cardiomyocyte; ryanodine receptor; cardiac muscle; excitation-contraction coupling; calcium signaling; sarcoplasmic reticulum
Potenza Duilio Michele, Janicek Radoslav, Fernandez‐Tenorio Miguel, Niggli Ernst (2020), Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes, in The Journal of Physiology
Illaste Ardo, Wullschleger Marcel, Fernandez-Tenorio Miguel, Niggli Ernst, Egger Marcel (2019), Automatic Detection and Classification of Ca2+ Release Events in Confocal Line- and Frame-scan Images, in Biophysical Journal
, 116, 383-394.
Potenza Duilio M., Janicek Radoslav, Fernandez-Tenorio Miguel, Camors Emmanuel, Ramos-Mondragón Roberto, Valdivia Héctor H., Niggli Ernst (2019), Phosphorylation of the ryanodine receptor 2 at serine 2030 is required for a complete β-adrenergic response, in Journal of General Physiology
, 151, 131-145.
Niggli Ernst, Fernandez-Tenorio Miguel (2019), Calcium-Binding Proteins of the EF-Hand SuperfamilyFrom Basics to Medical Applications, in Heizmann Claus W. (ed.), Springer New York, New York, NY, 53-71.
Niggli Ernst, Shirokova Natalia (2018), Caged Compounds: Applications in Cardiac Muscle Research, in Kästner Lars (ed.), Springer International Publishing, Cham, 75-95.
Shirokova Natalia, Niggli Ernst (2018), Duchenne Muscular Dystrophy: Emerging Pathological Role for Cardiac Myopathy, in Kardiologiia: vid nauky do praktyky
, 4 (33), 27-40.
Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes
|Persistent Identifier (PID)
Each ZIP file contains the confocal or Western blot images (.tif, .oib, .oif) as well as voltage-clamp recordings (.pxp files - Igor, Wavemetrics). It also contains the analyses of the data used in that figure. Data are organised by dates (cell isolations) and by measured cells.
1.1. Background and rationale:In cardiac muscle, contraction is activated by transient elevations of the intracellular Ca2+ concentration. The mechanisms governing these Ca2+ signals are referred to as excitation-contraction (EC) coupling. A small amount of Ca2+ entering the myocytes via voltage-dependent Ca2+ channels is amplified several-fold by the mechanism of Ca2+-induced Ca2+ release (CICR) from the sarcoplasmic reticulum (SR) via Ca2+ release channels (called ryanodine receptors or RyRs). The RyRs are thus the gatekeepers of EC-coupling. Opening and closing of the RyRs is modulated by a variety of post-translational modifications, and by cellular constituents and ions. The overarching goal of this project is to define the function of the RyRs in health and disease, and to characterize how these channels shape Ca2+ signaling and can cause arrhythmogenicity. We will concentrate our experimental efforts on 2 parameters, carefully selected because of their importance in physiological regulation of cardiac muscle activity, but also because of their pathophysiological and clinical relevance. The following mechanisms will be examined: 1) Regulation and mis-regulation of RyR activity by RyR phosphorylation. 2) Mis-regulation of RyR activity resulting from a arrhythmogenic RyR mutation. In addition, we will define how the two mechanisms interact, i.e. whether and how RyR phosphorylation may trigger the life-threatening arrhythmias in patients harboring this RyR mutation.1.2. Working hypothesis: We hypothesize that phosphorylation of the RyR, particularly at the under-explored S2030 site, affects the Ca2+ sensitivity of the RyR channel (aim 1). This increase of Ca2+ sensitivity would also have strong repercussions in channels with the RyRR420Q mutation, and could actually precipitate the catecholaminergic polymorphic ventricular tachycardia (CPVT) during physical or emotional stress (aim 2). The simultaneously occurring SERCA stimulation could have additional ramifications for such changes of RyR Ca2+ sensitivity.1.3. Specific aims: The proposal hast two connected aims. Aim 1: To define the functional role of the RyR2-S2030 phosphorylation site from the near-molecular to the cellular level, and its interaction with other known phosphorylation sites. Aim 2 is to characterize altered Ca2+ signaling (cytosolic, SR) in the presence of the RyRR420Q CPVT mutation and, as a sub-aim, to define the role of SERCA stimulation and RyR phosphorylation (occurring in parallel during stress) in precipitating the actual arrhythmia.1.4. Experimental design: We will use transgenic mouse models and a broad variety of approaches to specifically interrogate Ca2+ signaling from the near molecular to the cellular level (in isolated cardiomyocytes). The RyR-S2814A/2808A double knock-in mouse has the two major RyR phosphorylation sites removed, but retains the S2030 site. These findings will be complemented by results from the running project on the S2030A mouse, in which the RyR lacks this site but retains the two others. Functional consequences of the arrhythmogenic RyRR420Q mutation will be examined in transgenic mice harboring this mutation and recapitulating the human disease. 1.5. Specific methods: Confocal imaging of Ca2+ signals (transients, sparks, waves) in isolated cardiomyocytes will be combined with electrophysiology techniques (patch-clamp) and photolysis of caged compounds, a combination of techniques that was pioneered by our laboratory. Intact and permeabilized myocytes will be used for the proposed studies, the latter mainly for recordings of Ca2+ signals from inside the SR. To derive information about the RyR function under specific conditions, we will analyze Ca2+ spark parameters (e.g. frequency, amplitude, restitution) and intra-SR Ca2+ concentration and wave thresholds. This will be complemented by Western blotting, to quantify protein expression and extent of site-specific RyR phosphorylation. Pharmacological tools will allow manipulation of signaling pathways. A recently adopted technique enables specific SERCA stimulation, using Fab fragments of a PLB antibody.1.6. Expected value of the proposed project:With the proposed experiments we expect to obtain new information about fundamental cellular and molecular mechanisms that enable the heart to regulate the produced force and how this regulation may be impaired in cardiac diseases, some of which lead to arrhythmias. The RyRs and the SERCAs are considered to be promising drug targets and new pharmacological compounds stabilizing the RyRs and activating SERCA are under development. Thus, besides our genuine interest to comprehend the functioning of cardiac Ca2+ signaling, a detailed mechanistic and patho-mechanistic understanding of the RyRs and their interplay with SERCA activity is of crucial importance. With the approach presented here we hope to gain key information about some of these important mechanisms and how they interact synergistically or deleteriously, by their ability to change RyR function in favorable or harmful ways, particularly in patients with HF or those carrying arrhythmogenic RyR mutations.