The Gut Microbiome Targeted Compound 3,3‐Dimethyl‐1‐butanol Attenuates In Vivo Aortic Stiffening with Aging

Abstract

Increases in aortic stiffness occur with aging and predict cardiovascular (CV) mortality, but the upstream mechanism(s) by which this occurs are not fully understood. Age-related aortic stiffening can occur due to both structural (i.e., mechanical) changes, as well as functional changes, including increases in blood pressure (BP), autonomic nervous system activity, and impairments in endothelial function with consequent effects on vascular tone. Our laboratory has shown that the gut microbiome-derived metabolite trimethylamine N-oxide (TMAO) increases in circulation with aging, and supplementation with TMAO directly induces aortic stiffening in mice. Gut microbiome-dependent TMAO production can be suppressed by the naturally-occurring compound 3,3-dimethyl-1-butanol (DMB). Purpose To determine if supplementation with DMB prevents the development of age-related aortic stiffening and elucidate potential underlying mechanisms. Methods and Results Middle-aged (18 mo) male C57BL/6 mice received normal drinking water (control) or drinking water with 1% DMB until sacrifice at 21, 24, or 27 mo of age (n=11-21/age/treatment). Aortic stiffness was assessed in vivo by aortic pulse wave velocity (aPWV). In control mice, there were no apparent age-related increases in aPWV at 18 or 21 mo (365±6 and 374±11 cm/s) compared to a reference group of young (5 mo) male C57BL/6 mice (n=11; 356±14 cm/s; 18 and 21 mo both p>0.32 vs. young), but aortic stiffening developed with advancing age (24 mo: 413±14, 27 mo: 444±14 cm/s, both p≤0.001 vs. 18 mo). The increase from 18 to 27 mo was attenuated by ~70% in mice treated with DMB (aPWV at 24 mo: 358±8, 27 mo: 388±9 cm/s, both p≤0.01 vs. control).To determine if attenuation of aortic stiffening with DMB was due to prevention of structural changes in the aorta with advancing age, we measured intrinsic mechanical stiffness (elastic modulus; EM) in aortic rings by wire myography in a subset of mice (n=5-6/group). In control mice, there was a progressive increase in aortic EM from 5 to 18 to 21 mo (2106±186, 2550±217, 2924±304 kPa; ANOVA time effect: p=0.053) but no further increase after 21 mo (27 mo: 2696±344 kPa; p=0.77 vs. 21 mo). DMB treatment did not affect this age-related intrinsic mechanical stiffening (i.e., no difference between control vs. DMB at any time point, all p>0.5), indicating the prevention of increased aPWV in DMB mice was likely due to functional (vs. structural) changes in the aorta. To investigate this, we measured BP by the tail-cuff method in a subset of mice (n=4-11/group) but found no differences between control and DMB mice at any timepoint (all p>0.12). Conclusion DMB prevented the development of aortic stiffening in vivo but had no effect on intrinsic mechanical (wall) stiffness or tail cuff BP. Thus, DMB may prevent age-related increases in aPWV via other in vivo contributing factors, such as endothelial function and vascular tone, autonomic nervous system activity, or central BP. DMB holds promise for attenuating the development of aortic stiffening with age and consequent risk of CV diseases.