Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca2+ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca2+ channel (LTCC) protein density. The pore-forming channel subunit Cavα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Cavα1 protein levels. Our findings show that Akt-dependent phosphorylation of Cavβ2, the LTCC chaperone for Cavα1, antagonizes Cavα1 protein degradation by preventing Cavα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca2+ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca2+ entry, Ca2+ handling, and contractility

Comprehensive characterization of glial cells in the urochordate Ciona intestinalis

There are two main classes of cells that are present in the nervous system: neurons and glia. Neurons are the signalling units of the nervous system and are responsible to transmit electrical and chemical signals to other cells, and their function heavily relies on specialized glial cells. Initially considered as the glue keeping neurons together in nervous tissues, decades of research have highlighted the importance of glial cells and the pivotal roles they have in the nervous system assembly and functions. Glial cells are involved in neurogenesis, neuronal differentiation and migration, axonal growing and myelination, synaptogenesis, regulation of the synaptic microenvironment controlling ions and neurotransmitter concentrations, synaptic pruning, structural and tropic support to neurons, and phagocytosis of cell debris or external pathogen in defence to neurons. Also, glia contributes to various neurological disorders such as autism, schizophrenia, and neurodegenerative diseases such as Alzheimer’s, Parkinson's, and amyotrophic lateral sclerosis. Given the number of processes in which they are involved, glial cells have attracted the attention of researchers, trying to unravel many aspects of their biology that are still elusive. Glial cells among vertebrates and invertebrates species display commonalities in their molecular fingerprint and in performing analogous functions, raising questions about their evolutionary origin. Whether glial cells originate from a common ancestor and have evolved together with the nervous system or have appeared multiple times during evolution is still unclear. Neither is known to which extend glial cells had contributed to the evolution of the nervous system as a whole. To gain more information about glia history and have a more complete picture of the evolution of the nervous system it is therefore fundamental to study glial cells in unexplored organisms. Invertebrate organisms provide suitable experimental models to explore glia functions in vivo because of their simplified nervous system architectures and the availability of several molecular toolkits. In our current understanding, glial cells are present in the nervous system of nematodes, arthropods, annelids, mollusks, and ambulacrarians, while little is known about glial cells in the invertebrate chordates. The limited and sparse evidence about glial cell types in urochordates, the sister group of vertebrates, has led to the hypothesis that glia have been mostly lost in this group. This thesis provides a comprehensive understanding of the glial cells present in the embryonic and larva CNS of the urochordate Ciona, exploring their molecular characteristic and investigating the roles they have in the development and functioning of the nervous system. This work shows that glial cells with specialize functions are present in the urochordate, the sister group of vertebrates, providing insight into the evolution of glial cells in the chordate lineage.Doktorgradsavhandlin

EGFR Kinase Regulates Volume-sensitive Chloride Current Elicited by Integrin Stretch via PI-3K and NADPH Oxidase in Ventricular Myocytes

Stretch of β1 integrins activates an outwardly rectifying, tamoxifen-sensitive Cl− current (Cl− SAC) via AT1 receptors, NADPH oxidase, and reactive oxygen species, and Cl− SAC resembles the volume-sensitive Cl− current (ICl,swell). Epidermal growth factor receptor (EGFR) kinase undergoes transactivation upon stretch, integrin engagement, and AT1 receptor activation and, in turn, stimulates NADPH oxidase. Therefore, we tested whether Cl− SAC is regulated by EGFR kinase signaling and is volume sensitive. Paramagnetic beads coated with mAb for β1 integrin were attached to myocytes and pulled with an electromagnet. Stretch activated a Cl− SAC that was 1.13 ± 0.10 pA/pF at +40 mV. AG1478 (10 μM), an EGFR kinase blocker, inhibited 93 ± 13% of Cl− SAC, and intracellular pretreatment with 1 μM AG1478 markedly suppressed Cl− SAC activation. EGF (3.3 nM) directly activated an outwardly rectifying Cl− current (0.81 ± 0.05 pA/pF at +40 mV) that was fully blocked by 10 μM tamoxifen, an ICl,swell blocker. Phosphatidylinositol 3-kinase (PI-3K) is downstream of EGFR kinase. Wortmannin (500 nM) and LY294002 (100 μM), blockers of PI-3K, inhibited Cl− SAC by 67 ± 6% and 91 ± 25% respectively, and the EGF-induced Cl− current also was fully blocked by LY294002. Furthermore, gp91ds-tat (500 nM), a cell-permeable, chimeric peptide that specifically blocks NADPH oxidase assembly, profoundly inhibited the EGF-induced Cl− current. Inactive permeant and active impermeant control peptides had no effect. Myocyte shrinkage with hyperosmotic bathing media inhibited the Cl− SAC and EGF-induced Cl− current by 88 ± 9% and 127 ± 11%, respectively. These results suggest that β1 integrin stretch activates Cl− SAC via EGFR, PI-3K, and NADPH oxidase, and that both the Cl− SAC and the EGF-induced Cl− currents are likely to be the volume-sensitive Cl− current, ICl,swell

