Mol Med 2023 revision lecture-1.pptx

Myocardial Ischaemia & Reperfusion O2 Supply O2 Demand Infarction and Mechanisms of Cell Death; Cardioprotection Jason N. Peart [email protected] G05 Rm 2.05 Molecular events in early atherogenesis Chemokines, Chemotactic factors Blood (lumen) Selectins Integrins Monocyte Endothelial c...

Myocardial Ischaemia & Reperfusion O2 Supply O2 Demand Infarction and Mechanisms of Cell Death; Cardioprotection Jason N. Peart [email protected] G05 Rm 2.05 Molecular events in early atherogenesis Chemokines, Chemotactic factors Blood (lumen) Selectins Integrins Monocyte Endothelial cells VCAM ICAM LDL NFkB TNF, IL-1 ROS TNF MF MCP Fibromatous plaque Proliferation of smooth muscle cells MCSF oxLDL PDGF Foam cell Vessel wall (intima) Mestas and Ley, Trends Cardiovasc Med, 2008 1. Selectins on ECs – tether rolling monocytes (via PS-glycoprotein ligand-1) to inflamed endothelium 2. Selectins activate Integrins on monocytes that mediate adhesion to endothelial cells by interaction with adhesion molecules (VCAM, ICAM – ligands for integrins) 3. Chemokines (interleukin-8) and chemotactic factors (MCP – monocyte chemotactic protein) stimulate extravasation of monocytes 4. ROS (reactive oxygen species) and some enzymes (15-lipoxygenase) promote LDL oxidation 5. Pro-inflammatory cytokines (IL-1, TNF) activate nuclear factor-kB that controls transcription of numerous genes: IL-1 and TNF, ICAM (intercellular adhesion molecule), VCAM (vascular cell adhesion molecule). 6. Activated macrophages also secrete PDGF (platelet-derived growth factor) causing recruitment into intima and proliferation of SMCs, that further stimulate macrophage activation (MCSF – monocyte colony stimulating factor). Major cellular effects of ischemia and reperfusion A, during ischemia, ê availability of molecular oxygen and metabolic substrates results = deficit of high-energy phosphates. -SR Ca2+ uptake mechanisms impaired = intracellular Ca2+ accumulation. -Anaerobic metabolism = intracellular accumulation of inorganic phosphate, lactate, and H+. -Activation of sodium-hydrogen exchanger (NHE) by intracellular acidosis = accumulation of intracellular Na+. -Na+ overload exacerbated by inhibition of the Na+ pump due to ATP depletion. -Increasing intracellular concentrations of solutes = osmotic swelling = sarcolemmal fragility or disruption, further exacerbated by activation of Ca2+-dependent proteases and phospholipases. B, at reperfusion, cell death occurs predominantly by necrosis. -Reintroduction of molecular oxygen causes reenergization of mitochondria and reactivation of the electron transport chain with massive production of ROS, which may stimulate further ROS production (ROS-induced ROS release) and generation of RNS in the presence of NO. ROS/RNS cause oxidative and nitrosative damage to cellular structures, including the SR leading to Ca2+ release. -With restored ATP production, activity of Na+/Ca2+ exchanger is restored, leading to extrusion of Na+ in exchange for Ca2+, and SR Ca2+ release is further accentuated by restoration of ATP leading to cytosolic Ca2+ overload. -The combined effects of Ca2+ accumulation in the Reperfusion Injury -A double-edged sword Scheme of osmotic control in the cardiomyocyte upon reperfusion in the cardiomyocyte. Ischemia creates an intra- and extracellular increase of osmolality. Upon reperfusion the extracellular osmolality is rapidly normalized (‘small Osm’) and thus a transsarcolemmal osmotic gradient is created. This leads to water influx and cell swelling. Circumstances of ischemia and reperfusion independently augment sarcolemmal fragility. Increased sarcolemmal fragility and cell swelling together favor rupture of the sarcolemma. Relation between Na+, Ca2+, and H+ • Similarly, acidosis will contribute to Na+ and Ca2+ overload since Na+ and H+ are exchanged: Myocardial Ischaemia Tissue Acidosis Reperfusion NHE Result of Ca2+ overload Diastolic Tension Rapid Na+/H+exchange leading to rise in cytsolic Ca2+ Relaxation NHE NCX Arrhythmias Cell Death In The Ischaemic Heart Myocardial cell death begins after 15 to 40 min of total ischaemia in experimental animals; after about 6 hours, few viable cells remain. Necrotic Membrane Changes Mechanical Disruption (swelling etc) Ca2+ activated phospholipases Catecholamines driving cAMP Uncontrolled Ca2+ entry (sarcolemmal damage) Overall, these changes contribute to cell membrane damage, Ca2+ overload and ultimately cell death. It is thought that the rise in Ca2+ is the ultimate mediator of cell death. 