Overexpression of endothelial β3 -adrenergic receptor induces diastolic dysfunction in rats

Abstract

Aims: Diastolic dysfunction is common in cardiovascular diseases, particularly in the case of heart failure with preserved ejection fraction. The challenge is to develop adequate animal models to envision human therapies in the future. It has been hypothesized that this diastolic dysfunction is linked to alterations in the nitric oxide (^• NO) pathway. To investigate this issue further, we investigated the cardiac functions of a transgenic rat model (Tgβ₃ ) that overexpresses the human β₃ -adrenoceptor (hβ₃ -AR) in the endothelium with the underlying rationale that the ^• NO pathway should be stimulated in the endothelium.

Methods and results: Transgenic rats (Tgβ₃ ) that express hβ₃ -AR under the control of intercellular adhesion molecule 2 promoter were developed for a specific expression in endothelial cells. Transcriptomic analyses were performed on left ventricular tissue from 45-week-old rats. Among all altered genes, we focus on ^• NO synthase expression and endothelial function with arterial reactivity and evaluation of ^• NO and O₂ ^•- production. Cardiac function was characterized by echocardiography, invasive haemodynamic studies, and working heart studies. Transcriptome analyses illustrate that several key genes are regulated by the hβ₃ -AR overexpression. Overexpression of hβ₃ -AR leads to a reduction of Nos3 mRNA expression (-72%; P < 0.05) associated with a decrease in protein expression (-19%; P < 0.05). Concentration-dependent vasodilation to isoproterenol was significantly reduced in Tgβ₃ aorta (-10%; P < 0.05), while ^• NO and O₂ ^•- production was increased. In the same time, Tgβ₃ rats display progressively increasing diastolic dysfunction with age, as shown by an increase in the E/A filing ratio [1.15 ± 0.01 (wild type, WT) vs. 1.33 ± 0.04 (Tgβ₃ ); P < 0.05] and in left ventricular end-diastolic pressure [5.57 ± 1.23 mmHg (WT) vs. 11.68 ± 1.11 mmHg (Tgβ₃ ); P < 0.05]. In isolated working hearts, diastolic stress using increasing preload levels led to a 20% decrease in aortic flow [55.4 ± 1.9 mL/min (WT) vs. 45.8 ± 2.5 mL/min (Tgβ₃ ); P < 0.05].

Conclusions: The Tgβ₃ rat model displays the expected increase in ^• NO production upon ageing and develops diastolic dysfunction. These findings provide a further link between endothelial and cardiac dysfunction. This rat model should be valuable for future preclinical evaluation of candidate drugs aimed at correcting diastolic dysfunction.

Keywords: Diastolic dysfunction; Endothelium; HFpEF; Nitric oxide production; Rat model; Transcriptome; β3-Adrenoceptor.

Figure 1

β‐Adrenergic receptor transcript expression. Transcript levels of hβ₃‐AR mRNA (A) and rat β₁‐AR, β₂‐AR, and β₃‐AR mRNA (B), in wild‐type (WT) (n = 8) and Tgβ₃ (n = 13) rats. Reverse transcription PCR assays were performed on whole heart, and de‐endothelialized or intact aorta tissue extracts (C), and isolated cardiomyocytes (D). hadrb3, human β₃‐AR. Data are expressed as mean ± standard error of the mean, ^* P < 0.05.

Figure 2

Transcriptome analysis on the left ventricle. Hierarchical clustering of microarray data based on the 241 transcripts differentially expressed between Tgβ₃ and wild‐type (WT) rats (A). Gene expression is presented as a coloured matrix, where each row represents a gene and each column represents a sample. Green, black, and red correspond to lower values, median values, and higher values, respectively. (B–E) Gene set enrichment analysis enrichment plots and heat maps based on gene expression profiles from Tgβ₃ and WT rats. Only genes from the gene set enrichment analysis core enrichment are displayed in the heat maps.

Figure 3

Changes in NOS transcripts and protein expression and phosphorylation on the left ventricle. nNOS (Nos1) (A), iNOS (Nos2) (B), and nNOS (Nos3) (C) mRNA levels in wild‐type (WT) (n = 8) and Tgβ₃ (n = 13) rats. Western blot analysis was performed to study total expression of nNOS (D), iNOS (E), and eNOS (F) and level of phosphorylated Ser1177 eNOS (G). Data are expressed as mean ± standard error of the mean. ^* P < 0.05.

Figure 4

Vascular reactivity analysis. Concentration–response curves to isoproterenol were obtained by measuring contractility of wild‐type (WT) (○, n = 4–5) and Tgβ₃ (■, n = 4–5) aorta (A), in the presence (plain line) or absence (dotted line) of endothelium. ^•NO (B) and O₂ ^•− (C) production were evaluated by electron paramagnetic resonance spectroscopy in aorta at 45 weeks of age in WT (n = 4) and Tgβ₃ (n = 4). Data are expressed as mean ± standard error of the mean. ^* P < 0.05.

Figure 5

Cardiac function. Evolution of systolic function (A) and diastolic function (B) between 15 and 45 weeks measured by echocardiography. Evaluation of the area of the left atrium at 45 weeks by echocardiography (C). Left ventricular pressure measurements at 45 weeks of wild‐type (WT) (n = 7) and Tgβ₃ (n = 8) rats (D). Data are expressed as mean ± standard error of the mean. ^* P < 0.05.

Figure 6

Isoproterenol concentration–response curve in isolated working heart. dP/dt _max, dP/dt _min (A), cardiac output (B), and aortic flow (C) were measured on wild‐type (WT) (○, n = 8) and Tgβ₃ (■, n = 8). The effects of an afterload increase on cardiac function of WT (○, n = 8) and Tgβ₃ (■, n = 8) rats were evaluated on dP/dt _max and dP/dt _min rate (D), cardiac output (E), and aortic flow (F). The effects of a preload increase on cardiac function of WT (○, n = 12) and Tgβ₃ (■, n = 13) rats were evaluated through the study of dP/dt _max and dP/dt _min rate (G), cardiac output (H), and aortic flow (I). BL, baseline. Data are expressed as mean ± standard error of the mean. ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001 for WT vs. Tgβ₃.