Martin Morad - US grants

This research proposal deals primarily with the processes which mediate electromechanical coupling in heart muscle. Specifically, experiments are proposed which will probe processes of Ca++ entry, Ca++ release and reuptake in the myocardial cell. Experimental work will proceed along six major directions: The first two projects deal with measurements of Ca-dye signal in single isolated cells and in intact ventricular trabeculae. Ca++ signals, as well as intrinsic optical indicators of SR activity, will be measured under voltage clamp conditions in different hearts with well known ultrastructural differences. In this way we hope to establish the contribution of various Ca++ transporting systems as well as the role of the SR in the Ca++ release and reuptake cycle. The third project deals directly with Ca++ current through the Ca++ channel. Single cell studies as well as clamp studies on isolated patches of myocardial membranes in both mammalian and frog heart are planned to identify whether the Isi channel is truly different in frog and mammalian heart, or if the difference is related to the presence of intracellular Ca++ stores in mammalian heart. The fourth project deals with experiments which probe the molecular mechanisms which mediate the relaxant effect of adrenaline on the heart. Specific experiments are planned to determine the role of Na+ pump stimulation and cyclic AMP in mediating this process. Light-sensitive caged cAMP and ion-selective Na+ and K+ electrodes will be used to test the alternate two schemes. The fifth project will probe the kinetics of myocardial contraction by using light-sensitive "caged" compounds such as EGTA, Ca++, cAMP and organic Ca++ antagonists. The use of these agents will make it possible to do step changes of ionic or drug concentrations to probe the kinetics of the Ca++ channel and development of tension. Performing such experiments on hearts with different ultrastructural components will provide structure-function information regarding the pathway of the Ca++ cycle and the source of activator Ca++. In the sixth project we shall continue our studies on the role of (Ca)i and releasable Ca++ stores in generation of pacemaker current in SA nodal cell. We hope that these studies will provide a better understanding of the processes which control myocardial excitation and contraction.

This conference will deal with mechanisms involved in modulation of cardiac contractility. A host of mechanisms and their modifications by pharmacological agents or physiological regulation will be considered. The conference will deal not only with the molecular mechanisms of Ca2+ transport across the membrane, the nature of signal transmitted information from the surface membrane to SR, the regulation of mechanism of Ca2+ release and re-uptake, the mechanism involved in modulation of myosin or phosphodiestrase isozymes, and the molecular biology of contractile proteins and membrane receptor involved in regulation of contractility, but also with a number of pharmacological agents (old and new) which are thought to modulate these processes. It is hoped that by bringing together active scientists from different disciplines we will develop some understanding of the mechanisms which regulate cardiac contractility, and get some insight into the mechanisms by which drugs alter these processes. The selection of speakers has been based on scientific excellence and proven leadership of the individual participant in the field. I have asked support only for the speakers and chairpersons of the scheduled sessions. Support for the rest of the participants (approx. 2/3 of expected participants) will come from personal funds, private resources and foreign research organizations - I hope!

