Background <p>Sodium–glucose cotransporter 2 inhibitors (SGLT2i) are now widely applied in treatment plans for heart failure with reduced ejection fraction (HFrEF) patients, regardless of the presence of diabetes. SGLT2i, such as empagliflozin (EMP), reduce the risk of cardiovascular death and heart failure hospitalizations in HFrEF patients. However, the underlying molecular mechanisms of action, by which SGLT2i benefit cardiomyocytes (CMs) in HFrEF hearts, are not well understood.</p> Methods <p>This study investigated the role of the SGLT2i, EMP, in a patient-specific human model of dilated cardiomyopathy (DCM), a primary cause of HFrEF, which is frequently caused by inherited mutations in sarcomere proteins. We employed DCM patient-specific induced pluripotent stem cell-derived CMs (iPSC-CMs) carrying the inherited sarcomere protein mutation tropomyosin (TPM1)-L185F together with high-speed motion traction and optical action potential mapping analysis, as well as biochemical methods to dissect molecular signaling dysfunctions.</p> Results <p>Our findings indicate that in DCM patient-specific iPSC-CMs, SGLT2i may act via different mechanisms. This includes the regulation of signaling pathways, to modulate sarcomere function via improving mitochondrial respiration and ATP production. As our results show, the SGLT2i, EMP, ameliorates primary sarcomere dysfunctions in DCM TPM1-L185F iPSC-CMs, such as disrupted sarcomere organization, reduced contractile function, and prolonged action potential duration. Moreover, we found that a subcellular signaling pathway dysregulated in DCM CMs, clathrin-mediated endocytosis (CME)-dependent signaling, may be recovered by EMP treatment in DCM TPM1-L185F iPSC-CMs. This pathway is essential for the uptake and distribution of critical cargo, such as transferrin-bound Fe, in CMs. In the presence of the DCM-causing mutation, EMP recovered mitochondrial Fe levels and mitochondrial functional output, which are reduced in DCM iPSC-CMs as part of pathologically defective CME-dependent signaling. These findings support that EMP modulates aspects of CME-signaling, and contributes to restored sarcomere functions. To further elucidate the effects of SGLT2i in DCM CMs, we investigated the molecular functions of a state-of-the-art combinatorial therapy for DCM (HFrEF) patients, the drug combination referred to as “fantastic four” (F4), including an SGLT2i, in DCM iPSC-CMs.</p> Conclusions <p>Our in-vitro results point to a significant benefit of F4 to be attributed to the SGLT2i, EMP, regarding sarcomere functions as well as aspects of CME-dependent recovery of molecular signaling. Moreover, studying the recovery of subcellular dysfunctions by EMP treatment, findings from an in-vivo porcine model of HF support improvements observed in the presence of EMP regarding contractile and electrophysiological parameters. Together, our findings provide molecular insights into the subcellular function of SGLT2i and their role as part of the F4 state-of-the-art combination therapy for HFrEF, via targeting different molecular signaling pathways in a human patient-specific 2D model of HFrEF.</p>