Increase in Ca2+ current by sustained cAMP levels enhances proliferation rate in GH3 cells

Aims: Ca2 + and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2 + currents and proliferation in pituitary tumor GH3 cells. Main methods: Effect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2 + current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. Key findings: Sustained forskolin treatment (24 and 48 h) induced a significant increase in total Ca2 + current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2 + current density. However, the maximum effect of dbcAMP occurred only after 72 h incubation, whereas forskolin showed maximal effect at 48 h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2 + channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. Significance: We conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2 + current density and this phenomenon impacts proliferation rate in GH3 cells

High-Capacity Adenoviral Vectors Permit Robust and Versatile Testing of DMD Gene Repair Tools and Strategies in Human Cells

Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle wasting disorder arising from mutations in the ~2.4 Mb dystrophin-encoding DMD gene. RNA-guided CRISPR-Cas9 nucleases (RGNs) are opening new DMD therapeutic routes whose bottlenecks include delivering sizable RGN complexes for assessing their effects on human genomes and testing ex vivo and in vivo DMD-correcting strategies. Here, high-capacity adenoviral vectors (HC-AdVs) encoding single or dual high-specificity RGNs with optimized components were investigated for permanently repairing defective DMD alleles either through exon 51-targeted indel formation or major mutational hotspot excision (>500 kb), respectively. Firstly, we establish that, at high doses, third-generation HC-AdVs lacking all viral genes are significantly less cytotoxic than second-generation adenoviral vectors deleted in E1 and E2A. Secondly, we demonstrate that genetically retargeted HC-AdVs can correct up to 42% ± 13% of defective DMD alleles in muscle cell populations through targeted removal of the major mutational hotspot, in which over 60% of frame-shifting large deletions locate. Both DMD gene repair strategies tested readily led to the detection of Becker-like dystrophins in unselected muscle cell populations, leading to the restoration of β-dystroglycan at the plasmalemma of differentiated muscle cells. Hence, HC-AdVs permit the effective assessment of DMD gene-editing tools and strategies in dystrophin-defective human cells while broadening the gamut of DMD-correcting agents

Cyclic nucleotide signalling in cardiac myocytes from the mdx model of Duchenne muscular dystrophy

Background and Aims: Duchenne muscular dystrophy (DMD), the most frequent muscular dystrophy, is caused by mutations in the dystrophin gene resulting in the absence of dystrophin. Loss of cardiac dystrophin eventually leads to dilated cardiomyopathy and congestive heart failure in at least 20&percnt; of patients. Previous studies have shown that inhibition of enzymes that degrade cGMP improves cardiac contractility, energy metabolism and cardiac myocyte integrity in the DMD mouse model mdx. The aim of this project is to explore whether defective cGMP and cAMP signalling is a feature of mdx cardiac myocytes and whether this may play a role in the pathogenesis of the cardiomyopathy associated with dystrophin deficiency. Methods: Live-cell fluorescent imaging of Fluoresence Resonance Energy Transfer (FRET) has been used to investigate real-time changes in cAMP and cGMP in specific subcellular compartments of neonatal and adult isolated cardiomyocytes. The IonOptix contractility and force transducer system were used to explore the contractile performance and force generation upon contraction in mdx adult cardiomyocytes. Results: cAMP and cGMP regulation and compartmentalisation seem to be significantly altered in mdx mice cardiomyocytes. The data suggest that the activity of several isoforms of phosphodiesterase and the ability to generate cGMP and cAMP in specific subcellular compartments is altered in neonatal cardiomyocytes (NCM) from the mdx model, indicating that not only the cGMP pathway might underpin the cardiomyopathy associated with DMD but disruption of cAMP signalling may also play a role. Analysis of cyclic nucleotide signalling in adult mdx cardiac myocytes indicated significantly higher levels of oxidised sGC and PDE mRNA levels. Additionally, adult mdx cardiomyocytes presented lower nNOS expression in the PM and in the ER. Basal levels of cyclic nucleotides significantly changed with age, suggesting that compensatory mechanisms may develop with age. Furthermore, mdx cardiomyocytes showed significantly impaired force of contraction which was abolished upon IBMX treatment suggesting that altered PDE activity might underpin defects in contractility. Conclusions: In summary, the data presented in this thesis supports the hypothesis that defective cyclic nucleotides signalling might play a role in the cardiomyopathy associated with DMD in the mdx.</p

Cyclic nucleotide signalling in cardiac myocytes from the mdx model of Duchenne muscular dystrophy

