Abstract Body: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) reduce mortality and heart failure (HF) hospitalizations in diabetic and non-diabetic individuals. It was previously reported that SGLT2 is not expressed in cardiac myocytes (CMC), but recent studies found SGLT2 mRNA and protein abundant in human CMC. Molecular mechanisms for SGLT2i-induced cardioprotection are unclear, but one proposed mechanism is the inhibition the of sodium/hydrogen exchanger 1 (NHE-1). We investigated whether SGLT2 is expressed in spontaneously contracting mouse CMCs and hypothesized that the SGLT2i ertugliflozin (ERTU) will lower cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) after adrenergic stimulation.
Neonatal CMCs were isolated from 0–2-day old mice (C57BL/6J), cultured for one week, and confluent and spontaneously contracting CMC were treated for 72 hours with either ERTU (100 nM or 1 uM), the NHE-1 inhibitor cariporide (CARI, 10 uM), ERTU (100 nM or 1 uM) plus CARI, or vehicle (Ctrl, 0.01% DMSO in H₂0). After 72 hours, cells were either A) loaded with fura-2 acetoxymethyl ester to record baseline [Ca²⁺]_cyt for 5 minutes and the responses to phenylephrine (PE, 100 µM) for additional 30 minutes, B) lysed for immunoblotting to detect SGLT2, total phospholamban (PLB), phosphorylated PLB, sarcomeric alpha-actinin2, tubulin, vinculin, and connexin 43 (Cx43), or C) fixed for immunohistochemistry.
In CMC 100 nM and 1 uM ERTU reduce [Ca²⁺]_cyt peaks after PE stimulation compared to vehicle-treated CMC. In contrast, CARI increases [Ca²⁺]_cyt at baseline and enhances peaks after PE. SGLT2 protein is expressed in neonatal CMC, but whole kidney control samples show higher expression. ERTU decreases protein levels of total PLB, increases S16 PLB phosphorylation, and upregulates sarcomeric alpha-actinin2. Microscopic analysis shows that ERTU improves sarcomeric z-line organization, increases sarcomeric alpha-actinin2 abundance, and induces re-localization of Cx43 to cell-to-cell contacts. CARI resulted in large Cx43-containing gap junctions, perinuclear Cx43 expression, and disturbed z-line arrangement.
Considering that disturbed cell-to-cell contacts and Ca²⁺ overload are hallmarks of HF, our findings in mechanically active CMCs indicate potential direct cardioprotective effects of the SGLT2i ERTU by lowering of adrenergic-driven increases in [Ca²⁺]_cyt, improving cytoskeletal organization, and enhancing Cx43-dependant intercellular communication. ERTU’s effects differed from NHE1 inhibition.