Skeletal Muscle Diseases: Techniques: Functional assessments to evaluate therapeutic efficacy.

Question 1

What causes duchenne’s muscular dystrophy?

Answer 1

Mutation in dystrophin gene on Xp21 resulting in deficiency in dystrophin expression.

Question 2

What is dystrophin?

Answer 2

427 kDa cytoskeletal protein associated with several structures nearby. Dystrophin associated proteins and DGC.

Question 3

What proteins is dystrophin closely associated with?

Answer 3

sacroglycan complex

dystroglycan complex

laminin

F-actin

Question 4

How are animal models used to test muscular dystrophy?

Answer 4

Various animal models are used to test several aspects of muscular dystrophy

Question 5

Why do several therapies fail when clinical trials are performed?

Answer 5

A more rigorous pre-clinical testing period is needed when evaluating therapeutic potential.

Several therapies are not as functional in humans as they are in animals due to differing physiology.

Many other unknown reasons are also contributing.

Question 6

What is a physiologists goal in treating muscle disorders?

Answer 6

Attenuating loss of muscle preserves function because increasing muscle mass optimises potential for improving muscle function.
Improving muscle function could improve quality of life for patients as well as quantity for people with dystrophic disorders.

Question 7

What are the factors that should be looked into when evaluating interventions for preclinical testing?

Answer 7

Good animal models of human diseases/conditions.

Rigorous assessment of muscle function.

Complementary physiological, histological, and molecular biochemical assessments.

*This is especially relevant for pharmocological, gene, cell, and bioengineering based therapies.

Question 8

How are muscles from mdx mice different to muscles of wild type mice?

Answer 8

Muscles from mdx mice are larger than wild type mice. But they produce less force per CSA than the wild type. This muscle is also more fragile.

Question 9

What factors are important during pre-clinical evaluation?

Answer 9

Non- or minimally invasive tests.

Overall health and functional capacity of the animal.

Track performance.

Question 10

What are cellular functional assays?

Answer 10

Mechanically skinned fibers

Chemically permeabilized fibers

Question 11

What do mechanically skinned fibers allow us to study?

Answer 11

Excitation contraction coupling.

SR Ca2+ release

Ca2+ uptake rates SR function

Question 12

What do chemically permeabilized fibers allow us to study?

Answer 12

Contractile apparatus

Force-pCa Force-pSr

Vmax

Contraction induced injury

Question 13

What are the pros and cons of using cellular functional assays?

Answer 13

Can study cellular level for mechanistic understanding.

Cons: Technically difficult and requires expensive equipment.

Question 14

What muscle is typically used when studying muscular dystrophy? Why?

Answer 14

In muscular dystrophy diaphragm muscle strips can be used because diaphragm is the most important muscle for survival.

Question 15

What other analyses are performed to evaluate muscle effectiveness?

Answer 15

Structure-function assessments (need to perform complementary analyses of histology, immunohistochemical, and molecular biochemical analyses).

Creatine Kinase levels

Assessment of muscle fiber size (CSA)

Biochemical pathway analyses

Question 16

What are the features of dystrophic muscles?

Answer 16

Dystrophic muscles are not only weaker but also more susceptible to contraction-induced injury.

Sarcolemmal fragility of fibers from dystrophic mice is worse than wild type.

Greater susceptibility to rupture following osmotic shock.

Greater susceptibility to injury following stretch.

Dystrophic models are deficient in force production following stretch

Question 17

What happens to dystrophic muscle relative to normal muscle?

Answer 17

Dystrophic muscle exhibts a small deficit until 30% after which the deficit is so much that the muscle can’t function adequately.

Question 18

What happens to calcium release in muscle cells during muscular dystrophy?

Answer 18

Normally control of calcium is strictly regulated by muscles. In dystrophic muscle the calcium intracellular levels are often leaking out of the SR and this is especially a problem during exercise.

Question 19

What happens to contraction strength over time in dystrophic muscle?

Answer 19

Damage to muscle causes decrease in contractility and as a result muscle strength diminishes.

Question 20

How are muscles damaged when DGC components are missing?

Answer 20

No universal agreement on which mechanism predominates.

Question 21

What does the mechanical hypothesis state about muscle damage during DGC malfunctions?

Answer 21

Mechanical hypothesis states that loss of DGC leads to contraction-induced rupture of muscle cell membrane.

Question 22

What evidence supports the mechanical hypothesis?

Answer 22

Exercise in DMD patients and mdx causes greater muscle damage that in unaffected controls.

Question 23

What does the calcium hypothesis state?

Answer 23

Influx of calcium into the cell overwhelms muscle cell’s ability to maintain physiologic Ca2+ levels.

Elevated Ca2+ levels causes programmed cell death via activation of proteases such as calpains.

*overexpression of calpastatin, an endogenous inhibitor of calpains, reduces necrosis.

Question 24

Why do people tend to die young with muscular dystrophy?

Answer 24

Initially the regeneration of muscle is adequate but it gets worse and worse with time. For this reason people die young with this condition.