2 HISTORY Ringer first discovered the dependency of the beating heart on extracellular Ca2+ (1882).Ebashi (1976) and Weber & Murray (1973) initially described the importance of the sarcoplasmic reticulum (SR) in skeletal muscleFabiato (1985) introduced the concept of CICR in the heart
6 SARCOMERESarcomere –bet. Two Z linesI BAND –Only thin filamentA BAND- Both thick& thin filamentsH zone – only myosinM line – centre point of sarcomere
7 Components of excitation contraction coupling Sarcolemma(1) SR coupling in the form of dyads by means of the T-tubule(2) Caveolae(3) Intercalated disc4)Ankyrins
8 SARCOPLASMIC RETICULUM The SR is an intracellular membrane-bounded compartment comprised of terminal, longitudinal, and corbular componentsThe free walls of the terminal cisternae are apposed to the walls of the T-tubules and form the dyadic cleftThe RyR2 receptors- located in the walls of the terminal cisternae (feet) and face the dyadic cleftLongitudinal SR is fairly homogenous and contains primarily the SR Ca2+-ATPase proteins, SERCA2 &phospholambanSR calcium is transported from the tubular lumen of the SR to the terminal cisternae, where it is stored mostly bound to calsequestrin.
9 Transverse Tubules and the Sarcoplasmic Reticulum The transverse (T) tubules are an extensive network of muscle cell membrane (sarcolemmal membrane) that invaginates deep into the muscle fiber. The T tubules are responsible for carrying depolarization from action potentials at the muscle cell surface to the interior of the fiberThe sarcoplasmic reticulum is an internal tubular structure, which is the site of storage and release of Ca2+ for excitation-contraction coupling
12 MYOSINThe myosin molecule consists of two heavy chains with a globular head, a long -helical tail, and four myosin light chains500,000 DaTransduction of chemical to mechanical energy and work is the function of myosin ATPase, located in the myosin heads.Two myosin light chains (the alkali or essential light chain [MLC1] and the phosphorylatable or regulatory light chain [MLC2]), are associated with each myosin head and confer stability to the thick filament.
13 ARRANGEMENT OF THICK AND THIN FILAMENTS Myosin molecules arranged in apolarized manner , leaving globular portions projecting outwardSo that they can interact with actin to generate force & shortening
15 TITINTitin is the largest protein which is extraordinarily long, flexible, and slender.Titin molecule extends from the Z-line, stopping just short of the M-lineIt has two distinct segments:1) An inextensible anchoring segment2) An extensible elastic segment that stretches as sarcomere length increases.
16 Titin – functions1) It tethers the myosin molecule to the Z-line, thereby stabilizing the contractile proteins2) As it stretches and relaxes, its elasticity explains the stress-strain relation of cardiac and skeletal muscle.3) Increased diastolic stretch of titin as the sarcomere length of cardiac muscle is increased causes the enfolded part of the titin molecule to straightenThis stretched molecular spring then contracts more vigorously in systole4) Titin may transduce mechanical stretch into growth signals.
18 MYOSIN BINDING PROTEIN C Myosin-binding protein C runs at approximately right angles to the myosin molecules to tether myosin molecules by linking the structures that lie around subfragments of the myosin heads. This binding protein, which stabilizes the myosin head, itself flexes and extends at the level of the light chainsDefects in the binding protein C may be involved in some types of hypertrophic cardiomyopathy.
19 Thin Filaments The backbone -helical double-stranded actin Tropomyosin is a long, flexible, double-stranded (largely -helix) protein that lies in the groove between the actin strands and inhibits the interaction between actin and myosinThe troponin complex is composed of1)A calcium-binding subunit- troponin C (TnC)2) An inhibitory subunit to actin- troponin I (TnI)3) A tropomyosin-binding subunit- troponin T (TnT)
21 EXCITATION CONTRACTION COUPLING The cascade of biological processes that begins with the cardiac action potential and ends with myocyte contraction & relaxation
22 Influx of extracellular calcium Calcium-induced calcium release (CICR) – Ryanodine receptorActivation of actin myosin cross bridgesCardiac muscle contrn.Decrease in intracellular calcium- SERCA /NCXCardiac muscle relxn.
