---start---- bbd 9/25 chacko There is a NEW exam question - it has not been given to us yet. Remodeling of smooth and cardiac muscles to meet the increased functional demand. Causes of increased functional demand: on cardiac muscle - valvular disease, hypertension, myocardial fibrosis, etc. anything that decreases Fc (strength of contraction) or CO (cardiac output) will lead to hypertrophy on smooth muscle such as bladder: outflow obstruction (as with prostatic hyperplasia in dog or more importantly in human, prostatic cancer, transitional cell tumor in dogs and in the past in people, bladder calculi) SMOOTH MUSCLE: contractile proteins: myosin, actin, tropomyosin; no troponin but it does have caldesmonand calponin which bind calmodulin, the Ca++ binding protein (present in thin filament, may have regulatory role). Ca++ regulation of contraction is mediated by Ca++ calmodulin dependent phosphorylation of the 20 kDa light chain. myosin - when it's dephosphorylated it interacts with actin. when it is phosphorylated, it has high ADPase activity and that produces contraction. contraction and relaxation of the smooth muscle is regulated by calcium and phosphorylation via Ca++ dependent kinases. myosin in smooth muscle unlike cardiac or striated muscle is produced by only one gene with several isoforms formed by alterative splicing of the pre-mRNA. remember myosin is a tetramer with two heavy chains and two pairs of light chains. there is a head region and a rod region. most of the active sites are in the head area, like the actin binding area, and the ATP binding area. so you put myosin into something and denature it, it will separate out. splicing at 3' end - there is aninsertion of 39 nucleotides - this produces a myosin heavy chain only 200 kD instead of 240 - this is due to a stop codon insertion. so there are 200 and 204 kD isoforms present in all smooth muscle. but ratio of them is different in differentmuscles. how are they different functionally? Not sure. these proteins (SM1 and SM2 may have something to do with filament assembly. splicing at 5' end, N terminal region, with insertion of 21 nucleotides encoding a 7 AA peptide, which is inserted near ATP bidning region - this myosin is called SM-B, and is in muscles that undergo phasic contraction, like the bladder (rapid contraction, like bladder). the isoform without the insert is SM-A, and is found in muscles that undergo tonic contraction (aorta). species differences exist - chickens have different one - and any change in this 7 AA peptide causes a difference in ATPase activity of myosin. Functional diff b/w SMB and SMA - SMB has a 2-3x higher actin activated ATPase activity than SMA, and maximum velocity of force is higher 2-3x higher in SMB muscles than SMa muscles. experiment was done, comparing aorta and saphenous ...something i couldn't understand. what changes are present during hypertrophy, in myosin isoforms? 1. overexpression of SM1, causing the 1:2 ratio of SM1 to SM2 in normal muscle to change to about 1:1 - not clear how this affects function 2. smooth muscle cells express SMA which isn't usually expressed in bladdersmooth muscle, smooth muscular arteries - overexpression of SMA occurs wih hypertrophy. myosin in thick filaments becomes mixture of SMB and SMA, SMA with low level of actin activated ATPase activity causes slow cycling of cross bridges since hydrolyzes atp slowly. this increases internal load, slows force generation, and the hypertrophied bladder muscle becomes more tonic - takes longer to generate force to expel urine. what changes are present in the thin filaments in hypertrophy associated with increased functional demand in the urinary bladder? remember major regulation is mediated via phosphorylation of thick filament; but there may be some thin filament regulation - it isn't well established. anyway, the changes: actin: a actin (predominant in vascular smooth muscle) increases slightly, b nonmuscle actin decreases, and g smooth muscle (predominant in visceral smooth muscle) actin increases slightly. we see a slight increase in a actin, maybe due to increased vascularity during remodelling and hypertrophy - more capillaries bud out, so this may account for it. interestingly we find a decrease in b nonmuscle actin, maybe because some of the nonmucle cells differentiate into muscle cells and stop making that b and start making g form. caldesmon - made by one gene; two isoforms - l-caldesmon, present in nonmuscle cells, and h-caldesmon, smooth muscle type. caldesmon is the troponin analog in smooth muscle. in bladder hypertrophy, following outlet obstruction, there is a decrease in h-caldesmon and an increase in l-caldesmon. hmm. we know that cells are differentiated smooth muscle cells b/c they make g actin and sm muscle myosin, but they do not make the h-caldesmon for some reason. how cann this affect muscle function? the C terminal region of caldesmon binds to actin, and its N terminal region binds to the region below the myosin head. these bindings tether the atin to myosin, and this is believed to play a role in force maintenance, in smooth muscle. (this protein has several functional domains. it binds actin, calmodulin, tropomyosin, and it inhibits actin activated ATPase.) we talked about, in order to generate force, myosinhas to be phosphorylated, but once it reaches the force, the phosphorylation goes away, and the force is maintained. ATPase activity is low at this time, ATP isn't being hydrolyzed. force is maintained with low ATP utilization in normal smooth muscle. we think the caldesmon could be promoting this maintenance of force with low ATP use. now, the l-caldesmon doesn't have a piece that is present in the other form, and it doesn't do this tethering thing. so the caldesmon property of force maintenance is lost. a decrease in smooth muscle type h-caldesmon will decrease the ability of bladder muscle to maintain force needed to empty the bladder completely - leading to residual volume in urinary bladder, and increased frequency of urination. it takes more time to develop force, and then once force is reached, it isn't maintained. the non muscle form probably plays a role in actin filament aggregation. it can bind actin and myosin, and keeps bundles together. CARDIAC MUSCLE: hypertrophy: increased synthesis of myosin and other contractile proteins. myosin: 3 heavy chain isoforms: bb homodimer (V3 isoform in fetal heart); ab V2 isoform (some in adult heart); aa V1 isoform (present in adult heart) V3 has low ATPase activity--> breaks down ATP much more slowly than V1 isoform, muscle containing V3 uses less O2; muscle with V3 form can maintain contraction longer. therefore hypertrophied muscle is more economical in force generation and ATP use. ---end---