Remote ischemic preconditioning prevents sarcolemmal-associated proteolysis by MMP-2 inhibition.

The death of myocytes occurs through different pathways, but the rupture of the plasma membrane is the key point in the transition from reversible to irreversible injury. In the myocytes, three major groups of structural proteins that link the extracellular and intracellular milieus and confer structural stability to the cell membrane: the dystrophin-associated protein complex, the vinculin-integrin link, and the spectrin-based submembranous cytoskeleton. The objective was to determine if remote ischemic preconditioning (rIPC) preserves membrane-associated cytoskeletal proteins (dystrophin and ß-dystroglycan) through the inhibition of metalloproteinase type 2 (MMP-2) activity. A second objective was to describe some of the intracellular signals of the rIPC, that modify mitochondrial function at the early reperfusion. Isolated rat hearts were subjected to 30 min of global ischemia and 120 min of reperfusion (I/R). rIPC was performed by 3 cycles of ischemia/reperfusion in the lower limb (rIPC). rIPC significantly decreased the infarct size, induced Akt/GSK-3 ß phosphorylation and inhibition of the MPTP opening. rIPC improved mitochondrial function, increasing membrane potential, ATP production and respiratory control. I/R increased ONOO- production, which activates MMP-2. This enzyme degrades ß-dystroglycan and dystrophin and collaborates to sarcolemmal disruption. rIPC attenuates the breakdown of ß-dystroglycan and dystrophin through the inhibition of MMP-2 activity. Furthermore, we confirm that rIPC activates different intracellular pathway that involves the an Akt/Gsk3ß and MPTP pore with preservation of mitochondrial function.

Myocardial remote ischemic preconditioning: from cell biology to clinical application.

Remote ischemic preconditioning (rIPC) is a cardioprotective phenomenon where brief periods of ischemia followed by reperfusion of one organ/tissue can confer subsequent protection against ischemia/reperfusion injury in other organs, such as the heart. It involves activation of humoral, neural or systemic communication pathways inducing different intracellular signals in the heart. The main purpose of this review is to summarize the possible mechanisms involved in the rIPC cardioprotection, and to describe recent clinical trials to establish the efficacy of these strategies in cardioprotection from lethal ischemia/reperfusion injury. In this sense, certain factors weaken the subcellular mechanisms of rIPC in patients, such as age, comorbidities, medication, and anesthetic protocol, which could explain the heterogeneity of results in some clinical trials. For these reasons, further studies, carefully designed, are necessary to develop a clearer understanding of the pathways and mechanism of early and late rIPC. An understanding of the pathways is important for translation to patients.

Thioredoxin-1 is required for the cardioprotecive effect of sildenafil against ischaemia/reperfusion injury and mitochondrial dysfunction in mice.

Sildenafil is a phosphodiesterase type 5 inhibitor which confers cardioprotection against myocardial ischaemia/reperfusion (I/R) injury. The aim of this study was to determine if Trx1 participates in cardioprotection exerted by sildenafil in an acute model of I/R, and to evaluate mitochondrial bioenergetics and cellular redox status. Langendorff-perfused hearts from wild type (WT) mice and a dominant negative (DN-Trx1) mutant of Trx1 were assigned to placebo or sildenafil (0.7 mg/kg i.p.) and subjected to 30 min of ischaemia followed by 120 min of reperfusion. WT + S showed a significant reduction of infarct size (51.2 ± 3.0% vs. 30 ± 3.0%, p < .001), an effect not observed in DN-Trx. After I/R, sildenafil preserved state 3 oxygen consumption from WT, but had a milder effect in DN-Trx1 only partially protecting state 3 values. Treatment restored respiratory control (RC) after I/R, which resulted 8% (WT) and 24% (DN-Trx1) lower than in basal conditions. After I/R, a significant increase in H2O2 production was observed both for WT and DN-Trx (WT: 1.17 ± 0.13 nmol/mg protein and DN-Trx: 1.38 ± 0.12 nmol/min mg protein). With sildenafil, values were 21% lower only in WT I/R. Treatment decreased GSSG levels both in WT and DN-Trx1. In addition, GSSG/GSH2 ratio was partially restored by sildenafil. Also, an increase in p-eNOS/eNOS even before the myocardial ischaemia was observed with sildenafil, both in WT (14%, p > .05) and in DN-Trx (35%, p < .05). Active Trx1 is required for the onset of the cardioprotective effects of sildenafil on I/R injury, together with the preservation of cellular redox balance and mitochondrial function.

Mitochondrial bioenergetics links inflammation and cardiac contractility in endotoxemia.

