SKELETAL, CARDIAC, AND SMOOTH MUSCLES Structural characteristics Electrical and mechanical activities Molecular mechanisms of contraction Biophysical properties of muscle as a whole Mechanisms of gradation/modulation of contraction SKELETAL, CARDIAC, AND SMOOTH MUSCLES Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL MUSCLE CARDIAC MUSCLE SMOOTH MUSCLE 20 m 30 m 3 m 1 intercalated discs sarcolemma (vascular system, airways, gastrointestinal and urogenital systems) 1.6-2 nm pH [Ca2+]i membrane voltage 2 „gap“ (extracellular space) ELECTRICAL CONNECTIONS „GAP JUNCTIONS“ MYOCARDIUM SMOOTH MUSCLE BASIC STRUCTURAL ELEMENTS OF FUNCTIONAL SYNCYTIUM CONNEXON 1 CONNEXON 2 GAP JUNCTION UNIT Structural characteristics Electrical and mechanical activity Molecular mechanisms of contraction Biophysical properties of muscle as a whole Gradation / modulation of contraction Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL, CARDIAC, AND SMOOTH MUSCLES -85 mV 50mV 200 ms 200 ms -35 mV 20 ms SMOOTH MUSCLE ICa regular pacemaker activity (SA, AV nodes) irregular pacemaker activities of unstable foci slow- type I 3 REPOLARIZATION slow wave spike HEART SKELETAL MUSCLE INa DEPOLARIZATION CONTRACTION IK(Ca)ICa inact INainact IK -60 mV -90 mV 50mV ICa inact family of K currentsphase 3 GREAT VARIETY IN REPOLARIZATION phase 2 ICa IK1 fast - type II 4a SMOOTH MUSCLE CELL TRIGGERING AND MODULATION OF MECHANICAL RESPONSES GREAT VARIETY IN ELECTRO-MECHANICAL RELATIONS 0-50 mVtension 0 -50 tension 4b mV mV tensiontensiontension mV time time time time SLOW IRREGULAR WAVES in membrane voltage with APs 1 agent x agent y frequency of APs 2 SLOW CHANGES in membrane voltage 3 -50 0 0 -50 TETANIC CONTRACTION (ureter, gall duct, uterus) SLOW CHANGES IN TONE (smooth muscles of eye, arterioles) SLOW WAVES IN CONTRACTION (GIT) e.g. via LIGAND-RECEPTOR activation pathways NEUROHUMORAL STIMULATION CONSTANT MEMBRANE VOLTAGE 4 SLOW CHANGES IN TONE (vascular smooth muscle) ELECTRO-MECHANICALCOUPLING 4c SMOOTH MUSCLE CELL by different NEUROHUMORAL STIMULATION MECHANICAL RESPONSES can be triggered/modulated NEUROTRANSMITTERS (acetylcholine, noradrenaline, …) by different patterns of ELECTRICAL ACTIVITY ELECTRO-MECHANICAL COUPLING HORMONES (e.g. progesterone, oxytocin, angiotensin II, … ) LOCAL TISSUE FACTORS (NO, adenosine, PCO2, PO2, pH, …) NEURAL STIMULATION ELECTRICAL STIMULATION HUMORAL STIMULATION MECHANICAL STIMULATION by STRETCH of the smooth muscle cell (STRETCHACTIVATED CHANNELS) Structural characteristics Electrical and mechanical activity Molecular mechanisms of contraction Biophysical properties of muscle as a whole Gradation / modulation of contraction Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL, CARDIAC, AND SMOOTH MUSCLES CROSS STRIATED MUSCLES THIN ACTIN FILAMENT tail (134 nm) N N C C 2 heavy chains 40 nm 5 2 heads 4 light chains ACTIN binding site ATP binding site CONTRACTILE ELEMENTS tropomyosin troponin complex REGULATORY PROTEINS (ATP → ADP + Pi) THICK MYOSIN FILAMENT TROPOMYOSIN –TROPONIN COMPLEX Ca2+ MOLECULE OF MYOSIN II G-ACTIN MOLECULES~400 CROSS STRIATED MUSCLES CROSS-STRIATED MUSCLE ADP Pi ADP. Pi 6 regulatory light myosin chains binding sites for actin ATP binding site Ca2+- troponin C complex Ca2+ RESTING state ‚cocked‘ position of the head ε ONE ELEMENTARY CYCLE OF CONTRACTION AND RELAXATION MOLECULAR LEVEL P P CROSS-STRIATED MUSCLE ONE ELEMENTARY CYCLE OF CONTRACTION AND RELAXATION