Genetic enhancement of mitochondrial Ca2+ buffering capacity prevents apoptotic signaling activation in response to cytosolic Ca2+ elevation
Authors: Yang Benjamin , Cypress Michael , Nieto Bridget , Jhun Bong Sook , O-Uchi Jin 
Published: May 2024

Introduction: Over the past decade, multiple groups have used genetically engineered mitochondrial Ca2+ (mtCa2+)-sensitive biosensors to delineate the role of mtCa2+ uptake in various cell types/tissues. It has been widely shown that inhibition of mtCa2+ influx via the main mtCa2+ channel, mtCa2+ uniporter (MCU), can mitigate increases in free mitochondrial-matrix Ca2+ concentration ([Ca2+]mt) in response to cytosolic Ca2+ elevation, which prevents overproduction of reactive oxygen species, opening of mitochondrial transition pore (mPTP), and activation of apoptotic signaling. However, chronic interruption of the mtCa2+ handling system may be unfavorable for maintaining physiological cellular functions. Inhibiting mtCa2+ influx may secondarily increase cytosolic Ca2+ concentration ([Ca2+]c) in the absence of MCU activity for cytosolic Ca2+ clearance, activating signaling pathways that cause cellular damage. [Ca2+]mt is determined by the balance of mtCa2+ influx, efflux, and Ca2+ buffering capacity at the mitochondrial matrix. However, the molecular identity of mitochondrial Ca2+ buffering machinery is still unknown. Hypothesis: Enhancement of mtCa2+ buffering capacity by overexpressing mtCa2+ biosensors can prevent mtCa2+ overload by chelating excessive free Ca2+ at the mitochondrial matrix and providing anti-apoptotic effects during cytosolic Ca2+ elevation. Methods: HEK293T cells and H9c2 cardiac myoblasts transfected with mtCa2+ biosensors underwent biochemical, cell biological, and physiological assays. In this experiment, we tested Mitycam, a mitochondria-targeted inverse pericam (Kettlewell S, et al, 2009), and its mutant Mitycam-E31Q, a lower Ca2+-binding affnity version of the original Mitycam (Haviland S, et al, 2014). Results: First, we used Western blotting with an anti-GFP antibody to confirm that the original and mutant Mitycams were expressed at a similar protein size in HEK293T cells and H9c2 cells. The expression level of Mitycam-E31Q was significantly higher than the original Mitycam (55.4±12.9% increase, n=3, p<0.05) incresedespite the equal amount of plasmids transfected. Next, we observed that both Mitycams were exclusively localized in mitochondria by confocal microscopy after co-transfection with mitochondrial matrix-targeted DsRed (mt-RFP) in HEK293T cells and H9c2 cells. A decrease in Mitycam fluorescence in response to transient [Ca2+]c elevation was detected in H9c2 cells following treatment with thapsigargin, an inhibitor of sarco/endoplasmic Ca2+-ATPase (SERCA). These changes were abolished when cells were co-transfected with a dominant-negative pore-forming subunit of MCU, MCUb, confirming that Mitycam and Mitycam-E31Q are capable of sensing [Ca2+]mt elevation by mtCa2+ uptake via MCU. Lastly, H9c2 cells were transfected with Mitycams or mitochondrial matrix-targeted GFP (mt-GFP) as a control, and exposed to prolonged [Ca2+]c elevation by ionomycin, a membrane-permeable Ca2+ ionophore. Cells expressing mt-GFP showed apoptotic signaling activation after ionomycin treatment as assessed by the increased the amount of cleaved caspase-3 by Western blotting (40.1 ±9.4% increase after treatment compared to before treatment, n=4, p<0.05). Importantly, both Mitycams significantly inhibited the increase in cleaved caspase-3 after ionomycin stimulation, with the largest inhibition from Mitycam-E31Q (25.0±9.8% and 47.1±10.1% inhibition in Mitycam and Mitycam-E31Q, respectively. n=4). Conclusion: Overexpressing mitochondrial Ca2+ biosensors may serve as additional artificial buffering machinery in the mitochondrial matrix, which can reduce mtCa2+ overload and prevent apoptotic signaling activation in response to [Ca2+]c elevation.

NIH R01HL136757 (to J.O.-U.), R01HL160699 (to B.S.J.), P30GM1114750 (Targeted project to J.O.-U.), P30GM110759 (Targeted project to J.O.-U.), U54GM115677 (Targeted project to B.S.J.), and American Heart Association (AHA) 18CDA34110091 (to B.S.J.).

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.