There is current awareness about the central role of mitochondrial dysfunction in the development of cardiac dysfunction in systemic inflammatory syndromes, especially in sepsis and endotoxemia. The aim of this work was to elucidate the mechanism that governs the link between the severity of the systemic inflammatory insult and mitochondrial function, analysing the consequences on heart function, particularly in cardiac contractile state. Female Sprague-Dawley rats were subjected to low-grade endotoxemia (i.p. injection LPS 0.5 mg kg-1 body weight) and severe endotoxemia (i.p. injection LPS 8 mg kg-1 body weight) for 6 h. Blood NO, as well as cardiac TNF-α and IL-1ß mRNA, were found increased as the severity of the endotoxemia increases. Cardiac relaxation was altered only in severe endotoxemia, although contractile and lusitropic reserves were found impaired in both treatments in response to work-overload. Cardiac ultrastructure showed disorientation of myofibrillar structure in both endotoxemia degrees, but mitochondrial swelling and cristae disruption were only observed in severe endotoxemia. Mitochondrial ATP production, O2 consumption and mitochondrial inner membrane potential decreases were related to blood NO levels and mitochondrial protein nitration, leading to diminished ATP availability and impairment of contractile state. Co-treatment with the NOS inhibitor L-NAME or the administration of the NO scavenger c-PTIO leads to the observation that mitochondrial bioenergetics status depends on the degree of the inflammatory insult mainly determined by blood NO levels. Unravelling the mechanisms involved in the onset of sepsis and endotoxemia improves the interpretation of the pathology, and provides new horizons for novel therapeutic targets.

Dyslipidemia in Ischemia/Reperfusion Injury.

Ischemic heart disease is the main cause of morbidity and mortality in the developed world. Although reperfusion therapies are currently the best treatment for this entity, the restoration of blood flow leads, under certain circumstances, to a form of myocardial damage called reperfusion injury. Several studies have shown that age, sex, smoking, diabetes and dyslipidemia are risk factors for cardiovascular diseases. Among these risk factors, dyslipidemias are present in 40% of patients with ischemic heart disease and represent the clinical factor with the greatest impact on the prognosis of patients with cardiovascular diseases. It is known that during reperfusion the increase of the oxidative stress is perhaps one of the most important mechanisms implicated in cell damage. That is why several researchers have studied protective mechanisms against reperfusion injury, such as the ischemic pre- and post- conditioning, making emphasis mainly on the reduction of oxidative stress. However, few of these efforts have been successfully translated into the clinical setting. The controversial results in regards to the relation between cardioprotective mechanisms and dyslipidemia/hypercholesterolemia are mainly due to the difference among quality, composition and the time of administration of hypercholesterolemic diets, as well as the difference in the species used in each of the studies. Therefore, in order to compare results, it is crucial that all variables that could modify the obtained results are taken into consideration.

Adenosine A1 receptors and mitochondria: targets of remote ischemic preconditioning.

Adenosine is involved in classic preconditioning in most species and acts especially through adenosine A1 and A3 receptors. The aim of the present study was to evaluate whether remote ischemic preconditioning (rIPC) activates adenosine A1 receptors and improves mitochondrial function, thereby reducing myocardial infarct size. Isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion [ischemia-reperfusion (I/R)]. In a second group, before isolation of the heart, a rIPC protocol (3 cycles of hindlimb I/R) was performed. Infarct size was measured with tetrazolium staining, and Akt/endothelial nitric oxide (NO) synthase (eNOS) expression/phosphorylation and mitochondrial function were evaluated after ischemia at 10 and 60 min of reperfusion. As expected, rIPC significantly decreased infarct size. This beneficial effect was abolished only when 8-cyclopentyl-1,3-dipropylxanthine (adenosine A1 receptor blocker) and NG-nitro-l-arginine methyl ester (NO synthesis inhibitor) were administered during the reperfusion phase. At the early reperfusion phase, rIPC induced significant Akt and eNOS phosphorylation, which was abolished by the perfusion with an adenosine A1 receptor blocker. I/R led to impaired mitochondrial function, which was attenuated by rIPC and mediated by adenosine A1 receptors. In conclusion, we demonstrated that rIPC limits myocardial infarct by activation of adenosine A1 receptors at early reperfusion in the isolated rat heart. Interestingly, rIPC appears to reduce myocardial infarct size by the Akt/eNOS pathway and improves mitochondrial function during myocardial reperfusion. NEW & NOTEWORTHY Adenosine is involved in classic preconditioning and acts especially through adenosine A1 and A3 receptors. However, its role in the mechanism of remote ischemic preconditioning is controversial. In this study, we demonstrated that remote ischemic preconditioning activates adenosine A1 receptors during early reperfusion, inducing Akt/endothelial nitric oxide synthase phosphorylation and improving mitochondrial function, thereby reducing myocardial infarct size.

