins can have positive effects on blood pressure. Normal aging also is associated with changes in oxylipin levels in the brain, vasculature, and other tissues, indicating that oxylipin changes with aging may be involved in age-related changes in these tissues. A small number of trials in humans and animals with interventions that contain either 18-carbon or 20- and 22-carbon ω-3 fatty acids have indicated that dietary-induced changes in oxylipins may be beneficial in slowing the changes associated with normal aging. In summary, oxylipins are an important group of molecules amenable to dietary manipulation to target cardiovascular disease and age-related degeneration. NEW & NOTEWORTHY Oxylipins are an important group of fatty acid metabolites amenable to dietary manipulation. Because of the role they play in cardiovascular disease and in age-related degeneration, oxylipins are gaining recognition as viable targets for specific dietary interventions focused on manipulating oxylipin composition to control these biological processes., (Copyright © 2017 the American Physiological Society.)

Dietary flaxseed can retard the progression of atherosclerotic plaques. However, it remains unclear whether these antiatherogenic effects extend to plaque regression. In the present study, the therapeutic potential of dietary flaxseed on atherosclerotic plaque regression and vascular contractile function was evaluated using a novel rabbit model. Rabbits were randomly assigned to receive either a regular diet for 12 wk (group I) or a 1% cholesterol-supplemented diet for 4 wk followed by a regular diet for 8 wk (group II). The remaining experimental animals were treated as in group II but were fed for an additional 14 wk with either a regular diet (group III) or a 10% flaxseed-supplemented diet (group IV). Animals in group II showed clear evidence of plaque growth stabilization. Their vessels also exhibited significantly lower norepinephrine-induced contraction and an impaired relaxation response to acetylcholine compared with animals in group I. Dietary flaxseed supplementation resulted in a significant ≈40% reduction in plaque formation (P = 0.033). Animals in both groups II and III displayed improved contraction and endothelium-dependent vessel relaxation. Dietary flaxseed is a valuable strategy to accelerate the regression of atherosclerotic plaques; however, flaxseed intervention did not demonstrate a clear beneficial effect on the vessel contractile response and endothelium-dependent vasorelaxation.

An important part of training the next generation of physicians is ensuring that they are exposed to the integral role that research plays in improving medical treatment. However, medical students often do not have sufficient time to be trained to carry out any projects in biomedical and clinical research. Many medical students also fail to understand and grasp translational research as an important concept today. In addition, since medical training is often an international affair whereby a medical student/resident/fellow will likely train in many different countries during his/her early training years, it is important to provide a learning environment whereby a young medical student experiences the unique challenges and value of an international educational experience. This article describes a program that bridges the gap between the basic and clinical research concepts in a unique international educational experience. After completing two semester curricula at Alfaisal University in Riyadh, Kingdom of Saudi Arabia, six medical students undertook a summer program at St. Boniface Hospital Research Centre, in Winnipeg, MB, Canada. The program lasted for 2 mo and addressed advanced training in basic science research topics in medicine such as cell isolation, functional assessment, and molecular techniques of analysis and manipulation as well as sessions on the conduct of clinical research trials, ethics, and intellectual property management. Programs such as these are essential to provide a base from which medical students can decide if research is an attractive career choice for them during their clinical practice in subsequent years. An innovative international summer research course for medical students is necessary to cater to the needs of the medical students in the 21st century.

Dietary intake of industrially hydrogenated trans fatty acids (TFA) has been associated with coronary heart disease. Dietary flaxseed can inhibit atherosclerosis induced by dietary cholesterol. The aim of this study was to determine whether supplementing the diet with flaxseed could protect against atherosclerosis induced by a diet enriched in TFA. Low-density lipoprotein receptor-deficient (LDLr(-/-)) mice were fed 1 of 14 experimental diets for 14 wk containing one of two fat sources [regular (pork/soy) or trans fat] at two concentrations (4 or 8%) and supplemented with or without dietary cholesterol (2%), whole ground flaxseed, or one of the components of flaxseed [α-linolenic acid (ALA), defatted fiber, or lignan]. Adding flaxseed to the diet partially mitigated the rise in circulating cholesterol levels induced by the cholesterol-enriched diet. Atherosclerosis was stimulated by TFA and/or cholesterol. Including milled flaxseed to an atherogenic diet significantly reduced atherosclerosis compared with the groups that consumed cholesterol and/or TFA. ALA was the only component within flaxseed that could inhibit the atherogenic action of cholesterol and/or TFA on its own. Dietary flaxseed protects against atherosclerotic development induced by TFA and cholesterol feeding through its content of ALA.

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.

