Flashcards in KIN 406 Midterm 2 Deck (275):
How much exercise can someone with McArdle's Disease tolerate?
Very low-low intensity exercise can be tolerated, however, under high intensity situations, muscle fatigue occurs within a few seconds-minutes after ATp-PCr stores and blood glucose are depleted
Does muscle fatigue in McArdle's disease come along with lactate accumulation?
NO, because glycoylsis is fully activated due to the inability to utilize glycogen.
What are the painful muscle contractures in McArdle's disease caused from?
Resemble rigor mortis. Fatigue and contractures are a result of ATP depletion---myosin fails to detach from actin. Fatigue and contractures are confounded by decreased Ca2+ pump activity due to a fall in ATP content in critical intracellular compartments (SR-glycogenolytic complex). Damage may also occur since elevated Ca2+ can lead to caspase activation (proteolytic enzyme) and signal apoptosis.
What is the role of the SR-glycogenolytic complex?
It provides compartmentalized ATP supply to the SERCA pump, so that myosin ATPase doesn't use all the ATP in the cell for itself
What are mitochondrial myopathies?
Includes disorders which show defects in mitochondrial function.
What type of exercise capacity do individuals with mitochondrial myopathies have?
Very limited because they cannot make ATP aerobically or the ability is severely limited. Even mild activity is associated with breathlessness and severe metabolic acidosis becuase they use more glyocolysis. Very pronounced when performing longer duration exercise. THere is normal glycolytic function for ATP production since large increases in lactate occur
Amount of individuals mtiochondrial myopathies affect?
What type of DNA do mitochondria have?
Both nuclear DNA and mitochondrial DNA
Where are the gene mutations and deletions in people with mitochondrial myopathies?
In NDNA and mtDNA
Most studies and most common mitochondrial myopathies effect what?
Electron Transport Chain
How are the mitochondria abnormal in mitochondrial myopathies?
Pateients with defects in Complex I have abnirmal mitochondria proliferation, and mitochondria accumulate around the edge of the fibers and appear red with trichrome stain ("ragged red fibers"). Frequency ranges from 1-40%.
What structures do patients with mitochondrial myopathies show?
Paracrystallin inclusions which are composed of mt creatine kinase
Why do the mitochondria proliferate and paracrystalline structures accumulate during mitochondrial myopathies?
To compensate for energy deficiency
What mtCK do?
Makes creatine phosphate and ships into the cytosol
What does cystolic CK do?
Takes phosphate group from creatine phosphate and transfers it to an ADP to make ATP
What do fat metabolism disorders affect?
Usually have a dysfunction or deficiency in carntine or carnitine palmitoyl transferase (CPT) enzymes
What are the symptoms of a fat metabolism disorder?
Patients lacking carnitine and/or CPT develop muscle weakness, pain and damage during exercise (ATP depletion, acidosis, elevated Ca2+)
Why are symptoms of a fat metabolism disorder more evident during prolonged, submax exercise?
The glycogen and blood glucose stores become depleted, and then all that is left is fat for energy. However, fat cannot be utilized as a fuel source, so the person fatiguges/
What do biopsies of fat metabolism disorder patients show?
Large fat droplets...which under severe conditions may also disrupt myofibril architecture
What is COPD?
Disease of the lungs that makes breathing difficult. Chronic bronchitis and emphasema. Exposure to an initiating factor (mainly cigarette smoke) causes enlargement of the mucus glands and an increase in the mucus-secreting goblet cells. Ciliated epithelium that lines the airway become damages leading to decreased mucus transport, leading to an increased likelihood of respiratory infections
Damage from COPD causes what?
An inflammatory response (infiltrating neutrophils, macrophages, lymphocytes), and release of inflammatory mediators (TNF-alpha, IL-8, elastases)
How does COPD relate to skeletal muscle?
It is associated with exercise intolerance, which is expected, but patients with atrophy fatigue occur much sooner. Loss of muscle mass is a major co-morbidity of COPD and a powerful predictor of mortality.
What fibre type is preferentially atrophied and lost in COPD?
What does COPD do to the oxidative capacity of muscle fibres, and MLC?
Reduces it...decreases in SDH, CS, capillary density, and decreased myoglobin (25% lower)...shift towards MLC fast form and in most extreme cases, MHC, too
Consequences of the muscle brought on my COPD?
1. decreased muscle strength due to atrophy 2. decreased muscular endurance and increased fatigability (atrophy and decrease in Type I) 3. Lower resting ATP, PCr, and glycogen concentrations 4. Accelerated PCr and ATP depletion, glycogen breakdown, and lactate accumulation during exercise becaue the lack of O2 to produce ATP aerobically
How are VO2 and muscle wasting related in COPD?
People with muscle wasting have a lower VO2 than people with normal BMI, suggesting that muscle wasting in addition to COPD causes a drop in VO2
What phenotype does fibers become more like in COPD?
A fast phenotype
What is CHF?
Chronic heart failure/congestive heart failure. Caused by any number of conditions (CAD, hypertension, MI). Condition of the heart where functional or structural changes impair the ability of the heart to fill with or pump sufficient blood to body. Can have an impact on peripheral tissues since distribution of nutrients, growth factors, and O2 from blood can be altered.
What are the consequences of CHF on muscle?
