Marisa Cornacchione

Inhibition of type 5 phosphodiesterase counteracts β2-adrenergic signalling in beating cardiomyocytes

AIMS Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (βARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed. METHODS AND RESULTS Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and βAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by βAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by β2AR, but not β1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP. CONCLUSION These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk.

From genetics to genomics of epilepsy.

The introduction of DNA microarrays and DNA sequencing technologies in medical genetics and diagnostics has been a challenge that has significantly transformed medical practice and patient management. Because of the great advancements in molecular genetics and the development of simple laboratory technology to identify the mutations in the causative genes, also the diagnostic approach to epilepsy has significantly changed. However, the clinical use of molecular cytogenetics and high-throughput DNA sequencing technologies, which are able to test an entire genome for genetic variants that are associated with the disease, is preparing a further revolution in the near future. Molecular Karyotype and Next-Generation Sequencing have the potential to identify causative genes or loci also in sporadic or non-familial epilepsy cases and may well represent the transition from a genetic to a genomic approach to epilepsy.

β-Adrenergic response is counteracted by extremely-low-frequency pulsed electromagnetic fields in beating cardiomyocytes

Proper β-adrenergic signaling is indispensable for modulating heart frequency. Studies on extremely-low-frequency pulsed electromagnetic field (ELF-PEMF) effects in the heart beat function are contradictory and no definitive conclusions were obtained so far. To investigate the interplay between ELF-PEMF exposure and β-adrenergic signaling, cultures of primary murine neonatal cardiomyocytes and of sinoatrial node were exposed to ELF-PEMF and short and long-term effects were evaluated. The ELF-PEMF generated a variable magnetic induction field of 0-6mT at a frequency of 75Hz. Exposure to 3mT ELF-PEMF induced a decrease of contraction rate, Ca(2+) transients, contraction force, and energy consumption both under basal conditions and after β-adrenergic stimulation in neonatal cardiomyocytes. ELF-PEMF exposure inhibited β-adrenergic response in sinoatrial node (SAN) region. ELF-PEMF specifically modulated β2 adrenergic receptor response and the exposure did not modify the increase of contraction rate after adenylate cyclase stimulation by forskolin. In HEK293T cells transfected with β1 or β2 adrenergic receptors, ELF-PEMF exposure induced a rapid and selective internalization of β2 adrenergic receptor. The β-adrenergic signaling, was reduced trough Gi protein by ELF-PEMF exposure since the phosphorylation level of phospholamban and the PI3K pathway were impaired after isoproterenol stimulation in neonatal cardiomyocytes. Long term effects of ELF-PEMF exposure were assessed in cultures of isolated cardiomyocytes. ELF-PEMF counteracts cell size increase, the generation of binucleated of cardiomyocytes and prevents the up-regulation of hypertrophic markers after β-adrenergic stimulation, indicating an inhibition of cell growth and maturation. These data show that short and long term exposure to ELF-PEMF induces a reduction of cardiac β-adrenergic response at molecular, functional and adaptative levels.

Identification of Murine Phosphodiesterase 5A Isoforms and their Functional Characterization in HL-1 Cardiac Cell Line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2, and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N‐terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT‐PCR analysis showed that mPde5a1, mPde5a2, and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL‐1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL‐1 areas, and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy.