Dietary flaxseed has been shown to have potent antiatherogenic effects in rabbits. The purpose of the present study was to investigate the antiatherogenic capacity of flaxseed in an animal model that more closely represents the human atherosclerotic condition, the LDL receptor-deficient mouse (LDLrKO), and to identify the cellular mechanisms for these effects. LDLrKO mice were administered a regular diet (RG), a 10% flaxseed-supplemented diet (FX), or an atherogenic diet containing 2% cholesterol alone (CH) or supplemented with 10% flaxseed (CF), 5% flaxseed (CF5), 1% flaxseed (CF1), or 5% coconut oil (CS) for 24 wk. LDLrKO mice fed a cholesterol-supplemented diet exhibited a rise in plasma cholesterol without a change in triglycerides and an increase in atherosclerotic plaque formation. The CS mice exhibited elevated levels of plasma cholesterol, triglycerides, and saturated fatty acids and an increase in plaque development. Supplementation of the cholesterol-enriched diet with 10% (wt/wt) ground flaxseed lowered plasma cholesterol and saturated fatty acids, increased plasma ALA, and inhibited plaque formation in the aorta and aortic sinus compared with mice fed a diet supplemented with only dietary cholesterol. The expression of proliferating cell nuclear antigen (PCNA) and the inflammatory markers IL-6, mac-3, and VCAM-1 was increased in aortic tissue from CH and CS mice. This expression was significantly reduced or normalized when flaxseed was included in the diet. Our results demonstrate that dietary flaxseed can inhibit atherosclerosis in the LDLrKO mouse through a reduction of circulating cholesterol levels and, at a cellular level, via antiproliferative and anti-inflammatory actions.

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.

Dietary flaxseed has significant anti-atherogenic effects. However, the limits of this action and its effects on vascular contractile function are not known. We evaluated the effects of flaxseed supplementation on atherosclerosis and vascular function under prolonged hypercholesterolemic conditions in New Zealand White rabbits assigned to one of four groups for 6, 8, or 16 wk of feeding: regular diet (RG), 10% flaxseed-supplemented diet (FX), 0.5% cholesterol-supplemented diet (CH), and 0.5% cholesterol- and 10% flaxseed-supplemented diet (CF). Cholesterol feeding resulted in elevated plasma cholesterol levels and the development of atherosclerosis. The CF group had significantly less atherosclerotic lesions in the aorta and carotid arteries after 6 and 8 wk than the CH animals. However, the anti-atherogenic effect of flaxseed supplementation was completely attenuated by 16 wk. Maximal tension induced in aortic rings either by KCl or norepinephrine was not impaired by dietary cholesterol until 16 wk. This functional impairment was not prevented by including flaxseed in the high-cholesterol diet. Aortic rings from the cholesterol-fed rabbits exhibited an impaired relaxation response to acetylcholine at all time points examined. Including flaxseed in the high-cholesterol diet completely normalized the relaxation response at 6 and 8 wk and partially restored it at 16 wk. No significant changes in the relaxation response induced by sodium nitroprusside were observed in any of the groups. In summary, dietary flaxseed is a valuable strategy to limit cholesterol-induced atherogenesis as well as abnormalities in endothelial-dependent vasorelaxation. However, these beneficial effects were attenuated during prolonged hypercholesterolemic conditions.

The Na+/Ca2+ exchanger (NCX) NCX1 exhibits tissue-specific alternative splicing. Such NCX splice variants as NCX1.1 and NCX1.3 are also differentially regulated by Na+ and Ca2+, although the physiological implications of these regulatory characteristics are unclear. On the basis of their distinct regulatory profiles, we hypothesized that cells expressing these different splice variants might exhibit unique responses to conditions promoting Ca2+ overload, such as during exposure to cardiac glycosides or simulated ischemia. NCX1.1 or NCX1.3 was expressed in human embryonic kidney (HEK)-293 cells or rat neonatal ventricular cardiomyocytes (NVC), and expression was confirmed by Western blotting and immunocytochemical analyses. HEK-293 cells lacked NCX1 protein before transfection. With use of adenoviral vectors, neonatal cardiomyocytes were induced to overexpress the NCX1.1 splice variant by nearly twofold, whereas the NCX1.3 isoform was expressed on the endogenous NCX1.1 background. Total expression was comparable for NCX1.1 and NCX1.3. Exposure of NVC to ouabain induced a significant increase in cellular Ca2+, an effect that was exaggerated in cells overexpressing NCX1.1, but not NCX1.3. The increase in intracellular Ca2+ was inhibited by 5 microM KB-R7943. Cardiomyocytes overexpressing NCX1.1 also exhibited a greater accumulation of intracellular Ca2+ in response to simulated ischemia than did cells expressing NCX1.3. Similar responses were observed in HEK-293 cells where NCX1.1 was expressed. We conclude that expression of the NCX1.3 splice variant protects against severe Ca2+ overload, whereas NCX1.1 promotes Ca2+ overload in response to cardiac glycosides and ischemic challenges. These results highlight the importance of ionic regulation in controlling NCX1 activity under conditions that promote Ca2+ overload.

