Molecular Medicine of the

Ch. Depre

Dept. Cell Biology and Molecular Medicine UMDNJ Medical School

[email protected] MOLECULAR MEDICINE OF THE HEART

1. General concepts 2. Physiology of contraction and relaxation 3. Electrophysiology 4. Calcium and contraction 5. Metabolism 6. Signal transduction and gene expression 7. Partial test 8. Coronary physiology 9. Atherosclerosis 10. Cardiac hypertrophy and the athlete’s heart 11. Cardiac ischemia, cell death and survival 12. Heart failure 13. Review 14. Final test Molecular Medicine of the Heart

Class 1. General concepts Overview of class 1: General concepts

1. Anatomy of the heart 2. Pump function of the heart 3. Systemic vasculature 4. Coronary arteries 5. The sarcomere 6. Conduction system 7. Questions 1. Anatomy of the heart

Two sides - Three arteries - Four chambers

• The heart is made of two sides, a right side and a left side • Right side and left side do not communicate directly, except abnormally in some forms of congenital heart disease • Each side is made of an atrium and a separated by atrio-ventricular valves, named “tricuspid” on the right side and “mitral” on the left side • Each atrium receives blood from veins, the venae cavae on the right side and the pulmonary veins on the left side, and ejects this blood into the corresponding ventricle • Each ventricle ejects blood in an artery, the on the right side and the aorta on the left side. Reverse flow from the artery is prevented by a valve between the ventricle and the artery • The first branches of the aorta are the coronary arteries, which provide the heart with blood supply

Structure of the heart

• Myocardium. made of cardiac myocytes, major part of the heart • Endocardium. Endothelial layer separating the myocardium from the blood • Pericardium. Protective sheet surrounding the myocardium • Coronary arteries. Arteries from the aorta supplying blood to the myocardium • Capillaries. Microvessels between the cardiac myocytes • Subendocardium Deep myocardial layers, adjacent to the endocardium • Subepicardium Superficial myocardial layers, adjacent to the pericardium Pathophysiological importance of the structures

• Myocardium. Cardiomyopathy. Describes any form of dysfunctional myocardium (ischemic, hypertensive, congenital, valvular…). Any impairment in myocardial contraction results in altered cardiac function. Insufficient cardiac function is defined as heart failure.

• Pericardium. Pericarditis. Inflammation of the pericardium, usually of viral origin. Induces strong pain that mimicks heart attack. Because the pericardium is fibrous and rigid, any effusion will compress the myocardium and impair its function. Pathophysiological importance of the structures

• Coronary arteries. Ischemia. Obstruction of the coronary arteries leads to an insufficient supply of blood to the myocardium.

• Capillaries. Angiogenesis. Myocardium submitted to chronic ischemic conditions stimulates the growth of neovessels and collaterals to improve oxygen supply. 2. Pump function of the heart

The basic function of the heart is to pump blood

The heart ejects blood from the thick-walled left ventricle to be propelled through the body, ultimately to reach the peripheral circulation, where oxygen is removed to nourish the various organs and tissues.

The deoxygenated venous blood flows back to the right side of the heart, to be ejected from the right ventricle to the lungs, where it is oxygenated before it is directed toward the left atrium and ventricle. Function of the heart

Deoxygenated blood Oxygenated blood

Venae Cavae Pulmonary Veins

Right Atrium Left Atrium

Tricuspid Valves (3) Mitral Valves (2)

Right Ventricle Left Ventricle

Pulmonary Artery Aorta

Lungs Periphery = the ventricle relaxes = the ventricle contracts A normal contractile function requires a tight coupling between cardiac myocytes Cardiac myocytes are physically bound by the intercalated disks

Gap Junctions. Tight coupling between myocytes by the connexons for easy passage of small molecules and current

Desmosomes. Protein complex that is linked to the sarcomeres by desmin, and which promotes force transfer Coordinated contraction of the cardiac muscle Coordinated contraction of the cardiac muscle 3. Systemic vasculature

Conductance – Resistance – Exchange - Return

• Conductance vessels. Large arteries with low resistance, used as conduits to carry oxygenated blood toward the organs.

• Resistance vessels. Arterioles with thick muscular wall and high resistance, directing the blood flow to the organs that need oxygen. Constitute the Peripheral .

• Capillaries. Exchange vessels made of an endothelial layer without muscular cells, where oxygen leaves the blood and enter the tissues by diffusion.

• Veins. Low-pressure, large-capacitance system containing most of the , constitute the Venous Capacitance System, returning the blood flow to the right side of the heart. Global scheme of blood circulation Principle of conductance vessels

Systole

Diastole

The pressure drops The pressure remains high to receive blood to keep pushing the blood from the LA Principle of resistance vessels

PVR: Peripheral Vascular Resistance Blood flow distribution through capillaries drops in resistance vessels 4. The coronary arteries

Coronary arteries are the leading cause of heart disease

The left ventricle receives blood from the left coronary artery, that divides in two main branches • the left anterior descending artery (LAD) supplies most of the anterior part of the myocardium

• The circumflex artery supplies the lateral and posterior part

The right ventricle receives blood from the right coronary artery

The posterior descending artery can originate from either the right or left coronary artery

5. The sarcomere

The sarcomere is the fundamental contractile unit of the cardiac myocyte

It is limited on each side by the Z line, on which actin filaments are attached.

Myosin filament originate from the middle of the sarcomere, or M line, but do not attach directly to the Z line.

The A band represents the zone of overlap between actin and myosin

The I band represents the zone which contains only actin filaments

The H band represents the zone which contains only myosin filaments

Myosin filaments are indirectly connected to the Z line by the macromolecule Titin, which limits the maximal stretch of the sarcomere and, therefore, of the whole cardiac myocyte. Ultrastructure of the cardiac myocyte The sarcomere is the contractile unit of the myocyte

Think “HAZIM” ! Composition of the sarcomere

Titin: binds Z line to M line, prevents “overstretching” of the sarcomere Tropomodulin: caps actin filament Nebulette: attaches actin filament to Z line MyBPC: attaches myosin to titin Z line: α-actinin, desmin, CapZ protein M line: myomesin, M line protein, creatine kinase Calcium-induced…

…Calcium-release A and I bands result from the interaction of actin and myosin The morphological aspect of the sarcomere depends on the contractile state Sarcomeres are connected to the plasma membrane 6. Cardiac conduction system

Rule #1: atria and ventricles cannot contract simultaneously Rule #2: the different parts of a cardiac cavity must contract simultaneously

The Sino-Atrial Node is the natural pace-maker of the heart

From this node, the current diffuses through both atria

The current cannot diffuse freely to the ventricles, because atria and ventricles are separated by fibrous tissue

The only point of electrical transmission is the atrio-ventricular node, which slows down the current to avoid simultaneous contraction of atria and ventricles

The AV node distributes the current to the His bundle, which separates in one right and two left branches

The bundles separate in multiple Purkinje fibers that distribute the current simultaneously to the different parts of the myocardium

Principle of conduction system

Natural pace-maker

Electric slow-down

Homogeneous distribution Current transmission is facilitated by gap junctions

Gap junctions result from the cell-cell interaction of transmembrane proteins forming the connexon. Connexin 43 is the major protein of the connexon 7. Questions

1. Describe the coronary circulation 2. What is the sarcomere? 3. Why is the blood supply more limited in the subendocardium? 4. What is the role of titin? 5. Which structure is the natural pace-maker of the heart? 6. What is the principle of resistance vessels? 7. What is the diastolic recoil of the aorta? 8. What is a connexon? 9. Describe the cardiac conduction system Molecular Medicine of the Heart

Class 2. Physiology of contraction and relaxation Overview of class 2: Physiology of contraction and relaxation

1. The 2. Determinants of cardiac function 3. Modulation of cardiac pressure 4. Modulation of cardiac volume 5. Neurohumoral regulation of cardiac function 6. Questions 1. The cardiac cycle

The seven phases of contraction and relaxation

a. Atrial contraction b. Isovolumic contraction c. Maximal ejection d. Reduced ejection/start of relaxation e. Isovolumic relaxation f. Rapid filling g. Slow filling

Description of the cardiac cycle – part 1

a. The atrium contracts to finish the left ventricular filling b. The left ventricle starts contracting, which rapidly closes the atrio- ventricular valve. The contraction is isovolumic because the aortic or pulmonary valve is still closed c. The pressure in the ventricle becomes superior to that in the aorta or the pulmonary artery, and therefore the corresponding valve opens. Pressure keeps rising in the ventricle while it ejects blood d. The ventricle starts to relax while it keeps ejecting. Its pressure gradually decreases but is still superior to that in the aorta or the pulmonary artery Description of the cardiac cycle – part 2

e. The pressure in the ventricle becomes inferior to that in the aorta or pulmonary artery, and the corresponding valve closes. Pressure inside the ventricle rapidly drops but remains higher than that in the atrium f. Pressure in the ventricle becomes smaller than that in the atrium, and the atrio-ventricular valve spontaneously opens. Because of the atrio- ventricular pressure gradient, the ventricle rapidly fills g. The atrio-ventricular gradient fades because pressure starts to rise in the filling ventricle, while it decreases in the emptying atrium a. The atrium contracts to finish the left ventricular filling Cardiac sounds

S1. The mitral valve (M1) and tricuspid valve (T1) close

S2. The aortic valve (A2) and the pulmonary valve (P2) close

S3. The ventricle rapidly fills (“rushing” blood, can be physiological when is increased)

S4. Late filling by the atrial contraction (when ventricular relaxation is decreased, therefore this sound is abnormal)

Note: the systole is between S1 and S2, the diastole is between S2 and S1 the M/T and A/P splits are due to the right-left difference in pressure 2. Determinants of cardiac function

Cardiac function is a story of pressures and volumes

The main function of the myocardium is to maintain a cardiac output

The cardiac output is the product of x

• The heart rate is determined by the firing rate of the sino-atrial node. It can be accelerated by catecholamines or slowed down by acetylcholine (neurohumoral regulation)

• The stroke volume is determined by changes in pressure and volume in the ventricular cavity A cardiac muscle is definitely different from a skeletal muscle

Skeletal Muscle Change length to develop tension

Cardiac Muscle Develop pressure to change volume Determinants of cardiac output

CO, cardiac output CO = HR x SV SV, stroke volume HR, heart rate

Heart rate: firing rate of the sino-atrial node pace-maker Can be adapted by changes in the firing frequency (chronotropy)

Stroke volume = end-diatolic volume – end-systolic volume Can be adapted by changes in pressures ( and ) and volumes (inotropy and lusitropy) Determinants of cardiac output- 1. Heart rate

1. It is obvious that an increased heart rate will increase the cardiac output for a constant stroke volume

2. In addition, an increase in heart rate induces an increase in force development due to an accumulation of cytosolic Ca2+ This property is called the Bowditch phenomenon (“treppe”) Determinants of cardiac output- 2. Stroke volume

The stroke volume can be adapted by changes in either cardiac pressure and/or volume

Pressure is mainly determined by

• the preload, or the pressure with which the heart is filled • the afterload, or the pressure against which the heart ejects These pressure parameters are independent of the myocardium

Volume is mainly determined by

• the inotropy, or contractile capacity of the myocardium • the lusitropy, or relaxation capacity of the myocardium These volume parameters are controlled by the myocardium Theoretical pressure/volume relation

A, Contraction B, Ejection C, Relaxation D, Filling PRESSURE

VOLUME Physiological pressure/volume relation (Starling’s curve)

A, Contraction B, Ejection C, Relaxation D, Filling Physiological pressure/volume relation (Starling’s curve)

Inotropy (contraction capacity)

Afterload (ejection pressure)

Stroke volume

A, Contraction B, Ejection Lusitropy Preload C, Relaxation (relaxation capacity) (filling pressure) D, Filling 3. Modulation of cardiac pressure

Preload and afterload modulate cardiac pressure

the preload represents the pressure with which the heart is filled the afterload represents the pressure against which the heart ejects

Preload and afterload are essentially independent of the , they are controlled by the vasculature. However, a poor contractile performance (such as heart failure) will modify the loading conditions as a consequence

A practical example: Exercise Increased preload results in increased developed pressure Increased preload stretches the sarcomeres, which increases contractile force A practical example: Hypertension The normal pressure / volume relation Increasing the preload will increase the stroke volume

SV2 > SV1 Stroke volume 2

Stroke volume 1

Increased preload Increasing the afterload will decrease the stroke volume

Increased afterload Stroke volume 2 SV2 < SV1

Stroke volume 1 4. Modulation of cardiac volume

Inotropy and lusitropy modulate cardiac volumes

Inotropy corresponds to the modulation of the cardiac ability to contract in order to eject more or less blood during systole (“contractibility”)

Lusitropy corresponds to modulation of the cardiac ability to relax in order to receive more or less blood during diastole (“relaxibility”)

Inotropy and lusitropy are mainly controlled by the force of actin-myosin interaction in the sarcomere. The amplitude of this contractile force is modulated by calcium. However, external factors (such as fibrosis after myocardial infarction) can modify the capacity of the heart to eject or relax Positive Inotropy Negative Inotropy Positive Lusitropy Negative Lusitropy A few examples

The Bowditch phenomenon is an example of increased inotropy

Arterial hypertension is an example of increased afterload

Post-infarction fibrosis induces negative lusitropy

Severe dehydration provokes a decreased preload

Increased venous return during exercise results in increased preload

Catecholamines induce positive lusitropy and inotropy

Vasodilation induces a decreased afterload

A massive pulmonary embolism can induce a sudden increase in afterload The volume/pressure relationship determines the cardiac work

External work: work needed to propel the blood into the vasculature

Most of the external work consists in Stroke Work, needed for ejection: Stroke Work = P x SV (P, pressure; SV, stroke volume)

Little of the external work consists in Kinetic Work, needed to speed the blood: Kinetic Work = 1/2 mv2 (m, mass of blood; v, speed of blood flow)

Internal work: work done inside the wall to contract the muscle

Catecholamine stimulation increases external work Heart failure increases internal work 5. Neurohumoral regulation of cardiac function

The “yin-yang” of norepinephrine and acetylcholine

Catecholamines. • Epinephrine (adrenaline), released by the adrenal medulla (systemic). Binds to β- adrenergic receptors (β1 in heart and β2 mainly in arterioles). Has a global cardio- vascular effect (“fight or flight”), resulting in increased cardiac output and decreased arterial resistance. • Norepinephrine (noradrenaline), released by the stellate ganglion (Paracrine). Binds to β-adrenergic receptors in heart, resulting in increased heart rate, inotropy and cardiac output. Can also bind to α-adrenergic receptors in arterioles, resulting in increased arterial resistance.

Acetylcholine, released by the vagal nerve. Binds to muscarinic receptors. Decreases heart rate (negative chronotropy), decreases AV conduction (negative dromotropy), decreases inotropy.

Adrenergic receptors

α1. Smooth muscle cell contraction- Stimulation of cell growth and proliferation

α2. Negative feed-back inhibition of the sympathetic drive in the CNS

β1. Positive inotropy, lusitropy and chronotropy Stimulation of cell growth and proliferation Interacts with Gsα

β2. Positive inotropy and chronotropy Smooth muscle cell relaxation- Interacts with Gsα and Giα

Acetylcholine controls the cardiac function at rest

Atropine inhibits the muscarinic receptor Propranolol inhibits the β- Respective roles of acetylcholine and catecholamines in cardiac function

CO = SV x HR

The CO at rest is about 5 L/min. The maximal CO during exercise is 25 L/min

• Normal individual at rest: 5 L/min = 70 ml/beat x 70 beats/min

Acetylcholine controls the heart rate at rest to maintain the appropriate CO

• Trained athlete at rest: 5 L/ min = 90 ml/min x 55 beats/min

The trained athlete has a bigger ventricular cavity and therefore has a bigger stroke volume. As a consequence, heart rate is slowed down by an increased amount of acetylcholine

• Trained athlete during maximal exercise: 25 L/ min = 125 ml/min x 200 beats/min

During exercise, the SV increases because the preload increases following recruitment of the venous capacitance because catecholamine release increases both ventricular lusitropy and inotropy In addition, catecholamine release increases heart rate 6. Questions

1. Describe cardiac inotropy and lusitropy 2. What are the different phases of the cardiac cycle? 3. What is the Bowditch phenomenon? 4. What is the global effect of epinephrine on cardiac function? 5. Define cardiac preload and cardiac afterload 6. What are the differences between epinephrine and norepinephrine? 7. What is the effect of beta-adrenergic stimulation on the pressure-volume curve? 8. Represent how heart failure affects internal and external cardiac work 9. Explain how increased preload increases contractile force

Molecular Medicine of the Heart

Class 3. Electrophysiology Overview of class 3: Electrophysiology

1. Cell polarization 2. The action potential 3. Ion channels, pumps and exchangers 4. Cardiac pace-maker 5. The electrocardiogram 6. Arrhythmias 7. Questions 1. Cell polarization

Each cell has a membrane potential

• The membrane potential is an electric potential resulting from a difference in charge distribution on both sides of the plasma membrane

• The cell is rich in K+, and poor in Na+, the opposite being true in the extracellular milieu. Through the Na+ / K+ pump, the cell extrudes Na+ and accumulates K+

• K+ tends to spontaneously leave the cell and does it relatively easily because its permeability (conductance) is high

• Na+ tends to spontaneously enter the cell and does it slowly because its permeability (conductance) is low

• Therefore, there are more positive charges outside than inside the cell, and the plasma membrane develops a membrane potential, which is negative inside the cell compared to outside

• In the case of a cardiac myocyte, this resting potential is –85 mV The resting cardiomyocyte is negatively charged

Na + (low conductance)

Na + Na+ / K+ pump K+

K+ (high conductance)

The membrane potential (Em) is calculated from the Nernst equation

Em = 61.5 ln (PK Ko/Ki + PNa Nao/Nai)

P, conductance; o, extracellular; i, intracellular K+, Na+ and Ca2+ balance of the cell at rest

K+ and Na+ determine the membrane potential, whereas Ca2+determines cell contraction

• At rest, the cell is rich in K+, and poor in Na+, the opposite being true in the extracellular milieu.

• The Na+ / K+ imbalance is maintained by the Na+ / K+ pump

• At rest, the plasma membrane is impermeable to Ca2+, which creates a 103-104 –fold gradient through the plasma membrane

• Intracellular Ca2+ is extruded through the Na+ / Ca2+ exchanger K+, Na+ and Ca2+ balance of the cell at rest

The Ca2+ gradient is by far the most impressive and is totally imperative for normal contraction. At rest, the cell must be devoid of free Ca2+ to relax properly. 2. The action potential

Specific currents control cell depolarization and repolarization

The action potential is the sequence of depolarization-repolarization that leads to cardiac cell contraction.

The action potential is controlled by the influx or efflux of specific ions during a specific period of time.

The action potential is transmitted from one cell to the next by a “domino effect”. Cardiac contraction is initiated by membrane depolarization

Cardiac contraction relies on an influx of Ca2+ but the plasma membrane is impermeable to Ca2+. To let Ca2+ coming in first requires the loss of the membrane potential, or membrane depolarization

Depolarization is initiated by an influx of Na+, rapidly followed by Ca2+ influx. Inflowing Ca2+ then triggers contraction by the release of endogenous Ca2+ (Ca2+ - induced Ca2+ release), which leads to cardiac contraction The five phases of the action potential

The action potential is the sequence of depolarization-repolarization that leads to cardiac cell contraction.

The action potential is controlled by the influx or efflux of specific ions during a specific period of time.

The action potential is divided in 5 phases.

