Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - STARS (Strategies Targeting Thyroid Hormone in Athrophy Related Syndromes)

Teaser

1. Overview of the action\'s implementation for this reporting period Skeletal muscle atrophy—also known as muscle wasting—is a debilitating syndrome that slowly develops with age (sarcopenia) or suddenly appears at the end of various chronic diseases. Despite its medical...

Summary

1. Overview of the action\'s implementation for this reporting period

Skeletal muscle atrophy—also known as muscle wasting—is a debilitating syndrome that slowly develops with age (sarcopenia) or suddenly appears at the end of various chronic diseases. Despite its medical relevance, there is, as yet, no effective treatment option for patients suffering from this condition. Thyroid hormone (TH) is a major determinant of muscle functions, and thyroid dysfunctions are leading causes of many myopathies whose mechanisms of action are unknown. We observed that local activation of TH 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), largely prevents prevents skeletal muscle atrophy induced by denervation or by cancer cachexia. Our working hypothesis is that “amplification” of TH action/signalling in muscle is critical in determining the accelerated muscle catabolism that causes muscle loss in numerous diseases. On the contrary, attenuation of the TH signal reduces atrophy.

Work performed

The main results achieved so far are reported below according to proposed specific aims.

Specific Aim 1. To analyze the functional impact of muscle-specific modulation of TH action in different models of muscle atrophy.

Task 1.1. C57BL/6 mice were treated with T3 and T4 (hyperthyroid mice) or with 0.1% methimazole (MMI) plus 1% KCl2 (hypothyroid mice) and used for denervation and cachexia studies. The analysis of body weight, muscle weight and atrogene expression demonstrated that systemic hypothyroidism protects from massive muscle wasting upon denervation and cachexia (Figure 1A), while systemic hyperthyroidism accelerates wasting processes.
We have completed this task and have presented the results in two conferences: SfE-BES2016 - British Society of Endocrinology, Brighton, and the Congresso dell’Associazione Italiana della Tiroide, Cagliari.

Task 1.2 We performed denervation and cachexia studies in MLC-RosaD3 mice. Analogous to D2KO mice, D3 overexpression in muscle protected mice from muscle atrophy (Figure 1B). Consequently, this task confirmed our working hypothesis. We are currently preparing muscle extracts from denervated and cachectic wild type and MLC-RosaD3 mice that will be used for large scale metabolomics profiling (Task 3.3).

Task 1.3: Dio3fl/fl mice were crossed with MLC-Cre mice and metabolic profile and thyroid status of the fiber is currently under investigation.

Specific Aim 2. Dissect the functional interplay between the TH and FoxO3 pathways in the promotion of massive protein breakdown mechanisms.

Task 2.1. To determine whether FoxO3 is required for the induction of muscle atrophy by TH, MLC-Cre-FoxO null mice were treated with T3 and T4 (hyperthyroid mice) or with 0.1% methimazole (MMI) plus 1% KCl2 (hypothyroid mice). We firstly characterized the metabolic and atrophic profile of these mice in the absence of induced-atrophy by denervation, fasting and cachexia. Preliminary data indicate that in the absence of FoxO3 TH-dependent shift of the fibers is preserved, thus indicating that TH acts up-stream of FoxO3 signal. We are currently confirming these data and performing denervation studies to test whether the same effects can be observed also during denervation.

Task 2.2 We crossed D2KO mice with FoxO3KO mice. Double D2-FoxO3 mice are currently under analysis for their degree of muscle wasting. The work is proceeding according to the plan and is expected to be completed by project month 36.

Task 2.3 We are collecting muscles from tasks 2.1 and 2.2 and preparing RNA for transcriptomic and ChIP-Seq analysis.

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.

Task 3.1. Satellite cells in culture were exposed to different amounts of TH or transfected with different amounts of D2-expressing plasmid. TH treatment and D2 transfection caused apoptosis of satellite cells. To test whether this apoptosis involves ROS production, we blocked ROS production using two different ROS inhibitors, namely, N-acetyl-L-cysteine (NAC) and diphenyleneiodonium (DPI). Preliminary data indicated that DPI treatment reduces T3-dependent apoptosis by 80%, while NAC reduces apoptosis by 65% compared to control TH-treated cells). To assess what ROS species are induced by TH, we are currently using the luminol-based method (LumiMax kit, Stratagene).

3) Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The data obtained so far have established the role of systemic alterations of TH in the pathogenesis of muscle atrophy. Systemic hyperthyroidism accelerates muscle wasting induced by denervation and cachexia, while systemic hypothyroidism reduces massive protein breakdown. Since one of the major goals of STARS is to provide proof-of-principle studies investigating t

Final results

Muscle wasting is associated with a wide range of catabolic diseases. This debilitating loss of muscle mass and functional capacity reduces quality of life and increases the risks of morbidity and mortality. Despite the prevalence and the devastating effects of this syndrome, there are currently no effective treatment options. STARS will lead to targeted evidence-based disease prevention measures and treatment by defining how TH-related signaling pathways underpin common muscle wasting diseases.
Hormones are likely to be major regulators of protein turnover in these conditions. Several endocrinopathies are characterized by muscle wasting and loss of muscle function. A common sign of hyperthyroidism is muscle wasting and weakness, mainly caused by increased protein degradation. In physiologic concentrations TH stimulates both the protein synthesis and degradation, whereas, in supraphysiological levels, protein catabolism predominates. Data generated by STARS will test the therapeutic potential of tissue-specific modulators of TH action for the treatment of musculoskeletal degenerative diseases. It will also will provide proof-of-concept for developing novel TH modulator-based peptide-conjugates for muscle-specific drug delivery in order to enhance efficacy and target organ specificity thereby reducing systemic side-effects.