Antiretroviral Drugs Regulate Epigenetic Modification of Cardiac Cells Through Modulation of H3K9 and H3K27 Acetylation

Abstract

Antiretroviral therapy (ART) has significantly reduced the rate of mortality in HIV infected population, but people living with HIV (PLWH) show higher rates of cardiovascular disease (CVD). However, the effect of antiretroviral (ARV) drug treatment on cardiac cells is not clear. In this study, we explored the effect of ARV drugs in cardiomyocyte epigenetic remodeling. Primary cardiomyocytes were treated with a combination of four ARV drugs (ritonavir, abacavir, atazanavir, and lamivudine), and epigenetic changes were examined. Our data suggest that ARV drugs treatment significantly reduces acetylation at H3K9 and H3K27 and promotes methylation at H3K9 and H3K27, which are histone marks for gene expression activation and gene repression, respectively. Besides, ARV drugs treatment causes pathological changes in the cell through increased production of reactive oxygen species (ROS) and cellular hypertrophy. Further, the expression of chromatin remodeling enzymes was monitored in cardiomyocytes treated with ARV drugs using PCR array. The PCR array data indicated that the expression of epigenetic enzymes was differentially regulated in the ARV drugs treated cardiomyocytes. Consistent with the PCR array result, SIRT1, SUV39H1, and EZH2 protein expression was significantly upregulated in ARV drugs treated cardiomyocytes. Furthermore, gene expression analysis of the heart tissue from HIV+ patients showed that the expression of SIRT1, SUV39H1, and EZH2 was up-regulated in patients with a history of ART. Additionally, we found that expression of SIRT1 can protect cardiomyocytes in presence of ARV drugs through reduction of cellular ROS and cellular hypertrophy. Our results reveal that ARV drugs modulate the epigenetic histone markers involved in gene expression, and play a critical role in histone deacetylation at H3K9 and H3K27 during cellular stress. This study may lead to development of novel therapeutic strategies for the treatment of CVD in PLWH.

Keywords: ROS; SIRT1; antiretroviral therapy; cardiovascular disease; cellular hypertrophy; histone deacetylase; human immunodeficiency virus; methyltransferase.

Figure 1

ARV drugs treatment suppresses active histone marks. (A,B) Western blots show acetylation of histone at H3K9 and H3K27 and graphs show its quantification (^***p < 0.001). NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for 4, 12, and 24 h and western blots were done with total protein lysate. (C,D) Representative images show immunofluorescence staining of NRVCs stained with H3K9ac (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images (n = 50 cells); (^*p < 0.05). (E,F) Representative images show immunofluorescence staining of NRVCs stained with H3K27ac (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images (n = 50 cells); (^*p < 0.05). (G,H) Western blot shows acetylation of NRVCs after drugs treatment. NRVCs were treated with individual ARV drug for 12 h and western blot was done with total protein lysate. Graphs show quantification of western blot (^***p < 0.001; ^**p < 0.01, ns, not significant).

Figure 2

ARV drugs treatment promotes repressive histone marks. (A,B) Western blot shows antiretroviral drug treatment increase the methylation of H3K27me3 and H3K9me3. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for 4, 12, and 24 h and western blots were done with total protein lysate. Graph shows quantification of western blots (^***p < 0.001; ^**p < 0.01, ns, not significant) (C) Representative images show immunofluorescence staining of NRVCs stained with H3K9me3 (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (D) Graph shows quantification of microscopic images (n = 50 cells); (^*p < 0.05). (E) Representative images show immunofluorescence staining of NRVCs stained with H3K27me3 (green), actinin (red), and nucleus stained with DAPI (blue). Cells were treated with ARV drugs for 12 h and fixed with 4% PFA. (F) Graph shows quantification of microscopic images (n = 50 cells); (^***p < 0.001).