1) muscle atrophy of both Type I and II fibers (greater in Type I) 2) Decreased Type I and increased Type I and increased Type II percentage (preferential loss of Type I fibers and fiber type switching Type I --> Type II). 3) inc. intracellular lipid droplets and some fibrosis 4. decreased capillary density 5. decreased mitochondria volume and size 5. decreased Kreb's cycle enzymes (SDH, CS) 6) Lower resting ATP, PCr, and glycogen concentrations 7) Decreased muscle strength, mass, faster onset of fatigue 8) Increased exercise tolerance and decreased VO2 max 9) Accelerated PCr and ATP depletion, glycogen breakdown, and lactate accumulation during exercise (lack of O2 for aerobic metabolism)
Preferential loss and atrophy of fibers in CHF?
Preferntial loss of Type I fibers and fiber type switching Type I --> Type II, increases fatigability and decreases capillary density in hypertensive animals and humans
Direct and indirect mechanism of COPD and CHF?
Direct: both lead to exercise intolerance, which leads to a reduced health status Indirect: both cause hypoxia, oxidative, nutritional stress, systemic inflammation, medication, and disuse, causing limb and respiratory muscle alterations, leading to exercise intolerance, which reduces health statis
What are the 6 most important mechanisms that play the biggest role in the skeletal muscle dysfunction and atrophy observed during aging and disease?
1. Decreased satellite cell number and function 2. Mitochondrial dysfunction and accumulation of mt DNA mutations 3. Increased free radical generation and oxidative stress 4. Increased apoptosis nad cell death and autophagy and protein degradation 5. Systemic and local inflammation
Imbalances in cell proliferation and death can result in?
Undesired tissue growth or atrophy. The cell cycle is integral in controlling cell proliferation and tissue turnover.
What are steroids?
Growth factors (testosterone, estrogen, and cortisol) that penetrate the cell membrane and act on steroid receptors directly effecting genomic regulatory mechanisms
How do polypeptide GFs work?
Cannot penetrate the membrane and instead act on membrane receptors to activate kinases, which leads to activation of certain transcription factors (IGF-I, IL-2)
What do growth factors do?
Stimulate the cell to enter the cell cycle (proliferate) but small amounts are needed to inhibit the cell from dying
Which of the following would lead to a mitochondrial myopathy? a) PFK deficiency b) carnitine deficiency c) creatine kinase deficiency d) a and b e) a,b,c
b) carnitine deficiency
Muscles of individuals with COPD show similar phenotypes to people with?
Inactivty atrophic myopathy and CHF
What are the 5 phases of the cell cycle?
Go, G1, S, G2, and M
What is G0?
A non-proliferation state where the cell is quiescent, waiting for GF to activate it
What are G1 and G2?
Gap phases; cellular components such as RNA, protein and enzymes are synthesized and accumulated to be used in subsequent phases
What is S phase?
Synthesis phase where DNA replication occurs
What are the G and S phases grouped as?
What is mitosis?
Involves the division of nuclear and cellular components.
6 subphases of mitosis?
1. Prophase: chromosal material condenses to form compact mitotic chromosomes 2. Prometaphase: chromosomes become organized and move towards spindle equator 3. Metaphase: chromosomes line up on equator 4. Anaphase: chromosomes move towards opposite poles and cell begins to split 5. Telophase: chromosomes cluster at opposite poles and new nuclear membrane is formed 6. Cytokinesis: formation of 2 genetically identical daughter cells
What causes the transition from G0 to G1?
Growth stimulation from hormones or GFs
What happens to G1 cells deprived of sufficient growth factor?
Can exit into G0
How ling is growth stimulation needed during G1?
Only during the first 2/3
What is the restriction point?
The point where growth stimulation is required (early G1) and an irreversible commitment for the cell to undergo one cell division (late G1-M)
What are checkpoints, and where are they found?
Are safeguards to ensure the evensts of the previous stage have been completed correctly and are found at the G1 to S, and G2 to M phase
What progresses the cell cycle and its specific stages?
Cyclins and cyclin dependent kinases
What are cdk's?
Normally inactive but become activated when they become associated with cylcins (active cyclin-cdk complex). Collectively, these complexes phosphorylate proteins which stimulate gene expression of transcription factors and other critical cell cycle components
What are cdk's regulated by?
Their expression level (depending on the phase of the cell cycle), but also by the expression of cdk inhibitors (p21, p27) and tumor suppressors genes (p53)
What is the Hayflick limit?
The finite number of cellular divisions a cell can undergo
How is DNA copied?
With the aid of a RNA primer and DNA polymerase
How does DNA polymerase add nucleotides?
5' --> 3' direction upstream of the primer. DNA polymerase cannot add nucleotides to the new 5' end.
What are telomeres?
DNA structures which form "caps" on the end of the chromosome that consist of 1000-1700 base pair double stranded sequence followed by a single strand overhang that protrudes the 3' end. Serve to protect the end of the chromosome from degradation.
What is the purpose of telomeres?
Serve to protect the end of the chromosome from degradation and as a means to compensate for chromosome shortening during replication. Instead of the chromosome becoming shorter with every replication, the telomere does by 50 base pairs every time
What is telomerase and what does it do?
A telomere specific enzyme that is important to telomere maintenance, and is a reverse transcriptase that adds base pairs to the telomere to allow replication to continue without shortening the telomere all the way to the chromosome
How can replication capacity of a cell be predicted?