23 Action Potential to Excitation-Contraction Coupling CICRL-type Ca+2 channelCalcium removal by Na/Ca exchangerCalcium movement into the cell during phase 2 begins the process of excitation-contraction couplingBrief overviewCalcium enters the cell through L-type channels located in the T-tubules of the sarcolemmaCalcium-induced calcium release (CICR) occurs when the “trigger” calcium from L-type channels is sensed by the ryanodine receptors (RyR). This causes Ca release from the sarcoplasmic reticulum (SR).Ca interacts with troponin-C (TN-C), causing troponin-I (TN-I) to uncover a myosin binding site on actinIntracellular Ca is reabsorbed into SR via the sarco-endoplasmic reticulum Ca ATPase (SERCA) pump and removed from the cell via other means such as Na/Ca exchanger and an ATP dependent Ca pump.As intracellular Ca decreases, Ca dissociates from TN-C and the binding site on actin is inhibited. ATP is required to unbind myosin from actin and reset the sarcomere to its normal length.Calcium movement from uptake to release siteCalcium uptake by SERCAForce production
25 Calcium sparks (localized [Ca2+]i transients) are the elementary SR Ca2+ release events that trigger E–C coupling in heart muscle.The basis for the generally accepted local control theory of E–C coupling is that Ca2+ sparks are triggered by a local [Ca2+]i established in the region of the RyR2s by the opening of a single L-type Ca2+ channel.The amplitude of Ca2+ sparks is determined by SR Ca2+ load and gating properties of the RyR2
26 Factors responsible for the [Ca2+]i transient amplitude include (1) the calcium current (ICa), primarily caused by Ca2+ influx through the L- type Ca2+ channel, but in small part caused by reverse mode NCX(2) SR [Ca2+]i content(3) the efficiency of E–C coupling(4) intracellular Ca2+ buffersThe decline of the [Ca2+]i transient is caused by(1) Ca2+ reuptake into SR by SERCA2 (a process modulated by a phosphorylatable regulatory protein termed phospholamban)(2) Ca2+ extrusion from the cell by the NCX(3) Ca2+ extrusion from the cell by the sarcolemmal Ca2+-ATPase(4) Ca2+ accumulation by mitochondria(5) Ca2+ binding to intracellular buffers (including fluorescent indicators that are used in experimental systems to measure the transient).
27 Calcium release & uptake Ryanodine ReceptorsThe calcium release channel is part of the complex structure known as the ryanodine receptor, so called because it coincidentally binds the potent insecticide ryanodine; it is often abbreviated to RyR2 to indicate the cardiac isoform.Ryanodine receptors have a dual function, both containing the calcium release channels of the SR and acting as scaffolding proteins that localize numerous key regulatory proteins to the junctional complexes.These proteins include the important stabilizing protein (technical term: FKBP-12.6) that responds to phosphorylation to coordinate opening of neighboring ryanodine calcium channels by the process of coupled gating.
29 Calcium Induced Calcium Release (CICR) Intracellular [Ca] 10-7 to 10-5 M1231. Ca enters the cell through voltage controlled L-type channels located in the T-tubules of the sarcolemma2. Ca entry further stimulates Ca release from the SR via sensing by the RyR. Intracellular Ca concentrations increase about 100x, from 10-7 to 10-5 M.3. The rise in intracellular Ca, caused by CICR from the SR, allows Ca to bind to TN-C in a concentration dependent manner.Ca++ enters the cell through L-type calcium channelsCa++ stimulates Ca++ release from the SR via RyRCa++ interacts with contractile proteins to initiate shortening of the myocyte
30 Pictorial E-C Coupling Na+Na+SarcolemmaNa+Ca++Na+/Ca++ ExchangerCa++L-Type Ca++ChannelCa++Ca++PlbCa++Ca++Ca++SERCACa++Ca++Ca++Ca++Ca++Ca++Ca++Ca++The L-Type (“long lasting”) Ca Channel is a voltage controlled gate located in the T-tubules of the sarcolemma. Ca enters the cell through this channel during phase 2 of the action potential.This Ca stimulates further Ca release from the SR via the RyR. This process is referred to as calcium induced calcium release (CICR).Once contraction is complete, Ca is dissociates from TN-C and is reabsorbed by the SR via the SERCA pump. Phosphorlyation of phospholamban (Plb) increases the activity of SERCA.Phosphorylation occurs when a phosphate group binds to a molecule.Ca is also removed through the Na/Ca exchanger in a ratio of 3 Na ions for each Ca ion. An ATP dependent Ca pump also helps remove Ca from the cell.Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++SRCa++Ca++RyRCa++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++Ca++
