Adaptive Modifications of Muscle Phenotype in High-Altitude Deer Mice Are Associated with Evolved Changes in Gene Regulation

Abstract

At high-altitude, small mammals are faced with the energetic challenge of sustaining thermogenesis and aerobic exercise in spite of the reduced O2 availability. Under conditions of hypoxic cold stress, metabolic demands of shivering thermogenesis and locomotion may require enhancements in the oxidative capacity and O2 diffusion capacity of skeletal muscle to compensate for the diminished tissue O2 supply. We used common-garden experiments involving highland and lowland deer mice (Peromyscus maniculatus) to investigate the transcriptional underpinnings of genetically based population differences and plasticity in muscle phenotype. We tested highland and lowland mice that were sampled in their native environments as well as lab-raised F1 progeny of wild-caught mice. Experiments revealed that highland natives had consistently greater oxidative fiber density and capillarity in the gastrocnemius muscle. RNA sequencing analyses revealed population differences in transcript abundance for 68 genes that clustered into two discrete transcriptional modules, and a large suite of transcripts (589 genes) with plastic expression patterns that clustered into five modules. The expression of two transcriptional modules was correlated with the oxidative phenotype and capillarity of the muscle, and these phenotype-associated modules were enriched for genes involved in energy metabolism, muscle plasticity, vascular development, and cell stress response. Although most of the individual transcripts that were differentially expressed between populations were negatively correlated with muscle phenotype, several genes involved in energy metabolism (e.g., Ckmt1, Ehhadh, Acaa1a) and angiogenesis (Notch4) were more highly expressed in highlanders, and the regulators of mitochondrial biogenesis, PGC-1α (Ppargc1a) and mitochondrial transcription factor A (Tfam), were positively correlated with muscle oxidative phenotype. These results suggest that evolved population differences in the oxidative capacity and capillarity of skeletal muscle involved expression changes in a small suite of coregulated genes.

Keywords: RNA-seq; capillarity; hypoxia adaptation; muscle fiber type; oxygen transport; physiological genomics.

Fig. 1.

Histological analysis of fiber type and capillarity in the gastrocnemius muscle of deer mice. Representative images from individuals sampled in their native environment are shown. Oxidative muscle fibers were identified by staining for succinate dehydrogenase activity, slow oxidative (type I) muscle fibers were identified by staining for slow myosin ATPase protein using immunohistochemistry, and capillaries were identified by staining for alkaline phosphatase activity. There were clear differences in staining intensity between highland and lowland deer mice.

Fig. 2.

The gastrocnemius muscle has a more oxidative phenotype in highland deer mice. There were significant effects of population altitude on the areal density of oxidative fibers (A_A(ox,m); area of oxidative fibers relative to the total transverse area of the muscle) (F_[1,37] = 13.37, P < 0.001), the numerical density of oxidative fibers (N_N(ox,m); number of oxidative fibers relative to the total number of fibers) (F_[1,37] = 5.143, P = 0.029), and the areal (A_A(type I,m); F_[1,37] = 9.329, P = 0.004) and numerical (N_N(type I,m); F_[1,37] = 6.379, P = 0.016) densities of slow oxidative fibers. The effects of rearing environment (native vs. common-garden F1 raised in the lab) were not significant (A_A(ox,m) F_[1,37] = 1.168, P = 0.287; N_N(ox,m) F_[1,37] = 1.708, P = 0.199; A_A(type I,m) F_[1,37] = 0.133, P = 0.718; N_N(type I,m) F_[1,37] < 0.001, P = 0.990). The interactions between population and rearing environment were also not significant (A_A(ox,m) F_[1,37] = 0.694, P = 0.410; N_N(ox,m) F_[1,37] = 1.032, P = 0.316; A_A(type I,m) F_[1,37] = 2.259, P = 0.141; N_N(type I,m) F_[1,37] = 0.724, P = 0.400). *Significant pairwise difference between highlanders and lowlanders within an experimental group (native vs. F1). Native lowlanders, n = 12; F1 lowlanders, n = 9; native highlanders, n = 9; F1 highlanders, n = 11.

Fig. 3.

