Microarray analysis of the temporal response of skeletal muscle to methylprednisolone: comparative analysis of two dosing regimens

Abstract

The transcriptional response of skeletal muscle to chronic corticosteroid exposure was examined over 168 h and compared with the response profiles observed following a single dose of corticosteroid. Male adrenalectomized Wistar rats were given a constant-rate infusion of 0.3 mg x kg(-1) x h(-1) methylprednisolone for up to 7 days via subcutaneously implanted minipumps. Four control and forty drug-treated animals were killed at ten different time points during infusion. Liver total RNAs were hybridized to 44 individual Affymetrix REA230A gene chips. Previously, we described a filtration approach for identifying genes of interest in microarray data sets developed from tissues of rats treated with methylprednisolone (MPL) following acute dosing. Here, a similar approach involving a series of three filters was applied sequentially to identify genes of interest. These filters were designed to eliminate probe sets that were not expressed in the tissue, not regulated by the drug, or did not meet defined quality control standards. Filtering eliminated 86% of probe sets, leaving a remainder of 2,316 for further consideration. In a previous study, 653 probe sets were identified as MPL regulated following administration of a single (acute) dose of the drug. Comparison of the two data sets yielded 196 genes identified as regulated by MPL in both dosing regimens. Because of receptor downregulation, it was predicted that genes regulated by receptor-glucocorticoid response element interactions would exhibit tolerance in chronic profiles. However, many genes did not exhibit steroid tolerance, indicating that present perspectives on the mechanism of glucocorticoid action cannot entirely explain all temporal profiles.

Fig. 1

Gene tree representation of all probe sets (15,967) on individual Affymetrix R230A gene chips hybridized to total RNA prepared from muscles taken from animals treated chronically with methylprednisolone (MPL; 0.3 mg•kg⁻¹•h⁻¹) for periods ranging from 6 to 168 h. the values for each individual probe set at each time point were normalized to the mean value of that probe set for vehicle-treated controls. The x-axis represents the 11 time points, including vehicle-treated controls (nominal time 0). The y-axis presents the list of probe sets grouped by similarity using Pearson correlation. Yellow indicates no change from controls, red indicates probe sets with enhanced expression relative to controls, and blue indicates suppressed expression relative to controls.

Fig. 2

Top: a magnification of 4 probe sets selected from Fig. 1 that show apparent enhanced regulation by MPL. The linear plots for these probe sets are presented at bottom.

Fig. 3

Top: a magnification of 5 probe sets selected from Fig. 1 that show apparent downregulation by MPL. The linear plots of all 5 probe sets are presented at bottom.

Fig. 4

Gene tree representation of the 9,816 probe sets remaining after removing probe sets (6,151) that did not give a call of present using MAS5.0 software in at least 3 of 44 chips in the data set.

Fig. 5

Gene trees of probe sets remaining after filtering for MPL regulation. Left: probe sets with potential downregulation (1,466). Middle: those with potential upregulation (1,412). Right: probe sets that met both criteria (60).

Fig. 6

Gene tree representation of probe sets remaining (2,316) after filtering the probe sets in Fig. 5 for high coefficients of variation.

Fig. 7

MPL concentrations in rat plasma following chronic (top) and acute (bottom) administration of drug. MPL concentrations were determined by normal-phase HPLC analysis of plasma samples obtained from individual animals.

Fig 8

Response profiles of 2 genes showing enhanced expression and tolerance with chronic dosing. Acute-dosing profiles are shown at top, and chronic-dosing profiles are shown at bottom. Mitogen-activated protein kinase-14 (MAPK14) expressions are shown at left and peroxisome proliferator-activated receptor-δ (PPARδ) at right.

Fig. 9

Response profiles of 2 genes showing enhanced expression but the absence of tolerance with chronic dosing. Acute-dosing profiles are shown at top, and chronic-dosing profiles are shown at bottom. Glutamine synthetase (GS) expressions are shown at left and eukaryotic translation initiation factor-4e binding protein-1 (Eif4ebp1) at right.

Fig. 10

Response profiles of 2 genes showing both primary and secondary enhanced expression. Acute-dosing profiles are shown at top, and chronic-dosing profiles are shown at bottom. Interleukin-6 receptor (IL6R1) expressions are shown at left and interferon-related developmental regulator-1 (Ifrd1) at right.

Fig. 11

Response profiles of Syndecan 2 core protein (Sdc2), which shows decreased expression following MPL administration and only partial tolerance with chronic dosing. Acute-dosing profiles are shown at top and chronic-dosing profiles at bottom.

Fig. 12

Response profiles of Na-K-Cl cotransporter (Nkcc1), which shows decreased expression following MPL administration and tolerance with chronic dosing. Acute-dosing profiles are shown at top and chronic-dosing profiles at bottom.

Fig. 13

Response profiles of 2 genes showing decreased expression but absence of tolerance with chronic dosing. Acute-dosing profiles are shown at top, and chronic-dosing profiles are shown at bottom. Myristoylated alanine-rich C-kinase substrate (Macs) expression is shown at left and extracellular signal-related kinase-3 (ERK3) at right.

Fig. 14

Response profiles of myogenic factor-6 (Myf6), which shows complex regulation of expression following acute (top) and chronic (bottom) MPL dosing. In the acute profile, there is an initial downregulation followed by a period of enhanced regulation.

Fig. 15

Response profiles of myostatin following acute dosing as determined from gene arrays (top) and following chronic dosing (bottom) as determined by both gene arrays (●) and by kinetic-based quantitative RT-PCR (○).

Fig. 16

Response profiles of ring finger protein-28 (MURF1) following acute (top) and chronic (bottom) dosing.