+ • Phase 0. Influx of Na (INa). Induces membrane depolarization

+ + • Phase 1. Efflux of K (Ito). Limits the Na spike

2+ 2+ 2+ • Phase 2. Influx of Ca (ICa). Ca enters the cell to trigger the Ca - induced Ca2+ release

+ • Phase 3. Efflux of K (IK). Repolarization starts

• Phase 4. Restoration of the resting potential through the Na+ / K+ pump The five phases of the action potential

1 2 0

3

-85 mV 4 Ca2+ influx triggers cardiac contraction

Membrane depolarization is induced by an entry of Na+ followed by Ca2+. When Ca2+ flows in, contraction begins by the Ca2+–induced Ca2+ release. This explains why the polarized membrane must be impermeable to Ca2+ Ion currents during the action potential Variations in the action potential

• In the conduction system, the action potential shows a sharp Phase 1 and a short Phase 2, because speed of transmission matters

• In the cardiomyocytes, the action potential shows a small Phase 1 and a prolonged Phase 2, because Ca2+ influx matters Action potential in conduction system

The influx must be transmitted very quickly, therefore the whole action potential must be short. A prominent Ito current in phase 1 limits ICa and therefore shortens Phase 2. Action potential in contractile cells

2+ The influx must be sufficiently prolonged for allowing ICa to trigger the Ca -induced 2+ Ca release. A minor Ito current in phase 1 maintains ICa and therefore prolongs Phase 2. The action potential spreads by a “domino effect” The action potential spreads through connexons

By their distribution, the connexons organize a longitudinal, rather than transversal, distribution of the action potential 3. Ion channels, pumps and exchangers

K+, Na+ and Ca2+ are transported in and out of the cell through specific carriers

• Cardiac contraction is initiated by membrane depolarization, resulting from ion fluxes through the plasma membrane. • Because of the relative impermeability of the plasma membrane to ions, ion channels transport the ions to generate a current. The proper function of the channels requires two characteristics:

• Specificity each specific ion crosses through specific channels

• Gating Opening of the channel is transient, which determines the duration of the current, whereas the intensity of the current is determined by the amount of opened channels General structure of an ion channel

• Selectivity filter: determines the ion that crosses the channel • Activation gate: opens to start the current • Inactivation gate: closes to interrupt the current • Voltage sensor: determines at which potential the channel opens and closes

• Channel activity can be stimulated by phosphorylation, a mechanism particularly important for the Ca2+ channel

• Channel activity can be stimulated by ligands, a mechanism particularly important for the K+ channel Ion channels alternate between three states

REST ACTIVATION

INACTIVATION Current intensity is determined by the number of opened channels

Intensity depends on recruitment

duration depends on gating Ca2+ channel activity can be modulated by phosphorylation

Stimulation of the β-adrenergic receptors by catecholamines increases the inotropy by increasing the Ca2+ current through the L-type Ca2+ channel (L for long-acting), thereby increasing the Ca2+-induced Ca2+ release K+ channel activity can be modulated by ligand binding

Stimulation of the muscarinic receptor by acetylcholine decreases the chronotropy by increasing the K+ current, thereby hyperpolarizing the membrane 4. Cardiac pace-maker

The SA node is the natural pace-maker of the heart

• The initiation of the action potential lies in the automatic pace-maker activity of the SA node, in which there is spontaneous depolarization. The whole function of the SA node relies on this automaticity, which is translated into a repetitive and spontaneous firing of action potentials.

• The automaticity relies on specific cells in the SA node, the Pacemaker cells or P cells Currents in the SA node

The shape of the action potential in the SA node is totally different from the shape described in conduction system and in myocytes, because the ion channels expressed in the P cells are different from other cardiac cells.

2+ 2+ • ICa is an inward Ca current depolarizing the P cell through a T-type Ca channel (T for transient). Catecholamines through the β-adrenergic receptor accelerate the depolarization of ICa and therefore accelerate heart rate (positive chronotropy).

• IK is a rectifier potassium current that repolarizes the cells after the ICa.

Acetylcholine through the muscarinic receptor increases the repolarization of IK and therefore decreases heart rate (negative chronotropy).

• If (“funny current”) is an inward sodium current specific of the SA node that destabilizes the resting potential and therefore underlies the automaticity of the P cells. Currents in the SA node

The P cells do not really have a resting potential. The lowest potential is –65 mV, compared to –85 mV in cardiac myocytes. ICa depolarizes the cell, which is repolarized by IK. If prevents the stability of the resting potential. Currents in the SA node

If destabilizes the “resting” potential

ICa depolarizes the P cell but is far more transient (T type) than in myocytes (L type)

IK repolarizes the P cell Myocytes P cells

Myocytes have a L-type (for long-acting) Ca2+ channel that remains open long enough to trigger the Ca2+-induced Ca2+ release. The P cells have a T-type (for transient) Ca2+ channel that remains open the short period of time needed to depolarize the cell. Ca2+ flux determines the duration of depolarization Neurohumoral regulation of the SA node

Negative chronotropy. Acetylcholine increases

IK by G protein-mediated opening of the K+ channel

Positive chronotropy. Catecholamines increase

ICa by G protein-mediated phosphorylation of the Ca2+ channel

The same mechanisms explain corresponding changes in dromotropy (or speed of influx conduction) 5. The electrocardiogram

The EKG is the external record of cardiac electrical activity

The cardiac electrical impulse is generated in the SA node, rapidly conducted through the atria to the AV node, where it undergoes filtration and delay. Then follows another phase of rapid conduction through the His bundle and its branches, finally leading to excitation-contraction coupling in the ventricular myocyte. The whole sequence is monitored externally by the electrocardiogram. Principle of EKG recording

Outside electrodes “see” the current and record it as a wave If the current is going to the electrode, the wave will be positive If the current is going away from the electrode, the wave will be negative The size of the wave is proportional to the intensity of the current General profile of the EKG

P wave. Atrial depolarization QRS complex. Depolarization of both ventricles . Ventricular repolarization

PR interval. Slow-down in the AV node ST interval. Isoelectric phase 2 of the action potential Generation of the EKG waves

Initiation of ventricular depolarization AV node slow-down

Full ventricular Atrial depolarization depolarization

Ventricular repolarization

Electrode The shape of the waves depends on the localization of the electrodes

The intensity of current is proportional to the ventricular mass V1 to V3 “see” mainly the right ventricle, the complex is globally negative V4 to V6 “see” mainly the left ventricle, the complex is globally positive Therefore, the shape in V1 to V6 will vary in case of right or left ventricular hypertrophy 6. Arrhythmias

Arrhythmias are abnormalities in the initiation or conduction of the electric current

Sinus tachycardia. SA node firing at more than 100 per minutes.

Sinus bradycardia. SA node firing at less than 60 per minutes.

SA block. Absence of firing from the SA node. A whole depolarization is missing.

Atrial fibrillation. High-frequency firing from the atrium, which prevents normal atrial contraction and therefore impairs ventricular filling.

AV block. Conduction defect in the AV node with downstream relay to conduct the ventricular impulse. The atrium remains under control of the SA node. Arrhythmias are not necessarily “abnormal”

Examples of “normal” arrhytmias:

• The Hering-Breuer reflex. The cardiac rhythm is irregular during the respiratory cycle.

• Exercise-induced tachycardia. Catecholaminergic stimulation can increase the heart rate to the maximum rate: 220-age.

• Vagal bradycardia. Cholinergic stimulation in the athlete’s heart can decrease the heart rate under 60. The Hering-Breuer reflex: a “normal” arrhythmia

During inspiration, the venous return is impaired by the inflation of the lungs, which decreases the ventricular stroke volume (SV). To maintain the cardiac output (CO), the heart rate (HR) must increase, because CO = SV x HR. Therefore, lung inflation creates a reflex that decreases acetylcholine discharge from the vagal nerve, thereby inducing a relative increase in adrenergic activity and a resulting increase in heart rate. Sinus tachycardia

Sinus tachycardia. SA firing at more than 100 per minutes (positive chronotropy)

Causes: exercise, stress, most forms of heart disease (especially heart failure) Sinus bradycardia

Sinus bradycardia. SA firing at less than 60 per minutes (negative chronotropy)

Causes: athlete’s heart, sleep, vagal stimulation (cause of syncope) The SA block

SA block. Absence of firing from the SA node. A whole depolarization is missing. Causes: fibrosis, aging. Can be temporary (one beat missing) or complete (no firing at all), which is followed by a relay from the AV node. The atrial fibrillation

Atrial fibrillation. High-frequency firing from the atrium, which prevents normal atrial contraction and therefore impairs ventricular filling. Ventricular beat is irregular. Causes: atrial dilatation in many forms of heart disease, aging. Accumulation of blood in the non-contracting atrium is a cause of thrombosis (coagulation) and embolism (dissemination of the blood clot in the circulation) The AV block

AV block. Conduction defect in the AV node with downstream relay to conduct the ventricular impulse. The atrium remains under control of the SA node. Causes: congenital, ischemia. If the downstream relay is too slow, it is accompanied by episodes of syncope that require the installation of an external pace-maker.

QRS is at regular interval but slower than P 7. Questions

1. Why are cells polarized? 2. Describe the general structure of an ion channel 3. What is the function of the sodium-potassium pump?

4. What is the role of ICa in the duration of the action potential? 5. What is the mechanism of spontaneous depolarization in the SA node? 6. What is the role of potassium currents in the action potential? 7. What is a QRS complex on the EKG? 8. Define the Hering-Breuer reflex 9. What are the causes of sinus tachycardia? 10. What is an atrial fibrillation?

Molecular Medicine of the Heart

Class 4. Calcium and contraction Overview of class 4: Calcium and contraction

1. The principle of Ca2+ -induced Ca2+ release 2. Ca2+ sequestration and release 3. Structure of contractile proteins 4. Actin-myosin interaction 5. Integrated view of Ca2+ metabolism 6. Questions 1. The principle of Ca2+ -induced Ca2+ release

Extracellular Ca2+ releases intracellular Ca2+

• The action potential is propagated by a depolarization of the plasma membrane, 2+ 2+ which opens the Ca channel and creates a inward Ca current (ICa)

• This Ca2+ channel (also called L-type channel or DHP receptor, which is different from the T-type channel of the P cells and conduction system) is particularly abundant on the T tubule, an intracellular extension of the plasma membrane. The intracellular part of the T tubule is wrapped by the extremity (cisterna) of the sarcoplasmic reticulum

• The cisterna contains a Ca2+ binding site that binds the Ca2+ ions crossing the Ca2+ L-type channel in the T tubules

• Stimulation of the Ca2+ binding site triggers the release of Ca2+ from the sarcoplasmic reticulum through a specific channel, called the ryanodine receptor, which initiates cardiac contraction

• Cardiac relaxation is initiated by a reuptake of Ca2+ in the sarcoplasmic reticulum by SERCA Mechanisms of Ca2+ influx in the cardiac cytosol

Ca2+ is released in the cytosol through two channels:

• The DHP (dihydropyridine) receptor or L-type Ca2+ channel. Lets Ca2+ from the extracellular milieu enter the cell when the plasma membrane is depolarized by the action potential. The inward current (ICa) through this channel is driven by the 103-fold gradient of [Ca2+] through the plasma membrane

• The ryanodine receptor. Releases Ca2+ from the sarcoplasmic reticulum into the cytosol upon stimulation by extracellular Ca2+. Exit of Ca2+ through this channel is driven by the 103-fold gradient of [Ca2+] through the membrane of the sarcoplasmic reticulum

• These two channels are called “receptors” because they bind specific drugs. For instance, DHPs are used in clinical practice to limit the activity of the L-type Ca2+ channel and thereby decrease cardiac contractility (e.g., for patients with ischemic heart disease). Blocking the ryanodine receptor has no clinical utility because it would totally block contraction. Mechanisms of Ca2+ influx in the cardiac cytosol

Ca2+ crosses the plasma membrane through the DHP receptor (L-type channel) Ca2+ crosses the sarcoplasmic reticulum membrane through the Ryanodine receptor

DHP receptor (L-type Ca2+ channel)

Action potential Plasma membrane

Cisterna

SR Ryanodine receptor

DHP = Dihydropyridine (e.g., nifedipine) Ca2+ fluxes follow huge gradients

Upon Ca2+ release from the sarcoplasmic reticulum, cytosolic [Ca2+] increases 100-fold Sensitivity of the contractile machine to Ca2+

Upon Ca2+ release from the sarcoplasmic reticulum, cytosolic [Ca2+] increases 100-fold. This increase from 0.1 μM to 10 μM is sufficient to maximally stimulate contraction. Therefore, cardiac relaxation requires a tight control of cytosolic [Ca2+] The Ca2+ -induced Ca2+ release is coupled by the T tubules

T tubules are tube-like invaginations of the plasma membrane inside the cardiac myocytes, where the DHP receptors (L-type Ca2+ channels) are highly expressed. Inside the cell, the T tubule is wrapped by the extremity of the sarcoplasmic reticulum (or cisterna), which expresses the ryanodine receptor

Three advantages of the T tubule (coupling, coupling, coupling)

• Excitation-contraction coupling. The T tubules increase the surface of the plasma membrane by 30%, which increases the density of DHP receptors and therefore the speed of excitation-contraction coupling

• Receptor coupling. The tight coupling between DHP receptors on the T tubule and ryanodine receptors on the sarcoplasmic reticulum optimizes the coupling between Ca2+ entry (through the DHP receptor) and Ca2+ release (through the ryanodine receptor)

• SR coupling. The deep invaginations of the T tubule inside the cell allows a synchronized release of Ca2+ from the entire sarcoplasmic reticulum compartment The Ca2+ -induced Ca2+ release is coupled by the T tubules

Calcium-induced…

…Calcium-release Mechanisms of Ca2+ reuptake

Ca2+ is extruded from the cytosol through pumps and exchangers:

• The sarcoplasmic reticulum Ca2+ ATPase (SERCA). Pumps Ca2+ back inside the sarcoplasmic reticulum, which represents about 90% of the reuptake mechanisms. This pump requires ATP to overcome the Ca2+ gradient

• The sarcolemmal Na+/Ca2+ exchanger. Pumps Ca2+ out of the cell through an exchange with Na+, which represents about 5% of the reuptake mechanisms. The exchanger does not use ATP because Na+ follows its spontaneous electrochemical gradient, which provides the energy, but thereafter Na+ has to be pumped back outside the cell by the Na+/K+-ATPase, which requires ATP

• The mitochondrial Na+/Ca2+ exchanger. Pumps Ca2+ in the mitochondria through an exchange with Na+, which represents about 4% of the reuptake mechanisms

• The sarcolemmal Ca2+ ATPase. Pumps Ca2+ back in the extracellular milieu, which represents about 1% of the reuptake mechanisms. This pump requires ATP to overcome the Ca2+ gradient Mechanisms of Ca2+ reuptake

5% 1%

90% 4% 2. Ca2+ sequestration and release

Intracellular Ca2+ is sequestered in the sarcoplasmic reticulum

• 90% of the Ca2+ needed for contraction is stored in the sarcoplasmic reticulum.

There are four molecules that control the fate of Ca2+ in this compartment.

• Upon stimulation, Ca2+ is released through the ryanodine receptor

• Upon relaxation, Ca2+ is pumped back into the sarcoplasmic reticulum by SERCA

• SERCA activity is modulated by

• In the sarcoplasmic reticulum, Ca2+ is bound to calsequestrin Structure of the ryanodine receptor

The Ca2+ release channel from the sarcoplasmic reticulum is a large protein made of two subunits • The “foot” links the sarcoplasmic membrane to the T tubule • The transmembrane subunit is the Ca2+ channel in itself

This channel is specifically inhibited by ryanodine

This channel is activated by caffeine, which empties the sarcoplasmic reticulum from Ca2+ and therefore increases contraction strength

Opening of the channel results from an alignment of the two subunits Structure of the ryanodine receptor

caffeine ryanodine Structure of SERCA

The Ca2+ reuptake in the sarcoplasmic reticulum is performed by the sarcoplasmic reticulum Ca2+ ATPase SERCA.

Therefore, cardiac relaxation is an active process that requires ATP and SERCA function is impaired during ischemia or heart failure

SERCA represents 40% of the SR protein component.

Two Ca2+ ions are accumulated for each ATP consumed

SERCA exists in two main isoforms: • SERCA 1 in fast-twitch skeletal muscles • SERCA 2a in heart and slow-twitch skeletal muscles • SERCA 2b in smooth muscle cells Ca2+ is pumped in the sarcoplasmic reticulum by SERCA

SERCA represents 40% of the protein content of the sarcoplasmic reticulum Role of phospholamban

The Ca2+ reuptake in the sarcoplasmic reticulum by SERCA is modulated by phospholamban (“the phosphate carrier”).

Phospholamban is a pentamer made of five identical subunits, which colocalizes in a 1:1 ratio with SERCA.

Phospholamban is an inhibitor of SERCA. Inhibition is relieved by phosphorylation of the pentamer, which thereby activates SERCA activity to fasten relaxation. This is one of the molecular mechanisms of positive lusitropy.

Phospholamban is phosphorylated by: • upon β-adrenergic stimulation (explains the lusitropic effect of catecholamines) • Calcium-calmodulin protein kinase, activated when cytosolic [Ca2+ ] is increased Role of phospholamban

Phosphorylation of phospholamban increases the opening probability of SERCA Regulation of phospholamban

Phospholamban is phosphorylated on: • Serine 16 by Protein kinase A • Threonine 17 by Calcium-calmodulin protein kinase

Both phosphorylations have cumulative effect and are most often combined in vivo Role of calsequestrin

Calsequestrin is a highly-charged storage protein of 50 KDa, mainly expressed in the cisterna of the sarcoplasmic reticulum

Calsequestrin maintains Ca2+ in a compact “releasable” pool, close from the ryanodine receptor

Its increased expression increases the pool of releasable Ca2+

Ca2+ storage is also performed by calreticulin or sarcalumenin 3. Structure of contractile proteins

Cardiac contraction relies on contractile proteins and regulatory proteins

Cardiac contraction is performed by contractile proteins

• Actin filaments slide to shorten the sarcomere • Myosin is the molecular motor moving actin

Cardiac contraction is controled by regulatory proteins

• Tropomyosin prevents actin-myosin interaction in the relaxation phase

• Troponins regulate actin-myosin interaction in the contraction phase • troponin C binds Ca2+ to activate contraction • troponin T moves tropomyosin to release its inhibitory effect • troponin I regulates Ca2+ release to initiate relaxation

The organization of these contractile proteins is maintained in the structure of the sarcomere Composition of the sarcomere

Titin: binds Z line to M line, prevents “overstretching” of the sarcomere Tropomodulin: caps actin filament Nebulette: attaches actin filament to Z line MyBPC: attaches myosin to titin Z line: α-actinin, desmin, CapZ protein M line: myomesin, M line protein, creatine kinase Structure of myosin

Myosin molecules (MW = 500 KDa) are regrouped in filaments, each composed of 300 individual molecules, which represents the thick filament of the sarcomere

Each myosin molecule is made of two heavy chains and four light chains

• The heavy chains are embedded in the body of the filament at one end and form the heads that emerge from the filament at the other end in a spiral fashion. The head contains the myosin ATPase responsible for actin-myosin dissociation. Trypsin digestion separates the heavy chains in light and heavy meromyosin.