The aim of the proposed research is to examine the role of Ca2+ as the transmitter signal in gating Ca2+ release channels of the SR (Ryanodine receptors) and to evaluate the role of phospholamban in regulation of relaxation in mammalian cardiomyocytes. Central to the question of Ca2+ gating is the role and effectiveness of various sarcolemmal Ca2+ transporters in gating the Ca2+ release from the SR. WE shall, therefore, first evaluate the gating efficiency of Ca2+ delivered by the Ca2+ channel versus that transported by the Na+-Ca2+ exchanger and the Ca2+-selective Na+ channel (transformed by Atrionatriuretic Peptide in atrial and ventricular myocytes of mammalian heart by quantifying the integral of Ca2+ charge traversing the sarcolemma sufficient to trigger Ca2+ release. The hypothesis at the core of this study is that there is privileged communication between the Ca2+ channels (DHP receptors) and the Ryanodine receptors. The "privileged access concept" will be further probed by testing the accessibility of Ryanodine receptor blockers (ruthenium red and caged Mg2+) and caged Ca2+-buffers (DM-Nitrophen and Diazo-2) to the Ca2+-sensing site of the SR release channels in intact myocytes. The implications of the limited access hypothesis on the inactivation of the Ryanodine receptor by Ca2+ will be explored by a novel "epi-axial" method to photorelease Ca2+ in sub-sarcolemmal space. The level of expression of various sarcolemmal Ca2+-transporting proteins in cardiac myocytes is critical to this evaluation, requiring quantification of gating efficiency of different Ca2+ delivery systems in other mammalian species. Thus, the effectiveness of Ca2+ signalling via the Na+-Ca2+ exchanger, compared to the Ca2+ channel, will be probed in hamster myocytes, which show a ten-fold higher exchanger current density than the rat myocytes. Two pathological and one development model of E-C coupling will be examined which represent up-regulated expression of either the exchanger (myopathic hamster) or Ca2+ channels (spontaneously hypertensive rats), and the low density of SR release channels (neonatal human and cat cardiomyocytes). Detailed examination of Ca2+ release and uptake in AT-1 atrial tumor cell line will be carried out, not only because AT-1 cells do not express phospholamban (Ca2+ pump regulatory protein), but also because these cells are good candidates for genetic manipulation of various molecular components of E-C coupling. Probing the role of phospholamban in regulation of Ca2+ uptake will include evaluation of the kinetics of phosphorylation of phospholamban by injection of 2D12 antibody and photorelease of caged cAMP vs. alterations in the Ca2+ sensitivity of the myofilaments in regulating the relaxant effects of isoproterenol in AT-1 cells and normal ventricular myocytes. Studies on the regulation of Ca2+ uptake will be carried out in AT-1 cells transfected with various fragments of phospholamban to pinpoint its structure-function relationship. A newly-developed single-cell spectrofluorometer will be used in some experiments to monitor the activity of more than one dye simultaneously and quantify the photorelease of caged Ca2+ prior to and following photorelease. By exploring the properties of Ca2+ release and uptake systems in their native environment, we shall, to some extent, follow the signalling pathways to their points of origin and characterize a) the adequacy of our experimental interventions, b) the role of the Ca2+ channel and Na+-Ca2+ exchanger and their differential expression in different animals and pathological states, and c) the regulation of the Ca2+-ATPase by phospholamban.

This research proposal aims to examine the fundamental molecular mechanisms that underlie the signaling of contraction in the mammalian heart. Previous studies have shown that cardiac signaling takes place in microdomains surrounding the dihydropyridine/ryanodine receptor complex, via interchange of Ca2+ signals criss-crossing between these two proteins and the Na+-Ca2+ exchanger. Such studies also show that the gain of ICa-gated release was voltage-dependent, suggesting that the Ca2+ release complex is either regulated by more than one mechanism or that there are different populations of Ca2+ release units with different gating modes. The central idea we propose to test is that the signaling of contraction in the heart might be multi-modally gated. Evaluation of this hypothesis requires not only precise identification of the duration, magnitude, and cellular location of such release sites, but also the ability to monitor many such sites simultaneously in order to differentiate between their gating, voltage dependence, and regulation by metabolic factors. Using a method that limits the diffusion of Ca2+ to about 50 nm, we propose to monitor 50-300 Ca2+ release sites simultaneously in areas of approximately 20 x 50 mum2 of a cardiac myocyte employing rapid two-dimensional confocal imaging. We shall specifically: a) characterize the kinetics of focal Ca2+ release sites as a function of voltage, ICa, Ca2+ load of the SR, phosphorylation, and pharmacological interventions; b) measure the distribution of focal Ca2+ release sites relative to ultrastructural determinants in both ventricular and atrial cells (i.e. cells with and without t-tubules); c) identify distribution of focal release sites relative to gating by voltage, ICa, INaCa, INa; d) measure the properties of focal release sites as a function of redox state, and temperature; e) explore whether the C-terminal tail of Ca2+ channel is the conduit to both Ca2+ and voltage signaling. It is our contention that techniques that can monitor 100s of release sites may be required to examine the multiplicity in the mechanisms of Ca2+ signaling in the heart. The proposed studies may provide a better insight into fundamental molecular mechanisms that regulate the signaling of contraction in normal and diseased heart.

