HDAC inhibition attenuates inflammatory, hypertrophic, and hypertensive responses in spontaneously hypertensive rats

Abstract

Reactive oxygen species and proinflammatory cytokines contribute to cardiovascular diseases. Inhibition of downstream transcription factors and gene modifiers of these components are key mediators of hypertensive response. Histone acetylases/deacetylases can modulate the gene expression of these hypertrophic and hypertensive components. Therefore, we hypothesized that long-term inhibition of histone deacetylase with valproic acid might attenuate hypertrophic and hypertensive responses by modulating reactive oxygen species and proinflammatory cytokines in SHR rats. Seven-week-old SHR and WKY rats were used in this study. Following baseline blood pressure measurement, rats were administered valproic acid in drinking water (0.71% wt/vol) or vehicle, with pressure measured weekly thereafter. Another set of rats were treated with hydralazine (25 mg/kg per day orally) to determine the pressure-independent effects of HDAC inhibition on hypertension. Following 20 weeks of treatment, heart function was measured using echocardiography, rats were euthanized, and heart tissue was collected for measurement of total reactive oxygen species, as well as proinflammatory cytokine, cardiac hypertrophic, and oxidative stress gene and protein expressions. Blood pressure, proinflammatory cytokines, hypertrophic markers, and reactive oxygen species were increased in SHR versus WKY rats. These changes were decreased in valproic acid-treated SHR rats, whereas hydralazine treatment only reduced blood pressure. These data indicate that long-term histone deacetylase inhibition, independent of the blood pressure response, reduces hypertrophic, proinflammatory, and hypertensive responses by decreasing reactive oxygen species and angiotensin II type1 receptor expression in the heart, demonstrating the importance of uncontrolled histone deacetylase activity in hypertension.

Figure 1

Effects of VPA treatment on MAP. VPA attenuated the increase in MAP seen in untreated SHR rats. VPA: valproic acid, MAP: mean arterial pressure. n=7–8 in each group, *P<0.05 vs WKY, #P<0.05 vs SHR.

Figure 2

Effects of VPA treatment on cardiac hypertrophy. VPA attenuated ventricular wall thickness as assessed by echocardiography (2A) during both diastole and systole. VPA also attenuated the HW/BW (indicating reduction in cardiac hypertrophy) and LW/BW (indicating reduction in lung edema due to reduced cardiac function) ratio in SHR rats (2B). SHR rats had increased levels of ANP and Collagen IV mRNA expression which were reduced with VPA treatment (2C). Graphs expressed as fold change versus WKY rats. VPA reduced cardiac fibrosis versus SHR rats as indicated by picrosirius red staining collagen versus total area (2D). Stained slides are representative of typical results for each group (n=3/group). VPA: valproic acid, LVPWT: left ventricular parietal wall thickness, d: diastole, s: systole, HW: heart weight, LW: lung weight, BW: body weight, ANP: atrial natriuretic peptide. n=7–8 in each group, *P<0.05 vs SHR, #P<0.05 vs WKY.

Figure 3

Effect of VPA on AT1-R mRNA and protein expression. Untreated SHR rats showed higher mRNA (3B) and protein expression (3A) levels of the AT1-R in the LV when compared to WKY controls. SHR+VPA attenuated this increase. Western blot protein expression bands were normalized to GAPDH. VPA: valproic acid, AT1-R: angiotensin II type 1 receptor. n=7–8 in each group, *P<0.05 vs SHR.

Figure 4

Effects of VPA on HDAC activity as assessed through a colorimetric detection assay and NFκB activity and expression via EMSA and real-time RT-PCR, respectively, in the LV. HDAC activity was elevated in both WKY and SHR rats versusWKY+VPA and SHR+VPA rats (4A). Untreated SHR HDAC activity was elevated versus untreated WKY rats. Untreated SHR rats had an increased activity (as determined by EMSA) and mRNA expression of NF-κB which was attenuated in SHR+VPA (4B). VPA: valproic acid, HDAC: histone deacetylase, NF-κB: nuclear factor-kappaB, EMSA: Electrophoretic Mobility Shift Assay. n=7–8 in each group, *P<0.05 vs SHR.

Figure 5

Effects of VPA treatment on TNF and IL-1β protein and mRNA expression. SHR rats had notably increased protein staining of TNF and IL-1β in the LV when compared to WKY rats as shown through immunohistochemical staining (5A). This staining increase was markedly reduced in SHR+VPA. Images shown represent results observed in preparations from 4 to 6 rats. Untreated SHR rats had increased levels of PIC expression compared to WKY rats (5B). SHR+VPA reduced this increased expression in the LV tissue. VPA: valproic acid, TNF: tumor necrosis factor, IL-1β: interleukin-1beta, IL-6: interleukin-6, PIC: pro-inflammatory cytokines. n=7–8 in each group, *P<0.05 vs SHR.

Figure 6

Effects of VPA treatment on tROS and gp91phox in the LV. SHR rats had increased tROS levels as assessed by EPR. These levels were normalized in SHR+VPA (6A). SHR rats also showed increased mRNA (6B) and protein (6C) expression of gp91phox, the catalytic subunit of NADPH oxidase, in the LV versus WKY rats. This was attenuated in SHR+VPA. Immunofluorescence of LV cardiomyocytes shows increased presence of gp91phox in SHR rats versus SHR+VPA. VPA: valproic acid, ROS: reactive oxygen species, EPR: electron paramagnetic spin resonance. n=7–8 in each group, *P<0.05 vs SHR.