Duchenne Muscular Dystrophy Lecture 2 PDF

Duchenne Muscular Dystrophy KINE 3P97, LECTURE 2 VAL A. FAJARDO, PHD Goals of today’s lecture 1. What is Duchenne muscular dystrophy (DMD)? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – frien...

Duchenne Muscular Dystrophy KINE 3P97, LECTURE 2 VAL A. FAJARDO, PHD Goals of today’s lecture 1. What is Duchenne muscular dystrophy (DMD)? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice  Duchenne Muscular Dystrophy (DMD) Ø Severe and debilitating disease that primarily affects boys (X-linked transmittance) Ø 1 in every 10,500 Canadian boys Ø 1 in 3,500 boys worldwide Ø Skeletal muscle weakness – wheelchair bound by early teens Ø Cardiorespiratory failure - early mortality with an average lifespan of 30-40 years Copyright © 2010 Pearson Education, Inc.  Caused by an absence of dystrophin Copyright © 2010 Pearson Education, Inc. Gorza et al., 2021. Cells 10(1), 61  Serum CK q Absence of dystrophin leads to muscle fragility and extensive muscle degeneration q Serum creatine kinase is a marker of muscle damage 2000 * 1500 Serum CK (U/L) creatine kinase 1000 into circulation 500 0 WT mdx H&E stain of the mdx diaphragm *mdx mice are the mouse model of DMD – they also lack dystrophin Copyright © 2010 Pearson Education, Inc.  Healthy muscle fibre Creatine kinase Creatine kinase Creatine kinase Damaged muscle fibre with DMD Creatine kinase Creatine kinase Creatine kinase CK is released into circulation Copyright © 2010 Pearson Education, Inc. Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice Mode of inheritance: X-linked recessive u Mutation on the X chromosome to the dystrophin gene. u Recessive means that in females where there are two X chromosomes; both have to be mutated in order for the disease to present itself. u If the female only has one mutated X chromosome, then she is otherwise healthy but is a carrier mother. u Males only have one X chromosome, and so if they receive the mutated X-chromosome from the mother carrier then they will have the disease. u Carrier mothers have a 50% chance of passing on the mutated dystrophin gene to their sons. u For DMD boys: 70% of cases are inherited from their mothers other 30% are sporadic (spontaneous mutation). Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Calcineurin activation Inflammation – healthy regeneration u Injury leads to orchestrated muscle repair. u Activation of satellite cells that proliferate and differentiate and fuse to damaged muscles. u Inflammation plays an important role in the early stages – helping to clear the debris. u In the later stages of repair, inflammation must be reduced to allow for differentiation and fusion.  This orchestrated inflammatory response must be acute! What do you think happens if inflammation persists? Secondary necrosis Yang and Hu. 2018. Journal of Orthopaedic Translation. 13:25-32  The absence of dystrophin causes constant degeneration and thus chronic inflammation - Blue dots around a fibre immune cells that have invaded - Blue dots inside a fibre mark a regenerating muscle fibre Proinflammation > antiinflammation = secondary necrosis Death of muscle cells due to ↑ inflammation Copyright © 2010 Pearson Education, Inc. Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice Calcium and muscle Calcium influx u Calcium levels outside of a muscle fibre is ~2 mM. u Calcium levels inside the muscle at rest is 100 nM. u In the sarcoplasmic reticulum it is about 1 mM u In DMD, damage to the muscle plasma membrane (because of the absence of dystrophin) causes an influx of calcium into the muscle fibre. u Too much calcium in the muscle fibre is bad! u Reactive oxygen species production u Calpain activation  ROS damage ROS damage can lead to a domino effect = oxidative stress Calpains u Proteolytic enzymes activated with sustained elevations in cytosolic calcium. u Though calcium is good for muscle contraction and metabolism, too much calcium is bad! u Calcium dysregulation can lead to hyperactivation of these enzymes leading to muscle atrophy. u Notice that they work with the ubiquitin proteasome (26S) system. SERCA pump restoration u The sarco(endo)plasmic reticulum calcium ATPase (SERCA) pump transports calcium into the SR using energy from ATP hydrolysis. ATP ADP u Active transport: moving something against its concentration gradient. u Can bring in 2 calcium ions into the SR for every 1 ATP hydrolyzed. u Augmenting SERCA function can protect against calcium overload!  Calcium uptake is severely impaired in the D2 mdx SERCA activity is severely impaired in the D2 mdx Copyright © 2010 Pearson Education, Inc. SERCA overexpression in mdx muscle: v Reduces central nuclei counts (marker of degeneration/regeneration) v Reduces muscle necrosis and inflammation v Reduces serum creatine kinase (CK) release. v Will talk about utrophin soon Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice Muscle Physiology – Fibre types Fibre type I IIA IIX IIB vehicle tideglusib v Muscles are heterogenous and are comprised of several different fibre types Contractile and metabolic properties Fibre type Contractile kinetics Metabolism Fatigability (speed of contraction and relaxation) (ATP production) I slowest oxidative least IIA IIX IIB fastest glycolytic most  EDL Fast-twitch powerful muscle (IIB, IIX, and IIA) 60 Soleus Slow-twitch postural muscle (IIA and I) Force (mN) 40 20 0 0 100 200 300 400 500 Time (ms) Copyright © 2010 Pearson Education, Inc.  