Contributing role and molecular basis of Vitamin D/Vitamin D receptor deficiency in hyperhomocysteinemia-induced cardiac hypertrophy

Hyperhomocysteinemia and vitamin D deficiency are known to promote cardiac hypertrophy, however, whether vitamin D signaling is involved in hyperhomocysteinemia-induced cardiac hypertrophy remains unexplored. This study aimed to address this question by clarifying the effect of homocysteine on vitamin D and vitamin D receptor (VDR), with further elucidation of the regulatory mechanisms. Methionine diet-induced hyperhomocysteinemic (HHcy) rats and homocysteine-incubated cardiomyocytes were used as in vivo and in vitro models of cardiac hypertrophy. Gain-and-loss-of function of VDR and miR-125b-5p were achieved by plasmid transfection and AAV9-mediated delivery. HHcy rats showed lowered serum and cardiac 1,25(OH)2D3 levels and increased 24-hydroxylase (CYP24A1) expression in kidney and myocardium. VDR expression was downregulated and miR-125b-5p was upregulated in the myocardium of HHcy rats and in homocysteine-incubated cardiomyocytes as well. Knockdown of VDR facilitated while overexpression mitigated homocysteine-induced cardiomyocyte hypertrophy, accompanied by activation and inhibition of calcineurin/nuclear factor of activated T cells 4 (NFATc4) respectively. Dual-luciferase reporter gene assay and gain-and-loss-of function of miR125b-5p in cardiomyocytes indicated the targeting and repressing of VDR by miR-125b-5p and its prohypertrophic effect. The role of miR-125b-5p-mediated VDR downregulation in homocysteine-induced cardiac hypertrophy was further demonstrated in vivo. Treatment with VDR agonist inhibited hypertrophic growth both in vivo and in vitro, resulting from VDR upregulation and consequent calcineurin/NFATc4 inhibition. These findings demonstrated that homocysteine reduces 1,25(OH)2D3 level in both plasma and myocardium via upregulating CYP24A1 and represses myocardial VDR expression via the mediation of miR-125b-5p. Vitamin D/ VDR deficiency contributes to hyperhomocysteinemia-induced cardiac hypertrophy via activating calcineurin/ NFATc4 pathway.