5. Acidosis NHE NCX Ca2+ 1 2 3 4 Myocardial Cytoprotection Temporal Properties of Preconditioning Protection % Protection 100 Post-Translational? 75 Transcriptional/ Translational? 50 25 Delay ed 0 0 Earl y 24 48 72 Hours Post Preconditioning Stimulus 96 Simple Model of Acute Preconditioning TRIGGER Trigger: GPCR Activation (together with changes in NO and Ca2+) * Membrane Receptors Mediators: Protein Kinase Signaling * Intracellular Effectors: Mitochondrial targets, including mPTP, KATP channel * Modulation of Mito channels* Peart J N , and Headrick J P Am J Physiol Heart Circ Physiol 2009;296:H1705-H1720 ***understand the basic path of GPCR, kinases and mitochondrial targets (Katp, MPTP)*** Potential Model of Delayed Protection Post-translational Modification Phosphorylation Transcriptional Induction Downstream Signalling Mitochondrial Permability Transition Pore Hypothetical scheme showing possible mechanisms of inhibition of MPTP opening at reperfusion. (A) Ischemia–reperfusion: at reperfusion the MPTP opens. (B) In the presence of ischemic preconditioning or mitochondrial K ATP channel activation: at reperfusion there is inhibition of MPTP opening.VDAC, voltage dependant anion channel; ANT, adenine nucleotide translocase; ROS, reactive oxygen species. Scheme Of Enzyme-signalingdependent Protection Against mPTPinduction Mitochondria he Signalosome Hypothesis Caveolar GPCR - GPCR activation induces formation of a vesicular caveolar signaling platform (termed the ‘signalosome’) that phosphorylates a receptor (R1 currently unidentified) on the mitochondrial outer membrane. The terminal kinase of the signalosome is PKG, which phosphorylates R1 (~P) at a serine/threonine residue. - On phosphorylation of R1, the signal is transmitted across the inter-membrane space to activate PKC- 1 on the inner membrane. PKC- 1 causes mito KATP opening and a consequent increase in H2O2 production, which activates a second PKC (PKC- to inhibit 2) ***understand the idea the of a GPCR Signalosom e Mito Outer Membrane Quinlan, C. L. et al. Am J Physiol Heart Circ Physiol 295: H953H961 2008 ‘signalosome’*** 4. Hypertrophy & Heart Failur Mechanotransduction – Load Stimulates PI3K/Akt Paths To Alter Transcription Laminin (basement membrane) a7 Stress 'sensed' by integrins b1D Integrins Melusin ? Transduced by talin/melusin (adapter proteins) Talin Mechanical Stretching Triggers PI3-K, Akt, mTOR, GSK3ß and MAPK activation MAPKs Hypertrophic Genes Leads to induction of hypertrophic gene programme via NF-AT/GATA4 Abbreviations: mTOR, mammalian target of rapamycin; GSK3ß, glycogen synthase 3 kinase 3ß; p70S6, ribosomal p70 S6 kinase; NF-AT, nuclear factor of activated T-cells; MAPK, mitogen activated protein kinase PATHOLOGICAL VS PHYSIOLOGICAL HYPERTROPHY A schematic overview of pathological and physiological hypertrophy outlining key differences in initiating stimuli, signaling pathways, cellular responses and cardiac function. For simplicity we have focussed on the best characterized signaling pathways implicated in mediating pathological (shaded red) and physiological (shaded green) cardiac hypertrophy. Ang II: angiotensin II, ET-1: endothelin-1, GPCR: G protein-coupled receptor, IGF-1: insulin-like growth factor 1, MAPK: mitogen-activated protein kinase, NE: norepinephrine, PI3K(p110α): Molecular Composition: ii) Signal transduction pathways a) In normal heart, with high amounts of b- adrenoceptors (ß-AR), stimulatory G proteins (G s) activate adenylate cyclase (AC), raising cAMP and inducing protein kinase A (PKA). Active PKA phosphorylates phospholamban (PLB), releasing its inhibitory function on SR Ca 2+ ATPase (SERCA) to enhances Ca2+ transport from cytosol to SR. Removal of Ca2+ augments relaxation, and release of Ca 2+ from SR stores increases contractility. b) In HF there is down-regulation of ß-ARs (mainly ß 1 subtype) and desensitisation by phosphorylation, which impairs ability of ARs to activate G s. Furthermore, binding of ß-arrestin to the AR results in uncoupling from Gs. Additionally, there is up-regulation of inhibitory G protein (Gi) and receptor internalisation. Together, these effects inhibit ß-AR/PKA signalling. Impaired Ca 2+ cycling decreases contractility and relaxation. Diabetes and Atherosclerosis Hyperglycaemia, Insulin Resistance, and Cardiovascular Disease (inc Atherosclerosis). AGE = advanced glycated endproducts; FFA = free fatty acids; GLUT4 = glucose transporter 4; HDL-C = high-density lipoprotein cholesterol; LDL = low-density lipoprotein particles; NO = nitric