Abstract 11423: Mitochondrial Calcium Flux Modulates Pacemaker Activity in Mouse Stem Cell Derived Cardiomyocytes

Abstract

INTRODUCTION: Ca2+ release from sarcoplasmic reticulum (SR) is known to contribute to the pacemaker activity in embryonic stem cells (ESC) derived cardiomyocytes (CMs). Mitochondria are known to participate in Ca2+ cycling. Nevertheless, the role of mitochondria in pacemaker activity is unclear. We studied the role of mitochondrial Ca2+ flux in spontaneously activity of ESC derived CMs. METHODS: CMs were derived from Wt and ryanodine receptor type 2 knockout (RYR2-/-) mouse ESC. Action potentials (APs) were recorded by perforated whole-cell current-clamp. Cytoplasmic and mitochondrial Ca2+ transients were determined by Fluo-4 and Rhod-2 respectively. Mitochondrial Ca2+ uniporter (MCU) siRNA was used. The mRNA level was evaluated by qPCR. RESULTS: As predicted, SR Ca2+ handling inhibitors, 10 μM ryanodine and 2 μM 2-APB, reduced spontaneous beating rate to 56% and 73% respectively in Wt CMs. Inhibition of mitochondrial Ca2+ flux by 10 μM Ru360 showed a similar inhibition effect on the pacemaker activity as 2 μM 2-APB in Wt CMs. To isolate the mitochondrial component, we used RYR2-/- CMs. In these cells, MCU inhibition by pharmacological or molecular biological means reduced beating rate. The MCU mRNA decreased by 96% after MCU siRNA silence 72 hrs (p<0.01). AP and mitochondrial Ca2+ transient synchronous recording revealed that the reduction of spontaneous beating rate accompanied with the depressed mitochondrial Ca2+ uptaking and releasing. In RyR2-/- CMs, 2 μM 2-APB could significantly lower the spontaneous beating rate. While 2 μM 2-APB was applied to MCU silenced RyR2-/- CMs, the beating rate couldn’t be slowed down further. This indicated IP3 receptors reduced spontaneous beating rate via MCU. Thapsigargin could substantially slow down beating rate like 2-APB. Caffeine depletion experiments showed other ryanodine receptor subtypes didn’t contribute Ca2+ release in RyR2-/- CMs. A L-type Ca2+ channel block, 10 μM nifedipine, couldn’t reduce beating frequency. This indicated spontaneous beating rate is Ca2+ influx independent in RyR2-/- CMs. CONCLUSIONS: Mitochondrial Ca2+ handling plays an important role in decreasing spontaneous beating rate. IP3R reduced spontaneous beating rate through MCU.