MOLECULAR LEVEL CROSS-STRIATED MUSCLE ADP Pi ADP. Pi 6 regulatory light myosin chains binding sites for actin ATP binding site Ca2+- troponin C complex Ca2+ Pi ADP release of Pi strong cross-bridge state STATE OF CONTRACTION ATP ATP DISSOCIATION of actin–myosin complex rigor mortis CROSS BRIDGE weak cross-bridge state ε ➢ presence of ATP ➢  [Ca2+ ]i RESTING state ‚cocked‘ position of the head ε ONE ELEMENTARY CYCLE OF CONTRACTION AND RELAXATION MOLECULAR LEVEL RELAXATION CONTRACTION ➢ presence of ATP ➢  [Ca2+ ]i P P HUXLEY´S sliding model Animated model of interaction of myosin head with actin filament („ paddling “ ) 8 troponin – tropomyosin complex Ca2+ MYOSIN HEAD Mg2+ATP CROSS-STRIATED MUSCLE vertical position at resting state It consumes chemical energy released from hydrolysis of ATP and converts it into the motion (mechanical work) Myosin – MOLECULAR MOTOR 7a MOLECULAR MECHANISM OF CONTRACTION Binding of Ca2+ to TROPONIN C  shift of troponin-tropomyosin complex → actin binding sites for myosin heads are uncovered Formation of CROSS BRIDGES between actin and myosin (weak cross-bridge state) A .M . ADP . Pi Release of Pi (strong cross-bridge state)  conformational changes in myosin head-neck junction → tilt of the myosin head (power stroke) → sliding of thin on thick filaments  SHORTENING OF SARCOMERE ADP is released → actomyosin complex is left in a rigid ’attached’ state A .M CROSS-STRIATED MUSCLE RELAXATION of the muscle cell results from the presence of ATP and [Ca2+]i (Ca2+ is pumped back into SR and pumped out of the cell) 7b Binding of ATP to myosin head  low affinity of myosin for actin → dissociation of ACTIN–MYOSIN complex A M . ATP CONTINUING CONTRACTION results from the repeated cycling due to maintained [Ca2+]i in the presence of ATP ATP-ase activity of myosin head  partial hydrolysis of ATP, the gained energy is used for re-cocking of the myosin head (analogy of the stretched spiral spring). A M . ADP . Pi ORGANIZATION OF CYTOSKELETON AND MYOFILAMENTS 9 SRcaveolae REGULATORY PROTEINS TROPOMYOSIN membrane dense area INTERMEDIATE filament ACTIN filament MYOSIN filament 2 heavy chains CALDESMON DB - DENSE BODIES MYOSIN II mechanical junctions between cells CELL 1 CELL 2 P gap junctions SLOW ISOFORMS OF ➢ myosin ATP-ase ➢ Ca2+ transport systems CALMODULIN (TNC) DELAY in E-C coupling SLOW DEVELOPMENT of contraction and relaxation ? SMOOTH MUSCLE TROPONIN IS ABSENT !! Ca2+ CALPONIN DB 4 light chains: 2 essential 2 regulatory 10 myosin LIGHT CHAINS MYOSIN ACTIN RESTING STATE 2 ROLES OF Ca2+-CALMODULIN COMPLEX CALMODULIN SMOOTH MUSCLE ↑[Ca2+]i MYOSIN LIGHT CHAIN KINASE MLCK tropomyosin P myosin LIGHT CHAINSP MYOSIN-ACTIN interaction Ca2+-CALMODULINcalponin-caldesmon complex Ca2+-CALMODULIN complex TROPONIN COMPLEX is not present CALMODULIN Ca2+-calmodulin-MLCK complex activated MLCK Ca2+/CALMODULIN–MLCK activated MLCK caldesmon calponin CONTRACTION VARIANTS OF SMOOTH MUSCLE CELL 11 PHASIC variant of CONTRACTION - mode of CYCLING1 time TONIC variant of CONTRACTION - LATCH BRIDGES2 time SUSTAINED TONIC CONTRACTION SLOW dissociation of M.A complex ATP is spared ? At the state of CONTRACTION RMLCs are dephosphorylated by MLCP P ATP is consumed of myosin light chains (RMLCs) is a prerequisite of PHASIC contraction P P SMOOTH MUSCLE PHOSPHORYLATION of myosin light chains is a prerequisite of PHASIC CONTRACTION ATP ADP ADP. Pi ADP. Pi ADP Pi 12a P P P PHASIC variant of