High-fat diet abolishes the cardioprotective effects of ischemic postconditioning in murine models despite increased thioredoxin-1 levels.

Ischemic postconditioning (PostC) reduces infarct size in healthy experimental models. However, if protective effects of PostC are abolished during early stages of atherosclerotic and if this is related with a disbalance in mitochondrial energetics and alterations in thioredoxin-1 (Trx1) is still unknown. The objectives were to generate a murine high-fat diet (HFD)-fed model that developed in a phenotype consistent with early stages of atherosclerosis to then evaluate whether HFD exposure increased oxidative stress and consequently abolished the cardioprotection conferred by PostC. We used C57/BL6 mice fed with control diet (CD) or HFD for 12 weeks. Isolated mice hearts were subjected to 30 min of ischemia and 120 min of reperfusion (I/R group). For PostC group, after ischemia, six cycles of reperfusion/ischemia were performed (10 s per cycle) at the onset of reperfusion. In CD group, the PostC reduced infarct size (CD-I/R: 52.14 ± 2.8 vs. CD-PostC: 36.58 ± 1.8, P < 0.05) and increased phosphorylation of GSK3ß (CD-PostC: 2.341 ± 1.03 vs. CD-Baseline: 0.923 ± 0.41 AUOD, P < 0.05), and this cardioprotection was abolished in HFD-exposed mice. HFD increased hydrogen peroxide levels, produced a shift towards an oxidized intracellular environment (GSSG/GSH2), and increased Trx1 expression with higher fractions of oxidized protein. State 3 mitochondrial oxygen consumption in basal conditions decreased 24% in HFD-exposed mice and PostC improved state 3 values only in CD mice. Cellular redox state and mitochondrial bioenergetics were altered in HFD-exposed mice. We demonstrated that alterations in redox state at early stages of atherosclerosis abolished cardioprotective mechanisms, such as those induced by PostC, even with increased Trx1 levels.

Diabetes impairs heart mitochondrial function without changes in resting cardiac performance.

Diabetes is a chronic disease associated to a cardiac contractile dysfunction that is not attributable to underlying coronary artery disease or hypertension, and could be consequence of a progressive deterioration of mitochondrial function. We hypothesized that impaired mitochondrial function precedes Diabetic Cardiomyopathy. Thus, the aim of this work was to study the cardiac performance and heart mitochondrial function of diabetic rats, using an experimental model of type I Diabetes. Rats were sacrificed after 28days of Streptozotocin injection (STZ, 60mgkg-1, ip.). Heart O2 consumption was declined, mainly due to the impairment of mitochondrial O2 uptake. The mitochondrial dysfunction observed in diabetic animals included the reduction of state 3 respiration (22%), the decline of ADP/O ratio (∼15%) and the decrease of the respiratory complexes activities (22-26%). An enhancement in mitochondrial H2O2 (127%) and NO (23%) production rates and in tyrosine nitration (58%) were observed in heart of diabetic rats, with a decrease in Mn-SOD activity (∼50%). Moreover, a decrease in contractile response (38%), inotropic (37%) and lusitropic (58%) reserves were observed in diabetic rats only after a ß-adrenergic stimulus. Therefore, in conditions of sustained hyperglycemia, heart mitochondrial O2 consumption and oxidative phosphorylation efficiency are decreased, and H2O2 and NO productions are increased, leading to a cardiac compromise against a work overload. This mitochondrial impairment was detected in the absence of heart hypertrophy and of resting cardiac performance changes, suggesting that mitochondrial dysfunction could precede the onset of diabetic cardiac failure, being H2O2, NO and ATP the molecules probably involved in mitochondrion-cytosol signalling.

Inhibition of endogenous thioredoxin-1 in the heart of transgenic mice does not confer cardioprotection in ischemic postconditioning.