Telomere length...varies between cell tpyes and from one chromosome to another
What type of cells have shorter telomeres? Longer?
Cells that proliferate faster and more often (leukocytes) have shorter telomere lengths than cells that replicate slower (skin fibroblasts)
What is telomerase activity like in adult cells?
Very low or absent. Cessation of a cell to prolierate is indicative of cellular senescence and aging
Why don't skeletal muscle cells undergo the cell cycle?
The shape and fucntion of skeletal muscle fibers give it a high degree of specialization, but this comes at a cost of losing the ability to proliferate
How doe muscle cells repair and adapt?
Adding new nuclei to the existing fiber via satellite cells
What are myonuclei like in adult mammalian skeletal muscle?
Very stable with very little turnover of myonuclei. Estimated that no more that 1-2% of the myonucleo are replaced every week in healthy individuals
Where does the source of myonuclei for mmuscle regeneration come from?
Expansion of satellite cells and myogenic stem cells
Are muscle datellite cells myoblasts?
Muscle satellite cells in adults are distinct from the myoblast population during embryonic and fetal development
What are satellite cells?
Undifferentiated mononuclear cells.
Where are satellite cells located?
Between the plasma membrane and basement membrane.
Compared to myonuclie, satellite cells have...
a high nuclear to cytoplasm ratio, decreased organelle content, and smaller nuclear size
Morphological difference between satellite cells and myonuclei?
Smaller size, chromatin composition is different (compact and unorganized because cell is not dividing), and less cytoplasmic portion
How can satellite cells be identified besides from locatioN/
Pax7 appears to be specific to satellite cells since it is not epxressed in other cell types including differentiated myotubes. Skeletal umscle from Pax7-/- mice are absent of satellite cells and muscle regeneration is greatly reduced
ARe there more myocnuclei or satellite cells?
Is the same satellite cell marker found the entire cell cylce?
No, depends on what stages of development the cell is in
What do Pax7-/- mice show?
Much smaller overall size and much smaller muscles...shows that at some point after fibre formation and grouping, Pax7 is needed for muscle growth.
What do Pax7-/- mice lack?
Do not express other markers found in satellite cells including Desmin and c-Met
When are satellite cells present in limb muscle?
18 days after the primary fiber has been formed (after weeks 7-9 of gestation)
What happens to the relative number or percentage of satellite cells in all muscle fibers after birth?
Falls rapidly...at birth, SC account for approx 32% of muscle nuclie, 5% at 2 months, and less than 2-3% in adults. This trend continues as we age though the change is much slower
Which fiber type has more satellite cells?
Why do Type I fibers have more satellite cells?
1) Since slow muscle (i.e. soleus) is used to a greater extent there may be more "wear and tear" compared to fast 2) Nuclei number (not including satellitecells) is greater in slow fibers, therefore, may need more satellite cells to replace nuclei 3) Satellite cells have also been found to be more abundant in close proximity to capillaries which would give these cells access to a transport system and growth factors
What activates satellite cells?
Disease, exercise and damage
What determines the time course and the magnitude of the response of satellite cells?
The severity of the injury
Upon injury/damage, what happens to satellite cells?
Become actiavted, enter the cell cycle, and proliferate. These cells will undergo a number of cycles of proliferation to form a pool of muscle precursor cells
Once satellite cells have expanded and exit the cell cycle, what happens?
They mugrate to the specific muscle site, terminally differneitate, and fuse to the myofiber. Satellite cell activation is not restricted to the site of damage as damage at one end of a fiber can activate cells of the entire fiber.
7 satellite cells can give rise to?
>100 new myofibers containing >25000 differentiated myonuclie...have a high proliferative potential and ability to provide new genetic matieral
2 divisions satellite cells can undergo?
Plana and apical-basal division
Division on a vertical plane with cells remaining in contact with the basal lamina and sarcolemma...typically will generate two identical "self" cells (satellite cells with proliferative potential)...symmetric division
Division on a horizontal plane with one cell maintaining contact with the basal lamina and the other losing contact...typically will generate one cell committed to becoming a myotube and one "self" cell with proliferative potential (satellite cell)...asymmetric division
How many of the satellite cells that divide will differentiate and form myotubes or be incorporated into existing fibers?
~80%, the remaining 20% grow very slowly and are reserve cells that become quiescent
Is the regenerative capacity of stem cells unlimited?
In order for the cell cycle to begin, a cell requires...
growth factor stimulation (which then inc. the expression of cyclin-cdk complexes, increases the expression of cell-cycle specific transcription factors, increased levels of 2nd messenger kinases)
Which of the following would be a cdk-inhibitor? a) p53 b) p21 c) CDK-2 d) cyclin E e) all the above
The process of muscle regeneration and satellite cell expansion requires....
the influence of growth factors in a concentration and time dependant manner
How long after injury do satellite cells become activated?
After activation, how long do satellite cells proliferate for?
2-3 days in response to various growth factors
What produces growth factors for the satellite cells?
Autocrine, paracrine, and endocrine sources
Main satellite cell growth factors?
Insulin-like growth factor I and II, hepatocyte growth factor, fibroblast growth factor, leukemia inhibitory factor. Other factos such as NO, testosterone, IL-6, platelet derived growth factor, endothelial derived growth factor also play a role in satellite cell activity.