oxide; PAI-1 = plasminogen activator inhibitor-1; PKC = protein kinase C; PPARy = peroxisome proliferator-activated receptor y; PI3K = phosphatidylinositide 3-kinase; RAGE = AGE receptor; ROS = reactive oxygen species; SR-B = scavenger receptor B; tPA = tissue plasminogen activator. Diabetes, Ischaemia and Cardioprotection abetes May Significantly Alter Inhibition of GSK-3β and Activation of mP Major signaling pathways of IPC- and Ipost-mediated protection against cardiac cell death. Myocardial protection of IPC and Ipost were proposed to be mediated by stimulation of the prosurvival signaling pathway—PI3K/Akt pathway to inhibit the GSK-3β activation either via PI3K pathway or JAK2/STAT3 pathway. Diabetes (DM) can inhibit the activation of STAT3 or Akt to consequently activate GSK-3β that in turn induces mitochondrial cell death that is the critical cellular event for ischemia/reperfusion-induced myocardial infarction. Additional Pathways Other than GSK-3β are likely Involved In The Abolition Of Cardioprotection In Diabetes. Akt: protein kinase B (PKB); GSK-3β: glycogen synthase kinase 3; mPTP: mitochondrial permeability transition; JAK 2: Janus kinase 2; CGRP: calcitonin gene-related peptide; MAPK: mitogen-activated protein kinases; HSP 72: heat shock protein 72; eNOS: endothelial nitric oxide synthase; ERK: extracellular signal-regulated kinases; PTEN: phosphatase and tensin homolog. A Rana et al. Perfusion 2014;30:94-105 Diabetes and Heart Failure is ‘Bi-directional’ in Natur - - Patients with heart failure demonstrate impaired glucose metabolism and insulin resistance is common. (diabetic state) heart failure is a condition associated with increased plasma norepinephrine levels. Moreover, norepinephrine has been recently demonstrated to affect glucose homeostasis by decreasing insulin sensitivity. Diabetes is often associated with cardiomyopathy and heart failure Cardiovascular Ageing The effects of aging and calorie restriction (CR) on oxidative stress and inflammation in the vasculature and their relationship to atherogenesis. Increasing age is associated with increased production of reactive oxygen species, which results in oxidative stress and damage. Aging is also associated with a reduction in the inhibitory complex that prevents the transcription factor NF-κB from entering the nucleus where it can induce inflammatory genes. Together these factors result in increased expression of adhesion molecules (ICAM-1) and the proinflammatory enzyme inducible nitric oxide synthase (iNOS), thereby promoting monocytes adhesion to the endothelium and intimal infiltration and consequently initiating atherogenesis. Signal Transduction With Ageing Age impairs phospho-activation of survival kinases (ratio of kinase phosphorylation during d-OR agonism in aged vs. young hearts; n=5- Conclusions – How might Age impact stress intolerance and Cardioprotection? • Aged myocardium is sensitised to I-R injury/death • Stress intolerance is associated with oxidative stress, mitochondrial/bioenergetic dysfunction, ion overload, and shifts in damage control (eg. mitophagy) • Key molecules modulating these processes are altered (Beclin, Parkin, Bax, Bcl2) • GPCR protective signalling targeting multiple aspects of cardiac ageing is impaired • Impaired signalling/control of cell survival involves altered signal transduction (eg. MAPK path), and shifts in expression/phosphorylation of MKK3/6, MAPKAPK2, p70s6K and GSK3ß • Aged heart can nonetheless be protected by targeting effective signal elements Cholesterol is Modified with Age and CR Caveolae and Cav-3 Modified with Age and CR Calorie Restriction Pathway leading from calorie restriction to longevity. North B J , and Sinclair D A Circulation Research 2012;110:10971108 Copyright © American Heart Association Model for a longevity network. The effects of aging and calorie restriction (CR) on oxidative stress and inflammation in the vasculature and their relationship to atherogenesis. Increasing age is associated with increased production of reactive oxygen species, which results in oxidative stress and damage. Aging is also associated with a reduction in the inhibitory complex that prevents the transcription factor NF-κB from entering the nucleus where it can induce inflammatory genes. Together these factors result in increased expression of adhesion molecules (ICAM-1) and the proinflammatory enzyme inducible nitric oxide synthase (iNOS), thereby promoting monocytes adhesion to the endothelium and intimal infiltration and consequently initiating