Thioredoxin-1 maintains the cellular redox status and decreases the infarct size in ischemia/reperfusion injury. However, whether the increase of thioredoxin-1 expression or its lack of activity modifies the protection conferred by ischemic postconditioning has not been yet elucidated. The aim was to evaluate if the thioredoxin-1 overexpression enhances the posctconditioning protective effect, and whether the lack of the activity abolishes the reduction of the infarct size. Wild type mice hearts, transgenic mice hearts overexpressing thioredoxin-1, and a dominant negative mutant (C32S/C35S) of thioredoxin-1 were used. The hearts were subjected to 30min of ischemia and 120min of reperfusion (Langendorff) (I/R group) or to postconditioning protocol (PostC group). The infarct size in the Wt-PostC group decreased in comparison to the Wt-I/R group (54.6±2.4 vs. 39.2±2.1%, p<0.05), but this protection was abolished in DN-Trx1-PostC group (49.7±1.1%). The ischemia/reperfusion and postconditioning in mice overexpressing thioredoxin-1 reduced infarct size at the same magnitude (35.9±2.1 and 38.4±1.3%, p<0.05 vs. Wt-I/R). In Wt-PostC, Trx1-I/R and Trx1- PostC, Akt and GSK3ß phosphorylation increased compared to Wt-I/R, without changes in DN-Trx1 groups. In conclusion, given that the cardioprotection conferred by thioredoxin-1 overexpression and postconditioning, is accomplished through the activation of the Akt/GSK3ß survival pathway, no synergic effect was evidenced. Thioredoxin-1 plays a key role in the postconditioning, given that when this protein is inactive the cardioprotective mechanism was abolished. Thus, diverse comorbidities or situations modifying the thioredoxin activity, could explain the absence of this strong mechanism of protection in different clinical situations.

Cardiac-specific overexpression of thioredoxin 1 attenuates mitochondrial and myocardial dysfunction in septic mice.

Sepsis-induced myocardial dysfunction is associated with increased oxidative stress and mitochondrial dysfunction. Current evidence suggests a protective role of thioredoxin-1 (Trx1) in the pathogenesis of cardiovascular diseases. However, it is unknown yet a putative role of Trx1 in sepsis-induced myocardial dysfunction, in which oxidative stress is an underlying cause. Transgenic male mice with Trx1 cardiac-specific overexpression (Trx1-Tg) and its wild-type control (wt) were subjected to cecal ligation and puncture or sham surgery. After 6, 18, and 24h, cardiac contractility, antioxidant enzymes, protein oxidation, and mitochondrial function were evaluated. Trx1 overexpression improved the average life expectancy (Trx1-Tg: 36, wt: 28h; p=0.0204). Sepsis induced a decrease in left ventricular developed pressure in both groups, while the contractile reserve, estimated as the response to ß-adrenergic stimulus, was higher in Trx1-Tg in relation to wt, after 6h of the procedure. Trx1 overexpression attenuated complex I inhibition, protein carbonylation, and loss of membrane potential, and preserved Mn superoxide dismutase activity at 24h. Ultrastructural alterations in mitochondrial cristae were accompanied by reduced optic atrophy 1 (OPA1) fusion protein, and activation of dynamin-related protein 1 (Drp1) (fission protein) in wt mice at 24h, suggesting mitochondrial fusion/fission imbalance. PGC-1α gene expression showed a 2.5-fold increase in Trx1-Tg at 24h, suggesting mitochondrial biogenesis induction. Autophagy, demonstrated by electron microscopy and increased LC3-II/LC3-I ratio, was observed earlier in Trx1-Tg. In conclusion, Trx1 overexpression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover (mitophagy and biogenesis), preserves contractile reserve and prolongs survival during sepsis.

Thioredoxin-1 Attenuates Ventricular and Mitochondrial Postischemic Dysfunction in the Stunned Myocardium of Transgenic Mice.

AIM: We evaluated the effect of thioredoxin1 (Trx1) system on postischemic ventricular and mitochondrial dysfunction using transgenic mice overexpressing cardiac Trx1 and a dominant negative (DN-Trx1) mutant (C32S/C35S) of Trx1. Langendorff-perfused hearts were subjected to 15 min of ischemia followed by 30 min of reperfusion (R). We measured left ventricular developed pressure (LVDP, mmHg), left ventricular end diastolic pressure (LVEDP, mmHg), and t63 (relaxation index, msec). Mitochondrial respiration, SERCA2a, phospholamban (PLB), and phospholamban phosphorylation (p-PLB) Thr17 expression (Western blot) were also evaluated. RESULTS: At 30 min of reperfusion, Trx1 improved contractile state (LVDP: Trx1: 57.4 ± 4.9 vs. Wt: 27.1 ± 6.3 and DN-Trx1: 29.2 ± 7.1, p < 0.05); decreased myocardial stiffness (LVEDP: Wt: 24.5 ± 4.8 vs. Trx1: 11.8 ± 2.9, p < 0.05); and improved the isovolumic relaxation (t63: Wt: 63.3 ± 3.2 vs. Trx1: 51.4 ± 1.9, p < 0.05). DN-Trx1 mice aggravated the myocardial stiffness and isovolumic relaxation. Only the expression of p-PLB Thr17 increased at 1.5 min R in Wt and DN-Trx1 groups. At 30 min of reperfusion, state 3 mitochondrial O2 consumption was impaired by 13% in Wt and by 33% in DN-Trx1. ADP/O ratios for Wt and DN-Trx1 decrease by 25% and 28%, respectively; whereas the Trx1 does not change after ischemia and reperfusion (I/R). Interestingly, baseline values of complex I activity were increased in Trx1 mice; they were 24% and 47% higher than in Wt and DN-Trx1 mice, respectively (p < 0.01). INNOVATION AND CONCLUSION: These results strongly suggest that Trx1 ameliorates the myocardial effects of I/R by improving the free radical-mediated damage in cardiac and mitochondrial function, opening the possibility of new therapeutic strategies in coronary artery disease. Antioxid. Redox Signal. 25, 78-88.

