Abstract Age is a major risk factor for cardiovascular associated diseases which are leading causes of death globally. Accordingly, 80% of all cardiovascular deaths occurred in patients aged 65 and over. Endothelial cell senescence, characterized by cell cycle arrest and a senescence-associated secretory phenotype (SASP), is a major contributor to age related cardiovascular dysfunction. However, the mechanism by which endothelial cells are subjected to senescence is still unclear. We have recently made novel findings that endothelial cell HSPA12B deficient (eHSPA12B-/-) mice exhibit cardiac endothelial cell senescence accompanied by severe cardiac hypertrophy, fibrosis, and persistent inflammation when compared with age- and gender-matched WT controls. Our findings suggest that senescent endothelial cells play an important role in the regulation of cardiac hypertrophy and fibroblast activation and that endothelial cell HSPA12B limits senescence of endothelial cells. Therefore, understanding the mechanisms by which HSPA12B regulates endothelial cell senescence would be important for seeking an approach to prevent or reverse senescent endothelial cells, thus reducing age-related cardiovascular disease. To elucidate how HSPA12B limits senescence of endothelial cells, we examined the effect of HSPA12B on ATF6 (Activating transcription factor 6) transcriptional activity and its target gene MANF (Mesencephalic Astrocyte Derived Neurotrophic Factor) expression. ATF6 is an important transcriptional factor that regulates the expression of genes involved in proper protein folding and cellular senescence. MANF is an evolutionarily conserved protective modulator for the maintenance of tissue immune and metabolic homeostasis. Interestingly, we observed that HSPA12B is critical for sustaining ATF6 transcriptional activity and MANF expression. The data suggest that ATF6 and MANF may be involved in the HSPA12B mediated protective effect on endothelial cell senescence. To investigate how endothelial cell senescence contributes to cardiac hypertrophy, we examined cardiac mitochondrial glucose oxidation (MGO) which is one of the major contributors to myocardial energy production. Compromised mitochondrial glucose oxidation leads to the development of cardiac hypertrophy and eventually heart failure. We observed that eHSPA12B-/- resulted in decreased Acetyl-CoA and increased lactate accumulation. The data indicate that eHSPA12B-/- impairs cardiac mitochondrial glucose oxidation. Our observation also suggests that senescent endothelial cells induce cardiac hypertrophy via impairment of cardiac myocyte MGO (mitochondrial glucose oxidation). To address how endothelial cell senescence impairs cardiac MGO, we induced endothelial cell senescence by ETO (etoposide), collected the medium as the senescent conditioned medium (SCM), and treated adult cardiac myocytes with the SCM. We observed that SCM promotes metabolic reprogramming from glucose oxidation to glycolysis i...