Suppression of Calcineurin Signaling and PGC-1α Expression During the Chronic Skeletal Muscle Atrophy Associated with Diabetes Mellitus: Implications for Muscle Function Open Access

Abstract

Skeletal muscle atrophy frequently indicates a poor prognosis for patients with systemic pathologies, including diabetes mellitus (DM). These patients demonstrate reduced muscle size as well as decreased strength and endurance, indicating that a link exists between muscle size and functional capacity in these conditions. PGC-1α is a transcriptional coactivator that controls energy homeostasis through regulation of glucose and oxidative metabolism. Both PGC-1α expression and oxidative capacity are decreased in skeletal muscle of diabetic patients and animals undergoing atrophy, suggesting that PGC-1α participates in the regulation of muscle mass. This dissertation focuses on elucidating the mechanisms that regulate PGC-1α expression in vivo in a model of DM as well as the potential physiological effects of PGC-1α downregulation.
Our work reveals that suppressed calcineurin (Cn) signaling contributes to decreased PGC-1α expression in chronic DM rat skeletal muscle and this may result in a muscle fiber-type switch from an oxidative phenotype to a more glycolytic phenotype. Specifically, we demonstrate that expression of Cn, a calcium-dependent phosphatase, was decreased in the skeletal muscle of rats with streptozotocin-induced diabetes (STZ-DM) for 21 days. PGC-1α expression is regulated by two Cn substrates, MEF2 and NFATc, both of which showed significantly reduced activity in the same muscles. MEF2 and NFATc activity as well as PGC-1α expression were also decreased in muscles of CnAα^-/- and CnAβ^-/- mice without diabetes indicating that decreased Cn signaling, rather than changes in other signaling pathways, were responsible for decreased PGC-1α expression. These findings demonstrate that Cn activity is a major determinant of PGC-1α expression in skeletal muscle during diabetes and possibly other conditions associated with loss of muscle mass. We also found that STZ-induced atrophy is associated with fiber type switching from MHCI and the oxidative phenotype towards MHCII
and the glycolytic phenotype along the MHC gene expression continuum. Furthermore, there was a preferential decrease in the cross-sectional area of MHCII fibers in the skeletal muscles of STZ-DM animals. These results indicate that the chronic muscle atrophy associated with DM predominantly affects MHCII fibers and that switching from MHCI to MHCII may serve as a mechanism to sustain atrophy. A more thorough understanding of the signaling pathways that regulate protein degradation in different fiber types will be important for the development of therapies to treat the chronic atrophy associated with DM and other systemic diseases.

Table of Contents

TABLE OF CONTENTS
CHAPTER 1 ...................................................................................................................... 1
INTRODUCTION............................................................................................................. 1
DIABETES: ATROPHY AND FIBER-TYPE SWITCHING ......................................................... 2
The role of Cn/NFAT/MEF2 signaling ....................................................................... 3
The role of PGC-1α .................................................................................................... 4
CHAPTER 2 ...................................................................................................................... 6
BACKGROUND AND SIGNIFICANCE ....................................................................... 6
SKELETAL MUSCLE STRUCTURE ...................................................................................... 8
SKELETAL MUSCLE FIBER TYPES .................................................................................. 11
PROTEIN DEGRADATION ................................................................................................ 14
The Lysosomal System .............................................................................................. 14
The Ubiquitin Proteasome System ............................................................................ 15
The Calpains ............................................................................................................. 22
The Caspases ............................................................................................................ 23
MOLECULAR MECHANISMS REGULATING PROTEIN HOMEOSTASIS ............................... 25
PI3K/Akt .................................................................................................................... 25
MEK/ERK ................................................................................................................. 28
NFκB ......................................................................................................................... 30
Calcineurin/NFAT/MEF2 signaling ......................................................................... 31
MEF2 ........................................................................................................................ 34
PGC-1α ..................................................................................................................... 35
CHAPTER 3 .................................................................................................................... 43
MATERIALS AND METHODS ................................................................................... 43
MATERIALS .................................................................................................................... 44
ANIMALS ....................................................................................................................... 44
STZ-DM rats ............................................................................................................. 44
STZ-DM transgenic mice: ......................................................................................... 46
Calcineurin knock-out mice ...................................................................................... 46
MEASUREMENT OF MUSCLE PROTEIN DEGRADATION ................................................... 46
Rate of Tyrosine release............................................................................................ 46
Actin degradation...................................................................................................... 47
WESTERN BLOT ANALYSIS ............................................................................................ 47
REAL-TIME RT-PCR ..................................................................................................... 49
IMMUNOHISTOCHEMISTRY ............................................................................................. 49
ENDOGENOUS NFATC ACTIVITY ASSAY ....................................................................... 50
SINGLE FIBER ANALYSIS................................................................................................ 50
Single Fiber Preparation .......................................................................................... 50
Single fiber force assay ............................................................................................. 51
Single fiber MHC-type determination ....................................................................... 51