What do satellite cell growth factors do to the satellite cell?
Activation, chemotaxis, proliferation, and differentiation.
Before activation, do satellite cells express markers such as MyoD, Myogenin, Myf5, Desmin, and MHC?
No, these markers are assicated with myogenic potential and terminal differentiation...cells are quiescent
Following activation/proliferation how is satellite cell morpholically changed?
1) Satellite cells swell and develop cytoplasmic processes which extend at the poles of the cell (cell flattens out so it can fuse to a myofiber) 2) There is a decrease in heterochromatin, an increase in cytoplasmic to nuclear ratio, and in increase in organelle content
During satellite cell activation what molecules are upregulated, and what do they do in terms of promoting attachment?
Neural cell adhesion molecule, vascular cell adhesion molecule, and M-cadherin are upregulated. These adhesion molecules aid in satellite cell migration to sites of injury/damage by promoting attachment of satellite cells to the membrane of the myofiber.
When growth factor levels are high so is the expression of cyclin-cdk complexes, what does this promote?
Promotes proliferation and inhibits differentiation
What is upregulated in the early stages of satellite cell proliferation?
Cyclin D and MyoD in response to growth factors
As the levels of cylin D-cdk4 and cyclinE-cdk2 increase in satellite cell prolferation, what happens to levels of myoD and Myf5?
Decrease in levels of myoD and increases in Myf5...this up and down pattern allows the celll cycle to progress
WHen is Ofxo1 activated, and what does it do?
Activated under low growth factor conditions. Foxo1 works by activating the cdk inhibitor p21. p21 inhibits cyclin E-cdk2 and prevents the cell cycle progress through G1. The cell remains quiescent and doesn't express MyoD and Myf5.
What does IGF-I do for satellite cell activation and proliferation?
Growth factors (like IGF-I) bind to their receptors and activate the mitogen activated protein kinase (MAPK), which inhibits Foxo1. Inhibition of Foxo1, relieves the inhibition of cycline-cdk2 by p21 and allows the cell cycle and proliferation to progress. IFG-1/growth factors also upregualtes MyoD and Myf5, which allow the cell cycle and proliferation to occur.
How long after injury do satellite cells begin to withdraw from the cell cycle and wither self renew or differentiate?
What is differentiation characterized by?
A downregulation of Pax7 (no longer a satellite cell), cell cycle exit, terminal differentiation. Many of the cyclins and cdk's that regulate proliferation and cell cycle are down regulated during differentiation.
What causes the dowwnregulation of cyclins and cdk's that regulate proliferation and the cell cycle during satellite cell differentiation?
1) change in growth factor levels and type 2) changes in intracellular factors that regulate proliferation and differentiation
p21 inhibits cyclin-dependenat kinases, primarily by binding to...
cdk2, cdk4, and cdk6. Other factors including p15, p16, p18, p19, p27, and p57 act in a similar manner to p21 in the muscle.
The upregulation of what molecules promotes differentiation?
MyoD, myogenin, and MRF4...and an inhibition of cyclin-cdk complexes by p21
How does IGF-1 (and other factors) promote satellite cell differenation?
Binds to its receptor and increaases phosphatidylinositol (PI3K). This activates p21 and leads to cell cycle arrest (inhibits cyclinE-cdk2). PI3K also leads to an upregulation in MyoD, Myogenin, and MRF4. These actions promote satellite cell differentiation by acting on transcription factors that promote differentiation.
What is myostatin?
Growth and Differentiation Factor 8, a cytokine that acts to inhibit satellite cell proliferation and differentiation
What is transforming growth factor-beta?
acts in a similar manner of myostatin to inhibit satellite cell proliferation and differentiation
Myostatin and TGF-beta work mainly by...
Upregulation of cdk inhibitors (p21) (proliferation) and inhibition of MyoD (differentiation and proliferation)
What is myostatin's receptor?
Activin Type IIB Receptor
How does mystatin work?
Actiavtes p21, which inhibits cyclinE-cdk2 and leads to cell cycle arrest. Also inhibits MyoD which decreases satellite cell proliferation and differentiation.
When do differentiated satellite cells mature and become incorporated into the existing myocyte?
At approx. 7-10 days. At this time, the architecture of the myocyte in restored.
Terminally differentiated myocytes express?
myogenin, demin, MHC, and creatine kinase. After fusion, the satellite cells (which are now a myocyte nuclei) have lost their ability to undergo mitosis and are continuous with the membrane of the fibre.
Satellite cell time course following injury?
Activation --> proliferation --> differentiation --> maturation
What are muscle side population cells, what do they do?
have myogenic protential. Play a minor role in muscle regeneration and maintenance. Likely and reasonable to suggest SP cells (bone marrow and muscle) support other cell types such as connective tissue, fat, nerves, smooth muscle, etc. and play a very minor role in supporting the myofiber.
What happens to satellite cells with age?
A decrease in their number and proliferative capacity have been demonstrated during aging and is suggested to play a tole in reduced muscle regeneration. Satellite cells also seem to form thinner and more fragile myotubes
The number of satellite cells is more reduced in what fibre type?
Type II fibres
What happens to the number of non-dividing (BrdU-) satellite cells during aging?
It increases...meaning less proliferation
Isolated satellite cells from old vs young rats have a lower...
