Premature expression of a muscle fibrosis axis in chronic HIV infection

Abstract

Background: Despite the success of highly active antiretroviral therapy (HAART), HIV infected individuals remain at increased risk for frailty and declines in physical function that are more often observed in older uninfected individuals. This may reflect premature or accelerated muscle aging.

Methods: Skeletal muscle gene expression profiles were evaluated in three uninfected independent microarray datasets including young (19 to 29 years old), middle aged (40 to 45 years old) and older (65 to 85 years old) subjects, and a muscle dataset from HIV infected subjects (36 to 51 years old). Using Bayesian analysis, a ten gene muscle aging signature was identified that distinguished young from old uninfected muscle and included the senescence and cell cycle arrest gene p21/Cip1 (CDKN1A). This ten gene signature was then evaluated in muscle specimens from a cohort of middle aged (30 to 55 years old) HIV infected individuals. Expression of p21/Cip1 and related pathways were validated and further analyzed in a rodent model for HIV infection.

Results: We identify and replicate the expression of a set of muscle aging genes that were prematurely expressed in HIV infected, but not uninfected, middle aged subjects. We validated select genes in a rodent model of chronic HIV infection. Because the signature included p21/Cip1, a cell cycle arrest gene previously associated with muscle aging and fibrosis, we explored pathways related to senescence and fibrosis. In addition to p21/Cip1, we observed HIV associated upregulation of the senescence factor p16INK4a (CDKN2A) and fibrosis associated TGFβ1, CTGF, COL1A1 and COL1A2. Fibrosis in muscle tissue was quantified based on collagen deposition and confirmed to be elevated in association with infection status. Fiber type composition was also measured and displayed a significant increase in slow twitch fibers associated with infection.

Conclusions: The expression of genes associated with a muscle aging signature is prematurely upregulated in HIV infection, with a prominent role for fibrotic pathways. Based on these data, therapeutic interventions that promote muscle function and attenuate pro-fibrotic gene expression should be considered in future studies.

Figure 1

Muscle age profiling. Heatmaps displaying the relative expression of the ten gene muscle aging profile are shown comparing young males to older males in a supervised manner using Affymetrix U133A microarray gene set data for GSE362 (A) and GSE1428 (B) and in a healthy aging dataset using a NimbleGen microarray data (C). The gene expression of the profile is displayed in a supervised manner where intensity reflects relative expression (green = higher, red = lower). Heatmaps were generated using the Heatplus package in the statistical software R 2.14.0

Figure 2

A. Muscle age profiling with HIV. Heatmap displaying the ten gene profile shown in Figure 1, with the HIV group added using GSE1428 (Figure 2A) and GSE362 (similar results, data not shown). The CAGED software was used to cluster samples (shown as rows of the heat map) based on the ten gene muscle aging profile (columns of the heat map). The analysis shows that the young men form a cluster, while HIV samples (designated by arrows and in bold, 36 to 51 years old) cluster with old subjects using the ten gene muscle aging profile. The CAGED cluster analysis uses a model-based procedure that assigns samples to the same cluster if their merging increases the posterior probability of the model. Numbers attached to the branches of the dendrogram represent the posterior odds of the model that merges the branches versus the model that does not. B. CAGED analysis of the expression of the ten gene muscle aging profile is an HIV negative dataset with young (20 to 25 years old), intermediate (40 to 45 years old designated by arrows), and old (70 to 75 years old) subjects (this entire dataset used the microarray NimbleGen platform). The gene expression of the profile is displayed in a supervised manner where intensity reflects relative expression (green = higher than average, red = lower than average). CAGED, Cluster Analysis of Gene Expression Dynamics

Figure 3

Expression of p21/Cip1, p16INK4a and TGFβ1 in the HIV transgenic rodent. A. Realtime RNA PCR validation was observed for p21/Cip1, Fez2, H3, MFL1, MT1F and MYH8 in muscle from HIV transgenic gastrocnemius (n = 4) muscle or wild type gastrocnemius muscle (n = 3) and shows an increase in genes in HIV Tg compared to age matched wild type controls. All bar plots show mean fold change with error bars indicating the standard deviation. The difference between wild type and HIV Tg is significant to P ≤ 0.05. B. RT-PCR shows an increase in the cell cycle arrest gene, p16INK4a, in HIV Tg rat (n =4) compared to wild type (n = 3). The difference is significant to P < 0.05. The bar plot indicates mean fold change with the error bars indicative of the standard deviation. C. TGFβ1 protein levels based on ELISA of muscle homogenates from HIV transgenic gastrocnemius muscle (n = 3) or wild type gastrocnemius muscle (n = 3) show increased levels in the HIV Tg rodent at a significance of P < 0.05. Bar plots indicate mean protein levels with error bars indicative of the standard deviation. TGFβ, transforming growth factor β

Figure 4

Expression of collagen genes in the HIV transgenic rodent model. A. The collagen transcriptional regulator, CTGF, is increased in HIV Tg rat muscle (n = 3) compared to wild type (n = 3) using realtime RNA PCR at a significance of P <0.05. The bar plots indicate mean fold change with error bars indicative of standard deviation. B. The collagen genes, COL1A1 and COL1A2 are increase in HIV Tg rat muscle (n =3) compared to wild type (n = 3) using realtime RNA PCR. The bar plots indicate mean fold change with the error bars indicative of standard deviation. The difference between wild type and HIV Tg rodents are significant to P < 0.05. CTGF, connective tissue growth factor

Figure 5

Collagen deposition and fiber type switching in the HIV transgenic rat. Histological muscle sections of the gastrocnemius muscle of HIV Tg (n = 6) and control wild type rats (n = 3) were stained using Picrosirius Red for collagen. Representative images are shown in A and B. The scale bar equals 200 μm. A. Tissue section of the gastrocnemius muscle in wild type rat shows collagen deposition in red in intracellular space. B. Tissue section of gastrocnemius muscle from HIV Tg rat shows increased collagen deposition in red in the intracellular spaces. C. Quantification of the area of picrosirius red staining using Image J shows that there is an increase in fibrotic index in the HIV Tg rat compared to the wild type at a significance of P < 0.05. The bar plot indicates mean percent area of collagen with the error bars indicative of the standard deviation. D. Quantification of fiber type was done using Image J on control wild type (n = 4) and HIV Tg (n = 4). The bar plot indicates the percentage of fibers of each type with error bars indicative of standard deviation