atherogenesis. CR attenuates the age-related loss of NF-κB inhibition, thereby keeping NF-κB in the inactive state. Furthermore, by activating transcription factor NF-E2-related factor 2 (Nrf2) and sirtuin 1 (SIRT1), CR induces proteins involved in protection against oxidative and free radical stress including glutathione-S-transferases (GST), NADPH:quinone oxidoreductase 1(NQO1), and heme oxygenase 1 (HO-1), manganese superoxide dismutase (MnSOD), and catalase. Together, these effects of CR prevent the NF-κB-mediated vascular inflammatory response and attenuate the agerelated increase in atherogenesis. Summary • Emergence of cardiac ischemic intolerance by middle-age in male C57/Bl6 mice; Moderate CR (40% for 14 weeks) restores ischemic tolerance. • Age-related post-ischemic dysfunction and cell damage associated with repressed expression of autophagy regulators (Beclin1, Parkin) and p70S6K, increased expression of active GSK3ß, and increased post-ischemic activation of p38-MAPK. • Cardioprotection with CR is associated with reversal of age-related changes in p70S6K, augmented Akt expression, pre-ischemic activation of p38-MAPK, partial inhibition of changes in Beclin1, and exaggeration of Bcl2 levels. • Transduction of Akt/mTOR signaling via p70S6K may represent a key point of regulatory convergence for age and CR. Exercise Illustration of several exercise-induced mitochondrial alterations that promote cardioprotection against IR injury. Exercise increases mitochondrial levels of the important antioxidant enzyme superoxide dismutase 2 (SOD2). Exercise training could also increase the expression of mitochondrial ATP-sensitive potassium channels along with other mitochondrial proteins that could contribute to cardioprotection. MAO-A, monoamine oxidase; SIRT3, sirtuin 3; MitoKATP, mitochondrial potassium ATP-sensitive channel. Exercise training decreases the expression of the mitochondrial enzyme monoamine oxidase A (MAO-A) in both SS and IMF mitochondria in the rat heart. This observation is important because MAO-A catalyzes the oxidative deamination of several monoamines, resulting in increased ROS production. Importantly, ROS production by MAO-A is a contributor to myocardial apoptosis during postischemia A putative sequence of events leading to exercise-induced protection against infarction. A putative sequence of events leading to exercise-induced protection against infarction. Postulated “triggers” of exercise-induced cardioprotection are denoted in green, with end-effectors labeled in red font. ROS, reactive oxygen species; AMPK, AMP-activated protein kinase; sarcKATP, sarcolemmal ATP-sensitive K+ channel. Sirtuin 1 SIRTUIN Signaling May Limit Atherogenesis Athero 42 SIRT1 is a NAD+-dependent class III histone deacetylase (HDAC). Additional to eNOS activation and ROS scavenging effects, recent studies demonstrate protective roles of SIRT1 in atherosclerosis – potential in pharmacotherapy? CR attenuates the age-related loss of NF-κB inhibition, thereby keeping NF-κB in the inactive state. Furthermore, by activating transcription factor NF-E2-related factor 2 (Nrf2) and sirtuin 1 (SIRT1), CR induces proteins involved in protection against oxidative and free radical stress including glutathione-S-transferases (GST), NADPH:quinone oxidoreductase 1(NQO1), and heme oxygenase 1 (HO-1), manganese superoxide dismutase (MnSOD), and catalase. Together, these effects of CR prevent the NF-κB-mediated vascular inflammatory response and attenuate the age-related increase in atherogenesis. SIRTUIN Signaling May Limit Atherogenesis Athero 43 SIRTUIN Signaling May Limit Atherogenesis Athero 45 Cardiovascular effects of Sirt1. Within the nucleus Sirt1 interacts with diverse transcription factors, inhibiting NFκB signalling, and consecutive proinflammatory cytokine expression, e.g. vascular cell adhesion molecule 1 as well as expression of the reverse cholesterol transporter LXR. Moreover, Sirt1 reduces plasma Pcsk9 levels, thereby increasing hepatic lowdensity lipoprotein-cholesterol receptor density and thus decreasing plasma low-density lipoprotein-cholesterol levels. Along with activation of endothelial nitric oxide synthase, these effects improve endothelial dysfunction and decrease atherosclerosis. In addition, Sirt1 deacetylates NFκB and inhibits tissue factor activity and thereby slows arterial thrombus formation. Sirt1-mediated tissue factor inhibition may further follow activation of peroxisome proliferator-activated receptor delta and Cox2-derived prostaglandin synthesis.