FOXO1 and p27 (less differentiation) and also form fewer and smaller myotubes
What happens to the ability to satellite cells to fuse following expansion and differentiation with age?
Decreases with age
Overall, what happens to satellite cells with age?
Lose ability to proliferate, differentiate, and fuse, all of which reduce the ability to regenerate/repair muscle with age. Lose overall number of them, too.
Which of the following is not a marker found on quiescent satellite cells? a) desmin b) c-met c) Pax-7 d) NCAM e) M-cadherin
a) desmin (a structural protein that hold myofibrils to the sarcolemma, so why would a quiescent satellite cell need this?)
Name 2 different ways to identify a stem cell
presence of PAX7 and location outside of the plasma membrane and below the basement membrane
Is there an exhaustion of the satellite cell pool in individuals with MD?
YES. individuals with MD undergo more skeletal muscle regeneration by the age of 4-5 than healthy individuals of 60 years of age. The proliferative lifespan of satellite cells from 9 year old MD patient was 1/3 of an age matched control
What is the telomere length like in individuals with DMD and LGMD?
Telomere length is much shorter than healthy people, which indicates that SC are proliferating much more and at some point these people will start degrading the chromosome
What happens to satellite cells during inactivity? How does it change with increased activity?
Total number of SC isolated from hindlimb muscle decreases following 14 days of inactivity. Decreased number of Desmin+ cells (satellite cells) and a decreased expression of the mRNA of several genes (M-cadherin, Myf5) important in satellite cell function is also seen. BUT, recovery quickly rstores number and gene expression
What do isolated cells from an inactivity atrophy fibre look like?
Isolated cells following inactivity are initially fewer, expand more slowly and form fewer and smaller myotubes.
In an ideal situation, how would satellite cells and muscle derived stem cells be used in a clinically relevant therapeutic application?
Approach would involve freshly isolating cells from limb muscle, identifying these cells using cell surface markers and culturing these cells. Cells would then be exposed to the appropriate conditions that promote their myogenic potential, and then transplanted to the appropriate spot in a person.
Why can't PAX7 be used as a satellite cell marker for theraputics?
It is inside the cell, so you would have to destroy the membrane to get to it, which would make it so that you couldn''t use it for therapy
What is the newest and fastest way to isolate adult stem cells or satellite cells from muscle?
Fluorescent Activated Cell SOrter
How can stem cells be identified in FACS analysis?
By their exclusion of Hoechst dye or other surface markers
How can FACS be used to isolate specific cells?
Cells that contain the markers you are interested in are put into a little droplet that carries a charge. Cells are then separated by their charges, so you are lest with a 99% pure population of a specific cell marker
What happens when mdx mice are given fluorescent green satellite cells from healthy donor mice?
the MDX mouse shows fluorescent green mature fibers and red dystrophin, showing that the injected SC proliferated, differentiated, and formed new fibres that now have dystrophin.
What happens to force generation following satellite cell treatment?
It increases because there is increased myofiber regeneration
What are 2 other therapeutic approaches that are being developed and tested to treat age and disease related muscle dysfunction?
1) Use of growth factors to simulate satellite cell activation, grwoth, and differentiation (IGF-I and testosterone) 2) Use of myostatin blockade (blocking peptides of antibodies) or knockdown (through gene manipulation) to promote satellite cell activation and proliferation as well as muscle growth
Roles of mitochondria?
1) generate ATP 2) role in signaling apoptosis 3) regulate, at least in part, cellular oxidative stress 4) encode for some of their own proteins through their own genome (mtDNA)
How is cytochrome-c related to apoptosis?
When it is released from its position in the ETC, it activates numerous proteolytic enzymes, eventually causing cell death
how does the ETC contribute to oxidative stress, which can cause mtDNA damage?
Leaks e-, causing generation of free radicals
How are mitochondria distributed throughout the cell?
Distributed in a non-random way that forms a branched reticular network (acts like a powerline between mitochondria, so they can send ATP and other intraorganelle factors to each other)
When were satellite cells discovered, and by whom?
1961 Alexander Mauro
Are mitochondria static organelles?
NO! Constantly, moving around and undergoing fusion and fission to share components like AP< DNA, and enzymes. Fission and fusion and super important to health of the cell and of the mitochondria...stop these processes = cell die
Upregulation of which of the following INTERNAL cellular factors would help with SC differentiation? a) myf5, myoD, cyclin E b) P21, myogenin, mrf4 c) cyclinE, myoD, myogenin d) P21, IGF, MAPK e) all of the above
b) P21, myogenin, mrf4
2 external factors (i.e. growth factors) that inhibit SC differentiation would be?
Myostatin (growth and differentiation factor 8) and Transforming Grwoth Factor Beta
Where do cells keep their mitochondria?
In areas of energy need..skeletal muscle near myofibrils and rapidly dividing cells near nucleus or ribsome
Typical size of a mitochondria?
0.5 micometers in diameter and 0.5-2 micrometers in length, can change depending on what's going on in cell
What is a mitochondria roughly the size of?
a typical bacterium
Basic structure of a mitochondria consists of?
an outer membrane, an intermembrane space, an inner membrane, and space within the inner membeane
What is the important of the cristae in mitochondria?