Hyperhomocysteinemia is an independent risk factor for cardiovascular disorders. Elevated plasma homocysteine (Hcy) concentration is associated with other cardiovascular risk factors. We previously reported that Hcy stimulated cholesterol biosynthesis in HepG2 cells. In the present study, we investigated the underlying mechanisms of Hcy-induced hepatic cholesterol biosynthesis in an animal model. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 wk. The mRNA expression and the enzyme activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase were significantly increased in livers of hyperhomocysteinemic rats. There were marked hepatic lipid accumulation and an elevation of plasma cholesterol concentration in hyperhomocysteinemic rats. Three transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element-binding protein (CREB), and nuclear factor Y (NF-Y) were activated in livers of hyperhomocysteinemic rats. Upon Hcy treatment of hepatocytes, there was a significant increase in HMG-CoA reductase mRNA expression in these cells. The activation of SREBP-2, CREB, and NF-Y preceded the increase in HMG-CoA reductase expression in Hcy-treated cells. Pretreatment of hepatocytes with inhibitors for transcription factors not only blocked the activation of SREBP-2, CREB, and NF-Y but also attenuated Hcy-induced HMG-CoA reductase mRNA expression. These results suggested that hyperhomocysteinemia-induced activation of SREBP-2, CREB, and NF-Y was responsible for increased cholesterol biosynthesis by transcriptionally regulating HMG-CoA reductase expression in the liver leading to hepatic lipid accumulation and subsequently hypercholesterolemia. In conclusion, the stimulatory effect of Hcy on hepatic cholesterol biosynthesis may represent an important mechanism for hepatic lipid accumulation and cardiovascular disorder associated with hyperhomocysteinemia.

Oxidized low-density lipoprotein (oxLDL) may be involved in atherosclerosis by stimulating proliferation of cells in the vessel wall. The purpose of this study was to identify the mechanism by which oxLDL induces proliferation. Quiescent human fibroblasts and rabbit smooth muscle cells were treated with 0, 10, or 50 microg/ml oxLDL for 24-48 h. This resulted in significant increases in total cell counts at both concentrations of oxLDL, at both time points, for both types of cells. Western blot analysis revealed that oxLDL-stimulated cell proliferation was associated with significant increases in the expression of proteins that regulate entry into and progression through the cell cycle [cell division cycle 2, cyclin-dependent kinase (cdk) 2, cdk 4, cyclin B1, cyclin D1, and PCNA]. Surprisingly, the expression of cell cycle inhibitors (p21 and p27) was stimulated by oxLDL as well, but this was to a lesser extent than the effects on cell cycle-activating proteins. OxLDL also induced nuclear localization of all cell cycle proteins examined. The similar effects of oxLDL on the translocation and expression of both cell cycle-activating and -inhibiting proteins may explain the controlled proliferative phenomenon observed in atherosclerosis as opposed to the more rapid proliferative event characteristic of cancer.

Myocardial ischemia-reperfusion activates the Na(+)/H(+) exchanger, which induces arrhythmias, cell damage, and eventually cell death. Inhibition of the exchanger reduces cell damage and lowers the incidence of arrhythmias after ischemia-reperfusion. The omega-3 polyunsaturated fatty acids (PUFAs) are also known to be cardioprotective and antiarrhythmic during ischemia-reperfusion challenge. Some of the action of PUFAs may occur via inhibition of the Na(+)/H(+) exchanger. The purpose of our study was to determine the capacity for selected PUFAs to alter cardiac sarcolemmal (SL) Na(+)/H(+) exchange. Cardiac membranes highly enriched in SL vesicles were exposed to 10-100 microM eicosapentanoic acid (EPA) or docosahexanoic acid (DHA). H(+)-dependent (22)Na(+) uptake was inhibited by 30-50% after treatment with > or =50 microM EPA or > or =25 microM DHA. This was a specific effect of these PUFAs, because 50 microM linoleic acid or linolenic acid had no significant effect on Na(+)/H(+) exchange. The SL vesicles did not exhibit an increase in passive Na(+) efflux after PUFA treatment. In conclusion, EPA and DHA can potently inhibit cardiac SL Na(+)/H(+) exchange at physiologically relevant concentrations. This may explain, in part, their known cardioprotective effects and antiarrhythmic actions during ischemia-reperfusion.

