Periodic Reporting for period 4 - STARS (Strategies Targeting Thyroid Hormone in Athrophy Related Syndromes)
Reporting period: 2020-02-01 to 2021-07-31
Thyroid hormones (THs) influence the differentiation, growth and energy metabolism of almost all cells and tissues. The biological action of thyroid hormones is mediated through the interaction of the active hormone, T3, with nuclear thyroid hormone receptors and their binding to chromatin. The thyroid gland produces T3 (the most biologically active form of thyroid hormone) and T4 (a weaker receptor ligand than T3) in a ratio of approximately 1:14. Thus, a crucial step for the activation of TH signaling is the T4-to-T3 conversion, catalyzed by the type 2 deiodinase enzyme (D2) in the target cells. Conversely, termination of TH action is mostly achieved through the action of the type 3 deiodinase (D3), which catalyzes the conversion of both T3 and T4 into inactive metabolites.
Skeletal muscle is a well-known target tissue of TH, which is a major determinant of muscle functions, and thyroid dysfunctions are leading causes of many myopathies by mechanisms. Our data have demonstrated that the local activation of TH action by the type 2 deiodinase (D2) enzyme is critical in triggering the accelerated muscle catabolism that causes muscle loss in multiple disease states. Inactivation of TH by genetic D2-depletion or by muscle-specific overexpression of the TH inactivating enzyme, type 3 deiodinase (D3), significantly prevents skeletal muscle atrophy induced by denervation or by cancer cachexia. Importantly, attenuation of TH signaling in vivo not only reduced muscle mass loss, but also increased the survival rates in cancer cachexia studies. Furthermore, expression of the E3 ligases Atrogin-1 and MuRF1, two mediators of muscle atrophy, was reduced in D2KO and D3-overexpressing mice, consistent with a critical role for TH in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy.
The overall objective of STARS is to address the functional role of TH in muscle weakness and atrophic syndromes and to determine the molecular mechanisms by which TH induces massive muscle wasting. Moreover, a key goal of STARS is to assess the therapeutic ability of TH modulators in the muscle atrophy diseases.
Skeletal muscle is a well-known target tissue of TH, which is a major determinant of muscle functions, and thyroid dysfunctions are leading causes of many myopathies by mechanisms. Our data have demonstrated that the local activation of TH action by the type 2 deiodinase (D2) enzyme is critical in triggering the accelerated muscle catabolism that causes muscle loss in multiple disease states. Inactivation of TH by genetic D2-depletion or by muscle-specific overexpression of the TH inactivating enzyme, type 3 deiodinase (D3), significantly prevents skeletal muscle atrophy induced by denervation or by cancer cachexia. Importantly, attenuation of TH signaling in vivo not only reduced muscle mass loss, but also increased the survival rates in cancer cachexia studies. Furthermore, expression of the E3 ligases Atrogin-1 and MuRF1, two mediators of muscle atrophy, was reduced in D2KO and D3-overexpressing mice, consistent with a critical role for TH in the pathology of muscle wasting and establishing it as an important clinical target for the treatment of muscle atrophy.
The overall objective of STARS is to address the functional role of TH in muscle weakness and atrophic syndromes and to determine the molecular mechanisms by which TH induces massive muscle wasting. Moreover, a key goal of STARS is to assess the therapeutic ability of TH modulators in the muscle atrophy diseases.
Specific Aim 1. To analyze the functional impact of muscle-specific modulation of TH action in different models of muscle atrophy.
As planned in the different tasks, we assessed the functional impact of systemic and muscle-specific hypothyroidism on different models of muscle atrophy. By performing experiments of cancer cachexia, fasting and denervation, we demonstrated that reducing the TH signal has beneficial effects on the massive wasting process associated with these pathological conditions. Importantly, in the last period, we generated peptide-rT3 and -NH3 conjugates that were injected in mice challenged with muscle wasting. These experiments provided proof of principles that tool attenuating TH in vivo can be exploited as therapeutic strategy for the muscle wasting syndromes.
Specific Aim 2. Dissect the functional interplay between the TH and FoxO3 pathways in the promotion of massive protein breakdown mechanisms.
Our experiments demonstrated that a cross-talk between TH signal and FoxO3 factor is a critical determinant of muscle catabolism. During the first and second reporting periods, we demonstrated that the combination of TH- and FoxO-interfering approaches is a potent strategy to prevent muscle atrophy. Moreover, our experiments demonstrated surprisingly, that differently from our initial prevision that TH accelerates ROS production and oxidative stress, the role of TH in skeletal muscle is to protect from aberrant production of ROS. This is achieved through the induction of ROS scavenger enzymes.
