Abstract 4140343: Role of a novel protein phosphatase 2A activating drug in the regulation of late sodium current.

Introduction: The voltage-gated Na + channel, Na v 1.5, plays a leading role in the regulation of myocyte excitability and cardiac function. However, a small “late” component of Na v current I Na ( I Na,L ) increases in response to enhanced adrenergic conditions and is linked to fatal arrhythmias. I Na,L regulation is controlled by a host of proteins, in physiological and pathological states. However, little is known regarding pathways that negatively regulate I Na,L. Our previous work showed that protein phosphatase 2A (PP2A) modulation regulates Na v 1.5 activity but the field still needs molecules that selectively target PP2A in vivo. Hypothesis: Our newly identified PP2A activating drug will negatively modulate I Na,L via regulation of Na v 1.5 Ser571 phosphorylation. Methods: We isolated and cultured ventricular myocytes (VMs) from 3 months old C57Bl/6 mice. First, we treated the VMs with 2µM of drug for 1, 2, 3 and 4 hours to determine PP2A activity (n=4/group), later PP1 activity was assessed 3-hour post treatment of drug/DMSO in the presence of 5nM okadaic acid (n=4/group). Further, protein expression was evaluated after addition of drug/DMSO to the VMs for 3 hours, followed by 10 min treatment with 1 or 2µM Isoproterenol (Iso) (2µM Iso: n=3-7, 1µM Iso: n=4-5). Furthermore, I Na current-voltage relationship and I Na,L recordings comparing drug and DMSO treated mouse VMs were performed at baseline and after Iso (DMSO: N=8; n=15, drug: N=5; n=10, N: mouse, n: cell). Lastly, immunoblots were performed using heart lysates from control and post transverse aortic constriction surgery mice (n=4/group). Results: We observed an increase in PP2A activity 3 hours post drug treatment (p=0.0326), however no change in PP1 activity was observed in drug/DMSO treated VMs. We further confirmed a reduction in Na v 1.5pSer571 (2µM Iso: p=0.02, 1µM Iso: p=0.0398) levels with no changes in Ca v 1.2pSer1927 and total Na v 1.5 expression in drug treated VMs vs. DMSO. Patch clamp recordings revealed no significant changes in I Na current-voltage relationship, instead an increase in I Na,L was seen in DMSO treated VMs post Iso (p<0.05). Drug treated VMs displayed a resistance to Iso-induced I Na,L prolongation at -35, -30, -25, -20, -15 and -10 mV voltage. Finally, increased expression of the PP2A inhibitor (I2PP2A/SET) was observed in failing heart vs non-failing control heart lysates (p=0.04). Conclusion: Our new compound activates PP2A via SET and specifically targets I Na,L in VMs.