31 Acto-myosin interaction. Acto-myosin interaction. The myosin head carrying the ATPase site combines with actin to produce force. Calcium binding to troponin C (TnC) results in a conformational change of tropomyosin, troponin I (TnI), and troponin T (TnT), allowing the myosin head to attach to actin, facilitating the acto-myosin cross-bridge to cycle (see also Figures 2 and 6).Hasenfuss G , Teerlink J R Eur Heart J 2011;eurheartj.ehr026
32 Interaction Between the "Activated" Actin Filament and the Myosin Cross-Bridges- The "Walk-Along" Theory of ContractionThe head attaches to an active site, this causes forces between the head and arm of its cross-bridge. this causes the head to tilt toward the arm and to drag the actin filament along with it. This tilt of the head is called the power stroke.Then, immediately after tilting, the head automatically breaks away from the active site. Next, the head returns to its extended direction. In this position, it combines with a new active site farther down along the actin filament; then the head tilts again to cause a new power stroke, and the actin filament moves another step
33 A, At the beginning of the cycle, no ATP is bound to myosin, and myosin is tightly attached to actin in a "rigor" position. In rapidly contracting muscle, this state is very brief. However, in the absence of ATP, this state is permanent (i.e., rigor mortis
34 B, The binding of ATP to a cleft on the back of the myosin head produces a conformational change in myosin that decreases its affinity for actin; thus, myosin is released from the original actin-binding site. C,
35 C, The cleft closes around the bound ATP molecule, producing a further conformational change that causes myosin to be displaced toward the plus end of actin. ATP is hydrolyzed to ADP and Pi, which remain attached to myosin
36 D, Myosin binds to a new site on actin (toward the plus end), constituting the force-generating, or power, stroke. Each cross-bridge cycle "walks" the myosin head 10 nanometers along the actin filament
37 E, ADP is released, and myosin is returned to its original state with no nucleotides bound (A). Cross-bridge cycling continues, with myosin "walking" toward the plus end of the actin filament, as long as Ca2+ is bound to troponin C.
40 muscle contraction occurs by a sliding filament mechanism This is caused by forces generated by interaction of the cross-bridges from the myosin filaments with the actin filaments.
41 MitochondriaMitochondria comprise approximately 35% of ventricular myocyte volume and according to their cellular location are designated as either subsarcolemmal or interfibrillar.Mitochondria are the sites of oxidative phosphorylation and ATP generationAlthough they have the capacity to buffer large amounts of Ca2+ and are a potential source of activator calcium, classical teaching is that their contribution to E–C coupling is minimal in view of the short time constants involvedIn addition, the ability to accumulate large amounts of Ca2+ under pathological conditions (eg, ischemia) can help protect against myocyte Ca2+ overload; however, Ca2+ accumulation by mitochondria ultimately slows ATP production
42 NONO is produced by the myocardium and regulates cardiac function through both vascular-dependent and independent effectsNO's positive effects on relaxation or lusitropy are likely to be caused by (cGMP)-mediated reduction in myofilament Ca2+ sensitivityMitochondrial NO reduces maximal venous oxygen (MVO2) consumption and increases mechanical efficiency (stroke work/MVO2), suggesting that NO regulates energy production as well influencing consumptionNOS III- T tubule caveole – cgmp mediated lusiotropyNOS I – cardiac SR – modulates Ca 2+ homeostasis
43 DIASTOLEDiastole is the summation of processes by which the heart loses its ability to generate force and shorten and returns to its precontractile state.Diastolic properties of the ventricle are complex and multifactorial.Diastole occurs in a series of energy-consuming steps beginning with release of calcium from troponin CDetachment of actin–myosin crossbridgesSERCA2a-induced calcium sequestration into the SR,NCX-induced extrusion of calcium from the cytoplasmReturn of the sarcomere to its resting length.
