Endogenous adenine is a potential driver of the cardiovascular-kidney-metabolic syndrome

Abstract

Mechanisms underlying the cardiovascular-kidney-metabolic (CKM) syndrome are unknown, although key small molecule metabolites may be involved. Bulk and spatial metabolomics identified adenine to be upregulated and specifically enriched in coronary blood vessels in hearts from patients with diabetes and left ventricular hypertrophy. Single nucleus gene expression studies revealed that endothelial methylthioadenosine phosphorylase (MTAP) was increased in human hearts with hypertrophic cardiomyopathy. The urine adenine/creatinine ratio in patients was predictive of incident heart failure with preserved ejection fraction. Heart adenine and MTAP gene expression was increased in a 2-hit mouse model of hypertrophic heart disease and in a model of diastolic dysfunction with diabetes. Inhibition of MTAP blocked adenine accumulation in the heart, restored heart dysfunction in mice with type 2 diabetes and prevented ischemic heart damage in a rat model of myocardial infarction. Mechanistically, adenine-induced impaired mitophagy was reversed by reduction of mTOR. These studies indicate that endogenous adenine is in a causal pathway for heart failure and ischemic heart disease in the context of CKM syndrome.

Fig. 1.. Mass spectrometry reveals increase in adenine in the diabetic patient hearts with LVH.

Volcano plot of molecular features identified from an untargeted LC-MS analysis of human donor heart tissues from diabetics with LVH (n=6) and healthy controls (n=6). Fold change cutoff = 1.5 fold, p-value cutoff = 0.05. (A). Targeted LC-MS/MS analysis demonstrating increased adenine levels in heart tissue from patients with diabetes and LVH compared to healthy control (B). Scatterplot demonstrating human total heart weight is strongly correlated to heart adenine among both diabetics with LVH and healthy control donors (C). Scatterplot of heart adenine concentration and serum creatinine levels (Pearson R²=0.960, ****p<0.0001) (D). Participants from the Singapore Study of Macro-Angiopathy and microvascular Reactivity in Type 2 Diabetes (SMART2D) study (n=653) had UAdCR measurements at the time of enrollment and were followed for 10 years. The participants in the top tertile for UAdCR had the highest risk for incident HFpEF (E).

Fig. 2.. Adenine localizes to cardiac blood vessels in the hearts of patients with diabetes and LVH.

H&E staining (A), autofluorescent image (B), and MALDI-MSI ion image of adenine ([C₅H₅N₅+H]⁺ m/z = 136.0617) (C) in a representative healthy control and diabetic patients with LVH. Representative H&E staining image of a blood vessel in the left ventricle from a healthy control patient and a diabetic patient with LVH (D). AF imaging of the same blood vessel from adjacent serial section (E). MALDI-MSI image of adenine ion ([C₅H₅N₅+H]⁺ m/z = 136.0617) (F). Overlay of adenine (MSI) and AF blood vessel image from the same tissue section (G). Black/white arrows = blood vessel. Bar graph of the adenine measurement from MSI data for 3 coronary blood vessels from each of control (n=4) and diabetics with LVH (n=4) (H) (t-Test, **p-value < 0.01). Pearson correlation coefficients between cell lineage specific MTAP gene expression and cardiac parameters (I). Enrichment of rapamycin sensitive genes from WikiPathways in cell lineage specific pseudobulks via univariate linear models (J).

Fig. 3.. Mass spectrometry reveals increase in heart adenine in mouse models of HFpEF and diabetes.

AF images (A) and MSI images of adenine ([C₅H₅N₅+Cl⁻]⁻ m/z = 170.0239) in mice treated with HFD + L-NAME and control mice (A-F). MALDI-MSI image of adenine ion ([C₅H₅N₅+Cl⁻]⁻ m/z = 170.0239) from the same tissue section (B). AF imaging of a coronary blood vessel from HFD + L-NAME-treated and control mice. Black arrows = blood vessel (C). Overlay of adenine (from MSI) and AF blood vessel image from the same tissue section (D and E). Measurement of adenine from MSI data for 3 coronary blood vessels from control (n=4) and HFD + L-NAME treated mouse hearts (n=4) (F). Mice treated with HFD + L-NAME show increased Mtap gene expression (G) and decreased Tfam (H) and VegfA (I) gene expression (mean + SE, *p<0.05, **p<0.01, ***p<0.001 by t-test). H&E staining image of a blood vessel in the left ventricle from a db/m and db/db mouse (J). Autofluorescence imaging of the same blood vessel from post-MALDI H&E staining (K). MALDI-MSI image of adenine ion (m/z = 136.0617) (L). Overlay of adenine mass spectrometry image and AF image of blood vessel. Black/white arrows = coronary blood vessel (M). Localization of adenine to 4 coronary blood vessels from db/m (n=3) and db/db mouse heart (n=3) (mean + SE) (N). Urine adenine/creatinine ratio is increased in db/db mice at 8–10 weeks of age (n=12/group, *p<0.05 (O).

Fig. 4.. Inhibition of adenine synthesis with MTDIA restores diabetic cardiac dysfunction and reduces heart mTOR activity in diabetic mice.

Mitral valve deceleration time was increased in MTDIA-treated db/db mice (A) (n=4–6 per group). Echo E/A ratio was reduced in MTDIA-treated db/db mice (B). The urine AdCR was correlated with E/A ratio using Spearman correlation (r = 0.699) (C). Heart adenine was correlated with E/A ratio for all db/db mice with Spearman correlation (n=6 per group) (r = 0.6154) (D). Western blot of db/db and MTDIA-treated db/db mouse heart measuring p-4EBP1 and 4EBP1 (E) demonstrating MTDIA-treatment of db/db mouse significantly reduced mTORC1 activity as measured by p-4EBP1 (n=6 per group) (F). Scatterplot of db/db and MTDIA-treated db/db mouse LV mass against heart adenine concentration with Pearson correlation (n=6 per group) (r = 0.674, *p < 0.05) (G). Bar graph demonstrating decreased urinary adenine to creatinine ratio among MTDIA-treated db/db mice (n=12 per group) (H). MSI analysis of adenine in 4 coronary blood vessels from db/db mouse heart and MTDIA-treated db/db mouse heart (n=3 per group) (I). Inhibition of MTAP (MTAPi) reduced the infarct size % in a rat model of myocardial infarction (J). (mean + SE, *p<0.05, **p<0.01, ***p<0.001 by t-test).

Fig. 5.. Adenine alters mitophagy in wild-type mice which is reversed with inhibition of mTOR.

H&E staining of human hearts from healthy donors and those with diabetes and LVH (A). Electron microscopy images of the same cohort. Black arrows point to damaged mitochondria associated with mitochondrial autophagy (B). Quantification of cardiomyocyte size from H&E (C). Frequency of damaged mitochondria in healthy control and diabetic donors with LVH (D). Western blot of whole cell lysate from adenine-treated wild-type mouse heart tissue (E) demonstrating that adenine treatment decreased the protein levels of Beclin1, LC3I, LC3II, p-p62, and mitochondrial protein ubiquitination, and increased Parkin expression versus control (F). Rapamycin blocked the adenine-induced effects on mitophagy (E and F). Values are expressed as mean ± SEM; *P < 0.05 control versus control (Ctrl) group, and ^#P < 0.05 Rapamycin versus Adenine group (n = 3/group).