Fig 2: Expression of MCU, SIRT3, and GSK3β, calcium homeostasis and phenotypic markers in cerebral arteries of rats with hindlimb unloading (HU) and angiotensin II (Ang II)‐induced hypertension. Rats were subjected to 21‐day HU to simulate microgravity effects and treated with a 21‐day Ang II infusion (0.7 mg/kg/day) to establish hypertension. (A) Noninvasive tail cuff monitoring of systolic blood pressure (SBP) and diastolic blood pressure (DBP) in rats with vehicle or Ang II for 21 days. (B–D) Western blot analyses of MCU, SIRT3, GSK3β and p‐GSK3β Ser9 in HU and hypertensive (HTN) rats. (E, F) Quantitative PCR analyses of SPP1 and MYH11 in the cerebral arteries of HU and HTN rats. (G, H) Cytoplasmic and mitochondrial Ca2+ levels in cerebral VSMCs of HU and HTN rats. n = 6. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 3: Expression of MCU, SIRT3, α‐SMA, OPN, GSK3β and β‐catenin in vascular smooth muscle cells (VSMCs) incubated In vitro with angiotensin II (Ang II). The VSMCs were incubated In vitro with Ang II (100 nmol/L). (A, B) Western blot analyses of MCU and SIRT3 in Ang II‐treated VSMCs after 12 and 24 h of incubation. (C) Western blot analyses of the contractile marker α‐SMA and synthetic marker OPN in Ang II‐treated VSMCs after 24 h of incubation. (D) Western blot analyses of nuclear and cytoplasmic β‐catenin and GSK3β in Ang II‐treated VSMCs after 24 h of incubation. n = 5. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 4: MCU regulates cerebrovascular remodeling in hindlimb unloading (HU) rats. Rats were subjected to 21‐day HU to simulate microgravity effects. (A) Representative Western blot images of cerebral arteries immunoprecipitated with a SIRT3 antibody and immunoblotted with ubiquitin (Ub) antibodies. (B) Western blot analysis of MCU, SIRT3, GSK3β, and β‐catenin in HU rat cerebral arteries. (C) Representative western blot images of cerebral arteries immunoprecipitated with a GSK3β antibody and immunoblotted with anti‐acetyl‐lysine (Ace‐K) antibodies. (D) Representative images of H&E staining and representative images of immunohistochemical staining for α‐SMA, OPN, and PCNA of basilar arteries in HU rats. (E) Quantitative analysis of wall thickness and wall‐to‐lumen ratio basilar arteries in HU rats. (F) Quantitative analysis of relative optical density of α‐SMA, OPN, and PCNA, calculated by normalizing the integrated optical density to vessel wall area. L, lumen; I, intima; M, media; and V, adventitia. n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: MCU regulates GSK3β acetylation via SIRT3 in vascular smooth muscle cells (VSMCs). The VSMCs were incubated In vitro with angiotensin II (Ang II) (100 nmol/L). (A) Representative western blot images of VSMCs immunoprecipitated with a GSK3β antibody and immunoblotted with GSK3β and SIRT3 antibodies. (B) Representative western blot images of VSMCs immunoprecipitated with a SIRT3 antibody and immunoblotted with GSK3β and SIRT3 antibodies. (C) Western blot analysis of MCU in VSMCs. (D) Western blot analysis of SIRT3 in VSMCs. (E) Representative western blot images of VSMCs immunoprecipitated with a GSK3β antibody and immunoblotted with anti‐acetyl‐lysine (Ace‐K) and GSK3β antibodies. n = 5. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.