Optogenetic manipulation of cGMP highlights PDE5 as the predominant cGMP-hydrolyzing PDE in megakaryocytes

Cyclic guanosine monophosphate (cGMP) signalling plays a fundamental role in many cell types including platelets. cGMP has been implicated in platelet formation, but mechanistic detail about its spatiotemporal regulation in megakaryocytes (MKs) is lacking. We expressed a photo-activated guanylyl cyclase, Blastocladiella emersonii Cyclase opsin (BeCyclop), after viral-mediated gene transfer in bone marrow (BM)-derived MKs to precisely light-modulate cGMP levels. BeCyclop-MKs showed a significantly increased cGMP concentration after illumination, which was strongly dependent on phosphodiesterase (PDE) 5 activity. This finding was corroborated by real-time imaging of cGMP signals which revealed that pharmacological PDE5 inhibition also potentiated nitric oxide (NO) triggered cGMP generation in BM MKs. In summary, we established for the first time optogenetics in primary MKs and identified PDE5 as the predominant PDE regulating cGMP levels in MKs. These findings also demonstrate that optogenetics allows for the precise manipulation of MK biology.

PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca 2+ Content

Rationale: Phosphodiesterase-5 (PDE5) inhibition reduces the occurrence of ventricular arrhythmias following myocardial ischemia. However, the mechanisms of the anti-arrhythmic effects of PDE5 inhibition are unknown. Diastolic calcium (Ca 2+ ) waves lead to arrhythmias by inducing delayed after-depolarizations. Ca 2+ waves are initiated when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold level and the SR releases Ca 2+ spontaneously and generates a depolarizing inward sodium-calcium exchange (NCX) current. Objective: To determine the effects of PDE5 inhibition on the propensity for ventricular arrhythmias in a pro-arrhythmic large animal model and establish the role of alterations of intracellular Ca 2+ cycling / SR Ca 2+ content. Methods and Results: Arrhythmia burden, monophasic action potentials and beat-to-beat variability of repolarization were measured in a sheep model using the I Kr inhibitor dofetilide to induce QT prolongation and arrhythmia. Ca 2+ transients, Ca 2+ waves and SR Ca 2+ content were measured in isolated ventricular myocytes. PDE5 inhibition was achieved using acute application of sildenafil and protein kinase G (PKG) was inhibited with KT5823. PDE5 inhibition reduced beat-to-beat variability of repolarization and suppressed after-depolarizations, premature ventricular complexes, and torsade de pointes in vivo. In single cells, dofetilide-induced DADs and triggered action potentials were suppressed by PDE5 inhibition. PDE5 inhibition decreased Ca 2+ wave frequency in all cells and abolished waves in 12/22 cells. A decrease in SR Ca 2+ uptake, increased trans-sarcolemmal Ca 2+ efflux and reduced trans-sarcolemmal Ca 2+ influx led to a reduction of SR Ca 2+ content, and Ca 2+ wave abolition. These effects were dependent on PKG activation. Conclusions: PDE5 inhibition acutely suppresses triggered ventricular arrhythmias in vivo and cellular data suggests this occurs via suppression of cellular Ca 2+ waves. These novel anti-arrhythmic properties of PDE5 inhibition are mediated by a reduction of SR Ca 2+ content and are PKG-dependent.

Phenotypical Effect of Phosphodiesterase 5 (PDE5) Inhibitor on Behavioral Activities of Fruit Fly Drosophila melanogaster

Phosphodiesterase 5 (PDE5) inhibitor is a class of drugs currently used to treat erectile dysfunction. Physiologically, inhibition of PDE5 may lead to vasodilation, blood flow increment, and penile erection. However, PDE5 inhibitors have been reported not only to modify the function of the male reproductive organ but also to influence other physiological systems. To explore the effect of PDE5 inhibitor on metazoan physiological systems, a fruit fly (Drosophila melanogaster) model organism is used since the catalytic domain of fruit fly PDE5/6 shares a high similarity of amino acid sequence (58%) with the PDE5 of humans. This study aimed to investigate whether the effect of PDE5 inhibition by sildenafil is phenotypically observable as changes in the behavioral states. Two behavioral phenotypes of D. melanogaster, negative geotaxis, and ethanol sensitivity, were used as test parameters in this explorative study. The results demonstrated that sildenafil had a significant effect on reducing locomotor activity, as reflected by negative geotaxis assay, but it had no influence on the fruit fly sensitivity to ethanol. Taken together, our results suggested that PDE5 inhibition might impair the physiological condition of the metazoan species. Also, an explorative study using D. melanogaster might offer valuable insight as a model organism in the discovery and repurposing approach of PDE5 inhibitor.

Aging-Related Increase of cGMP Disrupts Mitochondrial Homeostasis in Leydig Cells

Since mitochondria play an essential role in the testosterone biosynthesis, serve as power centers and are a source of oxidative stress, a possible mitochondrial dysfunction could be connected with decreased activity of Leydig cells and lowered testosterone production during aging. Here we chronologically analyzed age-related alterations of mitochondrial function in Leydig cells correlated by the progressive rise of cGMP signaling and with respect to testosterone synthesis. To target cGMP signaling in Leydig cells, acute or long-term in vivo or ex vivo treatments with sildenafil (phosphodiesterase 5 [PDE5] inhibitor) were performed. Aging-related accumulation of cGMP in the Leydig cells is associated with mitochondrial dysfunction illustrated by reduced ATP and steroid production, lowered O2 consumption, increased mitochondrial abundance and mtDNA copies number, decreased expression of genes that regulate mitochondrial biogenesis (Ppargc1a/PGC1a-Tfam-Nrf1/NRF1), mitophagy (Pink1), fusion (Mfn1, Opa1), and increased Nrf2/NRF2. Acute in vivo PDE5 inhibition overaccumulated cGMP and stimulated testosterone but reduced ATP production in Leydig cells from adult, middle-aged, and old rats. The increased ATP/O ratio observed in cells from old compared to adult rats was diminished after stimulation of cGMP signaling. Opposite, long-term PDE5 inhibition decreased cGMP signaling and improved mitochondrial function/dynamics in Leydig cells from old rats. Mitochondrial abundance in Leydig cells decreased while ATP levels increased. Chronic treatment elevated Tfam, Nrf1, Nrf2, Opa1, Mfn1, Drp1, and normalized Pink1 expression. Altogether, long-term PDE5 inhibition prevented age-related NO and cGMP elevation, improved mitochondrial dynamics/function, and testosterone production. The results pointed on cGMP signaling in Leydig cells as a target for pharmacological manipulation of aging-associated changes in mitochondrial function and testosterone production.