Co-administration of isoprenaline and phenylephrine induced a new HFrEF mouse model through activation of both SNS and RAAS

Figures

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  Figure 1. Experimental design and a comparation among 3 groups in terms of systolic BP, heart rate and HW/TL. (A) Experimental design: mouse model was constructed using subcutaneous implantation of micro-osmotic pump, divided into control group, ISO (30 mg/kg/day) group and ISO/PE (30 mg/kg/day) group; 6 mice in each group. Cardiac ultrasound monitoring and blood pressure monitoring were performed before and 3, 7 and 14 days after surgery, respectively; mice were executed at 1 and 2 weeks, respectively, and the heart-to-tibial length (HW/TL) ratio was measured. (B) Systolic pressure and (C) heart rate both monitored by blood pressure monitors through the tail root. (D) HW/TL ratio was obtained by measuring heart weight and tibia length after execution of mice on days 0, 3, 7, and 14 of modeling. Individual data, mean ± SEM of n = 6 animals are shown. P values were determined by repeated measures one-way ANOVA. *P < 0.05.

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  Figure 2. Echocardiographic evaluation of left ventricle EF and FS. (A) Echocardiography analysis of cardiac function in control (NaCl), isoprenaline (ISO) and isoprenaline/phenylephrine (ISO/PE) treated mice. Showing ultrasound results after 1 week and 2 weeks of dosing. (B–E) Left ventricular ejection fraction (LVEF) and short-axis fractional shortening (% FS) results, statistical analysis was performed at 1 week and 2 weeks after drug administration as monitoring points. Individual data, mean ± SEM of n = 6 animals are shown. P values were determined by repeated measures one-way ANOVA. *P < 0.05, ***P < 0.001, ****P < 0.0001.

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  Figure 3. Contribution of RAAS activation in ISO/PE induced hypertrophic growth. (A) Mean BP measured by tail-cuff 24 h after minipump implantation and 48 h our losartan was added to the drinking water. (B) Percent increase in heart weight/tibia length (HW/TL) ratios renormalized to naCl. (C) Percent increase in lung weight/tibia length (LW/TL) ratios renormalized to NaCl. (D–F) Gene regulation (qPCR, whole heart tissue) in mice subjected to chronic perfusion of either ISO or ISO/PE (30 mg/kg/day). Values are shown as mean ± SEM; one-way-ANOVA, plus Sidak post-test, *p < 0.05 vs. respective controls, #p < 0.05 vs. ISO, $p < 0.05 vs. ISO/PE.

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  Figure 4. Cardiac hypertrophy growth and collagen deposition. (A,B) The mice were drug-molded at 1 and 2 weeks (n = 6 in each group), and the hearts were harvested for paraffin-embedded sections and stained with HE and Masson. (C,D) Using Image J to quantify the degree of fibrosis in the Masson stain, the ISO/PE group exhibited a significant fibrotic phenotype. Data are presented in the form of mean ± SEM. P values were determined by repeated measures one-way ANOVA. *P < 0.05, **P < 0.01, ****P < 0.0001.

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  Figure 5. HF associated genes were significantly upregulated in the ISO/PE group. (A,B) Two-week post-modeling cardiac tissues were subjected to qPCR (n = 6 in each group). Fibrosis-related genes Postn and Col1a1; (C,D) hypertrophy-related gene Myh7 and heart failure marker gene Nppb were clearly upregulated in the ISO/PE group compared with the ISO and control groups. (E) The expression of the inflammation-related gene IL-6 tended to be more significantly upregulated in the ISO group. Data are presented in the form of mean ± SEM. P values were determined by repeated measures one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 6. ISO/PE group showed α 1-activation induced phosphorylation of ERK1/2. (A–D) The phosphorylation and total protein levels of ERK, JNK and p38, the major protein components of the MAPKs family, were measured after lysis of the tissues using lysis solutions with protease inhibitors and phosphatase inhibitors in the 1-week and 2-week modeling groups (n = 6 in each group), respectively. Vinculin was used as an internal reference. Data are presented in the form of mean ± SEM. P values were determined by repeated measures one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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  Figure 7. Gene expression changes were more significant in the ISO/PE group. (A,B) Myocardial tissues from the control, ISO and ISO/PE groups were sequenced for transcriptomic sequencing (n = 3 in Control group, n = 4 in ISO group, n = 3 in ISO/PE group); a two-by-two comparison between the three groups showed that the ISO/PE group showed the most significant up-regulation and down-regulation of gene expression compared with the control group, followed by the inter-group comparison between the ISO/PE and ISO groups, followed by the difference between the ISO and control groups. (C,D) Compared to the control and ISO groups, the hypertrophy gene Myh7 and the heart failure marker gene Nppb were clearly upregulated in the ISO/PE group.

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  Figure 8. KEGG terms identified by pathway enrichment analysis of the indicated subgroups. (A) KEGG gene enrichment analysis of the differential genes among the control, ISO and ISO/PE groups revealed that the differential genes were heavily enriched in RAAS, AMPKs and other pathways. (B) Gene list taken from the indicated GO terms summarized as myocardial hypertrophy, fibrosis, oxidative stress and lipid metabolism.