Cardiac sarcolemmal Na(+)/H(+) exchange is critical for the regulation of intracellular pH, and its activity contributes to ischemia-reperfusion injury. It has been suggested that the membrane phospholipid environment does not modulate Na(+)/H(+) exchange. The present study was carried out to determine the effects on Na(+)/H(+) exchange of modifying the endogenous membrane phospholipids through the addition of exogenous phospholipase D. Incubation of 0.825 U of phospholipase D with 1 mg of porcine cardiac sarcolemmal vesicles hydrolyzed 34 +/- 2% of the sarcolemmal phosphatidylcholine and increased phosphatidic acid 10.2 +/- 0.5-fold. Treatment of vesicles with phospholipase D resulted in a 46 +/- 2% inhibition of Na(+)/H(+) exchange. Na(+)/H(+) exchange was measured as a function of reaction time, extravesicular pH, and extravesicular Na(+). All of these parameters of Na(+)/H(+) exchange were inhibited following phospholipase D treatment compared with untreated controls. Passive efflux of Na(+) was unaffected. Treatment of sarcolemmal vesicles with phospholipase C had no effect on Na(+)/H(+) exchange. We conclude that phospholipase D-induced changes in the cardiac sarcolemmal membrane phospholipid environment alter Na(+)/H(+) exchange.

Vascular smooth muscle cells in atherosclerotic vessels proliferate and change from a contractile to a synthetic phenotype. To determine whether oxidized low-density lipoprotein (oxLDL) is involved in this transformation, we chronically incubated cultured smooth muscle cells with native and oxidized LDL. Western blot analysis detected a decrease in actin and myosin content in treated cells. This was dependent on the time and concentration of oxLDL employed. Confocal microscopic images of cells immunostained for smooth muscle-specific alpha-actin and myosin showed a normal, elongated alignment of myofilaments in cells after incubation with native LDL. Surprisingly, when the cells were treated with oxLDL, actin and myosin filaments underwent a striking process of disorganization and accumulation into ball-shaped aggregates. These changes were dependent on the duration and concentration of oxLDL employed. Our results demonstrate that oxLDL has the capacity to decrease the content of myofilaments in smooth muscle cells. The loss in myosin and actin protein may be associated with an unusual formation of large cellular aggregates that appear to be in the process of being expelled from the cell.

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.

Depressed myofibrillar Ca2+-ATPase activity and sarcoplasmic reticulum (SR) Ca2+ uptake are important mechanisms that are responsible for the cardiac dysfunction exhibited by insulin-deficient (type I) diabetic animals. The JCR:LA-cp rat is a model for type II non-insulin-dependent diabetes mellitus (NIDDM). This rat is insulin resistant, obese, and has high levels of circulating glucose, cholesterol, insulin, and triglycerides. The purpose of this study was to determine whether changes in cardiac myofibrillar, SR, and cardiomyocyte function exist in this model of type II diabetes. Myofibrils and SR were isolated from hearts by differential centrifugation. Surprisingly, we found that myofibrillar Ca2+-ATPase activities were unaltered in these animals. Ca2+ uptake in isolated SR fractions was increased in diabetic cp/cp rats, whereas Ca2+-ATPase activity and ryanodine binding were unchanged. Cardiomyocytes isolated from hearts of control and experimental animals had similar active cell shortening and intracellular Ca2+ concentration under basal conditions and in response to caffeine. Our data argue against the presence of a cardiomyopathy in this diabetic model and suggest that insulin may be an important factor in the cardiomyopathy observed in type I diabetic models.

Amiloride analogs block Na+/H+ exchange and thereby protect the heart from myocardial ischemia-reperfusion injury. It is unclear whether drugs must be present before ischemia to be cardioprotective. After 60 min of global ischemia in the coronary-perfused right ventricular wall (RVW), as little as 1 min of exposure to dimethyl amiloride (DMA) immediately at the time of reperfusion protected the RVW. Delaying the drug attenuated the cardioprotection. If DMA was introduced in an ischemic solution near the end of ischemia, the cardioprotective effects were augmented. If the drug was washed out of the RVW vascular space before ischemia, cardioprotection was not observed. In contrast, in whole hearts, preischemic perfusion of the drug was necessary for cardioprotection and the cardioprotection remained even if the drug was washed out before ischemia. We conclude that Na+/H+ exchange is active and contributes to contractile dysfunction during the first seconds of reperfusion. This is difficult to detect in the perfused whole heart, and the washout data suggest that this may be due to a limitation in drug delivery across the vascular wall. The data also suggest that the exchanger is not as active during ischemia itself as it is during reperfusion.

Hearts from hyperinsulinemic, insulin-resistant JCR:LA-cp rats do not properly regulate intracellular Ca2+ concentration. We hypothesized, therefore, that these hearts may be unusually sensitive to ischemic insults in which Ca2+ overload would be expected. We investigated the response to global ischemia of hearts from JCR: LA-cp animals at three different ages. At 3 mo of age, isolated hearts from insulin-resistant cp rats were mildly resistant to both mild and severe ischemic insults in comparison to the lean control rat hearts. However, at 6 and 9 mo of age, the cp rats demonstrated a poorer recovery of developed tension after ischemia and/or a higher level of resting tension during reperfusion than the lean controls. Postischemic glycogen and ATP contents were significantly lower and lactate content was higher in hearts from 6-mo-old cp rats compared with controls. The results demonstrate that an insulin-resistant animal model exhibits an increased sensitivity to ischemic myocardial injury that develops with advancing age. The mechanism responsible for the enhanced sensitivity may involve augmented glycolytic metabolism. The data also emphasize the importance of the type of diabetes when cardiac dysfunction is examined.