44 DIASTOLEAdequate ATP must be present for these processes to occur at a sufficient rate and extent.The P–V relationship during early diastole reflects the lusitropic (relaxation) state of the heart, analogous to the inotropic (contraction) state measured during systoleThe rate of LV relaxation can be estimated from the maximal rate of pressure decay (–dP/dtmax) and indices (eg, relaxation half-time [RT1/2]) that are related to the time necessary for ventricular relaxation,
45 CALCIUM UPTAKE -SERCACalcium uptake is mediated by SERCA ATPase in the sarcoplasmic reticulum.This is an active process and for each ATP -2 Ca2+ taken upThis uptake is regulated by phospholambanPhosphorylated phospholamban activates SERCACalcium taken up is stored in the junctional SR with calsequesterin and calreticulinSERCA down regulated in heart failure
46 SR Calcium UptakePhospholambanSR removes Ca++ through an ATP dependent pump (SERCA)Disinhibition of phospholamban increases the rate of calcium uptakeCytosolic Ca++ decreases and Ca++ is removed from TN-CExcess Ca++ is removed from the cell by other processesCa entry diminishes near the end of the AP. CICR stops and Ca uptake begins through SERCA. Phosphorylation of phospholamban removes its inhibitory effects and increases the activity of SERCA. This pump uptakes intracellular Ca, which is released from TN-C, and stores it in the SR for release during subsequent contractions. Intracellular Ca can also be removed by an ATP dependent Ca pump or by the Na/Ca Exchanger.
48 Turning Off Calcium Release: Role of Calmodulin Kinase Calmodulin is activated when calcium binds to its high-affinity binding sites to become calcium-calmodulin (CaM) . The latter in turn also activates the calmodulin kinase II (CaMKII) system.Activation of CaM closes the previously open L channels to help shut off calcium ion entry into the cell.Effects of CaMKII - complex and appear to play a physiologic role in acute beta-adrenergic stimulation.There is a converse pathologic role especially in chronic heart failure, when the major effects appear to include(1) increased inflow of the slow sodium current(2) promotion of calcium leak through the ryanodine receptors to promote release of calcium ions(3) enhancement of calcium entry into the SR by stimulation of the Ca uptake pump, SERCA.The overall effect is calcium dysregulation, especially in chronic beta- adrenergic stimulation .
50 Sarcolemmal Control of Calcium and Sodium Ions L&T type calcium channelsT – denotes transient Ca2+ entryL type channels seen in myocardium and T tubules- inhibited by CCBLtype- activation causes entry of Ca2+It is inactivated by1) increasing depolarization2)rising intracellular Ca2+
51 Ion exchangers & pumpsThe calcium homeostasis is maintained by Na/Ca exchanger which pumps calcium outThis removes 25% of intracellular calciumAt times there is reverse transport of Na and Ca that occurs during rapid depolarization increasing the Ca intracellularilyNa /K pumpNa entry during rapid depolarization and during calcium exchange is maintained by this pump which extrudes 3 Na and takes in 2 K.