• The light chains are of two different types. MLC-1 (myosin light chain-1) or ELC (essential light chain) participates in the interaction with actin and is indispensable for the protein function (hence, “essential”). MLC-2 (myosin light chain-2) or RLC (regulatory light chain) is a regulatory subunit that controls the intensity of contraction (e.g., positive inotropy by catecholamines). There are two of each MLC-1 and MLC-2 per myosin molecule Structure of myosin molecule

HEAD TAIL Structure of myosin filament

Myosin heads spiral from the filament

Two essential motifs in the myosin head • The actin-binding cleft • The ATP-binding pocket Structure of actin

Monomeric actin, or actin G, is a globular protein of MW = 42 KDa

Monomeric actin polymerizes into a filament, or actin F, which represents the thin filament of the sarcomere, attached to the Z line

In the sarcomere, two actin filaments are twisted around each other like two strings of beads that follow the spiral of myosin heads

The extremity of the actin filament is capped by tropomodulin, which prevents excessive growth of actin F Structure of tropomyosin

Tropomyosin is a twisting backbone supporting the actin filaments, lying in the groove between the two strands of actin and thereby offering rigidity to the thin filament

Tropomyosin is also made of two peptide chains (MW = 35 KDa) bound in a coiled-coil structure

In the sarcomere, tropomyosin inhibits the actin-myosin interaction. Through cooperative interaction with the troponins, this inhibition is relieved when [Ca2+] increases, which allows Ca2+ to activate muscle contraction Structure of troponins

The troponin complex is made of three subunits

• Troponin C (C=Calcium) is is dumbbell-shaped protein of the EF-hand family (Ca2+- binding proteins), which allows TnC to recognize a rise in [Ca2+] as a signal to activate muscle contraction

• Troponin I (I=Inhibitory) is an inhibitor of actin-myosin interaction that induces cooperative interactions in the thin filament to reduce Ca2+ affinity for TnC, and thereby initiates relaxation. Such effect is enhanced when TnI is phosphorylated by protein kinase A upon catecholamine stimulation (another molecular mechanism of positive lusitropy, together with phosphorylation of phospholamban)

• Troponin T (T=Tropomyosin) glues the whole troponin complex together and links it to tropomyosin. Upon TnC stimulation, TnT moves the tropomyosin filament to allow actin-myosin interaction Structure of troponins 4. Actin-myosin interaction

The crossbridge cycle

The crossbridge represents the attachment of the myosin heads to a binding site on the actin filament

The crossbridge cycle consists of the repetitive attachment and detachment of myosin heads to and from actin F

The crossbridge is initiated by the binding to TnC of the Ca2+ released from the sarcoplasmic reticulum and is followed by four steps:

• The myosin head attaches to actin • The power stroke bends the myosin head and actin slides • ATP binds to the myosin head, which is thereby released from actin • ATP is hydrolyzed, which energizes the myosin head to bind actin The crossbridge cycle is dictated by nucleotide binding

Interaction of actin and myosin is dictated by the occupancy of the ATP-binding pocket in the myosin head

• When the ATP-binding pocket is occupied by ATP, the actin-myosin affinity is low, does not produce force and oscillates between attached and detached states. Therefore, the muscle is relaxed and can be stretched

• When the ATP-binding pocket is occupied by ADP, the myosin head strongly binds to actin (crossbridge), which sets the muscle under tension

• When the ATP-binding pocket is empty, the myosin head bends over the tail (power stroke), which slides the actin filament and creates contraction

132

1. ATP = Relaxation 2. ADP = Tension 3. Empty = Contraction Molecular basis of the crossbridge cycle

• In diastole, ATP binds to myosin, which cannot interact with actin because tropomyosin is in the way and because ATP induces a weak actin-myosin binding. ATP is rapidly hydrolyzed into ADP + Pi by the myosin ATPase, which extends the myosin head.

• Upon Ca2+ stimulation, the troponin complex moves tropomyosin. The myosin head is still extended. The ADP resulting from ATP hydrolysis creates a strong actin-myosin binding, and Pi is released.

• De-energized myosin bends its head to come back to a rest position and thereby creates the power stroke that slides the actin filament (rigor state, or maximal sliding). The change in conformation extrudes ADP.

• ATP binds to the myosin head, restores a weak actin-myosin bond and re- extends the myosin head. ATP is rapidly hydrolyzed by the myosin ATPase. A new cycle can be repeated if TnC still binds Ca2+. If Ca2+ reuptake by SERCA has begun, tropomyosin comes back to original position and the actin-myosin complex remains in a relaxed state Molecular basis of the crossbridge cycle

ATP releases myosin ATP hydrolysis relaxes head from actin myosin head

ADP extrusion leads to rigor state

ADP initiates ADP binds myosin power stroke head to actin Molecular basis of the crossbridge cycle

Tropomyosin masks actin ATP detaches myosin head ATP is hydrolyzed by ATPase but stays as ADP + Pi

TnC removes tropomyosin Pi is extruded myosin head attaches to actin myosin head can restart a cycle or go back to rest position if Ca2+ is pumped back by SERCA ADP–bound myosin head makes power stroke to return to resting position ATP extends myosin head, which faces a new actin

ADP is extruded from myosin head ATP detaches myosin and extends myosin head myosin head detaches from actin Optimization of the actin-myosin interaction by the sarcomeric proteins

Titin: prevents sarcomere overstretching Nebulette: attaches actin filaments to the Z line Tropomodulin: Prevents actin filament overgrowth MyBP-C: attaches myosin filaments to titin 5. Integrated view of Ca2+ metabolism

Ca2+ is both the trigger and the effector of contraction

Normal Ca2+ movements include the following steps: influx through L-type channel, release through ryanodine receptor, binding to TnC, release by TnI, pumping by SERCA and sequestration by calsequestrin

Ca2+ metabolism is largely controlled by ATP, which maintains cardiac cell relaxation

Modification of Ca2+ metabolism by catecholamines explains their positive inotropic, lusitropic and effects Structures controling Ca2+ metabolism Structures controling Ca2+ metabolism Importance of ATP in excitation-contraction coupling

In the excitation-contraction coupling, ATP is used by three main ATPases:

• The Na+ / K + pump that maintains the membrane potential. Lack of ATP results in membrane depolarization and increased risk of arrhythmias

• SERCA that maintains 90% of Ca2+ stores in the sarcoplasmic reticulum. Lack of ATP results in cytosolic Ca2+ accumulation and activation of the myofilament.

• The myosin ATPase that detaches the myosin head from the actin filament. Lack of ATP results in irreversible actin-myosin interaction and hypercontracture (rigor) of the sarcomere.

Therefore, ATP is responsible for cardiac cell relaxation rather than contraction, and a lack in ATP will result in cardiac contracture. Importance of ATP in excitation-contraction coupling

ATP: membrane potential

ATP: Ca2+ sequestration

ATP: filament relaxation Effects of catecholamines on Ca2+ metabolism

• Positive chronotropy:

2+ • activates ICa by phosphorylating the T-type Ca channel in the SA node, which increases the firing rate

• Positive inotropy:

2+ • activates ICa by phosphorylating the L-type Ca channel in the T tubules, which increases the release of Ca2+ from the ryanodine receptor • activates MLC-2 by phosphorylation, which stimulates the myosin power stroke

• Positive lusitropy: • activates actin-myosin dissociation by phosphorylation of TnI • activates Ca2+ reuptake by phosphorylation of phospholamban Effect of catecholamines on Ca2+ metabolism

A+C+E: inotropy ; F+D: lusitropy Increased inotropy accelerates the speed of contraction and thereby reduces the time of isovolumic contraction. The ventricle can empty faster because the aortic valve opens earlier. The pressure increases faster in the ventricle during systole, which, in physiological terms, represents an increase in +dP/dt (amplitude of pressure increase per unit of time). Increased inotropy increases the extent of contraction by making more Ca2+ available for TnC. The ventricle can eject more (increased stroke volume) because the extent of myofilament sliding during the crossbridge cycling is increased Increased lusitropy accelerates the speed of relaxation and thereby reduces the time of isovolumic relaxation. The ventricle can fill faster because the AV valve opens earlier. The pressure drops faster in the ventricle during diastole, which, in physiological terms, represents an increase in –dP/dt (amplitude of pressure drop per unit of time) Increased lusitropy increases the extent of relaxation by pulling more Ca2+ out of the cytosol. The ventricle can fill more because it is more relaxed and therefore its diastolic pressure is lower. The increased diastolic volume will result in increased stroke volume CO = HR x SV

By combining positive inotropy and increased lusitropy, catecholamines markedly increase the stroke volume. By adding a positive chronotropic effect (increased heart rate), catecholamines markedly increase cardiac output. 6. Questions

1. Describe the molecular mechanism of Ca2+ -induced Ca2+ release 2. What is a T tubule and what are its three advantages? 3. Describe the four molecules controlling Ca2+ uptake and release from the SR 4. What is the role and regulation of phospholamban? 5. Define DHP receptor and ryanodine receptor 6. Describe the structure of myosin 7. What is the role of ATP in excitation-contraction coupling? 8. What is the function of the different troponin subunits? 9. Describe the mechanisms by which catecholamines affect Ca2+ metabolism 10. What is the molecular basis of the crossbridge cycle? Molecular Medicine of the Heart

Class 5. Metabolism Overview of class 5: Metabolism

1. Determinants of cardiac metabolism 2. Glycolysis 3. Glycogen metabolism 4. Fatty acid metabolism 5. ATP production 6. Anaerobic metabolism 7. Questions 1. Determinants of cardiac metabolism

Cardiac metabolism is determined by workload, substrates and oxygen

WORKLOAD • Due to its constant contractile activity, the heart requires a considerable amount of ATP to feed the ATPase activities of the myosin head, SERCA and the Na+ / K + pump

SUBSTRATES • To sustain its energy supply, the heart is a”metabolic omnivore”, using every substrate that provides ATP. Substrates interact with each other to coordinate the metabolic activity

• The two main substrates for the heart are fatty acids in fasting conditions and glucose in fed condition. The heart also uses lactate during exercise

OXYGEN • A sufficient supply of ATP can be achieved only through oxidative phosphorylation, which requires an aerobic metabolism

• In conditions of oxygen deprivation (ischemia), the heart must shift toward an anaerobic metabolism, which provides far less ATP per mole of substrate Lactate Glucose Free fatty acids

GLUT

G-6-P Glycogen FFA Triglycerides Cytosol

Pyruvate Acyl-CoA

β-oxidation CPT Pyruvate Acyl-CoA

CO 2 O 2 Mitochondria Acetyl-CoA NADH + H + ADP FADH 2 + Pi Citrate Cytochromes CO 2 NAD + CO FAD ATP TCA cycle 2 H O 2 25% ++ ADP Ca ++ ATP Ca Ca++ + Pi Ca++ ATP

Cytosol SERCA ADP ATP 50% ADP ADP ATP 15% Na+/K+-ATPase

K+ Na+

%: percentage of total ATP utilization Glucose metabolism

• Glucose concentration is high in the circulating blood particularly in the fed state and glucose utilization is stimulated by insulin

• Glucose is carried into the cardiomyocyte through specific glucose transporters (GLUT). The cardiac myocyte expresses the ubiquitous GLUT1 and the insulin- regulated GLUT4

• Inside the cardiac cell, glucose is immediately phosphorylated to prevent any exit

• Phosphorylated glucose can be stored in the form of glycogen or degraded through the glycolytic pathway that ends up with pyruvate. A minor component enters the pentose-phosphate pathway

• Pyruvate is taken in the mitochondria, where it is oxidized through the tricarboxylic acid (Krebs) cycle Fatty acid metabolism

• Fatty acid concentration is high in the circulating blood particularly in the fasted state

• Protein-bound fatty acids are taken by lipoprotein lipase and cross the plasma membrane

• Inside the cardiac cell, fatty acids can be stored in the form of triglycerides

• Most of fatty acids are transferred to the mitochondria upon binding to the specific carrier carnitine by carnitine palmitoyltransferase (CPT)

• Inside the mitochondria, fatty acids are oxidized by the β-oxidation chain that produces acetyl-CoA to feed the tricarboxylic acid (Krebs) cycle

• When fatty acids are oxidized, glucose oxidation is inhibited and glucose taken up by the cardiomyocyte is converted into glycogen Lactate metabolism

• During acute exercise, blood lactate concentration increases

• Lactate diffuses through a monocarboxylate carrier in the plasma membrane of the cardiac myocyte

• Inside the cardiac cell, lactate is reduced into pyruvate by lactate dehydrogenase. The NADH produced is transferred into the mitochondria by the malate-aspartate shuttle

• Pyruvate is taken in the mitochondria, where it is oxidized through the tricarboxylic acid (Krebs) cycle

• Lactate utilization inhibits the utilization of both glucose and fatty acids Substrate interactions

FASTED STATE FED STATE

The heart permanently adapts its substrate utilization according to the plasma concentration of substrates. It is not an “all-or-nothing” phenomenon, the substrates constantly interact 2. Glycolysis

Glycolysis is the only source of anaerobic ATP

The regulatory steps of glycolysis are:

• Glucose uptake through specific transporters (GLUT) • Glucose phosphorylation through hexokinase (HK) • Hexose phosphate phosphorylation through phosphofructokinase (PFK-1) • Triose phosphate oxidation through glyceraldehyde-3-phosphate dehydrogenase (GAPDH) • Pyruvate oxidation through pyruvate dehydrogenase (PDH) • Glycolysis can be fed from glycogen, which is regulated by glycogen phosphorylase Overview of glycolysis Regulatory steps of glycolysis

Glycogen Glucose e GLUT

PH OS Glucose i HK G-6-P

PFK-1

Fru-1,6-P2

GAPDH

Lactate Pyruvate

PD H

Acetyl-CoA Cardiac glucose transporters

The uptake of glucose from plasma through the sarcolemma is controlled by the glucose transporters GLUT1 and GLUT4. This transport does not require energy because it follows the concentration gradient of glucose through the plasma membrane

• GLUT1 is an ubiquitous transporter expressed in every tissue. Its expression on the cardiac sarcolemma is stimulated by exercise, β-adrenergic stimulation and oxygen deprivation

• GLUT4 is the insulin-sensitive transporter expressed only in heart, skeletal muscle and adipose tissue. It is mainly expressed intracellularly in the microsomial fraction. Upon insulin stimulation in the fed state, GLUT4 translocates to the plasma membrane and becomes active until glycemia returns to normal values. GLUT4 also translocates upon β-adrenergic stimulation Hexokinase

Hexokinase irreversibly transforms glucose into glucose 6-phosphate and thereby “traps” glucose inside the cell because the sarcolemma is impermeable to phosphate. This phosphorylation costs one ATP per molecule of glucose. Hexokinase can be inhibited by the reaction product, glucose 6-phosphate, which matches offer and demand

Glucose

- ATP ADP

Glucose-6-P

Glycogen Glycolysis Phosphofructo-1-kinase

PFK-1 catalyzes the transformation of fructose 6-phosphate into fructose 1,6- bisphosphate, which marks the first irreversible step of glycolysis in itself. PFK-1 is the main regulatory step of the glycolytic pathway and the perfect sensor of glucose needs, as a function of oxygen availability, workload and substrate availability. • It is regulated by the energetic status: inhibited by ATP and activated by AMP • It is activated by fructose 2,6-bisphosphate upon β-adrenergic stimulation or insulin stimulation • It is inhibited by citrate when fatty acids or lactate are oxidized

Oxygen Alternative substrates Workload Fructose-6-P

+- AMP ATP Fru-2,6-P2 Citrate

Fructose-1,6-P PDH is an interconvertible enzyme controlled by energy demand

PDH catalyzes the conversion of pyruvate into acetyl-CoA. Its activity is under tight control by effectors and phosphorylation/dephosphorylation, the active form being dephosphorylated. • PDH kinase is activated by acetyl-CoA upon fatty acid oxidation, whereas it is inhibited by pyruvate. • PDH phosphatase is activated by Ca2+, which increases PDH activity upon β- adrenergic stimulation or during cardiac hypertrophy.

inactive ADP H 2 O ++ acetyl-CoA + PDH 2+ PDH PDH Ca phosphatase kinase pyruvate Pi active PDH ATP Pyruvate is oxidized in acetyl-CoA to enter the Krebs cycle

Glycolysis

NAD+

NADH + H+

Pyruvate NAD+

NADH + H+

acetyl-CoA

oxaloacetate citrate

malate CO2 + 2-oxoglutarate NADH + H CO 2 3. Glycogen metabolism

Glycogen is the cardiac storage form of glucose

Glycogen is the endogenous form of glucose storage in animal cells. In the heart, • glycogen synthesis is stimulated by alternative substrates (fatty acids, lactate) through an inhibition of glycolytic flux, and by insulin through an overall stimulation of glucose metabolism • glycogen breakdown is stimulated by catecholamines, which is accompanied by an overall stimulation of glucose metabolism

The main roles of glycogen are • an emergency fuel for ATP production in conditions of severe oxygen deprivation (ischemia) • a rapidly mobilized source of energy upon adrenergic stimulation

Glycogen metabolism is controlled by two enzymes: • glycogen synthase is the regulator of glycogen synthesis • glycogen phosphorylase is the regulator of glycogen breakdown Glycogen turn-over

Insulin Catecholamines Alternative fuels Increased workload

+ +

Glycogen Glycogen Synthase phosphorylase Coordinated regulation of glycogen turn-over

Catecholamines (increased glucose utilization) and insulin (increased glucose storage) reciprocally regulate both synthase and phosphatase

catecholamines catecholamines H O inactive ADP 2 + + Synthase Synthase PKA PI - + phosphatase GSK-3 - Pi active - insulin Synthase ATP insulin catecholamines ADP catecholamines H 2 O active + Phosphorylase + Phosphorylase Phosphorylase PI - + PKA phosphatase kinase - insulin Pi inactive ATP Phosphorylase

PI, phosphatase inhibitor PKA, cAMP-dependent protein kinase GSK-3, glycogen synthase kinase-3 Effects of fatty acid on glucose metabolism

Glycogen Glycogen synthesis Glucose e GLUT - 50% inhibition

SYNTH Glucose i Glucose HK uptake H-6-P

PFK-1 - 70% inhibition Glycolytic flux Fru-1,6-P2

GAPDH

Pyruvate Glucose oxidation PD H - 90% inhibition

Fatty acids Acetyl-CoA

Citrate Effects of insulin on glucose metabolism

Synthase Translocation Glycogen phosphatase Glucose + e + GLUT

SYNTH Glycogen Glucose i Glucose synthesis HK uptake H-6-P

Fru 2,6-P2 + PFK-1 Glycolytic Fru-1,6-P2 flux

GAPDH

Pyruvate

Pyruvate + PD H Glucose Acetyl-CoA oxidation

Citrate Effects of catecholamines on glucose metabolism

Phosphorylase Translocation kinase Glycogen Glucose e + + GLUT

PH OS Glycogen Glucose i Glucose degradation HK uptake H-6-P

Fru 2,6-P2 + PFK-1 Glycolytic Fru-1,6-P2 flux

GAPDH

Pyruvate

Ca2+ + PD H Glucose Acetyl-CoA oxidation

Citrate 4. Fatty acid metabolism

Fatty acids are the main source of energy for the heart

• Most abundant fatty acids are oleic acid and palmitic acid

• Insoluble, albumin-bound (from adipose tissue) or lipoprotein-bound (from intestine) fatty acids are cleaved by the endothelial lipoprotein lipase and linked up in the cardiac myocytes by the fatty acid-binding protein (FABP)

• Fatty acids need to be activated as fatty acyl-CoA to be released from the FABP, which requires ATP, and are transferred to the mitochondria

• Transfer into the mitochondria is performed through a specific carrier, the carnitine palmitoyltransferase (CPT), which is the regulated step of fatty acid metabolism

• Inside the mitochondria, fatty acids are degraded through the β-oxidation into acetyl-CoA units that enter the tricarboxylic acid (Krebs) cycle Overview of fatty acids metabolism The β-oxidation of fatty acids

The purpose of the β-oxidation is to degrade the fatty acyl-CoA into acetyl-CoA through reactions that produce protons and electrons to feed the oxidative chain. Acetyl-CoA enters the Krebs cycle and produces more protons

2 steps of proton production at each cycle of β-oxidation

o Carnitine palmitoyltransferase is the regulatory step of fatty acid metabolism

Fatty acid CoA-SH

Fatty acyl-CoA CYTOSOL

CPT- 1 outer mitochondrial carnitine membrane CoA-SH Fatty acyl-carnitine inner mitochondrial membrane CPT- 2 carnitine CoA-SH MITOCHONDRION Fatty acyl-CoA Carnitine palmitoyltransferase is regulated by malonyl-CoA

malonyl-CoA Fatty acid glucose/ lactate

AMPK - - Fatty acyl-CoA acetyl-CoA pyruvate CYTOSOL

CPT- 1 outer mitochondrial membrane Fatty acyl-carnitine inner mitochondrial membrane CPT- 2

MITOCHONDRION Fatty acyl-CoA

β - oxidation

acetyl-CoA 5. ATP production

ATP is produced by proton flux through ATP synthase

ATP production relies on two basic mechanisms:

• Proton production, which is essentially made by the tricarboxylic acid (Krebs) cycle and the β-oxidation (in the mitochondria), and, in smaller amount, by GAPDH and lactate dehydrogenase (in the cytosol)

• Proton accumulation and transfer, through the mitochondrial membrane

The Krebs cycle is the common mechanism of oxidation of glucose, fatty acids and lactate. Because it is a cycle rather than a linear pathway, its activity (“speed”) can be rapidly adapted to the ATP demand Overview of the Krebs cycle

2H

3 NADH2 =3x3 ATP

α-ketoglutarate CoA

2 ATP 1 ATP Important characteristics

• 2 carbons are lost that generate CO2 • 8 protons are produced that generate 11 ATP through oxidative phosphorylation • 1 ATP is produced from GTP • It is a cycle, so there is no accumulation of end-product • It can be accelerated by Ca2+ which enters the mitochondria through the Ca2+/Na+ exchanger The Krebs cycle rapidly adapts energy offer to the demand