DESCRIPTION (Adapted from Investigator's Abstract): This research proposal is designed to advance a study of heterogeneity, patterns of expression and molecular correlates for regulation of the class C voltage-gated L-type Ca2+ channel using molecular and electrophysiological approaches. The long-term objective is to pursue the study of the structure-functional alterations of the Ca2+ channel pore-forming alpha 1C subunit due to alternative splicing, and to explore the affected molecular correlates for the channel inactivation. The molecular mechanisms of channel inactivation will be studied using two of the identified alpha1C channel isoforms, one deprived of inactivation and the other lacking the Ca2+-dependent inactivation. The hypothesis states that Ca2+-induced inactivation of the channel is mediated by the interaction of the pore-associated site(s) with Ca2+ sensors recently discovered in the cytoplasmic C-terminal tail of alpha1C. To examine molecular correlates for the regulation of the alpha1C channel by Ca2+ sensors, the P.I. will investigate whether they are differentially targeted by the pore-permeating and cytoplasmic Ca2+. Studies will determine if Ca2+ sensors contribute to the mechanisms controlling the conductance and ion selectivity of the channel. The P.I. will directly identify the molecular target for the Ca2+ sensor(s)-controlled inactivation gates. In addition, the P.I. will examine which Ca2+-sensor of the alpha1C channel is involved in the local Ca2+ signaling with the ryanodine receptor of the cardiac myocytes. Results may give important insights into the fundamental principles of Ca2+ signaling underlying excitation-contraction coupling in human cardiac and vascular muscle cells and provide useful clues for the molecular diagnostics and drug developments.

DESCRIPTION (provided by applicant): The overall goal of this proposal is to examine whether protons can serve as co-transmitters in cholinergic neuronal synaptic signaling. The rationale for this possibility carne from our recent findings that acidification uniquely modulated the recombinant neuronal nicotinic acetylcholine receptors (nAChRs) in part based on their desensitization kinetics and their beta subunit composition. In sharp contrast to reports that acidification generally inhibits ligand- and voltage-gated cation channels, we found that rapid (approximately 20 ms) acidification enhanced the alpha3/beta4 neuronal nAChR current and accelerated its activation and decay kinetics. Since it is well known that: a) repetitive firing in the brain causes brief acidic shifts, b) ACh is stored in acidic vesicles, and c) release of vesicular protons inhibits presynaptic Ca2+ channels, we hypothesize that brief intermittent acidification of synaptic cleft modulates the postsynaptic nAChRs providing focal plasticity to synaptic signaling. The experiments proposed here are aimed at: 1) examining, on a time scale approximating synaptic transmission, how acidification modulates the native neuronal nAChRs in primary culture of chromaffin cells, cortical, and cerebellar neurons; 2) imaging the microdomains of acidification and probing for presence of "proton sparks" with transmitter release in PC12 and chromaffin cell cultures; 3) evaluating directly the roles of released protons in modulation of synaptic signaling in cortical and cerebellar neurons. To test this hypothesis under feasible experimental conditions, we will use two models of neurosecretion: 1) PC12 and chromaffin cells and 2) cultures of cortical and cerebellar neurons, where agonist and protons may be applied rapidly (approximately 2 ms) and briefly, and membrane currents measured simultaneously. In chromaffin cells and cortical neurons, we shall quantify the effect of protons in modulation of nAChRs, and image the acidification profiles resulting from the release of secretory vesicles into paracellular space, using custom-made pH-sensitive dyes, in combination with rapid (240-480 f/s) confocal microscopy and a novel high-resolution optical technique (Total Internal Reflection Fluorescence Microscopy, TIRF), developed in our laboratory. Our finding, we believe, will provide novel insights into synaptic signaling that may be critical in the pathogenesis of neurodegenerative diseases such as Alzheimer's, where proton content of synaptic vesicle may be altered leading to impaired cholinergic signaling.

