A number of protocols and drugs have been shown to reduce cell death following ischemia-reperfusion. These agents initiate a signalling cascade that leads to alterations in mitochondrial proteins. Cardioprotection involves translocation of proteins such as Cx43, PKCε, and AKT to the mitochondria, which initiate a signalling cascade leading to post-translational modifications such as phosphorylation and S-nitrosylation. Unphosphorylated (active) GSK phosphorylates cyclophilin D promoting activation of the MPT. AKT phosphorylate and inhibit GSK, thus inhibiting MPT opening. PKCε phosphorylates ALDH2 and αKGDH resulting in less ROS, an activator of the MPT. Nitric oxide is reported to increase SNO of complex I, thereby reducing ROS generation. Thus, post-translational modifications of mitochondrial proteins alter their function and ultimately lead to inhibition of the MPT. The MPT is a large conductance channel which is opened in the mitochondria by death signals and its opening results in cell death.
Abbreviations: Cx43= connexin 43; PKC= protein kinase C; SNO=S-nitrosylation; CypD= cyclophilin D; GSK = glycogen synthase kinase; MPT= mitochondrial permeability pore transition; VDAC = voltage dependent anion channel; ROS= reactive oxygen species; ALDH2= aldehyde dehydrogenase 2; aKGDH= alpha keto-glutarate dehydrogenase; PRX=peroxiredoxin; AKT=a serine-threonine kinase.
Expression of skeletal muscle sodium channel (Nav1.4) or connexin32 prevents reperfusion arrhythmias in murine heart
Overexpressing the acidosis-resistant connexin32 (Cx32) maintains normal conduction in myocardium exposed to a low pH environment. Intracellular acidification induced by acute myocardial ischemia can close pH-sensitive Cx43 gap junctions and contribute to conduction slowing and reentrant arrhythmias. We studied right ventricular epicardium from mice whose hearts had been injected 4 days earlier with either adenoviral Cx32 or naked adenovirus. We simulated acidosis using a low pH solution containing Na-acetate. This decreased intracellular pH (measured with pH-sensitive microelectrodes) from 7.0 to 6.4. At pH 6.4, most Cx43 gap junctions are closed, while significant numbers of Cx32 junctions should remain open and support conduction.
In representative maps of conduction times (A – D) the pacing site is marked with a cross and isochrones are drawn at 3 ms intervals. Low pH had no effects on maximum diastolic potential (MDP) (E) and maximum upstroke velocity of the action potential (Vmax) (F) in both groups. Acidosis induced a significant decrease in conduction velocity (CV) in Shams and had no effect in Cx32 (G). As a result, at low pH, CV was higher in Cx32-expressing mice. *P<0.05 vs. pH 7.4 in the same group, +P<0.05 vs. Sham at the same pH (n=8 for both groups).
Insulin-initiated PI3K–Akt–eNOS–NO survival signalling and cardiovascular protection. Insulin binds to cell membrane insulin receptor, leading to the activation of mainly two signalling pathways: Ras–MAPK, which results in cell proliferation; and PI3K–Akt–eNOS, which results in metabolic modulation and cardiovascular protection. Among the insulin-activated signalling cascades, PI3K–Akt–eNOS–NO represents a special link between insulin and the cardiovascular system with regard to health and pathology. Activation of this signalling cascade, together with other Akt-activated molecules (such as GSK-3β, mTOR and p70S6 kinase), elicits pro-survival and cardiovascular protective effects, including vasodilatation, anti-apoptosis, anti-inflammation, and anti-oxidative/nitrative stress.
Abbreviations: Akt, protein kinase B; eNOS, endothelial nitric oxide synthase; ET-1, endothelin-1; GLUT4, glucose transporter 4; GSK, glycogen synthase kinase; IRS, insulin receptor substrate; MAPK, mitogen-activated protein kinase; mTOR, the mammalian target of rapamycin; PI3K, phosphatidylinositol 3′-kinase; PMN, polymorphonuclear neutrophil; and ROS, reactive oxygen species.
Metabolic reprogramming induced by HIF-1.