Specific Aim 3. To determine the effects of TH on cellular metabolism and cellular stress in the pathogenesis of muscle wasting through unbiased high-throughput molecular and metabolic profiling.
We performed metabolic profiling of atrophic and control muscles by GC/MS. The mass spectrometry and liquid chromatography analysis were successfully completed. Principal Component Analysis (PCA) and Dendrograms confirmed that hyperthyroid muscle present similar metabolic landscapes when compared to D3KO muscles and hypothyroid muscles have similar metabolic shifts as the D3 overexpressing. As expected, denervation and cachexia processes affected the intracellular levels of several metabolites. We are currently analyzing different pathways. Notably, among the metabolites mostly modified during atrophy and rescued by local or systemic hyperthyroidism, we identified Glutamine. These interesting results suggest the possible involvement of TH in the metabolism of glutamine. This completely novel aspect on metabolic action of TH will open new avenues on the effects of TH manipulation for the metabolic pathways activated in skeletal muscle during physiological and pathological conditions.
As planned in the different tasks, we assessed the functional impact of systemic and muscle-specific hypothyroidism on different models of muscle atrophy. By performing experiments of cancer cachexia, fasting and denervation, we demonstrated that reducing the TH signal has beneficial effects on the massive wasting process associated with these pathological conditions. Importantly, in the last period, we generated peptide-rT3 and -NH3 conjugates that were injected in mice challenged with muscle wasting. These experiments provided proof of principles that tool attenuating TH in vivo can be exploited as therapeutic strategy for the muscle wasting syndromes.
Specific Aim 2. Dissect the functional interplay between the TH and FoxO3 pathways in the promotion of massive protein breakdown mechanisms.
Our experiments demonstrated that a cross-talk between TH signal and FoxO3 factor is a critical determinant of muscle catabolism. During the first and second reporting periods, we demonstrated that the combination of TH- and FoxO-interfering approaches is a potent strategy to prevent muscle atrophy. Moreover, our experiments demonstrated surprisingly, that differently from our initial prevision that TH accelerates ROS production and oxidative stress, the role of TH in skeletal muscle is to protect from aberrant production of ROS. This is achieved through the induction of ROS scavenger enzymes.
Specific Aim 3. To determine the effects of TH on cellular metabolism and cellular stress in the pathogenesis of muscle wasting through unbiased high-throughput molecular and metabolic profiling.
We performed metabolic profiling of atrophic and control muscles by GC/MS. The mass spectrometry and liquid chromatography analysis were successfully completed. Principal Component Analysis (PCA) and Dendrograms confirmed that hyperthyroid muscle present similar metabolic landscapes when compared to D3KO muscles and hypothyroid muscles have similar metabolic shifts as the D3 overexpressing. As expected, denervation and cachexia processes affected the intracellular levels of several metabolites. We are currently analyzing different pathways. Notably, among the metabolites mostly modified during atrophy and rescued by local or systemic hyperthyroidism, we identified Glutamine. These interesting results suggest the possible involvement of TH in the metabolism of glutamine. This completely novel aspect on metabolic action of TH will open new avenues on the effects of TH manipulation for the metabolic pathways activated in skeletal muscle during physiological and pathological conditions.
The main goal of STARS was to understand the mechanisms by which the TH pathway contributes to common muscle wasting diseases. As master regulators of transcription, nuclear receptors, including TRs constitute the second largest category of druggable targets. TH is 1 of the 2 most prescribed drugs; 3% of women > 50 years receive T4 replacement therapy worldwide. However, potent off-target effects create an urgent need to develop technology for tissue/cell-type specific action and delivery (for diagnosis and therapy).
STARS-generated information for tissue/cell-type specific action and delivery of TH modulators will open avenues for the pharmaceutical industry to develop new products or further exploit existing products based on novel information on effectiveness of thyroid inhibiting drugs in prevention of muscle wasting diseases.
The knowledge that STARS will generate can open new directions in clinical research for better risk identification and stratification for disease susceptibility and therapy development.
STARS-generated information for tissue/cell-type specific action and delivery of TH modulators will open avenues for the pharmaceutical industry to develop new products or further exploit existing products based on novel information on effectiveness of thyroid inhibiting drugs in prevention of muscle wasting diseases.
The knowledge that STARS will generate can open new directions in clinical research for better risk identification and stratification for disease susceptibility and therapy development.