Urocortin 2 Gene Transfer Reduces the Adverse Effects of a Western Diet on Cardiac Function in Mice

Abstract

Diabetes mellitus is associated with increased risk of heart failure. It has been previously demonstrated in mice that a single injection of adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal in models of insulin resistance and improves the function of the failing heart. The present study tested the hypothesis that UCn2 gene transfer would reduce diabetes-related left ventricular (LV) dysfunction. Eight-week-old C57BL6 male mice were fed a Western diet (WD; 45% fat, 35% carbohydrate) for 40 weeks. At week 30, they received saline or AAV8.UCn2 (2 × 10¹³ genome copies/kg) via intravenous injection. Ten weeks after gene transfer, fasting blood glucose, glucose tolerance, and cardiac function were measured via echocardiography and in vivo measurement of LV contractile function, and the results were compared to those of mice fed normal chow (NC; 10% fat; 70% carbohydrate). The contents of key LV signaling proteins were also measured to probe mechanisms. WD increased 12 h fasting glucose (WD: 190 ± 11 mg/dL, n = 8; NC: 105 ± 12 mg/dL, n = 7; p = 0.0004). WD tended to reduce LV peak +dP/dt (p = 0.08) and LV peak -dP/dt (p = 0.05). LV ejection fraction was unchanged. Among WD-fed mice, UCn2 gene transfer reduced 12 h fasting glucose (WD-UCn2: 149 ± 6 mg/dL, n = 8; WD-Saline: 190 ± 11 mg/dL, n = 8; p = 0.012), increased LV peak +dP/dt (p < 0.001) and LV peak -dP/dt (p = 0.013), and reduced Tau (p < 0.02), indicating beneficial effects on systolic and diastolic LV function. In addition, among WD-fed mice, UCn2 gene transfer increased LV ejection fraction (p < 0.005) and the velocity of circumferential fiber shortening (p = 0.0005). Finally, a reduction was seen in fatty infiltration of the liver in WD-fed mice that had received UCn2 gene transfer. LV samples from WD-UCn2 mice showed increased phosphorylation of the protein kinase A catalytic domain (p = 0.03). In conclusion, UCn2 gene transfer increased LV systolic and diastolic function and reduced blood glucose in mice with diabetes-related LV dysfunction, indicating that UCn2 gene transfer may be of potential therapeutic benefit.

Keywords: adeno-associated virus; gene therapy; insulin resistance; type 2 diabetes.

Figure 1.

Experimental protocol, adeno-associated virus 8 encoding urocortin 2 (AAV8.UCn2) plasma UCn2 after gene transfer. (A) Experimental protocol. Eight-week-old C56BL6 mice were fed a Western diet (WD; fat: 45%, carbohydrate 35%, protein 20%) for 40 weeks. At 30 weeks, mice were randomized to receive UCn2 gene transfer or saline intravenously (i.v.). Ten weeks later (week 40 of WD), animals underwent assessment of glucose disposal (fasting glucose and glucose tolerance testing) and assessment of left ventricular (LV) function. Tissues were collected for biochemical assessment. (B) AAV8.mUCn2 vector map. ITR, inverted terminal repeat; CMV.en, human cytomegalovirus enhancer; mUCn2, murine urocortin 2; RbGpA, rabbit beta-globin poly A. (C) Plasma UCn2 concentration. Ten weeks after delivery of AAV8.UCn2 (2 × 10¹³ genome copies/kg, i.v.) or saline (i.v.), plasma was obtained to measure UCn2 concentration. Plasma UCn2 was increased 20-fold compared to saline-treated mice. Data are the mean ± standard error (SE). p-values are from unpaired Student's t-tests (two-tailed). Color images are available online.

Figure 2.

WD-fed mice: effects of UCn2 gene transfer on body, LV, and liver weight and glucose disposal. (A) Body weight: mice gained weight after 40 weeks on the WD (NC-Con vs. WD-Sal; p < 0.0001). However, no group difference was seen in body weight 10 weeks after UCn2 gene transfer. (B) LV weight: mice fed on the WD for 40 weeks had higher LV–tibial length ratios (NC-Con vs. WD-Sal; p = 0.03). However, no group difference was seen in LV-tibial length ratio 10 weeks after UCn2 gene transfer. (C) Liver weight: mice fed on the WD for 40 weeks had higher liver–tibial length ratios (NC-Con vs. WD-Sal; p = 0.0006). However, no group difference was seen in liver–tibial length ratio 10 weeks after UCn2 gene transfer. (D) Fasting blood glucose: Mice fed on the WD for 40 weeks had higher 12 h fasting blood glucose (NC-Con vs. WD-Sal; p = 0.0004). However, 10 weeks after UCn2 gene transfer, fasting glucose was reduced (WD-Sal vs. WD-UCn2; p = 0.0004). (E) Glucose tolerance test: mice fed on the WD for 40 weeks showed impaired glucose disposal (NC-Con vs. WD-Sal; p < 0.003). However, 10 weeks after UCn2 gene transfer, glucose disposal tended to improve (WD-Sal vs. WD-UCn2; p < 0.06). Symbols indicate mean values; bars indicate SE; Con, Control; Sal, Saline; UCn2, urocortin 2; NC, Normal chow; WD, Western diet. p-Values are from unpaired Student's t-tests (two-tailed), with Bonferroni correction (D and E). GTT analysis (E) was performed by area under the curve analysis. Color images are available online.

