Signaling and cellular mechanisms in cardiac protection by ischemic and pharmacological preconditioning

Ischemic preconditioning (IPC) is a defensive adaptive cellular phenomenon. Brief ischemic stimuli render the heart resistant to subsequent similar stress. Signaling for IPC and pharmacologically induced preconditioning involves several G-protein coupled cell surface receptors, second messengers, specific Ser-Thr-protein kinase-C isoforms, Tyr-kinases, and finally, results in activation of ATP-dependent potassium channels (inward rectifiers) at the sarcolemma and in the mitochondria. In cardiomyocytes these channels regulate cytosolic and mitochondrial Ca2+ levels. K+ influx into mitochondria proves to be a key factor for keeping the mitochondrial permeability transition pore closed. This ensures continuous energy production and prevents cell death by apoptosis or necrosis. Molecular structure, function, and pharmacological properties of the ATP-dependent potassium channels and of the mitochondrial permeability transition pore are discussed. Channel activating agents mimic IPC and also affect reactive oxygen species producing enzymes involved in mitochondrial respiration. Volatile anesthetics, among other drugs, mimic the cardioprotective effects of IPC. Their intracellular signaling and clinical application are briefly discusse

Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms

Cardiac hypertrophy is an adaptive response that normalizes wall stress and compensates for increased workload. It is accompanied by distinct qualitative and quantitative changes in the expression of protein isoforms concerning contractility, intracellular Ca2+-homeostasis and metabolism. Changes in the myosin subunit isoform expression improves contractility by an increase in force generation at a given Ca2+-concentration (increased Ca2+-sensitivity) and by improving the economy of the chemo-mechanical transduction process per amount of utilised ATP (increased duty ratio). In the human atrium this is achieved by partial replacement of the endogenous fast myosin by the ventricular slow-type heavy and light chains. In the hypertrophic human ventricle the slow-type β-myosin heavy chains remain unchanged, but the ectopic expression of the atrial myosin essential light chain (ALC1) partially replaces the endogenous ventricular isoform (VLC1). The ventricular contractile apparatus with myosin containing ALC1 is characterised by faster cross-bridge kinetics, a higher Ca2+-sensitivity of force generation and an increased duty ratio. The mechanism for cross-bridge modulation relies on the extended Ala-Pro-rich N-terminus of the essential light chains of which the first eleven residues interact with the C-terminus of actin. A change in charge in this region between ALC1 and VLC1 explains their functional difference. The intracellular Ca2+-handling may be impaired in heart failure, resulting in either higher or lower cytosolic Ca2+-levels. Thus the state of the cardiomyocyte determines whether this hypertrophic adaptation remains beneficial or becomes detrimental during failure. Also discussed are the effects on contractility of long-term changes in isoform expression of other sarcomeric proteins. Positive and negative modulation of contractility by short-term phosphorylation reactions at multiple sites in the myosin regulatory light chain, troponin-I, troponin-T, α-tropomyosin and myosin binding protein-C are considered in detai

Tissue Expression and Actin Binding of a Novel N-Terminal Utrophin Isoform

Utrophin and dystrophin present two large proteins that link the intracellular actin cytoskeleton to the extracellular matrix via the C-terminal-associated protein complex. Here we describe a novel short N-terminal isoform of utrophin and its protein product in various rat tissues (N-utro, 62 kDa, amino acids 1–539, comprising the actin-binding domain plus the first two spectrin repeats). Using different N-terminal recombinant utrophin fragments, we show that actin binding exhibits pronounced negative cooperativity (affinity constants K1 = ∼5 × 106 and K2 = ∼1 × 105 M−1) and is Ca2+-insensitive. Expression of the different fragments in COS7 cells and in myotubes indicates that the actin-binding domain alone binds exlusively to actin filaments. The recombinant N-utro analogue binds in vitro to actin and in the cells associates to the membranes. The results indicate that N-utro may be responsible for the anchoring of the cortical actin cytoskeleton to the membranes in muscle and other tissues

Phosphoproteome analysis of isoflurane-protected heart mitochondria: phosphorylation of adenine nucleotide translocator-1 on Tyr194 regulates mitochondrial function

Aims Reversible phosphorylation of mitochondrial proteins is essential in the regulation of respiratory function, energy metabolism, and mitochondrion-mediated cell death. We hypothesized that mitochondrial protein phosphorylation plays a critical role in cardioprotection during pre and postconditioning, two of the most efficient anti-ischaemic therapies. Methods and results Using phosphoproteomic approaches, we investigated the profiles of phosphorylated proteins in Wistar rat heart mitochondria protected by pharmacological pre and postconditioning elicited by isoflurane. Sixty-one spots were detected by two-dimensional blue-native gel electrophoresis-coupled Western blotting using a phospho-Ser/Thr/Tyr-specific antibody, and 45 of these spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Eleven protein spots related to oxidative phosphorylation, energy metabolism, chaperone, and carrier functions exhibited significant changes in their phosphorylation state when protected mitochondria were compared with unprotected. Using a phosphopeptide enrichment protocol followed by liquid chromatography-MS/MS, 26 potential phosphorylation sites were identified in 19 proteins. Among these, a novel phosphorylation site was detected in adenine nucleotide translocator-1 (ANT1) at residue Tyr194. Changes in ANT phosphorylation between protected and unprotected mitochondria were confirmed by immunoprecipitation. The biological significance of ANT phosphorylation at Tyr194 was further tested with site-directed mutagenesis in yeast. Substitution of Tyr194 with Phe, mimicking the non-phosphorylated state, resulted in the inhibition of yeast growth on non-fermentable carbon sources, implying a critical role of phosphorylation at this residue in regulating ANT function and cellular respiration. Conclusions Our analysis emphasizes the regulatory functions of the phosphoproteome in heart mitochondria and reveals a novel, potential link between bioenergetics and cardioprotectio