Increased offer

Increased demand

Offer - demand matching Proton generation from glycolysis

oxaloacetate aspartate NADH + H+

NAD+ glutamate α keto-glutarate malate CYTOSOL

MITOCHONDRION NAD+ NADH + H+ glutamate aspartate

malate oxaloacetate α keto-glutarate

Protons generated by glycolysis in the cytosol are transferred inside the mitochondria by the malate-aspartate shuttle The proton-motive force of ATP synthesis

Enzymes of the Krebs cycle and the β-oxidation are located in the mitochondrial matrix. Electrons from NADH and FADH are taken by the electron transport chain, which assembles in four complexes crossing the inner mitochondrial membrane. Accompanying protons are ejected in the inter-membrane space, which creates an electrical gradient through the membrane

Mitochondrial matrix

Inner II membrane

Inter-membrane space The proton-motive force of ATP synthesis

Protons accumulating in the inter-membrane space will spontaneously tend to cross the inner mitochondrial membrane to the mitochondrial matrix because of the electrical gradient. They can do so only through a specific carrier, the ATP synthase. Energy released by the crossing protons couples ADP and Pi into ATP. Protons eventually bind to O2 to form H2O Importance of cytochrome c

All the proteins of the electron transport chain are organized in four multi-protein complexes that cross the inner mitochondrial membrane to extrude protons from one side to the other. The only exception is cytochrome c, a very small protein (12.5 kDa, 100 amino-acids) that is loosely attached on the outer surface of the inner membrane. This structure allows cytochrome c to move freely in order to transfer electrons from complex III to complex IV. In case of mitochondrial damage, cytochrome c is easily released and can migrate to the cytosol, where it is the main trigger of the mitochondrial pathway of apoptosis The role of creatine phosphate

Creatine phosphate is the shuttle of high-energy phosphates between their point of synthesis (mitochondria) and their point of utilization (cytosol and sarcomere)

Creatine phosphate (PCr) is synthesized upon phosphorylation of creatine (Cr) by ATP by the enzyme creatine phosphokinase (CPK)

CPK ATP + Cr PCr + ADP

CPK is expressed on the outer surface of the inner mitochondrial membrane to transfer the high-energy phosphate from ATP to Cr

Another isoform of CPK is expressed in the cytosol and in the M line of the sarcomere to transfer the high-energy phosphate from PCr to ADP. The re-synthesized ATP can now be used by myosin ATPase in the sarcomere and by SERCA in the cytosol 6. Anaerobic metabolism

In absence of oxygen, the heart triggers metabolic emergency mechanisms

• In conditions of oxygen deprivation (ischemia), the heart must shift toward an anaerobic metabolism, which provides far less ATP per mole of substrate

• In case of ATP depletion, its degradation products trigger emergency mechanisms: • Adenosine decreases energy demand by a negative chronotropic and inotropic effect

• AMP-dependent protein kinase optimizes energy production from glucose, the only endogenous anaerobic source of ATP Production of adenosine

In normoxic conditions, the ATP used by ATPases is quickly reformed in the mitochondria ATPase ATP synthase ATP ADP + Pi ATP

In hypoxic conditions, ADP accumulates. To regenerate ATP from ADP by the enzyme myokinase, the cell must also produce AMP

Myokinase ADP + ADP ATP + AMP

AMP is subsequently broken down to adenosine in several enzymatic steps

AMP Adenosine

Because adenosine is 1,000-fold less abundant than ATP, even a small breakdown of ATP is sufficient to trigger adenosine signaling Effects of adenosine

Adenosine binds to the specific receptor A1 in cardiac tissue and A2 in vascular tissue

• Receptor A1

+ • Stimulates an adenosine-dependent K channel (KAdo) in the SA node and AV node, which results in bradycardia (negative chronotropy) and negative dromotropy

• Blocks the L-type Ca2+ channel, which results in a negative inotropy

• Triggers ischemic preconditioning (cardioprotective mechanism against ischemia)

• Receptor A2

• Induces vasodilation of the coronary arteries to improve oxygen and substrate supply (coronary flow reserve). This is the main mechanism through which an atherosclerotic artery maintains coronary flow AMP-dependent protein kinase

AMP-dependent protein kinase (AMPK) is a metabolite-sensing kinase, activated by changes in energy status resulting from • Increased workload • Decreased substrate supply • Decreased oxygen supply

Overall, AMPK activates glucose metabolism. It also stimulates fatty acid oxidation (which, by feed-back, replenishes glycogen stores) by inhibiting the production of malonyl-CoA Effects of AMPK on cardiac metabolism

ATP inactive Workload - AMPK

Low O2 + AMP AMPK kinase

active AMPK

ACTION EFFECT

Increases GLUT 1 and 4 translocation Stimulates glucose uptake Increases glucose 6-phosphate Stimulates glycogen synthesis Increases fructose 2,6-bisphosphate Stimulates glycolytic flux Inhibits malonyl-CoA production Stimulates fatty acid oxidation Effects of AMPK on cardiac metabolism

Synthase Translocation Glycogen phosphatase Glucose + e + GLUT

SYNTH Glycogen Glucose i Glucose synthesis HK uptake H-6-P

Fru 2,6-P2 + PFK-1 Glycolytic Fru-1,6-P2 flux

GAPDH

Pyruvate

PD H

Fatty acid Fatty acids + Acetyl-CoA oxidation

Citrate 7. Questions

1. What are the regulatory steps of glucose metabolism in the heart? 2. What are the characteristics of cardiac glucose transporters? 3. What are the regulatory mechanisms of pyruvate dehydrogenase? 4. What is the role of creatine phosphate in the heart? 5. What is the particularity of cytochrome c in the electron transport chain? 6. Explain the regulation of glycogen metabolism and the roles of glycogen in the heart 7. Explain the mechanism of production and the actions of adenosine in the heart 8. Describe the mechanisms of control of catecholamines on glucose metabolism 9. Describe the role and action of AMP-dependent protein kinase in the heart 10. What are the effects of fatty acids on cardiac glucose metabolism? Molecular Medicine of the Heart

Class 6. Signal transduction and gene expression Overview of class 6: Signal transduction and gene expression

1. General principles 2. Catecholamines 3. Acetylcholine 4. Nitric oxide 5. Ca2+-dependent cardiac cell growth 6. Ca2+-independent cardiac cell growth 7. Questions 1. General principles

Adapting cellular activity to the extracellular world

Signal transduction is the process by which an extracellular stimulus activates an intracellular messenger that modifies cellular activity. This modification can be:

• Acute: enzyme phosphorylation, ion channel opening (e.g., catecholamines, acetylcholine)

• Chronic: phosphorylation of transcription factors resulting in an adaptation of gene expression (e.g., growth signaling pathways)

Gene expression is regulated by the sum of extracellular stimuli channeled to the nucleus by transduction pathways. Gene expression is therefore the end- point of information integration, as well as the starting point of protein translation General principles

Stimulus

Sensor

Gene as an Transducer “end-point”

Effector

Gene expression

Post-transcriptional regulation Gene as a “starting point” Translation

Post-translational modifications General principles

Examples of signal transduction

• Acute • phosphorylation of phospholamban and MLC-2 by catecholamines • opening of K+ channel by acetylcholine

• Chronic • cardiac cell survival by PI-3-kinase/Akt • stimulation of protein synthesis by mTOR • activation of transcription factors by MAP kinases

Most of the acute signaling regulates cardiac function Most of the chronic signaling regulates cardiac growth and survival Modes of transduction

Most of the extracellular stimuli bind specific receptors on the plasma membrane, which triggers the synthesis of an intracellular transducer (second messenger). This mode of transmission is used by most messengers of small molecular weight (e.g., adrenaline) and peptides (e.g., growth factors). The intracellular messenger usually activates a kinase and/or a phosphatase that mediate the effects. Some receptors can have opposite effects on the same intracellular messenger, thereby creating transduction antagonisms (e.g., catecholamines and acetylcholine)

Some messengers of lipidic origin can cross the plasma membrane and bind to an intracellular adapter. The complex usually migrates to the nucleus and affects gene expression by binding to a specific nucleotide sequence (e.g., steroid hormones) Receptor-mediated transduction

Examples Agonist Effector

catecholamines Gs protein acetylcholine Gi protein NO guanylate cyclase Gq protein insulin IRS-1 2. Catecholamines

Catecholamines are the major mediators of the cardiac response to increased work

Catecholamines are synthesized from the amino acid tyrosine

• Epinephrine (adrenaline) is released by the adrenal medulla (systemic). Binds to β-adrenergic receptors (β1 in heart and β2 mainly in arterioles). Has a global cardio-vascular effect (“fight or flight”), resulting in increased cardiac output and decreased arterial resistance.

• Norepinephrine (noradrenaline) is released by the stellate ganglion (Paracrine). Binds to β-adrenergic receptors in heart, resulting in increased heart rate, inotropy and cardiac output. Can also bind to α-adrenergic receptors in arterioles, resulting in increased arterial resistance. Control of adrenergic receptors by receptor specificity

α1. Smooth muscle cell contraction- vasoconstriction Stimulation of cell growth (heart) and proliferation (smooth muscle cell). Interacts with the G protein Gq

α2. Negative feed-back inhibition of the sympathetic drive in the CNS.

β1. Positive inotropy, lusitropy and chronotropy Stimulation of cell growth and proliferation Interacts with the G protein Gs

β2. Positive inotropy and chronotropy Smooth muscle cell relaxation- vasodilation Interacts with the G proteins Gs and Gi

α And β adrenergic receptors will have different effects despite a similar ligand because they are conjugated to different transducers (G proteins) that generate different effectors (cAMP, diacylglycerol…) Control of adrenergic receptors by transducer specificity

Epinephrine is systemic: it plays all the effects at the same time Norepinephrine is paracrine: effects depend on the site of release Structure of β-adrenergic receptors

The beta-adrenergic receptor is made of:

• Seven transmembrane domains that bind the catecholamines and their chemical antagonists (β-blockers)

• An extracellular domain that can prevent the access to the transmembrane domain

• A cytoplasmic domain that is responsible for the coupling of the receptor to the effector G protein and for the phosphorylation of the receptor by BARK Structure of β-adrenergic receptors Function of β-adrenergic receptors

Upon binding of catecholamines to the transmembrane domain, the cytoplasmic tail activates the trimeric protein Gs. The subunit α of the G protein detaches from the complex β−γ and activates the membrane protein adenylate cyclase.

Adenylate cyclase produces cyclic AMP (cAMP) from ATP. cAMP is an activator of the cAMP-dependent protein kinase (PKA) that phosphorylates the targets of catecholamines, including:

L-type Ca2+ channel MLC-2 Phospholamban Troponin I Glucose transporters PFK-2, the enzyme producing fructose 2,6-bisphosphate Glycogen synthase kinase, phosphorylase phosphatase cAMP-responsive element binding protein (CREB) Function of β-adrenergic receptors

The G protein is a GTPase that spontaneously degrades GTP into GDP. When binding GDP, the G protein is in an inactive, trimeric state. β-receptor stimulation displaces GDP by GTP and the subunit α translocates to adenylate cyclase. Inactivation of the system is performed by the GTPase hydrolysis of GTP into GDP and reunification of the trimeric protein Function of β-adrenergic receptors

Cyclic AMP releases the catalytic subunit of PKA from the regulatory subunit, which activates the enzyme Substrates of PKA

L-type Ca2+ channel Increased inotropy MLC-2

Phospholamban Increased lusitropy Troponin I

Glucose transporters PFK-2, the enzyme producing fructose 2,6-bisphosphate Increased production of Glycogen synthase kinase, phosphorylase kinase ATP from glucose

cAMP-responsive element binding protein (CREB) Increased heart mass (hypertrophy) Function of β-adrenergic receptors

PL, phospholamban TnI, troponin I

PKA acts simultaneously on the different proteins presenting the appropriate aminoacid consensus sequence to be phosphorylated Function of β-adrenergic receptors

If catecholamine stimulation is excessive, the β-receptor cytoplasmic tail is phosphorylated by the β-agonist receptor kinase (BARK), which uncouples the receptor from the Gs protein. The receptor can be reactivated by a phosphatase or it can be internalized in specific vesicles. Internalized receptors can be returned to the plasma membrane or degraded in the lysosomes 3. Acetylcholine

Acetylcholine controls the cardiac activity at rest

The chief function of acetylcholine is to act as an antagonist of the sympathetic system, by lessening the formation of cAMP in response to catecholamine stimulation.

Acetylcholine acts by two main mechanisms:

• negative inotropic effect in the myocardium

• negative chronotropic effect in the sino-atrial node

Acetylcholine is the main modulator of cardiac contractile activity at rest, whereas catecholamines are the main modulator of cardiac activity when workload increases Function of muscarinic receptor

Acetylcholine is released from the vagal nerve and binds to its specific receptor, the muscarinic receptor, which is inhibited by atropine.

The receptor is coupled to a trimeric protein Gi. Binding of acetylcholine to the receptor dissociate the trimeric Gi protein in an α subunit and a β−γ complex.

In the myocardium, the α subunit binds to adenylate cyclase and decreases its enzymatic activity. The β−γ complex acts as a GTPase that hydrolyses the GTP bound to Gsα. The combined action decreases the β-adrenergic stimulation and thereby decreases cardiac contractility (negative inotropy).

In the SA node, the Gi β−γ complex opens an acetylcholine-dependent K+ channel that inhibits the rate of spontaneous depolarization and thereby decreases the heart rate (negative chronotropy) Function of muscarinic receptor

G

Activation of the cholinergic receptor dissociates the protein Gi, and the subunit Giα inhibits adenylate cyclase. This effect is reinforced by an increased GTPase activity of Gsα by the β−γ subunit Control of adrenergic receptors by antagonism

Both catecholamines and acetylcholine “compete” on the same effector to create diametrically opposite actions. The global activity of the effector depends on the balance between the stimuli. However, the concentration of catecholamines is usually low when acetylcholine is released, therefore the negative inotropic effect of acetylcholine is very moderate. By far, the most important effect of acetylcholine in the heart is to decrease heart rate (negative chronotropy). The β2-AR can also activate the Gi protein to avoid an overload of Ca2+. 4. Nitric oxide

NO diffuses freely because it is a gas

Nitric oxide (NO) is a freely diffusible gas produced from the aminoacid arginine by three different isoforms of the enzyme NO synthase (NOS): endothelial, neuronal and inducible. All three isoforms are expressed in the cardiac myocyte and have different kinetic properties and different subcellular compartmentation, which helps to limit the free diffusion of the gas. The main activator of NOS in the myocyte is the muscarinic receptor, upon acetylcholine stimulation. NOS can also be activated by a subtype of adrenergic receptor, the β3-AR.

NO can be produced by the cardiac myocyte or can diffuse from the endothelial cells. The second messenger of NO is cGMP, which is produced upon activation of guanylate cyclase by NO. cGMP in the myocyte is mostly an antagonist of cAMP. Activation of the NO pathway is therefore a way to limit adrenergic stimulation, and is therefore a mediator of the acetylcholine pathway. Synthesis of NO in the cardiac myocyte

Acetylcholine Catecholamines

Muscarinic receptor β3-adrenergic receptor

NO synthase

NO

Guanylate cyclase

cGMP Effects of NO in the cardiovascular system

In the myocardium, NO has different effects:

• Negative inotropic effects. Decreases L-type Ca2+ channel current and antagonize the effects of PKA thereupon

• Positive lusitropic effects. Increases the activity of TnI to dissociate Ca2+ from the myofilament

• Negative chronotropic effects. Antagonizes the effects of catecholamines on the T- type Ca2+ channel in the SA node

• Metabolic effects. Increased fatty acid oxidation and decreased glucose uptake

In the vasculature, NO produced by endothelial cells migrates to the vascular smooth muscle cells and induces the principal mechanism of vasodilation 5. Ca2+-dependent cardiac cell growth

Ca2+ is both a transducer and a target of cell signaling

• Ca2+ as a transducer. Increased [Ca2+] can trigger the activation of kinases (such as Ca2+ /calmodulin protein kinase or protein kinase C) and phosphatases (such as calcineurin)

• Ca2+ as a target. Ca2+ release from the SR can be stimulated by specific signals, such as inositol 3-phosphate (IP3). This represents the main mechanism of initiation of contraction in arteries. This released Ca2+ can in turn work as a transducer

• Ca2+ is a perfect stimulator of cardiac cell growth because prolonged increase in free Ca2+ concentration that accompanies increased workload is used as a signal that tells the cardiac myocyte to increase its contractile capacity (hypertrophy) The phosphatidylinositol system

• Phosphatidylinositol bisphosphate (PIP2) is a normal component of the plasma membrane, which is the substrate of phospholipase C

• Phospholipase C cleaves PIP2 in inositol triphosphate (IP3) and diacylglycerol (DAG)

• Activity of phospholipase C is stimulated by the G protein Gq upon binding of stimuli of cardiac hypertrophy and stress (angiotensin, endothelin-1, α-adrenergic receptor)

• IP3 will bind on a specific receptor of the sarcoplasmic reticulum in vascular smooth muscle cell to stimulate Ca2+ release. This is the main mechanism of activation of contraction in vascular smooth muscle cells, not in cardiac myocytes

• DAG together with Ca2+ will stimulate the family of protein kinases C by recruiting these kinases to the plasma membrane (translocation) The phosphatidylinositol system The phosphatidylinositol system

PIP2 IP3 PLC

DAG

α And β adrenergic receptors have different effects despite a similar ligand because they are conjugated to different G proteins that generate different transducers (cAMP for Gs, diacylglycerol for Gq) The phosphatidylinositol system

• Protein kinase C (PKC) is the main effector of the phosphatidylinositol system in the cardiac myocytes. PKC activation requires an anchoring to DAG and a stimulation by Ca2+.

• There are at least 11 different isoforms of PKC, with different sensitivity to DAG and Ca2+. At least five isoforms are expressed in the heart (α,β,δ,ε,ζ). These isoforms differ by their substrate specificity and their anchoring mechanisms to the plasma membrane

• The main effects of PKC are to stimulate cell growth during hypertrophy through an activation of the MAP kinase pathway and through phosphorylation of transcription factors

• In vascular smooth muscle cells, PKC phosphorylates caldesmon to allow actin- myosin interaction Ca2+/calmodulin-dependent protein kinase (CamK)

CamK is a kinase activated by a CamK kinase upon increased cytosolic [Ca 2+].

