Differential regulation of PDE5 expression in left and right ventricles of feline hypertrophy models

Abstract

Background: Though long known to affect smooth muscle biology, recent studies indicate that phosphodiesterase 5 (PDE5) is also expressed in myocardium. Recognizing that the regulation of PDE5 in hypertrophy is not well understood, we assessed the response of PDE5 expression and the level of cGMP-dependent kinase I (cGKI) in the left and right ventricles of feline hypertrophy models.

Methodology/principal findings: Using a cDNA library of feline aortic smooth muscle cells, we identified and cloned PDE5 cDNA for the first time in this species. The sequence shares 98% identity with its human orthologue at the amino acid level. E. coli expression of the cloned allele allowed selection of antibodies with appropriate specificity, facilitating the analysis of PDE5 expression in feline models created by selective proximal aortic (Ao) or pulmonary artery (PA) banding that resulted in hypertrophy of the left ventricle (LV) and right ventricle (RV), respectively. We demonstrated that PDE5 expression responded differentially with a decreased expression in the LV and an increased expression in the RV in the Ao-banded model. Similarly, in the PA-banded model, LV showed reduced expression while the RV expression was unaltered. In addition, the expression of cGKI was significantly decreased in the RV of Ao-banded group, correlating inversely with the increase in PDE5 expression.

Conclusions/significance: The differential regulation of PDE5 and cGKI expression suggests that the mechanisms involved in hypertrophy could be different in RV vs. LV. Reciprocal PDE5 and cGKI expression in the RV of Ao-banded model suggests functional significance for PDE5 up-regulation.

Figure 1. Sequence and Structural features of feline PDE5.

(a) Similarity between feline PDE5 and PDE5s from other species. The feline PDE5 sequence was obtain from the translation of the cloned cDNA sequence. Sequences of PDE5 from other species were obtained from GenBank. Multi-sequence alignments were calculated using Clustal W. The % identities between feline and other species are in bold. (b) Domain structure of the feline PDE5 protein. GAF indicates cGMP binding domain, and HDc the phosphohydrolase catalytic domain. Structure prediction was performed using the SMART software. (c) Alignment of protein sequences from the N-terminal region of feline and human PDE5 variants. The shaded area indicates identical residues and the residues in feline PDE5 that are different from those of human variants are noted with asterisks. The start codons are in bold faces and amino acid residue numbers are indicated.

Figure 2. Immunoblotting and enzyme activity measurement of feline PDE5 expressed in E. coli.

(a) Immunoblot analysis and coomassie blue staining of whole cell extracts made from E. coli cells carrying feline PDE5 expression plasmid pT7-PDE5 or a control vector pSNAP-tag(T7). Lanes 1 and 3 are extracts from cells carrying pSNAP-tag(T7). Lanes 2 and 4 are extracts from cells carrying pT7-PDE5. Lanes 1 and 2 are Coomassie blue staining of the gel. Lanes 3 and 4 are immunoblot using an anti-human PDE5 antibody. (b) Measurement of PDE5 activity. PDE activity of the extracts from cells carrying pT7-PDE5 in the presence or absence of a PDE5 inhibitor, MY5445, was shown (see Method for details). Basal level PDE activity determined with extract from cells carrying pSNAP-tag(T7) was subtracted. The error bars indicate standard deviation (n = 4).

Figure 3. Detection of PDE5 expression in myocytes of feline RV by IHC staining.

Consecutive paraffin embedded RV tissue sections from control feline hearts were used to examine PDE5 expression. The section in a) was incubated first with a rabbit polyclonal anti-PDE5 antibody followed by HRP conjugated anti-rabbit secondary antibody and then developed using ImmPACT NovaRED as a substrate to visualize the bonded antibodies. Section b) was processed in parallel with a) but omitted the primary anti-PDE5 antibody. Hematoxylin QS was used as nuclear counterstaining.

Figure 4. Detection of cGMP specific PDE activities in feline cardiac tissues.

Tissue homogenates from RV and LV of control animals were prepared as described in Methods. cGMP specific PDE activity in these samples were measured in the presence or absence of 120 uM MY5445, a specific inhibitor of PDE5, as described in Methods. The error bars indicate standard deviation (n = 4).

Figure 5. Determination of PDE5 expression in feline hypertrophy models.

Tissue homogenates from LV and RV of control, PA-banded and Ao-banded animal models were analyzed for PDE5 expression by immunoblotting. (a) Homogenates from LV samples. (b) Homogenate from RV samples. Samples from control, PA-banded and Ao-banded groups were indicated in the figure. E. coli extracts from strains carrying pT7-PDE5 and pSNAP-tag(T7) were used as positive and negative controls (shown as+and -). GAPDH levels in the same set of tissue samples were also determined by immunoblotting, and used for normalization of PDE5 levels. (c) Normalized average PDE level in LV tissues from each group (n = 4). (d) Normalized average PDE level in RV tissues from each group. The error bars indicate standard deviations. Student t-test p values that show statistical significance are indicated in the figure.

Figure 6. cGKI expression in feline hypertrophy models.

Tissue homogenates from LV (a) and RV (b) of control, PA-banded and Ao-banded animal models were analyzed for cGKI expression by immunoblotting, as described in Figure 5. GAPDH levels in the same set of samples were used to normalize the cGKI levels. The normalized average cGKI levels in each group (n = 4) are shown in (c) for LV and (d) in RV. The error bars indicate standard deviations. Student t-test p values that show statistical significance are indicated in the figure.