Genomics in cardiac metabolism

Cell biology is in transition from reductionism to a more integrated science. Large-scale analysis of genome structure, gene expression, and metabolites are new technologies available for studying cardiac metabolism in diseases known to modify cardiac function. These technologies have several limitations and this review aims both to assess and take a critical look at some important results obtained in genomics restricted to molecular genetics, transcriptomics and metabolomics of cardiac metabolism in pathophysiological processes known to alter myocardial function. Therefore, our goal was to delineate new signalling pathways and new areas of research from the vast amount of data already published on genomics as applied to cardiac metabolism in diseases such as coronary heart disease, heart failure, and ischaemic reperfusio

Cardiac remodelling hinders activation of cyclooxygenase-2, diminishing protection by delayed pharmacological preconditioning: role of HIF1α and CREB

Aims We tested whether delayed pharmacologic preconditioning elicited by isoflurane is protective in infarct-remodelled hearts. Methods and results Male Wistar rats were treated with the preconditioning drug isoflurane 6 weeks after permanent ligation of the left anterior descending coronary artery. Twenty-four and 48 h later, hearts were perfused on the Langendorff system and treated with cyclooxygenase-2 or 12-lipoxygenase inhibitors before exposure to 40 min of ischaemia followed by 90 min of reperfusion. Infarct size was determined by triphenyltetrazolium chloride staining and lactate dehydrogenase release. Cyclooxygenase-2 expression and activity were measured by Western blotting and colorimetric assay. Nuclear translocation of cyclooxygenase-2-inducing transcription factors HIF1α, CREB, STAT3, and NFκB was determined. Post-infarct, remodelled hearts exhibit alterations in cellular signalling, time course and extent of isoflurane-induced late protection. While remodelled, preconditioned hearts exhibited protection exclusively at 24 h, healthy hearts showed sustained protection for up to 48 h, which correlated with cyclooxygenase-2 protein expression and enzymatic activity. The cyclooxygenase-2 inhibitors celecoxib and NS-398, but not the 12-lipoxygenase inhibitor cinnamyl-3,4-dihydroxycyanocinnamate, abolished delayed protection in both healthy and remodelled hearts, identifying cyclooxygenase-2 as a key mediator of late protection in both models. Isoflurane induced nuclear translocation of HIF1α in all hearts, but CREB was exclusively activated in healthy but not remodelled myocardium, which expressed higher levels of the CREB antagonist ICER. Delayed protection by isoflurane in remodelled hearts was more vulnerable to inhibition by celecoxib. Conclusion Isoflurane failed to mobilize cyclooxygenase-2-inducing CREB in ICER-overexpressing, remodelled hearts, which was associated with a shortening of the second window of protectio

First evidence of nanoparticle uptake through leaves and roots in beech (Fagus sylvatica L.) and pine (Pinus sylvestris L.)

Trees have been used for phytoremediation and as biomonitors of air pollution. However, the mechanisms by which trees mitigate nanoparticle pollution in the environment are still unclear. We investigated whether two important tree species, European beech (Fagus sylvatica L.) and Scots pine (Pinus sylvestris L.), are able to take up and transport differently charged gold nanoparticles (Au-NPs) into their stem by comparing leaf-to-root and root-to-leaf pathways. Au-NPs were taken up by roots and leaves, and a small fraction was transported to the stem in both species. Au-NPs were transported from leaves to roots but not vice versa. Leaf Au uptake was higher in beech than in pine, probably because of the higher stomatal density and wood characteristics of beech. Confocal (3D) analysis confirmed the presence of Au-NPs in trichomes and leaf blade, about 20–30 μm below the leaf surface in beech. Most Au-NPs likely penetrated into the stomatal openings through diffusion of Au-NPs as suggested by the 3D XRF scanning analysis. However, trichomes were probably involved in the uptake and internal immobilization of NPs, besides their ability to retain them on the leaf surface. The surface charge of Au-NPs may have played a role in their adhesion and uptake, but not in their transport to different tree compartments. Stomatal conductance did not influence the uptake of Au-NPs. This is the first study that shows nanoparticle uptake and transport in beech and pine, contributing to a better understanding of the interactions of NPs with different tree species