Under hypoxic conditions, the activation of HIF-1 induces the expression of a number of genes involved in the adaptation of tissues to hypoxia. Low oxygen concentrations reduce PHD activity, leading to HIF-α accumulation, its dimerization with HIF-β, and the subsequent induction of the transcription of specific genes. Increased HIF-1 activity leads to increased expression of glucose transporters and glycolytic enzymes. HIF-1 also induces PDK expression, which inhibits PDH, the enzyme that converts pyruvate into AcCoA, therefore inhibiting oxidative metabolism of AcCoA derived from glucose and reducing ATP synthesis. Moreover, HIF-1 increases LDH expression and consequently the conversion of pyruvate into lactate. Another adaptation to reduced oxygen levels is a subunit switch that occurs in complex IV whereby the COX4-1 regulatory subunit is replaced by the COX4-2 isoform. This switch maintains efficient electron transport and minimizes ROS production in hypoxia. In addition, HIF-1 induces the active destruction of mitochondria by mitochondrial autophagy.
Abbreviations: AcCoA, acetyl coenzyme A; COX, cytochrome c oxidase; ETC, electron transport chain; HIF-1, hypoxia-inducible factor- 1; LDH, lactate dehydrogenase; PDH, pyruvate dehydrogenase; PDK, pyruvate dehydrogenase kinase; PHD, prolyl hydroxylase; Pyr, pyruvate; ROS, reactive oxygen species; TCA, tricarboxylic acid cycle.
A scheme depicting aldehyde-induced mitochondrial damage and how ALDH2 reduces this aldehydic toxicity.
(Left) Ischaemia and reperfusion and other oxidative stress in the heart increase ROS production, which triggers lipid peroxidation and the accumulation of reactive aldehydes, such as 4-HNE. Other xenogenic aldehydes from the environment, from ethanol metabolism, and from food additives can also increase the cellular ‘aldehydic load’ (depicted as a grey cloud in the figure). Aldehydes induce inactivation of a number of macromolecules including the proteasome, the electron transport chain (ETC) in the mitochondria, as well as inactivation of ALDH2 itself. This aldehyde-induced macromolecule inactivation contributes to mitochondrial impairment and increases in oxidative stress, thus leading to cell damage. Particularly relevant to cardiac disease, the use of nitroglycerin (GTN) can further contribute to ALDH2 inactivation, thus decreasing the cell's natural ability to reduce ROS-induced aldehydic load and cytotoxicity. Finally, a common mutation in ALDH2 (ALDH2*2) in humans further impairs the ability to reduce the aldehydic load under oxidative stress conditions.
(Right) Agents that increase ALDH2 activity and protect ALDH2 from inactivation by aldehydes and by GTN will decrease the aldehydic load by enhancing the conversion of aldehydes to non-reactive acid (blue cloud in the scheme), thus leading to cytoprotection. Such one potential agent is Alda-1, an aldehyde dehydrogenase 2 activator. Alda-1 increases ALDH2 activity by about two-folds, blocks ALDH2 inactivation by both aldehydes and GTN, and thus increases the cell's natural ability to protect from oxidative stress, leading to 60% reduction from cardiac damage in an animal model of AMI. The ability of Alda 1 to increase the activity of the mutant ALDH2*2 may be of particular importance for over >0.5 billion humans who carry this mutation. (See text for details.) Reduction in aldehydic load decreases mitochondrial structural and functional damages and increases ATP generation, thus leading to cardiac protection from oxidative stress.
The process of mitochondrial fission is under the control of the mitochondrial fission proteins Drp1 and Fis1. Drp1 is located mainly in the cytosol and comprises a GTPase, a central region, and a GTPase effector domain (GED) or assembly domain. Fis1 is localized in the outer mitochondrial membrane with most of the protein facing into the cytosol, acting as a docking station for Drp1. On activation, Drp1 translocates to the mitochondria (a process which is regulated by phosphorylation and sumoylation), oligomerizes, and constricts the mitochondrial scission site, a process which requires GTPase, thereby resulting in mitochondrial fission.