Figure 3

ARV drugs treatment modulates expression of epigenetic regulating enzyme. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 4, 12, and 24 h and expression of epigenetic enzyme was measured by RT² PCR array profiling. (A) Clustergram showing differentially expressed genes (p < 0.05, Fold Change >1.2) obtained on RT² PCR array profiling, green represent minimum and red represent maximum magnitude of expression. (B) Scatter Plot representing normalized expression of genes between 4, 12, and 24 h ARV drugs treated vs. control group. Central diagonal line represents no change, whereas, outer diagonal lines indicate the fold regulation threshold (>1.2). Genes with data points beyond the outer lines in the upper left and lower right corners are up-regulated or down-regulated, respectively. (C) Graphical representation of differentially expressed chromatin modifying enzymes against expression regulation on drug treatment at 4 h (red), 12 h (green), and 24 h (blue).

Figure 4

Expression of epigenetic chromatin enzymes affected by ARV drugs treatment. (A) Graphs showing mRNA expression of Sirt1, Ezh2, and Suv39h1 in NRVCs treated with drugs. Cells were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir and Lamivudine) for different time point (4, 12, and 24 h) and expression was checked by qRT-PCR. (B,C) Western blots showing protein expression of SIRT1, SUV39H1, and EZH2 enzymes and graph show quantification. NRVCs were treated with ARV drugs (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 4, 12, and 24 h and expression was checked in total protein lysate by western blot with respective antibody (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, ns, not significant).

Figure 5

Expression of epigenetic regulating enzymes dysregulated in HIV + patients have history of ART treatment. (A–C) Graphs show expression of SIRT1, SUV39H1, and EZH2 enzymes in human cardiac tissue. Total RNA was isolated from the frozen heart tissue and expression was checked by qRT-PCR (^*p < 0.05, ^**p < 0.01).

Figure 6

Expression of SIRT1 is critical in ARV drugs mediated modulation of cellular acetylation. (A,B) Western blots show that knockdown of SIRT1 upregulate acetylation of histone at H3K9. NRVCs were treated with SIRT1 siRNA for 48 h and followed by ARV drugs treatment (5 μM of Ritonavir, Abacavir, Atazanavir, and Lamivudine) for 12 h. Graph shows quantification of western blot. (^***p < 0.001; ^**p < 0.01, ^*p < 0.05, ns, not significant). (C,D) Western blots show that over expression of SIRT1 can significantly decreases the H3K9ac in rat cardiomyocytes. SIRT1 and GFP protein were over expressed in the cardiomyocytes for 48 h by adenoviral transduction and treated with the ARV drugs for another 24 h. Graph shows the quantification of H3K9ac (^*p < 0.05). (E) Drug treatment upregulates the SIRT1 enzyme activity. NRVCs were treated with ARV drugs for 4–24 h and enzyme activity was determined in total protein lysate. (^***p < 0.001; ^****p < 0.0001, ns, not significant). (F) ARV drugs treatment reduces cellular viability in SIRT1 knockdown cells. NRVCs were treated with SIRT1 siRNA and ARV drugs and viability was measure by CellTiter-Glo (^**p < 0.01; ^*p < 0.05, ns, not significant). (G) Graph shows that overexpression of SIRT1 improves cellular viability during ARV mediated cellular stress. NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. Cellular viability was determined by the CellTiter-Glo (^***p < 0.001; ^**p < 0.01, ^*p < 0.05, ns, not significant). (H,I) Representative microscopy images show that ARV drugs treatment induces cellular hypertrophy and SIRT1 over expression significantly reduces the cellular hypertrophy in ARV drugs treated cells (^*p < 0.05). NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. Drug treated cells were fixed with 4% PFA and stained with actinin antibody (red) and DAPI for nucleus. Cell size was determined by Image J software (National Institute of Health, USA). Graph shows the measurement of cell size (^***p < 0.001, ns, not significant). (J,K) Representative images show that ARV drugs treatment induces cellular ROS level and SIRT1 over expression significantly reduces the ROS level of drug treated cells. Graph shows the quantification of ROS. NRVCs were transfected with adenovirus for 48 h and then treated with ARV drugs for another 24 h. ROS level of the cells were determined by DHE staining. Live imaging was done under fluorescence microscope (^***p < 0.001, ns, not significant).