Pericyte K_ATPChannel Hyperactivity Contributes to Layer IV Cortex Hypoperfusion in Cerebral Small Vessel Disease

Background: Cerebral small vessel diseases (SVDs) cause brain circulation dysfunction, leading to vascular dementia and stroke. The most common genetic SVD is CADASIL, characterized by NOTCH3 mutations, which result in receptor extracellular domain accumulation and vascular dysfunction. There are no treatments for CADASIL, and patients experience transient ischemic attacks, cognitive decline, and premature death.Pericytes have been largely overlooked in CADASIL research despite high levels of NOTCH3 expression. Pressure-induced constriction is known to be impaired by CADASIL at the arteriolar level, but the impact on pericyte reactivity is unknown. Single-cell RNA-seq studies have identified the K ATP channel Kir6.1 as highly expressed in pericytes, and functional studies have revealed the K ATP channel plays a key role in regulating pericyte contractility. However, the K ATP channel and its role in SVD remains unexplored. We hypothesize that the K ATP channel is hyperactive in pericytes within the arteriole to capillary transition (ACT) zone, causing aberrant hyperpolarization and impaired contractility. Objective: To interrogate pericyte participation in CADASIL by determining the impacts on pressure-induced constriction, vasoreactivity, and cerebral blood flow distribution. Methods: We utilized the well-established CADASIL mouse model TgNotch3 R169C and TgNotch3 WT controls. Ex vivo assays that recapitulate the brain microenvironment in isolated microvasculature include membrane potential recordings, advanced pressure myography, and patch-clamp electrophysiology, with K ATP pharmaceutical interrogation. We also employed acute cranial window implantation combined with two photon laser scanning microscopy to investigate changes in microvascular diameter, tone, and K ATP channel activity in vivo. Finally, we completed in silico vascular modeling to query impacts of K ATP channel hyperactivity on perfusion throughout the cortex. Results: Our research, conducted both in vivo and ex vivo, demonstrated a 50% decrease in pressure-induced constriction in the CADASIL ACT zone. Membrane potential recordings indicated that CADASIL ACT zone pericytes were notably more hyperpolarized (-51 mV) than control pericytes (-32 mV). Our ex vivo pressure myography and in vivo cranial window experiments revealed that the CADASIL ACT zone minimally responded to K ATP agonist pinacidil, whereas controls exhibited significant dilation (17% versus 44%). Conversely, the CADASIL ACT zone exhibited a robust constriction in response to K ATP antagonist glibenclamide, unlike controls (28% versus 10%). Further investigation into K ATP channel hyperactivity in CADASIL using intact microvascular perforated patch revealed that ACT zone pericytes had a significantly higher basal current, which was sensitive to the K ATP antagonist glibenclamide. In silico modeling using membrane potential, patch-clamp, and ex vivo pressure myography data suggested that K ATP channel hyperactivity increases blood flow in the upper cortical layers at the expense of layer IV, leading to significant hypoperfusion in that region. Conclusion: These results support the novel concept that CADASIL’s impact on arteriolar reactivity further extends to the capillary level and ACT zone pericytes. K ATP channel hyperactivity contributes to impaired cortical blood flow distribution across different layers, enhancing perfusion in the upper layers while reducing it in layer IV. Overall, these results add valuable contributions to our understanding of SVDs and introduce new targets that could aid in rescuing the cerebral blood flow dysregulation and devastating pathological remodeling seen in CADASIL patients. This research was funded by NHLBI: DAJ F31HL170645, FD R01HL136636, NINDS: FD RF1/R01NS129022, R01/RF1NS140137 and cureCADASIL. This abstract was presented at the American Physiology Summit 2025 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.