Figure 3.

Echocardiography. (A) Ejection Fraction (EF). Mice fed on the WD for 40 weeks showed no change in EF (NC-Con vs. WD-Sal; p = not significant [n.s.]). However, 10 weeks after UCn2 gene transfer, EF was increased (WD-Sal vs. WD-UCn2; p < 0.005). (B) Velocity of circumferential fiber shortening corrected for heart rate (VCFc). Mice fed on the WD for 40 weeks showed no change in VCFc, an estimate of contractile function (NC-Con vs. WD-Sal; p = n.s.). However, 10 weeks after UCn2 gene transfer, VCFc increased (WD-Sal vs. WD-UCn2; p = 0.0005). (C) Heart rate. There were no group differences in heart rate. (D and E) LV end diastolic diameter (EDD) and end systolic diameter (ESD). Mice fed on the WD for 40 weeks showed no change in EF (NC-Con vs. WD-Sal; p = n.s.). However, 10 weeks after UCn2 gene transfer, EDD and ESD were lower (WD-Sal vs. WD-UCn2; EDD, p = 0.01; ESD, p = 0.004). Symbols indicate mean values; bars indicate SE. p-Values are from unpaired Student's t-tests (two-tailed). Color images are available online.

Figure 4.

WD-fed mice: effects of UCn2 gene transfer on LV function. (A) LV peak +dP/dt. The peak rate of LV pressure development (+dP/dt), a measure of contractile function, tended to be lower after 40 weeks on the WD (NC-Con vs. WD-Sal; p = 0.08). However, 10 weeks after UCn2 gene transfer, LV +dP/dt was higher and attained normal levels (WD-Sal vs. WD-UCn2; p < 0.001). (B) LV peak –dP/dt. The peak rate of LV pressure decay (–dP/dt), a measure of LV relaxation, tended to be lower after 40 weeks on the WD (NC-Con vs. WD-Sal; p = 0.05). However, 10 weeks after UCn2 gene transfer, LV –dP/dt was higher and attained normal levels (WD-Sal vs. WD-UCn2; p = 0.013). (C) Tau. The time constant of –dP/dt, Tau, was unchanged after 40 weeks on the WD (NC-Con vs. WD-Sal; p = n.s.) but was more rapid 10 weeks after UCn2 gene transfer (WD-Sal vs. WD-UCn2; p < 0.02). (D and E) LV pressure and heart rate. There were no group differences in LV developed pressure or heart rate after 40 weeks on the WD (NC-Con vs. WD-Sal; p = n.s.); 10 weeks after UCn2 gene transfer, no group differences in LV pressure or heart rate were seen in WD-fed mice. (F) LV end diastolic pressure (EDP). LV EDP was unchanged after 40 weeks on the WD (NC-Con vs. WD-Sal; p = n.s.). However, 10 weeks after UCn2 gene transfer, LV EDP tended to be lower, consistent with improved LV function (WD-Sal vs. WD-UCn2; p < 0.06). Symbols indicate mean values; bars indicate SE. p-Values are from unpaired Student's t-tests (two-tailed), with Bonferroni correction (A and B). Color images are available online.

Figure 5.

Histological analysis and immunoblotting. (A) Liver fatty infiltration. Area of lipid droplet in liver tissue sections stained with hematoxylin and eosin was measured. UCn2 gene transfer in WD-fed mice decreased liver fatty infiltration compared to the WD-Sal group (p < 0.008). (B) Fibrosis in liver and LV. Liver and LV tissue sections were stained with Masson's trichrome. The fibrotic area in liver was increased in WD-fed mice receiving saline compared to NC-fed mice (p = 0.02), and UCn2 gene transfer had no effect. No group differences were found in LV fibrosis. (C) Immunoblotting. The content of signaling proteins in LV homogenates was examined by immunoblotting. UCn2 gene transfer increased phosphorylation of PKA catalytic subunit in WD-fed mice compared to WD-fed mice receiving saline. Scale bars: 100 μm. Data are the mean ± SE. p-Values are from unpaired Student's t-tests (two-tailed), with Bonferroni correction. Color images are available online.