Celia Fernandez-Sanz

The SR–mitochondria interaction: a new player in cardiac pathophysiology

Mitochondria are essential for energy supply and cell signalling and may be triggers and effectors of cell death. Mitochondrial respiration is tightly controlled by the matrix Ca(2+) concentration, which is beat-to-beat regulated by uptake and release mainly through the mitochondrial Ca(2+) uniporter and Na(+)/Ca(2+) exchanger, respectively. Recent studies demonstrate that mitochondrial Ca(2+) uptake is more dependent on anatomo-functional microdomains established with the sarcoplasmic reticulum (SR) than on cytosolic Ca(2+). This privileged communication between SR and mitochondria is not restricted to Ca(2+) but may involve ATP and reactive oxygen species, which has important implications in cardiac pathophysiology. The disruption of the SR-mitochondria interaction caused by cell remodelling has been implicated in the deterioration of excitation-contraction coupling of the failing heart. The SR-mitochondria interplay has been suggested to be involved in the depressed Ca(2+) transients and mitochondrial dysfunction observed in diabetic hearts as well as in the genesis of certain arrhythmias, and it may play an important role in myocardial reperfusion injury. During reperfusion, re-energization in the presence of cytosolic Ca(2+) overload results in SR-driven Ca(2+) oscillations that may promote mitochondrial permeability transition (MPT). The relationship between MPT and Ca(2+) oscillations is bidirectional, as recent data show that the induction of MPT in Ca(2+)-overloaded cardiomyocytes may result in mitochondrial Ca(2+) release that aggravates Ca(2+) handling and favours hypercontracture. A more complete characterization of the structural arrangements responsible for SR-mitochondria interplay will allow better understanding of cardiac (patho)physiology but also, and no less important, should serve as a basis for the development of new treatments for cardiac diseases.

Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption

Connexin 43 (Cx43) is present at the sarcolemma and the inner membrane of cardiomyocyte subsarcolemmal mitochondria (SSM). Lack or inhibition of mitochondrial Cx43 is associated with reduced mitochondrial potassium influx, which might affect mitochondrial respiration. Therefore, we analysed the importance of mitochondrial Cx43 for oxygen consumption. Acute inhibition of Cx43 in rat left ventricular (LV) SSM by 18α glycyrrhetinic acid (GA) or Cx43 mimetic peptides (Cx43‐MP) reduced ADP‐stimulated complex I respiration and ATP generation. Chronic reduction of Cx43 in conditional knockout mice (Cx43Cre‐ER(T)/fl + 4‐OHT, 5–10% of Cx43 protein compared with control Cx43fl/fl mitochondria) reduced ADP‐stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O2/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O2/min.*mg protein in Cx43fl/fl mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43Cre‐ER(T)/fl+4‐OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43fl/fl mice (19.8 ± 1.3%, n = 8, P < 0.05); complex II activities were similar between genotypes. Supporting the importance of Cx43 for respiration, in Cx43‐overexpressing HL‐1 cardiomyocytes complex I respiration was increased, whereas complex II respiration remained unaffected. Taken together, mitochondrial Cx43 is required for optimal complex I activity and respiration and thus mitochondrial ATP‐production.

Defective sarcoplasmic reticulum-mitochondria calcium exchange in aged mouse myocardium.

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria–sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5–6 months). Mitochondrial membrane potential and resting O2 consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca2+, we investigated the effect of age on mitochondrial Ca2+ uptake. Although no age-dependent differences were found in Ca2+ uptake kinetics in isolated mitochondria, mitochondrial Ca2+ uptake secondary to SR Ca2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca2+ handling. Age-dependent alterations in SR Ca2+ transfer to mitochondria and in Ca2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR–mitochondria communication underlies inefficient interorganelle Ca2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.

Altered FoF1 ATP synthase and susceptibility to mitochondrial permeability transition pore during ischaemia and reperfusion in aging cardiomyocytes