Ischemic postconditioning confers cardioprotection and prevents reduction of Trx-1 in young mice, but not in middle-aged and old mice.

Thioredoxin-1 (Trx-1) is part of an antioxidant system that maintains the cell redox homeostasis but their role on ischemic postconditioning (PostC) is unknown. The aim of this work was to determine whether Trx-1 participates in the cardioprotective mechanism of PostC in young, middle-aged, and old mice. Male FVB young (Y: 3 month-old), middle-aged (MA: 12 month-old), and old (O: 20 month-old) mice were used. Langendorff-perfused hearts were subjected to 30 min of ischemia and 120 min of reperfusion (I/R group). After ischemia, we performed 6 cycles of R/I (10 s each) followed by 120 min of reperfusion (PostC group). We measured the infarct size (triphenyltetrazolium); Trx-1, total and phosphorylated Akt, and GSK3ß expression (Western blot); and the GSH/GSSG ratio (HPLC). PostC reduced the infarct size in young mice (I/R-Y: 52.3 ± 2.4 vs. PostC-Y: 40.0 ± 1.9, p < 0.05), but this protection was abolished in the middle-aged and old mice groups. Trx-1 expression decreased after I/R, and the PostC prevented the protein degradation in young animals (I/R-Y: 1.05 ± 0.1 vs. PostC-Y: 0.52 ± .0.07, p < 0.05). These changes were accompanied by an improvement in the GSH/GSSG ratio (I/R-Y: 1.25 ± 0.30 vs. PostC-Y: 7.10 ± 2.10, p < 0.05). However, no changes were observed in the middle-aged and old groups. Cytosolic Akt and GSK3ß phosphorylation increased in the PostC compared with the I/R group only in young animals. Our results suggest that PostC prevents Trx-1 degradation, decreasing oxidative stress and allowing the activation of Akt and GSK3ß to exert its cardioprotective effect. This protection mechanism is not activated in middle-aged and old animals.

Loss of myocardial protection against myocardial infarction in middle-aged transgenic mice overexpressing cardiac thioredoxin-1.

Thioredoxin-1 (Trx1) protects the heart from ischemia/reperfusion (I/R) injury. Given that the age at which the first episode of coronary disease takes place has considerably decreased, life at middle-aged (MA) emerges as a new field of study. The aim was determine whether infarct size, Trx1 expression and activity, Akt and GSK-3ß were altered in young (Y) and MA mice overexpressing cardiac Trx1, and in a dominant negative (DN-Trx1) mutant of Trx1. Langendorff-perfused hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (R). We used 3 and 12 month-old male of wild type (WT), Trx1, and DN-Trx1. Trx1 overexpression reduced infarct size in young mice (WT-Y: 46.8±4.1% vs. Trx1-Y: 27.6±3.5%, p < 0.05). Trx1 activity was reduced by 52.3±3.2% (p < 0.05) in Trx1-MA, accompanied by an increase in nitration by 17.5±0.9%, although Trx1 expression in transgenic mice was similar between young and middle-aged. The expression of p-Akt and p-GSK-3ß increased during reperfusion in Trx1-Y. DN-Trx1 mice showed neither reduction in infarct size nor Akt and GSK-3ß phosphorylation. Our data suggest that the lack of protection in Trx1 middle-aged mice even with normal Trx1 expression may be associated to decreased Trx1 activity, increased nitration and inhibition of p-Akt and p-GSK-3ß.

Role of thioredoxin-1 in ischemic preconditioning, postconditioning and aged ischemic hearts.