The process of mitochondrial fusion is under the control of the mitochondrial fusion proteins Mfn1 and 2 and OPA-1. Mitochondrial membrane fusion has been shown to be a distinct two-step process which occurs separately for the inner and outer membrane, but in chronology. Both the outer and inner membranes of the mitochondria must fuse properly in order for the matrix contents to mix properly. (A) The mitochondrial fusion proteins Mfn1 and Mfn2 are located on the outer mitochondrial membrane with a cytosolic GTPase domain and two hydrophobic heptad repeat (HR) regions separated by a transmembrane repeat. The C-terminal HR region (HR2) mediates oligomerization between Mfn molecules on adjacent mitochondria, allowing the membranes to fuse. GTP hydrolysis facilitates the fusion process. (B) The mitochondrial fusion protein OPA1 comprises an N-terminal mitochondrial import sequence (MIS), hydrophobic heptad repeat (HR) segments, coiled-coil domain (C C), a GTPase domain, a central domain, and a GTPase effector domain (GED) at the C-terminus. OPA1 mediates the fusion of the inner mitochondrial membranes.
Mechanisms of endothelial barrier failure following ischaemia–reperfusion (I/R).
The endothelial barrier failure elicited by I/R is accompanied by oxidative stress and the recruitment of leucocytes and platelets. The blood cells that are recruited into the post-ischaemic microvasculature appear to contribute to both the endothelial barrier dysfunction and enhanced production of reactive oxygen species (ROS) via mechanisms that require adhesive interactions between blood cells and vascular endothelium. The available evidence suggests that products of blood cell activation, including ROS, cytokines, and chemokines, play a major role in mediating the adhesion-dependent increase in vascular permeability caused by I/R. Whether these agents act directly on endothelial cells or do so indirectly by activating perivascular cells such as mast cells and macrophages remain unclear. Despite these uncertainties about the nature of the involvement of different cell populations and molecular mediators in the I/R-induced endothelial barrier dysfunction, there is mounting evidence that the well-established risk factors for cardiovascular disease amplify the inflammatory and oxidative responses elicited by I/R, with a corresponding exacerbation of the barrier failure.
Abbreviations: O2-, superoxide; H2O2, hydrogen peroxide; IL-12, interleukin-12; IFN-γ, interferon-γ PSGL-1, P-selectin glycoprotein ligand-1; IL-1, interleukin-1; TNF-α, tumor necrosis factor-α ONOO-, peroxynitrite anion; MIP-1α, monocyte inhibitory peptide-1; IL-6, interleukin-6
MMP-2 is present in discrete intracellular compartments within the cardiac myocyte (sarcomere, nuclei, caveolae, and mitochondria) as a 72 kD zymogen. It can be activated in two ways that likely dictate its diverse biological roles. Its secretion and proteolytic removal of its autoinhibitory propeptide domain by MT1-MMP together with TIMP-2 results in a 64 kD form that targets extracellular matrix proteins. Oxidative stress, particularly as ONOO- in the presence of glutathione, causes the S-glutathiolation of a critical cysteine residue in the propeptide and conformational change and activation of the 72 kD form, allowing access of intracellular substrates (troponin I, α-actinin, myosin light chain-1, and titin are thus far known) to its catalytic zinc centre. MMP-2 is also a phosphoprotein (both 72 and 64 kD forms) and phosphorylation markedly reduces its activity (FASEB J 2007;21:2486). The kinases and phosphatases that regulate its activity in vivo are unknown; however, PKC can phosphorylate MMP-2 in vitro. Thus, MMP-2 can ‘remodel’ both intracellular and extracellular protein substrates. The cleavage of intracellular substrates by MMP-2 is an early response to enhanced oxidative stress that results in acute contractile dysfunction.
