Hypertension Is a Conditional Factor for the Development of Cardiac Hypertrophy in Type 2 Diabetic Mice

<div><p>Background</p><p>Type 2 diabetes is frequently associated with co-morbidities, including hypertension. Here we investigated if hypertension is a critical factor in myocardial remodeling and the development of cardiac dysfunction in type 2 diabetic db/db mice.</p><p>Methods</p><p>Thereto, 14-wks-old male db/db mice and non-diabetic db/+ mice received vehicle or angiotensin II (AngII) for 4 wks to induce mild hypertension (n = 9–10 per group). Left ventricular (LV) function was assessed by serial echocardiography and during a dobutamine stress test. LV tissue was subjected to molecular and (immuno)histochemical analysis to assess effects on hypertrophy, fibrosis and inflammation.</p><p>Results</p><p>Vehicle-treated diabetic mice neither displayed marked myocardial structural remodeling nor cardiac dysfunction. AngII-treatment did not affect body weight and fasting glucose levels, and induced a comparable increase in blood pressure in diabetic and control mice. Nonetheless, AngII-induced LV hypertrophy was significantly more pronounced in diabetic than in control mice as assessed by LV mass (increase +51% and +34%, respectively, p<0.01) and cardiomyocyte size (+53% and +31%, p<0.001). This was associated with enhanced LV mRNA expression of markers of hypertrophy and fibrosis and reduced activation of AMP-activated protein kinase (AMPK), while accumulation of Advanced Glycation End products (AGEs) and the expression levels of markers of inflammation were not altered. Moreover, AngII-treatment reduced LV fractional shortening and contractility in diabetic mice, but not in control mice.</p><p>Conclusions</p><p>Collectively, the present findings indicate that type 2 diabetes in its early stage is not yet associated with adverse cardiac structural changes, but already renders the heart more susceptible to hypertension-induced hypertrophic remodeling.</p></div

Cardiac phospho-AMPK levels are decreased in hypertensive, diabetic mice.

<p>Western blot analysis of LV pAMPK levels in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks. In the top panel, the quantification of LV pAMPK levels from 4–6 animals per group is shown. Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM. In the bottom panel, representative examples of pAMPK and GAPDH signals are shown for 2 animals per group. * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and # refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Echo pulse-doppler determination of changes in cardiac dimensions and cardiac performance in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or angiotensin II (Ang) for 4 weeks.

<p>Data are expressed as means ± S.E.M. * refers to effect of angiotensin II in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and ^# refers to effect of diabetes in vehicle-treated and Angiotensin II-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Effect of AngII-induced hypertension on cardiac function in non-diabetic and diabetic mice.

<p>Left ventricular fractional shortening (A) and ejection fraction (B) as determined by echocardiography in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks (age 18 weeks). Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM (n  =  7–9 per group). * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05) and ^# refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Left ventricular mRNA levels of genes signifying changes in cardiac hypertrophy, metabolism, fibrosis, inflammation and AGE signalling in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks.

<p>Data are expressed relative to non-diabetic vehicle-treated mice as mean±SEM. Abbreviations are explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085078#pone.0085078.s001" target="_blank">Table S1</a>. * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and ^# refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Effect of hypertension on cardiac AGE metabolism in non-diabetic and diabetic mice.

<p>Cardiac levels of GLO-1 protein (A), GLO-1 activity (B), CML (C), CEL (D), and MG-H1 (E) in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks (age 18 weeks). Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM (n  =  4–6 per group) * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and ^# refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Effect of AngII treatment on blood pressure in non-diabetic and diabetic mice.

<p>Body weight (A), blood glucose levels (B) and systolic arterial blood pressure (Pa_syst; C) of non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) just prior to treatment (age 14 weeks) and at age 16 and 18 weeks. Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM (n  =  7–11 per group). * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and # refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

Effect of AngII-induced hypertension on left ventricular function during dobutamine stimulation.

<p>Hemodynamic analysis of left ventricular function in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks (age 18 weeks), at baseline and during dobutamine stimulation. A) heart rate, B) peak systolic left ventricular pressure (LVSP), C) maximal positive pressure development (+dP/dt_max) and D) maximal negative pressure development (–dP/dt_max). Data are analyzed by two-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM (n  =  7–9 per group). * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and ^# refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p

AngII-induced hypertension enhances cardiac hypertrophy in diabetic mice.

<p>Cardiac structural remodeling in non-diabetic (Cn) and diabetic (DM) mice treated with vehicle or AngII (Ang) for 4 weeks. Left ventricle weight/Tibia length (LV/TL – Panel A) and diastolic LV wall thickness (WT – Panel B). Representative pictures depicting cardiomyocyte size (C) and extent of fibrosis (D). Quantification of cardiomyocyte cross-sectional surface area (E) and collagen fraction (F). Data are analyzed by one-way ANOVA with Bonferroni post-hoc testing and expressed as mean±SEM (n  =  7–9 per group). * refers to effect of AngII in non-diabetic and diabetic mice (* P<0.05, ** P<0.01, *** P<0.001) and ^# refers to effect of diabetes in vehicle-treated and AngII-treated mice (# P<0.05, ## P<0.01, ### P<0.001).</p