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. 2014 Dec 3;15(1):1058.
doi: 10.1186/1471-2164-15-1058.

Patterns of Gene Expression Associated With Recovery and Injury in Heat-Stressed Rats

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Free PMC article

Patterns of Gene Expression Associated With Recovery and Injury in Heat-Stressed Rats

Jonathan D Stallings et al. BMC Genomics. .
Free PMC article

Abstract

Background: The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model.

Results: We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response.

Conclusions: Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

Figures

Figure 1
Figure 1
Heat stress experimental design. (A) Core temperature (Tc) was monitored by telemetry probe in real time in heated rats [X] or time-matched controls [C], and animals were sacrificed at Tc,Max (41.8°C), 24 hours, or 48 hours. Tissues were preserved for histopathology, transcriptomics, and proteomics. (B) Blood chemistries and histopathology were evaluated. Differential gene expression, gene set analysis, and KEGG enrichment were examined in all organs at Tc,Max to characterize the consensus gene response to heat stress. Self-organizing maps were constructed to identify genes that clustered in individual responders. We evaluated proteomic changes in the heart at Tc,Max, 24 hours, and 48 hours. Animals in the 48-hour cohort were subdivided by histopathology (injured and uninjured). Venn diagrams indicate the numbers of genes (left) and proteins (right) identified in each respective analysis. Red text denotes statistically significant changes in the same genes and proteins.
Figure 2
Figure 2
Histopathological evidence of cardiomyopathy and inflammation around cardiomyofibers at 48 hours in heat-stressed rats (hematoxylin and eosin staining). Minimal cardiomyopathy in unheated control rats is observed at (A) 20× and (B) 200× magnification. Arrows represent focal areas of cardiomyopathy with few macrophages and rare neutrophils. (C) Cardiac sections from heat-stressed animals show moderate inflammatory infiltrate (arrows) at 48 hours (20× magnification). (D) Inflammation separates and surrounds the cardiomyofibers with occasional degenerative cardiomyofibers (200× magnification).
Figure 3
Figure 3
Heart self-organizing map identifies genes that anchor to histopathological changes at 48 hours. Node A (1351 down-regulated genes) and Node B (887 up-regulated genes) were common to the three animals (9, 11, and 12) with the most severe histopathological injury. See Additional file 8 for a full list of transcript IDs and the KEGG analysis of Nodes A, B, and C.
Figure 4
Figure 4
Differentially expressed genes and proteins and KEGG pathway enrichment analysis in hearts at T c,Max . Proteins were mapped to gene probes to compare differentially expressed proteins and genes in the heart. Heat shock proteins and proteins related to HSPs are bolded. Proteins expressed at ±1.3-fold and their respective genes (±2-fold) are represented by red squares. Proteins and genes which mapped together but did not meet significance at the gene level are represented as blue diamonds. Genes (green triangles) or proteins (purple triangles) that were expressed and significant, but did not map together, are plotted along the axes. For clarity, only the gene designations are listed.
Figure 5
Figure 5
Differentially expressed genes and proteins and KEGG pathway enrichment analysis at 24 hours. Proteins were mapped to gene probes to compare differentially expressed proteins and genes in the heart. Insets show the results of the KEGG pathway enrichment analysis for concordant gene and protein up- and down-regulation. Genes and proteins with the greatest concordance with the pathway enrichment analysis are depicted. Proteins expressed at ±1.3-fold and their respective genes (±2-fold) are represented by red squares. Proteins and genes which mapped together but did not meet significance at the gene level are represented as blue diamonds. Genes (green triangles) or proteins (purple triangles) that were expressed and significant, but did not map together, are plotted along the axes. For clarity, only the gene designations are listed.
Figure 6
Figure 6
Differentially expressed genes and proteins and the KEGG pathway enrichment analysis in hearts with evidence of histopathological injury 48 hours after heat stress. Proteins were mapped to gene probes to compare differentially expressed proteins and genes in the heart. The KEGG pathway analysis results for genes and proteins with the greatest concordance are depicted. Heat shock proteins and proteins related to HSPs are bolded. Proteins expressed at ±1.3-fold and their respective genes (±2-fold) are represented by red squares. Proteins and genes which mapped together but did not meet significance at the gene level are represented as blue diamonds. Genes (green triangles) or proteins (purple triangles) that were expressed and significant, but did not map together, are plotted along the axes. For clarity, only the gene designations are listed.
Figure 7
Figure 7
Differentially expressed genes and proteins in uninjured hearts 48 hours after heat stress. Proteins were mapped to gene probes to compare differentially expressed proteins and genes in animals with uninjured hearts. Proteins expressed at ±1.3-fold and their respective genes (±2-fold) are represented by red squares. Proteins and genes which mapped together but did not meet significance at the gene level are represented as blue diamonds. Genes (green triangles) or proteins (purple triangles) that were expressed and significant, but did not map together, are plotted along the axes. For clarity, only the gene designations are listed.
Figure 8
Figure 8
Aggregation propensities for modulated pathways. Proteins and genes with concordant differential expression in heat-injured animals at 48 hours were mapped to subcellular localization (see Figure 6). Up-regulated proteins/gene pairs mapped to the KEGG pathways Ribosome, Antigen Processing and Presentation, Regulation of Actin Cytoskeleton, Complement and Coagulation Cascade, and Pentose Phosphate Pathway. Down-regulated protein/gene pairs mapped to Hypertrophic/Dilated Cardiomyopathy and Cardiac Muscle Contraction, Fatty Acid Metabolism, and Oxidative Phosphorylation. Gene/protein pairs are mapped to target subcellular organelles. For simplicity in presentation, only protein designations are shown.
Figure 9
Figure 9
Preferential increase in propensity to aggregate in proteins modulated by heat stress at 24 and 48 hours with histopathological evidence of cardiac injury. Median-corrected supersaturation scores (σf) were calculated for proteins which changed (white bars) or remained unchanged in abundance as measured by mass spectrometry after heating as described by Ciryam et al.[79].

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