CONTRACTION - mode of cycling1 time RESTING STATE Pi MLCP MYOSIN-LC PHOSPHATASECa2+-CaM-MLCK MYOSIN-LC KINASE activated CROSS BRIDGE low energy conformation state state of contraction ATP ATP dissociation of actin–myosin complex Ca2+-calmodulin complex P P P ε high energy conformation state RESTING STATEε P P P Adapted from Berne and Levi (2004) ATP is consumed Ca2+-CaM –CALPONINCALDESMON complex MLCK* / MLCP Ca2+-CaM-MLCK MLCP ATP ATP ATP 12 b LATCH BRIDGE mechanism of sustained TONIC CONTRACTION P RESTING STATE RESTING STATE STATE OF CONTRACTION P P P P P ε TONIC variant of CONTRACTION - LATCH BRIDGE2 time ε P P P Adapted from Berne and Levi (2004) very slow DISSOCIATION of M.A complex MLCK* DEPHOSPHORYLATION of MLCs at the STATE OF CONTRACTION ↓ MLCK* / MLCP ↑ MLCK* / MLCP SMOOTH MUSCLE ATP is spared REPEATING CYCLING PHOSPHORYLATION of myosin light chains is maintained SUSTAINED TONIC CONTRACTION -“LATCH BRIDGE” mechanism; DEPHOSPHORYLATION of myosin light chains at the state of contraction 13 Binding of Ca2+ to CALMODULIN  Ca2+-CaM complex Phosphorylation of MYOSIN LIGHT CHAINS and simultaneous conformational changes of Ca2+-CaM-calponin-caldesmon-ACTIN-TROPOMYOSIN complex  formation of CROSS BRIDGES ATP is consumed ATP is spared Activation of MYOSIN LIGHT CHAIN KINASE Ca2+-CaM-MLCK complex Conformational changes in MYOSIN molecule  TILT of MYOSIN HEAD  SLIDING of ACTIN on MYOSIN filaments  SHORTENING of the myocyte SMOOTH MUSCLE Structural characteristics Electrical and mechanical activity Molecular mechanisms of contraction Biophysical properties of muscle as a whole Gradation / modulation of contraction Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL, CARDIAC, AND SMOOTH MUSCLES ISOMETRIC AND ISOTONIC CONTRACTION SKELETAL MUSCLE RESTING STATE PE, SE - parallel and series elasticity (in relation to contractile elements) PE SE IMC ISOMETRIC CONTRACTIONIMC at constant LENGTH ITC ITC ISOTONIC CONTRACTION at constant TENSION CE CE – contractile elements HEART ISOVOLUMIC PHASE (ISOMETRIC) EJECTION PHASE (ISOTONIC) AUXOTONIC AUXOTONIC CONTRACTION changes in TENSION are measured by tensiometer changes in LENGTH are measured 14 SMOOTH MUSCLE TONIC CONTRACTION (tone of blood vessels) PHASIC CONTRACTION (contraction of urinary bladder) RESTING TENSION PE – extracellular and intracellular elasticity (titin connecting Z and M lines in the sarcomere) SE – elasticity of fibrous tissue - tendon TENSION-LENGTH RELATIONSHIP 15 SKELETAL MUSCLE increase in muscle length (in cm)muscletension TOTAL tension TOTAL tension ISOMETRIC CONTRACTIONS of stimulated muscle at gradually increased initial (resting) length ACTIVE tension ACTIVE tension difference between TOTAL and PASSIVE tension curves at any length (tension actually generated by contractile elements) PASSIVE tension PASSIVE tension tension of unstimulated muscle at gradual stretching (ELASTIC COMPONENTS) resting length in vivo ACTIVE TENSION of cross striated muscles as a function of INITIAL LENGTH of SARCOMERE initial sarcomere length [m] activetension(%) 1.65 1.9 2.05 2.2 3.65 16 maximumtensionarea CARDIAC MUSCLE SKELETAL MUSCLECARDIAC MUSCLE stretch-dependent sensitivity of ACTIN filaments (TnC) to Ca2+ 2.9 physiological working area STRETCH-ACTIVATED membrane channels FRANK-STARLING´S LAW dependence of ventricular contraction on blood volume at the end of diastole SMOOTH MUSCLE 17a GREAT EXTENSIBILITY PLASTICITY (e.g. myocytes of urinary bladder can lengthen up to 200%, myocytes of uterus even up to 1000% at the end of pregnancy in relation to their original state) CHARACTERISTIC FEATURES No direct relation between the LENGTH and TENSION in smooth muscle cells. Stretch-induced increased tension almost immediately spontaneously decreases. Analogous relation is valid between VOLUME and PRESSURE in hollow organs (stomach, intestines, urinary bladder, …). volume pressure CYSTOMETROGRAM STRETCH-activated Ca2+-channels (mechano-sensitive channels) PLASTICITY OF SMOOTH MUSCLE  TENSION LAPLACE LAWP = 2T/r 17b 1 2 micturition reflex is triggered 3 time time lengthtension calcium-activated [Ca2+]i-sensitive K+-channels IKCa REPOLARIZATION IKCa  [Ca2+]i TENSION Ca2+ K+ ? ISOLATED MYOCYTE (human jejunum) T tensiometer PLASTICITY electrophysiological measurements depolarization repolarization voltage time ICams  [Ca2+]i DEPOLARIZATIONICa ms ? urinary bladder Structural characteristics Electrical and mechanical activity Molecular mechanisms of contraction Biophysical properties of muscle as a whole Gradation / modulation of contraction Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL, CARDIAC, AND SMOOTH MUSCLES 18 MAIN FACTORS IN GRADATION OF CONTRACTION  frequency of discharges in motor neuron  FREQUENCY SUMMATION of contractions in skeletal muscle fibre (TETANIC CONTRACTION)  number of activated MOTOR UNITS by increasing voluntary effort  SPATIAL SUMMATION (multiple fibre summation) - RECRUITMENT OF MOTOR UNITS motor unit 5-1000 SKELETAL MUSCLE frequency of stimulation (Hz) strengthofcontraction SKELETAL MUSCLE SINGLE MUSCLE FIBRE ? complete (smooth) tetanus TETANIC CONTRACTION The decreased time interval at high frequency limits the time for relaxation Summation of individual releases of Ca2+ from SR into sarcoplasm Short refractory period of AP of skeletal muscle fibre enables to copy the high frequency of APs in motor neuron 1 Hz = 1 impulse/sec 19 GRADATION of CONTRACTION by  FREQUENCY of STIMULATION single twitches incomplete (undulatory) tetanus ↑[Ca2+]i RANGE OF SUMMATION physiological behaviour of skeletal myocyte 20 CARDIAC MUSCLE ↑ DIASTOLIC FILLING of ventricles in vivo („preload“)  ↑contraction of ventricles proportionate to the stretching of cardiomyocytes at the end of diastole FRANK-STARLING´S LAW LIGAND-RECEPTOR ACTIVATION CASCADES leading to ↑ [Ca2+]i (noradrenalin, adrenalin, thyroxine, …) ↑ FREQUENCY of electrical activity of cardiac cells via modulation of pacemaker activity of SA node by sympathetic nerves - positive FREQUENCY EFFECT ↑ [ Ca2+]i MAIN FACTORS IN GRADATION OF CONTRACTION 21 SMOOTH MUSCLE Ligand-receptor activation cascades leading to ↑ [Ca 2+]i (e.g. via activation of PLC  ↑ IP3 releasing Ca2+ from SR) Stretching of the smooth muscle cell  opening of the stretch-activated channels  ↑ [Ca 2+]i DEPOLARIZATION of the smooth muscle membrane with or without triggering of action potentials via opening of the voltage dependent calcium channels  ↑ [Ca 2+]i FACTORS independent on membrane depolarization ↑ Ca2+-calmodulin complex MAIN FACTORS IN GRADATION OF CONTRACTION / TONUS Structural characteristics Electrical and mechanical activity Molecular mechanisms of contraction Biophysical properties of muscle as a whole Gradation / modulation of contraction Overview of characteristic properties of skeletal, cardiac, and smooth muscles SKELETAL, CARDIAC, AND SMOOTH MUSCLES 22 MAIN