Abbreviations: matrix metalloproteinase-2 (MMP-2); tissue inhibitor of metalloproteinase-2 (TIMP-2); membrane-type-1 matrix metalloproteinase (MT1-MMP); glutathione (GSH); peroxynitite (ONOO-); protein kinase A (PKA); protein kinase C (PKC)
Free ubiquitin proteins are generated from the processing of ubiquitin precursors or ubiquitin chains by deubiquitylation enzymes (DUBs). An enzymatic cascade involving the E1 (ubiquitin activase), E2 (ubiquitin conjugase), and E3 (ubiquitin ligase) enzymes covalently conjugates ubiquitin chains to lysine residues in target proteins. Proteins deemed for degradation are singled out by E3 enzymes through the presence of a degradation signal (degron). The ubiquitylated substrate is recognized by a large proteolytic complex, the proteasome. The proteasome contains of 19S regulatory particles and the 20S core particle, which contains several proteolytic active subunits. The 19S regulatory particle binds, deubiquitylates, unfolds, and translocates the substrate into the proteolytic chamber of the 20S particle where the protein is degraded into short peptide fragments.
During myocardial ischaemia and reperfusion (I/R) there is accumulation of cytosolic Ca2+ due to defects in multiple Ca2+ handling proteins such as SERCA2a, NCX, LTCC, and RyR2. Importantly, the resultant cytosolic Ca2+ overload with myocardial reperfusion causes an increased influx of Ca2+ into mitochondria and results in the opening of the mitochondrial permeability transition pore (mPTP). In addition to Ca2+ overload, I/R is associated with increased generation of myocardial reactive oxygen species (ROS). While small amounts of Ca2+ and oxygen are necessary for optimal cardiac function, cytosolic free Ca2+ overload and increased oxidative stress are thought to be major contributors to myocardial I/R-induced injury and myocyte death. The potential targets to modulate Ca2+ overload and reduce myocardial ischaemia-reperfusion injury are: (1) Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), (2) Na+/Ca2+ exchanger (NCX), (3) SR Ca2+ release channel RyR, (4), L-type Ca2+ channel (LTCC), (5) Na+-H+ exchanger (NHE), and (6) Ca2+ and calmodulin-dependent protein kinase II (CaMKII).
Activation of the survivor activating factor enhancement (SAFE) pathway, as represented by the binding of a low concentration of endogenous or exogenous tumour necrosis factor alpha (TNFα) to its TNF receptor 2 (TNFR2) at the onset of reperfusion with the subsequent activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3), initiates a cardioprotective signalling cascade in both ischaemic pre- and postconditioning that is activated independently of the well-known reperfusion injury salvage kinases (RISK) pathway. The delineation of the SAFE pathway further emphasizes the importance of RISK-independent pathways in cardioprotection, which may have potential therapeutic application in the mitigation of ischaemic-reperfusion injury.
Abbreviations: RISK: Reperfusion Injury Salvage Kinases; SAFE: Survivor Activating Factor Enhancement; S1P: sphingosine-1-phosphate; TNFα: tumour necrosis factor alpha; GPCR: green protein coupled receptors; S1P R1/R3: sphingosine-1-phosphate receptors 1 or 3; TNFR2: tumour necrosis factor alpha receptor 2; MEK: mitogen-activated protein kinase; PI3K: phosphoinositide 3- kinase; Erk1/2: extracellular regulated kinases 1/2; Akt: protein kinase B; GSK-3β: glycogen synthase kinase-3 beta; JAK: janus kinase; STAT-3: signal transducer and activator of transcription-3; mPTP: mitochondrial permeability transition pore; P: phosphorylation.
This schematic provides a simplified overview of the intracellular transduction pathways underlying cardioprotection elicited by the growth factors: transforming growth factor-β1 (TGF-β1), cardiotrophin-1 (CT-1), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin, insulin-like growth factor (IGF), and urocortin. Ligand binding to their respective cell-surface receptors on the cardiomyocyte activates intracellular signalling kinase cascades including Raf-Ras-Mek1/2-Erk1/2 and PI3K-Akt of the reperfusion injury salvage kinase (RISK) pathway, the JAK-STAT pathway, and various anti-apoptotic mechanisms (including the phosphorylation and inhibition of Bax and BAD as well as the inhibition of cytochrome C release).