52 FACTORS AFFECTING LV CHAMBER STIFFNESS Physical properties of the LV LV chamber volume and mass .Composition of the LV wall ViscosityStress relaxationIntrinsic factors Myocardial relaxation Coronary turgorExtrinsic factors Pericardial restraintRV interaction Atrial contraction Pleural and mediastinal pressure
53 Factors affecting ventricular relaxation First, the cytosolic calcium level must fall to cause the relaxation phase, a process requiring ATP and phosphorylation of phospholamban for uptake of calcium into the SRSecond, the inherent viscoelastic properties of the myocardium are of importanceThird, increased phosphorylation of troponin I enhances the rate of relaxationFourth, relaxation is influenced by the systolic load
54 Measurement of Isovolumic Relaxation The rate of isovolumic relaxation is best measured by negative dP/dtmax during invasive catheterizationTau, the time constant of relaxation, describes the rate of fall of LV pressure during isovolumic relaxation and also requires invasive techniques for precise determinationTau is increased as the systolic LV pressure rises.Other indices of isovolumic relaxation can be obtained echocardiographically or from tissue Doppler measurements to monitor the peak rate of wall thinning
55 Diastolic suctionLV suction by active relaxation could increase the pressure gradient from left atrium to left ventricle during the early filling phase is now well supported by dataThe suction effect can be found by carefully comparing LV and left atrial pressures, and it occurs especially in the early diastolic phase of rapid filling as a result of the LV elastic recoilIn early diastole, myosin is pulled into the space between the two anchoring segments of titin to lower the intraventricular pressure to below that in the atriumVentricular suction, by propagating a dominant backward pressure wave, is also responsible for diastolic coronary filling and attenuated in LV hypertrophy
56 Beta adrenergic signal system Beta 1 ,beta 2(20%) & beta 3Predominant is beta 1It acts through G protein coupled
60 Contractile Function Versus Loading Conditions Contractile function or contractility is the inherent capacity of the myocardium to contract independently of changes in the preload or afterload.At a molecular level, an increased contraction is called ionotropic effectAn increased contractile function is often associated with enhanced rates of relaxation called the lusitropic effect.Factors that increase contractile function include exercise, adrenergic stimulation, digitalis, and other inotropic agents
61 PreloadAny change in the contractile state should be independent of the loading conditions.The preload is the load present before contraction has started, at the end of diastoleThe preload reflects the venous filling pressure that fills the left atrium, which in turn fills the left ventricle during diastole.When the preload increases, the left ventricle distends during diastole, and the stroke volume rises according to Starling's lawClinically ,preload is assesed by the PCWP and the LVEDP
62 FRANK STARLING’S LAWThe force of contraction is directly proportional to the lenghth of the muscle fibre upto a physiological limit1918, Related venous pressure in the right atrium to the heart volume in dog heart – lung preparationIncreased diastolic filling – STARLINGS LAWIncreased ionotropic state – FRANK’S findingsIt is due to1)E nhanced calcium binding to troponin I2) Narrower interfilament gaps at long sarcomere length3)Increased SR calcium release and uptake at increased length
64 AfterloadThis is the systolic load on the left ventricle after it has started to contractIn the nonfailing heart, the left ventricle can overcome any physiologic acute increase in loadChronically, however, the left ventricle must hypertrophy to overcome sustained arterial hypertension or significant aortic stenosisArterial blood pressure can be taken as the afterload
65 Anrep Effect Abrupt Increase in Afterload When the aortic pressure is elevated abruptly, a positive inotropic effect rapidly follows.Proposed mechanism - increased LV wall tension could increase cytosolic sodium and then, by Na+/Ca2+ exchange, the cytosolic calcium increases
66 WALL STRESSA more exact definition of the afterload is the wall stress during LV ejectionFirst, the bigger the left ventricle and the greater its radius, the more is the wall stress.Second, at any given radius (LV size), the greater the pressure developed by the left ventricle, the greater the wall stress.
67 BOWDITCH STAIRCASE PHENOMENON An increased heart rate progressively enhances the force of ventricular contraction, even in an isolated papillary muscle preparation (Bowditch staircase phenomenon)Alternative names are the treppe (German, steps) phenomenon, positive inotropic effect of activation, and force- frequency relationshipConversely, a decreased heart rate has a negative staircase effect. When stimulation becomes too rapid, force decreases
68 Optimal measurement of LV function should assess LV twist motion, which results from apical counterclockwise and basal clockwise rotation of the left ventricle, both essential for generation of LV pumping power.The extent of the twist can now be measured noninvasively by speckle tracking echocardiography, which closely correlates with dP/dtmax in a variety of experimental conditions and is superior to the global ejection fraction
69 CONTRACTION OF RIGHT VENTRICLE RV contraction is sequential, starting with the contraction ofthe inlet and trabeculated myocardium and ending with thecontraction of the infundibulum (approximately 25 to 50 ms apartContraction of the infundibulum is of longer durationthan contraction of the inflow region.The RV contracts by 3 separate mechanisms:(1) Inward movement of the free wall, which produces a bellows effect(2) Contraction of the longitudinal fibers, which shortens thelong axis and draws the tricuspid annulus toward the apex(3) Traction on the free wall at the points of attachment secondary to LV contraction.
71 Shortening of the RV is greater longitudinally than radially. In contrast to the LV, twisting and rotational movements do not contribute significantly to RV contraction.Moreover, because of the higher surface-to- volume ratio of the RV, a smaller inward motion is required to eject the same stroke volume.
