Calcium Homeostasis of Cardiomyocytes PDF

Calcium homeostasis of cardiomyocytes  Introduction – ic. Ca2+ secondary messenger Plays a role in specific cell functions (muscle contraction, fertilization, synaptic transmission, cell division, haemostasis, apoptosis, etc.) Changes can be quite fast (eg. Contraction...

Calcium homeostasis of cardiomyocytes  Introduction – ic. Ca2+ secondary messenger Plays a role in specific cell functions (muscle contraction, fertilization, synaptic transmission, cell division, haemostasis, apoptosis, etc.) Changes can be quite fast (eg. Contraction – ms time scale) Can not be measured directly in living cells Calcium cycling in cardiomyocytes NCX ICa NCX  Calcium cycling in cardiomyocytes Resting [Ca2+]i ≈ 150 nM 50 % force generation: Δ[Ca]total ≈ 70 μM [Ca2+]i ≈ 600 nM !!! Heavy (100:1) Ca2+ buffering in the cytosol! Calcium cycling in cardiomyocytes  Calcium cycling in cardiomyocytes Origin of calcium ions: Extracellular: ICa,L, ICa,T, NCX, ICa,TTX; „slip-mode” Intracellular: SR CICR: RyR2 Voltage dependent Ca2+ release Inozitol-1,4,5-trisphosphate mediated Ca2+ release  Calcium cycling in cardiomyocytes Origin of calcium ions: Extracellular: ICa,L, ICa,T, NCX, ICa,TTX; „slip-mode” Intracellular: SR ICa,L mediated CICR Stop of Ca2+ release: RyR inactivation and adaptation SR (local) calcium depletion  Calcium cycling in cardiomyocytes Functional CICR unit („couplon”): 10-25 DHPR/100 RyR Calcium cycling in cardiomyocytes  Calcium cycling in cardiomyocytes Species dependent! Short AP (eg: mouse) ~ patkány Long AP (guinea pig, dog, human) ~ rabbit Mitochondrial Ca2+ uptake: regulation relaxation Calcium cycling in cardiomyocytes [Ca2+]i ≠ [Ca2+]sm ≠ [Ca2+]cleft How to measure calcium concentration? Non-optical methods Ionselective electrode Electrophysiological methods Optical methods Fluorescence Epifluorescent microscopy Confocal microscopy Luminescence (Bioluminescence) etc.  Jablonski diagram Lowest vibration energy level Excited state excited state Excited state lifetime: 10-9-10-8 s Excitation Fluorescence Stokes shift Emission hEX - h Ground state Fluorescence spectrum Theory of fluorescent measurements Confocal microscopy  SELECTION CRITERIA FOR Ca2+ INDICATORS Ca2+ concentration range of interest (dissociation constant Kd; detectable response 0.1Kd to 10Kd) The method of delivery of the indicator to the cell Measurement mode (quantitative vs. qualitative ion concentration data, type of instrument, source of noise etc.) The intensity of the light emitted from the indicator Simultaneous recordings of other physiological parameters  SCHEMATIC DIAGRAM OF LOADING THE CELLS USING ACETOXYMETHYL (AM) ESTER DERIVATIVE FURA-2/AM PROBLEMS: Compartmentalization Incomplete AM ester hydrolysis Leakage  FLUORESCENT Ca2+ INDICATORS UV Fura-2, Indo-1 and derivatives Low-affinity calcium indicators (Fura-FF, BTC, Mag-Fura-2, Mag-Fura-5, Mag-Indo-1) VISIBLE LIGHT Fluo-3, Fluo-4, Rhod-2 and related derivatives Low-affinity calcium indicators: Fluo-5N, Rhod- 5N, X-Rhod-5N and related derivatives FLUORESCENCE EXCITATION SPECTRA FURA-2 Kd~135 nM (Mg2+-free) Kd~224 nM (Mg2+ 1mM) FLUO-3 Kd~ nM (Mg2+-free) Kd~ nM (Mg2+ 1mM) FLUORESCENT Ca2+ INDICATORS EXCITED WITH VISIBLE LIGHT - ADVANTAGES Efficient excitation with most laser-based instrumentation, including confocal laser scanning microscope Reduced interference from sample autofluorescence Less cellular photodamage and light scatter Higher absorbance of the dye Compatibility with UV probes and “caged” probes Microscope for epifluorescent measurement  Electrically stimulated skeletal muscle fibers filled with Aquaeorin Ashley & Ridgway (1970) The Journal of Physiology, 209, 105-130 Isolated cardiac myocytes  Elementary calcium release events spark ember F0 3 F0 200 ms 250 p Epifluoreszcent image Epifluoreszcent calcium measurement Epifluoreszcent image Confocal image  Calcium release events Calcium sparks and waves 200 ms 250 p Confocal calcium measurement Ca2+ – Calmodulin – CaMKII mediated signalization  Structure of calmoduline (a) Apo-CaM (b) Ca-CaM (4 Ca binding site) (c) CaM:smMLCK peptide complex  Model of the interaction of CaM with KCNQ channels Yus-Najera, E. et al. J. Biol. Chem. 2002;277:28545-28553 CaMK mediated signalization  Domain and molecular structure of CaMKIIδ Thr 286/287 306 307 N’ Catalytic Regulatory / Autoinhibitory Association C’ ATP Substrate CaM  Domain and molecular structure of CaMKIIδ Thr 286/287 306 307 N’ Catalytic Regulatory / Autoinhibitory Association C’ ATP Substrate CaM milliseconds P minutes Autophosphorylation on Thr 286/287 8-14 subunits  CaMK isoforms α ß I γ B CaMKIIδB CaMK II δ C CaMKIIδC IV D H I Role of CaMKII in β-Adrenergic receptor signaling HDAC: histone deacetylase EPAC: exchange protein activated by cAMP  Parallel pathways of CaMKII signalization Ca2+ release/entry Cytosolic Ca2+ ↑ CaMKIIδB CaMKII δ C Nucleus Cytosol + membrane ??? Proliferation: RyR: Ion channels and pumps: Apoptosis Hypertrophic program Ca2+ “leak” LTCC Facilitacion Myocardial dysfunction → Heart failure Zhang, T. et al. Cardiovasc Res 2004 63:476-486  Ca2+ homeostasis of a cardiac myocyte An Integrated Map Contractility Rhythm generator Growth/Differentiation Regulatory roles of calcium on ion channels Calcium cycling in cardiomyocytes [Ca2+]i ≠ [Ca2+]sm ≠ [Ca2+]cleft Pacemaker activity Pacemaker activity in the SA node „Membrane clock” Local Calcium Realeses (LCRs) in different [Ca]e Lakatta AnnNYAS 2008 LCRs during hyperpolarization  Correlation between LCR and spontaneous beating frequencies „Calcium clock” The coupled-clock pacemaker system Lakatta E G et al. Circulation Research. 2010;106:659-673 Lakatta E G et al. Circulation Research. 2010;106:659-673 Copyright © American Heart Association, Inc. All rights reserved. Na+ homeostasis of cardiac myocytes  Interaction between Na+ and Ca2+ homeostases of cardiac myocytes  Afterdepolarizations Early afterdepolarization Delayed afterdepolarization (EAD) (DAD) AP AP long AP EAD-DAD-triggeredAP_ATX-II_0.2Hz triggered AP ATX-II 100 nM 50 # EAD # # # Vm (mV) 0 DAD -50 -100 0 5000 10000 15000 idő (ms) # : paced APs at 0.2 Hz : APs triggered by suprathreshold DADs Changes in calcium homeostasis in heart failure Depolarizing currents INa,late and ICa,L  Depolarizing currents INa,late és ICa,L 0.0 0 * (A/F) … -50 * * csúcsérték -50 * ICa,L peak (A/F) Q - INa,late (µC/F) -0.5 -1 Q - ICa,L (µC/F) -150 -150 INa,latepeak -1.0 -2 -1.5 -250 -3 -250 INa,late -2.0 -350 -4 -350 AMC HF AMC HF AMC HF AMC HF Calcium transients  Calcium transients 1.7 1.7 0.3 1.5 NS 1.5 * * peak F340/F380 dia F340/F380 0.2 taurelax (s) 1.3 1.3 0.1 1.1 1.1 0.9 0.9 0.0 AMC HF AMC HF AMC HF Pathology Electrical dysfunction: arrhythmogenesis Mechanical dysfunction: heart failure Molecular structure  Arrhythmogenesis Normal cardiac cycle Increased Increased calcium entry phosphorylation Increased Calcium sensitivity Altered LTCC overload of RyR Spontaneous release  Topics 1. Calcium measurements, methodology 2. Calcium cycling 3. Transduction of the calcium signal 4. Role of calcium in 1. Electrophysiology 2. Pacemaker activity 3. Excitation contraction coupling 4. Contraction 5. Beat to beat regulation 6. Cardiac memory 7. Energy consumption 8. Transcriptional regulation 9. Apoptosis 5. Pathology 1. Electrical dysfunction: arrhythmogenesis 2. Mechanical dysfunction: heart failure Confocal microscopy Calcium as a universal intracellular messenger -Electrophysiology (electric signalization) -E-C coupling (governs the calcium transient) -Contraction (myofilaments) -Energy consumption (mitochondrial ATP production) -Cell death (apoptosis/necrosis) -Transcriptional regulation (calmodulin/calcineurin) -….. Calcium cycling  Mechanisms of Ca2+ signalization 1. Direct (Tnc) 2. Calmodulin mediated (LTCC) 3. CaMK mediated (RyR, SERCA, LTCC) Components of Calcium homeostasis in cardiac myocytes Entry 1. L-type Ca2+ channel 2. T-type Ca2+ channel 3. Na/Ca exchange (NCX) 4. TTx-sensitive Ca2+ channel 5. Slip mode conductance Release 1. Ryanodine receptors (RyR) 2. IP3 receptors (IP3s ) Extrusion 1. Na/Ca exchange (NCX) 2. Sarcolemmal Ca2+ pump Sequestration 1. SR Ca2+ pump (SERCA) Buffers 1. SR (Calsequestrin) (dynamics) 2. Cytosolic 3. Mitochondria  1. Direct Ca2+ regulation Mechanisms of Ca2+ entry The structure of LTCC Subunits: C C    conductive pore 2: E.C. drug-binding site, regulatory site : drug-binding site, regulatory site cAMP dependent PKA phosphorilation : I.C.  