Empagliflozin mitigates cardiac hypertrophy through cardiac RSK/NHE-1 inhibition

Background: SGLT2i reduce cardiac hypertrophy, but underlying mechanisms remain unknown. Here we explore a role for serine/threonine kinases (STK) and sodium hydrogen exchanger 1(NHE1) activities in SGLT2i effects on cardiac hypertrophy. Methods: Isolated hearts from db/db mice were perfused with 1 µM EMPA, and STK phosphorylation sites were examined using unbiased multiplex analysis to detect the most affected STKs by EMPA. Subsequently, hypertrophy was induced in H9c2 cells with 50 µM phenylephrine (PE), and the role of the most affected STK (p90 ribosomal S6 kinase (RSK)) and NHE1 activity in hypertrophy and the protection by EMPA was evaluated. Results: In db/db mice hearts, EMPA most markedly reduced STK phosphorylation sites regulated by RSKL1, a member of the RSK family, and by Aurora A and B kinases. GO and KEGG analysis suggested that EMPA inhibits hypertrophy, cell cycle, cell senescence and FOXO pathways, illustrating inhibition of growth pathways. EMPA prevented PE-induced hypertrophy as evaluated by BNP and cell surface area in H9c2 cells. EMPA blocked PE-induced activation of NHE1. The specific NHE1 inhibitor Cariporide also prevented PE-induced hypertrophy without added effect of EMPA. EMPA blocked PE-induced RSK phosphorylation. The RSK inhibitor BIX02565 also suppressed PE-induced hypertrophy without added effect of EMPA. Cariporide mimicked EMPA's effects on PE-treated RSK phosphorylation. BIX02565 decreased PE-induced NHE1 activity, with no further decrease by EMPA. Conclusions: RSK inhibition by EMPA appears as a novel direct cardiac target of SGLT2i. Direct cardiac effects of EMPA exert their anti-hypertrophic effect through NHE-inhibition and subsequent RSK pathway inhibition.