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Inserte, J., G. Taimor, B. Hofstaetter, D. Garcia-Dorado, and H. M. Piper. Influence of simulated ischemia on apoptosis induction by oxidative stress in adult cardiomyocytes of rats. Am. J. Physiol. Heart Circ. Physiol. 278: H94-H99, 2000.--Oxidative stress may cause apoptosis of cardiomyocytes in ischemic-reperfused myocardium. We investigated whether ischemia-reperfusion modifies the susceptibility of cardiomyocyte induction of apoptosis by oxidative stress. Ischemia was simulated by incubating isolated cardiomyocytes from adult rats in an anoxic, glucose-free medium, pH 6.4, for 3 h. Annexin V-fluorescein isothiocyanate/ propidium iodide staining and the detection of DNA laddering were used as apoptotic markers. [H.sub.2][O.sub.2] (7.5 [micro]mol/l) induced apoptosis in 20.1 [+ or -] 1.8% of cells under normoxic conditions but only 14.4 [+ or -] 1.6% (n = 6, P [is less than] 0.05) after ischemia-reoxygenation. This partial protection of ischemic-reoxygenated cells was observed despite a reduction in their cellular glutathione content, from 11.4 [+ or -] 1.9 in normoxic controls to 2.9 [+ or -] 0.8 nmol/mg protein (n = 3, P [is less than] 0.05). Elevation of end-ischemic glutathione contents by pretreatment with 1 mmol/lN-acetylcysteine entirely protected ischemic-reoxygenated cells against induction of apoptosis by [H.sub.2][O.sub.2]. In conclusion, ischemia-reperfusion can protect cardiomyocytes against induction of apoptosis by exogenous oxidative stress. This endogenous protective effect is most clearly demonstrated when control and postischemic cardiomyocytes are compared at similar glutathione levels. simulated ischemia-reoxygenation; hydrogen peroxide; glutathione

Objective: We have recently shown that spontaneous Ca2+ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca2+ through the reverse mode of the Na+/Ca2+ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. Methods: Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pHo 6.4) and 10 min reoxygenation (pHo 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca2+ (control), with nominally Ca2+-free medium or with medium containing 10 μmol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. Results: In reoxygenated cardiomyocytes rapid Ca2+ oscillations occurred which were reduced under Ca2+-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca2+-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 μmol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1±9.9 (control) to 49.3±8.8 U/60 min/g dry weight. Conclusion: Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury. [ABSTRACT FROM PUBLISHER]

Background: Microtubules are highly dynamic polymers present in eukaryote cells that are essential components of cell cytoskeleton. They play an important role in intracellular transport, in maintaining organelle organization and function, and in transmitting mechanical forces within the myocardium. However, this intracellular network becomes disrupted during myocardial ischemia/reperfusion, and it has been proposed that microtubule disruption is an early sign of irreversible ischemic injury, and that prevention of microtubule disruption during ischemia-reperfusion could be beneficial. In this study we aimed to assess the effects of prevention of microtubule disruption with paclitaxel on ischemia/reperfusion injury in both isolated rat cardiomyocytes and isolated, Langendorff-perfused, rat hearts.Methods and results: Isolated rat cardiomyocytes were submitted to normoxia (1h), or to 50 min of anoxia (pH 6.4, 0% O2, 37° C) and 15 min reoxygenation, without or with treatment during anoxia with the microtubule stabilizer paclitaxel (10 μM) or the inhibitor of microtubule polymerization colchicine (5 μM). The condition of microtubule network was assessed by immunofluorescence detection of α-tubulin. Anoxia/reoxygenation leads to disruption of the microtubule network before the onset of ischemic contracture (650±21 vs. 328±11 arbitrary units of fluorescence (auf), p≤0.05). Paclitaxel attenuated both microtubule disruption (377±15 auf, p≤0.05 vs. anoxia/reoxygenation) and the incidence of hypercontracture (31±7 % vs. 56±8 % in anoxia/reoxygenation, p≤0.05) induced by anoxia/reoxygenation, whereas treatment with colchicine mimicked the effects of anoxia/reoxygenation (fluorescence: 188±16 auf, p≤0.01 vs. anoxia/reoxygenation; hypercontracture: 65±7 %). In isolated rat hearts under normoxic conditions, paclitaxel induced a concentration-dependent increase in perfusion pressure and a decrease in heart rate and left ventricular developed pressure. Despite protection against cell hypercontracture, paclitaxel pretreatment did not modify infarct size (60.37±2.27 % in control hearts vs. 58.75±10.25, 55.44±10.32 and 50.06±10.14 % in hearts pretreated with 10-6, 3 10-6 and 10-5 M, of paclitaxel respectively), LDH release or functional recovery after 60 min of global ischemia and reperfusion in isolated rat hearts.Conclusions: Microtubule stabilization with paclitaxel reduces hypercontracture in isolated rat cardiomyocytes but does not protect against infarction in isolated rat hearts. [ABSTRACT FROM AUTHOR]

Cysteine proteases calpains contribute to heart failure (HF), but it remains unknown whether their inhibition provides any benefit compared to standard pharmacological treatment for HF. Here, we characterize the pharmacological properties of NPO-2270 (NPO) as a potent inhibitor of cysteine proteases. Then, we describe that acute administration of NPO in rodent models of transient ischemia at the time of reperfusion reduces myocardial infarction, while its chronic oral administration attenuates adverse remodeling and cardiac dysfunction induced by ischemic and non-ischemic pathological stimuli more effectively than enalapril when given at the same dose. Finally, we provide evidence showing that the effects of NPO correlate with calpain inhibition and the preservation of the T-tubule morphology, due at least in part to reduced cleavage of the calpain substrate junctophilin-2. Together, our data highlight the potential of cysteine protease inhibition with NPO as a therapeutic strategy for the treatment of heart failure., Competing Interests: Dr. J. Inserte received a grant from Landsteiner Genmed SL., (© 2024 The Author(s).)