As a first step in addressing the question of function for basic fibroblast growth factor (bFGF) in the adult myocardium, expression of bFGF receptors by adult rat myocytes was investigated. Cross-linking of 125I-labeled bFGF to purified sarcolemmal vesicles from adult hearts indicated specific binding to 90- to 104-kDa proteins, whereas equilibrium binding studies revealed the presence of "low"-affinity (1 nM) and "high"-affinity (115 pM) sites. Adult myocytes were found to express short and long variants of bFGF receptor 1 (FGFR-1, tyrosine kinase) mRNA. Adult heart overall levels of FGFR-1 mRNA were decreased by about one-third of corresponding fetal values. Several lines of evidence indicated that bFGF receptors in adult cardiomyocytes in situ and/or in isolation are functional. Isolated adult myocytes were found to be capable of heparin-resistant internalization of 125I-labeled bFGF, to lose their viability after interaction with bFGF-saporin (a mitotoxin known to kill cells after entry via the bFGF receptor), and to respond to bFGF by activation of mitogen-activated protein kinase. In addition, introduction of exogenous bFGF into the myocardium by Langendorff perfusion resulted in stimulation of tyrosine phosphorylation in association with cardiomyocyte intercalated disks, as assessed by immunofluorescence. It is concluded that adult cardiomyocytes express functionally coupled high-affinity bFGF receptors and that they are capable of a biologic response to bFGF in vivo.

We previously demonstrated that ATP-sensitive K+ channels (KATP) protect the guinea pig myocardium against ischemia-reperfusion injury (Cole et al., Circ. Res. 69: 571-581, 1991), but the cellular alterations leading to ischemic injury affected by KATP remain to be defined. This study investigates the relationship between activation of KATP and preservation of high-energy phosphates during global no-flow ischemia in arterially perfused guinea pig right ventricular walls. Electrical and mechanical activity were recorded via intracellular microelectrodes and a force transducer. Glibenclamide (10 and 50 microM) and pinacidil (10 microM) were used to modulate KATP. ATP and creatine phosphate (CP) levels were determined at the end of no-flow ischemia by enzymatic analysis. Preparations were subjected to 1) 20 min no-flow +/- glibenclamide (10 or 50 microM), 2) 30 min no-flow +/- pinacidil (10 microM) or pinacidil (10 microM) and glibenclamide (50 microM), or 3) 40 or 50 min of control perfusion before rapid freezing in liquid nitrogen. Pinacidil (10 microM) enhanced ischemic shortening of action potential duration (APD) and early contractile failure, prevented ischemic contracture, and inhibited high-energy phosphate depletion during ischemia. Glibenclamide (50 microM) inhibited the effects of pinacidil (10 microM) on electromechanical function and preservation of ATP and CP. Glibenclamide (10 microM) alone inhibited the early decline in APD and produced earlier ischemic contracture but did not enhance ATP or CP depletion compared with untreated tissues during 20 min of no-flow. Glibenclamide (50 microM) produced a greater inhibition of APD shortening in early ischemia, further decreased the latency to ischemic contracture, and caused enhanced ischemic depletion of ATP. The data indicate the changes in electrical activity induced by KATP indirectly preserve high-energy phosphates and reduce injury associated with ischemia. However, the data also suggest the possible presence of additional mechanisms for cardioprotection by KATP.

The purpose of the present study was to compare the protective effects of amiloride and three of its derivatives (which are selective inhibitors of Na(+)-H+ exchange) during postischemic reperfusion. Previously, amiloride has been shown to have a protective effect on ischemia-reperfusion injury. However, because of its nonselective actions, the mechanism of its effect is unclear. 5-(N,N-dimethyl)-amiloride (DMA) is also protective and appears to act via inhibition of the Na(+)-H+ exchanger. However, corroborative effects using other selective Na(+)-H+ exchange blockers are needed. Amiloride, DMA, ethylisopropyl amiloride (EIPA), and 5-(N,N-hexamethylene)-amiloride (HMA) were included for 10 min in the reperfusion period after 60 min of global ischemia in the rat right ventricular wall. Peak developed tension and the rates of tension generation and relaxation were significantly improved during reperfusion in the presence of 100 microM amiloride, 10 microM DMA, 2.5 microM HMA, or 1 microM EIPA compared with those of drug-untreated muscles. Contracture formation was significantly depressed in the presence of these drug concentrations as was release of creatine kinase from the ventricular wall into the coronary effluent. The efficacy of these drugs for protecting the right ventricular wall from postischemic contractile dysfunction correlates well with their potency as blockers of Na(+)-H+ exchange. The results provide further evidence in support of a role for Na(+)-H+ exchange. The results provide further evidence in support of a role for Na(+)-H+ exchange in determining ischemia-reperfusion injury in the heart.