72 APPLIED PHYSIOLOGY 1) IONOTROPES 2) CHANGES IN E-C DURING HEART FAILURE3) MUTATIONS AFFECTING CONTRACTILE PROTEINS
74 Inotropic mechanisms and current inotropic interventions. Inotropic mechanisms and current inotropic interventions. Activation of the β-adrenoceptor stimulates adenylyl cyclase to produce cAMP, which activates protein kinase A (PKA) to phosphorylate intracellular calcium-cycling proteins. Phosphodiesterases (PDEs) degrade cAMP. Phosphodiesterases are inhibited by Phosphodiesterase inhibitors. Digitalis inhibits transport of three sodium ions for two potassium ions through Na/K-ATPase. Calcium sensitizers increase the affinity of troponin C for calcium.Hasenfuss G , Teerlink J R Eur Heart J 2011;eurheartj.ehr026
75 Mode of action of cardiac myosin activators. Mode of action of cardiac myosin activators. The agents promote actin-dependent phosphate release (Pi–release) moving the cross-bridge into its strongly bound force-producing state (see text). A, actin; M, myosin.Hasenfuss G , Teerlink J R Eur Heart J 2011;eurheartj.ehr026
76 Future inotropic compounds: the ryanodine receptor (RyR) stabilizers reduce sarcoplasmic reticulum leak through the ryanodine receptor and reconstitute ryanodine receptor channel function. Istaroxime inhibits sodium-potassium-ATPase and stimulates SERCA2a. Cardiac myosin activators promote transition of cross-bridges from the weakly to the strongly bound force-producing state. Energetic modulators improve myocardial energetics through switching from fatty acid to glucose oxidation or by other mechanisms including means to increase the cellular phosphorylation potential. Virus-mediated sarcoplasmic reticulum calcium pump gene transfer (AV-SERCA) increases sarcoplasmic reticulum calcium uptake. Nitroxyl (HNO) may increase sarcoplasmic reticulum calcium uptake by modification of sarcoplasmic reticulum calcium pump and/or phospholamban (PL).Hasenfuss G , Teerlink J R Eur Heart J 2011;eurheartj.ehr026
78 Ryanodine 2 mutation causes – CPVT,ARVD2 Calsequestrin mutation causes –AR variety of CPVTFKBP mutation can cause cardiomyopathy
79 Abnormalities in E-C Coupling CICR – Ion ChannelsChannels operate differently and conductances change, possibly due to the effect of heart failure on membrane characteristicsLess Ca++ may move across membrane during each AP
80 Abnormalities in E-C Coupling CICR - SRThe RyR channel undergoes changes and calcium leaks out.SR contains less calcium for release during each AP.
81 Abnormalities in E-C Coupling PhospholambanPlb protein levels increase due the continued stimulation by the sympathetic nervous system.SERCA2 protein levels decreaseThis will lead to elevated diastolic calcium levelsDonald M. Bers.”Cardiac excitation-contraction coupling” Nature Vol January 2002
82 Overview of E-C Coupling Changes in the Failing Heart :Regulation of Intracellular Calcium 1. Reduced Ca++ trigger thru L-type channel2. Reduced RyR function (Calcium leaks from SR)53. Decreased sensitivity of TN-C to Ca++244. Reduced Ca++ uptake due to loss of SERCA function and increased Plb15. Increased Na/Ca exchanger function3
83 BIBILOGRAPHY 1.HURST’S THE HEART ,13TH EDITION,VOLUME 1 2.BRAUNWALD HEART DISEASES,9TH EDITION3.CARDIAC EXCITATION CONTRACTION COUPLING ;ROLE OF MEMBRANE POTENTIAL IN REGULATION OF CONTRACTION : Am J Physiol Heart Circ Physiol 280: H1928– H1944, 20014. Ludwig W. Eichna, RICHARD J. BING and K. KAKO;Contractile Proteins of Heart Muscle in Man Circulation ;24:5. Olga M. Hernandez, Philippe R. Housmans and James D. Potter thin filament regulationcontraction and relaxation as a result of alterations inInvited Review: Pathophysiology of cardiac muscleJ Appl Physiol 90: , 2001.