Allows for lots of SA which is important for more space for ETC enzymes and complexes and pumping hydrogens...lose cristae = lose function
What are some role o the outer membrane in mitochondria?
protein imports, metabolite influx/efflux, fission/fusion.distribution, apoptosis factors (Bcl-2, Bax), signalling molecules
What are some roles of the intermembrane space?
ETC (cytochrome C), cristae remodelling, redox enzymes, protein imports, apoptosis factors (cytochrome X, Smac/Diablo)
What are some role of the inner membrane?
Oxidative phosphorylation, metabolic transport, protein import, protein assembly, protein degradation
What are some roles of the matrix?
TCA cycle enzymes, fatty0acid oxidationn, mtDNA replication, mtDNA transcription/translation
Where did mitochondria come from?
Thought to have arise from a mutually dependent relationship between a primitive glycolytic eukaryotic cell and an oxidative bacterium. The union took place 2 billion years ago when the earth's atmosphere became more oxidative. This relationship allowed for a place of "residence" for the bacterium and for enhanced ATP delivery and oxygen detoxification for the eukaryotic cell.
What has happened to mtDNA throughout evolution?
Has lost many genes (no longer needed due to residence in the cell) and/or transferred some essential genes to the nucleus
When was mtDNA discovered?
How many base pairs does human mtDNA have?
16,569...consistent across most mammals (sheep ~16500, cows ~16338, rat ~16298, donkey ~16670)
What is the structure of mtDNA?
A double standed circular molecule (like bacteria) not a souble helix like nDNA
What is the genome of mtDNA like?
Very compact and contains only one non-coding region of significant length
How much of the mtDNA genome is noncoding?
less than 6%
How many base pairs does nDNA have, and how many proteins does it code for?
~3 billion base pairs which codes for ~25000 proteins
How much of nDNA is noncoding?
How many copies of genes does mtDNA have?
have multiple copies, whereas nDNA has 2 copies
How is mtDNA inherited?
all mtDNA is inherited from the maternal line
How many copies of mtDNA are there per mitochondria?
2-10 copies are found per mitochondria x 1000s of mitcohondria = thousand of copies of mtDNA
Is mtDNA linked to the cell cycle?
NO! Can make mtDNA whenever, unlike nDNA that is only copied during cell division
How does copy number of mtDNA differ between tissues?
Depends on oxidative potential...cardiac muscle 6-70/cell, skeletal uscle 3650/cell, platelet 4/cell, RBS 0/cell
Approximate mitochondrial cell volume for cardiac muscle, fast muscle, and slow muscle?
20%, 2%, 6%
How many genes does the mtDNA have, and what are they needed for?
13 genes needed for synthesis of 13 essential polypeptide subunits of the oxidative phosphorylation system. Also contains 22 tRNA and 2 rRNA genes require for protein synthesis to occur.
Of the 13 proteins encoded by mtDNA code for NADH dehydrogenase (complex I)?
Of the 13 proteins encoded by mtDNA code for cytochrome c reductase (Complex III)?
Of the 13 proteins encoded by mtDNA code for cytochrome c oxidase (Complex IV)?
Of the 13 proteins encoded by mtDNA code for ATP synthase (complex V)?
Does mtDNA code for Complex 2?
nDNA for Complex I, Complex III, Complex II, Complex IV, and Complex V?
39, 10, 4, 10, 14
Why is mtDNA used in anthropology and forensic situations?
Decreased number of base pairs and greater number of copies as compared tonDNA. Also, very little variation occurs between generations.
What is a mutation?
A permanent change in the DNA sequence
Most individuals have ____________ absent from mutations
The mutation rate in mtDNA is ___________ higher than nDNA.
What are the 4 factors that cause the mutation rate to be so high in mtDNA?
1) no protective histone cover 2) mtDNA repair is less efficient 3) mtDNA has very few non-coding regions 4) close proximity to ETC, so highly susceptible to oxidative stress
If an individual has a mtDNA mutation will all mitcohondria have it?
No, the mutatued genome will always co-exist with normal :wild-type" mtDNA
What is heteroplasmy?
Differences in mtDNA (maybe 3+ mutations, along with 1 normal). Can occur within and between tissues depending on how the mtDNA segregates (replicative segregation)
What determines the seriousness of a mtDNA mutations?
The percentage of mutant DNA present determines the seriousness of the disease (wild type: mutant)
Ratio of wild-type mutant:mutant DNA can range from...
100:0 to 0:100. In contrast, in Mendelian genetics, a mutation is only heterozygous or homozygous.
What is the threshold for symptoms in mtDNA mutations?
usually above 70% mutated mtDNA to show signs of a problem. If sufficient normal mitochondria is present then no impairment
What types of cells are mtDNA mutations most common in?
POst-mitotic tissues such as the brain, heart, skeletal muscle (cytopathies)
Why are mtDNA mutations more common in post-mitotic tissues?
Cells can't divide, so mtDNA mutations accumulate over time.
What can mtDNA mutations result in?
Exercise intolerance, deafness, diabetes, and infant death
Is the phenotype of mtDNA (ratio of wild:mutant) always the same when the mother passes it on? Why or why not?
NO...there is a random distribution of mitochondria in fertilized eggs (not all the same). Therefore, offspring of the same mother can differ greatly on their percentage of mutant mtDNA and severity of the disease.
Although men and women can have mutated mtDNA, who passes on the mutation?
