R14del-PLN in Cardiac Function

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

2. 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

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

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

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

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

7. 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

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

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

10. R14del-PLN Inhibits Calcium Cycling in RV NO effects in LV RV R14del PLN A Force or Ca Ca B Ca SERCA SR LV 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

11. R14del-PLN Binds and Inhibits SERCA more than WT or Normal PLN (Athens Lab) 0 Calcium SERCA SERCA Calcium R14del PLN Force Force Time Time

12. 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

13. R14del-PLN Induces Spontaneous Contractions (indicative of Arrhythmias) 40 WT 30 Sponaneous Contractions ( % Cells) 20 R14del 10 0 RV +KN93 R14del+KN93 CaMKII inhibitor KN-93InhibitsSpontaneous Contractions

14. P R14del-PLN Triggers Arrhythmias through Super-Inhibition of SERCA and SR Ca-leak; KN-93 Inhibits Arrhythmias 3Na 1) R14del-PLN inhibits SERCA and this increases cytosolic Calcium2) High Calcium activates CaMKII, which phosphorylates RyR3) P-RyR leaks Calcium from SR and triggers arrhythmias 3 Na Arrhythmia Trigger Ca KN93 Ca leak CaMKIIdc RyR R14del PLN Ca Ca SERCA SR

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

16. P • 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 CaMKIIdc RyR SERCA Ca R14del PLN: Aggregates R14del PLN SR ER Stress, Autophagy Nucleus

17. 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

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