Abstract 545: A Multifunctional Genetic Probe for Investigating Mitochondrial Calcium Dynamics in Cardiac Cells

Abstract

Introduction: Calcium cycling plays a critical role in regulating function of cardiomyocytes (CMs) under both physiological and pathological conditions. Among their many important functions, mitochondria are known to involve in calcium handling. However, little is known about the calcium dynamics of human stem cell-derived cardiomyocytes (hiPSC-CM) and what little is known shows much greater variability than what is seen in adult cardiomyocytes. Methods: We produced adenovirus encoding the red Ca 2+ indicator RCaMP prepended with a mitochondrial targeting sequence fused to the green Ca 2+ indicator GCaMP with a cleavable linker. We then used this virus to induce expression of this calcium probe in CMs & recorded calcium activity with a confocal microscope. Results & Discussion: Mitochondrial targeting was confirmed by confocal imaging showing GCaMP (green), mRCaMP (red), and costaining with Mitotracker Deep Red (Magenta) (Figure 1A, 1B). Probe functionality was validated by observing beat-by-beat calcium dynamics in paced neonatal rat CMs with and without blocking mitochondrial calcium uptake with the mitochondrial calcium uniport inhibitor Ru360. Recording GCaMP and mRCaMP dynamics in adult CMs (Figure 1C) showed synchronous beat-by-beat calcium activity in the cytosol and mitochondria. However, iPSC-CMs (Figure 1D) showed variable mitochondrial activity, with some synchronous mitochondria and some showing inverse activity. Conclusions: hiPSC-CMs and adult CMs show differences in beat-by-beat mitochondrial calcium dynamics, offering a potential investigatory target for improving the therapeutic effect of stem cell-derived heart therapies.