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Principal Investigator: Donald M Bers
Organization: UNIVERSITY OF CALIFORNIA AT DAVIS
Fiscal Year: 2022
Award: $760,168
Funding agency: National Heart Lung and Blood Institute
Project Summary
Our overall goal is to understand the structural mechanisms for the functional dysregulation of the cardiac
ryanodine receptor (RyR2) Ca2+-release channels in the sarcoplasmic reticulum (SR). RyR2 contributes to and
is a therapeutic target for treating heart failure (HF) and arrhythmias. The proposed studies take the new
knowledge obtained in the current award period to new levels of insight with novel methods and goals (below).
Our work has developed a new integrated mechanistic paradigm explaining how RyR2 becomes dysfunctional
and promotes arrhythmias, and systolic and diastolic dysfunction in HF. We discovered a common pathological
RyR2 structural state that causes diastolic SR Ca leak, identified key physiological modulators of this state –
calmodulin (CaM), ROS, Ca/CaM-dependent kinase (CaMKII) –, and devised novel drug discovery strategies
to target leaky RyR2 conformation. The core observation: CaM inhibits (a) RyR2 Ca leak and (b) RyR2 binding
of DPc10 biosensor peptide. In HF, RyR2 readily binds DPc10 but CaM affinity is reduced and Ca leak is high.
These 3 HF RyR2 properties can be induced by CaMKII and ROS (both increased in HF) and DPc10, but
dantrolene or high [CaM] reverses each of them. Combining this conformation-shifting paradigm with our
FRET-based molecular tools has enabled us to start a drug discovery program to seek novel therapeutics for
HF and CPVT, and this is already yielding promising compounds.
The influence of CaM-CaMKII-RyR2 interactions on cardiac function and dysfunction are widely appre-
ciated. Here, we extend our mechanistic progress to determine the structural basis of functional CaM-CaMKII-
RyR2 effects. We develop a new FRET-based dynamic conformational approach with functioning RyRs, to test
hypotheses suggested by cryo-EM static structural snapshots of frozen, detergent-purified RyR. For example,
cryo-EM and FRET-based mapping place apo(Ca-free)-CaM in similar locations on RyR1 (skeletal muscle)
and RyR2 (heart), but the RyR2 position of Ca-CaM is controversial. We found that human CaM mutants linked
to CPVT outcompete WT-CaM/RyR2 binding, but promote (rather than inhibit) arrhythmogenic SR Ca leak.
This apo-CaM effect is akin to RyR1 activation by WT-CaM. So structure-function analysis of WT and CPVT-
CaMs (as we plan) may be the key to expose the molecular mechanism of CaM-RyR regulation. We will test
this using our evolving RyR-targeted FRET toolkit to correlate state-of-the-art confocal studies in relatively
intact cardiomyocytes (Bers Lab) with fluorescence lifetime detection of FRET within RyR2 in SR vesicles
(Cornea Lab) and emerging high-resolution structural information. Our success with RyR-CaM-FKBP, the
importance of CaMKII in RyR regulation, and our parallel CaMKII FRET tool development, drive us to enhance
understanding of dynamic CaMKII with new mechanistic clarity. Specific Aims are to resolve the (1) structural
basis of CaM regulation of RyR2 vs. RyR1, (2) Ca-dependent kinetics of CaM-RyR2 structure-function in SR
and myocytes, (3) differences in CaMKII physical states upon direct CaM and autonomous activation.
Terms: <21+ years old><Address><Adult><Adult Human><Affinity><American><Arrhythmia><Award><Back><Binding><Biosensor><Ca Release Channel-Ryanodine Receptor><CaM KII><CaM PK II><CaM kinase II><CaMKII><Calcium><Calcium-Dependent Activator Protein><Calcium-Dependent Regulator><Calcium-Ryanodine Receptor Complex><Calmodulin><Cardiac><Cardiac Arrhythmia><Cardiac Muscle Cells><Cardiac Myocytes><Cardiocyte><Cats><Cats Mammals><Cell Body><Cells><Complement><Complement Proteins><Complex><Cornea><Cryo-electron Microscopy><Cryoelectron Microscopy><Cytoplasm><Dantrolene><Data><Detection><Detergents><Diastole><Disease><Disorder><Dissociation><Domestic Cats><Dorsum><Dysfunction><Electron Cryomicroscopy><Engineering><Exposure to><FK-506-Binding Protein><FK506 Binding Proteins><FKBP><FKBP Rotamase><FRET><Feline Species><Felis catus><Felis domestica><Felis domesticus><Felis sylvestris catus><Fluorescence><Fluorescence Resonance Energy Transfer><Freezing><Functional disorder><Förster Resonance Energy Transfer><Goals><Health><Heart><Heart Arrhythmias><Heart Muscle Cells><Heart failure><Heart myocyte><Human><Isoforms><Kinases><Kinetics><Knowledge><Label><Link><Location><Maps><Measures><Memory><Methods><Modeling><Modern Man><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Muscle Cells><Myocardial depression><Myocardial dysfunction><Myocytes><Nodal><Pathologic><Pathology><Peptides><Phosphodiesterase Activating Factor><Phosphodiesterase Protein Activator><Phosphotransferase Gene><Phosphotransferases><Physiologic><Physiological><Physiopathology><Position><Positioning Attribute><Property><Protein Isoforms><Public Health><Publications><Recombinants><Regulation><Reporter><Research><Resolution><RyR1><Ryanodine Receptor><Ryanodine Receptor Calcium Release Channel><Sarcoplasmic Reticulum><Scientific Publication><Site><Skeletal Muscle><Structure><Systole><Tacrolimus Binding Proteins><Testing><Therapeutic><Time><Transphosphorylases><Ventricular><Vesicle><Voluntary Muscle><Work><adulthood><base><biological sensor><calcium-dependent CaM kinase II><calmodulin-dependent protein kinase II><cardiac dysfunction><cardiac failure><cardiac function><cardiomyocyte><conformation><conformational state><corneal><cryo-EM><cryoEM><design><designing><drug discovery><function of the heart><heart dysfunction><heart function><innovate><innovation><innovative><insight><mutant><new drug treatments><new drugs><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><next generation therapeutics><novel><novel drug treatments><novel drugs><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutics><novel therapy><novel therapy approach><oxidation><pathophysiology><physical state><programs><receptor binding><receptor bound><receptor structure function><response><success><temporal measurement><temporal resolution><therapeutic target><time measurement><tool><tool development>