Background and Aims: Duchenne muscular dystrophy (DMD), the most frequent muscular dystrophy, is caused by mutations in the dystrophin gene resulting in the absence of dystrophin. Loss of cardiac dystrophin eventually leads to dilated cardiomyopathy and congestive heart failure in at least 20&amp;percnt; of patients. Previous studies have shown that inhibition of enzymes that degrade cGMP improves cardiac contractility, energy metabolism and cardiac myocyte integrity in the DMD mouse model mdx. The aim of this project is to explore whether defective cGMP and cAMP signalling is a feature of mdx cardiac myocytes and whether this may play a role in the pathogenesis of the cardiomyopathy associated with dystrophin deficiency. Methods: Live-cell fluorescent imaging of Fluoresence Resonance Energy Transfer (FRET) has been used to investigate real-time changes in cAMP and cGMP in specific subcellular compartments of neonatal and adult isolated cardiomyocytes. The IonOptix contractility and force transducer system were used to explore the contractile performance and force generation upon contraction in mdx adult cardiomyocytes. Results: cAMP and cGMP regulation and compartmentalisation seem to be significantly altered in mdx mice cardiomyocytes. The data suggest that the activity of several isoforms of phosphodiesterase and the ability to generate cGMP and cAMP in specific subcellular compartments is altered in neonatal cardiomyocytes (NCM) from the mdx model, indicating that not only the cGMP pathway might underpin the cardiomyopathy associated with DMD but disruption of cAMP signalling may also play a role. Analysis of cyclic nucleotide signalling in adult mdx cardiac myocytes indicated significantly higher levels of oxidised sGC and PDE mRNA levels. Additionally, adult mdx cardiomyocytes presented lower nNOS expression in the PM and in the ER. Basal levels of cyclic nucleotides significantly changed with age, suggesting that compensatory mechanisms may develop with age. Furthermore, mdx cardiomyocytes showed significantly impaired force of contraction which was abolished upon IBMX treatment suggesting that altered PDE activity might underpin defects in contractility. Conclusions: In summary, the data presented in this thesis supports the hypothesis that defective cyclic nucleotides signalling might play a role in the cardiomyopathy associated with DMD in the mdx.</p

Modulation of Compartmentalised Cyclic Nucleotide Signalling via Local Inhibition of Phosphodiesterase Activity

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors

Modulation of Compartmentalised Cyclic Nucleotide Signalling via Local Inhibition of Phosphodiesterase Activity

Cyclic nucleotide phosphodiesterases (PDEs) are the only enzymes that degrade the cyclic nucleotides cAMP and cGMP, and play a key role in modulating the amplitude and duration of the signal delivered by these two key intracellular second messengers. Defects in cyclic nucleotide signalling are known to be involved in several pathologies. As a consequence, PDEs have long been recognized as potential drug targets, and they have been the focus of intense research for the development of therapeutic agents. A number of PDE inhibitors are currently available for the treatment of disease, including obstructive pulmonary disease, erectile dysfunction, and heart failure. However, the performance of these drugs is not always satisfactory, due to a lack of PDE-isoform specificity and their consequent adverse side effects. Recent advances in our understanding of compartmentalised cyclic nucleotide signalling and the role of PDEs in local regulation of cAMP and cGMP signals offers the opportunity for the development of novel strategies for therapeutic intervention that may overcome the current limitation of conventional PDE inhibitors

Multi-Compartment, Early Disruption of cGMP and cAMP Signalling in Cardiac Myocytes from the mdx Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is the most frequent and severe form of muscular dystrophy. The disease presents with progressive body-wide muscle deterioration and, with recent advances in respiratory care, cardiac involvement is an important cause of morbidity and mortality. DMD is caused by mutations in the dystrophin gene resulting in the absence of dystrophin and, consequently, disturbance of other proteins that form the dystrophin-associated protein complex (DAPC), including neuronal nitric oxide synthase (nNOS). The molecular mechanisms that link the absence of dystrophin with the alteration of cardiac function remain poorly understood but disruption of NO-cGMP signalling, mishandling of calcium and mitochondrial disturbances have been hypothesized to play a role. cGMP and cAMP are second messengers that are key in the regulation of cardiac myocyte function and disruption of cyclic nucleotide signalling leads to cardiomyopathy. cGMP and cAMP signals are compartmentalised and local regulation relies on the activity of phosphodiesterases (PDEs). Here, using genetically encoded FRET reporters targeted to distinct subcellular compartments of neonatal cardiac myocytes from the DMD mouse model mdx, we investigate whether lack of dystrophin disrupts local cyclic nucleotide signalling, thus potentially providing an early trigger for the development of cardiomyopathy. Our data show a significant alteration of both basal and stimulated cyclic nucleotide levels in all compartments investigated, as well as a complex reorganization of local PDE activities

An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain

An essential cell-autonomous role for hepcidin in cardiac iron homeostasis

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain