Hypothyroidism impairs skeletal muscle regeneration after injury by altering myogenic and nonmyogenic pathways
Paola Aguiari, Valentina Villani, Yan-Yun Liu, Gianni Carraro, Gregory A. Brent, Laura Perin, Anna Milanesi
Paola Aguiari, Valentina Villani, Yan-Yun Liu, Gianni Carraro, Gregory A. Brent, Laura Perin, Anna Milanesi
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Research Article Cell biology Endocrinology Muscle biology

Hypothyroidism impairs skeletal muscle regeneration after injury by altering myogenic and nonmyogenic pathways

Abstract

Thyroid hormone signaling is an essential regulator of skeletal muscle development, function, and metabolism, yet the specific signaling pathways required for muscle regeneration are not yet defined. We used scRNA-seq and the FUCCI (fluorescent ubiquitination-based cell cycle indicator) reporter mouse model to examine how hypothyroidism impacts repair processes after cardiotoxin-induced injury in mice. During regeneration, and up to 2 months after injury, hypothyroid muscles displayed smaller myofibers and a shift to slower oxidative fiber types. scRNA-seq of tibialis anterior muscle during regeneration revealed that hypothyroidism reduced myogenic-lineage diversity. Cell cycle analysis confirmed delayed cell cycle progression at 5 and 14 days after injury, with skeletal muscle stem cells stalled at the G1/S transition, hindering differentiation. Transcriptomic data revealed altered nonmyogenic dynamics, including elevated activated fibro-adipogenic progenitors (FAPs) early in repair and persistent proinflammatory macrophages. Integrative regulon and ligand-receptor analysis further demonstrated that triiodothyronine acted through dual modes: a direct transcriptional control of myogenic cell cycle and oxidative programs and an indirect paracrine remodeling mediated by FAP and immune signaling networks. This study identified what we believe to be novel effects of hypothyroidism on myogenic heterogeneity and impaired tissue repair, offering insights into muscle-wasting mechanisms relevant to hypothyroidism-associated myopathy and sarcopenia.

Authors

Paola Aguiari, Valentina Villani, Yan-Yun Liu, Gianni Carraro, Gregory A. Brent, Laura Perin, Anna Milanesi

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Figure 6

Thyroid hormone deficiency alters the immune and FAP landscape during skeletal muscle regeneration.

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Thyroid hormone deficiency alters the immune and FAP landscape during sk...
(A) t-SNE visualization of integrated scRNA-seq datasets from control and hypothyroid immune cells, with clusters distinguished by color. (B) Proportions of cells (graph, top panel) in each cluster shown as a percentage of control and hypothyroid immune cell muscle before injury and 5 and 14 dpi. Bottom panel: Fold change in cell proportion in hypothyroid compared with control samples for each cluster. In uninjured (NI) datasets, the immune cell samples were too small to be compared (/). Number of cells for each sample: CTRL NI, 128; CTRL 5d, 3,381; CTRL 14d, 548; HYPO NI, 72; HYPO 5d, 2,052; HYPO 14d, 572. (C) t-SNE visualization of control and hypothyroid immune cell scRNA-seq data colored by cluster and grouped by time points. (D) Table showing fold change of the number of proinflammatory (Ccl6+) and antiinflammatory (C1qa+) macrophages in hypothyroid versus control muscles at 5 and 14 dpi. In uninjured (NI) datasets, the immune cell samples were too small to be compared (/). (E) t-SNE visualization of immune cells at 5 dpi in control and hypothyroid scRNA-seq datasets colored by expression of Ccl6 (proinflammatory macrophages, blue), C1qa (antiinflammatory macrophages, red), and Cd75 (patrolling macrophages, green). Cells negative for the 3 markers are shown in black. (F) t-SNE plot of integrated scRNA-seq datasets from control and hypothyroid FAPs, with clusters distinguished by color. (G) Table describing FAP clusters and their functional roles based on gene expression classification, cluster labeling, and fold change (FC) in cell number between hypothyroid and control. Roman numerals represent the cluster numbers used in the text; asterisks indicate FC values for clusters of more than 100 cells, which are underlined, FC > 1.5 in red; FC < –1.5 in blue. (H) Histogram bar graph showing relative abundance of FAPs and muscle before injury and 5 and 14 dpi. Number of cells for each sample: CTRL NI, 1,648; CTRL 5d, 919; CTRL 14d, 3,597; HYPO NI, 655; HYPO 5d, 1,784; HYPO 14d, 1,875. (I) Condition-specific t-SNE plots illustrating distribution of FAP clusters in control and hypothyroid muscles before injury and 5 and 14 dpi. C, control; H, hypothyroid; CTRL, control; HYPO, hypothyroid.