• In the cytosol, CamK increases contractility (positive lusitropy and inotropy) by phosphorylating phospholamban and MLC-2. It also activates glucose utilization by phosphorylating glycogen phosphorylase (increased glycogenolysis) and PFK-2 (increased fructose 2,6 bisphosphate and therefore, increased glycolysis). These effects are cumulative with the similar effects of PKA. In vascular smooth muscle cells, CamK is the main mechanism of actin- myosin interaction and contraction

• In the nucleus, CamK phosphorylates transcription factors (such as CREB or SRF), which mediate the gene adaptation to hypertrophy. These effects are cumulative with similar effects of calcineurin, MAP kinases…

• Excessive activation of CamK leads to apoptosis and heart failure Ca2+/calmodulin-dependent protein kinase (CamK)

Chronic adrenergic stimulation

β1-AR β3-AR

β2-AR NO [Ca 2+]

Gi PKG Ca 2+ - calmodulin

ICa Cam KK Excessive activation ASK-1 Cam K NFΚB Nuclear translocation APOPTOSIS

glycogen phosphorylase CREB 6-phosphofructo-2-kinase SRF MLC2 GROWTH phospholamban

INOTROPY / METABOLISM Calcineurin

Calcineurin is a serine/threonine phosphatase that is activated by sustained elevation in [Ca2+]. It is therefore not stimulated by the transient rise in cytosolic [Ca2+] during systole. The enzyme is activated by the Ca2+/calmodulin complex

The main role of calcineurin is to adapt the gene response to increased workload by activating transcription factors that increase the expression of genes encoding contractile proteins

Calcineurin dephosphorylates the shuttle protein NFAT (nuclear factor of activated T lymphocytes), which promotes the binding of the transcription factors GATA4 and MEF2. The complexes NFAT-GATA4 or NFAT-MEF2 migrate to the nucleus and activate gene expression Calcineurin

Stimuli of hypertrophy

Akt

NFAT - P calcineurin GSK-3 [Ca2+] NFAT GATA4 Nuclear translocation MEF2

NFAT-GATA4 NFAT-MEF2

ANF TnI BNP CPK βMHC myoglobin 6. Ca2+-independent cardiac cell growth

Chronic signaling regulates cardiac cell growth and survival

Long-term stimulation of the cardiac cell from an extracellular sensor results in an adaptation of gene and protein expression. In the heart in vivo, this adaptation consists in two main consequences:

• Growth. Cardiac cell growth is stimulated when the cardiac cell is submitted to increased external work, such as cardiac hypertrophy • Survival. Survival mechanisms are stimulated when the viability of the cardiac cell is threatened, such as myocardial ischemia and heart failure

These adaptations are performed by two main signaling pathways: • The MAP kinase pathway • The PI-3 kinase pathway MAP kinases

The MAP kinases (mitogen-activated protein kinases) can be divided in three cascades • Extracellular-signal regulated kinases 1 and 2 (ERK1 / ERK2) • c-Jun N-terminal kinase (JNK), isoforms 1 to 3 • p38-MAP kinase (p38), isoforms α to δ

The three cascades function on a same model: a MAPK kinase kinase phosphorylates a MAPK kinase, which phosphorylates and activates a MAPK. The MAPK phosphorylates mainly transcription factors, although they can phosphorylate also other protein kinases

• ERK1/ERK2 are mainly triggered by increased workload to mediate the adaptation of gene and protein expression to hypertophy. They mainly promote cell growth • JNK and p38 can be activated by hypertrophic stimuli but also by sensors of cell stress. They can promote cell growth and/or cell death (apoptosis) depending on the isoform that is activated MAP kinases

Overload stimuli Overload and stress stimuli

Gq RTK

PKC Ras MEKKs Raf

MEK 1,2 MEK 4,7 MEK 3,6

ERK1/ERK2 JNK p38

Transcription Translation Transcription Transcription GATA4 P70S6K Ets-1 ATF2 Ets-1 UBF Elk-1 CHOP Elk-1 ATF2 MEF2 STAT cJun CREB cMyc NFAT CBP GROWTH GROWTH / APOPTOSIS Mechanism of ERK activation

Hypertrophic stimuli (angiotensin, endothelin, epinephrin)

G protein-coupled receptor Hypertrophic stimuli (growth hormones)

Dissociation of Gq α

Receptor Tyrosine Kinase Activation of phospholipase C

Phosphatidylinositol Diacylglycerol Ras bisphosphate IP3 Activation of PKC

Raf PI-3 kinase

Upon stimulation by growth factors (FGF, IGF, cardiotrophin, insulin) or hypertrophic stimuli (through the protein Gq), a receptor-linked tyrosine kinase (RTK) phosphorylates and activates phosphatidylinositol 3-kinase (PI-3K).

PI-3K is a lipid kinase that adds a phosphate on the carbon 3 of phosphatidylinositol bisphosphate in the plasma membrane. 3- Phosphatidylinositol phosphate activates PDK1, which in turn activates Akt/PKB. Akt participates in:

• stimulation of protein synthesis • regulation of glucose metabolism • cell survival mechanisms

3-Phosphatidylinositol phosphate is dephosphorylated by the tumor suppressor PTEN. Inactivation of PTEN creates a constitutively active PI-3K signaling pathway, which induces cancer PI-3 kinase

P

PI-3-Kinase PI-3 kinase

Growth factors insulin G proteins

RTK

PtdInsP PI-3K 2 PtdInsP3 PTEN PDK1

Akt

mTOR GSK3 Bad Caspase-9 eNOS P70S6K Glycogen FKH synthase

Protein Glycogen Cell survival synthesis synthesis 7. Questions

1. What are the targets of PKA in cardiac cells? 2. Explain the mechanism of action of acetylcholine 3. How is the beta-adrenergic receptor deactivated? 4. Describe the distribution and function of the four adrenergic receptors 5. Describe the role of nitric oxide in cardiac myocyte 6. Describe the role and regulation of protein kinase C 7. Describe the role and regulation of Ca2+/calmodulin protein kinase 8. Describe the role and regulation of calcineurin 9. Describe the PI-3-kinase pathway 10. Describe the activation and effects of the ERK1/ERK2 pathway in the heart 11. Why is the β2-AR coupled to the Gi protein? Molecular Medicine of the Heart

Class 7. Coronary physiology Overview of class 7: Coronary physiology

1. Vascular structure 2. Vasomotion 3. Vasorelaxation 4. Vasoconstriction 5. Regulation of coronary flow 6. Coronary 7. Questions 1. Vascular structure

Coronary arteries adapt oxygen supply to the demand

Coronary arteries are the vascular system of the heart. Their function is: • To supply enough blood to match the high energy demand of the heart • To adapt blood delivery to the variation in energy needs

Coronary arteries are extremely receptive to signals from the heart and the vasculature that warn of changes in energy demand (such as adenosine, NO…). This adaptability of coronary flow is defined as coronary autoregulation

When coronary arteries are obstructed by atherosclerosis, the imbalance between oxygen demand and supply creates myocardial ischemia Anatomy of coronary arteries Structure of coronary arteries

• vascular intima, made of the endothelium, a monolayer of thin cells supported by a basement membrane • produces most of the vasoactive substances (NO, prostacyclin, endothelin…) • maintains a laminar blood flow and prevents thrombosis • produces growth factors for angiogenesis

• vascular media, made of vascular smooth muscle cells, a multilayer of contractile cells separated by the extracellular matrix • the smooth muscle cells are responsible for the control of the arterial diameter (vasomotion) • the extracellular matrix contains elastin that is responsible for the expansion/recoil of conductance vessels during the cardiac cycle • the extracellular matrix contains collagen that offers resistance to vessel expansion from blood pressure

• vascular adventitia, made of connective tissue, contains nerve endings and small vessels feeding the arterial wall Structure of coronary arteries 2. Vasomotion

Cardiac contraction is phasic, vascular contraction is tonic

Like the heart, vascular smooth muscle cells contract by a mechanism of Ca2+ - induced Ca2+ release. However, there are major differences compared to the heart

• Similarities with the heart: • contraction is initiated by the entry of extracellular Ca2+ • extracellular Ca2+ can stimulate the release of endogenous stores of Ca2+ from the sarcoplasmic reticulum • Ca2+ interacts with the myofilament to initiate contraction • Ca2+ is taken up by the sarcoplasmic reticulum to initiate relaxation

• Differences with the heart: • cAMP stimulates the heart to contract and the vessel to relax • Ca2+ release can be induced by IP3 independently from Ca2+ entry • vascular contraction is tonic and maintained, whereas cardiac contraction is short-lived • smooth muscle cells do not express troponins, including troponin C. Instead, Ca2+ binds to calmodulin to control the actin-myosin interaction Contrast between cardiac contraction and vasomotion

The smooth muscle cell is “smooth” because it has no sarcomere. The sarcomeric organization is made to prevent overstreching of the contractile proteins, which happens in the heart due to big variations in pressure, but which does not happen in the vasculature The phosphatidylinositol system

• Phosphatidylinositol bisphosphate (PIP2), which is a normal component of the plasma membrane, is the substrate of phospholipase C

• Phospholipase C cleaves PIP2 in inositol triphosphate (IP3) and diacylglycerol (DAG)

• Activity of phospholipase C is stimulated by the G protein Gq upon binding of specific stimuli (angiotensin, endothelin-1, α-adrenergic receptor)

• IP3 will bind on a specific receptor of the sarcoplasmic reticulum in vascular smooth muscle cell to stimulate Ca2+ release. This is the main mechanism of activation of contraction in vascular smooth muscle cells, not in cardiac myocytes

• DAG together with Ca2+ will stimulate the family of protein kinases C by recruiting these kinases to the plasma membrane (translocation) The phosphatidylinositol system The phosphatidylinositol system

PIP2 IP3 PLC

DAG Mechanisms of vascular contraction

As in cardiac muscle, vascular contraction is operated by Ca2+. Ca2+ enters the vascular smooth muscle cell through two different channels:

• The voltage-operated Ca2+ channel (VOC) is similar to the cardiac L-type Ca2+ channel. Its is activated by membrane depolarization and operates contraction by a Ca2+ –induced Ca2+ release mechanism. This mechanism is responsible for peristaltic (transient) contraction.

• The receptor-operated channel Ca2+ (ROC). It is activated by ligand binding, essentially endothelin, angiotensin II and α1 adrenergic receptor stimulation. In addition, activators of the ROC produce IP3 through phospholipase C activation by the G protein Gq. IP3 directly releases Ca2+ from the sarcoplasmic reticulum, which is quantitatively more important than the Ca2+ –induced Ca2+ release. This mechanism is responsible for tonic (maintained) contraction.

• Activation of phospholipase C also produces diacylglycerol (DAG), an activator of protein kinase C (PKC). A target of PKC in smooth muscle cell, that does not exist in striated muscles, is caldesmon. Caldesmon binds to actin and compete with the myosin head. Phosphorylation by PKC removes this competition and allows actin- myosin interaction. Mechanisms of Ca2+ entry in smooth muscle cell Mechanisms of vascular contraction

Ca2+ released form the sarcoplasmic reticulum binds to calmodulin (instead of troponin C, as in the heart). The Ca2+-calmodulin complex activates Cam Kinase, which phosphorylates the regulatory chain of myosin light chain (MLC-2). This phosphorylation positions the myosin head in front of the actin filament for crossbridge formation

Once myosin and actin are joined by MLC-2, they latch and do not relax until further signal is given, which does not require ATP. When Ca2+ level starts to decrease, CamK is deactivated and MLC-2 is dephosphorylated by a phosphatase, which, together with the binding of ATP to the myosin ATPase, detaches the crossbridge Mechanisms of vascular contraction

ROC VOC Mechanisms of vascular relaxation

In vascular smooth muscle cells, relaxation is transduced by both cAMP and cGMP, which therefore do not have a “yin-yang” effect as in the myocardium

• cAMP is produced by β-receptor stimulation, adenosine and prostacyclin. Through PKA, it inhibits Cam Kinase and accelerates the reuptake of Ca2+ in the sarcoplasmic reticulum

• cGMP is produced by NO or by ANF released from the heart during overload. Via the cGMP-dependent protein kinase (PKG), it deactivates Cam Kinase, stimulates MLC-2 phosphatase and inhibits the voltage-operated Ca2+ channel

+ • In addition, K channels can inhibit ICa by hyperpolarization of the plasma membrane. The ATP-dependent K+ channel is activated by adenosine and is responsible for rapid vasodilation during ischemia. The Ca2+-dependent K+ channel is a negative feed-back mechanism activated by Ca2+ itself Mechanisms of vascular relaxation Mechanisms of vascular relaxation 3. Vasorelaxation

The endothelium controls vascular relaxation

Most of the physiological vasoactive substances are synthesized and released by the endothelium, then diffuse to the smooth muscle cells

The basic function of the endothelium in vasomotion is to promote vasodilation. Thereby, the endothelium protects the vascular wall against the vasoconstrictory influence of circulating substances (especially serotonin or thromboxane from platelets)

Most importantly, the endothelium is the mediator of the vasodilatory effect of acetylcholine through the production of nitric oxide (NO). In addition, the endothelium promotes vasodilation by the release of the prostaglandin prostacyclin. An adenosine receptor also allows maximal vasodilation in conditions of ischemia through opening of the K+ channel Vasorelaxation by adenosine

Adenosine is formed from AMP within the myocardial cells when, as a result of ischemia or vigorous heart work, the high-energy ATP is broken down. The enzyme controlling this pathway is 5’-nucleotidase, the sarcolemmal enzyme converting AMP into adenosine.

Adenosine leaves the cell and binds to its purinergic receptors (A1-myocardial and A2-vascular). The A1 receptor is coupled to the Gi protein, which hyperpolarizes the plasma membrane by activating the K+ channel. The A2 receptor is coupled to the Gs protein, which activates PKA

Adenosine is degraded into inosine by adenosine deaminase, an enzyme expressed in red blood cells and the vessel wall

In case of extreme stress, ATP can leave the cell and forms adenosine under the action of the enzyme ecto-ATPase. ATP itself can also bind to vascular purinergic receptors inducing vasodilation Vasorelaxation by adenosine Role of prostaglandins in vasomotion

Prostaglandins are derived from unsaturated fatty acids, usually the arachidonic acid. The two main prostaglandins in vasomotion are prostacyclin and thromboxane

• Prostacyclin is synthesized by the endothelium and has vasodilatory properties through the stimulation of adenylate cyclase. It also inhibits platelet aggregation

• Thromboxane is produced by the platelets and promotes vasoconstriction through the IP3 messenger system. It also promotes platelet aggregation

An enzymatic step in prostaglandins synthesis is performed by cyclooxygenase (COX), which exists in two isoforms. The isoform responsible for the synthesis of thromboxane is highly sensitive to inhibition by aspirin, whereas the isoform responsible for prostacyclin synthesis shows a limited sensitivity. This is why aspirin is used in the prevention against platelet aggregation and vasoconstriction in patients at risk for myocardial ischemia and/or stroke Role of prostaglandins in vasomotion COX-2 inhibitors increase the risk of cardiovascular disease 4. Vasoconstriction

Smooth muscle cells control vascular contraction

The receptors for the main vasoconstrictive agents are on the smooth muscle cell and balance the effects of the endothelium. The main stimuli of vasoconstriction are controlled by norepinephrine, angiotensin II and endothelin

• α-adrenergic receptor. They are stimulated by the release of norepinephrine from nerve endings. They are coupled to the protein Gq and stimulate vasoconstriction by the IP3/DAG pathway

• angiotensin II. The kidney hormone renin stimulates the synthesis of angiotensinogen, which is converted into angiotensin II (ATII) in the lungs. ATII is a powerful vasoconstrictory agent through stimulation of the IP3/DAG pathway

• endothelin. When the endothelium is damaged, such as in atherosclerosis or hypertension, it looses this vasodilatory effect and promotes vasoconstriction by excessive production of endothelin Roles of angiotensin II

Angiotensin II is mainly responsible for the control of vascular blood pressure. During hypotension or hyponatremia, there is an adrenergic stimulation of the kidney to release renin, a glycoprotein enzyme that cleaves the liver protein angiotensinogen into angiotensin 1. Angiotensin I is released in the blood and cleaved into angiotensin II by the angiotensin-converting enzyme (ACE). Angiotensin II increases blood pressure by stimulating the Gq protein in vascular smooth muscle cells (which in turn activates contraction by an IP3- dependent mechanism). ACE inhibitors are major drugs against hypertension Roles of angiotensin II Roles of angiotensin II Role of endothelin in vasomotion

Endothelin is a 21 aminoacid peptide synthesized in the endothelium from a precursor (pre-pro-ET)

At low dose, ET binds to the receptor ETB on endothelial cells and promotes relaxation by release of NO

At higher dose, ET binds to the receptor ETA on smooth muscle cells and promotes vasoconstriction by opening the ROC and stimulating the production of IP3 by the protein Gq

At high dose, ET also promotes smooth muscle cell growth and division (by DAG- mediated activation of MAP Kinases), a process involved in the development of atherosclerosis (upon the influence of TGFβ) and in hypertension (upon the influence of angiotensin II) Role of endothelin in vasomotion

normal damaged Role of endothelin in vasomotion Role of endothelin in vasomotion Integration of vasomotion mechanisms

Vasodilatory and vasoconstrictory mechanisms are constantly integrated depending on the intensity of each stimulus

• Basal mechanisms of vasodilation involve acetylcholine and prostacyclin. These mechanisms are further activated by ANF (cardiac overload), adenosine (ischemia) and β2-adrenergic stimulation (exercise)

• Basal mechanisms of vasoconstriction involve α1-adrenergic stimulation, angiotensin II and endothelin-1. These mechanisms are excessively activated when the endothelium is damaged and can not fulfill its vasodilatory function, such as during hypertension and atherosclerosis Integration of vasodilatory mechanisms

Cardiac Basal Cardiac Exercise Basal overload conditions ischemia conditions

ANF Acetylcholine Adenosine β2-receptor Prostacyclin

Nitric oxide ATP-dependent Adenylate cyclase K+ channel

cGMP cAMP

Ca2+ influx PKG PKA

Vasodilation Integration of vasoconstrictory mechanisms

Basal Impaired conditions endothelium

Endothelin-1 Angiotensin II α1-receptor

Phospholipase-C

IP3 Diacylglycerol

Ca2+ release PKC

Cam K Caldesmon

Vasoconstriction 5. Regulation of coronary flow

Coronary blood flow is mainly regulated by oxygen demand

Coronary blood flow is tightly coupled to the energy status of the myocardium. During increased workload or during ischemia, myocardial metabolism self-regulates the coronary flow by the release of adenosine, which dilates the small resistance arterioles through the activation of ATP-dependent K+ channel and adenylate cyclase. This mechanism is called the coronary autoregulation. In conditions of maximal need, the coronary flow can be as much as 5-fold higher than in basal condition. This recruited flow represents the coronary blood flow reserve

In basal conditions, the coronary tone is balanced by the release of acetylcholine, which vasodilates through the production of NO, and the α-adrenergic mediated constriction through the IP3 messenger pathway. If the endothelium is damaged, the effect of NO is lost and vasoconstriction is increased Regulation of coronary flow by the cardiac cycle

During systole, subendocardial coronary arteries are compressed by the increased left ventricular pressure. Therefore, most of subendocardial coronary flow occurs during diastole (phasic coronary flow). In subepicardium, the coronary flow is maintained throughout the cardiac cycle

If cardiac relaxation is impaired (e.g., ischemic heart disease and heart failure), the diastolic component of coronary is decreased

In case of atherosclerosis, the phasic flow is perturbed, which induces the synthesis of thromboxane form the platelets and increases the risk of platelet aggregation Regulation of coronary flow by the cardiac cycle Regulation of coronary flow by the cardiac cycle

Coronary flow is decreased in systole 6. Coronary autoregulation

Increased resistance does not necessarily impair coronary blood flow

Coronary autoregulation = The process by which the myocardial oxygen demand regulates coronary blood flow independently of the arterial perfusion pressure. This mechanism allows to keep constant the blood flow to the myocardium, independently of changes in blood pressure. Although blood pressure remains constant in a normal artery, it can be dramatically affected in presence of an obstruction, such as atherosclerosis

The main mechanism of autoregulation is an increased vascular tone proportional to an increase in perfusion pressure, creating a coronary flow reserve. In case of increased need (exercise, hypoxia), this vascular tone is released and flow increases for the same pressure, using the coronary flow reserve Coronary autoregulation Coronary autoregulation in diseased artery

The main form of coronary artery disease is the obstruction (stenosis) by atherosclerosis. The stenosis creates a resistance (R) that limits the flow (ΔQ) by creating a pressure gradient (ΔP)

The resistance increases by a power of 4 as the radius decreases (Poiseuille’s law)

R Q ΔP ΔP = ΔQ . R Coronary autoregulation in diseased artery

Because of the mechanism of autoregulation, the coronary artery can maintain a constant flow (Q) despite large pressure gradients (ΔP) by using the coronary flow reserve. Therefore, the stenosis will become significant and limit blood flow only when at least 70% of the diameter (90% of the vessel surface) is obstructed

ΔP 1. Normal artery 2. Mild stenosis – flow is maintained 3. Significant stenosis – flow is reduced 1 2 4. Capillaries close 3

4 The slope is sharp. When a ! ! stenosis becomes significant, it can quickly be critical. Coronary autoregulation in diseased artery

Because of the mechanism of autoregulation, the coronary artery can maintain coronary flow (Q) despite large pressure gradients (ΔP). Therefore, the stenosis (R) will become critical and limit blood flow only when at least 70% of the diameter (90% of the vessel area) is obstructed 6. Questions

1. Explain the antagonism between prostacyclin and thromboxane in the vasculature 2. Explain the regulation of ROC and VOC in the vasculature 3. Explain the mechanism of action of aspirin in the vasculature 4. What are the mechanisms of vasoconstriction? 5. Describe the integration of the different mechanisms of vasodilation 6. What is the main mechanism of Ca2+ release in the smooth muscle cell? 7. What is the role and mechanism of action of adenosine in coronary arteries? 8. What is the coronary autoregulation and its role in a diseased artery? 9. What is the role and mechanism of action of endothelin in the vasculature? 10. Name four differences between cardiac and vascular mechanisms of contraction