DESCRIPTION (provided by applicant): In working myocardium, acute blockage of blood flow is followed by a rapid drop in oxygen tension that within minutes causes irreversible tissue damage. The onset of ischemic infarction is marked by a cascade of events that at the cellular level includes reduced energy production (-AMP, / ATP, -gycolysis, /pH, -ROS), altered ion channel activity (/ICa, -[K+]o, -[Na+]i), and impaired Ca2+ signaling (/ICa, -diastolic [Ca2+]i) leading eventually to arrhythmia, cardiomyopathy, and heart failure. We hypothesize that the onset of cardiac hypoxia (<60 s) is first detected by a Ca2+ channel regulatory mechanisms leading to rapid channel current suppression long before the global cellular metabolic manifestations (/ATP, /pH, -ROS etc.). To test this hypothesis, we shall perform experiments on single cardiomyocytes exposed to step changes in oxygen tension while ICa and [Ca2+]i are monitored using voltage-clamp and Ca2+-imaging techniques. The changes in pO2 will be implemented with a rapid perfusion system (<50 ms), and will be monitored in the immediate vicinity of the cells. The specific aims are: 1) To characterize the ionic-, voltage-, and phosphorylation- dependence of suppression of ICa in response to acute hypoxia, and 2) To identify the molecular entity that detects the loss of oxygen and the signaling pathway that leads to the modulation of the Ca2+ channel. Significance and Impact: The proposed research might be directly relevant to the management of patients who suffer periods of cardiac hypoxic ischemia. The results may establish hypoxia-induced suppression of ICa as an inherent first line of defense that preserves metabolic energy and delays Ca2+ overload. In a wider perspective, it is important to sort out the various regulatory pathways that are triggered by hypoxia and/or converge to control ICa, force of contraction, and expenditure of ATP. In turn, recognition of the independence or interdependence of these pathways may serve to identify prophylactic and therapeutic options that are relevant to all stages of acute and chronic cardiac hypoxia including e.g. the onset of reperfusion where suppression of ICa is already clinically used to prevent ensuing arrhythmias. If the proposed O2 sensor does indeed contribute significantly to the control of the Ca2+ channel, it may lead to development of new class of therapeutics for treatment of cardiac injury in general. Innovation: It is a novel idea that the suppression of ICa by acute hypoxia can be triggered by a rapid regulatory pathway long before significant occurrence of changes in the cellular energy metabolism, ionic gradients and redox state. To test this idea, we use an array of electrophysiological, optical, and molecular technique that provide simultaneous measurements of key signaling parameters and are suited for kinetic studies. To explore clinical relevance we shall expand the experimental scope from standard animal models to also include available human cardiac cells and cells from the right and left ventricles. PUBLIC HEALTH RELEVANCE: The human heart suffers irreversible damage when its supply of oxygenated blood is interrupted even briefly by coronary thrombosis. We shall explore an inherent, potentially protective, mechanism whereby heart cells sense oxygen deprivation and respond rapidly to husband their energy resources by down-regulating their calcium channels, which are essential in maintaining the rhythm and strength of the heart beat. This project may provide insight into the multi-faceted function of one of the key proteins of the heart, and help us identify the oxygen sensor of the heart and develop new therapeutic strategies for treatment of the diseased heart.

