The longevity gene mIndy (I’m Not Dead, Yet) affects blood pressure through sympathoadrenal mechanisms

Reduced expression of the plasma membrane citrate transporter INDY (acronym I’m Not Dead, Yet) extends life span in lower organisms. Deletion of the mammalian Indy (mIndy) gene in rodents improves metabolism via mechanisms akin to caloric restriction, known to lower blood pressure (BP) by sympathoadrenal inhibition. We hypothesized that mIndy deletion attenuates sympathoadrenal support of BP. Continuous arterial BP and heart rate (HR) were reduced in mINDY-KO mice. Concomitantly, urinary catecholamine content was lower, and the decreases in BP and HR by mIndy deletion were attenuated after autonomic ganglionic blockade. Catecholamine biosynthesis pathways were reduced in mINDY-KO adrenals using unbiased microarray analysis. Citrate, the main mINDY substrate, increased catecholamine content in pheochromocytoma cells, while pharmacological inhibition of citrate uptake blunted the effect. Our data suggest that deletion of mIndy reduces sympathoadrenal support of BP and HR by attenuating catecholamine biosynthesis. Deletion of mIndy recapitulates beneficial cardiovascular and metabolic responses to caloric restriction, making it an attractive therapeutic target.

(a) Body length of mINDY KO mice and WT littermate controls (WT n = 3; 9.3±0.03cm; mINDY KO n = 4; 9.1±0.05cm) at the age of 13-14 weeks fed a regular chow diet. Results were determined by a two-sided student´s ttest. Data represent the mean ±SEM (*P<0.05). (b) Diastolic and (c) systolic arterial blood pressure of mINDY KO and WT littermate control mice. Results were determined by a two-way ANOVA (***P<0.001).

Supplementary figure 2. Western Blot analysis of TH, StAR and CYP11B2 in adrenal glands of mINDY KO mice.
Protein expression by western blot analysis and quantification of TH, StAR (a, c, d) and CYP11B2 (b, e) in adrenal glands of mINDY KO (n=3) and WT control mice (n=7). Proteins were normalized to the protein expression of vinculin. TH=tyrosine hydroxylase, StAR=steroidogenic acute regulatory protein, CY11B2=cytochrome P450 family 11 subfamily B member 2.

Supplementary figure 3. Confirmation of effective separation of adrenal medulla from cortex.
(a) Tyrosin hydroxylase (TH) gene expression in adrenal medulla (n = 5) and cortex (n = 5) of WT mice fed a regular chow diet. (b) Steroidogenic acute regulatory protein (StAR) gene expression in adrenal medulla (n = 4) and cortex (n = 5) of WT mice fed a regular chow diet. Results were determined by a two-sided student´s ttest. Data represent the mean ±SEM (**P<0.01;***P<0.01).

Supplementary figure 4. Endothelial function in aortae of WT and mINDY KO mice.
Endothelial function in aortae of WT (n = 8) and mINDY KO (n = 8) mice. Endothelial function was assessed using cumulative increasing concentrations of acetylcholine (ACh) in phenylephrineprecontracted aortic rings. Data represent the mean ±SEM.  individual genes with Z ratio > 1.5 in both directions, ANOVA P value < 0.05, and false discovery rate < 0.30 were considered significantly changed as described (1, 2). Gene Ontology analysis and parametric analysis of gene set enrichment (PAGE) method were conducted using tools that are part of DIANE 6.0 software (http://www.grc.nia.nih.gov/branches/rrb/dna/diane_software.pdf) (3). For each Z (pathway), a p value was computed in JMP 6.0 to test for the significance of the Z score obtained.
First-strand cDNA was synthesized from 1 ug RNA using High-Capacity reverse transcription kit with random hexamers, according to the manufacturer's instructions (Applied Biosystems, Foster City, CA).

Adrenal cortex and medulla isolation
Adrenal glands were first explanted from mice, fried from surrounding fat and kept in a sterile phosphate-buffered saline (PBS) on ice. Adrenals were then put on Petri dishes in sterile ice-cold PBS and the medulla was carefully cut out of the surrounding cortex with a safety scalpel (Brown) under the dissection microscope as described previously (4)(5)(6). Only clearly visual separated medulla and cortex were collected in pre-chilled separate Eppendorf tubes containing RLT-Plus RNA extraction buffer (RNeasy Micro Kit) for further RNA isolation.
Adrenal cortex and medulla RNA extraction, cDNA synthesis, and quantitative PCR RNA isolation was performed using RNeasy Micro Kit and reverse transcribed using iScript RT Kit