Only the mother
How does mitochondrial content vary throughout fiber types?
Slow fibers have the highest amount, and fast fibers have the lowest amount.
How can mitochondrial content in skeletal muscle be increased? Decreased?
Increased in response to contractile activity and disease. Decreased in response to inactivity and disease.
2 distinct locations of mitochondria in muscle?
Immediately under the sarcolemma (subsarcolemma mitochondria) and interspersed between the myofibrils (intermyofibrillar mitochondria)
Percentage of mitochondria in skeletal muscle that are IMF? SS?
75% IMF, 25% SS
Why are there more IMF mitochondria than SS?
Higher energy needs at the myofibril (myosin ATPase, Ca2+ ATPase) compared to the sarcolemma (Na+/K+ ATPase)
How do IMF mitochondria differ from SS in terms of ATP production and concentration?
IMF mitochondria have a higher rate of respiration (greater rate of ATP production) and higher resting ATP concentration
How are IMF and SS mitochondria different?
Location, membrane composition (cardiolipin), rates of ROS production, and susceptibility to apoptotic signaling
Which of the following is false? a) mitochondria originated from bacteria b) mtDNA has a greater mutation rate than nDNA c) mtDNA has more protein coding regions than nDNA d) mtDNA deletions are most common in post mitotic tissues e) none of the above
The threshold of mutation rate for symptoms arising from mtDNA mutations is usually above ____ %.
4 things that stimulate mitochondria biogenesis?
exercise, cold/fasting, nitric oxide, energy deprivation/exercise
How does exercise stimulate mitochondria biogenesis?
Increases cystolic calcium while exercise is happening --> stimulates CaMKIV --> activates CREB --> activates PCG-1alpha --> upregulates transcription factosr for numerous proteins involved in mitochondrial biogenesis
How does cold/fasting increase mitochondrial biogenesis in tissues (more so in brown adipose than muscle)?
Activates cAMP --> activates PKA --> activates CREB --> activates PCG1alpha --> upregulates numerous transciption factors for numerous proteins involved in mitochondrial biogenesis
How does NO increase mitochondrial biogenesis?
Activates cGMP --> PCG1alpha --> transciption factors for proteins involved in mitochondrial biogenesis
How does energy deprivation increase mitochondrial biogenesis?
Depleted ATP and increased ADP concentrations, tells the cell that more ATP is needed and ADP needs to be taken care of --> activates AMPK --> PCG1aplha --> mitochondrial biogenesis
What is the main molecule that upregulates mitochondrial biogenesis and promotes fibre type switching from Type II to Type I?
What happens to fibres in a Tg mouse (overexpression of PCG1-alpha)?
They become more oxidative by increasing myoglobin, TN I (slow), and cytochrome C oxidase.
What tissues are most sensitive to altered mitochondrial function?
Ones with high energy demands...like muscle?
Why are mtDNA mutations and mitochondria myopathies most frequently diagnosed in skeletal muscle?
1) high energy demands so clinical manifestations are easily noticed 2) post-mitotic tissue with little regeneration so mtDNA mutations can accumualte or develop if not inherited 3) muscle biopsies are a frequent procedure so diagnostic testing is much easier
How many proteins does the oxidative phosporylation system consist of? How many are fomr mtDNA? How many from nDNA?
90 proteins, 13 from mtDNA, 77 from nDNA
What type of mutations can affect oxidative phosphrylation and other important mitochondrial functions?
Both nDNA and mtDNA mutations
The severity of symptoms in a mtiochondrial myopathy is usually dependent on?
the energy requirements of the tissue or process affected
What would the muscle biopsy of a patient with mtDNA single deletion is the muscle was stained fro SDH and COX?
There would be extensive SDH staining in some fibres (a Krebs Cycle enzyme entirely encoded by nDNA) and that same fiber with the mtDNA mutation would show no staining for cytochrome c oxidase (an enzyme containing 3 subunits encoded by mtDNA).
Would all the fibers in a patient with a mtDNA mutation stain the same for SDH and COX?
NO! Not all fibers are afected the same because they don't all have the same mtDNA due to random distribution of mtDNA early on in development when myoblasts are dividing.
Why would a biopsy from a patient with a mitochondrial myopathy show increased numbers of mitochondria?
Compensation for the lack of ATP production
Why would you stain for both SDH and COX in someone you suspected to have a mitocohdrial myopathy?
To determine where the mutation is...nDNA or mtDNA
How many mitochondrial proteins are nuclear encoded?
Percentage of genes in nucleus that encode for mitochondrial proteins?
Proteins that are encoded for mitocohdrial proteins in the nDNA are synthesized where?
In the cytosol as infolded precursor proteins
What 2 major cystolic chaperones that bring unfolded precursor proteins from the cytsol to the mitochondria?
Heat Shock Protein 70 (Hsp70) and Mitochondrial Import Stimulation Factor (MSF)
How do the precursor proteins get imported into the mitochondria?
Translocases of the outer membrane (TOM) and Translocases of the inner membrane (TIM)
What is TOM?
Translocases of the outer membrane (TOM40, TOM20, TOM22, TOM37, TOM70)...pores on the outer membrane of the mitochondria that allows passage of precursor proteins from the cytosol into the inner membrane space
What is TIM?
