Effects of empagliflozin and target-organ damage in a novel rodent model of heart failure induced by combined hypertension and diabetes

Abstract

Type 2 diabetes mellitus and hypertension are two major risk factors leading to heart failure and cardiovascular damage. Lowering blood sugar by the sodium-glucose co-transporter 2 inhibitor empagliflozin provides cardiac protection. We established a new rat model that develops both inducible diabetes and genetic hypertension and investigated the effect of empagliflozin treatment to test the hypothesis if empagliflozin will be protective in a heart failure model which is not based on a primary vascular event. The transgenic Tet29 rat model for inducible diabetes was crossed with the mRen27 hypertensive rat to create a novel model for heart failure with two stressors. The diabetic, hypertensive heart failure rat (mRen27/tetO-shIR) were treated with empagliflozin (10 mg/kg/d) or vehicle for 4 weeks. Cardiovascular alterations were monitored by advanced speckle tracking echocardiography, gene expression analysis and immunohistological staining. The novel model with increased blood pressure und higher blood sugar levels had a reduced survival compared to controls. The rats develop heart failure with reduced ejection fraction. Empagliflozin lowered blood sugar levels compared to vehicle treated animals (182.3 ± 10.4 mg/dl vs. 359.4 ± 35.8 mg/dl) but not blood pressure (135.7 ± 10.3 mmHg vs. 128.2 ± 3.8 mmHg). The cardiac function was improved in all three global strains (global longitudinal strain - 8.5 ± 0.5% vs. - 5.5 ± 0.6%, global radial strain 20.4 ± 2.7% vs. 8.8 ± 1.1%, global circumferential strain - 11.0 ± 0.7% vs. - 7.6 ± 0.8%) and by increased ejection fraction (42.8 ± 4.0% vs. 28.2 ± 3.0%). In addition, infiltration of macrophages was decreased by treatment (22.4 ± 1.7 vs. 32.3 ± 2.3 per field of view), despite mortality was not improved. Empagliflozin showed beneficial effects on cardiovascular dysfunction. In this novel rat model of combined hypertension and diabetes, the improvement in systolic and diastolic function was not secondary to a reduction in left ventricular mass or through modulation of the afterload, since blood pressure was not changed. The mRen27/tetO-shIR strain should provide utility in separating blood sugar from blood pressure-related treatment effects.

Figure 1

Characteristic of the new animal model. (A) Rat model for hypertensive and diabetic heart failure by breeding a hypertensive rat (mRen27), with an inducible diabetic rat (tetO-shIR) resulting in the hypertensive diabetic rat (mRen27/tetO-shIR). (B) Kaplan–Meier survival analysis of SD, diabetic (tetO-shIR), hypertensive (mRen27) and hypertensive diabetic rats (mRen27/tetO-shIR). At week 9, only 30% of hypertensive diabetic rats (mRen27/tetO-shIR) survived. Survival curves are statistically different by the log-rank test (p < 0.0001). No SD, diabetic (tetO-shIR) and hypertensive (mRen27) rats died before end of study. (C) Tail-cuff mean arterial pressure (MAP) was increased in mRen27 rats compared to SD, tetO-shIR and mRen27/tetO-shIR rats. The hypertensive diabetic rats (mRen27/tetO-shIR) showed significantly elevated MAP compared to SD and tetO-shIR rats. SD and tetO-shIR rats were normotensive. (D) Blood glucose level were significantly higher in tetO-shIR and mRen27/tetO-shIR rats compared to SD and mRen27 rats. 1-way ANOVA, *p < 0.05, ***p < 0.001, ****p < 0.0001; data expressed as mean ± SEM.

Figure 2

Functional analysis by Speckle Tracking Echocardiography. (A) Hypertensive rats (mRen27) showed an increased wall thickness (IVSd + LVPWd) compared to SD, tetO-shIR and mRen27/tetO-shIR rats. Diabetic (tetO-shIR) and hypertensive diabetic rats (mRen27/tetO-shIR) had no thickened cardiac walls. (B) Ejection fraction and (C) fractional shortening were significantly reduced in tetO-shIR, mRen27 and mRen27/tetO-shIR rats compared to SD. (D) Global longitudinal strain was significantly reduced in tetO-shIR, mRen27 and mRen27/tetO-shIR rats compared to SD, as were (E) global radial strain and (F) global circumferential strain. 1-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; data expressed as mean ± SEM.

Figure 3

Study design and outcome after empa treatment. (A) Experimental flow chart of EMPA study design. (B) Kaplan–Meier survival analysis showed no effect of empa treatment on survival and no effect on (C) mean arterial blood pressure. (D) Blood glucose level were significantly reduced by empa during the treatment period. (E) Empa treatment had no effect on the drinking volume. 2-way ANOVA, **p < 0.01, ***p < 0.001; data expressed as mean ± SEM.

Figure 4

Speckle Trackle Echocardiography and gene expression analysis after empa treatment. (A) No effects of empa treatment on wall thickness could be detected. (B) Ejection fraction and (C) fractional shortening were significantly increased by empa. (D) Global longitudinal strain was significantly elevated by empa treatment, as were (E) global radial strain and (F) global circumferential. (G) Heatmap of gene expression values in relation to 18S housekeeper are shown for every single animal. Relative gene expression is illustrated in corresponding color. Statistical outliers are marked with X. There is no change in cardiac atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), fibronectin (Fn) or connective tissue growth factor (Ctgf) mRNA expression due to empa treatment. The ratio of alpha (Mhy6) and beta myosin heavy chain (Mhy7) mRNA expression is increased by treatment. Students t-test, *p < 0.05 **p < 0.01; data expressed as mean ± SEM.

Figure 5

Protein expression after empa treatment. (A) Representative image of ED-1 IHC staining for macrophages in the heart from empa and vehicle-treated rats. The number of macrophages was decreased in the heart of empa treated rats. (B) Representative image of fibronectin IHC staining in the hearts from empa and vehicle-treated rats. Interstitial cardiac fibrosis was estimated as fibronectin-positive area per view field. Heart sections of empa and vehicle-treated rats showed no differences in cardiac interstitial fibrosis. (C) Representative image of collagen I IHC staining in the hearts from empa and vehicle-treated rats. Perivascular fibrosis area was normalized to vessel media cross-sectional area. Heart sections of empa and vehicle-treated rats showed no differences in cardiac perivascular fibrosis. Students t-test, *** p < 0.001; data expressed as mean ± SEM.