Abstract Body (Do not enter title and authors here): Background and Research Question: We have previously shown that mitochondrial fission protein 1 (Fis1) is over-expressed in endothelial cells obtained from humans with diabetes. However, whether Fis1 is mechanistically linked to impaired microvascular endothelial function, either health or cardiometabolic disease, remains unknown.
Methods: Fresh resistance arterioles from 73 healthy individuals and 24 individuals with type 2 diabetes (T2DM) by gluteal adipose tissue biopsy or discarded surgical tissue. Endothelium-dependent vasoreactivity was tested with increasing doses of acetylcholine (10^-10 to 10^-5 M) following two different molecular manipulations: (1) intraluminal lentiviral transfection for endothelial-specific Fis 1voverexpression vs. control or (2) intraluminal Fis1 siRNA transfection vs. scrambled control. We developed a small peptide Fis1 inhibitor, pep213, and tested if pep213 improved endothelial function in vessels from T2DM patients and healthy individuals exposed to high glucose (HG, 33mM). The impact of molecular manipulations on mitochondrial superoxide (mtO₂^●-) function were performed using mitoNeoD.
Result: Fis1 knockdown significantly improved vasodilation to Ach in an endothelial nitric oxide synthase (eNOS)-dependent manner (N=6, P<0.0001) in T2DM vessels and healthy vessels exposed to high glucose (HG=33 mM, N=5, P<0.0001), but had no effect Ach vasodilation in healthy vessels exposed to normal glucose concentrations (NG=90 mg/dL., N=3, P=NS). Fis1 overexpression impaired Ach vasodilation in an eNOS-dependent manner in healthy vessels (N=5, P<0.0001) compared to control transfection. NONOate and papaverine controls for these studies showed effects on smooth muscle reactivity. Intraluminal pep213 (1 µM for 1 hour), reversed impaired eNOS-dependent vasodilation in T2DM vessels (N=5, P<0.001), healthy vessels exposed to HG (N=5, P<0.001), and healthy vessels over-expressing Fis1 (N=5, P<0.05), while a scrambled version of pep213 without ability to bind Fis1 had no effects in any of these scenarios. Pep213 had no effect on vasodilation to NONOate. Pep213 reduced mtO₂^●- in T2DM and healthy vessels over-expressing Fis1 (N=5, P<0.001).
Conclusion: Fis1 mechanistically regulates eNOS-dependent vasoreactivity in human vessels, potentially through reducing mtO₂^●- levels. Fis1 over-expression may be particularly important in T2DM, and pharmacological inhibition of Fis1 can reverse endothelial dysfunction in T2DM human vessels.