IF IN THE HEART, PDE5 IS ∼10NM 30 AND DURING FAILURE, IS UPREGULATED BY 2-FOLD (OUR STUDY), PDE5 WOULD BE ∼20NM, ...
Our results demonstrating that PDE5 was not detected in the murine LV samples are in agreement with those of others 11 , 30 , 31 The lack of detectable PDE5 expression in the murine myocardium is also supported by data demonstrating that Fildena does not influence cardiac contractility 29 , 31 , and the inhibition of PDE5 with Fildena does not increase LV cGMP content 8 , 9 Additionally, without a positive control loaded on the same gel (refer to Fig. Others have reported that PDE5 is expressed in murine myocardium 7 - 10 , and in addition, have demonstrated that Fildena reduces cardiac hypertrophy in the TAC model of LV failure 8 However, LV hypertrophy following TAC was not increased in the cardiac specific PKG KO mouse 11 These investigators 11 demonstrated that PDE5 expression was not detected in murine cardiomyocytes, but PDE5 was detected in the lung and smooth muscle, which is similar to our results ( Fig. (A) PDE5 is not detected by immunoblotting of human LV samples (heart failure (n = 6) and controls (n = 8); bovine lung is used as a positive control (rabbit polyclonal anti-PDE5 antibody provided by Joseph A Beavo, 26 ). Upper blot—HF 1-3, Control 1-4 and lower blot—HF 4-6, Control 5-8 (refer to Table 1 ). (B) Western blots of 2-D gels with the anti-PDE5 antibody (Cell Signaling) demonstrate that PDE5 is detected in the bovine lung as 2 isoelectric variants, while PDE5 is not detected in cardiac tissue lysates of human LV samples (control (Control 2)) and heart failure (HF1)).
Fig 3. PDE5 is not detected in LV samples from human controls or patients with heart failure. For LV samples from normal (control), heart failure and compensated hypertrophy, there is non-specific binding of the anti-PDE5 antibody from Santa Cruz at a MW near the positive control, while PDE5 is not detected with the anti-PDE5 antibody from Cell Signaling or the rabbit polyclonal anti-PDE5 antibody provided by Joseph A Beavo, 26 (B) Western blots of 2-D gels with the anti-PDE5 antibody (Cell Signaling) demonstrate that PDE5 is detected in the lung as 2 isoelectric variants, while PDE5 is not detected in cardiac tissue lysates (normal mice). 1A ). Although the anti-PDE5 antibody from Cell Signaling and the rabbit polyclonal anti-PDE5 antibody 26 recognized a single band in the lung, these antibodies did not identify any protein in cardiac muscle lysates ( Fig.
To examine the specificity of the anti-PDE5 antibodies, we performed immunoblotting with cardiac LV cell lysates obtained from mouse heart (control, HF (TAC-HF), compensated hypertrophy (TAC-Comp)), and mouse lung (positive control) using the three different anti-PDE5 antibodies.