Succinate accumulates during myocardial ischemia and is rapidly oxidized during reperfusion, leading to reactive oxygen species (ROS) production through reverse electron transfer (RET) from mitochondrial complex II to complex I, and favoring cell death. Given that connexin 43 (Cx43) modulates mitochondrial ROS production, we investigated whether Cx43 influences RET using inducible knock-out Cx43 Cre-ER(T)/fl mice. Oxygen consumption, ROS production, membrane potential and coenzyme Q (CoQ) pool were analyzed in subsarcolemmal (SSM, expressing Cx43) and interfibrillar (IFM) cardiac mitochondria isolated from wild-type Cx43 fl/fl mice and Cx43 Cre-ER(T)/fl knock-out animals treated with 4-hydroxytamoxifen (4OHT). In addition, infarct size was assessed in isolated hearts from these animals submitted to ischemia-reperfusion (IR), and treated or not with malonate, a complex II inhibitor attenuating RET. Succinate-dependent ROS production and RET were significantly lower in SSM, but not IFM, from Cx43-deficient animals. Mitochondrial membrane potential, a RET driver, was similar between groups, whereas CoQ pool (2.165 ± 0.338 vs. 4.18 ± 0.55 nmol/mg protein, p < 0.05) and its reduction state were significantly lower in Cx43-deficient animals. Isolated hearts from Cx43 Cre-ER(T)/fl mice treated with 4OHT had a smaller infarct size after IR compared to Cx43 fl/fl , despite similar concentration of succinate at the end of ischemia, and no additional protection by malonate. Cx43 deficiency attenuates ROS production by RET in SSM, but not IFM, and was associated with a decrease in CoQ levels and a change in its redox state. These results may partially explain the reduced infarct size observed in these animals and their lack of protection by malonate., (© 2024. Springer-Verlag GmbH Germany, part of Springer Nature.)

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 μL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion., Competing Interests: The authors declare no conflicts of interest.

TRPV4 channels, which respond to mechanical activation by permeating Ca 2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca 2+ -dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca 2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca 2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca 2+ -dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

Cardiovascular diseases are the leading cause of death globally and more than four out of five cases are due to ischemic events. Cardiac fibroblasts (CF) contribute to normal heart development and function, and produce the post-ischemic scar. Here, we characterize the biochemical and functional aspects related to CF endurance to ischemia-like conditions. Expression data mining showed that cultured human CF (HCF) express more BCL2 than pulmonary and dermal fibroblasts. In addition, gene set enrichment analysis showed overrepresentation of genes involved in the response to hypoxia and oxidative stress, respiration and Janus kinase (JAK)/Signal transducer and Activator of Transcription (STAT) signaling pathways in HCF. BCL2 sustained survival and proliferation of cultured rat CF, which also had higher respiration capacity and reactive oxygen species (ROS) production than pulmonary and dermal fibroblasts. This was associated with higher expression of the electron transport chain (ETC) and antioxidant enzymes. CF had high phosphorylation of JAK2 and its effectors STAT3 and STAT5, and their inhibition reduced viability and respiration, impaired ROS control and reduced the expression of BCL2, ETC complexes and antioxidant enzymes. Together, our results identify molecular and biochemical mechanisms conferring survival advantage to experimental ischemia in CF and show their control by the JAK2/STAT signaling pathway. The presented data point to potential targets for the regulation of cardiac fibrosis and also open the possibility of a general mechanism by which somatic cells required to acutely respond to ischemia are constitutively adapted to survive it., (© 2021 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Despite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca 2+ -dependent cysteine proteases calpains in regulating the hypertrophic process and highlight the feasibility of their long-term inhibition as a pharmacological strategy. In this review, we discuss the existing evidence implicating calpains in the development of cardiac hypertrophy, as well as the latest advances in unraveling the underlying mechanisms. Finally, we provide an updated overview of calpain inhibitors that have been explored in preclinical models of cardiac hypertrophy and the progress made in developing new compounds that may serve for testing the efficacy of calpain inhibition in the treatment of pathological cardiac hypertrophy.

Information about heart failure with reduced ejection fraction (HFrEF) in women and the potential effects of aging in the female heart is scarce. We investigated the vulnerability to develop HFrEF in female elderly mice compared to young animals, as well as potential differences in reverse remodeling. First, HF was induced by isoproterenol infusion (30 mg/kg/day, 28 days) in young (10-week-old) and elderly (22-month-old) female mice. In a second set of animals, mice underwent isoproterenol infusion followed by no treatment during 28 additional days. Cardiac remodeling was assessed by echocardiography, histology and gene expression of collagen-I and collagen-III. Following isoproterenol infusion, elderly mice developed similar HFrEF features compared to young animals, except for greater cell hypertrophy and tissue fibrosis. After beta-adrenergic withdrawal, young female mice experienced complete reversal of the HFrEF phenotype. Conversely, reversed remodeling was impaired in elderly animals, with no significant recovery of LV ejection fraction, cardiomyocyte hypertrophy and collagen deposition. In conclusion, chronic isoproterenol infusion is a valid HF model for elderly and young female mice and induces a similar HF phenotype in both. Elderly animals, unlike young, show impaired reverse remodeling, with persistent tissue fibrosis and cardiac dysfunction even after beta-adrenergic withdrawal.