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. 2012;7(9):e45243.
doi: 10.1371/journal.pone.0045243. Epub 2012 Sep 21.

Anti-inflammatory and cardioprotective effects of tadalafil in diabetic mice

Amit Varma  1 , Anindita Das, Nicholas N Hoke, David E Durrant, Fadi N Salloum, Rakesh C Kukreja
Affiliations

Anti-inflammatory and cardioprotective effects of tadalafil in diabetic mice

Amit Varma et al. PLoS One. 2012.

Abstract

Background: Insulin resistance impairs nitric oxide (NO) bioavailability and obesity promotes a state of chronic inflammation and damages the vascular endothelium. Phosphodiesterase-5 inhibitors restore NO signaling and may reduce circulating inflammatory markers, and improve metabolic parameters through a number of mechanisms. We hypothesized that daily administration of the PDE-5 inhibitor, tadalafil (TAD) will attenuate inflammation, improve fasting plasma glucose and triglyceride levels, body weight, and reduce infarct size after ischemia/reperfusion injury in obese, diabetic mice.

Methods: Twenty leptin receptor null (db/db) mice underwent treatment with TAD (1 mg/Kg) or 10% DMSO for 28 days. Body weight and fasting plasma glucose levels were determined weekly. Upon completion, hearts were isolated and subjected to 30 min global ischemia followed by 60 min reperfusion in a Langendorff model. Plasma samples were taken for cytokine analysis and fasting triglyceride levels. Infarct size was measured using computer morphometry of tetrazolium stained sections. Additionally, ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia and reoxygenation. Necrosis was determined using trypan blue exclusion and LDH release assay and apoptosis was assessed by TUNEL assay after 1 h or 18 h of reoxygenation, respectively.

Results: Treatment with TAD caused a reduction in infarct size in the diabetic heart (23.2 ± 1.5 vs. 47.8 ± 3.7%, p<0.01, n = 6/group), reduced fasting glucose levels (292 ± 31.8 vs. 511 ± 19.3 mg/dL, p<0.001) and fasting triglycerides (43.3 ± 21 vs. 129.7 ± 29 mg/dL, p<0.05) as compared to DMSO, however body weight was not significantly reduced. Circulating tumor necrosis factor-α and interleukin-1β were reduced after treatment compared to control (257 ± 16.51 vs. 402.3 ± 17.26 and 150.8 ± 12.55 vs. 264 ± 31.85 pg/mL, respectively; P<0.001) Isolated cardiomyocytes from TAD-treated mice showed reduced apoptosis and necrosis.

Conclusion: We have provided the first evidence that TAD therapy ameliorates circulating inflammatory cytokines and chemokines in a diabetic animal model while improving fasting glucose levels and reducing infarct size following ischemia-reperfusion injury in the heart.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Experimental Protocol.
Mice were treated with TAD (1 mg/Kg) or an equivalent volume of DMSO for 28 days. The hearts were, isolated and subjected to 20 min stabilization, followed by 30 min of no-flow global ischemiaand 60 min of reperfusion in a Langendorff model. At the time of sacrifice, serum samples were also collected for inflammatory cytokine and chemokine analysis. A second subset of db/db mice were treated and hearts were collected. The ventricular cardiomyocytes were isolated and subjected to 40 min of simulated ischemia (SI) followed by either 1 h or 18 h of reoxygenation (RO) for evaluation of necrosis and apoptosis as described under Methods.
Figure 2
Figure 2. Effect of TAD on fasting blood glucose and body weight.
The db/db mice were treated with TAD or DMSO as described in Figure 1 A: Fasting blood glucose levels. B: Triglycerides. There was a significant decline in plasma triglyceride levels in the TAD group compared to control. C: Body weight. TAD treatment had significant effect in reducing blood glucose levels.
Figure 3
Figure 3. Effect of TAD on myocardial infarct size.
Treatment with TAD significantly reduced infarct size when compared to the DMSO-treated control group, p = 0.011; n = 6/group.
Figure 4
Figure 4. Cardiac function and coronary flow.
A: Cardiac function presented as the double product of the heart rate and ventricular developed force (percentage of the pre-ischemic baseline) is shown. B: Coronary flow. The post-ischemic cardiac function remained unchanged whereas coronary flow was significantly improved in the TAD group (as a percentage of pre-ischemia baseline, p = 0.005).
Figure 5
Figure 5. Effect of TAD in protection of ventricular cardiomyocytes against necrosis and apoptosis following simulated ischemia/reoxygenation.
Necrosis was determined by trypan-positive (A) and LDH release in the medium (B); apoptosis was determined by TUNEL staining (C). TAD treated group had significantly lower trypan-blue positive cardiomyocytes and LDH release in the medium as compared with control (n = 4/group). Similarly, TAD treatment significantly reduced apoptotic cells when compared to DMSO, p<0.001, n = 4/group. (D): PKG activity. PKG activity was significantly higher in the TAD group, p<0.001, n = 4/group.
Figure 6
Figure 6. Effect of TAD on plasma inflammatory cytokines and chemokines.
TAD treatment caused reductions in the cytokines IL-1β, TNF-α, IFN-γ, and chemokines MIP-1β, MCP-1, and RANTES (p<0.01, compared to DMSO; n = 6/group) as shown in ‘A”. The levels of IL-10 was higher in TAD treated mice than control, (p<0.001; n = 6/group) as shown in ‘B’. No significant changes in the IL-6 levels were observed between the two groups as shown in ‘C’.
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