Critical role of phosphodiesterase 2A in mouse congenital heart defects

Aims Phosphodiesterase 2 A (Pde2A), a cAMP-hydrolysing enzyme, is essential for mouse development; however, the cause of Pde2A knockout embryonic lethality is unknown. To understand whether Pde2A plays a role in cardiac development, hearts of Pde2A deficient embryos were analysed at different stage of development. Methods and results At the stage of four chambers, Pde2A deficient hearts were enlarged compared to the hearts of Pde2A heterozygous and wild-type. Pde2A knockout embryos revealed cardiac defects such as absence of atrial trabeculation, interventricular septum (IVS) defects, hypertrabeculation and thinning of the myocardial wall and in rare cases they had overriding aorta and valves defects. E14.5 Pde2A knockouts showed reduced cardiomyocyte proliferation and increased apoptosis in the IVS and increased proliferation in the ventricular trabeculae. Analyses of E9.5 Pde2A knockout embryos revealed defects in cardiac progenitor and neural crest markers, increase of Islet1 positive and AP2 positive apoptotic cells. The expression of early cTnI and late Mef2c cardiomyocyte differentiation markers was strongly reduced in Pde2A knockout hearts. The master transcription factors of cardiac development, Tbx, were down-regulated in E14.5 Pde2A knockout hearts. Absence of Pde2A caused an increase of intracellular cAMP level, followed by an up-regulation of the inducible cAMP early repressor, Icer in fetal hearts. In vitro experiments on wild-type fetal cardiomyocytes showed that Tbx gene expression is down-regulated by cAMP inducers. Furthermore, Pde2A inhibition in vivo recapitulated the heart defects observed in Pde2A knockout embryos, affecting cardiac progenitor cells. Interestingly, the expression of Pde2A itself was dramatically affected by Pde2A inhibition, suggesting a potential autoregulatory loop. Conclusions We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

Model of Murine Ventricular Cardiac Tissue for In Vitro Kinematic-Dynamic Studies of Electromagnetic and β-Adrenergic Stimulation

In a model of murine ventricular cardiac tissue in vitro, we have studied the inotropic effects of electromagnetic stimulation (frequency, 75 Hz), isoproterenol administration (10 μM), and their combination. In particular, we have performed an image processing analysis to evaluate the kinematics and the dynamics of beating cardiac syncytia starting from the video registration of their contraction movement. We have found that the electromagnetic stimulation is able to counteract the β-adrenergic effect of isoproterenol and to elicit an antihypertrophic response.

Critical role of phosphodiesterase 2A in congenital heart disease

cAMP and cGMP levels are regulated in a highly specific and stimulus-dependent manner by cyclic nucleotide degrading phosphodiesterases (PDEs). Among the different PDEs, PDE2A is unique because it responds to elevated cGMP levels increasing its cAMP hydrolytic activity. PDE2A is essential for mouse development and Pde2A null mice die in utero (1). Pde2A-/- embryos display a nuchal edema which is associated with congenital heart defects in other mouse models (2). To investigate if the absence of PDE2A is associated with heart defects, macroscopical and microscopical observations of hearts isolated from Pde2A-/- E14.5 embryos were performed. Pde2A-/- hearts appeared enlarged compared to the wild type counterparts and displayed interventricular septum defect, hypertrabeculation, myocardial wall defects and atrial trabeculae loss. Increased apoptosis was detected in specific areas of Pde2A-/- hearts. Compromised expression pattern of critical genes involved in heart development (T-box genes) was observed. RT-PCR analysis revealed that Tbx1, Tbx2, Tbx18 and Tbx20 expression was significantly down-regulated in hearts from Pde2A-/- mice. To investigate whether cAMP might regulate T-box gene expression, fetal cardiomyocytes were treated with a PDE2A inhibitor (EHNA) and the β-adrenergic agonist Isoproterenol (Iso). T-box gene expression was down-regulated in cardiomyocytes treated with EHNA and Iso. Taken together these data suggest that PDE2A activity cannnot be replaced by other phosphodiesterases and its specific function could be the modulation of T-box gene expression during embryonic development. To our knowledge, this is the first time that the genetic deletion of a phosphodiesterase is associated with the establishment of congenital heart defect.

Low frequency electromagnetic field reduces β-adrenergic response in murine cardiomyocytes