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.

Perfusion of the rat right ventricular wall muscle for 4 min with a Ca2(+)-free medium followed by perfusion with a Ca2(+)-containing solution resulted in a 42% recovery of developed tension, contracture, and a massive release of creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) from the muscle. High concentrations (1-5 mM) of amiloride partially protected the ventricular wall from Ca2+ paradox-induced dysfunction. The inclusion of benzamil, an amiloride analogue, 2 min before and during the Ca2(+)-free perfusion period prevented contracture development, restored force development, and almost totally eliminated the release of CPK and LDH from the muscle. Contractile function was best protected by 10-50 microM benzamil. The results demonstrate the efficacy of benzamil as a protective agent against Ca2+ paradox-induced myocardial dysfunction and damage. In view of the known capacity of benzamil to block transsarcolemmal Na(+)-Ca2+ exchange, this study supports the involvement of elevated intracellular Na+ and a stimulation of Na(+)-Ca2+ exchange in this model of cardiac pathology.

Intracellular pH and [Na+] in the heart are regulated by the sarcolemmal membrane Na(+)-H+ exchange pathway. No data are currently available regarding the adaptation of this system to pathological conditions in the heart. Because ionic interactions with the heart are altered in cardiomyopathy during chronic experimental diabetes, it was hypothesized that Na(+)-H+ exchange may become abnormal. In addition, the effects of treating diabetic rats with daily injection of L-propionylcarnitine were investigated to determine whether alterations in lipid metabolism may be involved in any potential changes in ion transport. Rats were injected with streptozotocin (65 mg/kg) and killed 8-10 wk later, and sarcolemmal membrane vesicles were isolated from pooled ventricles. Significant depressions in Na(+)-K(+)-adenosinetriphosphatase (ATPase) activity and Na(+)-Ca2+ exchange were observed in the diabetic preparations in comparison to control. L-Propionylcarnitine treatment of the diabetic rats partially normalized these activities. A striking depression in cardiac sarcolemmal Na(+)-H+ exchange was observed in the diabetic animals in comparison to control, and this was not a result of a nonspecific increase in membrane permeability. L-Propionylcarnitine treatment of the diabetic rats did not improve sarcolemmal Na(+)-H+ exchange.

Heart sarcolemmal membranes were isolated by the hypotonic shock-LiBr treatment from rats with chronic diabetes induced by a streptozotocin (65 mg/kg, iv) injection. Sarcolemmal Mg2+-dependent ATPase activity was elevated, whereas 5'-nucleotidase and K+-p-nitrophenylphosphatase activities in diabetic heart were depressed in comparison to control preparations. Although patent Na+-K+-ATPase and patent ouabain-sensitive Na+-K+-ATPase activities were unaltered, latent Na+-K+-ATPase activities, as determined in membranes after alamethicin or deoxycholate treatments, were found to be significantly depressed in diabetic animals. A depression in the latent Na+-K+-ATPase activity in diabetic preparations was also observed in membranes prepared by the sucrose density gradient method. Insulin-treated diabetic rats were observed to have normalized latent Na+-K+-ATPase activities. Total phospholipid content did not differ, but cholesterol content of the sarcolemmal membranes was significantly increased in diabetic heart preparations. Sarcolemmal Na+-K+-ATPase activity in diabetic heart was more resistant to treatments with filipin, an agent known to bind with cholesterol residues. These results suggest that chronic experimental diabetes is associated with some defects in sarcolemmal enzymatic activities and composition.

The effects of verapamil on cardiac myofibrillar adenosinetriphosphatase (ATPase) activity, myosin ATPase, and myosin isoenzyme profile as well as sarcoplasmic reticular Ca2+ uptake and ATPase activities were examined in Sprague-Dawley rats made diabetic with a single injection of streptozotocin (65 mg/kg). Myofibrillar ATPase activity and myosin Ca2+ ATPase activity as well as Ca2+ uptake and Ca2+-stimulated ATPase activities of the sarcoplasmic reticulum were significantly decreased in diabetic hearts in comparison to the control values. The myosin isoenzyme component V3 was prominent in diabetic hearts, whereas V1 isoenzyme was the major myosin component in control hearts. Chronic treatment of diabetic rats with verapamil (8 mg/kg daily for 4-8 wk) resulted in an improvement of the altered myofibrillar ATPase activity, myosin ATPase, myosin isoenzyme distribution, and sarcoplasmic reticular Ca2+-pump activities in ventricular tissue. The ability of verapamil to normalize the observed defects in the subcellular organelles in diabetic cardiomyopathy may be related to its effects in controlling the entry of Ca2+ into the cardiac cell.