Many of the acute cardioprotective mechanisms manifested at the time of reperfusion converge on the mitochondria and include the inhibition of the mitochondrial permeability transition pore (mPTP), which can be achieved through several different mechanisms including the phosphorylation and inhibition of GSK3β; the opening of the ATP-sensitive mitochondrial potassium (Mito KATPM) channel by the eNOS-NO-PKG-PKC-ε cascade which produces mitochondrial ROS, which inhibits mitochondrial permeability transition pore opening; and the intracellular calcium modulation due to augmented SERCA uptake of calcium into the sarcoplasmic reticulum. More long-term cardioprotection may be achieved through the genetic transcription of various cardioprotective mediators such as iNOS, NFκB, MMP-1, phospholipase-1, and so on (not shown on diagram, see text for details).
The core structure of the mPTP remains unresolved. Known mPTP regulatory elements are depicted on the left side of the figure, whereas the right side indicates symbolically the threshold for mPTP-induction by oxidant stress. The middle row (horizontally) depicts the basal state of ANT and CyP-D as they relate to the basal threshold for mPTP induction by oxidant stress. The top row reflects factors that facilitate mPTP induction: atractyloside, Ca2+, and indirect effects of Pi. The bottom row includes factors that are known to inhibit mPTP induction: genetic deletion of ANT (ANT is dispensable for mPTP formation per se; inhibition of CyP-D by CsA remains protective), ADP, or bongkrekic acid (requirement/role of CyP-D under these conditions is unknown), CsA and genetic deletion of CyP-D in the presence of Pi (atractyloside, CsA and Ca2+ are no longer effective when compared with WT). Note the opposing mechanisms of Pi in mPTP induction: (i) Pi as a direct mPTP desensitizer (bottom row) is opposed by CyP-D binding (top row), whereas (ii) Pi may also act as an indirect mPTP sensitizer (through regulation of Mg2+ and/or polyphosphate levels; top row). Note that Ca2+ is not a major factor in mPTP induction in intact cardiomyocytes and neurons.
mPTP mitochondrial permeability transition pore
ANT adenine nucleotide translocator
BKA bongkrekic acid
CyP-D cyclophilin D
Pi inorganic phosphate
CsA cyclosporin A
ADP adenosine diphosphate
Ppif gene encoding CyP-D in mouse
There are three major signalling cascades of protein kinase activation in cardioprotection: (A) the GPCR/NPR-AKT-eNOS-PKG pathway, (B) the reperfusion-injury salvage kinase (RISK) pathway, and (C) the survival activating factor enhancement (SAFE) pathway, which centrally involves gp130-JAK-STAT signalling. In each system, there are molecules that are decreased in expression and/or activity with advancing age (marked in yellow) and possibly contribute to the loss of cardioprotection with aging. Such loss of cardioprotection with aging is one major problem in the translation of experimental data from (usually young and healthy) animals to the clinical situation in elderly humans.
Abbreviations: AMPK, AMP-activated kinase; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CB-R, cannabinoid receptor; Cx43, connexin 43; eNOS, endothelial NO synthase; ERK, extracellular regulated kinase; FGF-2, fibroblast growth factor 2; gp130, glycoprotein 130; GPCR, G-protein-coupled receptor; GSK3β, glycogen synthase kinase 3 β; H11K, H11 kinase; IGF, insulin-like growth factor 1; IL-6, interleukin 6; iNOS, inducible NO synthase; JAK, janus kinase; KATP, ATP-dependent potassium channel, MnSOD, manganese superoxide dismutase; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NPR, natriuretic peptide receptor; p38, p38 mitogen activated protein kinase; P70S6K, p70 ribsosomal S6 protein kinase; pGC, particulate guanylyl cyclase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKG, protein kinase G; ROS, reactive oxygen species; sGC, soluble guanylyl cyclase; SIRT1, sirtuin 1; STAT3, signal transducer and activator of transcription 3; TNF-R, tumour necrosis factor receptor; UCN, urocortins.
Vicious relationship between wall stress and ventricular remodelling to aggravate postinfarction heart failure
(A) Transverse ventricular sections taken from mouse hearts on Day 3, 7, or 28 postinfarction and stained with Masson's trichrome. Left ventricular remodelling progresses with time following myocardial infarction (MI). (B) Photomicrographs of infarct tissue collected from mouse hearts on Day 3, 7, or 28 post-MI, showing, respectively, acute inflammation, granulation, and scar. (C) With the passage of time after the onset of MI, the infarct length and left ventricular cavity become larger, whereas the infarct wall thickness decreases. Wall stress is proportional to the cavity diameter and intracavitary pressure and inversely proportional to the wall thickness (Laplace's law). Thus, wall stress and ventricular remodelling (dilatation and wall thinning) have a vicious relationship, aggravating one another and exacerbating post-infarction heart failure.
