Body temperature, metabolism and heart rate
As previously reported, hibernating bears had core body temperatures of 34.2 ± 0.5°C (mean ± SD, n = 6) and minimum rates of oxygen consumption of 0.083 ± 0.008 ml g-1
(n = 5), when measured over at least a 0.5 h interval 2-9 hours before euthanasia [11
]. Heart rate (HR) derived by counting beats within a 240 s on ECG recording during this period was 14.4 ± 2.4 b/min. (mean ± SD, n = 6) and showed pronounced respiratory related sinus arrhythmia. In four hibernating bears for which post-immobilization measurements were made, body temperature decreased from 34.2 ± 0.7°C to 33.3 ± 1.1°C (p < 0.05) and HR increased from 13.4 ± 2.2 to 82.9 ± 22.8 b/min (p < 0.05). Sinus arrhythmia was absent in anesthetized bears. Bears lost 4.7 ± 0.8% (n = 6) of their body mass per month during the 4-5 month hibernation period. Metabolic rates measured in two fasted and anesthetized summer active bears were 0.252 and 0.213 ml g-1
and averaged 0.233 ml g-1
; body temperatures were 37.18 and 37.13°C [11
] and HR was 102.3 and 97.2 b/min and averaged 99.7 b/min. Immediately prior to tissue sampling, metabolic rate of hibernating bears was 36-49%, and body temperature averaged 3.75°C lower compared to values in summer bears [11
] while HR was at about the same level due to the effect of anesthesia.
Difference in gene expression
For heart, signals from 2,594 of 3,200 probes (81.1%) on the first bear array showed median intensities that were above the level of two backgrounds, whereas 6,860 of 9,600 (71.5%) probes showed significant signals on the second bear array (see Material and Methods for the array description). In order to define genes that were differentially expressed in hibernating compared to summer active bears, we used P < 0.01 and log2
FC > 0.5 where FC is fold change, the mean expression value in the hibernating bears divided by the mean expression value in the summer active bears, as the cutoff for differentially expressed genes (as described in Methods). A total of 245 genes, 3.3% of all unique genes with significant signals (7,359 genes), were differentially expressed in heart during hibernation (Additional file 1
, Table S1). The maximal change of 7.28 fold (log2
FC = 2.86) was detected for RNA binding protein motif 3 (Rbm3
), but most genes (74%) demonstrated moderate changes in expression less than two fold (log2
FC < 1). Of the significantly differentially expressed genes, we identified 183 (75%) that were over-expressed and 62 (25%) genes that were under-expressed in heart during hibernation.
For liver, 6,860 of 9,600 (71.5%) probes showed significant signals on the second bear array, and 319 genes of these, 6.2% of all unique genes with significant signal (5,092 genes), were differentially expressed in liver during hibernation (Additional file 1
, Table S1). There were 165 (52%) significantly over-expressed genes and 154 (48%) under-expressed genes in liver sampled during hibernation compared to in summer. Phosphoenolpyruvate carboxykinase (Pck1
) showed the largest positive expression change of 10.91 fold (log2
FC = 3.448), and aldehyde dehydrogenase (Aldh1l1
) was 17.94 fold down regulated (log2
FC = -4.168), but most genes (74%) demonstrated modest expression changes that did not exceed two fold (|log2
FC| < 1).
To validate the microarray results, we conducted quantitative real-time PCR tests for 32 randomly selected genes that were identified as differentially expressed by the array hybridizations. Expression changes of 28 (87.5%) out of 32 genes identified on the array were confirmed by the RT PCR tests (Table ) with significant positive correlation (r = 0.83, p < 0.001) between fold change values of supported genes (Table , Figure ). High consistency between microarray experiments and RT PCR tests is in agreement with the mean false discovery rate of 12% for the list of differentially expressed genes.
Gene expression differences tested by microarray and real-time PCR in bear heart and liver
Figure 1 Selection of genes differentially expressed during hibernation in heart (A) and liver (B) tissue in black bears. Expression values are normalized to the mean in summer active animals. Solid bars show expression values obtained in real-time PCR, open bars (more ...)
The expression of 33 genes was changed in common in both liver and heart (Table ) of which 24 genes were elevated in both tissues. RNA binding motif protein 3 (Rbm3) showed high positive expression change in both liver and heart during hibernation. Most genes (70%) up regulated in both tissues are involved in lipid catabolism (3 genes) and protein biosynthesis (14 genes, Table ). Among five genes down regulated in both heart and liver, three genes are involved in amino acid catabolism. Five genes demonstrated transcriptional changes in opposite directions in liver and heart.
Differentially expressed genes shared between liver and heart in hibernating bears
The Gene Ontology analysis revealed a highly significant enrichment of the protein biosynthesis (translation) category in the biological processes and the RNA binding in the molecular function by over-expressed genes in both heart and liver during hibernation (Tables , ). Four additional categories demonstrated significant enrichment in liver: up regulated genes in the fatty acid beta-oxidation pathway and under expressed genes in amino acid catabolism, cholesterol metabolism and cellular respiration categories during hibernation. The molecular function categories with significantly elevated proportion of down regulated genes in liver included catalytic processes: oxidoreductase and transaminase activities (Table ).
Gene Ontology categories significantly enriched with differentially expressed genes
Genes in significant Gene Ontology categories of the biological processes
Significant enrichment of the biological processes categories by differentially expressed genes was validated and supported by the results of gene set enrichment analysis (GSEA; Figures , ). GSEA ranks all genes with significant signals on the array therefore its results are not affected by the selection of genes above cutoffs for significance of expression differences and false discovery rate [13
Figure 2 Gene set enrichment analysis results for the translation category. The translation category is enriched by up regulated genes in heart (A, false discovery rate (FDR) of <0.001) and liver (B, FDR = 0.046) of hibernating black bears. An expression (more ...)
Figure 3 Gene set enrichment analysis results for the fatty acid β oxidation and amino acid catabolism categories. The fatty acid β oxidation category (A) is enriched by up regulated genes (FDR = 0.043) and amino acid catabolism (B) is enriched (more ...)
Differential expression of selected genes in liver
Some important genes involved in metabolic pathways demonstrated expression differences between hibernating and summer active bears. Among 116 genes involved in lipid biosynthesis, expression of diacylglycerol O-acyltransferase (Dgat2), 7-dehydrocholesterol reductase (Dhcr7), emopamil binding protein (Ebp), farnesyl diphosphate synthase (Fdps ), propionyl Coenzyme A carboxylase (Pccb) all decreased during winter hibernation. GSEA revealed significant enrichment (FDR = 0.006) of the lipid biosynthesis category by genes with lower expression levels in hibernating bears as compared to summer active animals. Expression of long-chain fatty acyl elongase (Elovl2) involved in biosynthesis of long chain fatty acids for catabolism through β oxidation increased during winter hibernation. Expressions of phosphoenolpyruvate carboxykinase (Pck1) and fructose-1,6-bisphosphatase 1 (Fbp1 ), main control enzymes in the regulation of gluconeogenesis, were elevated during hibernation. In contrast, an important glycolytic gene, pyruvate dehydrogenase beta (Pdhb) and transketolase (Tkt) involved in the pentose phosphate pathway were down regulated in liver during hibernation.
Genes involved in protein catabolism did not show coordinated transcriptional change during hibernation. Among 140 genes involved in protein catabolism, leucine aminopeptidase 3 (Lap), calpastatin (Cast), proteasome 26S subunit (Psmc2) and proteasome subunit, beta type (Psmb1) were down regulated while YME1L1 gene (Yme1l1), ubiquitin protein ligase E3A (Ube3a), ubiquitin specific peptidase 7 (Usp7) and F-box and leucine-rich repeat protein 4 (Fbxl4) were over expressed in liver during hibernation.
Differential expression of selected genes in heart
In common with liver (Table ), there were three over expressed genes (Acadvl, Acadm, Hadha) that are involved in fatty acid beta oxidation in the heart of hibernating bears. Among 44 genes involved in amino acid catabolism, three genes shared with liver (Aldh6a1, Got1, Lap3) and glutathione transferase zeta 1 (Gstz1) were under expressed in heart during hibernation.
Although five out of 190 genes involved in protein catabolism were up regulated in heart during hibernation, similar to liver, this category did not demonstrate significant enrichment (FDR = 1.00). Over expressed protein catabolism genes included selenoprotein S (Sels), ornithine decarboxylase antizyme 1 (Oaz1), proteasome (prosome, macropain) 26S subunit (Psmc3), proteasome (prosome, macropain) subunit alpha type 3 (Psma3) ubiquitin specific peptidase 11 (Usp11).