In the modern society, concerns have been raised about possible adverse effects of exposure to low frequency electromagnetic field (ELF) on biological systems. Many research groups are working in this field, but the obtained results are often contradictory and cannot be reproduced by an experimental point of view in different laboratories, preventing definitive conclusions (Kheifets L. et al., 2007). In this work, the ELF was generated by a bioreactor that produced a variable magnetic field of 0-3.3 mT at a frequency of 75 Hz. The ELF was applied to cultures of murine neonatal cardiomyocytes to study several biological parameters. Neonatal cardiomyocytes are able to form spontaneously beating syncytia (Fassina L. et al., 2011). In basal conditions and after a β-adrenergic stimulation with Isoproterenol, a short exposure to ELF induced a decrease of contraction rate, Ca2+ transients, force of contraction and energy consumption. ELF did not modify frequency of contraction after stimulation with Forskolin and Norepinephrine, suggesting a specific effect of ELF on β-adrenergic receptors. Isolated cardiomyocytes were cultured in monolayer for 48 hours in the presence or absence of ELF, to investigate the effects of the ELF exposure on cell viability, cell size and differentiation. Immunofluorescence experiments showed that the ELF decreased the percentage of mature cardiomyocytes without having effects on cell size and morphology. Moreover, the ELF caused a decrease of binucleated cardiomyocytes, indicating possible differentiation defects. In conclusion these data show that a short exposure to ELF induces a decrease on β-adrenergic response while a long exposure to ELF influences the cell viability and the differentiation of cardiomyocytes.

Effects of the hydrostatic pressure in in vitro beating cardiac syncytia in terms of kinematics (kinetic energy and beat frequency) and syncytia geometrical-functional classification

Many important observations and discoveries in heart physiology have been made possible using the isolated heart method of Langendorff, e.g. the discovery of the very famous Frank-Starling law of the heart. Nevertheless, the Langendorffs method has some limitations and disadvantages such as the probability of preconditioning and a high oxidative stress, leading to the deterioration of the contractile function. To avoid the preceding drawbacks associated to the use of a whole heart, we have alternatively used beating mouse cardiac syncytia cultured in vitro in order to assess the ergotropic and chronotropic effects of both increasing and decreasing hydrostatic pressures. To achieve the preceding aim, we have developed a method based on image processing analysis to evaluate the kinematics of that pressure-loaded beating syncytia starting from the video registration of their contraction movement. We have verified the Frank-Starling law of the heart in in vitro beating cardiac syncytia and we have obtained their geometrical-functional classification. The present method could be used in in vitro studies of beating cardiac patches, as alternative to the Langendorffs heart in biochemical, pharmacological, and physiology studies, and, especially, when the Langendorffs technique is inapplicable. Furthermore, the method could help, in heart tissue engineering and bioartificial heart researches, to “engineer the heart piece by piece”.

Posphodiesterase 5 regulates the beating rate in murine neonatal cardiomyocytes

Heart rate is fi nely regulated by the sympathetic nervous system through beta adrenoreceptors (βARs) signaling. β-ARs stimulation induces cAMP/cGMP synthesis whereas phosphodiesterases (PDE) catalyze the hydrolysis of the cyclic nucleotides, however their precise interaction is not well defi ned (1, 2). The experimental model used to investigate the role of PDE5 inhibition on the heart function are the spontaneously beating cardiac myocytes from neonatal mice. Preliminary experiments were performed to determine whether neonatal cardiomyocyte cultures show the same PDEs expression pattern of adult mice hearts. We analyzed through RT-PCR and WB experiments the mRNA and protein levels of PDE1C, PDE2, PDE3A, PDE4 and PDE5 and we observed that are expressed both in hearts and in cultured neonatal cardiomyocytes. These data suggest that the cardiomyocyte is a suitable model to investigate the PDEs role in cardiac function. Experiments performed to evaluate the contraction rate stimulated by β-AR signaling activation show: that PDE5 is a positive modulator through hydrolysis of cGMP, and the inhibition of PDE5 causes a positive chronotropic eff ects reduction by the PDE2 activation through the increase of cGMP level. Notably, the use of PDE2 knockout mice reverts the negative chronotropic eff ects obtained by PDE5 inhibition. Finally, we observed that PDE5 selectively impacts heart rate interfering with β2AR signaling in neonatal cardiac myocytes, with little or no eff ect on β1AR signaling. These data show a novel role of PDE5 on the sympathetic regulation of cardiac beating and highlight the mechanicistic pathways of the long term eff ect of PDE5 inhibition in cardiac hypertrophy.