Molecular Medicine of the Heart

Class 8. Atherosclerosis Overview of class 8: Atherosclerosis

1. General overview 2. Pathogenesis of atherosclerosis 3. Plaque rupture 4. Cholesterol metabolism 5. Risk factors 6. Treatment of coronary artery disease 7. Questions 1. General overview

Atherosclerosis is the leading cause of cardiovascular disease

• Atherosclerosis is a progressive disease characterized by the development of an atherosclerotic plaque resulting from the accumulation of cholesterol-rich lipids (atheroma) recovered by fibrous tissue (fibrous cap) in the intima of large arteries and coronary arteries, together with the loss of normal endothelial function and the proliferation of smooth muscle cells

• In coronary arteries, the development of the atheroma reduces the artery lumen diameter and reduces blood perfusion, which induces ischemia. Progression of atherosclerosis can lead to the occlusion of the diseased coronary artery and stop blood perfusion, which induces myocardial infarction

• Atherosclerosis represents the leading cause of cardiovascular disease, which is the leading cause of death and illness in developed countries and becomes the leading cause in developing countries Structure of the atherosclerotic plaque

The atherosclerotic plaque is made of a lipid pool (atheroma) recovered by fibrous tissue (fibrous cap). The plaque is eccentric at first, then starts protruding in the lumen of the coronary artery as the atheroma accumulates

Normal artery Atherosclerotic artery

Fibrous cap

Lipid pool

The plaque is eccentric at first to preserve the lumen diameter A quick reminder (1)…

The main form of coronary artery disease is the obstruction (stenosis) by atherosclerosis. The stenosis creates a resistance (R) that limits the flow (ΔQ) by creating a pressure gradient (ΔP)

The resistance increases by a power of 4 as the radius decreases (Poiseuille’s law)

R Q ΔP ΔP = ΔQ . R A quick reminder (2)…

Because of the mechanism of autoregulation, the coronary artery can maintain a constant flow (Q) despite large pressure gradients (ΔP) by using the coronary flow reserve. Therefore, the stenosis will become significant and limit blood flow only when at least 70% of the diameter (90% of the vessel surface) is obstructed

ΔP 1. Normal artery 2. Mild stenosis – flow is maintained 3. Significant stenosis – flow is reduced 1 2 4. Capillaries close 3

4 The slope is sharp. When a ! ! stenosis becomes significant, it can quickly be critical. General mechanisms of atherosclerosis

• Atherosclerosis is initiated by the accumulation of cholesterol-rich macrophages (fatty streak) and activated lymphocytes in the arterial wall

• These inflammatory cells trigger a reaction leading to the proliferation of smooth muscle cells and accumulation of extracellular matrix, which forms a fibrous cap covering the fatty streak

• Macrophages accumulating cholesterol will die of overload. Necrotic debris and released lipids are the molecular basis of the atheroma. The atheroma is fluid and retained by the fibrous cap, but progressively accumulates as more macrophages die and protrudes in the artery lumen. Lowering circulating cholesterol level is the only way to slow down this process

• If the surrounding fibrous cap ruptures, the atheroma is exposed to circulating coagulation factors, which triggers the formation of a thrombus that can occlude the vessel. This phenomenon predominates in coronary arteries (myocardial infarction) and cerebral arteries (stroke) General mechanisms of atherosclerosis

A. Diffusion of macrophages and lymphocytes through the vessel wall into the intima forms the fatty streak

B. Accumulation of cholesterol in macrophages, neo-intimal proliferation of smooth muscle cells, formation of a fibrous cap. In coronary arteries, this is the cause of angina

C. Rupture of the fibrous cap, thrombosis and vessel occlusion. In coronary arteries, this is the cause of myocardial infarction Forms of atherosclerotic plaque

• Early atheroma. First phase of the disease. Small lipid pool without repercussion on blood flow

• Stable plaque. Large plaque protruding in the lumen, with a thick fibrous cap and a small lipid pool

• Lipid-rich plaque. Large atheroma and multiple inflammatory cells with thin fibrous cap.

• Ruptured plaque. Interruption of the fibrous cap followed by thrombosis Forms of atherosclerotic plaque

Early atheroma Stable plaque Lipid-rich plaque Ruptured plaque 2. Pathogenesis of atherosclerosis

Atherosclerosis is a form of inflammatory disease

• Atherosclerosis is initiated by the excessive transfer of cholesterol-rich lipoproteins through endothelial cells

• To eliminate this cholesterol, endothelial cells attract macrophages that absorb the lipoproteins

• Accumulation of macrophages initiates an inflammatory reaction attracting more cells

• Excessive accumulation of lipids in the macrophages makes them die and release the lipids in a fluid pool, or atheroma

• The lipid pool is maintained by a fibrous cap that can rupture, which is followed by thrombosis Pathogenesis of atherosclerosis

• Cholesterol-rich lipoproteins (LDL) induce the expression of the receptor VCAM-1 (vascular cell adhesion molecule-1) on endothelial cells

• VCAM-1 is used as a receptor for monocytes, which cross the endothelial layer from the arterial lumen to the intima. This migration (diapedesis) is stimulated by the chemoattractant protein –1 (MCP-1)

• Once in the intima, monocytes express scavenger receptors to phagocyte the lipoprotein particles, and become macrophages. By accumulating lipid droplets in their cytosol, the macrophages take the aspect of foam cells and initiate the early atherosclerotic lesion (fatty streak). These foam cells proliferate by the release of macrophage colony-stimulating factor (M-CSF)

• Upon lipid accumulation, foam cells will die, and their debris accumulate in the form of atheroma

• Macrophages/foam cells release other growth factors that stimulate the proliferation of smooth muscle cells in the intima and the synthesis of extracellular matrix that will form the fibrous cap Pathogenesis of atherosclerosis Progression of atherosclerosis

Atherosclerosis progresses through seven stages:

• Normal artery. No lesion detected • Adhesion of macrophages to endothelial cells. Endothelial cells express VCAM-1 • Diapedesis. MCP-1 gradient in the intima of the vulnerable artery • Accumulation of atheroma. Phagocytosis of LDL particles by macrophages and subsequent death forming the atheroma • Plaque rupture. Rupture of a fragile fibrous cap, with contact between atheroma and circulating blood, and thrombosis • Healing fibrosis. Non-occlusive thrombus with plaque fibrosis • Occlusive thrombus. Lumen occlusion and interruption of blood flow Progression of atherosclerosis Example of human coronary atherosclerotic plaque

Atheroma

Fibrous cap Phenotypes of coronary plaques

Fibrotic = stable Lipid-rich = unstable

Collagen staining 3. Plaque rupture

Plaque rupture precipitates acute coronary syndromes

The fibrous cap separates the thrombogenic atheroma in the plaque from coagulation factors in the bloodstream. Fissure of the fibrous cap allows a contact between atheroma and coagulation factors, which triggers the formation of an intravascular thrombus. Plaque rupture is usually due to plaque inflammation

• In case of occlusive thrombus, the arterial lumen is totally obstructed, which interrupts blood flow and causes acute myocardial infarction.

• In case of a limited mural thrombus, blood flow persists. The resorption of the thrombus will engender a healing response that leads to fibrous tissue formation. Because the lesion becomes predominantly fibrotic, it is stabilized, but the residual lumen diameter after healing is far smaller than it was before thrombus formation. Occlusive versus non-occlusive thrombus

1. The thrombus is not occlusive. After healing, Fibrous plaque rupture the plaque becomes fibrotic Atheroma contacts coagulation factors but the residual lumen is Thrombus formation ensues small. This situation requires angioplasty

2. The thrombus is occlusive. Blood flow is interrupted, which engenders myocardial infarction. This situation requires thrombolysis Plaque thrombosis and vessel occlusion leads to myocardial infarction

Thrombus

Atheroma

This vessel needs to be repermeabilized ASAP to restore blood flow. Repermeabilization is performed with drugs digesting the thrombus, or thrombolytics Plaque thrombosis and partial vessel occlusion leads to plaque fibrosis

Atheroma is fluid. Upon plaque rupture, it will empty into the arterial lumen and be replaced by “healing” fibrosis Mechanisms of plaque rupture

• Lymphocytes can also penetrate the intima by binding to VCAM-1

• They are also attracted by specific chemokines (IP-10, Mig, I-TAC)

• In the atherosclerotic plaque, lymphocytes are activated by interaction with LDL particles and produce pro-inflammatory cytokines (interleukin, TNF-α, IFN- γ), which favor plaque instability by blocking collagen synthesis and promoting the production of matrix metalloproteases

• Matrix metalloproteases (collagenases, stromelysin, elastases) are enzymes specialized in the digestion of specific proteins of the extracellular matrix Mechanisms of plaque rupture Mechanisms of plaque rupture

The strength of the fibrous cap depends on the balance between collagen synthesis and degradation • Collagen synthesis is performed by smooth muscle cells, which can be blocked by interferon-γ (IFN- γ) produced from activated lymphocytes • Collagen degradation is performed by matrix metalloproteases (collagenases, gelatinases, stromelysin) produced by endothelial cells and macrophages • When inflammation prevail, cytokine expression (interleukin, TNF-α, IFN- γ) is high, and repair and maintenance of the fibrous cap decreases, which renders this structure weak and susceptible to fracture by hemodynamic stress 4. Cholesterol metabolism

Cholesterol is the primary cause and principal risk factor of atherosclerosis

• Function. Cholesterol increases the fluidity of the plasma membrane by its insertion between the hydrocarbon chains of the lipid bilayer. By increasing the fluidity of the membrane, it decreases its rigidity and improves the membrane capacity to deform (e.g., the membrane of red blood cells, which are “squeezed” in capillaries, is made of 25% cholesterol). Cholesterol is found only in animal cells. Bacteria and plants do not have cholesterol

• Origin. Cholesterol can be synthesized by the liver and is absorbed from food. Excess of circulating cholesterol (hypercholesterolemia) can result from excessive intake and/or excessive endogenous synthesis.

• Transport. Because of its relative insolubility, cholesterol is transported in the bloodstream by specific lipoproteins. Synthesis of endogenous cholesterol

• Endogenous cholesterol is mainly synthesized by the liver from acetyl-CoA

• Three molecules of acetyl-CoA associate to form hydroxy-methyl-glutaryl-CoA (HMG-CoA)

• HMG-CoA is reduced in mevalonate by the enzyme HMG-CoA reductase. This enzyme is the limiting step of cholesterol synthesis and the target of cholesterol- lowering drugs (lovastatin, simvastatin)

• Cholesterol is exported from the liver to peripheral tissues by the lipoprotein VLDL (Very-Low Density Lipoprotein) Synthesis of endogenous cholesterol Uptake of exogenous cholesterol

• Exogenous cholesterol is absorbed in the intestine

• Because of its water insolubility, cholesterol, together with fat, is bound to protein complexes in large lipoproteins called chylomicrons circulating in the lymph

• From the lymph, chylomicrons reach the general circulation, where fatty acids are removed from the lipoprotein by lipoprotein lipase expressed on endothelial cells. The chylomicron releases about 90% of its fat but keeps the cholesterol and becomes a remnant

• The remnant is taken up by the liver by receptor-mediated endocytosis, thus delivering cholesterol from the intestine to the liver Uptake of exogenous cholesterol Lipoprotein metabolism

• The liver exports cholesterol and fat to peripheral tissues in the form of specific lipoproteins, the VLDL (Very-Low Density Lipoprotein). Fat is distributed mainly to muscles for oxidation and to adipose tissue for storage. Due to fat release, the size of the lipoprotein decreases and becomes a LDL (Low-Density Lipoprotein)

• LDL lipoprotein is therefore rich in cholesterol. This LDL lipoprotein is taken up by receptor-mediated endocytosis in peripheral tissues for membrane renewal. If cholesterol concentration is in excess, LDL particles accumulate in the blood and become the trigger of atherosclerosis

• Cholesterol from peripheral tissues is picked-up by the HDL (High-Density Lipoprotein), which brings it back to the liver for elimination as bile acid. HDL can also remove cholesterol from foam cells and decrease atherosclerotic damage

• LDL Cholesterol is referred to as the “bad cholesterol” because it is the smoking gun of atherosclerosis. HDL cholesterol is referred to as the “good cholesterol” because it is the house-keeping mechanism that protects against atherosclerosis Lipoprotein metabolism Fish or chips?

• The omega-3 (Ω-3 or ω-3) fatty acids • Docosahexaenoic acid (DHA, 22:6, n-3) and eicosapentaenoic acid (EPA, 20:5, n-3) • Found in fatty fish, fish oil and algae • Decrease triglyceridemia by reducing lipogenesis and increasing lipid oxidation in liver and muscle (transcriptional effect on SRBPC, PPAR and HNF) • Decrease cholesterolemia by decreasing the substrates for VLDL synthesis and accelerating apoB degradation • Decrease neo-intimal proliferation and vasoconstriction, and improve exercise tolerance

• Trans fats • Fatty acids produced in technological (industry) and biological (ruminants) processes of isomerization of a cis double bond into a trans configuration • The most common are trans-octodecanoic acid (80:10, margarines) and vaccenic acid (milk) • Increase total cholesterol and decrease HDL cholesterol, due to an increase in rate of cholesterol synthesis • The exact clinical relevance is still a matter of debate and depends largely on the overall intake of fat 5. Risk factors

Atherosclerosis is precipitated by specific risk factors

Risk factors are independent variables that increase the risk and probability of developing atherosclerosis and cardiovascular disease

•Risk factors that can not be changed: • Age • Gender • Heredity

• Risk factors that can be changed: • High blood cholesterol • High blood pressure • Smoking • Obesity • Diabetes Risk factors that can not be changed

• Age. More than half of those who have heart attack are 65 or older. This is due to the slow progression of coronary artery disease through decades

• Gender. Men are more likely than women to develop coronary heart disease as a result of atherosclerosis. Estrogens have a predominant protective role against atherosclerosis. Therefore, the risk of heart attacks in women increases dramatically after menopause

• Heredity. Familial history of heart disease, especially before 55 years of age, constitutes a greater likelihood of having cardiovascular disease. Ethnic background also plays a role (more lipid plaques in Caucasians than African-Americans) Example of increased risk linked to heredity

A mutation in the LDL receptor prevents LDL uptake by endocytosis and thereby leads to increased circulating concentration of LDL particles Risk factors that can be changed

• High blood pressure. About 25% of the American population has hypertension. Increased blood pressure increases workload and vascular shear stress.

• Smoking. Counts for 40% of the death toll linked to coronary artery disease. Increases oxidative stress, decreases insulin sensitivity, impairs endothelial function, induces coronary spasms, increases circulating lipids and cholesterol, stimulates coagulation factors.

• Obesity. Multiplies the risk of coronary artery disease by three. Is often accompanied by diabetes and hypertension. Creates an excessive level of circulating lipids.

• Diabetes. Elevated insulin accelerates the deposit of cholesterol-rich particles in the arterial wall. Therefore, atherosclerotic plaques are most often lipid-rich and unstable. Diabetes is the most common cause of multiple atherosclerotic lesions on the coronary trees, which require bypass surgery. 6. Treatment of coronary artery disease

The best treatment of atherosclerosis is prevention

• Non-invasive treatment

• Treat the risk factors. The simplest way to avoid problems. Exercise, stop smoking, weight watch, diet, medical check-up, familial history • Cholesterol-lowering drugs. If LDL remains high despite diet, it must be lowered therapeutically • Vasodilators. In cases of symptoms of angina, open up the coronary arteries to a maximum

• Invasive treatment

• Angioplasty. In case of severe stenosis, crush the atherosclerotic plaque. Recurrence of the stenosis (30%) is limited by the addition of stenting • Thrombolysis. In case of acute occlusion of the vessel by a thrombus, thrombolytic therapy must be applied in emergency to avoid myocardial infarction • Bypass surgery. In case of multiple atherosclerotic plaques, a mammary artery can be sutured downstream of the stenosis Invasive treatment of coronary syndromes

Angiographic aspect of atherosclerotic plaque

Normal coronary artery Diseased coronary artery Principle of angioplasty and stenting

Symptomatic coronary lesion

The catheter with deflated balloon is introduced through the stenosis

The balloon is inflated to crush the lesion

Deflation and removal of the balloon. The patency of the artery is restored

A rigid stent can be placed at the lesion site to prevent recurrence Example of angioplasty

LAD occluded Angioplasty / stenting Reperfused artery Example of bypass surgery 7. Questions

1. What is the definition of atherosclerosis? 2. What are the different forms of atherosclerosis? 3. Explain the role of macrophages in the development of atherosclerosis 4. What are the seven stages of progression of an atherosclerotic plaque? 5. What is the mechanism of atherosclerotic plaque rupture? 6. What is the function and origin of cholesterol? 7. Describe the metabolism of LDL and HDL cholesterol 8. What are the risk factors of coronary atherosclerosis? 9. What is the treatment of coronary syndromes? 10. Describe the principle of angioplasty and stenting Molecular Medicine of the Heart

Class 9. Cardiac Hypertrophy and Athlete’s Heart Overview of class 9: Cardiac Hypertrophy

1. Definition 2. Mechanisms of hypertrophy 3. Characteristics of the hypertrophied heart 4. The athlete’s heart 5. Questions 1. Definition

Cardiac hypertrophy is an adaptation to increased workload

Hypertrophy is the mechanism by which cells increase in size. This is opposed to hyperplasia, which is the mechanism leading to increased cell number.

Increased cardiac workload requires an increase in the contractile capacity of the heart. Because of their limited mitotic capacity, if any, cardiac myocytes respond to increased workload by hypertrophy. A hypertrophied cardiac myocyte accumulates more sarcomeres, which improves its contractile capacity but also increases its energy needs

Hypertrophy is initially an adaptive mechanism by which an increased contractile demand is matched by an increased number of sarcomeres in the cardiac cell. Chronically, however, this increased contractile capacity becomes maladaptive and leads to cardiac dysfunction, or heart failure. Chronic cardiac hypertrophy therefore represents the most common cause and origin of heart failure

Hypertrophy is triggered by stress

The goal of cardiac hypertrophy is to restore normal mechanics through the operation of the Laplace’s law. In case of increased workload, cardiac stress is increased. Subsequently, stress can be normalized by an increase in wall thickness

When cardiac hypertrophy is followed by heart failure, the ventricular dilation will be accompanied by a marked increase in wall stress Causes of cardiac overload

Cardiac hypertrophy is an adaptive response to overload. The most common causes of overload are:

• Hypertension. Increased stiffness in conductance vessels and/or increased peripheral vascular resistance require a higher ventricular systolic pressure development

• Myocardial infarction. The irreversible damage of a part of the myocardium requires the remaining myocytes to work more

• Valve dysfunction. Aortic stenosis increases developed pressure. Aortic and mitral regurgitations increase cardiac volume

• Congenital diseases. Abnormal communications between cardiac cavities require a higher blood flow. Genetic mutations in contractile proteins require more sarcomeres Concentric versus eccentric hypertrophy

The causes of cardiac hypertrophy create either a condition of pressure overload (such as hypertension) or (such as mitral regurgitation).

