Background and Aim
The Takotsubo syndrome (TTS) is a transient cardiac disorder characterized by acute left ventricular dysfunction, typically occurring in the absence of coronary artery obstruction. Mitochondrial Ca2+ concentration ([mtCa2+]) represents a potential key factor in its pathophysiology, as it has been shown to be altered in various cardiac diseases. A reduction in [mtCa2+] may impair mechano-energetic coupling leading to an imbalance between energy supply and demand, whereas excessively high [mtCa2+] can trigger cell death. The aim of this project was to investigate the role of [mtCa2+] in the development of TTS using a human stem cell-based TTS model and a genetically encoded Ca2+ indicator (GECI).
Methods and Results
We used induced pluripotent stem cell-derived ventricular cardiomyocytes (iPSC-CM) from a TTS patient and compared them with two healthy controls. First, we established a ratiometric [mtCa2+] measurement method using the GECI ratiometric-Pericam-mt in iPSC-CMs. The [mtCa2+] was significantly higher in TTS iPSC-CM compared to control iPSC-CM under basal conditions. Moreover, isoprenaline treatment (100nM) enhanced [mtCa2+] significantly in TTS iPSC-CM compared to basal conditions, but not in control cells suggesting a higher sensitivity of TTS iPSC-CM to β-adrenergic activation. Elevated [mtCa2+] in TTS is in line with increased mitochondrial membrane potential using TMRM in TTS compared to control as higher levels lead to increased oxidative phosphorylation in mitochondria causing higher mitochondrial membrane potential.
In order to analyze mitochondrial Ca2+ transporters and associated proteins, we used qPCR for gene expression analysis. The Ca2+ influx-regulating proteins MICU2 and the Voltage-Dependent Anion Channel 2 (VDAC2), as well as the NCLX-regulating gene TMEM65 were downregulated in expression in TTS compared to control.
Since TMEM65 enhances [mtCa2+] efflux and MICU2 functions as a gatekeeper for [mtCa2+] influx, this might be a potential reason for increased [mtCa2+] in TTS. VDAC2 enables [mtCa2+] influx and may be downregulated in TTS as a compensatory mechanism. No change in gene expression was observed for MCU, MICU1, NCLX and Mfn2 between TTS and control.
We demonstrated further that nocodazole-induced microtubule disruption significantly reduced [mtCa2+], an effect that was no longer observed after rescue with the MCU enhancer ezetimibe. These findings indicate that microtubules are crucial for [mtCa2+] regulation and that ezetimibe enhances [mtCa2+] levels.
Conclusion
In conclusion, we demonstrated elevated [mtCa2+] levels and increased mitochondrial membrane potential in TTS iPSC-CMs, which may be explained by changes in the abundance of mitochondrial Ca2+ transporters. Altogether, these findings make [mtCa2+] an interesting potential therapeutic target in future TTS research.