STATISTICAL ANALYSIS. ................................................................................................. 52
CHAPTER 4 .................................................................................................................... 53
CALCINEURIN SIGNALING AND PGC-1α EXPRESSION ARE SUPPRESSED
DURING MUSCLE ATROPHY DUE TO DIABETES .............................................. 53
INTRODUCTION .............................................................................................................. 54
RESULTS ........................................................................................................................ 56
Rats treated with streptozotocin experience skeletal muscle atrophy due to increased
protein degradation. ................................................................................................. 56
PGC-1α expression is decreased in muscle from STZ-treated rats. ......................... 56
Decreased PGC-1α transcription is not due to decreased CREB activity. .............. 60
Calcineurin signaling is down-regulated in muscle from STZ-treated rat. .............. 60
Loss of calcineurin signaling results in decreased PGC-1α transcription in skeletal
muscle. ...................................................................................................................... 68
DISCUSSION ................................................................................................................... 71
CHAPTER 5 .................................................................................................................... 75
SINGLE SKELETAL MUSCLE FIBER TYPE SPECIFICITY AND SWITCHING
IN ATROPHY ASSOCIATED WITH STREPTOZOTOCIN-INDUCED INSULIN-
DEFICIENCY ................................................................................................................. 75
INTRODUCTION .............................................................................................................. 76
RESULTS ........................................................................................................................ 78
STZ reduced body weight by elevating protein breakdown in skeletal muscle. ........ 78
STZ did not affect specific force or structural integrity of fibers. ............................ 81
STZ caused muscle fiber type switching from MHCI to MHCII. .............................. 81
STZ caused a preferential reduction of CSA of MHCII fibers. ................................. 81
DISCUSSION ................................................................................................................... 87
CHAPTER 6 .................................................................................................................... 90
DISCUSSION AND CONCLUSIONS .......................................................................... 90
CELLULAR MECHANISMS REGULATING PGC-1α IN STZ-DM ........................................ 92
CREB signaling is abnormal in STZ-DM ................................................................. 92
Cn signaling is supressed in STZ-DM ...................................................................... 99
PHYSIOLOGICAL IMPACT OF SUPPRESSED PGC-1α EXPRESSION IN STZ-DM ............... 100
Muscle fibers transition from MHCI to MHCII in STZ-DM ................................... 100
MHCII fibers are more susceptible to atrophy in STZ-DM .................................... 101
The link between fiber-type and atrophy ................................................................ 104
CONCLUSIONS .............................................................................................................. 106
REFERENCES .............................................................................................................. 109

FIGURES
2.1 Schematic of human skeletal muscle sarcomere structure and protein
components...9
2.2 The ubiquitin proteasome system...16
2.3 Insuling signaling activates both the PI3K/Akt and MEK/ERK pathways...26
2.4 Transcriptional regulation of PGC-1α...41
4.1 The rate of protein degradation and ubiquitin expression are increased in 21day
STZ-treated rat muscle...58
4.2 PGC-1α expression is decreased in 21day STZ-treated rat muscle...59
4.3 CREB signaling is abnormal in 21day STZ-treated rat muscle...61
4.4 Cn catalytic A subunit protein is decreased in 21day STZ-treated rat muscle...63
4.5 NFATc activity is decreased in 21 day STZ-treated rat muscle...64
4.6 GSK-3β signaling is unchanged in 21day STZ-treated rat muscle...66
4.7 MEF2 activity is decreased in 21day STZ-treated rat muscle...69
4.8 MEF2 and NFATc signaling and PGC-1α mRNA are decreased in muscles of
CnAα^-/- and CnAβ^-/- mice...70
5.1 STZ-treated rats have elevated blood glucose, decreased body mass, increased
protein degradation...79
5.2 Fibers from STZ-treated rats maintained structural integrity...82
5.3 Fiber type switching from MHCI to MHCII in soleus muscle of STZ-treated
rats...83
5.4 MHCII fibers are more susceptible to STZ-induced atrophy than MHCI fibers in
both the soleus and gastrocnemius...84
6.1 Phosphorylation-dependent regulation of TORC...95
6.2 Hypothetical model showing the role of fiber-type switching in sustaining skeletal
muscle atrophy...102

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Biochemistry, Cell & Developmental Biology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Physiology Biology, Cell
Keyword	PGC-1 diabetes mellitus skeletal muscle atrophy Calcineurin
Committee Chair / Thesis Advisor	Price, Stephen Russ, Emory University
Committee Members	Gooch, Jennifer, Emory University Saxe, Charles, American Cancer Society Moreno, Carlos, Emory University Pavlath, Grace K, Emory University

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