Dysregulation of the HSF1-Mediated UPRmt Pathway in Colonic Smooth Muscle Cells Drives Motility Dysfunction in Functional Constipation.

Mitochondrial dysfunction in colonic smooth muscle cells (SMCs) is closely associated with impaired gut motility in functional constipation (FC), but the underlying molecular mechanisms remain incompletely understood. The mitochondrial unfolded protein response (UPRmt) is a critical pathway for maintaining mitochondrial proteostasis, and heat shock factor 1 (HSF1) acts as an important upstream regulator of this response. In the present study, we employed a loperamide-induced FC mouse model, combined with single-cell transcriptomic, molecular, and functional analyses to characterize the HSF1-UPRmt pathway in colonic SMCs and to investigate its role in FC. Single-cell transcriptomic analysis of colon tissue from FC mice revealed marked downregulation of UPRmt-associated genes in colonic SMCs. Immunofluorescence, Western blotting, and RT-qPCR analyses of colonic tissue confirmed that HSF1 expression was reduced in colonic SMCs, along with the downregulation of the UPRmt components, including HSP60, mtHSP70, and LONP1. These molecular changes were accompanied by mitochondrial structural damage, seen by transmission electron microscopy, and by functional impairments, including reduced mitochondrial membrane potential, elevated mtROS production, decreased ATP levels, and diminished activities of respiratory chain complexes I-V. AAV9-mediated overexpression of HSF1 reactivated the UPRmt pathway, improved mitochondrial function, and ameliorated constipation, whereas shRNA-mediated knockdown of HSF1 further suppressed UPRmt activity and aggravated mitochondrial damage, indicating that HSF1 bidirectionally regulates this pathway. Complementary experiments in primary colonic SMCs confirmed that this regulatory mechanism operates in a cell-autonomous manner, as modulation of HSF1 expression produced corresponding changes in the UPRmt pathway, in the expression of mitochondrial respiratory chain complex subunits (ATP5A, NDUFA9, COX1, SDHA, UQCRC1), and in ATP production, mirroring the in vivo findings. Collectively, these results demonstrate that HSF1 plays a pivotal role in maintaining mitochondrial homeostasis in colonic SMCs through regulation of the UPRmt pathway and that HSF1 dysfunction is closely associated with slowed gut motility in FC. These findings offer a new mechanistic perspective on FC and point to the HSF1-UPRmt axis as a potential therapeutic target.