Loss of proteostasis as a substrate for atrial fibrillation:Defining novel targets for therapy

Boezemfibrilleren is de meest voorkomende en hardnekkige hartritmestoornis die gepaard gaat met een verhoogde kans op sterfte. De huidige behandelingen van boezemfibrilleren zijn niet effectief genoeg en kunnen het voortschrijden van boezemfibrilleren niet voorkomen. Om nieuwe behandelingen van boezemfibrilleren te ontwikkelen is het noodzakelijk te begrijpen welke processen in de hartspiercel bijdragen aan het ontstaan, het behoudt en de verergering van boezemfibrilleren. In dit proefschrift is onderzocht welke processen in de hartspiercel hieraan bijdragen, met daarbij een centrale rol voor het verlies van eiwit homeostase. Verlies van eiwit homeostase kan leiden tot veranderingen in de lokalisatie, de activiteit en/of de afbraak van eiwitten betrokken bij de normale hartfunctie en kan daarmee bijdragen aan het ontstaan van boezemfibrilleren. Daarnaast leidt boezemfibrilleren zelf tot een verder verlies van eiwit homeostase, wat uiteindelijk electrische en structurele veranderingen van de hartspiercel tot gevolg heeft, hetgeen weer leidt tot verergering van het boezemfibrilleren. Nieuwe therapeutische aangrijpingspunten gericht op het behoudt van de eiwit homeostase in de hartspiercel waren succesvol in het voorkomen van boezemfibrilleren in de gebruikte experimentele modellen. Het beschreven onderzoek laat zien dat remmers van HDAC6, RhoGTPase, ER stress en HSP inducerende middelen interessante kandidaten zijn voor de toekomstige behandeling van patiënten met boezemfibrilleren

The role of PTEN in cardioprotection against ischaemia-reperfusion injury

Activation of the PI3K/AKT pathway protects the heart from ischaemia-reperfusion injury. Phosphatase and Tensin Homolog deleted on Chromosome10 (PTEN) is a negative regulator of this pathway. The hypothesis on which this thesis was based stated that inhibition of PTEN would confer protection against ischaemia-reperfusion injury. PTEN was reduced using: 1) a PTEN inhibitor, bpV(HOpic), 2) a mouse model of PTEN haploinsufficiency and 3) PTEN siRNA. The effects of PTEN reduction on ischaemia-reperfusion injury were investigated by using: 1) an isolated perfused heart model of ischaemia-reperfusion injury, 2) an isolated cardiomyocyte model of ROS induced mitochondria damage and 3) a cellular model of hypoxia-reoxygenation injury. No protection against ischaemia-reperfusion was observed in isolated perfused myocardium from C57BL/J6 mice, which were perfused with bpV(HOpic), or from PTEN+/-mice. Likewise, no protection against ROS induced mitochondrial damage was observed in isolated cardiomyocytes from the PTEN+/- mice. In these models an increase in AKT activity was recorded, however, this was not sufficient to confer cardioprotection. Similarly, H9c2 rat myoblast cells, silenced for PTEN expression using siRNA, were not protected against hypoxia-reoxygenation injury. Nevertheless, in isolated C57BL/J6 hearts perfused with bpV(HOpic) and in myocardium from PTEN+/- mice, when the PI3K/AKT pathway was stimulated by the cardioprotective intervention of ischaemic preconditioning a reduced threshold for protection was achieved. To conclude, the level of PTEN inhibition achieved in this study was not sufficient to bestow protection against simulated ischaemiareperfusion injury. However, it appears that reductions in PTEN can increase the sensitivity towards cardioprotection

Cyclic Nucleotide Signaling and the Cardiovascular System

The cyclic nucleotides 3',5'-adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) play important roles in the control of cardiovascular function under physiological and pathological conditions. In this book, which is a reprint of a Special Issue of the Journal of Cardiovascular Development and Disease entitled "Cyclic Nucleotide Signaling and the Cardiovascular System", internationally recognized experts give an overview of this vibrant scientific field. The first series of articles deal with the localization and function of membrane-bound and soluble adenylate cyclases, followed by articles on the roles of phosphodiesterase isoforms in the heart. Cyclic nucleotide signaling takes place in nanodomains and the A-kinase anchor proteins (AKAPS) are essential for the compartmentalized assembly of signaling proteins into functional complexes. Reviews on the role of AKAP proteins in the physiology and pathophysiology of the heart are also included in this book. Cyclic nucleotides act through effector proteins and articles on EPAC and POPDC proteins inform the reader of recent developments on these topics. A major advancement in our understanding of cyclic nucleotide signaling came through the use of genetically encoded cAMP sensor molecules, and a series of articles review the current insight that these reporter molecules have provided. The final set of articles in this book deals with the association of the cyclic nucleotide pathway and cardiovascular disease as well as the development of novel therapeutic approaches. Thomas Brand and Enno Klussmann Special Issue Editor