----start physio 1.14.97----- Lee Sweeney lsweeney@mail.med.upenn.edu 80485 STRIATED MUSCLE the components which drive muscle are the same things that drive other motile cells Cytoskeletal Elements: microtubules (tubulin) microfilaments (actin) intermediate filaments the top two provide tracks for directed movement...molecular motors move on microtubules (dynein and kinesin) and microfilaments (myosin) dynein and kinesin involved in vesicular movement, chromosome movement etc. myosin moves on actin, involved in muscle contraction et al. multiple classes of myosin..muscle, vesicular movement...at least 12-13 classes of motors in cells....retina, auditory system cilia, etc. may be some overlapping functions with microtubule motors. also forms cleavage furrow, drives cleavage part of cell division. 4 polypeptide subunits, 2 heavy chains, two light chains. the part of myosin common to all myosin classes is the motor. muscle myosin is the only one with the long alpha helical coiled coil. see diagram in handout (fig 3) the lever arm amplifies small motion that occurs up in the active region. it moves about 10 nm (100 angstroms) which is quite large. motion driven by ATP hydrolysis. it binds to active site and is hydrolyzed, causing the lever arm to "prepare" then when actin binds to actin binding site phosphate and ADP are released and lever arm swings back to pre-ATP binding position. this is called the power stroke. so this cycle is seen in fig 4 p 4 of handout "myosin crossbridge cycle" it's important to realize that it is a cycle, and that during isometric contraction it still occurs... it's called crossbridge because of what it looks like under electron microscope. the cycle doesn't end til you run out of ATP, at which point you end up between steps 3 and 4, which is where you end up when you die and enter rigor mortis, where the myosin is on the actin and can't be released because you're out of ATP, all the actin and myosin is crosslinked and muscle is very rigid. slide: myosin molecule...central bare zone with no myosin heads, because this part is where the filaments are stacked end to end, and on either side you have the heads coming foff the filaments. many many heads...looks like (to me) two of those wheat things end to end. so you have a bipolar myosin filament, and the actin filament is just made of actin monomers by having bipolar myosin filaments, you can interact with actin filaments of opposite polarity, and [can't see slide at all, at the bottom. argh] throughout vertebrates, the system that turns contraction on and off in skeletal/cardiac muscle is the troponin/tropomyosin system, aka thin filament regulation. actin is the thin filament/myosin is thick filament. tropomyosin is fairly ubiquitous in eukaryotic systems. it sits on actin and alone can enhance actin/myosin interactions. as you might imagine there is a specific site on actin that myosin has to bind to. troponin locks tropomyosin over the binding site, so it can only interact very weakly with actin, but can't generate a power stroke. BUT if Ca++ levels go up in the cell, the troponin/tropomyosin complex changes. throughout all myosin II systems, Ca++ is the messenger which activates actin/myosin interaction, it seems. troponin complex= troponin T (tropomyosin), troponin I (inhibitory, locks it onto actin), and troponin C (calcium, the Ca++ binding subunit). troponin C is of the calmodulin superfamily. undergoes structural change such that the actin/myosin binding site becomes exposed, as long as Ca++ is bound to the troponin C. so this gates muscle contraction, as long as Ca++ level is high, can contract, then can lower ca++ level to stop ability to contract. so the contractability is a function of [Ca++] see fig 5, 6. fig 6: force generated as function of [Ca++]. skeletal muscle has steeper relationship than cardiac muscle, needs more ca++ to activate skeletal than cardiac. the [ca++] influences the number of available actin/myosin binding sites, see. vertebrate muscle: striated: skeletal, cardiac -these appear striped under the light microscope. this is due to actin and myosin filaments being put together in highly ordered pattern. smooth: actin and myosin are present but are not ordered, so you don't see a pattern. skeletal and cardiac muscle have very similar contractile apparatus. most of today's talk is applicable to both. skeletal muscle cardiac muscle smooth muscle ______________ _____________ _____________ att. to bone the heart surrounds hollow organs and blood vess. moves bones pumps blood contracts/repels/vascular resistance somatic NS inn autonomic NS autonomic ns striated striated smooth multinucleated 1,2 or 3 nuclei mononuclear electrically alone connected electrically either [excuse me. there is an extremely large cockroach moving around back here. we're going to call it spot, i think. it's causing somewhat of a disturbance.] in skeletal muscle, there are cells in the basal lamina which have not differentiated, which can be activated by HGF (hepatic growth factor) - these are satellite cells, unfused precursors, which can fuse and repair damaged muscle. so it can regenerate. in cardiac muscle if you lose muscle, you lose it, and it scars over. you don't regenerate it. cardiac muscle cells are usually 20-30 microns in diameter, and are somewhat branched. look quite different from skeletal muscle cells. skeletal muscle cells are electrically isolated fused fibers. if one cell (fiber) depolarizes, its neighbor does not. in the heart, all cells are electrically connected via gap junctions in the intercalated disks. smooth muscle can be connected or isolated from neighbor cells. so cardiac cells form functional syncitium. in heart, all cells beat at once so the main way to change the force the heart generates is changing where you are on the force/Ca++ curve (which is why digitalis works, right? -hg) so when heart needs more force, it releases more ca++ in skeletal muscle, you just recruit more muscle fibers to contract - eg, you fire more nerves / activate more motor units. basic ultrastructure of skeletal muscle: the actin and myosin filaments are bundled into myofibrils which contain sarcomeres. the myofibrils are surrounded by highly specialized structure made partly of ER which is called SR in the muscle cell. the SR stores Ca++. so it contains binding proteins and Ca++ channels which when open allow calcium to flood out and bathe myofibrils. this evolved because muscle cells can achieve diameters of 100 microns or more. if you waited for ca++ to come in through plasma membrane, would take too long to diffuse to centerofcell. so myofibrils are surrounded with this stored, bound, calcium. how do you tell SR to release Ca++? that's the basis of the T tubular system. the t tubular system, formed by invaginations of plasma membrane so. muscle will fire action potentials, and the T tubular system propagates them deep into the muscle to communicate the depolarization to the SR causing it to release calcium. T tubule comes near the "terminal cisternae" of the SR where the calsequestrin is sequestered. there's a lot of Ca++ bound to it. also in these regions is where most of the Ca++ release channels are located. this is key for skeletal muscle because it is thought that the ca+= release proteins interact with something in the T tubules. so it isn't just the electrical interaction, there are interacting proteins. in cardiac muscle it is thought that the calcium release proteins are also sensitive to ca++ levels, with increaing ca++ level providing + feedback to release more calcium. so you depolarize membrane, then t tubule, coupling to SR opens ca release channels in SR, ca++ floods out, contraction occurs. now, there are also ATP driven ca++ pumps to pump calcium back into SR, and thse pumps work even while calcium is being released - they just can't keep up with the release channels, but they're always working to put calcium back into the SR. also lots of mitochondria around myofibrils, to supply ATP. now, the AP on the surface membrane is triggered because muscle is hooked into somatic NS. every muscle fiber has to have nervous input to be activated. input comes from alpha motor neurons, which generally speaking have many branches which innervate a variety of muscle fibers. the neuron and it's population of muscle fibers is called a motor unit; the basic unit of muscle function. [neat slide: motor neuron coming into muscle fibers, arborizing into synapses on a number of fibers] the nature of the synapse is that ACH = the NT, the nerve ending synapses with the motor end plate and what you see there is that the ACH vesicles are released from nerve, and ACH receptors are present in that end plate, which when activated cause a graded depolarization. if it reaches threshold potential, will cause AP. so the motor end plate is right in the middle ofthe fiber, so APs can propagate rapidly around whole cell. why do muscle fibers stop at length they do? length seems limited by speed of propagation of AP and timing of contraction. they're short enough to depolarize the cell at a rate that is rapid compared to speed of contraction. see fig 9 for AP graphs. fairly normal looking sodium spike and potassium repolarization. the AP is conducted through Ttubular system (see fig 10) so the coupling between the depolarization of t tubules and SR is not truly electrical but seems to be mechanical coupling involving proteins in t tubular membrane and ryanodine receptor in SR membrane, see figs 11A and B and 12. IP3 may act as second messenger in skeletal and cardiac muscle as well. in skeletal muscle, mechanical coupling is major factor, and in cardiac muscle, calcium induced calcium release is the major player. ---break--- ok, so moving right along (and note it is STILL freakin' ice cold in this room; i do NOT understand it) see figure 14 on p 16 of the striated muscle handout. note: ACH gates Na+/K+ ligand gated channels in motor end plate; doesn't directly depolarize membrane. the voltage gated channels are responsible for the AP. so, as a result of all this, there is a calcium surge within the cell and then the calcium is resequestered....see fig 14 p 16 to see calcium spike. associated with this is a slightly delayed tension wave. this is called a twitch, a response to a single AP. the timing is different depending on the type of cell, etc. some are faster than others. if MORE than one APs are fired right close together, you see instead of single twitch, a larger force response (fig 15) indicating the response to twitch didn't activate all the ca+= binding sites. you have more force generated with increasing number of APs close together untl you saturate all the calcium binding sites on troponin C, at which point you have tetanus, and you can maintain steady force until the muscle fatigues. if your APs are farther apart you can get unfused tetanus - see diagram fig 15. as yyou fire APs faster and faster you increase the Ca++ in cytoplasm and increase Fc. the faster the muscle fiber is, the faster it contracts and relaxes, the closer together APs must be to acheive tetanus. so there isa relationship between frequency of APs and resultant Fc. so you can change the force by changing AP frequency as well as by recruiting more fibers in skeletal muscle. "rate coding". but the major way to control Fc is to change # of activated motor units. an there is a defined recruitment order...smallest motor units activated first, because they are easier to bring to potential. the first motor unit you activate activates a few fibers...the bigger units activate a larger # of fibers, so you get a smooth recruitment...you don't go from having 5 active fibers to 500 active fibers. you add 5, then 5, then 6, 8, etc. force isn't only function of Ca++ level, also a function of the length of the fiber. fig 16 p 18 . if you stretch muscle passively it will act sort of like rubber band. as you stretch a passive tension builds up, has elastic recoil. if you electrically stimulate isolated muscle to acheive fused tetanus and measure that force at different lengths, you get fig A - so you see that force increases as length increases, but part of the force is due to passive force, not actin/myosin interaction. the active force from actin/myosin is shaped more like a hill, it peaks and then falls again, because there is a length which optimizes the number of actin/myosin interactions. see fig 17. when muscle length changes the sarcomeres are stretched or compressed, so different amts of actin and myosin overlap. at the length which allows maximum # of interactions of actin and myosin you can generate the greatest force. the bare zone in the middle of the filament is whyyou see a plateau where changing length doesn't change force. if you overshorten, you have actin fibers on the "wrong side" of the filament: actin filaments have polarity. can't interact well on the wrong side; if they do would be in the wrong direction. so force starts to drop. eventually there is total reverse overlap and force totally drops off. eg, at short lengths actin is wrong polarity and runs into z lines and generates force in wrong direction. so skeletal muscle works at a specific length as set up in the body for optimum contraction. cardiac muscle tends to be lower on the length tension curve when at reast. if the heart overfills, it can generate more force - part of frank/starling mechanism: if you give itmore blood, it can pump harder. skeletal muscle works over a narrow range and tends to be set up closer to optimum length at rest. see fig 18. another way force changes is whether it's shortening or not, or how fast it is shortening. eg, keep load constant and measure speed of contraction. as you increase load, speed slows, at zero load, speed = Vmax, at maximum load, it can't contract any more, but when you add more load, you stretch the muscle. some types of exercise eg running downhill causes stretching and generates forces much higher than could generate against a load on their own. so the forces are much higher than that normally seen, and tends to damage the muscle. so don't just go out and start running downhill! :) it will hurt. isometric contraction: load is of magnitude that muscle can't move, so muscle generates force as large as possible, with as much myosin as possible on the actin. if you do a shortening contraction, the bridges tend to come off faster...therefore steady state force is lower. speed of contraction actually depends on how fast bridges let go, so that other bridges can get on and move the filament. isometricly, this involves larges number of bridges being on most of the time. when you pull on actively contracting muscle, bridges resist being pulled off, but this requires an outside force pulling on the activated muscle (eg the dotted part of fig 18 doesn't happen on its own unless something elseis pulling on the muscle - eccentric contraction - eg running downhill) if you look at the heart the same phenomena are at work; length/tension, force/velocity, calcium/force.... fig 19 the higher the venous pressure, the more venous return, the more ventricular filling, the more force generated by contraction, increased stroke volume, but heart doesn't EMPTY as completely as before. cardiac muscle has a length tension curve similar to that of skeletal muscle. cardiac muscle is more active than skeletal, has more mitochondria, so has fewer myofibrils per unit volume, so generates less force per unit volume, but can always generate enough ATP to power its contraction. if heart becomes ischemic youdevelop problems. but you have the same shape length tension curve even though peak is lower. also similar active curve. but in cardiac muscle the resting/passive curve is shifted to the left and is steeper, because the heart passively resists overstretch to a much greater degree than skeletal. skeletal is limited by joints so it doesn't need to resist as much. but heart needs to resist overfilling; isn't limited by anything except innate properties. so as end diastolic volume goes up, end diastolic pressure goes up in a non linear way as determined by passive elastic elements of the cardiac muscle. within the cell the main elastic element is a giant protein called titin which connects myosin filaments directly to A line and acts as spring, so at lengths where there's no passive tension it isn't stretched, but when you stretch the filaments you stretch this stuff, and it's stiffer in cardiac muscle than skeletal. or something like that. it's a very very large protein. so the length tension curve is very important in the heart but also it responds to HR...when heart rate increases you get increased force of contraction. even in absence of neuroendocrine input (eg in isolated heart) because you are increasing the amount of free calcium. heart can also change sensitivity to ca++. troponin I component is different: can respond to second messengers by getting phosphorylation of troponin I and changing the sensitivity to ca++ by lowering the affinity (eg ca++ comes off faster.) so it takes more ca++ to give the same amt of force. so heart will relax faster as well. so if heart rate increases, you need more calcium to get a given force, and you phosphorylate the troponin I because you need the faster relaxation for good filling time, and you're releasing tons of extra calcium anyway. to get higher CO you need more force, greater rate of rise of force, and greater rate of relaxation. cardiac c protein and light chain of myosin both involved in rate of rise of force. another important thing about cardiac vs skeletal is % of ca++ avail for contraction in heart does come in from surface membrane ca+= channels, where in skeletal it ALL comes from SR. in heart, most comes from SR but loading into SR requires it coming in from outside, so sympathetic input bringing Ca in through external channels is importt. drugs bloscking Na+/K+ atpase slow removal of calcium so you get more forceful contraction. ----end----