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mito midterm.docx

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York University
Kinesiology & Health Science
KINE 4516
David Hood

 Increase in mito= biogenesis and function leading to positive health effects  Less biogenesis and dysfunction causes INCREASE in apoptosis, DECREASE in energy supple, and INCREASE in ros (reactive oxygen species) production  This can be caused by aging (which decreases mito content and function, and disease which can be metabolic, neurodegenerative, cardiovascular, and muscular)  Ex. Of diseases= alzheimer’s, type 2 diabetes, ALS, mitochondrial myopathies, huntington’s, cancer  Mito theory of aging= free radicals cause aging  2 main factors= absolute count of mito and function of mito  Microtubules move the mitochondria around the cell  Muscle reps about 40% of body mass in non obese individuals  Fibre type is determined by myosin (the isoform) thick filament  Thin filament contains the actin, troponin, and tropomyosin  Troponin has 3 different binding sites (tropomyosin, calcium, and actin)  Low cytosol in muscle cell allows more room for muscle fibres and organelles  Peripheral nuclei are on the edge of the fiber, if they are in the middle it is a sign of injury  Muscle fibers are long multinucleated cells, packed with myofibrils  Cardiac cells have one or two nuclei, and a lot more mitochondria (due to aerobic demand)  30% mito in heart cell, only 3% in muscle cell  Muscle development: myoblast—fuses together to become ->myotube-->differentiated muscle fiber (type 1 or II), all through the process of myogenesis and differentiation  Evolution? Used to rely more on glycolysis to make ATP for survival, 1.5 billion yeasr ago, O2 became more dominant in the atmosphere and bacteria entered cells, and it has double membrane  Plasma membrane is a fluid mosaic model, has integral vs. peripheral proteins as well as signaling proteins  Extracellular vs. cytosolic vs. transmembrane domains  5 kinds of phospholipids found in membrane (cardiolipin is found only in mitochondria)  3 types of structures to cytoskeleton: 1. Microtubules (tubulin isoforms)… 2. Actin filaments ( cytoskeletal y-actin; not involved in muscle contraction, and microfilaments)…3. Intermediate filaments (esp in the nucleus, the lamin protein)  Kinesin is a motor protein, that basically moves the mitochondria along the microtubule  Kinesin connects to Milton which connects to miro (protein that goes inside mito)  Nuclear pores regulate entry and exit  Chromatin (DNA and histones which prevent transcription from taking place) and nucleolus (for rRNA synthesis)  Glycosylation= adding carb molecules to a protein structure on the RER after it has been translated  Free ribosomes become cytosolic proteins  Autophagosome fuses with lysozome for degradation using proteolytic enzymes  Autophagy= splitting organelles  Mitophagy= breakdown of mito  Synthesis of the mitochondria includes transcription, protein import, intra mito protein synthesis and fusion  Degradation of the mito includes fission, mitophagy, and intra mito proteolysis  Folds in the mito are called cristae  The inner membrane is made of the ETC and select membranes that are pretty tight  The matrix is the inner most part of the mito, it has the krebs cycle and contains many enzymes  The outer membrane is permeable and porous  The intermembrane space has proteins that can be released into the cytoplasm  Mito undergo shape changes: 1. Swelling and opening pores for protein release…and 2. For motility, either fusion or fission  Lipid extraction from mito: muscle homogenation, buffer and extract lipids  Cardiac muscle have more inner membrane proteins than skeletal muscle  More cardiolipin, more enzyme, the more cardiolipin, the higher the conc. Of enzyme per gram of CL  Proteins are visualized using immunofluorescence: we permeabilize with detergent, add antibody, tag the antibody, and look under miscroscope at a specific wavelength  The mito is more of a membrane network  FTW= very little mito (more glycolytics)  FTR= many mito  Brooks study in 1986 found that there are differences between species in terms of mito in different fiber types…soleus muscle or FTR or white FTW  16% in FTR muscle, 9% FTW but it depends what species we're talking about...this is a rat  Takuro ogata, in cell tissue respiration journal, found that we have 5-7% mito in STR, 4-6% in FTR, and 2-3% in FTW  HUMANS  Compared to a mouse or a rat we have a lot less mito  SS mito (close to sacrolemma) are much more adaptable to disease and exercise than IMF mito  SS mito are more oval  How to isolate and separate? Mince muscle and homogenize to remove the sarcolemma, SS becomes liberated. Then centrifuge, treat the remainder with a protease to loosen themyofivrils and liberate the IMG, then centrifuge again.  