The effects of insulin and thyroid hormone treatments on cardiac sarcoplasmic reticular function were investigated in chronic streptozotocin-induced diabetes in rats. ATP-dependent Ca2+ transport and Ca2+-stimulated ATPase activities were depressed significantly in microsomal samples from diabetic rats in comparison with control (P less than 0.05). This defect was seen at various times of incubation (1-20 min) and different concentrations of free Ca2+ (10(-7) to 10(-5) M Ca2+) and was accompanied by changes in the protein composition and phospholipid contents of the microsomal fraction. The defect in calcium transport in microsomal vesicles was not evident until 28 days after streptozotocin (65 mg/kg iv) injection, whereas increases in plasma glucose levels due to insulin-deficiency occurred within 3 days. All changes in function and composition of the sarcoplasmic reticulum were reversed by insulin administration to the diabetic rats. Although the plasma level of thyroid hormone was decreased in the diabetic rat, thyroid hormone treatment did not restore microsomal calcium transport in the diabetic animals. The results of this study provide some evidence that the depression in cardiac sarcoplasmic reticular calcium accumulation during diabetes is a consequence of insulin deficiency and associated chronic metabolic changes but the hypothyroid condition that accompanies experimental diabetes does not appear to play any role in this defect.

Cardiac contractile function is dependent on the integrity and function of the sarcolemmal membrane. Swimming exercise training is known to increase cardiac contractile performance. The purpose of the present study was to examine whether a swimming exercise program would alter sarcolemmal enzyme activity, ion flux, and composition in rat hearts. After approximately 11 wk of exercise training, cardiac myosin and actomyosin Ca2+-adenosinetriphosphatase (ATPase) activity was significantly higher in exercised rat hearts than in sedentary control rat hearts. Glycogen content was increased in plantaris and gastrocnemius muscles from exercised animals as was succinic dehydrogenase activity in gastrocnemius muscle of exercised rats in comparison to sedentary rat preparations. Sarcolemmal vesicles were isolated from hearts of exercise-trained and control rats. Sarcolemmal Na+-K+-ATPase and K+-p-nitrophenylphosphatase activities, Na+-Ca2+ exchange, and passive Ca2+ binding did not differ between the two groups. ATP-dependent Ca2+ uptake and 5'-nucleotidase activity were elevated in the cardiac sarcolemmal vesicles isolated from exercised animals compared with sedentary control rats. Sarcolemmal phospholipid composition was not altered by the exercise training. Our results demonstrate that swimming training in rats does not affect most parameters of cardiac sarcolemmal function or composition. However, the elevated sarcolemmal Ca2+ pump activity in exercised rats may help to reduce intracellular Ca2+ and augment cardiac relaxation rates. The enhanced 5'-nucleotidase activity may stimulate adenosine production, which could affect myocardial blood flow. The present results further our knowledge on the subcellular response of the heart to swimming training in the rat.

Selected substrate and hormonal responses to exercise were compared in two phases of the menstrual cycle. Exercise-induced changes in substrate [glucose, lactate, free fatty acids (FFA), glycerol] and hormonal patterns [luteinizing hormone (LH), follicle-stimulating hormone (FSH), insulin, progesterone (P), growth hormone (GH), cortisol] were compared in the follicular and luteal phases of the menstrual cycle in 24-h-fasted (n = 5), glucose-loaded (n = 6; 1.50 g/kg, 20% solution), and control subjects (n = 8). A treadmill walk was maintained for 60 min (30 min, 40% VO2 max; 30 min, 80% VO2 max). Blood samples were obtained 5 min before, 15, 30, 45, and 60 min during, and 30 min after exercise. In the glucose group a blood sample was also taken 20 min before exercise, and glucose was ingested 15 min before exercise. Within each nutritional group the metabolic and endocrine responses to exercise were similar in the two phases for glucose, lactate, glycerol, LH, FSH, and cortisol (P greater than 0.05). In the glucose group the FFA response was lower in the luteal phase (P less than 0.05). In the fasted subjects insulin and GH responses were elevated in the luteal phase (P less than 0.05). P responses in the control and glucose groups were markedly greater in the luteal phase (P less than 0.05). In the fasted subjects no alteration in P occurred in either phase (P less than 0.05), and the LH concentration was lower in these subjects relative to the control groups (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes was induced in rats by an intravenous injection of streptozotocin (65 mg/kg body wt), and animals were killed 8 wk later. Some animals were maintained in a diabetic state for 6 wk and then given 2 wk of insulin treatment in vivo. Myofibrils were isolated and ATPase activities measured. Mg2+-ATPase and Ca2+-stimulated ATPase activities were depressed in diabetic rat hearts in comparison to control; insulin treatment normalized these activities. The depression in myofibrillar ATPases was of gradual onset as no changes were detected 2 wk after inducing diabetes. Treatment of diabetic animals with thyroid hormone did not restore changes in myofibrillar ATPase activities. Marker enzyme activities did not reveal any detectable contamination by cardiac membranes. Mg2+-ATPase activity of myofibrillar preparations from control and diabetic hearts responded differently to N-ethylmaleimide modification. Furthermore, myofibrillar sulfhydryl reactivity to 5,5'-dithiobis(2-nitrobenzoic acid) was significantly depressed in diabetic preparations in comparison to control and insulin-treated diabetic animals. These results suggest that the defect in myofibrillar ATPase activities in chronic diabetes may be due to some modification of sulfhydryl groups.

