Phosphodiesterase-5 Inhibitor Sildenafil Preconditions Adult Cardiac Myocytes against Necrosis and Apoptosis

Abstract

We investigated the effect of sildenafil in protection against necrosis or apoptosis in cardiomyocytes. Adult mouse ventricular myocytes were treated with sildenafil (1 or 10 M) for 1 h before 40 min of simulated ischemia (SI). Necrosis was determined by trypan blue exclusion and lactate dehydrogenase release following SI alone or plus 1 or 18 h of reoxygenation (RO). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay and mitochondrial membrane potential measured using a fluorescent probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). Sildenafil reduced necrosis as indicated by decrease in trypan blue-positive myocytes and leakage of lactate dehydrogenase compared with untreated cells after either SI or SI-RO. The number of terminal deoxynucleotidyl transferase-mediated nick end labeling-positive myocytes or loss of JC-1 fluorescence following SI and 18 h of RO was attenuated in the sildenafil-treated group with concomitant inhibition of caspase 3 activity. An early increase in Bcl-2 to Bax ratio with sildenafil treatment was also observed in myocytes after SI-RO. The increase of Bcl-2 expression by sildenafil was inhibited by nitric-oxide synthase (NOS) inhibitor, L-nitro-amino-methyl-ester. The drug also enhanced mRNA and protein content of inducible NOS (iNOS) and endothelial NOS (eNOS) in the myocytes. Sildenafil-induced protection against necrosis and apoptosis was absent in the myocytes derived from iNOS knock-out mice and was attenuated in eNOS knock-out myocytes. The up-regulation of Bcl-2 expression by sildenafil was also absent in iNOS-deficient myocytes. Reverse transcription-PCR, Western blots, and immunohistochemical assay confirmed the expression of phosphodiesterase-5 in mouse cardiomyocytes. These data provide strong evidence for a direct protective effect of sildenafil against necrosis and apoptosis through NO signaling pathway. The results may have possible therapeutic potential in preventing myocyte cell death following ischemia/reperfusion.

Long-term continuous sildenafil treatment ameliorates corporal veno-occlusive dysfunction (CVOD) induced by cavernosal nerve resection in rats

“…12,33,37,38 This assumption is based on the known inhibitory effects of cGMP on collagen synthesis and the TGFb1 pathway, and its vasculoprotective effects on arterial SMC. [14][15][16][17]20,[45][46][47] However, the anti-apoptotic and pro-proliferative effects of sildenafil that we found in the corporal smooth muscle do not agree with the fact that cGMP inhibits vascular SMC proliferation. 48,49 Since the effects of BCNR on the corporal histology and pharmacokinetics are the same as those seen in the aged rat, we assume that cGMP effects on corporal SMC may be modulated by some specific features in the corporal SMC themselves or the tissue milieu, that are not operative in the vascular SMC.…”

“…[11][12][13] It is likely that this occurs through the maintenance of high levels of cGMP, since this compound reduces collagen synthesis and the activation of the pro-fibrotic TGFb1 pathway and protects SMC from apoptosis, while stimulating the spontaneous induction of inducible nitric oxide synthase (iNOS, also known as NOS2). [14][15][16][17][18][19][20][21][22][23] The expression of iNOS in certain non-immunological tissues is assumed to be a defense mechanism against fibrosis. [24][25][26][27][28][29] The nitric oxide produced by iNOS, besides inhibiting collagen synthesis and the TGFb1 pathway, also quenches reactive oxygen species, and in some cases, the differentiation of fibroblasts to myofibroblasts, the cells that produce collagen in many fibrotic conditions.…”

Increased cyclic guanosine monophosphate production and endothelial nitric oxide synthase level in mononuclear cells from sildenafil citrate-treated patients with erectile dysfunction

“…8 In this work, 12 weeks after sildenafil citrate administration, mononuclear cells from vascular patients with ED showed a significant increase in the level of eNOS protein. Accordingly to our results, Das et al 23 also showed an enhanced level of eNOS protein in sildenafilincubated mouse ventricular myocytes that also contain PDE5.However, as occurs in this study, the molecular mechanisms responsible for the increased level of eNOS protein related to sildenafil citrate administration remained to be established. …”

A standalone perfusion platform for drug testing and target validation in micro-vessel networks

“…25 Typical pharmaceutical drugs, such as sildenafil, are orally administered and dissolve into blood reaching concentration above 1 lM. 26 Other studies have tested sildenafil in vitro at 10 lM on primary cardiac cells 27 to 0.1 mM on isolated arteries. 28 On our platform, pronounced responses were seen at 0.1 mM concentration.…”