The gastrocnemius muscle has a higher capillarity in highland deer mice than in lowland deer mice. There were significant effects of population altitude on capillary surface density (CSD, μm of capillary surface per μm² of transverse muscle area) (F_[1,35] = 18.92, P < 0.001), the ratio of capillary surface to fiber surface (CS:FS) (F_[1,35] = 19.20, P < 0.001), the density of capillaries (CD, capillaries per mm² of transverse muscle area) (F_[1,35] = 4.525, P = 0.041), and the number of capillaries per muscle fiber (C:F) (F_[1,35] = 4.223, P = 0.0474). The effects of rearing environment (native vs. common-garden F1 raised in the lab) were not significant (CSD F_[1,35] = 0.781, P = 0.383; CS:FS F_[1,35] = 0.071, P = 0.792; CD F_[1,35] = 1.688, P = 0.202; C:F F_[1,35] = 0.517, P = 0.477). The interactions between population and rearing environment were also not significant (CSD F_[1,35] = 0.942, P = 0.339; CS:FS F_[1,35] = 1.337, P = 0.255; CD F_[1,35] = 0.321, P = 0.575; C:F F_[1,35] = 0.430, P = 0.516). *Significant pairwise difference between highlanders and lowlanders within an experimental group (native vs. F1). Native lowlanders, n = 12; F1 lowlanders, n = 9; native highlanders, n = 8; F1 highlanders, n = 10.

Fig. 4.

Capillarity in the gastrocnemius muscle is greater in highland deer mice than expected from the variation in muscle oxidative phenotype. (A) There was a strong linear correlation between capillary surface density (CSD) and the areal density of oxidative fibers (A_A(ox,m)) (CSD = 0.0540 A_A(ox,m) + 0.0133, P < 0.001). Dashed lines represent the 95% confidence intervals of the regression. Symbols are as follows: F1 lab-raised lowlanders, black upwards triangles; native lowlanders, white upwards triangles; F1 lab-raised highlanders, dark gray downwards triangles; native highlanders, light gray downwards triangles. (B) There was a significant effect of population altitude on the residual CSD from the regression in (A) (*F_[1,35] = 5.558, P = 0.024), but there was no significant effect of rearing environment (F_[1,35] = 2.453, P = 0.126) and no significant interaction between population and rearing environment (F_[1,35] = 0.337, P = 0.566).

Fig. 5.

Correlated transcriptional modules. (A) Clustering of the 68 transcripts with significant population effects into two transcriptional modules. (B) Clustering of 589 transcripts with significant effects of rearing environment into five transcriptional modules.

Fig. 6.

Altitudinal variation in the expression of transcriptional modules that are statistically associated with muscle phenotypic traits. Module expression was summarized using PCA and PC1 scores are shown. There were significant effects of population altitude on modules P2 (F_[1,17] = 238.0, P < 0.001) and T5 (F_[1,17] = 10.19, P = 0.005). There was also a significant effect of rearing environment (native vs. common-garden F1 raised in the lab) on module T5 (F_[1,17] = 29.64, P < 0.001) but not module P2 (F_[1,17] = 0.937, P = 0.347). Transcriptional modules are shown in figure 5, and the genes that compose each module are presented in supplementary tables S1 and S2, Supplementary Material online. *Significant pairwise difference between highlanders and lowlanders within an experimental group (native vs. F1). Native lowlanders, n = 6; F1 lowlanders, n = 5; native highlanders, n = 5; F1 highlanders, n = 5.

Fig. 7.

Some individual genes involved in metabolism and angiogenesis were more highly expressed in highlanders than in lowlanders. Reaction norms for gene expression are shown with native environment on the left and lab environment on the right, and data are shown relative to the average normalized read count for native lowlanders. There was a statistically significant effect of population altitude on all genes shown (see supplementary table S1, Supplementary Material online). The mean normalized read counts (cpm) for native lowlanders were as follows: enoyl-CoA hydratase (Ehhadh), 3.7; acetyl-Coenzyme A acyltransferase 1A (Acaa1a), 4.5; mitochondrial creatine kinase 1 (Ckmt1), 2.9; mitochondrial ribosomal protein L22 (Mrpl22), 8.5; aldehyde dehydrogenase 1A1 (Aldh1a1), 41.8; aldehyde dehydrogenase 1A7 (Aldh1a7), 20.2; mannose receptor C type 1 (Mrc1), 23.7; cadherin-7 (Cdh7), 3.8; and Notch-4 (Notch4), 45.8. There were also significant effects of rearing environment on Aldh1a1 and Cdh7 (†) (see supplementary table S2, Supplementary Material online). n = 6 for all groups.

Fig. 8.

There was a strong negative association between the expression of fibroblast growth factor receptor 2 (Fgfr2) and the abundance of slow oxidative fibers. There was a significant linear correlation between Fgfr2 transcript abundance and both the numerical (R² = 0.574, P = 0.0001) and areal (R² = 0.446, P = 0.0013) densities of slow oxidative fibers in the gastrocnemius muscle. Dashed lines represent the 95% confidence intervals of the regression. Symbols are as follows: F1 lab-raised lowlanders, black upwards triangles; native lowlanders, white upwards triangles; F1 lab-raised highlanders, dark gray downwards triangles; native highlanders, light gray downwards triangles.