This study characterizes the passive Ca2+-binding capacity of a crude homogenate fraction prepared from rabbit ventricular tissue. The soluble and particulate fractions of the homogenate were separated by centrifugation, and the Ca2+-binding capacity of each fraction was measured separately with Ca2+-selective electrodes and then summed to obtain the Ca2+-binding capacity of the total tissue homogenate. Qualitatively, the Ca2+ bound to the homogenate exhibited both relatively high- (Km less than 1 microM) and low-affinity (Km greater than 10 microM) sites over the physiological range of free Ca2+ concentrations. The total homogenate bound 72, 128, and 308 mumol Ca2+/kg wet wt tissue at 1, 10, and 50 microM free Ca2+ concentrations, respectively. The Ca2+ bound to the homogenate was mostly La3+ displaceable, which suggests that it represents a largely noncompartmentalized, rapidly exchangeable fraction of Ca2+. Myofibrillar ATPase activity was examined as a function of free Ca2+ concentration in an ionic medium similar to that used in the Ca2+-binding experiments. At free Ca2+ concentrations that stimulated myofibrillar ATPase activity half maximally, the measured rise in Ca2+ bound to the homogenate above resting values was greater than 100 mumol/kg wet wt. These data provide base-line values for the passive Ca2+-buffering capacity of the myocardium, although relevance to the physiological setting must be interpreted with caution.

Studies have been performed to examine the effects of different concentrations of adriamycin (10(-9)-10(-3) M) on sarcolemmal ATP-independent Ca2+ binding, contractility, ultrastructure, energy [ATP and creatine phosphate (CrP)] stores, and lipid peroxide activity in the rat myocardium. Low-affinity Ca2+ binding was stimulated, whereas high-affinity Ca2+ binding was inhibited by adriamycin in sarcolemmal vesicles isolated from ventricles. These alterations were accompanied by changes in the number of Ca2+ binding sites without affecting the membrane affinity for Ca2+. Peak developed force as well as positive and negative dF/dt in the isolated papillary muscle were depressed. The percent changes in contractility due to adriamycin were not affected by extracellular Ca2+ concentration. Ultrastructural changes in the papillary muscles due to adriamycin included mitochondrial swelling, presence of intramitochondrial granules, increased lysosomes, and disruption of myofibrils. ATP and CrP content of the papillary muscles was significantly reduced by the drug. Lipid peroxides, estimated by quantitating malondialdehyde content, were elevated by adriamycin. Most of these changes due to adriamycin became apparent at 10 microM or higher concentrations of the drug, and the maximum effect was seen at 1 mM of adriamycin. The in vitro depressant effect of adriamycin may not be related to changes in the low-affinity Ca2+ binding sites, but instead it appears to be associated with a lower energy state and myocardial cell damage.

This study investigated alterations in glycogen, catecholamines, and the function of various subcellular membranes of the heart after exhaustive swimming in rats. The rats were exhausted by daily exercise over 1, 3, or 7 consecutive days. Glycogen content of the heart and three selected skeletal muscles was depleted after a single bout of exhaustive exercise. Repeated bouts of exhaustive swimming elicited a depletion of glycogen in only the plantaris and gastrocnemius skeletal muscles. Plasma norepinephrine and epinephrine levels were highly elevated, and cardiac concentrations of these hormones were significantly depleted immediately after all exercise sessions. Cardiac sarcoplasmic reticulum (SR) Ca2+ transport was depressed after a single exhaustive exercise period. After three exercise bouts SR Ca2+ accumulation remained depressed; however, mitochondrial Ca2+ transport was found to be augmented. If the exhaustive exercise protocol was continued up to seven days, only mitochondrial Ca2+ accumulation was depressed. Various parameters of sarcolemmal membrane function were observed to be unaltered after exhaustive exercise. These findings demonstrate that exhaustive swimming exercise in rats is capable of producing significant alterations in the Ca2+ transport capacity of the SR and mitochondrial membrane systems of the heart but is without apparent effect on the sarcolemmal membrane.