Project Summary/Abstract: Cardiac contractility is regulated by transient release of Ca2+ form the sarcoplasmic reticulum through type-2 ryanodine receptor (RyR2), a tetrameric ~5000 amino acids protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase and phosphatase, and RyR2-stabilizing protein, FKBP12.6. Since a number of RyR2 missense mutations have been reported to associate with lethal cardiomyopathies, a better understanding of regulatory mechanisms of RyR2 is essential for prevention and treatment of these pathologies. Two major research strategies, heterologous cell expression in HEK293 cell lines carrying RyR2 mutations and transgenic mouse models expressing mutant RyR2, have been thus far used to study structure/function relationship of RyR2 and its pathological consequences. These approaches have advanced the understanding of RyR2 regulatory mechanisms, but suffer from inherent drawbacks of cells with non-cardiac genetic backgrounds or size and electrophysiological differences between human and mice. Thus, functional consequences of RyR2 mutagenesis remain to be fully explored in human myocardial model system. In this proposal, we aim to establish a new research platform where RyR2 mutagenesis is carried out in cardiomyocytes derived from human-induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 gene editing directed to Ca2+ and caffeine binding sites associated with cardiac pathology. Toward this end, we set three specific aims: (1) Establish the RyR2 mutagenesis in hiPSC-derived cardiomyocyte as a reliable platform to reproduce the calcium signaling aberrancies associated with the arrhythmia-linked mutations, by comparing the Ca2+ signaling aberrancies of gene-edited F2483I-RyR2 mutation carrying myocytes with cells derived directly from patient biopsies harboring the same mutations, and previously characterized by us, (2) Characterize functional consequences of mutating the potential Ca2+ and caffeine binding site of RyR2, recently identified in the high resolution cryo-electron microscopy studies, (3) Characterize the Ca2+ signaling phenotypes of RyR2 mutations associated with cardiac pathology in the three structurally distinct domains of RyR2. The membrane currents and global and focal intracellular Ca2+ signals of wild type and mutant hiPSC-derived cardiomyocytes will be quantified in patch-clamped myocytes imaged by confocal/TIRF microscopy using genetically engineered Ca2+ fluorescent probes targeted to various nodes of Ca2+ signaling proteins. This novel approach may enable us to systematically characterize the phenotype of the mutant RyR2 in cells with more relevant genetic background of human cardiac cells in time-effective manner, leading hopefully to better understanding of molecular mechanism of RyR2 regulation and cardiac excitation-contraction coupling based on the near-atomic structural model of RyRs.

Project Summary/Abstract: Cardiac contractility is regulated by Ca2+ release form the sarcoplasmic reticulum through ryanodine receptor (RyR2), a protein with multiple regulatory domains for Ca2+, Mg2+, protein kinase, caffeine and FKBP12.6. Since a number of RyR2 missense mutations associate with lethal cardiomyopathies, a detailed understanding of regulatory mechanisms of RyR2 is essential for treatment of these pathologies. Two strategies of heterologous expression of recombinant RyR2 mutants in HEK293 cells and transgenic mouse models, have been used to study structure/function relationship of RyR2 and the functional consequences of disease-linked RyR2 mutations. Although these approaches have provided new insights into RyR2 regulatory mechanisms, they have inherent drawbacks of cells with non-cardiac genetic background and differences in human and mice hearts. We have therefore established an alternate research platform where RyR2 mutations are introduced in human induced pluripotent stem cells (hiPSCs)-derived cardiomyocytes (CMs) using CRISPR/Cas9 gene-editing. Mutant myocytes are then cultured in media that matures them structurally and functionally toward adult cardiomyocyte state. Using this human myocyte platform, we propose to examine molecular mechanisms underlying Ca2+, caffeine, and FKBP regulation of RyR2 associated with CPVT1 pathology. Specifically we aim: 1) To compare Ca2+-signaling consequences of domain specific CPVT1-associated RyR2 mutations expressed in ?mature? hiPSC-CMs , rescue their phenotype by back-mutagenesis, and determine their drug specificity; 2) To characterize the functional consequence of mutating the RyR2 Ca2+ and caffeine binding sites, predicted from near atomic structure and determine their interaction; and 3) To characterize mechanisms underlying loss-of-function CPVT1-associated RyR2 mutations and identify the difference between Ca2+ leaky and non-leaky mutations. To accomplish these aims we propose to create multiple mutant lines of our more mature hiPSC-CMs carrying the different RyR2 mutations and examine their Ca2+ signaling aberrancies. Membrane currents and intracellular Ca2+ signals of wild type and mutant hiPSC- derived cardiomyocytes will be quantified in patch-clamped myocytes imaged by confocal/TIRF microscopy using genetically encoded Ca2+ probes targeted to various nodes of Ca2+ signaling pathway. We will also use [3H]ryanodine binding assay, to determine possible alterations in affinities of Ca2+, caffeine and accessory proteins. To assure the reliability of our hiPSC-platform, we will compare the Ca2+ signaling aberrancies of mutagenesis in hiPSC-CMs with in vivo knock-in of RyR2 mutations in mouse models. We hope that our novel approach will make it possible to systematically characterize the phenotype of the CPVT1 mutants, as well as non-CPVT1 mutants with implication to atomic structure of RyR2, in human myocardium, thus providing a novel and synergistic human platform for studies of RyR2 regulation.