Cardiomyocyte-specific deletion of TLR4 attenuates angiotensin II-induced hypertension and cardiac remodeling

Abstract

Toll-like receptor 4 (TLR4) is an integral factor in the initiation of the innate immune response and plays an important role in cardiovascular diseases such as hypertension and myocardial infarction. Previous studies from our lab demonstrated that central TLR4 blockade reduced cardiac TLR4 expression, attenuated hypertension, and improved cardiac function. However, the contribution of cardiac specific TLR4 to the development of hypertension and cardiac remodeling is unknown. Therefore, we hypothesized that cardiomyocyte specific knockdown of TLR4 would have beneficial effects on hypertension, cardiac hypertrophy, and remodeling. To test this hypothesis, cardiomyocyte-specific TLR4 knockdown (cTLR4KO) mice were generated by crossing floxed TLR4 mice with Myh6-Cre mice, and subjected to angiotensin II (Ang II, 1 µg/kg/min or vehicle for 14 days) hypertension model. Blood pressure measurements using radio telemetry revealed no differences in baseline mean arterial pressure between control littermates and cTLR4KO mice (103 ± 2 vs. 105 ± 3 mmHg, p > 0.05). Ang II-induced hypertension (132 ± 2 vs. 151 ± 3 mmHg, p < 0.01) was attenuated and cardiac hypertrophy (heart/body weight; 4.7 vs. 5.8 mg/g, p < 0.01) was prevented in cTLR4KO mice when compared with control mice. In addition, the level of myocardial fibrosis was significantly reduced, and the cardiac function was improved in cTLR4KO mice infused with Ang II. Furthermore, cardiac inflammation, as evidenced by elevated gene expression of TNF, IL-6, and MCP-1 in the left ventricle, was attenuated in cTLR4KO mice infused with Ang II. Together, this data revealed a protective role for cardiomyocyte-specific deletion of TLR4 against Ang II-induced hypertension and cardiac dysfunction through inhibition of proinflammatory cytokines.

Keywords: TLR4; angiotensin II; cardiac hypertrophy; hypertension; inflammation.

Figure 1

Cardiac TLR4 expression is increased in angiotensin II-induced hypertension. (A) Gene expression measured by real time RT-PCR shows a significant increase in cardiac TLR4 mRNA after 14 days of angiotensin (Ang) II treatment (n = 9/group, unpaired, 2-tailed t-test, *p < 0.05 vs. saline). (B) Representative images from western blot analysis and quantification reveals an increase in TLR4 protein expression in heart lysates from hypertensive mice (n = 4/group, unpaired, 2-tailed t-test, *p < 0.05 vs. saline controls). (C) Immunofluorescence staining reveals a largely elevated expression of TLR4 (red) in hypertensive mice compared with saline infused controls.

Figure 2

Cardiomyocyte-specific TLR4 deletion attenuates angiotensin II-induced hypertension. (A) Mouse genotyping PCR using genomic DNA extracted from tail snips confirms that cardiomyocyte-specific TLR4 deletion (cTLR4KO) mice contain only the cTLR4KO band (300 bp, samples 4–7), as wild-type (WT) mice do not exhibit this band (samples 1–3.9). ntc, no template control; M, molecular weight marker. (B) Representative western blot image and quantification data indicate a significant reduction in cardiac TLR4 expression in cTLR4KO mice compared to WT mice (n = 4–6/group, unpaired, 2-tailed t-test, *p < 0.05 vs. WT + Saline). (C) Time course of blood pressure measurement using radiotelemetry showing angiotensin II (Ang II) infusion significantly increased the mean arterial pressure (MAP) in WT mice, however this effect in cTLR4KO mice was significantly attenuated (n = 10, repeated measures two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). (D) MAP at day 14 of Ang II-infusion (n = 10, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II).

Figure 3

Cardiomyocyte-specific TLR4 deletion abrogates angiotensin II-induced cardiac hypertrophy. (A) Cardiac hypertrophy was measured by calculating heart weight/body weight (HW/BW) ratio. Ang II increased heart weight to body weight in WT mice, but this effect was blunted in cTLR4KO mice (n = 10, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). (B) To visualize cardiomyocyte hypertrophy, we used wheat germ agglutinin immunofluorescence staining to measure cardiomyocyte cross-sectional area (µM²). Quantification data showing a significant increase in cardiomyocyte cross-sectional area in WT mice treated with Ang II, which was attenuated in cTLR4KO mice (n = 4 mice/group, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). (C) Representative heart images showing immunofluorescence staining with wheat germ agglutinin. (D) Real time RT-PCR was used to measure gene expression of natriuretic peptides ANP, BNP and β-MHC. In hypertensive WT mice there is a significant elevation in these natriuretic peptides, which is attenuated in cTLR4KO mice (n = 9, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II).

Figure 4

Cardiac-specific TLR4 deletion attenuates angiotensin II-induced cardiac fibrosis. (A) Gene expression of profibrotic markers (Collagen I, Collagen III, and CTGF) in the left ventricle tissue was measured using real time RT-PCR. Increased profibrotic gene expression was observed in hypertensive WT mice, but this increase was attenuated in cTLR4KO mice with Ang II-infusion. (B) Changes in collagen deposition leading cardiac fibrosis was analyzed by Masson's Trichrome staining. Mean interstitial collagen content in the left ventricular sections indicate an increased fibrosis in WT mice infused with Ang II. This increase in collagen deposition in the heart was attenuated in cTLR4KO mice receiving Ang II infusion. (n = 3 mice/group, Two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). (C) Representative images of left ventricle sections stained with Masson's Trichrome staining indicating collagen fibers (blue in color).

Figure 5

Cardiomyocyte-specific TLR4 deletion attenuates angiotensin II-induced cardiac dysfunction. Echocardiography was used to evaluate cardiac function following Ang II infusion in hypertensive mice. In WT mice, Ang II significantly increased (A) interventricular septal thickness end diastole (IVSd) and (B) left ventricular posterior wall thickness end diastole (LVPWTd), while decreasing (C) fractional shortening %. However, these changes were reversed in cTLR4KO mice with Ang II infusion. (n = 6 mice/group, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II).

Figure 6

Cardiomyocyte-specific TLR4 knockdown reduces mRNA expression of pro-inflammatory markers. (A) Angiotensin II (Ang II) infusion for 2 weeks significantly increased TNF, IL-1β, IL-6, and MCP-1 mRNA expression in the left ventricle cardiac tissue indicating an increased inflammatory state. This effect was attenuated in cTLR4KO mice (n = 9, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). ELISA analysis of left ventricular lysates showed that Ang II increased (B) TNF and (C) IL-1β in WT mice but these increases were blunted in cTLR4KO mice (n = 6–9, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II). (D) NF-κB binding activity measured in the left ventricle nuclear extracts exhibited a significantly increased cardiac NF-κB p65 activity in WT mice but not in cTLR4KO mice with Ang II-infusion (n = 6, two-way ANOVA, *p < 0.05 vs. WT + Saline, †p < 0.05 vs. WT + Ang II).