PROJECT SUMMARY/ABSTRACT In atrial myocytes excitation-contraction coupling (ECC) and Ca release from the sarcoplasmic reticulum (SR) have unique features that result from the lack or the irregular organization of the transverse tubule membrane system. Atrial myocytes have two types of SR, junctional (j-SR) and non-junctional (nj-SR). Ca release from j- SR is controlled by Ca entry through voltage-gated L-type Ca channels (ICa,L) whereas release from nj-SR occurs by subsequent propagating wave-like Ca-induced Ca release (CICR) driven by the newly identified 'fire- diffuse-uptake-fire' (FDUF) mechanism. IP3 receptor-induced Ca release (IICR) contributes to ECC by enhancing inotropy, but also leads to arrhythmogenic Ca release and alternans. Cardiac alternans has been linked to cardiac arrhythmia, including atrial fibrillation. Alternans is defined as beat-to-beat alternations in action potential (AP) duration (APD, electrical alternans), contraction strength and Ca transient (CaT) amplitude, and thereby generates a dynamic arrhythmia substrate. Disturbances of the bi-directional coupling of [Ca]i and membrane voltage (Vm) regulation ([Ca]i↔Vm coupling) are responsible for alternans occurrence. Sex differences in cardiac structural and electrical properties have been linked to differences in arrhythmia susceptibility and determine alternans inducibility. Focusing on the FDUF mechanism, the overall goals are to establish a mechanistic model of atrial ECC, Ca release and atrial alternans and its sex-specific attributes at cellular, cell pair and organ level. Specific aim 1. Determine FDUF-dependent mechanisms of atrial alternans. We will determine the critical role of the novel FDUF paradigm in atrial alternans, testing the hypotheses that 1) uncoupling of j-SR and nj- SR Ca release promotes 'reverse' FDUF and triggers alternans; 2) the FDUF trigger signal (ICa,L, junctional CaT) has Vm dependence, and that 3) SERCA dependent Ca uptake; 4) mitochondrial Ca buffering, energetics and redox signaling and 5) IICR modulate FDUF and alternans. Specific aim 2. Determine FDUF alternans mechanisms in cell pairs. Alternans is either Vm- or Ca-driven. Vm-driven alternans is spatially homogeneous, while Ca-driven alternans can be spatially discordant where over short distances regions alternate out-of-phase. Cell pairs define the elementary structural and functional unit of cell-cell communication. We will test 1) the spatio-temporal organization of CaT and APD alternans in cell pairs, 2) how during Ca-driven alternans the FDUF mechanism, SERCA, mitochondrial signaling and IICR determine cell pair alternans; and 3) how Vm-driven alternans precipitates CaT alternans in adjacent cells. Specific aim 3. Determine the spatio-temporal organization and mechanisms of Ca- and Vm-driven tissue alternans. We will determine at organ level (perfused hearts, live animals) the mechanisms, manifestations and spatio-temporal organization of 1) Ca-driven and 2) voltage-driven alternans, and test pharmacological interventions to reduce pro-arrhythmic alternans risk.

NARRATIVE Cardiac alternans is a recognized risk factor for cardiac arrhythmia, including atrial fibrillation, and sudden cardiac death. This study seeks to investigate the conditions that cause and sustain atrial alternans, and to determine cellular mechanisms of alternans in individual atrial myocytes, in multicellular tissue and at organ level, and to explore therapeutic interventions that reduce alternans risk.