molecular mechanisms and signaling pathways in muscle atrophy in immobilization and aging marina...
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Molecular Mechanisms and Signaling Pathways in Muscle Atrophy in Immobilization and Aging
Marina Bar- Shai
Abraham Z. Reznick
Department of Anatomy and Cell Biology
The Bruce Rappaport Faculty of Medicine
Technion – Israel Institute of Technology
Haifa, Israel
Summary of the main topics
1. Introduction to aging and muscle protein degradation
2. In vivo model of immobilization and the stages of skeletal muscle breakdown
3. In vitro model of the involvement of RNS in activation of NF- κB in muscle cells
Changes in the neural system and
stimuli
Changes in the muscle apparatus
with aging
Environmental and external
influences
Loss of motor units*
*Changes in the neuromuscular junction
Atrophy and loss of
muscle fibers
*Decrease in physical activity
* Disuse and immobilization
*Nutrition and food restriction
Diseases* *Injuries
Medications*
Decline in muscle mass (Sarcopenia)
Loss of force generating capacity and functional
decline
Changes in CNS
Neural molecular and cellular changes
Intrinsic molecular and
cellular changes
Factors in aging of skeletal muscle
Immobilization (first 24 -48h)
CaCa+2+2 influx influx
Increased CaIncreased Ca+2+2 dependent proteolysis by calpains dependent proteolysis by calpains
Initiation of myofibrillar proteins degradation and
Z- disk disintegration
The fast phase of muscle breakdown due to immobilization
Infiltration of monocytes and differentiation into Infiltration of monocytes and differentiation into macrophagesmacrophages
Macrophages activationMacrophages activation
Synthesis of cytokinesSynthesis of cytokines
IL-1, IL-6, TNF- IL-1, IL-6, TNF- αα by the macrophages by the macrophages
Oxidative stressOxidative stress
Activation of NF-kB andActivation of NF-kB and
AP-1 (?) transcription factorsAP-1 (?) transcription factors
Upregulation of stress and inflammation Upregulation of stress and inflammation genes including iNOSgenes including iNOS
NO, ONOONO, ONOO--
RNSRNS
Increased muscle wastingIncreased muscle wasting
Biphasic regulation of the Biphasic regulation of the transcription factors by NOtranscription factors by NO::
Low levels activate, high Low levels activate, high levels shut downlevels shut down
Ubiquitin- proteasome- dependent Ubiquitin- proteasome- dependent proteolysisproteolysis
Lysosomal proteolysis Lysosomal proteolysis
CaCa+2+2 dependent proteolysis dependent proteolysis
The slow phase of muscle breakdown due to immobilization (2-30 days)
Experimental design
6-8 months old female Wistar rats (250-300gr) and 24 months old female Wistar rats (300-350gr)
Immobilization periods : one, two, three and four weeks
Right limbs were immobilized, left limbs served as controls
At the end of each immobilization period the muscles were removed for biochemical and histological studies
Muscle proteolytic systems
Intracellular:
CaCa+2+2 – dependent proteases (calpains) – dependent proteases (calpains)
Ubiquitin- proteasome systemUbiquitin- proteasome system
Intracellular lysosomal proteases (Cathepsins D, H, L, B., nucleases, lipases, Intracellular lysosomal proteases (Cathepsins D, H, L, B., nucleases, lipases, glycosidases, ACP)glycosidases, ACP)
Extracellular:Extracellular:
Macrophage lysosomal proteasesMacrophage lysosomal proteases
Matrix Metalloproteases (MMPs): MMP-2, MMP-9Matrix Metalloproteases (MMPs): MMP-2, MMP-9
Ubiquitination of muscle proteins following immobilization of young rats
Protein staining Immunostaining (anti- Ubiquitin AB.)
L-control leg
R- immobolized leg
Acid phosphatase activity in normal vs. immobilized (30 days of E.F) muscle of young animals (histochemical staining)
Zymography of gastrocnemius muscles of five
young rats after 21 and 30 days of immobilization
L-control leg
R- immobolized leg
ObservationsIn the slow phase of muscle atrophy due to limb immobilization, the kinetics of activation of the extracellular and the intracellular degradation systems are very similar.
ConclusionConclusion
There appears to be a link There appears to be a link between the activation of the between the activation of the extracellular and intracellular extracellular and intracellular proteolytic systemsproteolytic systems
Infiltration of monocytes and differentiation into Infiltration of monocytes and differentiation into macrophagesmacrophages
Macrophages activationMacrophages activation
Synthesis of cytokinesSynthesis of cytokines
IL-1, IL-6, TNF- IL-1, IL-6, TNF- αα by the macrophages by the macrophages
Oxidative stressOxidative stress
Activation of NF-kB andActivation of NF-kB and
AP-1 (?) transcription factorsAP-1 (?) transcription factors
Upregulation of stress and inflammation Upregulation of stress and inflammation genes including iNOSgenes including iNOS
NO, ONOONO, ONOO--
RNSRNS
Increased muscle wastingIncreased muscle wasting
Biphasic regulation of the Biphasic regulation of the transcription factors by NOtranscription factors by NO::
Low levels activate, high Low levels activate, high levels shut downlevels shut down
Ubiquitin- proteasome- dependent Ubiquitin- proteasome- dependent proteolysisproteolysis
Lysosomal proteolysis Lysosomal proteolysis
CaCa+2+2 dependent proteolysis dependent proteolysis
The slow phase of muscle breakdown due to immobilization (2-30 days)