Single-Nucleus Transcriptome Reveals Cellular Heterogeneity and Transcriptional Response to Heat Stress in Skeletal Muscle

Abstract

Background

Heat stress can induce skeletal muscle injury. Typical characteristics of heat-exposed muscle tissues include apoptosis, oxidative stress and autophagy. The understanding of molecular mechanisms underlying heat stress-induced muscle injury is limited, especially at the single-cell transcription level.

Methods

We collected skeletal muscles from 12-week-old female C57BL/6J mice in control (NC) and four heat stress groups. The experimental scheme comprised five groups: NC (25.5°C ± 0.5°C), HS0 (after ~3-h heat exposure, 41.5°C ± 0.5°C), HS8, HS16 and HS24 group (recovery at 25.5°C ± 0.5°C for 8, 16 and 24 h, respectively). Skeletal muscles were subjected to HE staining (n = 6), TUNEL staining (n = 3) and transmission electron microscopy (n = 3). Transcripts were measured at the tissue (n = 5 or 6) and single-nucleus levels (n = 2).

Results

Histologically, myofibrillar structure deformation, mitochondrial swelling and fusion, intramuscular triglyceride accumulation and autophagy occurred in the muscles subjected to heat stress. At the tissue level, a gene cluster associated with the response to heat exhibited an increasing trend of transcription in the HS0 versus NC groups, and the levels decreased to those in the NC group after 16 h. At the single-cell level, 134 320 high-quality myonuclei were collected from the muscles and annotated as seven cell types, including myonuclei, muscle stem cells (MuSCs) and immune cells. We identified the larger number of differentially expressed genes in the myonuclei. After heat stress, new cell clusters appeared in type IIa/IIx (HS8 group) and IIb (HS0 group) myonuclei but not in type I myonuclei. Generally, immediate early genes, highly expressed genes and transcription factor regulons identified in new cell clusters induced by heat were related to responses to heat, heat shock, oxidative stress and antioxidative stress. To repair the injured muscles, MuSCs highly expressed the development-related genes, such as Atp2a1, Ckm, Myh1, Aldoa, Pde4d and Pdlim5. Analysis of cell–cell communication showed that Dag1 and Egf signals associated with myonuclei were the underlying pathways that participated in repair tissues.

Conclusions

We constructed the largest transcriptomic dataset, to date, for heat-exposed skeletal muscles. At tissue resolution, the response of muscles to heat stress was eliminated after a recovery period of 16 h. At single-nucleus resolution, the myofibre was the most heat-sensitive cell type, and type IIb myonuclei were the most heat-sensitive subtype of myonuclei. To our knowledge, this is the first study to reveal the molecular mechanisms underlying heat-induced muscle injury and repair at the single-cell transcriptional level.