Type 2 diabetes has very little SS mito, SS is closer to the membrane, closer to substrates and oxygen  When there is an increase in insulin resistance, there is a greater loss of SS function  SS mito are closer to the delivery site of relatively insoluble substrates like FFA  Training increases SS mito in type 2 fibers by 6 fold, and in IMF by 3 fold (6-8 weeks at 60-70% of your VO2 max)  SS mito have ROS production, and adapt more to muscle use and disuse…as well as RRF and disease  IMF mito are responsible for state 3 and 4 respiration, protein import into matrix, and protein synthesis (at rest and activity)  In order for the reticulum to expand, we need a balance between fusion and fission  Fis1, and Drp promote fission (which favours CHO metab and apoptosis and creates more ROS) which breaks the mito up into pieces  Mfn-1, Mfn-2, and OPA1 promote fusion, which occurs with training and favours lipid metabolism  CHO and lipid metabolism:  Glucose can go into muscle cell using glut4 transport, and it can either be used right away or stored as glycogen  Glycogen converted to pyruvate which goes into the mito and undergoes the kreb’s cycle which makes citrate (citrate inhibits PFK)  PFK is the rate limiting step in glycolysis (NEED PFK TO BREAK SUGAR)  2 sources of FFA, one from blood, and one from TG. FFA in muscle cell goes into beta oxidation, which makes acetyl CoA and ultimately inhibits PFK again  Beta oxidation makes NADH and FADH…as well as kreb’s makes it  NADH and FADH can inhibit PDH which inhibits CHO metabolism  Insulin inhibits HSL (which breaks down TG into FFA and glycerol) …whereas epineph and norepineph stimulate glycogen breakdown in muscle  Glut4 is the glucose traffic regulator  Glucose undergoes glycolysis and is split into 2 molegules of carbon (CO2 produced as byproduct) and before it enters kthb’s it is decarboxylated into 2 C, Acetyl CoA  5 complexes in ETC (5 is ATPsynthase)  Outer membrane of mito doesn’t do much regulation of what comes in and out  Pyruvate enters cell, makes NADH and FADH  When we exercise, ADP and Pi are dependent on ATPase  Pi comes in through a carrier and ADP comes in using the ANC protein, they make ATP, which can then be exchanged out for another ATP (direct exchange of ANC 1:1)  pH is lower outside because there are more H+ outside, this represents the potential energy for ATP synthesis  you want a gradient on the outside, so that the flow drives the ATPase  When ADP binds to the complex, it acts as a gatekeeper and allows the H+ to flow through  Cyanide will inhibit complex 4, backing up the whole system and causing cell death  If you are not exercising (you don’t have a lot of ADP) and have a high proton motive force, you need to make ATP but there needs to be a way to dissipate the gradient  UCP dissipates the gradient and allows more electrons to flow …leaky membrane  Peter Mitchell in 61 was the first to propose that an ion gradient had potential energy, and that phosphotylation was due to the transfer of protons across the membrane  Wallace in 2005 said the main 3 functions of mito include: 1. energy production during state 3 and 4 respiration 2. Reative oxygen species generation (electrons from the ETC can become loose and can attract to O2, making MnSOD which is a free radical)…there are 2 dangerous radicals, the SOD, and the hydroxyl ion that is made from the iron 3. Mediation of cell death…Certain intermembrane proteins cause apoptosis if they are released (there are 6 proteins, including CytC)  There are more ATP in the cell at rest, and more ADP in the cell during exercise (because ATP is broken down when you need it, and a flux of ADP is created plus free phosphate)  The steps of the ETC: 1.NADH donates electron to NADH-Q reductase, which then passes it off to ubiquinone 2. Coenzyme Q (not a protein) in the inner membrane of the mito can move freely and in the oxidized form (when it is missing an electron) it comes in contact with complex 1 and accepts an electron to become reduced 3.The free energy released from this electron transfer is used to pump protons (H+) through the NADHQ reductase complex into the IM space where a proton concentration gradient gets built up  Ubiquinone only carries electrons  NADHQ reductase is an electron carrier and a proton pump 4. Cytochrome reductase accepts electrons into a subunit called cytochrome b first and then passes the electron to the other subunit called cytochrome c1 5. Another electron carrier, cytochrome c picks up the electron from cytochrome reductase 6. Cytochrome c carries the electron on its heme group (which it only has one of) until it reaches the final protein carrier, cytochrome c oxidase. Cytochrome reductase acts as a proton pump (just like the NADHQ reductase 7. COX is the funal electron carrier. It is similar to NADHQ reductase and cytochrome reductase. It accepts an electron from cytochrome c and passes it to O2, the final acceptor of electrons in the chain.  