Thioredoxin is one of the most important cellular antioxidant systems known to date, and is responsible of maintaining the reduced state of the intracellular space. Trx-1 is a small cytosolic protein whose transcription is induced by stress. Therefore it is possible that this antioxidant plays a protective role against the oxidative stress caused by an increase of reactive oxygen species concentration, as occurs during the reperfusion after an ischemic episode. However, in addition to its antioxidant properties, it is able to activate other cytoplasmic and nuclear mediators that confer cardioprotection. It is remarkable that Trx-1 also participates in myocardial protection mechanisms such as ischemic preconditioning and postconditioning, activating proteins related to cellular survival. In this sense, it has been shown that Trx-1 inhibition abolished the preconditioning cardioprotective effect, evidenced through apoptosis and infarct size. Furthermore, ischemic postconditioning preserves Trx-1 content at reperfusion, after ischemia. However, comorbidities such as aging can modify this powerful cellular defense leading to decrease cardioprotection. Even ischemic preconditioning and postconditioning protocols performed in aged animal models failed to decrease infarct size. Therefore, the lack of success of antioxidants therapies to treat ischemic heart disease could be solved, at least in part, avoiding the damage of Trx system.

Papel de la tiorredoxina-1 en el atontamiento cardíaco en ratones transgénicos / Role of Thioredoxin-1 on Myocardial Stunning in Transgenic Mice

Introducción: La disfunción ventricular posisquémica (miocardio atontado) involucra un aumento del estrés oxidativo. En este sentido, la célula cuenta con mecanismos de defensa, como la tiorredoxina-1, un antioxidante que protege al miocardio de la lesión por isquemia/reperfusión, reduciendo el tamaño del infarto. Objetivo: Evaluar el comportamiento de la función ventricular sistólica y diastólica, particularmente estudiando la rigidez miocárdica y la relajación isovolúmica en el miocardio atontado en diferentes ratones transgénicos. Material y métodos: Se utilizaron corazones de ratones que sobreexpresan tiorredoxina-1 y de ratones transgénicos que sobreexpresan tiorredoxina-1 mutada en su sitio activo (dominante negativo), comparados con los de ratones no transgénicos, los cuales fueron sometidos a 15 minutos de isquemia global y 30 minutos de reperfusión utilizando la técnica de Langendorff. Se evaluó la función ventricular sistólica y diastólica y se calculó el t63 y el t93 como índice de relajación isovolúmica. Resultados: Las mediciones a los 30 minutos de reperfusión mostraron una mejoría significativa del estado contráctil en los ratones tiorredoxina-1 (57,4 ± 4,9 mm Hg; p ≤ 0,05 vs. no transgénicos) y también en la rigidez (11,8 ± 2,9 mm Hg; p ≤ 0,05 vs. no transgénicos). Por otra parte, en los ratones dominantes negativos se observó un aumento de la rigidez (37,7 ± 5,5 mm Hg; p ≤ 0,05 vs. no transgénicos) y un enlentecimiento de la relajación a los 30 minutos de la reperfusión (78,2 ± 9,8 mseg; p ≤ 0,05 vs. no transgénicos). Conclusión: Este trabajo evidencia el rol protector de la tiorredoxina-1 en el miocardio atontado y su importancia fisiopatológica en ratones que sobreexpresan este antioxidante.

Background: Postischemic ventricular dysfunction (myocardial stunning) involves increased oxidative stress. In this sense, the cell has defense mechanisms, as thioredoxin-1, an antioxidant that protects the myocardium from ischemia/reperfusion injury, reducing infarct size. Objective: The aim of this study was to evaluate systolic and diastolic ventricular function, specifically analyzing myocardial stiffness and isovolumic relaxation, during myocardial stunning in different transgenic mice. Methods: Hearts from mice overexpressing thioredoxin-1 and transgenic mice overexpressing thioredoxin-1 with gene mutation in its active site (dominant negative) were compared with hearts from non-transgenic mice after 15-minute global ischemia and 30-minute reperfusion using the Langendorff technique. Systolic and diastolic ventricular function was evaluated and t63 and t93 were calculated as ventricular relaxation index. Results: At 30-minute reperfusion, thioredoxin-1 mice showed a significantly improved contractile state (57.4±4.9 mmHg; p≤0.05 vs. non-transgenic mice) and stiffness (11.8±2.9 mmHg; p≤0.05 vs. non-transgenic mice). Conversely, at the same reperfusion time, dominant negative mice exhibited increased stiffness (37.7±5.5 mmHg; p≤0.05 vs. non-transgenic mice) and slower relaxation (78.2±9.8 ms; p≤0.05 vs. non-transgenic mice). Conclusion: This study reveals the protective role of thioredoxin-1 on myocardial stunning and its pathophysiological importance in mice overexpressing this antioxidant.

Selective TNF-α targeting with infliximab attenuates impaired oxygen metabolism and contractile function induced by an acute exposure to air particulate matter.

