Abstract Rationale Asthma in the elderly (AIE) is characterized by hyperreactive and thickened, fibrotic airways (remodeling) that are not adequately addressed by current therapies. Thus, understanding mechanisms that drive AIE becomes important. Here, airway smooth muscle (ASM) cells play important roles in normal airway physiology and asthma pathophysiology. Recent discoveries have highlighted the significance of mechanosensitive Piezo (Pz)-Piezo1 and Piezo2 in the lung. While Pz channels are highly expressed, their specific contributions to ASM function remains largely unexplored. Moreover, Pz channels play essential roles in regulating the intracellular calcium homeostasis, which further drives mitochondrial dynamics. Whether Pz signaling influences mitochondrial structure and function-a key determinant of ASM energetics and contractility is unknown and therefore of significant interest. In the present study, we hypothesize that Pz1 channel activation regulates mitochondrial structure and function via an increase in intracellular calcium in ASM, leading to metabolic changes and remodeling, characteristic of AIE. Methods Primary human ASM cells were isolated from lungs of young (45y) and AIE (≥65y) individuals obtained post-surgery (Mayo Clinic IRB approved). Cells were exposed to mechanical stretch (mimicking breathing) in the presence of either Yoda1/Jedi2 (agonist) or GsMTx4 (inhibitor) for 24 hrs. Mitochondrial fractionation was performed, and protein expression evaluated for mitochondrial dynamics targets (MFN1, MFN2, OPA1, DRP1 and FIS1) and transcriptional markers (mt-Cox1, mt-Cox2, ATP5b and TFAM). The colocalization of mitochondria and Pz channels was observed by immunofluorescence staining. Mitochondrial morphology was examined with MitoTracker staining while function was assessed using Seahorse XF analysis. Intracellular calcium (Ca2+i) and mitochondrial calcium (Ca2+m) were measured using 5µM Fura-2/AM and 1µM Rhod-2 AM respectively with fluorescence microscopy. Results Confocal microscopy revealed that Piezo1 localizes to mitochondria in ASM. Pz1 activation promoted mitochondrial fission, evidenced by an upregulation of DRP1/FIS1 and downregulation of MFN1/OPA1, confirming a fission-dominant phenotype. Pz1 activation also increased mitochondrial branching while reducing branch length, indicating enhanced fragmentation, particularly in AIE ASM. Yoda1 treatment reduced mitochondrial oxygen consumption rates, suggesting a lower energy demand. Furthermore, Yoda1 increased both Ca2+i and Ca2+m responses to histamine (10µM), with a more pronounced effect in AIE ASM. Conclusion Overall, our findings suggest that mechanotransduction through Pz channels, particularly Pz1 regulates intracellular calcium regulation and mitochondrial structure and function in ASM, linking extracellular mechanical forces to intracellular energy homeostasis. This mechanism may contribute to the altered energetics and remodeling observed in AIE airway pathophysiology. This abstract is funded by: 24POST1241979 (NAB), R01- HL056470 (YSP), R01-HL177837 (CMP)
Borkar et al. (Fri,) studied this question.