Muscle types and fatigability 7 0 H z fo r 3 5 0 m s , e v e r y 2 s , fo r 5 m in ( 3 0 0 s ) 100 P e r c e n t o f in itia l fo r c e S o le u s EDL 50 0 0 100 200 300 T im e (s ) Copyright © 2010 Pearson Education, Inc.  Contractile and metabolic properties Fibre type Contractile kinetics Metabolism Fatigability Dystrophic pathology (speed of contraction and relaxation) (ATP production) (susceptibility) I slowest oxidative least least IIA IIX IIB fastest glycolytic most most Copyright © 2010 Pearson Education, Inc. Another key difference between fibre types u For DMD specifically: the fast glycolytic fibres are MORE susceptible to the dystrophic pathology. u This is due to a number of reasons including: u More force produced – stronger contractions lead to more stress u Less PGC-1a – the master regulator of mitochondrial biogenesis u Less utrophin PGC-1alpha u Peroxisome proliferator- activated receptor γ coactivator 1α u Transcription factor u Master regulator of mitochondrial biogenesis u Activated by exercise and can promote the slow oxidative phenotype PGC-1a treatment increase type I% …reduces necrosis …improves performance Tideglusib is a GSK3 inhibitor that promotes the oxidative fibre type and muscle strength in mdx mice ↓necrosis ↓serum CK ↑Force production (Fajardo lab results, under review) Another key difference between fibre types u For DMD specifically: the fast glycolytic fibres are MORE susceptible to the dystrophic pathology. u This is due to a number of reasons including: u More force produced – stronger contractions lead to more stress u Less PGC-1a – the master regulator of mitochondrial biogenesis u Less utrophin Dystrophin and utrophin – do they look the same? Dystrophin Utrophin Utrophin in the mdx mouse u Upregulated in the mdx mouse to try and compensate. u Knocking out utrophin in the mdx mouse worsens the disease (Grady et al., 1997; Deconinck et al., 1997 – both published in Cell back-to-back). Overexpressing utrophin in mdx mouse u Improves muscle structure and function! Utrophin is found more in slow fibres u Controlled by calcineurin signalling – a calcium dependent signaling pathway u Not all calcium signalling is bad! Inhibit calcineurin Activate calcineurin § Calcineurin promotes the oxidative fibre type, which have more utrophin! § Increasing calcineurin signaling in the mdx (DMD mouse model) increases utrophin. § Improves the structure of the muscle: § Less central nuclei – meaning less damage and less need for regeneration § Less fibre size variability because of less degeneration/regeneration cycling Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice Physical activity friend or foe? Friend Foe u Activate pathways that stimulate the u Muscles are weak and fragile. oxidative phenotype (ie. calcineurin u Won’t exercise just cause more and PGC1a). damage? u Increase utrophin and improve muscle u Eccentric exercise causes damage – performance. muscle is activated in while it’s lengthening. Eccentric contraction u Eccentric action q Downward phase of the bicep curl. q Biceps produce force as the muscle lengthens. u Relatively most damaging. u Downhill running and mdx mice causes extensive muscle damage. Copyright © 2010 Pearson Education, Inc. Kiriaev et al., JGP 2023  However, low-to-moderate intensity endurance exercise may be beneficial Copyright © 2010 Pearson Education, Inc. Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice  Skeletal muscle plasticity u Muscle fibres are plastic and they can alter their phenotype depending on external/environmental cues Fibre type Contractile/kinetics Metabolism Fibre type (speed*of*contraction*and*relaxation) (ATP*production) I slowest oxidative I Least susceptible to dystrophic pathology IIA IIA Muscle unloading and disuse (i.e., bedrest and spaceflight) IIX IIX Most susceptible to dystrophic pathology IIB fastest glycolytic IIB Combats disease Endurance training Fibre type switching with exercise and clinical implications! q Calcium: activates calcineurin to promote the slow-oxidative phenotype directly and/or through increased PGC-1α expression. q AMPK: enhances PGC-1α expression and thus the slow-oxidative phenotype. AMPK is our energy sensor! q Slow oxidative fibres q Duchenne Muscular Dystrophy: The fast glycolytic fibres are more prone to dystrophic pathology for several reasons. Thus, promoting the slow oxidative fibres in this disease could be beneficial!! q Obesity: Use more fat as fuel and are more insulin-sensitive and could be used to combat obesity! No calcineurin(CsA – calcineurin inhibitor) = no protection I - Myh7 IIA - Myh2 Without calcineurin activation = ↓ slow oxidative fibres! Goals of today’s lecture 1. What is it? 2. Mode of inheritance 3. Pathophysiology u Inflammation u Calcium overload u Fibre type differences 4. Physical activity – friend or foe? 5. Endurance exercise and calcineurin activation 6. Isometric training in mdx mice Assigned paper u Isometric training in ambulant boys with DMD u Based on promising results from mdx mice 2019 Lindsay et al. Methods u mdx mice were subjected to isometric training u Foot was placed on a metal plate apparatus and the anterior muscles of the hindlimb (TA and EDL) were electrically stimulated u Kept the foot in place (isometric) and stimulated the muscle to mimic a maximal voluntary contraction u Study 1: 3 sessions of training over 1 week u Study 2: 6 sessions of training over 4 weeks Results from Study 1 Results from Study 2 ↓ fibrosis and increased regeneration! Muscle hypertrophy = ↑ CSA small big ↑ Satellite cell number ↑ Resistance to eccentric contractions