Protein kinase D regulates mitochondrial Ca ²⁺ uptake by changing mitochondrial morphology

Introduction: Changes in the size and shape of mitochondria play a crucial role in regulating key mitochondrial and cellular functions. We previously showed that activation of the stress-responsive protein kinase D (PKD) induces mitochondrial fragmentation by phosphorylating the mitochondrial fission protein dynamin-related protein 1 (DRP1), which subsequently promotes proliferation in primary cardiac fibroblasts. We also showed that genetic inhibition of mitochondrial Ca 2+ (mtCa 2+ ) uniporter reduces fibroblast proliferation in response to Gq protein-coupled angiotensin II type 1 (AT1) receptor stimulation. Although small and fragmented mitochondria exhibited larger and more sustained mtCa 2+ transients compared to basal tubular mitochondria when mitochondrial membrane potential was maintained (O-Uchi J et al, Am J Physiol Heart Circ Physiol. 2013), it remains unclear whether PKD activity specifically regulates mtCa 2+ uptake and cellular Ca 2+ handling. Hypothesis: PKD modulates mtCa 2+ uptake via changing mitochondrial morphology. Methods: HEK293T cells stably expressing a dominant-negative mutant of PKD (PKD-DN), a mitochondrial outer membrane (OMM)-targeted PKD-DN (mt-PKD-DN, generated by adding an OMM-targeting sequence [amino acid 1-33 of human TOM20] to the N-terminus of PKD-DN), or an empty pcDNA3.1(+) were generated and used for biochemical and cell biological assays. Results: First, mitochondrial digestion assays (using combinations of proteinase K and various concentrations of the mild detergent digitonin) revealed that endogenous PKD is present at the OMM but not in the mitochondrial intermembrane space and matrix. Second, mt-PKD-DN was specifically expressed in mitochondria, whereas PKD-DN was expressed in both the cytosol and mitochondria, as assessed by Western blotting using fractionated proteins and confocal microscopy. Additionally, mt-PKD-DN inhibited PKD activation exclusively in mitochondrial fractions after Gq protein-coupled receptor (GqPCR) stimulation. Importantly, cells expressing mt-PKD-DN showed elongated mitochondria under basal conditions and smaller mtCa 2+ uptake in response to GqPCR stimulation compared to control cells. Conclusion: PKD modulates mtCa 2+ uptake, potentially via changing DRP1-dependent mitochondrial fission activity. Funding: The work was partially supported by NIH/NHLBI R01HL160699 (to B.S.J.), NIH/NHLBI R01HL171710 (B.S.J. and J.O.-U.), and NIH/NHLBI R01HL136757 (to J.O.-U.). This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.