electron affinity increases across the chain(stronger magnet as you move)  4 complex is the COX  3 spots for proton movement across the membrane  FADH donates H to cytochrome reductase c to help, but it doesn’t contribute to much of the ATP production  What regulates mitochondrial respiration is free ADP and its interaction with the ATP synthase (both Pi and O2 are in plentiful supply)  The energy status of the cell is measured by ATP/ADP+Pi  The less ATP, the more ADP and Pi…so during exercise, the ratio is small  Control strength theory= control is thought to be shared at multiple points, of ADP to O2 consumption  The gradient created by all of the protons that are transferred from the inside to the outside makes the outside a lot more positive (proton motor force)  At rest= high ATP and low ADP  Knowing the concentrations can give us info about the status of the muscle tissue  The pmf that drives the ATP synthesis is composed of two parts, the voltage difference and the ph difference  The voltage difference is caused by the positive charges being on the outside of the cell  The lower pH outside (more acidic environment) is caused by the H conc.  The higher the pmf, the lower the rate of electron transport because of backpressure  Under resting conditions, the demand for ATP synthesis is limited, and although the PMF may be high, the flow of protons back into the mito is minimal  When demands are increased (like in exercise), cytosolic ADP increases, and it is exchanged with ATP via the ANT. More ADP causes the PMF to become discharged as protons pour through the ATP synthase generating a pool of ATP.  ADP conc is what determines the rate of electron transport  State 4 is the respiratory rate= when energy charge is high, concentration of ADP is low, and electron transport is limited by ADP  In state 4 there is little or no adp, oxygen consumption is low, and if you add ADP, it increase electron flow, and o2 consumption becomes faster, making the mito go into state 3 respiration  There is a leak in the membrane because of the porous nature of it, and it allows protons to dissipate and allows electrons to flow to O2 and be reduced to H2O  The dissipation of the proton gradient in this way produces heat, instead of ATP, because it uncouples the source of potential energy from ATP production via ATP synthase  Mito resp activity cycles between state 3 and 4 respiration  Respiratory control ratio is between state 3 and 4.  Clark electrode= measures oxygen on a catalytic platinum surface (mito are isolated and incubated in a chamber with an electrode that measures O2)  How fast does mito consume o2?? Substrate must be a source of NADH or FADH (so used malate and glutamate…as well as succinate) and then since there was no ADP, the UCP had to pump H+ inside of mito  If you add mito, over time, endogenous substrates cause O2 levels to go down a little bit, until substrate is added, and more O2 is used up. When ADP is added, we enter state 3 respiration, and once it is used up we can resume state 4 respiration (which doesn’t use ADP)  State 3 uses up a lot more O2 and is at a greater slope, unless mito doesn’t function properly  Uncoupling proteins= provide relief of proton flow without ADP  Increased PMF means an increase in ROS production  Clark electrode= measures oxygen on a catalytic platinum surface (mito are isolated and incubated in a chamber with an electrode that measures O2)  Decreasing the PMF reduced ROS production  UCP1 (found mostly in BAT, causing it to generate lots of heat) 2, and 3 lower ROS production and membrane potential compared to knockout  The mobile electron carriers of the ETC are coenzyme Q and cytochrome c  Some inhibitors can discharge the proton gradient, they can block the oxidation of cytochrome b, but cytochrome c remains oxidized as do the downstream cytochromes  Uncoupling agenst act as weak acids that associate with protons on the exterior or mito, passing through the membrane with bound proton and dissociating it on the interioir of the mito. They cause max resp rates but ETC generates no ATP since the protons don’t return to the interior through ATP synthase (generate heat vs. energy)  85 proteins total in the ETC, 13 make up COX (3 of which are encoded by the mito)…most genes encoded by nucleus  Heme groups found in both complex 1 and 2 (iron deficiency diet affects ETC)  Cytochrome c oxidase (first three subunits out of the 13 are endoded by mtDNA)  Iron and copper (the two metal ions) play a role in enabling the acceptance of four electrons in reducing molecular oxygen to water  Transfer of electrons within the COX complex to oxygen:  4 electrons enter from cytochrome c and bind to the heme groups that are in close proximity to a copper atom. The o2 is trapped between the two metals until it is completely reduced to h2o, after which it can leave the protein complex  The net process uses 4 reduced cytochrome c’s and 4 protons to reduce o2 to two water molecules  Dimers (protein compl
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