• In case of pressure overload, the myocardium will respond by a concentric hypertrophy

• In case of volume overload, the myocytes will be stretched and respond by an eccentric hypertrophy. In chronic stages however, such as hypertensive heart failure, both forms are mixed Pressure overload Volume overload 2. Mechanisms of hypertrophy

The end-point of cardiac hypertrophy is an adaptation in the rate of protein synthesis

Cardiac hypertrophy results from an accumulation of sarcomeres in the cardiac cell, which is made possible by the coordinated activation of different mechanisms

• Increased protein synthesis, necessary to accumulate more sarcomeric proteins

• Adaptation of gene expression, to upregulate the transcription of genes encoding contractile proteins

• Activation of signaling pathways, to trigger the adaptation of gene expression (eg, calcineurin), the increased capacity of protein synthesis (eg, PI-3- kinase), or both (eg, MAP kinases)

• Activation of sensors and receptors, to activate the signaling pathways in response to overload (eg, G proteins or integrins) Increased wokload

Stretch sensors Receptors (integrins, ion channels) (G proteins, RTKs, gp130)

Signaling pathways (MAPK, PI-3-K, calcineurin, CamK)

Adaptation of Increased gene expression protein synthesis

HYPERTROPHY Sensors of hypertrophy

• Stretch-activated ion channels are specifically activated by increased load. These gadolinium-sensitive channels modify both Na+ and K + currents, which results in an opening of the L-type Ca2+ channel and an influx of Ca2+ that activates intracellular Ca2+-sensitive signaling pathways (calcineurin, Cam Kinase…)

• Integrins are transmembrane proteins that transduce inside the cell any changes in extracellular tension. An important intracellular transducer of integrins is focal adhesion kinase, a tyrosine kinase that can activate the PI-3- kinase pathway and the ERK 1/2 pathway Integrin signaling -1 Integrin signaling -2

Integrin clustering

Talin

FAK FAK Grb2 Src

Nuclear ERK1/ERK2 PI-3K translocation

Transcription Translation GATA4 P70S6K mTOR Akt Ets-1 UBF Elk-1 Transcription STAT Protein Metabolism cMyc synthesis CBP Receptors of hypertrophy

• Gq protein. The main stimuli of Gq protein in the heart are angiotensin II, endothelin-1 and the α1 adrenergic receptor. Through phopsholipase C, the Gq protein leads to the formation of diacylglycerol that activates PKC, an activator of the MAP kinase pathway

• Gs protein. The main stimulus of the Gs protein in the heart is the β adrenergic receptor. Through cAMP, this pathway activates CREB (cAMP-response element binding protein), a transcription factor that participates in the gene adaptation of hypertrophy. Cyclic AMP through PKA also increases free cytosolic [Ca2+], which activates Ca2+-dependent pathways

• Growth factor receptors. The main growth factors stimulating cardiac growth are insulin, IGF-1, TGFβ, PDGF, growth hormone and FGF. These receptors activate receptor-coupled tyrosine kinases that activate both the MAP kinase pathway and the PI-3-K pathway

• gp 130 receptor. Binds interleukin-6 or cardiotrophin and modifies gene expression through the activation of the JAK/STAT pathway Receptors of hypertrophy

PI-3-K CamK Calcineurin Roles of angiotensin II

In normal conditions, angiotensin II is mainly responsible for the control of vascular blood pressure. During hypotension or hyponatremia, there is an adrenergic stimulation of the kidney to release renin, a glycoprotein enzyme that cleaves the liver protein angiotensinogen into angiotensin 1. Angiotensin I is released in the blood and cleaved into angiotensin II by the angiotensin- converting enzyme (ACE). Angiotensin II increases blood pressure by stimulating the Gq protein in vascular smooth muscle cells (which in turn activates contraction by an IP3-dependent mechanism). ACE inhibitors are major drugs against hypertension

When cardiac cell growth is stimulated, a local (autocrine-paracrine) renin- angiotensin system is activated in the cardiac myocyte, which stimulates cardiac cell growth through PKC. The same system can be activated in smooth muscle cells in conditions of hypertension, atherosclerosis or endothelial damage. Angiotensin II is the most important Gq stimulator of the heart during hypertrophy, compared to endothelin-1 and α1 adrenoreceptors Roles of angiotensin II Roles of angiotensin II Roles of angiotensin II The gp 130 receptor

JAK = Janus Kinase STAT = Signal Transducer and Activator of Transcription Genes of hypertrophy

Signaling pathways

Immediate/early genes

Growth genes

Contractile Ca2+ Heat-shock Mitochondria proteins handling proteins Genes of hypertrophy - 1

Adaptation of gene expression in cardiac hypertrophy is both quantitative and qualitative

• Immediate/early genes. So called because their activation is very fast (within 30 minutes) and does not require protein synthesis. They are activated by signaling pathways (JAK/STAT, MAPK, calcineurin…) and encodes proto-oncogenes (fos, jun, myc…), the products of which are used as transcription factors to trigger the expression of the other genes of hypertrophy

• Heat-shock proteins. Chaperones that protect the structure of other proteins against denaturation. These proteins improve the translation efficiency by decreasing the rate of formation of denatured proteins. These proteins also have strong survival properties that protect the cardiac cell against apoptosis

• Mitochondrial genes. The expression of oxidative enzymes is increased, mainly by CREB, to stimulate the metabolic capacity of the heart Genes of hypertrophy - 2

Adaptation of gene expression in cardiac hypertrophy is both quantitative and qualitative

• Contractile proteins. All the genes encoding contractile proteins are stimulated during hypertrophy to increase the number of sarcomeres, and thereby increase the contractile capacity of the heart. These genes are controlled by immediate/early genes and by the transcription factors regulated by the signaling pathways. In rodents, there is an isoform switching from highly efficient contractile proteins to the form found in the fetal heart (“fetal gene program”). In larger mammals, including humans, the normal heart expresses already these slower isoforms and they are upregulated during hypertrophy.

• Ca2+ metabolism. There is a decreased expression of SERCA and phospholamban to increase free intracellular Ca2+ and thereby enhance contractility. There is an increase in L-type Ca2+ channel to stimulate the Ca2+-induced Ca2+ release Protein synthesis in hypertrophy

The end-point of the hypertrophic response is to increase the synthesis of proteins. This is done mainly by the mTOR pathway (mTOR= mammalian target of rapamycin).

• mTOR is mainly under the control of PI-3-K, but can also be stimulated by the MAP kinase pathway, and is also transcriptionaly stimulated during hypertrophy

• The two main phosphorylation targets of mTOR are p70S6 kinase and 4EBP1. The purpose of these two molecules is to improve the interaction of messengers RNA with the ribosomes to accelerate translation

• This adaptation is accompanied by an increased rate of ribosome synthesis upon activation of the transcription factor UBF (upstream binding factor) Protein synthesis in hypertrophy 3. Characteristics of the hypertrophied heart

The adaptation of the hypertrophied heart improves contractile efficiency

Characteristics of the hypertrophied myocardium include

• Increased duration of the action potential

• Increased tension development

• Changes in ion channels and pumps density

• Improved contact between cardiac myocytes and capillaries

• Increased content of interstitial collagen, which will impair diastolic relaxation Increased action potential in hypertrophy

• The density of L-type Ca2+ channels is not increased in hypertrophied myocyte, but is maintained, which requires an upregulation of the corresponding gene to match the increase in membrane surface

+ • However, the channel controlling the Ito K current is decreased. Therefore the phase 1 of the action potential is limited, which increases the duration of phase 2

• An increased phase 2 increases the duration of Ca2+ influx and therefore the duration of Ca2+ release from the sarcoplasmic reticulum. This increases the force of contraction, especially because Ca2+ reuptake by SERCA is decreased The five phases of the action potential

1 2 0

3

-85 mV 4 Increased action potential in hypertrophy Hypertrophy promotes angiogenesis and fibrosis

• Because the hypertrophied myocytes increase the space between capillaries, this stimulates neocapillarization, which in turn improves oxygen supply to the hypertrophied muscle

• The interstitium will be filled with more dense collagen produced from interstitial fibroblasts. This collagen maintains a better tension of the myocardium during systole but impairs its relaxation during diastole 4. The athlete’s heart

Exercise induces an increased demand in blood flow, not an overload

• During dynamic exercise, the cardiac output automatically increases to match the increased demand of the skeletal muscles and the heart itself. The resting value of the cardiac output is about 5 L/min and can reach up to 25 L/min for an Olympic athlete at maximal exercise

• Cardiac output = Heart Rate x Stroke Volume. An increase in cardiac output can be generated by

• a change in heart rate by positive chronotropy

• a change in stroke volume by change in preload • a change in stroke volume by a change in afterload • a change in stroke volume by a positive inotropy/lusitropy Cardiac adaptation to exercise Cardiac adaptation to exercise

CO = HR x SV

• Increased heart rate. Catecholaminergic stimulation of the SA node increases the firing rate up to 200 beats per min

• Increased preload. Improved venous return by α1 stimulation of the venous bed increases the diastolic volume and, therefore, the stroke volume

• Decreased afterload. Decreased peripheral vascular resistance by increased β2 stimulation of the arterioles also increases the stroke volume

• Positive inotropy/lusitropy. Gs protein stimulation by catecholamines activates PKA (L-type Ca2+ channel, TnI, phospholamban, MLC-2…), which improves both the end-systolic and end-diastolic pressure/volume relation

Flow distribution during exercise Cardiac adaptation to exercise

Dynamic exercise (aerobic or isotonic). Examples: running, bike riding, swimming. A rapid β-adrenergic stimulation increases heart rate and inotropy, which substantially increases the cardiac output. The total peripheral resistance decreases by β2 stimulation and by the release of adenosine from muscles. The systolic blood pressure increases because of the increased cardiac contractility. α1 stimulation of the venous bed increases the cardiac preload and, therefore, the stroke volume. The splanchno-renal area is vasoconstricted to redistribute the blood to the muscles

Static exercise (isometric). Examples: weight lifting, wrestling. Both systolic and diastolic pressures increase because of the increased muscular resistance. Heart rate increases only modestly. Stroke volume remains unchanged, so cardiac output increases proportionately to the heart rate. Oxygen demand increases more than the external work performed The normal pressure / volume relation

Effects of exercise on cardiac metabolism

Phosphorylase Translocation kinase Glycogen Glucose e + + GLUT

PH OS Glycogen Glucose i Glucose degradation HK uptake H-6-P

Fru 2,6-P2 + PFK-1 Glycolytic Fru-1,6-P2 flux

GAPDH

Lactate Uptake Lactate Pyruvate

Ca2+ + PD H Pyruvate Acetyl-CoA oxidation

Citrate Physiologic hypertrophy of the athlete’s heart

The exercised heart undergoes a process of hypertrophy to match the increased demand. At the opposite of pressure- or volume-induced hypertrophy, exercise hypertrophy is not the consequence of a dysfunction but a pure physiological adaptation to a change in workload. Therefore, the athlete’s heart at rest is characterized by bradycardia, because its increased contractile capacity (increased stroke volume) requires a lower heart rate, which is maintained by a higher production of acetylcholine

The stimulus for hypertrophy comes from both changes in workload and catecholaminergic stimulation. The gene and protein response resembles that found in overload hypertrophy and is accompanied by collateral development

The hypertrophy is not accompanied by accumulation of extracellular collagen, which preserves the diastolic properties of the myocardium. An athlete’s heart therefore does not evolve into heart failure

Exercise decreases the risk of heart disease

• Decreases LDL cholesterol • Increases HDL cholesterol • Decreases blood pressure • Decreases heart rate and limits arrhythmias • Increases collateral vascularization and neocapillaries • Increases vagal tone • Stimulates coronary vasodilation • Stimulates glucose metabolism, which limits diabetes and obesity 5. Questions

1. What are the most common causes of cardiac overload? 2. What is the definition of cardiac hypertrophy? 3. Describe the sensors and receptors of cardiac hypertrophy 4. Describe the gene response to cardiac hypertrophy 5. Describe the adaptation of protein synthesis during cardiac hypertrophy 6. Describe the roles of angiotensin II in normal heart, vascular damage, hypertrophy and heart failure 7. Explain the changes in action potential that occur during hypertrophy 8. What are the physiological parameters of the heart that are affected by exercise? 9. Explain the physiological adaptation of the heart to dynamic exercise 10. Why does exercise limit the risk of heart disease? Molecular Medicine of the Heart

Class 10. Cardiac Ischemia, Cell Death and Survival Overview of class 10: Cardiac Ischemia

1. Definition 2. Functional consequences of ischemia 3. Death mechanisms 4. Survival mechanisms 5. Post-ischemic reperfusion 6. Ischemic cardioprotection 7. Questions 1. Definition

Myocardial ischemia results from an imbalance between supply and demand

Myocardial ischemia represents an imbalance between oxygen and substrate supply and demand resulting in a dysfunction of the myocardium

The most likely cause of ischemia is an obstruction of coronary arteries by atherosclerosis resulting in:

• stable (exercise) angina. The coronary stenosis resulting from atherosclerosis uses the coronary flow reserve to maintain normal flow at rest, but the extra-flow available during exercise becomes insufficient, which therefore creates ischemia

• unstable (rest) angina. The atherosclerotic plaque fissured, creating a sub- occlusive thrombus. There is still some residual flow, but it is insufficient to sustain the needs at rest

• myocardial infarction. The atherosclerotic plaque fissured, creating an occlusive thrombus. There is no residual flow at all. The damaged myocardium can still be perfused marginally by collaterals Forms of angina Coronary autoregulation in diseased artery

Because of the mechanism of autoregulation, the coronary artery can maintain a constant flow (Q) despite large pressure gradients (ΔP) by using the coronary flow reserve. Therefore, the stenosis will become significant and limit blood flow only when at least 70% of the diameter (90% of the vessel area) is obstructed

1. Normal artery ΔP 2. Mild stenosis – Exercise angina- rest flow is maintained 3. Significant stenosis – Unstable angina- rest flow is reduced 1 4. Occlusion – Myocardial infarction - 2 3 Capillaries close

4 The slope is sharp. When a stenosis ! ! becomes significant, it is quickly critical Parameters of oxygen demand Imbalance between oxygen demand and supply Symptoms of cardiac ischemia

• Pain. Due to the stimulation of nerve endings by adenosine release. Crushing pain on the left part of the chest, extending typically to the left arm, and creating a feeling of anxiety (“angina”)

• Syncope. Due to arrhythmias, vagal stimulation or massive myocardial dysfunction

• Nausea. Due to an irritation of the diaphragm by an inflammatory reaction. Sometimes, nausea/vomiting is the only symptom of a heart attack

• Irregular heart beats. Signs of arrhythmias created by ischemia of the conduction system

• Dyspnea. Difficulty to breathe. Due to the accumulation of blood in the right circulation following a dysfunction of the left ventricle. Can be followed by pulmonary edema (effusion of plasma inside the lungs)

Any of these symptoms requires immediate medical attention (physical, blood test, EKG) 2. Functional consequences of ischemia

Ischemia results in both systolic and diastolic dysfunction

Cardiac ischemia induces both a systolic and diastolic cardiac dysfunction:

• Systolic dysfunction is characterized by impaired contraction. Contraction decreases because the lack of oxygen and blood flow leads to an accumulation of end-products (inorganic phosphate, protons, lactate…) and a loss of K+ that impair the function of contractile proteins. In addition, coronary pressure distends the sarcomeres (“garden hose” effect). In case of decreased blood flow, this effect disappears and the sarcomere length decreases, which reduces contractile efficiency

• Diastolic dysfunction is characterized by impaired relaxation. The lack of ATP impairs the function of the myosin ATPase, SERCA and the Na+/K+ ATPase. Impaired myosin ATPase leads to impaired relaxation of the myofilament. Impaired SERCA activity leads to increased [Ca2+] in diastole, and therefore, to impaired relaxation (“contracture”). Impaired Na+/K+ pump activity leads to accumulation of cytosolic Na+ that is extruded by an exchange with Ca2+ through the Na+/Ca2+ exchanger. This further increases cytosolic Ca2+ Causes of systolic dysfunction

• Metabolic inhibition

• The garden hose effect

• Potassium loss Effects of ischemia on cardiac metabolism

Catecholamines

Phosphorylase Translocation Glycogen kinase Glucose e + + GLUT

PH OS Glycogen Glucose i Glucose degradation HK uptake H-6-P

AMPK Fru 2,6-P2 PFK-1 PKA + Glycolytic Fru-1,6-P2 flux

GAPDH

H+ Lactate release Lactate Pyruvate

PD H - Pyruvate oxidation Fatty Acids Acetyl-CoA

Citrate Metabolic inhibition

Impaired energy production

Proton accumulation

Systolic dysfunction The “garden hose” effect

Normal flow. The coronary pressure distends the sarcomeres

Decreased flow. The sarcomeres “shrink” Potassium loss

Slowing of the Co-efflux with Opening of Na+/K+ pump phosphate ATP-inhibited channels

Potassium loss Accumulates in the extracellular space

Decreased resting membrane potential The membrane potential (Em) is calculated from the Nernst equation

Em = 61.5 ln (PK Ko/Ki + PNa Nao/Nai)

Decreased action potential Potassium loss

• In normal conditions, a K+ loss means hyperpolarization because the cell looses positive charges, which are immediately washed away in the bloodstream. This is the main mechanism of repolarization after the action potential and the mechanism of hyperpolarization by acetylcholine and adenosine

• In the ischemic heart, because of the reduced blood flow, the lost K + accumulates in the extracellular space. According to the Nernst equation, the membrane potential decreases because the K+ gradient decreases, and therefore the membrane depolarizes partially. It results that the influx of Na+ and Ca2+ during the action potential will be lower, and therefore the contraction of the myocyte will be decreased. Altogether, this is a protective mechanism of the ischemic heart to decrease energy demand. However, a partially depolarized membrane is particularly vulnerable to the development of arrhythmias

The membrane potential (Em) is calculated from the Nernst equation

Em = 61.5 ln (PK Ko/Ki + PNa Nao/Nai)

P, conductance; o, extracellular; i, intracellular Potassium loss

Normal conditions

Myocardial ischemia

The membrane potential (Em) is calculated from the Nernst equation

Em = 61.5 ln (PK Ko/Ki + PNa Nao/Nai) P, conductance; o, extracellular; i, intracellular Causes of diastolic dysfunction

Because of the slow-down of the Na+/K+-ATPase during ischemia, Na+ accumulates in the cell, which creates an osmotic swelling of the cell directly responsible for membrane rupture and death by necrosis. A mechanism to limit Na+ accumulation is an activation of the Na+/Ca2+ exchanger, which leads to an accumulation of diastolic Ca2+ and an increased diastolic tension of the myocardium called ischemic contracture. This contracture is amplified by the decreased capacity of SERCA to pump Ca2+ and by the decreased ATPase activity of the actin-myosin crossbridge Causes of diastolic dysfunction

ATP

Actin-myosin SERCA Na+/K+ pump

Reduced dissociation Reduced Ca2+ reuptake Accumulating Na+ is of myofilaments in diastole exchanged with Ca2+

Decreased lusitropy Contracture Example of ischemic contracture 3. Death mechanisms

Myocardial ischemia induces both necrosis and apoptosis

Prolonged severe ischemia leads to cardiac cell death, which occurs by necrosis and apoptosis. Irreversible cardiac cell loss is defined as Myocardial Infarction

• Necrosis results from severe loss in high-energy phosphates. The loss of ionic gradients by the impaired function of ion pumps leads to a swelling of the cardiac cell and mitochondria, and a rupture of the plasma membrane. This mode of cell destruction triggers an inflammatory response, essentially characterized by an attraction of polymorphonuclear leukocytes. This inflammatory reaction is in turn responsible for triggering the accumulation of fibrosis and scar formation following myocyte necrosis

• Apoptosis induces a serial activation of intracellular mechanisms that lead to the cleavage of specific intracellular proteins and DNA breakdown. This is followed by cell shrinkage, resulting in an apoptotic body which is absorbed by macrophages. The inflammatory reaction is close to none because the plasma membrane is preserved. Triggers of apoptosis are milder reductions in oxygen, which disrupt cytochrome C from the mitochondria, or the activation of specific receptors on the plasma membrane Mechanisms of cell death in the heart Importance of cytochrome c

All the proteins of the electron transport chain are organized in four multi-protein complexes that cross the inner mitochondrial membrane to extrude protons from one side to the other. The only exception is cytochrome c, a very small protein (12.5 kDa, 100 amino-acids) that is loosely attached on the outer surface of the inner membrane. This structure allows cytochrome c to move freely in order to transfer electrons from complex III to complex IV. In case of mitochondrial damage, cytochrome c is easily released and can migrate to the cytosol, where it is the main trigger of the mitochondrial pathway of apoptosis Ischemia

Oxygen deprivation Substrate deprivation

Anaerobic metabolism

Lack of ATP Acidosis

SERCA activity Na/H exchange Contraction Na/Ca exchange

Ca2+ accumulation

Mitochondrial damage Membrane damage

Apoptosis Necrosis

Cell death NECROSIS

Bax

ATP

Bax Cytochrome C - HYPOXIA Bad/Bcl-2 Bcl-2 + -

Cytochrome C

Bad P- Bad HSPs PKB APAF-1

Caspase-9

APOPTOSIS Consequences of myocardial infarction

The lost myocytes are replaced by non-contractile and dense connective tissue that stretches the ventricle. As a consequence, wall stress is increased and contraction is impaired. This progressively leads to a dilation of the ventricle followed by heart failure. 4. Survival mechanisms