Inflammation plays a central role in the onset and progression of cardiovascular diseases associated with the exposure to air pollution particulate matter (PM). The aim of this work was to analyze the cardioprotective effect of selective TNF-α targeting with a blocking anti-TNF-α antibody (infliximab) in an in vivo mice model of acute exposure to residual oil fly ash (ROFA). Female Swiss mice received an intraperitoneal injection of infliximab (10 mg/kg body wt) or saline solution, and were intranasally instilled with a ROFA suspension (1 mg/kg body wt). Control animals were instilled with saline solution and handled in parallel. After 3 h, heart O2 consumption was assessed by high-resolution respirometry in left ventricle tissue cubes and isolated mitochondria, and ventricular contractile reserve and lusitropic reserve were evaluated according to the Langendorff technique. ROFA instillation induced a significant decrease in tissue O2 consumption and active mitochondrial respiration by 32 and 31%, respectively, compared with the control group. While ventricular contractile state and isovolumic relaxation were not altered in ROFA-exposed mice, impaired contractile reserve and lusitropic reserve were observed in this group. Infliximab pretreatment significantly attenuated the decrease in heart O2 consumption and prevented the decrease in ventricular contractile and lusitropic reserve in ROFA-exposed mice. Moreover, infliximab-pretreated ROFA-exposed mice showed conserved left ventricular developed pressure and cardiac O2 consumption in response to a ß-adrenergic stimulus with isoproterenol. These results provides direct evidence linking systemic inflammation and altered cardiac function following an acute exposure to PM and contribute to the understanding of PM-associated cardiovascular morbidity and mortality.

Ischemic postconditioning: mechanisms, comorbidities, and clinical application.

Since ischemic heart disease (IHD) is a major cause of mortality and heart failure, novel therapeutic strategies are expected to improve the clinical outcomes of patients with acute myocardial infarction. Brief episodes of ischemia/reperfusion performed at the onset of reperfusion can reduce infarct size; a phenomenon termed "ischemic postconditioning." Extensive research has determined that different autacoids (e.g., adenosine, bradykinin, opioid, etc.) and cytokines, their respective receptors, kinase signaling pathways, and mitochondrial modulation are involved in ischemic conditioning. Modification of these factors by pharmacological agents mimics the cardioprotection by ischemic postconditioning. Here, the potential mechanisms of ischemic postconditioning, the presence of comorbidities, and the possible extrapolation to the clinical setting are reviewed. In the near future, large, multicentered, randomized, placebo-controlled, clinical trials will be required to determine whether pharmacological and/or ischemic postconditioning can improve the clinical outcomes of patients with IHD.

Ischemic postconditioning reduces infarct size through the α1-adrenergic receptor pathway.

The α1-adrenergic receptors (α1-ARs) are involved in preconditioning. Given that certain intracellular pathways seem to be shared by preconditioning and postconditioning, it is possible that postconditioning could also be mediated by α1-ARs. The objective was to evaluate, by analyzing infarct size, if α1-ARs activation could trigger postconditioning and also determine Akt and glycogen synthase kinase 3ß (GSK-3ß) phosphorylation. Langendorff-perfused rat hearts were subjected to 30 minutes of ischemia and 120 minutes of reperfusion (I/R; n = 8). After 30 minutes of global ischemia, we performed 6 cycles of reperfusion/ischemia of 10 seconds each, followed by 120 minutes of reperfusion [ischemic postconditioning group (postcon); n = 9]. In another postcon group, we administered prazosin during postcon protocol (postcon + prazosin; n = 7). Finally, we repeated the I/R group, but prazosin (prazosin; n = 7), phenylephrine (PE; n = 5) and clonidine (CL; n = 6) were administered during the first 2 minutes of reperfusion. Infarct size was measured using the triphenyltetrazolium chloride technique. Total and phosphorylated Akt and mitochondrial GSK-3ß expression were measured by Western blot. Infarct size was 58.1 ± 5.1% in I/R. Postcon and PE reduced infarct size to 40.1 ± 2.9% and 35.3 ± 5.5%, respectively (P < 0.05 vs. I/R). Postcon + prazosin administration abolished the beneficial effect on infarct size (61.6 ± 4.5%; P < 0.05 vs. postcon). Cytosolic Akt phosphorylation and mitochondrial GSK-3ß phosphorylation were higher in the postcon and PE groups compared with the I/R and postcon + prazosin groups. Prazosin or clonidine administration did not modify neither protein expression nor infarct size. Our data demonstrate that postconditioning decrease infarct size by activation of the α1-AR pathway through Akt and GSK-3ß phosphorylation.

