R14del-PLN in Cardiac Function Litsa Kranias, Ph.D. University of Cincinnati College of Medicine

What is PLN? PLN Is a Regulator of the Heart’s Pumping Action Heart beats: ~70x/min or 100,000x per day 80ml or 1/3 cup per beat When we run and exercise: there is a signal to the heart to pump stronger and send more blood to periphery; PLN plays a key role Relax Contract

PLN Regulates Calcium Cycling and Contractility in Cardiac Cells channel 5 6 7 8 pCa RR Pump PLN Myofillaments SR

R9C, R9L, R9H, R14del, R25C, L39stop, and V49G Human PLN Mutations: R9C, R9L, R9H, R14del, R25C, L39stop, and V49G R V I IB II L R R IB C II L V 4

Dead, no cardiomyopathy 2006: PLN-R14Deletion in a Greek Family I II III IV (+) index V (+) (+) (+) (+) VI (+) VII Dead, no cardiomyopathy PLN-R14 Deletion PLN-Normal Dead, cardiomyopathy + Cardiomyopathy Not determined

PLN-R14Del Mutation in a Subfamily Pedigree II I III IV V DCM Dead, no cardiomyopathy PLN-R14Del Not tested Dead, CM PLN-Normal

Greek Patients with PLN-R14Del Mutation 10yrs-mid 30s yrs: asymptomatic, abnormal characteristic EKG 30yrs-on: heart failure symptoms, contractile dysfunction, ventricular arrhythmias Arrhythmogenic RV cardiomyopathy and DCM for every Greek carrier Netherlands: Van der Swaag et al., 2012

How Does R14del-PLN Cause Arrhythmias and HF? Generate Animal Models to mimic the human disease Humanized models: Roger’s team WT-PLN R14del-PLN

Isolate and study Right and Left Ventricular Myocytes RV LV RV cells LV cells RA LA RV LV Oxygenated blood Deoxygenated blood

R14del-PLN Inhibits Calcium Cycling in RV NO effects in LV RV R14del PLN A Force or Ca Ca B Ca LV SR SERCA Time A: Ca removal is slow; Relaxation and filling of RV is SLOW B: Ca remains high; RV does not fully relax to fill in with blood Time

R14del-PLN Binds and Inhibits SERCA more than WT or Normal PLN (Athens Lab) Calcium Calcium SERCA SERCA PLN R14del Force Force Time Time

R14del-PLN Increases Ca2+ Sparks: Defects in Calcium Cycling WT R14del 50 µm 1 s Ca-sparks Ca-sparks RyR RyR WT-PLN R14del PLN Ca Ca SERCA SERCA SR SR

R14del-PLN Induces Spontaneous Contractions (indicative of Arrhythmias) 40 WT 30 Sponaneous Contractions ( % Cells) R14del 20 10 R14del+KN93 RV +KN93 CaMKII inhibitor KN-93 Inhibits Spontaneous Contractions

R14del-PLN Triggers Arrhythmias through Super-Inhibition of SERCA and SR Ca-leak; KN-93 Inhibits Arrhythmias 3Na 3 Na Arrhythmia Trigger Ca KN93 Ca leak P CaMKIIdc RyR R14del PLN Ca SERCA Ca SR 1) R14del-PLN inhibits SERCA and this increases cytosolic Calcium 2) High Calcium activates CaMKII, which phosphorylates RyR 3) P-RyR leaks Calcium from SR and triggers arrhythmias

The Function of PLN and Mutant-PLN SR Ca leak; Arrhythmias P JNK TRI CamKII JNK RyR RyR TRI CSQ SERCA CSQ SERCA PLN R14del-PLN Ca Ca SR SR WT-PLN R14del-PLN

Two Steps in r14del-pln pathology: Early: serca inhibition and calcium -leak Late:aggregate formation 3Na 3 Na Arrhythmias Autophagy, Remodeling. Lipid droplets Ca Ca leak P CaMKIIdc RyR SERCA Ca R14del PLN: Aggregates SR R14del PLN ER Stress, Autophagy Nucleus

The future looks bright Many cardiovascular diseases have extended windows for effective therapeutic treatments as the disease can take months or even years to reach a level sufficient to produce symptoms. This provides an opportunity to first model the particular disease, test the most effective treatments in an animal model, and then personalize a treatment that can be delivered at any stage, or even prophylactically before symptoms present. J James and J Robbins, Circ Res 2016

Team Effort Towards Therapy University of Cincinnati Stanford University The Mount Sinai Medical Center The Netherlands Heart Institute University of Gottingen Biomedical Research Foundation Academy of Athens