Rational Design from Cryo-EM Structures of High-Affinity Ryanodine Receptor Ligands Based on Natural Peptides

SUMMARY Ryanodine receptors (RyRs), are the Ca2+ release channels of sarcoplasmic reticulum (SR) that play an essential role in excitaton-contraction coupling of cardiac and skeletal muscle cells. Mutations in the cardiac RyR gene (RYR2) are associated with catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic syndrome characterized by the development of adrenergically-mediated ventricular tachycardia and sudden death. CPVT is clearly an arrhythmogenic disorder stemming from intracellular Ca2+ mishandling caused by RyR2 dysfunction. As such, insights gained from CPVT syndromes benefit our mechanistic understanding of other cardiomyopathies where RyR2-linked Ca2+ mishandling plays a pivotal role. RyR2 dysfunction brings about deleterious effects in the heart by inducing SR Ca2+ “leak” and/or spontaneous Ca2+ release (SCR), a sudden, diastolic Ca2+ bolous of critical mass that triggers arrhythmic activity. Thus, reigning in RyR2 hyperactivity to mitigate SR Ca2+ “leak” and avoid SCR is a primary goal of therapeutic regimes for major cardiomyopathies. A group of globular peptides termed calcins target RyRs with high affinity and specificity. Imperacalcin (IpCa), the founding member of the calcin family, engages RyRs by entering through their wide vestibule and binding with exquisite precision to a site deep in the cytosolic “cap” of the channel, adjacent to the transmembrane region, acting as a wedge that “pushes” laterally the transmembrane helices that line the channel pore, causing it to open and giving rise to a long-lived subconductance state. In animal models of CPVT, IpCa penetrates the external membrane of ventricular myocytes and induces a partial depletion of SR Ca2+, thus preventing SCR and in effect relieving the adrenergically-mediated Ca2+ overload that triggers Ca2+-dependent arrhythmias. Thus, IpCa behaves naturally as an agonist of RyRs, with anti-arrhytmic effect in cardiomyopathies where SCR is the primary trigger of arrhythmias, and by rational design of IpCa analogs capable of closing the natural “grooves” it creates with RyRs, it has the tangible potential to become the first high-affinity RyR blocker that may prevent SR Ca2+ “leak”. Using cryo-EM structures of calcin-RyR complexes and a series of functional assays, aim 1 will determine the structural domains of calcins that allow them to bind to RyRs with exquisite affinity and specificity, and to generate calcin analogs capable of blocking RyR ion conduction. Aim 2 will use CPVT mouse lines that readily present SCR during sympathetic stimulation and a novel rabbit CPVT model that presents constitutive SR Ca2+ leak and pathological cardiac remodeling to determine whether native and mutant calcins are capable of preventing RyR dysfunction-triggered arrhythmias. Our multi-disciplnary program, with well-defined deliverables and milestones and led by two PIs experts in structural (Van Petegem) and functional (Valdivia) biology of RyRs, will produce a novel paradigm for the treatment of Ca2+-dependent arrhythmias, an area severely underserved by existent pharmacotherapy.