Second_Messengers_in_CV_2023.10.12_Student.pptx

Second Messengers and CICR in the Cardiovascular System Steven J. Ontiveros, MBA, PhD Objectives • Describe the mechanism of excitation-contraction coupling and calcium-induced-calciumsignaling in cardiomyocytes. • Describe the mechanism of calcium release by the sarcoplasmic reticulum via the rya...

Second Messengers and CICR in the Cardiovascular System Steven J. Ontiveros, MBA, PhD Objectives • Describe the mechanism of excitation-contraction coupling and calcium-induced-calciumsignaling in cardiomyocytes. • Describe the mechanism of calcium release by the sarcoplasmic reticulum via the ryanodine receptor 2 (RyR2) in cardiac cells, and the regulatory roles of calsequestrin (CSQ). • Describe the mechanism associated with the removal of cytosolic calcium via the SERCA2, and the regulatory roles of phospholamban (PLB). • Describe the control measures regulatory molecules such as protein kinase A (PKA), calcium, and calmodulin (CaM) play in regulating cytosolic calcium levels. • Describe the signaling mechanisms of β-adrenergic and cholinergic stimulation in cardiac cells. • Describe the signaling mechanisms and pathways associated with vasoconstriction and vasodilation. • Compare and contrast muscle contraction between vascular smooth muscle cells and skeletal muscle. • Describe the roles myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) play in regulating muscle contraction in vascular smooth muscle cells. Acronyms • • • • • • • • • • cAMP – cyclic adenosine monophosphate cGMP – cyclic guanosine monophosphate SR – Sarcoplasmic reticulum DHPR – Dihydropyridine receptor RyR2 – Ryanodine receptor 2 SERCA2 – Sarco/endoplasmic reticulum calcium ATPase 2 CICR – Calcium-induced-calcium-release CSQ2 – Calsequestrin 2 J-SR – Junctional sarcoplasmic reticulum L-SR – Longitudinal sarcoplasmic reticulum • • • • • • • • • • PKA – Protein kinase A PKG – Protein kinase G PLB/PLN - Phospholamban CaM - Calmodulin CaMKII – Calmodulin-dependent Kinase 2 NO – Nitric Oxide eNOS – endothelial nitric oxide synthase MLC – Myosin light chain MLCK – Myosin light chain kinase MLCP – Myosin light chain phosphatase Microanatomy of Contractile Cells and Proteins • Cardiomyocytes are morphologically different from skeletal muscle: • Shorter • Branched • Connected via intercalated discs (disks) • Intercalated discs: • Cell-to-cell contacts • Adhesion and communication • Synaptic input: • Sympathetic and parasympathetic of ANS • Do not initiate contraction…only modulate • Pacemaker cells: • SA node • Initiate electrical excitation • Action potential flows through gap junctions Medical Physiology 3rd Edition 2017 Intercalated Discs • 2 major regions: • Longitudinal • Transverse Longitudinal Region Transverse Region • Intercalated discs Intercalated Discs • Longitudinal • Communication • Gap junctions (communication) • Desmosomes (adhesion) • Transverse • Adhesion and transmission of forces • Enriched in desmosomes • Fascia adherence • Similar to adherens junctions • Found proximate to sarcomeres Trends id Cardiovasc Med; 19(6): 182-190, 2009 Functional Aspects of Intercalated Discs • Cellular communication • Ions/second messengers/small molecues are shared between cardiomyocytes • Adhesion Gray’s Anatomy 42nd Ed, 2021 • Fascia adherence and desmosomes adhere cells and transmit forces across the sarcolemma Clinical Significance of Intercalated Discs • Plakophilin-2 (PKP2) • Linker protein for desmosomes and IF • Expressed in skin and cardiomyocytes • Mutations in PKP2 • Causes cardiomyopathies and heart failure • Arrhythmogenic right ventricular cardiomyopathy (ARVC) • Exercise-induced ARVC • Presentation within intense endurance athletes in the absence of mutations Clinical Significance