Dissertation
ULK1 and ULK2 modulate skeletal muscle homeostasis and whole-body metabolism
University of Iowa
Doctor of Philosophy (PhD), University of Iowa
Autumn 2020
DOI: 10.17077/etd.005643
Abstract
Skeletal muscle is an important tissue as it plays vital roles in multiple aspects of our daily life raging from movement and locomotion to thermoregulation and whole-body metabolism. Unfortunately, however, skeletal muscle homeostasis becomes a major concern during a variety of metabolic and myopathic disorders, neurodegenerative diseases, and with aging. In fact, skeletal muscle mass and strength are major predictors of positive prognosis in several disease settings such as in cancer and heart failure. Therefore, a better understanding of the cellular and molecular mechanisms required to preserve skeletal muscle contractile and metabolic functions is of utmost clinical importance. Maintenance of skeletal muscle homeostasis requires appropriate protein turnover, which is dependent on both protein synthesis and degradation. It turns out that skeletal muscle presents a unique challenge to proteolytic systems due to the large number of long-lived and cytoskeletal proteins present therein, which are prone to misfolding and aggregation. Autophagy is a catabolic process that is primarily responsible for the degradation of these long-lived and aggregate proteins and is essential for maintenance of skeletal muscle health. Of note, skeletal muscle autophagy is often compromised under myopathic and metabolic disorders, and with aging. Therefore, dissecting the molecular mechanisms involved in the regulation of autophagy in skeletal muscle will likely prove to be beneficial in the development of therapies to preserve skeletal muscle functions in such circumstances. With this in mind, we postulated that essential factors modulating skeletal muscle autophagy would be enriched in muscle under normal, healthy conditions. Through data mining and bioinformatics, we discovered that ULK1 and ULK2, two autophagy related protein kinases, were enriched in skeletal muscle, and yet their function was poorly understood. Therefore, we hypothesized that ULK1 and ULK2 would be required for maintenance of skeletal muscle mass, strength, and metabolic homeostasis. We conducted two new studies (reported in Chapters 2 and 3 of the present dissertation) in mice to test this prospect as well as to gain initial insights into the potential cellular pathways and molecular events that these kinases might regulate in muscle.
In Chapter 1, we provide a brief review on what is currently known about ULK1 and ULK2 with a particular focus on their role in skeletal muscle. We highlight the importance of ULK1 and UKL2 in autophagy and point out that understanding their unique and complementary functions in skeletal muscle will provide important insight for conditions where skeletal muscle autophagy is impaired.
In Chapter 2, we report experiments where we established that ULK2 is the predominant ULK in skeletal muscle. We used electroporations to transfect micro RNAs targeting either the ULK1 or ULK2 genes in the tibialis anterior muscle to compare and contrast the independent roles of these proteins. We demonstrate that muscle ULK2 is necessary for the degradation of insoluble ubiquitinated protein aggregates associated with autophagy adaptors p62 and NBR1. Interestingly, these alterations occurred despite no obvious impairments in autophagy indicating that ULK2 modulates recognition and/or sequestration of ubiquitinated protein aggregates in muscle. This is important because these insoluble protein aggregates can become toxic as they accumulate in cells. In fact, just within weeks of ULK2 deficiency muscles atrophied becoming weak and presenting clear signs of myofiber damage. These findings were not observed in muscles deficient of ULK1, demonstrating a unique role for ULK2 in the degradation of protein aggregates and maintenance of skeletal muscle homeostasis.
These results were interesting and novel, but potential redundant functions of muscle ULK1 and ULK2, as well as their potential impact on whole-body metabolic homeostasis, would require further investigations using a model where both ULKs were deficient in the entire musculature. Therefore, in Chapter 3 we show that mice deficient of both ULK1 and ULK2 in skeletal muscle (mDKO mice) present a lean whole-body phenotype with decreased body fat accumulation as they age when compared to their WT littermates. The reduced fat mass is associated with an increased basal metabolic rate, which is at least in part due to increased carbohydrate-driven, uncoupled respiration in muscle mitochondria. Interestingly, mDKO mice have larger and yet weaker muscles than WT. Myofiber damage was also observed in mDKO muscle, likely contributing to the impaired maximal force production of these muscles. Of note, deficiency of both ULK1 and ULK2 impaired basal skeletal muscle autophagy, something that was not observed with either ULK1 or ULK2 deficiency alone. In addition, protein synthesis was also impaired in mDKO muscle.