Tranlocase of the inner membrane...TIM23 and TIM44...pores on the inner membrane of the mitochondria that allow the passage of precursor proteins from the inner membrane space to the matrix
When are presursor proteins refolded into their functional, 3D conformation?
Once they have crossed into the matrix
The level and function of TOM and TIM influence?
Mitochondrial protein import, mitochondrial function, and mtDNA maintenance
What is uncoupled respiration?
When protons leak across the inner membrane without forming ATP....this process is exactly the same as normal metabolic respiration (substrate breakdown, electron donation, electron transport, O2 utilization) but hydrogens pass through uncoupling proteins (UCP) and not ATP synthase
Where are UCP most prominent?
in the liver and skeletal muscle
Uncoupling process can increase metabolic rate by?
What are the 2 main roles on UCPs?
play an important role in both tissue metabolic rate and thermogenesis
How many isoforms are UCPs are there?
Where is UCP1 mainly found?
brown adipose tissues (infants and other mammals, not abundant in adult humans)
UCP2 mainly found?
a variety of tissues
UCP3 found mainly in?
What UCP is predominant in skeletal muscle?
What are some characteristics of mice who overepxress UCP?
Higher metabolic rate/oxygen consumption. Lower resting fat stores and increased resistance to obesity following a high fat diet.
What is the preferred source of energy in muscle?
How does fat enter the muscle?
FAT/CD36 and FABP
In order to be oxidized in the mitochondria, fat must be...
activated, forming a fatty acyl-CoA
Reaction for the activation for fatty acid activation?
fatty acid + ATP + CoA --acyl-CoA synthethase--> fatty acyl-CoA + AMP + PPi
Where is acyl-CoA synthetase found?
Mitochondrial outer membrane
4 mitochondrial proteins that facilitate the transport of fatty acyl-CoA in the mitochondria, because the inner membrane is impermeable to CoA and its derivatives?
carnitine, carntine palmitoyl transferase I (CPTI), carnitine palmitoyl transferase II (CPTII), carnitine acylcarnitine translocase
Only fatty acids attached to ________ can enter mitochondrial matrix?
What does CPTII do?
found on the inner mitochondrial membrane, facing that matrix, that exchanges carnitine for CoA, reforming fatty-acyl CoA
How is carntine transported back to the intermembrance space?
A deficiency in any of the fattty acid transport proteins can lead to?
metabolic and muscle dysfunction
Steps in fatty acid transport
1) FFA is activated into acyl-CoA via acyl-CoA synthetase 2) Carnitine palmitoyl transferase I on the outer mito. membrane switches a carnitine for a CoA, forming acyl-carntine 3) Acyl-carnitine crosses into the matrix via the carntine acylcarnitine translocase 4) Carnitine palmitoyl transferaae II exchanges the carnitine for a CoA, reforming acyl-coA 5) Acyl-CoA is turned into acetyl CoA via beta oxidation and carntine is reshuttled into intermembrane space by carntine-acylcarnitine translocase
Where is carnitine acylcarnitine translocase found?
where is CPTII found?
Where is acyl-CoA synthetase found?
Where is CPT I found?
what does CPT stand for?
canritine palmitoyl transferase
Where is VDAC?
outer mitochondrial membrane
What is VDAC?
Voltage Dependent Anion Channel. Also known as porin. Is the primary route of entry of metabolites and ions across the outer mitochondrial membrane. Alllows passage of ATP, ADP, Pi, Ca2+, K+, Na+, as well as nonelectrolytes (<6KDa)
Isoform of VDAC in skeletal muscle?
What is ANT?
Adenine Nucleotide Translocator. Located in inner mitochondrial membrane. One of the most abundant proteins in the membrane. Regulate the movement of ATP out of matrix and ADP into matrix.
How many isoforms of ANT are there?
What is ANT isoform is found in skeletal muscle?
Loss of ANT causes what?
over proliferation of mitochondria and leads to ragged red fibers in skeletal muscle
Besides transport, what else do ANT and VDAC do?
participate in apoptotic cell death
Which of the following is false regarding mtDNA? a) contains 37 coding regions b) contains 13 protein coding regions c) found only in mitochondrial matrix d) # of base pairs is similar across species e) none of the above
e) none of the aboce
Mitochondria contain approximately _____________ proteins?
What does exercise do in terms of PCG1alpha and COX activity? What does denervation do?
Exercise increases both, deinnervation decreases both
What happens to oxidative phosphorylation capacity with age?
What happens to mitochondrial content with age?
What 2 things cause the decrease in oxidative phosphrylation with age?
mitchondrial content decreases and each mitochondria produces less ATP independent of content
How does the number of dysfunctional mitochondria change with age?
What does the increase in the number of dysfunctional mtiochondria with age cause?
Compensatory proliferation of mitochondria and accumulation of mitochondria is some muscle fibers. Number of ragged red fibers increases with aging.
Why does mtDNA copy number decrease with age?
decreased mitochondrial content but also due to decreased copy number per mitochondria. With againg there is also an increase in the number of mtDNA mutations present in skeletal muscle
D357A/D257 transgenic mice show?
An accumulation of mtDNA mutations and show an aging phenotype. Also show accelerated muscle wasting and greater cell death, and decreased survival
What does exercise do in terms of mtDNA mutations?
prevents mtDNA mutations, mitochondrial alterations, atrophy.