Aging is a major determinant of the incidence and severity of ischaemic heart disease. Preclinical information suggests the existence of intrinsic cellular alterations that contribute to ischaemic susceptibility in senescent myocardium, by mechanisms not well established. We investigated the role of altered mitochondrial function in the adverse effect of aging. Isolated perfused hearts from old mice (> 20 months) displayed increased ischaemia-reperfusion injury as compared to hearts from adult mice (6 months) despite delayed onset of ischaemic rigor contracture. In cardiomyocytes from aging hearts there was a more rapid decline of mitochondrial membrane potential (Δψm) as compared to young ones, but ischaemic rigor shortening was also delayed. Transient recovery of Δψm observed during ischaemia, secondary to the reversal of mitochondrial FoF1 ATP synthase to ATPase mode, was markedly reduced in aging cardiomyocytes. Proteomic analysis demonstrated increased oxidation of different subunits of ATP synthase. Altered bionergetics in aging cells was associated with reduced mitochondrial calcium uptake and more severe cytosolic calcium overload during ischaemia-reperfusion. Despite attenuated ROS burst and mitochondrial calcium overload, mitochondrial permeability transition pore (mPTP) opening and cell death was increased in reperfused aged cells. In vitro studies demonstrated a significantly reduced calcium retention capacity in interfibrillar mitochondria from aging hearts. Our results identify altered FoF1 ATP synthase and increased sensitivity of mitochondria to undergo mPTP opening as important determinants of the reduced tolerance to ischaemia-reperfusion in aging hearts. Because ATP synthase has been proposed to conform mPTP, it is tempting to hypothesise that oxidation of ATP synthase underlie both phenomena.

Effects of a reduction in the number of gap junction channels or in their conductance on ischemia-reperfusion arrhythmias in isolated mouse hearts

A transient reduction of cell coupling during reperfusion limits myocardial necrosis, but little is known about its arrhythmogenic effects during ischemia-reperfusion. Thus, we analyzed the effect of an extreme reduction in the number of gap junction channels or in their unitary conductance on ventricular arrhythmias during myocardial ischemia-reperfusion. Available gap junction uncouplers have electrophysiological effects independent from their uncoupling actions. Thus, isolated hearts from Cx43(Cre-ER(T)/fl) mice treated with 4-hydroxytamoxifen (4-OHT), from Cx43KI32 mice [in which connexin (Cx)43 was replaced with Cx32], and from control animals were submitted to regional ischemia and reperfusion, and spontaneous and induced ventricular arrhythmias were monitored. In additional hearts, changes in activation time and electrical impedance during global ischemia-reperfusion were assessed. In contrast to treatment with 4-OHT, replacement of Cx43 with Cx32 did not modify baseline activation time or electrical impedance. However, the number of extrasistole and ventricular tachyarrhythmias was higher in isolated hearts from Cx43KI32 and 4-OHT-treated Cx43(Cre-ER(T)/fl) animals versus wild-type animals during normoxia, ischemia (12.29 ± 3.26 and 52.17 ± 22.51 vs. 3.00 ± 1.46 spontaneous tachyarrhythmias, P < 0.05), and reperfusion. The impairment in conduction during ischemia was steeper in isolated hearts from Cx43KI32 animals, whereas changes in myocardial impedance were attenuated during ischemia in both transgenic models, suggesting altered cell-to-cell coupling at baseline. In conclusion, both reduction of Cx43 with 4-OHT and replacement of Cx43 by less-conductive Cx32 were arrhythmogenic under normoxia and ischemia-reperfusion, despite no major effects on baseline electrical properties. These results suggest that modifications in gap junction communication silent under normal conditions may be arrhythmogenic during ischemia-reperfusion.

Connexin and pannexin as modulators of myocardial injury

Multicellular organisms have developed a variety of mechanisms that allow communication between their cells. Whereas some of these systems, as neurotransmission or hormones, make possible communication between remote areas, direct cell-to-cell communication through specific membrane channels keep in contact neighboring cells. Direct communication between the cytoplasm of adjacent cells is achieved in vertebrates by membrane channels formed by connexins. However, in addition to allowing exchange of ions and small metabolites between the cytoplasms of adjacent cells, connexin channels also communicate the cytosol with the extracellular space, thus enabling a completely different communication system, involving activation of extracellular receptors. Recently, the demonstration of connexin at the inner mitochondrial membrane of cardiomyocytes, probably forming hemichannels, has enlarged the list of actions of connexins. Some of these mechanisms are also shared by a different family of proteins, termed pannexins. Importantly, these systems allow not only communication between healthy cells, but also play an important role during different types of injury. The aim of this review is to discuss the role played by both connexin hemichannels and pannexin channels in cell communication and injury. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells.

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Microtubule stabilization with paclitaxel does not protect against infarction in isolated rat hearts

What is the central question of this study? The microtubule network is disrupted during myocardial ischaemia–reperfusion injury. It was suggested that prevention of microtubule disruption with paclitaxel might reduce cardiac infarct size; however, the effects on infarction have not been studied. What is the main finding and its importance? Paclitaxel caused a reduction in microtubule disruption and cardiomyocyte hypercontracture during ischaemia–reperfusion. However, it induced a greater increase in cytosolic calcium, which may explain the lack of effect against infarction that we have seen in isolated rat hearts. The large increase in perfusion pressure induced by paclitaxel in this model may have clinical implications, because detrimental effects of the drug were reported after its clinical application.

P141Prevention of microtubule disruption with paclitaxel does not protect against infarction in isolated rat hearts

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.