CHARACTERISTIC FEATURES Multinucleated long cylindrical cells (max. length up to 20 cm) Rich sarcoplasmic reticulum Regular arrangement of thick and thin filaments in sarcomeres (cross striation) Activity strongly dependent on motor nerve supply (excitation transmitted via motor end-plate) Activity under voluntary control Summation of contractions (tetanus) is a physiological property of muscle fibre Without intercellular connections (no gap junctions between muscle cells) Motor neurons branch to innervate more muscle cells (motor unit defined as one motor neuron with 5-1000 myocytes) motor unit 5-1000 SKELETAL MUSCLE 23 MAIN TYPES OF SKELETAL MUSCLE FIBRES High OXIDATIVE CAPACITY and high resistance to fatigue Slow (posture-maintaining) contractions Motor units contain slowly conducting motor neurons e.g. muscles of the back, soleus m.SLOW - REDTYPE I TYPE II FAST (RED /WHITE) e.g. extraocular muscles, muscles of the hand Short twitches for fine skilled movements Motor units with rapidly conducting motor neurons Proportion of OXIDATIVE and GLYCOLYTIC metabolism determines the resistance to fatigue Sport activities cause gradual transformation from IIb into IIa TYPE IIa (FAST-RED) and TYPE IIb (FAST-WHITE) 24 Branched and interconnected cells with one nucleus in the centre (length ~100 μm) Well (moderately) developed sarcoplasmic reticulum Regular arrangement of thick and thin filaments in sarcomeres (cross striation) Excitations (contractions) are independent on nerve supply (specialized pacemaker cells) Functional syncytium (electrical connections - gap junctions) Long refractory period prevents cells from tetanic contraction (which would be life threatening) Activity ís not under voluntary control Receptors for neurotransmitters (released from neuron endings) and hormones (brought by circulation); activity is modulated by local mediators MAIN CHARACTERISTIC FEATURES CARDIAC MUSCLE 25 Thin spindle-shaped cells of various length (20-200 m) with one nucleus in the centre Poorly developped sarcoplasmic reticulum, TT system is missing Irregular arrangement of thick and thin filaments; no cross striation Slow phasic, often tonic, even tetanic contractions Numerous receptors for neurotransmitters (released from neuron endings) and hormones (brought by circulation). Activity is greatly modulated by local mediators (local tissue factors) Contractions of visceral muscles can be triggered independently on nerve supply (slow irregular unstable pacemaker activity); functional syncytium (gap junctions) Activity without voluntary control Activity can be triggered by stretch (stretch activated channels) MAIN CHARACTERISTIC FEATURES SMOOTH MUSCLE Great extensibility and plasticity 26 TYPES OF SMOOTH MUSCLE Functional syncytium (gap junctions) Excitation and contraction can be evoked in the absence of nerve supply (slow irregular pacemakers in multiple foci shifting from place to place, gap junctions) Contraction evoked by stretching (stretch-activated channels) e.g. stomach, intestine, uterus, ureter, …VISCERAL „SINGLE UNIT“ MULTIUNITstimulated by neurons e.g. arterioles, m. ciliaris, muscle of iris, … Cells are not interconnected by gap junctions, APs are not triggered Contractions are finely graded and localized Myocytes need the stimulation by autonomic “motor” neurons releasing acetylcholine / norepinephrine, … (AUTONOMIC „MOTOR UNITS“) Synapses „en passant“ in the course of the neuron endings