Main facts -Location: sarcolemma, T-tubule -Physiologiacal role: mean Ca2+ entry -Slow response (slow AP) upstroke (SA, AV node) -Atrioventricular nodal conduction (Slow AP) -Excitation-contraction coupling (CICR) -Plateau depolarization -Regulation: Adrenergic, Ca2+-Calmodulin-CaMKII, PKA -Pharmacology -Inhibitors -Phenylalkylamines (verapamil) IIIS6, IVS6 -Benzothiazepines (diltiazem) IIIS5, IIIS6, IVS6 -Dihydropiridines (nifedipine) IIIS5, IIIS6, IVS6 -Activators: - Dihydropiridines (BayK 8644)  The LTCC during Action Potential F07092402 50 0 Vm (mV) -100 0 ICa,L (pA/pF) -1 -2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Time (s)  Electrophysiology of LTCC 0 mV -40 mV 500 pA 100 ms Differences in the structures of L- and T-type Ca2+ channels Cardiac specific subunits: 1H, 1G Larger E.C. loop in domain I, larger citoplasmatic loops between I-II and II-III domains (differences in voltage sensitivity). Longer carboxi terminal of 1C subunit, having important role in Ca2+-dependent inactivation. I-V characteristics of LTCC and TTCC  Main facts Localisation: sarcolemma Fast activation and inactivation (both in ms range) Vakt and Vinakt much more negative, than in case of ICa-L, LVA Ligands: Mibefradil, nickel (low concentration) Physiologic role: Depolarization of Pacemaker cells EC-coupling? Governing cell growth and differentiation Found in pacemaker cells (SA, AV node) and Purkinje cells. Not present in most atrial and ventricular cells. Expressed in dedifferentiated cells. Arrhythmogenesis  TTx sensitive Ca2+ channel Identified in rat ventricular cells Permeable to both Na+ and Ca2+ Localization: sarcolemma Physiological role – Augmenting depolarization – Excitation-Contraction coupling Pharmacology: – Inhibitor: TTX Pathophysiological role: Arrhythmogenesis  Slip mode conductance Voltage dependent fast sodium channels Selectivity Na+/Ca2+=3000/1 – The amount of Ca2+ entering into cardiac myocytes during upstroke is not known Certain drugs (isoproterenol, digitalis) can reduce Na+/Ca2+ to 1 No physiological role is known Arrhythmogenesis?  IP3 signalization of cardiac myocytes Catecholamines/ET IGF/FGF EC GPCR PLC TKR IC Gα Gβγ Nuclear membrane IP3 SR -Atria CaMKIIδB CaM Ca2+ IPR -Nucleus -Heart Failure Transcription CaN Sparks and waves Movie Calcium extrusion & sequestration 1. NCX  Na/Ca exchange (NCX) Forward Reverse mode mode Na+ Na+ Na+ Extracellular Intracellular Ca2+ Driving force: Electrochemical potential gradient 3-2=1  Na/Ca exchange (NCX) ?  Na/Ca exchange (NCX) Electrogenic Na+/Ca2+ transport: 3 Na+: 1Ca2+ Forward mode (Na+ in and Ca2+ out) Reverse mode (Na+ out and Ca2+ in) Transport rate is determined by Na+ and Ca2+ concentration (cardiac glycosides) Membrane potential (pH?) Inhibitors: XIP, lanthanum (not specific), Ni 5-8 mM (not specific) Physiological role: - removal of Ca2+ from cytoplasm - Ca2+ entry during early phase of systole Pathophysiologic role: arhythmogenesis (DADs)  2. Sarcolemmal Calcium Pump Ubiquitous ATP dependent calcium pump -Localized in the sarcolemma -Homology with SERCA, several isoenzymes and splice variants -Stochiometry: 1 Ca2+/ATP -Regulated by CaM (!!!), PKA, PKC -Transport rate is only 3-10 % of NCX (species dependent) Minor contribution to Ca2+ turnover  3. SR Calcium Pump (SERCA) ATP dependent P type calcium pump -Localized in the SR -3 major form (SERCA1-2-3) -Stochiometry: 2Ca2+/ATP -Regulated by Phospholamban (!!!), PKC, CaMKII  When calcium homeostasis goes wrong Main forms of defective calcium handling in the heart - Insufficient contractility/pump function → Heart Failure (acute/chronic) - Pathologic activation/excitability → Arrhythmia - Morphological disorders → Hypertrophy / Dilation Heart Failure (acute or chronic) is the one single leading cause of death in western societies - More than 2 million Americans suffer from Heart Failures - 200,000 Americans die annually, 50% is Sudden Cardiac Death (SCD) - Incidence of cardiac disease is >400,000/year - 15% of HF patients dies within 1 year - 6 year mortality is >80%