In Chapter 4, we then summarize our major findings and briefly outline future directions. In short, our studies reveal unique and redundant functions of muscle ULK1 and ULK2, which may affect whole-body metabolic homeostasis. Particularly, ULK2 is required for the degradation of ubiquitinated aggregate proteins in skeletal muscle thereby promoting overall muscle quality and function. In addition, we show that ULK1 and ULK2 have redundant roles in the maintenance of basal skeletal muscle autophagy. Our findings also indicate that muscle ULKs are required for coupling respiration to ATP synthesis in muscle mitochondria, which affects whole-body metabolism. Together these findings provide novel and important insights into the functions of ULK1 and ULK2 in skeletal muscle. Our results also open new avenues for future studies determining the precise mechanisms modulating protein turnover and muscle metabolism downstream of ULKs. These may identify new proteins to be targeted for therapy in conditions of skeletal muscle contractile and metabolic dysfunction, such as in several metabolic and myopathic disorders and in aging.
Details
- Title: Subtitle
- ULK1 and ULK2 modulate skeletal muscle homeostasis and whole-body metabolism
- Creators
- Jordan D Fuqua
- Contributors
- Vitor Lira (Advisor)Warren Darling (Committee Member)Ling Yang (Committee Member)Christopher Adams (Committee Member)Sue Bodine (Committee Member)
- Resource Type
- Dissertation
- Degree Awarded
- Doctor of Philosophy (PhD), University of Iowa
- Degree in
- Health and Human Physiology
- Date degree season
- Autumn 2020
- DOI
- 10.17077/etd.005643
- Publisher
- University of Iowa
- Number of pages
- xiii, 103 pages
- Copyright
- Copyright 2020 Jordan D. Fuqua
- Language
- English
- Description illustrations
- color illustrations
- Description bibliographic
- Includes bibliographical references (pages 96-103).
- Public Abstract (ETD)
Skeletal muscle is an important tissue as it makes up ~35-40% of our total body mass and plays vital roles in multiple aspects of our daily life and overall health. For example, skeletal muscle is responsible not only for movement and locomotion, but also contributes to respiration, body temperature regulation, metabolism, and preservation of bone mass. In addition, maintenance of muscle mass and function are strong predictors of independence and mobility in the elderly, rate of recovery in patients at intensive care units, and positive outcomes in cancer and heart failure patients. Therefore, preservation of skeletal muscle mass and quality are of upmost importance.
One major regulator of skeletal muscle mass and quality is the cellular process called autophagy. Autophagy is essentially the recycling center of the cell; it degrades old or dysfunctional cellular materials which the cell can then reuse to help maintain cellular health. Autophagy often becomes impaired in aging or diseased skeletal muscle, contributing to disease progress and muscular dysfunction. Therefore, understanding how autophagy is regulated at a cellular level will likely provide potential therapies to treat such conditions.
In these studies, our lab investigated the role of the autophagy related proteins ULK1 and ULK2 in skeletal muscle. We show that ULK2 is important for degrading toxic aggregate proteins, thereby promoting overall health and function of muscle. In addition, we also demonstrate that both ULKs are necessary for normal skeletal muscle autophagy and maintenance of muscle quality. The loss of both ULK1 and ULK2 in skeletal muscle leads to increased whole-body metabolism and reduced body fat mass. Together, our findings demonstrate critical roles for ULK1 and ULK2 in regulating skeletal muscle autophagy, muscle mass, contractile function, and whole-body metabolic homeostasis. These findings lay the foundation for future studies where ULK1 and ULK2 may serve as candidate targets for therapy in myopathic/metabolic disorders, and in aging.
- Academic Unit
- Health, Sport, and Human Physiology
- Record Identifier
- 9984036086802771
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