La tiorredoxina-1 no atenúa la disfunción ventricular postisquémica a pesar de disminuir el tamaño del infarto / Thioredoxin-1 Reduces Infarct Size But Does Not Improve Postischemic Ventricular Dysfunction

Introducción La tiorredoxina-1 es un potente antioxidante endógeno que participa en la protección miocárdica frente a la injuria por isquemia/reperfusión. Sin embargo, no se ha estudiado si esta protección se mantiene en la edad media de la vida y si se produce una disociación entre el efecto sobre la función ventricular y el infarto de miocardio. Objetivo Evaluar el tamaño del infarto y la función ventricular en ratones transgénicos jóvenes y de edad media que sobreexpresan tiorredoxina-1, comparándolos con sus respectivos controles no transgénicos. Material y métodos Se utilizaron corazones aislados de ratones FVB machos de 3 meses (jóvenes) y de 12 meses (edad media) que fueron sometidos a una isquemia global de 30 minutos seguida por 120 minutos de reperfusión según la técnica de Langendorff. Se conformaron cuatro grupos experimentales: no transgénico jóvenes, no transgénico edad media, tiorredoxina-1 jóvenes y tiorredoxina-1 edad media. Se evaluó la función del ventrículo izquierdo y también se midió el tamaño del infarto (trifenil tetrazolio). Resultados La función ventricular no mostró cambios significativos entre los grupos estudiados. Sin embargo, se observó una disminución del tamaño del infarto en ratones tiorredoxina-1 jóvenes (27,6% ± 3,5% vs. 42,9% ± 6,1% en no transgénico jóvenes); en cambio, el grupo tiorredoxina-1 de edad media no presentó cambios frente al control (49,1% ± 6,4% vs. 52,6% ± 5,2%). Conclusiones Los datos obtenidos sugieren que la tiorredoxina-1 es capaz de reducir el tamaño del infarto en los ratones jóvenes que la sobreexpresan; sin embargo, no se han evidenciado cambios en la función ventricular. Además, el efecto protector del antioxidante se abole en los ratones transgénicos en edad media de la vida.

Background Thioredoxin-1 is a potent endogenous antioxidant involved in myocardial protection from ischemic/reperfusion injury. However, it is unknown whether this protection is preserved in middle age or whether there is dissociation between the effect on ventricular function and infarct size. Objective The purpose of this study was to compare infarct size and ventricular function in young and middle age transgenic mice overexpressing thioredoxin-1 with their corresponding wild-type controls. Methods Isolated hearts of 3-month (young) and 12-month (middle-age) FVB male mice were submitted to 30-minute global ischemia and 120 minutes reperfusion using the Langendorff technique. Four experimental groups were considered: young wild-type, middle age wild type, young thioredoxin-1 and middle age thioredoxin-1. Left ventricular function was assessed and infarct size was measured with phenyl triphenyl tetrazolium. Results Ventricular function showed no significant differences between the studied groups. However, young thioredoxin-1 mice reduced infarct size (27.6%±3.5% vs. 42.9%±6.1% in young wild-type mice); conversely, the middle age thioredoxin-1 group was not significantly different from its wild-type control (49.1%±6.4% vs. 52.6%±5.2%). Conclusions Results suggest that young mice overexpressing thioredoxin-1 reduce infarct size, but without changes in ventricular function. Moreover, the protective antioxidant effect is abolished in middle age transgenic mice.

High cholesterol diet effects on ischemia-reperfusion injury of the heart.

Ischemic heart disease is the leading cause of morbi-mortality in developed countries. Both ischemia-reperfusion injury and mechanisms of cardioprotection have been studied for more than 50 years. It is known that the physiopathological mechanism of myocardial ischemia involves several factors that are closely related to its development, of which hypercholesterolemia is one of the main ones. Therefore, the objective of this review was to elucidate the effects of a high-cholesterol diet on normal ventricular function and ischemia-reperfusion injury associated phenomenon such as post-ischemic ventricular dysfunction (stunned myocardium). Although there exist many studies considering several aspects of this physiopathological entity, the majority were carried out on normal animals. Thus, experiments carried out on hypercholesterolemic models are controversial, in particular those evaluating different mechanisms of cardioprotection such as ischemic preconditioning and postconditioning, and cardioprotection granted by drugs such as statins, which apart from exerting a lipid-lowering effect, exert pleiotropic effects providing cardioprotection against ischemia-reperfusion injury. These controversial results concerning the mechanisms of cardioprotection vary according to quality, composition, and time of administration of the high-cholesterol diet, as well as the species used in each experiment. Thus, to compare the results it is necessary to take all of these variables into account, since they can change the obtained results.