Differential expression of PDE5 in failing and nonfailing human myocardium

Abstract

Background: Recognizing that inhibitors of phosphodiesterase type 5 (PDE5) are increasingly employed in patients with pulmonary hypertension and right ventricular (RV) failure, we examined PDE5 expression in the human RV and its impact on myocardial contractility.

Methods and results: Tissue extracts from the RV of 20 patients were assayed for PDE5 expression using immunoblot and immunohistochemical staining. Tissues were selected from groups of nonfailing organ donors and transplant recipients with endstage ischemic cardiomyopathy or idiopathic dilated cardiomyopathy. Among dilated cardiomyopathy patients, subgroups with mild or severe RV dysfunction and prior left ventricular assist devices were analyzed separately. Our results showed that PDE5 abundance increased more than 4-fold in the RVs of the ischemic cardiomyopathy compared with the nonfailing group. In dilated cardiomyopathy, PDE5 upregulation was more moderate and varied with the severity of RV dysfunction. Immunohistochemical staining confirmed that cardiac myocytes contributed to the upregulation in the failing hearts. In functional studies, PDE5 inhibition produced little change in developed force in RV trabeculae from nonfailing hearts but produced a moderate increase in RV trabeculae from failing hearts.

Conclusions: Our results showed the etiology- and severity-dependent upregulation of myocyte PDE5 expression in the RV and the impact of this upregulation on myocardial contractility. These findings suggest that RV PDE5 expression could contribute to the pathogenesis of RV failure, and direct myocardial responses to PDE5 inhibition may modulate the indirect responses mediated by RV afterload reduction.

Figure 1. PDE5 Expression in RV tissues of ICM, DCM and NF Human Hearts

Immunoblot analysis was carried out to examine PDE5 protein expression levels in the RV tissue extracts using an anti-PDE5 antibody. In each blot, a group of NF samples was included as a control for the disease groups and abundance of GAPDH was also determined to control for total protein content in each sample. A) DCM with mRVD vs. NF hearts; C) DCM with sRVD without or with antecedent VAD support vs. NF hearts; and E) ICM vs. NF hearts. In B), D) and F) GAPDH normalized PDE5 protein levels, measured by densitometry of protein bands in the blots, were used to compare the relative abundance of the protein in the samples. Sample orders in each group are listed in the Table. Abbreviations: NF-Non-failing, ICM-ischemic cardiomyopathy, DCM-dilated cardiomyopathy, mRVD-mild RV dysfunction, sRVD-severe-RV dysfunction, and VAD-left ventricular assist device.

Figure 2. PDE5 Expression in Different Categories of Failing Human Hearts

A) Right ventricular PDE5 abundance was elevated in heart failure (HF) compared to non-failing controls (NF); Mann-Whitney P = 0.003. B) Comparison of different RV failure subgroups suggests heterogeneity in the degree of PDE5 elevation in the failing RV depending on the degree of RV failure; Kruskal-Wallis P = 0.004. Data are in arbitrary units normalized to GADPH abundance (see Fig. 1). PDE5 expression level in the LV was determined in the same manor as that of RV’s. The GAPDH normalized PDE5 levels in paired RV and LV samples from each heart was plotted in Figure 2C demonstrating a lack of correlation of RV and LV PDE5 expression levels; Spearman rho = 0.04; P = 0.85. See Figure 1 for definitions of abbreviations.

Figure 3. PDE5 Expression in Myocytes of NF, DCM and ICM Human Hearts

Consecutive paraffin embedded RV tissues sections from NF, DCM, and ICM hearts were used to examine PDE5 expression. The sections in A) were incubated first with a rabbit polyclonal anti-PDE5 antibody followed by HRP conjugated goat anti-rabbit secondary antibody and then developed by using DAB-Ni as a substrate to visualize the bonded antibodies. Section B) was processed in parallel with A) but without using the primary anti-PDE5 antibody. An antibody against striated muscle myosin heavy chain was used to replace the anti-PDE5 antibody in A) to reveal the myocyte location in the tissue with DAB as substrate for HRP-conjugated goat anti-mouse IgG secondary antibody C). Hematoxylin nuclear counterstaining was only applied to section in C) to avoid false positives that could be introduced to A) due to the staining color similarity between hematoxylin and DAB-Ni.

Figure 4. Contractile Response to Acute PDE5 Inhibition

Representative twitch tracings are shown in A) non-failing and B) failing human RV trabeculae before and after administration of the PDE5 inhibitor MY-5445. Mean steady-state developed force C), diastolic force D), rate of force development E) and decline F) before and after application of the PDE5 inhibitor MY-5445 in non-failing and failing human right ventricular trabeculae were also determined. Three RV trabeculae from two non failing hearts, and nine trabeculae from four failing hearts (3 ICM and 1 DCM) were used. Non-bolded P-values compare parameters within each patient group before and after PDE5 inhibition, and bolded P-values compare the temporal changes in heart failure with the temporal change in non-failing hearts. Error-bars indicate 95% confidence intervals.