of Intercalated Discs • Clinical Presentation • May not have symptoms • Right ventricle weakens first • Arrhythmias (ventricular tachycardia, palpitations, syncope) • Sudden death may be the first indication • Occurs in adolescence or older adults • Histological Observations • Hypercontracted alternated with hyperdistended cells • Square or irregular-shaped nuclei • Detached sarcomeres Subcellular Structures in Cardiomyocytes • Sarcoplasmic reticulum (SR): • Continuous with T-tubules • Network surrounding myofibrils • Calcium storehouse • Activity: Braunwald’s Heart Disease 11th Eidtion, 2019 • Releases Ca2+ during contraction (systole) • Reabsorbs Ca2+ during relaxation (diastole) Front. Cell Dev. Biol. (2015) Excitation-Contraction Coupling in Cardiac Muscle 1. Cardiac Node Excitation • Membrane depolarization 2. Ca2+ Channels (DHPR) open • Ca2+ enters through L-Type Ca2+ channels • Serves as trigger 3. Ca2+ activates cardiac Ryanodine Receptor-2 (RyR2) • Ca2+ release into cytosol 4. Contraction Dyad • Dyad Components: • Plasma membrane of the T-tubule • Junctional-SR (J-SR) & Ryanodine receptor (RyR2) • Dyad membranes (Plasma membrane & Sarcoplasmic Reticulum): • Close proximity (12-15 nm) • Important for facilitating Ca2+ signaling • SR membrane: Up to 250 RyR2 • PM: 20 to 40 L-Type Ca2+ channels (DHPR) • Localized cytoplasmic Ca2+: • Acts on the RyR2 receptors • Facilitates calcium-induced calcium release (CICR) ECC-CICR in Cardiac Muscle • Calcium-induced Calcium Release (CICR): • Ca2+ enters through the L-Type Ca2+ channel (DHPR) • Causes an induction of Ca2+ release from RyR2 • RyR2 is activated by low levels of Ca2+ • RyR Receptor: • Skeletal – RyR1 is activated by tugging motion from DHPR • Cardiac – RyR2 and L-Type Ca2+ Channel (DHPR) are not linked • RyR2 is activated by Ca2+ • Calcium Spark: • CICR initiates calcium spark • Positive feedback mechanism • Increases concentration of cytosolic Ca2+ Cold Spring Harbor 2015; 7:a006023 Skeletal vs Cardiac Muscle ECC Cold Spring Harbor 2015; 7:a006023 Skeletal vs Cardiac Muscle Skeletal • Action potential • Conformational change on Dihydropyridine receptor (DHPR) • DHPR physically linked to RyR1 • RyR1 opens • Ca2+ release from Sarcoplasmic Reticulum (SR) • Ca2+  contraction Cardiac • Electrical excitation • L-Type Ca2+ Channel (DHPR) opens • Ca2+ enters the cell from extracellular space • Cytoplasmic Ca2+ binds the RyR2 receptor on the cytosolic side • RyR2 opens • Ca2+ release from SR • Ca2+  contraction (systole) Calcium Spark Ca2+ T-Tubule L-Type Ca2+ Channel • Calcium Spark: • Localized Ca2+ release from SR • Significantly increases cytosolic Ca2+ concentration Cytosol RyR2 • Positive feedback mechanism • First RyR2s (activated by Ca2+ channel) activate additional RyR2 • Spark causes dramatic increase in cytosolic Ca2+ • Sources of cytosolic Ca2+ • Ca2+ from SR-CICR • Ca2+ from extracellular space Ca2+ Ca2+ Sarcoplasmic Reticulum Ca2+ Ryanodine Receptor • Isoforms: • Type I (RyR1) – skeletal (physically tethered to DHPR) • Type II (RyR2) – cardiac • RyR2: • Located on the Junctional-SR (J-SR) • Tetramer with hollow channel • Binds: • Calmodulin (CaM) • FK-506 Binding Proteins (FKBPs) • Kinases (PKA & CaMKII) • Regulated on the cytosolic and luminal sides Braunwald’s Heart Disease, 10th Edition 2015 Cytosolic side: • Similar to skeletal muscle RyR regulation!!! RyR2 Regulation • PKA: • Phosphorylates (activates) receptor • Ca2+/Calmodulin (CaM): • Activate/inhibit RyR2 • Low cytosolic Ca2+  activates (binds high affinity sites) • High cytosolic Ca2+  inhibits (binds low affinity sites) Luminal side: • Ca2+ binding site: • Ca2+ binds RyR2 (luminal side) • Luminal “sensor” that detects level of Ca2+ in the SR • CSQ also regulates RyR2 from lumen side