The ischemic heart develops endogenous mechanisms of cardioprotection

In response to myocardial ischemia, the cardiac myocyte activates endogenous mechanisms of cytoprotection that delay and limit irreversible cell loss

The major mechanisms of cardioprotection are:

• Akt / PKB. Primarily a mechanism controling proteins synthesis under the control of PI-3-kinase, the phosphorylation targets of Akt also includes molecules regulating apoptosis

• Heat shock proteins. Primarily a mechanism maintaining the correct structure of proteins and preventing protein denaturation, HSPs can also inactivate different steps of the apoptotic cascade

• Hypoxia-inducible factor- 1α. Transcription factor that is constantly produced but immediately degraded by the proteasome when oxygen tension is normal. Under hypoxia, HIF-1α becomes stabilized and activates the expression of cytoprotective genes Mechanisms of survival in the heart

Akt/PKB Stimulate growth pathways (mTOR) Activate metabolism (glucose) Inactivates pro-apoptotic molecules (Bad, caspase-9) Activates eNOS

Heat-shock proteins Renature proteins Block apoptosis Preserve cell structure

HIF-1 Activates metabolism Activates angiogenesis Cardioprotective effects of PKB/Akt

PI-3-K / CamK

Forkhead PKB / Akt eNOS NO

Bim Bad Caspase-9 GSK-3

Bcl-2 Bcl-2 Caspase-3 HSF-1

HSPs Cardioprotective effects of Heat-Shock Proteins

Role #1. Anti-apoptosis Role #2. Preservation of cell structure

Cytochr. c HSP-60 Protects mitochondria

HSP-90 Protects plasma membrane Apaf-1

HSP-70 Protects nucleus/transcription Caspase-9 aB-crystallin Protects myofilaments Caspase-3

Role #3. “Chaperones”

All HSP preserve other proteins against denaturation Cardioprotective effects of HIF-1α

O2 Ubiquitination HIF-1α HIF-1α -OH proteasome degradation

HIF-1α

Glucose transporter VEGF Glycolytic enzymes IGF

Sustains metabolism Improves cell growth Prevents necrosis Activates survival pathways Prevents apoptosis 5. Post-ischemic reperfusion

Ca2+ and free radicals are the main causes of reperfusion injury

Myocardial reperfusion after ischemia can further enhance irreversible cellular damage through two main mechanisms

Ca2+. Accumulates in the cell through the Na+/Ca2+ exchanger. Its main deleterious effects are an increased diastolic tension and an activation of membrane-damaging phospholipases

• free radicals. Highly reactive molecules derived from oxygen, including . - . - superoxide radical ( O2 ), hydroxyl radical ( OH) and peroxynitrite (ONOO ). These molecules are formed by capture of electrons when the oxidative phosphorylation is reactivated at reperfusion in damaged mitochondria. Free radicals bind to phospholipids and degrade the plasma membrane Ca2+ and reperfusion injury

The washout of Na+, protons and lactate at reperfusion leads to a further increase in cytosolic Ca2+ which further impairs cardiac relaxation. Contracture of the cardiac cells blocks the capillaries (“no-reflow” phenomenon), which disturbs the reoxygenation, substrate supply and washout of degradation products in the ischemic territory. In addition, high [Ca2+] activates phospholipases that degrade phospholipids of the plasma membrane and cause further cell death Free radicals and reperfusion injury Example of reperfusion injury 6. Ischemic cardioprotection

Preconditioning, stunning and hibernation are three forms of adaptation to ischemia

• Ischemic preconditioning Several short episodes of ischemia/reperfusion confer resistance against irreversible damage during a subsequent longer period of ischemia. The main effect of ischemic preconditioning is to decrease infarct size

• Myocardial stunning A short episode of ischemia (such as angina) is followed by transient dysfunction at reperfusion, then full functional recovery. The main effect of myocardial stunning is to allow full functional recovery after brief ischemia

• Myocardial hibernation Prolonged ischemia leads to phenotypic changes of the cardiac myocyte that decrease its contractile activity. Upon reperfusion (bypass surgery or PTCA), such myocardium functionally recovers. The main effect of myocardial hibernation is to maintain cell viability in a context of chronic ischemia Example of Preconditioning in Mouse

CTRL 45 min 24 h reperfusion TTC 6 x 4/4 no- flow PC

TTC

CONTROL PRECONDITIONING Physiological effect of Preconditioning Mechanisms of Preconditioning

Early phase of preconditioning: protection immediately after preconditioning protocol, due to adenosine-mediated opening of ATP-dependent K+ channels

Late phase of preconditioning: protection 24-72 hours after preconditioning protocol, due to PKC-dependent activation of survival genes Example of Stunning in Swine

Stenosis Reperfusion 140 120 Blood Flow 100 Flow 80 Probe STUNNING 60

40 Wall Thickness

% of control value control of % 20

LV Pressure 0 Gauge -90 -60 03060+12 h Time after reperfusion (min) Mechanism of stunning

Both free radicals and Ca2+ participate in the desensitization of crossbridge cycling. In addition, the different troponins can be degraded during ischemia Example of hibernating myocardium in patients

Normal Ischemic

Normal

Ischemic

Hibernating myocardium is fed through collaterals, which prevents cell death Mechanisms of myocardial hibernation

• Loss of myofilaments (decreases energy demand)

• Decreased content of mitochondria (decreases O2-dependent metabolism) • HIF-1α-mediated activation of glucose metabolism • Accumulation of glycogen • Activation of Akt (survival and glucose metabolism) • Activation of survival genes (heat-shock proteins, growth factors) • Repression of pro-apoptotic pathways 7. Questions

1. What is the definition of myocardial ischemia? 2. Explain the mechanisms and consequences of K+ loss during myocardial ischemia 3. Compare cardiac cell necrosis and apoptosis 4. Describe the three main molecular survival mechanisms of the ischemic heart 5. Explain the role of heat-shock proteins in normal and ischemic heart 6. Explain the role and mechanism of action of HIF-1α 7. Explain the role of Ca2+ in both ischemic and reperfusion injury 8. Explain the role of free radicals in reperfusion injury 9. Define and explain myocardial preconditioning 10. What are the symptoms of myocardial ischemia? 11. Define and explain myocardial hibernation

Molecular Medicine of the Heart

Class 11. Heart failure Overview of class 11: Heart failure

1. Definition 2. Physiology of heart failure 3. Molecular basis of heart failure 4. Clinical aspect of heart failure 5. Treatment of heart failure 6. Questions 1. Definition

Heart failure is the end stage of all forms of heart disease

Heart failure represents a condition in which the heart fails maintaining its physiological function, which is to supply enough blood to the entire body. Rather than a disease in itself, heart failure represents the end-stage consequence of most forms of heart disease

Heart failure results from three potential mechanisms: pressure overload, volume overload or primary myocardial disease

All forms of heart failure are characterized by four physiological parameters: negative inotropy, increased diastolic dimension, increased preload and increased afterload

All forms of heart failure include both a depressed contraction and an impaired relaxation

At the molecular level, heart failure results from impaired energy production, increased energy consumption, altered Ca2+ fluxes, decreased crossbridging and cell death The basic principle of heart failure is a myocardial congestion resulting from a dramatically reduced capacity of blood ejection. As a result, the preload of the failing heart increases. The afterload also increases by vasoconstriction

NORMAL FAILING Some numbers

Each year, there are 400,000 new cases of heart failure in USA

There are currently 5 million patients with heart failure in USA

The total cost of heart failure is $18 billion per year

The mortality of advanced heart failure is 30% per year

The overall 5-year survival rate is 25% Causes of heart failure

Pressure load • Aortic stenosis • Hypertension

Volume load • Myocardial infarction • Aortic valve regurgitation • Mitral valve regurgitation • Congenital Heart disease

Primary myocardial disease • Hypertrophic cardiomyopathy • Dilated cardiomyopathy • Aging • Metabolic disease Global mechanism of heart failure

• Any form of cardiac overload leads to increased catecholaminergic stimulation, increased energy demand, increased ventricular pressure and cavity dilation, which all result in increased wall stress

• After a phase of compensation (such as hypertrophy), these parameters lead to focal cell death and fibrosis. For instance, mitochondrial dysfunction due to increased energy demand can lead to cytochrome c release and focal apoptosis, or Ca2+ overload can lead to necrosis

• Fibrosis increases ventricular stiffness, which further increases wall stress and therefore leads to more ventricular dilation. Increased wall stress further increases the parameters of overload: increased catecholaminergic stimulation, increased energy demand, increased ventricular pressure and cavity dilation

• The heart enters a vicious cycle that leads to further dilation, cell loss, Ca2+ alterations and energy imbalance Global mechanism of heart failure 2. Physiology of heart failure

The failing heart is like a tired horse that is constantly whipped

The four physiological characteristics of the failing heart, their mechanisms and their consequences are:

• Decreased inotropy: due to alterations in myocardial energetics, cross bridging and Ca2+ metabolism. decreased cardiac output

• Increased end-diastolic volume: due to ventricular dilatation, which increases wall stress. cavity enlargement

• Increased afterload: there is a reflex vasoconstriction due to decreased cardiac output. increased peripheral resistance

• increased preload: due to the decreased cardiac output, blood accumulates in the pulmonary circulation. venous congestion of the failing heart Decreased inotropy

Contractile function in the failing heart is insufficent and does not respond to beta- adrenergic stimulation anymore. It creates a negative inotropic effect that results in a decreased stroke volume

Decreased inotropy Increased cardiac volume

The increase in wall stress results in ventricular dilation. Theoretically, an increased end-diastolic volume should result in an increased stroke volume. However, because of the negative inotropic effect, the stroke volume is actually further decreased

Decreased inotropy with

Increased cardiac volume Increased afterload

Because of the decreased cardiac output, there is a vasoconstrictory reflex that further increases the afterload, resulting in a further decrease in stroke volume

Decreased inotropy with

Increased cardiac volume with

Increased afterload Increased preload

Because of the limited ejection capacity, the returning blood accumulates upstream the left ventricle, which increases the pulmonary vein pressure. As a consequence, the end-diastolic pressure of the ventricle increases, which further dilates the ventricle and increases wall stress

Decreased inotropy with

Increased cardiac volume with

Increased afterload

with

Increased preload

= HEART FAILURE Pressure / volume relationship of the failing heart

Compared to a normal heart, the failing heart shifts to higher volumes because of the cavity enlargement and increased preload, but decreased output due to the negative inotropic effect and increased afterload Pressure / volume relationship of the failing heart

The decrease in contractility and increase in diastolic pressure combine to dramatically reduce cardiac performance and increase internal work The contractile function is decreased by wall stress

Stress = Tension per surface

Tension = Forces pulling an object apart (cfr. a spring)

Laplace’s Law: Wall stress is proportional to • pressure • radius

And inversely proportional to • wall thickness wall stress wall stress wall stress

Heart failure by pressure load

Heart failure by volume load wall stress

Pressure x Radius Stress = Thickness

Pressure overload: increase in pressure requires an increase in wall thickness to maintain normal stress. If the cause of pressure is not treated, heart failure will ensue when wall thickness can not increase further and contractile function deteriorates

Volume overload: increase in radius requires an increase in wall thickness to maintain normal stress. If the cause of dilation is not treated, heart failure will ensue when wall thickness can not increase further and contractile function deteriorates

Exercise: Chronic exercise increases wall thickness without affecting pressure or volume. The wall stress is therefore decreased 3. Molecular basis of heart failure

Profound alterations of cellular function lead to cell death in the failing heart

The causes of contractile dysfunction of the failing heart are:

• Decreased crossbridging of the myofilaments • Energy deprivation • Increased energy consumption • Beta-adrenergic uncoupling • Decreased Ca2+ storage in the SR • Cell death • Increased afterload Decreased crossbridging

Increased ventricular pressure and diameter alter the actin/myosin interaction and thereby decrease the number of crossbridges. Titin is overstretched, which rises the resting tension of the sarcomeres and, therefore, of the myocytes (resulting in decreased lusitropy)

Titin prevents further extension…

… therefore pressure rises Energy starvation

The increased internal work requires more ATP to be produced but the progressive mitochondrial dysfunction impairs energy provision. The lack of ATP will result in an accumulation of cytosolic Ca2+ by the slowing of ion pumps and exchangers Depressed inotropy

There is an uncoupling of the beta-receptors because of their chronic and constant stimulation. BARK expression and activity are increased, which uncouples the receptor from Gsα. By feed-back, the gene expression of β receptors and Gsα increase. In addition, the Gi protein expression increases, which further blocks the Gs system. As a result, the production of cAMP is markedly inhibited. The heart is therefore continuously stimulated by catecholamines, but does not respond. Depressed inotropy

There is an uncoupling of the beta-receptors because of their chronic and constant stimulation, and therefore the level of cAMP decreases. Therefore, phospholamban is not phosphorylated and impairs Ca2+ reuptake in the SR. In addition, SERCA gene expression is decreased as an attempt to increase contractile force. Therefore, the SR progressively “empties”. Because of this impairment in Ca2+ release/reuptake during the cardiac cycle, [Ca2+] remains more or less constant, which impairs the actin/myosin interaction Alteration of Ca2+ transients in the failing heart

In the failing heart, Ca2+ transients between systole and diastole are severely blunted. The sarcomere is therefore never really stimulated to contract or relax. This effect has a net negative inotropic effect and also impairs diastolic relaxation. The Ca2+ that empties from the SR is taken up by the mitochondria, which impairs their function and leads to cell necrosis Cell death in the failing heart

The Ca2+ that empties from the SR is taken up by the mitochondria to limit its cytosolic accumulation, which impairs their function and leads to cell necrosis. Apoptosis is also activated in the failing heart, both by the release of cytochrome c from dysfunctional mitochondria and by the increased production of cytokines (such as TNFα). Roles of angiotensin II – part 1

In normal conditions, angiotensin II is mainly responsible for the control of vascular blood pressure. During hypotension or hyponatremia, there is an adrenergic stimulation of the kidney to release renin, a glycoprotein enzyme that cleaves the liver protein angiotensinogen into angiotensin 1. Angiotensin I is released in the blood and cleaved into angiotensin II by the angiotensin- converting enzyme (ACE).

In hypertension, angiotensin II increases blood pressure by stimulating the Gq protein in vascular smooth muscle cells, which in turn activates contraction by an IP3-dependent mechanism and promotes cell proliferation. ACE inhibitors are major drugs against hypertension

When cardiac cell growth is stimulated, a local (autocrine-paracrine) renin- angiotensin system is activated in the cardiac myocyte, which stimulates cardiac cell growth through PKC. The same system can be activated in smooth muscle cells in conditions of hypertension, atherosclerosis or endothelial damage. Angiotensin II is the most important Gq stimulator of the heart during hypertrophy, compared to endothelin-1 and α1 adrenoreceptors Roles of angiotensin II – part 2

In heart failure, both low renal perfusion pressure and adrenergic stimulation lead to renin release from the renal glomerula, which results in angiotensin production and activation. Angiotensin II • increases the vascular tone, which is already stimulated by adrenergic stimulation • stimulates the production of aldosterone, which is responsible for Na+ and fluid retention, and thereby hemodilution (decreased concentration of red blood cells)

The low renal perfusion pressure is due to a low cardiac output, not to a low plasma volume. Therefore, the retention of Na+ and water leads to excessive circulating fluid and to the formation of edema.

The net effects are an accumulation of fluid together with a vasoconstriction, which both increase the afterload

Angiotensin-II also activates the production of collagen by cardiac fibroblasts, which increases cardiac fibrosis Roles of angiotensin II Molecular basis of increased afterload

Low renal perfusion and adrenergic stimulation increase the release of renin, which in turn activates angiotensin and aldosterone Molecular basis of increased afterload

The vasoconstrictory effect results from both angiotensin II and α1-adrenergic stimulation Catecholamines and heart failure

Noradrenaline whips the heart…

… and increases vascular resistance 4. Clinical aspect of heart failure

Heart failure results in global dysfunction of the organism

Heart failure results in a dysfunction of many other organs than the heart, including lung, liver, kidney, brain, blood and muscles. Most of these organs are affected by the increased preload and/or the low cardiac output

The main symptoms of heart failure result from both a “backward” and a “forward” failure. “Backward” failure are the extra-cardiac consequences of heart failure upstream of the failing heart. “Forward” failure are the extra-cardiac consequences of heart failure downstream of the failing heart Heart failure is both “backward” and “forward”

Backward heart failure. Characterized mainly by an increased pulmonary capillary pressure, due to the incapacity of the failing heart to eject the venous return. The pressure in the pulmonary circulation can increase to such a level that plasma effuses into the pulmonary alveoli, which is known a pulmonary edema. The venous blood also accumulates in the liver, which becomes congested (hepatomegaly) and can suffer irreversible damage (cardiac cirrhosis). The venous pressure can also increase in the kidney circulation, leading to renal damage

Forward failure. Due to poor ejection. Poor muscle perfusion impairs physical activity. Poor general perfusion impairs brain functions. Decreased cardiac output induces a reflex vasoconstrictory response, including angiotensin II (AT II). AT II stimulates the production of aldosterone, which retains Na+ in the kidneys. This increases plasma volume and provokes leg edema. The accumulation of plasma also decreases the concentration of red blood cells (hemodilution) “backward” heart failure “forward” heart failure Symptoms of heart failure

Symptoms Causes

Dyspnea Pulmonary congestion Distended liver Venous congestion Limb fatigue Low cardiac output Cold extremities Peripheral vasoconstriction Tachycardia Reflex to low cardiac output Edema Aldosterone activation Oliguria Fluid retention Pale skin Hemodilution, low output 5. Treatment of heart failure

Heart failure eventually requires cardiac transplantation

The therapeutic options for the failing heart are:

Drug therapy • Diuretics • ACE inhibitors • Vasodilators • Beta-blockers

Palliative surgery • Left ventricular assist device (LVAD) • Cardiomyoplasty

Cardiac transplantation Drug therapy

• Diuretics: limit fluid retention by eliminating Na+ and water. The side-effect is the possibility of hypotension

• ACE inhibitors: block the effects of excessive angiotensin II, by limiting its vasoconstrictory and fluid-accumulating effects

• Vasodilators: decrease the afterload by limiting the vasoconstricting effects of angiotensin II and adrenergic stimulation

• Beta-blockers: the beta-adrenergic system in the failing heart is uncoupled (non-responsive) because constantly stimulated. Low-dose beta-blockers will interrupt this stimulation and thereby reactivate the system. The result is an improved inotropy of the failing heart. It is important to use low doses of beta- blockers, otherwise the system would be totally inhibited Palliative surgery

• Left ventricular assist device (LVAD): Mechanical pump that takes the blood from the failing myocardium and ejects it into the aorta. The failing heart is unloaded and its dimensions decrease. The LVAD is used as a “bridge” for patients awaiting cardiac transplantation, but can sometimes improve significantly the function of the failing heart

• Cardiomyoplasty: Skeletal muscle wrapped around the failing heart and stimulated by a pace-maker. This external muscle improves the geometry of the failing ventricle and supports the contraction Example of left ventricular assist device Example of cardiomyoplasty Cardiac transplantation

Cardiac transplantation is the only cure of advanced heart failure and is indicated when conventional drug therapy fails. It is performed by grafting the ventricles of a donor to the atria of the recipient.

Advantage: the patient is cured and has a new heart

Disadvantages: • fear of rejections • the correct function of the graft must be controlled often • anti-rejection drugs have multiple side-effects • most of all: cruel lack of donors 6. Questions

1. What is the definition of heart failure? 2. What are the causes of heart failure? 3. Describe the four physiological characteristics of the failing heart 4. Describe the pressure/volume relationship of the failing heart 5. What are the molecular causes of contractile dysfunction in the failing heart? 6. Explain the molecular basis for depressed inotropy in the failing heart 7. Explain the molecular basis for increased afterload in the failing heart 8. Describe the “backward” heart failure 9. What are the symptoms of heart failure? 10. What are the therapeutic options for the failing heart? 11. What is the rationale of beta-blockade in patients with heart failure? 12. Explain the left ventricular assist device