Nitrite homeostasis is determined by nitric oxide (NO) generation from NO synthases and dietary consumption of nitrate. Nitrate enters the stomach and then circulates in the blood and is converted into nitrite via salivary bacteria containing nitrate reductase. Nitrite derived from the diet and NOS activity rapidly accumulates in the plasma and is transported into tissues such as the heart. Nitrite is then stored in the myocardium and is metabolized into NO during hypoxia or ischaemia.
Schematic diagram showing the proposed mechanisms by which calpains participate in reperfusion injury and in the cardioprotective effects of preconditioning and postconditioning. NCX, Na+/Ca2+ exchanger; NBC, Na+/HCO3− cotransporter; NHE, Na+/H+ exchanger.
Mechanisms and consequences of altered Ca2+ handling in cardiomyocytes during initial reperfusion. Main events are connected through black lines, whereas red lines indicate important modulating factors. GCPR, G-coupled protein receptors; IP3, inositol trisphosphate; NOS, nitric oxide synthase; ROS, reactive oxygen species.
Pathophysiological role of SR–mitochondria functional units on lethal reperfusion injury. Calcium overload and re-energization cause calcium oscillations. ROS favour oscillations and trigger MPT. mNCX, mitochondrial Na/Ca exchanger; MCU: mitochondrial calcium uniporter.
Myocardial injury in ischemia–reperfusion develops in two phases. Reperfusion injury adds to the injury developed during initial ischemia (resulting in the red curve). The extent of reperfusion injury can be influenced by protective procedures, such as postconditioning or protective agents, applied during the first minutes of reperfusion (resulting in the blue curve). When the myocardium is not reperfused, it becomes entirely subject to ischemic cell death (broken black curve). While the past dogma was that protection against ischemia–reperfusion injury achieved by the pre-ischemic application of preconditioning is solely achieved by an effect on ischemic injury, it is now thought that this protection is also largely due to an effect on the causes of reperfusion injury (blue arrows).
Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia–reperfusion injury
Scheme summarizing the potential roles of Cx43 in the pathophysiology of ischaemia–reperfusion. Solid lines indicate roles for which there is experimental evidence. Broken lines indicate phenomena for which available evidence has been obtained under conditions other than ischaemia–reperfusion. PK, protein kinases; Src, Src tyrosine kinase.
Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia–reperfusion injury
Potential mechanisms by which mitochondrial Cx43 could participate in ischaemic pharmacological (diazoxide) preconditioning. Monomeric Cx43 (in blue) could modulate mitochondrial K+ATP channels (in brown), but also the effects of diazoxide on the respiratory chain (in dark gray).103 Cx43 hemichannels could favor H+ and K+ leak resulting in protective mild uncoupling104 and swelling.105,106
Scheme of the pathogenesis of acute reperfusion injury. Reperfusion reactivates ATP production in mitochondria (Mito). Recovering energy production (High ATP) activates the Ca2+ pump (SERCA) of the sarcoplasmic reticulum (SR), which clears the cytosol from Ca2+ overload accumulated during ischemia. Repetitive release of Ca2+ through the ryanodine receptor Ca2+ release channel (RyR) and reuptake into the SR leads to Ca2+ oscillations with high cytosolic peak Ca2+ concentrations. This high Ca2+ together with ATP provokes myofibrillar hypercontracture (Ca2+ contracture) and subsequent disruption of cells (Necrosis). Ca2+ uptake through the uniporter into mitochondria causes the opening of mitochondrial permeability transition pores (mPTP) and cytochrome c (Cyt c) release. The former leads to failure of energy production (low ATP), and the latter activates apoptosis. Low ATP induces rigor contracture of the myofibrils, again leading to cell disruption. Protection by reperfusion injury salvage kinase pathways (RISK) may interfere favourably at the SR or at mitochondria.