Eur Biophys J (2009) 39:19–26 • 2 Isoforms: Calsequestrin 2 • CSQ1 – skeletal muscle • CSQ2 – cardiac muscle • Function of CSQ2: • Buffering agent for Ca2+ • Similar to CSQ1 • Luminal RyR2 regulator • Mechanism: • Inhibitory complex: Junctin/Triadin/CSQ2 • Low levels of SR-Ca2+ • CSQ2-RyR2 binding occurs • Closes RyR2 • High SR-Ca2+: • CSQ becomes bound to Ca2+ • Free RyR2 (Open) Am J Physiol Heart Circ Physiol (2012); 302(6):H1250-H1260 Myofilament Relaxation  Plasma membrane Ca2+ ATPase (PMCA):    Found on PM Na+/Ca2+ Exchanger (NCX):  Found on PM  Exchanges Ca2+ for Na+ Sarco/Endoplasmic Reticulum Ca2+ ATPase Type 2 (SERCA2):  Found on the Longitudinal-SR (L-SR)  Same as skeletal, primary transporter of Ca2+ in cardiomyocytes  Removes  majority of cytosolic Ca2+ Regulated by phospholamban Expert Opin Biol Ther (2011) Phospholamban • Phospholamban (PLB or PLN): • Transmembrane protein of the cardiac SR • Binds and regulates (inhibits) SERCA2 Ca2+ uptake • When bound it inhibits SERCA2 Braunwald’s Heart Disease, 10th Edition 2015 Expert Opin Biol Ther (2011) What other factors affect heart rate and muscle contraction? Adrenergic Receptors • Adrenergic receptors (adrenoceptors) • GPCRs • Found in many cells • Respond to catecholamines (E, NE) • β-AR • All 3 subgroups are coupled to Gs that activates AC • β1 • Increased HR, CO (heart) • β2 • α-AR • α1 – coupled to Gq activates PLC • Vasoconstriction of peripheral vascular smooth muscle • α2 – coupled to Gi inhibits AC • Sympathetic presynaptic nerve endings • Inhibit the release of neurotransmitter (NE) • Bronchodilation (lungs) • Vasodilation (skeletal muscle) • β3 • Increased lipolysis G-Proteins β1-AR • G stimulatory (Gαs) – β1-AR • Activates adenylyl cyclase Cholinergic Receptor • G inhibitory (Gαi) – ACh receptor (muscarinic) • Inhibits adenylyl cyclase ***Note: Both Gs and Gi modulate AC and the levels of cAMP • Gs – Increases cAMP levels…PKA is on • Gi – Decreases cAMP levels…PKA is off α1-AR • IP3 signaling pathway • Gαq (stimulatory)  Activates PLC • Increases the levels of IP3 Heart: β1-AR • Sympathetic stimulation: • Catecholamines: Epinephrine/Norepinephrine • GPCR: β-Adrenergic Receptor (β1-AR) • β1-AR: • Heart • Activates Gs • Gs activates adenylyl cyclase (AC) • Increased cAMP & PKA • PKA: • Regulates Ca2+ cycle in cardiomyocytes • Increases HR Braunwald’s Heart Disease, 10th Edition 2015 Heart: Cholinergic Receptor • Parasympathetic (cholinergic) stimulation: • Acetylcholine (ACh) • GPCR: Muscarinic acetylcholine receptor (mAchR): • Heart • Activates Gαi • Inhibits AC • Causes a decrease in cAMP levels and active levels of PKA • Decreases HR Braunwald’s Heart Disease, 10th Edition 2015 PKA • Protein Kinase A (PKA) • PKAII isoform is dominant in cardiomyocytes Function in cardiomyocytes: • Activates Ca2+ channels • Increased Ca2+ influx • Activates RyR2 • Increased SR-Ca2+ efflux • Inhibits PLB/PLN • Increased Ca2+ uptake by SR Cold Spring Harbor 2015; 7:a006023 SERCA2/PLB Regulation by PKA & Ca SERCA  PLB SERCA2 Activation   2+ High cytosolic Ca2+ activates SERCA2 PLB Inhibition  PKA phosphorylates (inhibits) PLB/PLN  SERCA2 become active OPEN Closed OPEN Nature Reviews (2004); 4:566 PKA Regulation of Cytosolic Calcium Cold Spring Harbor 2015; 7:a006023 Calmodulin (CaM) • Calmodulin Pathway: • Calmodulin – Ca2+ responsive protein • Can activate Ca2+ /calmodulindependent protein kinases (CaMKs) • CaM: • Capable of regulating many targets (ion channels, transcription machinery) • Contains 4 Ca2+ binding sites • 90% of CaM in myocytes is pre-bound to cellular targets Medical Physiology 3rd Edition, 2017 Calmodulin (CaM) and CaMKII Regulation of Intracellular Calcium in Cardiomyocytes When CaM is bound to these molecules: • RyR2 (activates/inactivates) • Ca2+ Channel (inactivates) • Phospholamban (inactivates) • Leads to SERCA activation (open) Braunwald’s Heart Disease, 10th Edition 2015 Summing it up Cold Spring Harbor 2015; 7:a006023 Braunwald’s Heart Disease, 10th Edition 2015 Second Messengers in vascular smooth muscle control Vascular Smooth Muscle – Cellular Communication • Smooth muscle: • Lining of the walls of various organs (stomach, intestines, bladder, uterus) • Tubular structures/passageways (arteries and veins) • Control: • Autonomic nervous system • Hormone, para/autocrine stimuli • Vascular Smooth Muscle (VSM): • Endothelial cells receive signal • Produce endothelial factors VSMC Endothelial cells • Endothelial factors act on target cells (VSM cells) Luniver Press (2008). Proceedings of the 2008 Workshop on Complex Systems Modeling, p182-196 Blood Vessel Lumen • Actin and myosin filaments: • Similar to striated muscle • Actin/myosin interaction the same Molecular Activation of Myosin in VSMC • VSMC do not contain: • Troponin complex • VSMC do use: • Kinases to activate myosin • Myosin light chain (regulatory subunit): • Small subunit of myosin • Regulates actin/myosin interaction • MLC states: • Phosphorylated (active) • Unphosphorylated (inactive) Medical Physiology 3rd Edition 2017 Molecular Modulation of Smooth Muscle Tone • Regulatory proteins: • Myosin light chain kinase (MLCK) • Phosphorylates myosin light chain • Myosin light chain phosphatase (MLCP) • Dephosphorylates myosin light chain • Myosin light chain • If phosphorylated • Becomes active  contraction • If dephosphorylated • Becomes inactive  Relaxation Medical Physiology 3rd Edition 2017 Myosin Light chain Myosin Light Chain Kinase Myosin Light Chain Phosphatase Where does the calcium come from for the activation of CaM and MLCK? • Phospholipase C (PLC) Pathway: • Gq protein • Activation leads to calcium release from SR • IP3: • Hydrophilic sugar phosphate • Binds and opens calcium channels on ER • Cytosolic Ca2+ levels increase • DAG: • Membrane-bound second messenger • Recruits protein kinase C (PKC) to the lipid membrane • PKC: • Similar to PKA Phospholipase C Pathway Vasoconstriction (α1-AR) • Modulators of vasoconstriction: • Angiotensin II, thrombin, vasopressin, endothelin • Intracellular Ca2+ increases by: • Efflux from SR • AngII acts on endothelial cells • Endothelial cells release endothelial factors • Endothelial factors activate GPCR (smooth muscle cells)  IP3  Ca2+ • Influx from plasma membrane Ca2+ channel • Ca2+ binds CaM: • Active CaM activates Myosin light chain Kinase (MLCK) • MLCK: • Phosphorylates light chain  vasoconstriction VSM Cell Endothelial Factors Vasodilation • Modulators of vasodilation: • Epinephrine (β2), acetylcholine, bradykinin, shear stress • Endothelial Cell • ACh  GPCR  PLC  IP3  Ca2+ • Ca2+ activates CaM • CaM activates endothelial nitric oxide synthase (eNOS) • Nitric Oxide (NO): • Arginine precursor • Rapid diffusion into VSM cell Vasodilation • Nitric Oxide diffuses into vascular smooth muscle (VSM) cell • VSM Cell: • Guanylyl cyclase & Cyclic GMP (cGMP): • NO activates Guanylyl cyclase • GTP  cGMP • Guanylyl cyclase similar to AC • cGMP activates protein kinase G (PKG) PKG: • Inhibits Ca2+ channel • Activates myosin light chain phosphatase (MLCP) • MLCP • Removes phosphate from myosin • Promotes relaxation of VSMC VSM Cell • ADP Stimulatoion of Endothelial cells • Endothelial cells secrete endothelium-derived hyperpolarizing factor (EDHF) and Prostaglanadin I2 (PGI2) • Stimulates 2 pathways in VSM cells • EDHF • Opens K+ ion channels on VSM cells • Hyperpolarization and VSM cell relaxation • PGI2 • Aka prostaclyclin • Stimulates cAMP-PKAmediated pathway • PKA directs SMC relaxation by inhibiting MLCK Vasodilation: Other Pathways