Empagliflozin Ammeliorates High Glucose Induced-Cardiac Dysfuntion in Human iPSC-Derived Cardiomyocytes

Abstract

Empagliflozin, a sodium-glucose co-transporter (SGLT) inhibitor, reduces heart failure and sudden cardiac death but the underlying mechanisms remain elusive. In cardiomyocytes, SGLT1 and SGLT2 expression is upregulated in diabetes mellitus, heart failure, and myocardial infarction. We hypothesise that empagliflozin exerts direct effects on cardiomyocytes that attenuate diabetic cardiomyopathy. To test this hypothesis, cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) were used to test the potential effects of empagliflozin on neutralization of cardiac dysfunction induced by diabetic-like cultures. Our results indicated that insulin-free high glucose culture significantly increased the size of and NPPB, SGLT1 and SGLT2 expression of hiPSC-derived cardiomyocytes. In addition, high glucose-treated hiPSC-derived cardiomyocytes exhibited reduced contractility regardless of the increased calcium transient capacity. Interestingly, application of empagliflozin before or after high glucose treatment effectively reduced the high glucose-induced cardiac abnormalities. Since application of empagliflozin did not significantly alter viability or glycolytic capacity of the hiPSC-derived cardiomyocytes, it is plausible that empagliflozin exerts its effects via the down-regulation of SGLT1, SGLT2 and GLUT1 expression. These observations provide supportive evidence that may help explain its unexpected benefit observed in the EMPA-REG trial.

Figure 1

Schematic representation of the proposed mechanisms underlying diabetic cardiomyopathy in relation to sodium-glucose co-transporter (SGLT). Upregulation of SGLT in cardiomyocytes in the presence of diabetes mellitus results in an increase in sodium influx into cardiomyocytes that in turn increases cytosolic calcium loading via the sodium-calcium exchanger (NCX). Intracellular calcium overload can lead to (1) delayed after depolarizations (DADs), triggers for tachycardia, (2) impaired excitation-contraction coupling, and (3) activation of calcium-sensitive signaling pathways, leading to pathological changes.

Figure 2

Hypertrophic Changes under High Glucose Condition and Empagliflozin to hiPSC-derived cardiomyocytes. (A) Schematic outline of the time and duration of high glucose treatment and empagliflozin application. (B) Representative immunofluorescence staining of KS1 hiPSC-derived cardiomyocytes cultured in normal glucose (5.5 mM); high glucose (22 mM); high glucose and empagliflozin; and high glucose for 7 days followed by empagliflozin (Red: α-Actinin; blue: DAPI); (C) The cell size; (D–G) Relative expressions of ACTA1, FHL, MLC2A and NPPB respectively. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. *p < 0.05; **p < 0.01.

Figure 3

Contractility of hiPSC-cardiomyocytes under High Glucose Condition and Empagliflozin. (A) Representative tracings of cell changes. (B) Percentage cell shortening; (C) Maximal velocity of cell shortening and (D) Maximal velocity of cell re-lengthening. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. *p < 0.05; **p < 0.01.

Figure 4

Calcium Handling Properties of hiPSC-derived cardiomyocytes under High Glucose Condition and Empagliflozin. (A) Representative tracings of whole-cell calcium transients in hiPSC-derived cardiomyocytes; (B) Amplitudes of whole-cell calcium transients; (C) Maximal upstroke velocity of whole-cell calcium transients; and (D) Maximal decay velocity of whole-cell calcium transients. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. (E) The expression of ATP2A2, RYR2 and NCX1 was evaluated by quantitative PCR analysis using the expression of TNNT2 as an internal control. Abbreviations: NG: normal glucose; HG: high glucose; HG + E: high glucose and empagliflozin; and HG, HG + E: high glucose for 7 days followed with empagliflozin. (F) Protein level of phospho-phospholamban and total phospholamban was evaluated by Western blot analysis, the protein level of β-actin was used as internal reference. *p < 0.05 and **p < 0.01. For the full-length image of gels and blots shown in this figure, please refer to Supplementary Figs S4 and S5.

Figure 5

Effects of High Glucose, Empagliflozin and insulin on the expression of glucose transporters in hiPSC-derived Cardiomyocytes. (A) Schematic outline of the times and durations of high glucose treatment and the application of empagliflozin and insulin. (B,C) Relative expressions of SGLT1 and SGLT2. (D and E) Relative expressions of GLUT1 and GLUT4. *p < 0.05 and **p < 0.01.

Figure 6

Expression of SGLT1 and SGLT2 in hiPSC-derived cardiomyocytes and human heart tissue. (A) Confirmation of the identity of the PCR product obtained from SGLT2-specific amplifications by DNA sequencing analysis. (B,C) Expression of SGLT1 and SGLT2 in human heart tissue were confirmed by PCR analysis and DNA sequencing analysis. (D) Protein levels of SGLT1 and SGLT2 in the hiPSC-derived cardiomyocytes were evaluated by Western blot analysis. *p < 0.05. For the full-length images of gels and blots shown in this figure, please refer to Supplementary Figs S4 and S5.

Figure 7

Effects of High glucose and empagliflozin on the protein level of caspase 3 and caspase 7. Protein level of caspase 3 and caspase 7 was evaluated by Western blot analysis using antibodies that could detect both full length and cleaved forms of the target protein. The protein level of β-actin was used as internal control for statistical analysis. *p < 0.05. (n = 4).

Figure 8

Effects of High glucose and empagliflozin on the functional alterations of cardiomyocytes derived from IMR90 hiPSCs. (A) Representative immunofluorescence staining, and (B) cell size of IMR90 hiPSC-derived cardiomyocytes cultured in normal glucose (5.5 mM); high glucose (22 mM); high glucose and empagliflozin; and high glucose for 7 days followed by empagliflozin (Red: α-Actinin; blue: DAPI). (C) Existence of SGLT1 and SGLT2 transcripts in the IMR90 hiPSC-derived cardiomyocytes were evaluated by PCR analysis. (D,E) Real-time quantitative PCR analysis of SGLT1 and SGLT2 expression. (F) % of cell shortening. (G) Amplitude of intracellular calcium trainsets. *p < 0.05; **P < 0.01.