Cardiac Microvascular Endothelial Enhancement of Cardiomyocyte Function Is Impaired by Inflammation and Restored by Empagliflozin

Figures

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  Figure 1. CMEC-CM Co-Culture and High-Throughput MultiCell CM Detection System (A) Cardiac microvascular endothelial cells (CMECs) were cultured on an insert placed above the cardiomyocytes (CMs) seeded on a well of a culture plate. (B, right panel) After a co-culture procedure, the CMEC compartment was removed, and (C) the CM compartment was placed into a temperature-controlled inverted microscope chamber and visualized. (B, left panel) The objective lens was positioned directly under the well containing CMs (A). (D) CMs were paced, and cell contraction and relaxation profiles were monitored.

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  Figure 2. CMECs Enhance CM Function CM contraction and relaxation were evaluated under basal conditions (control) and after co-culture with untreated (endothelial cells [ECs]) or tumor necrosis factor (TNF)-α pre-treated CMEC (EC+TNF). (A and D) Co-culture of CMs with ECs increased CM contraction (proportion sarcomere shortening), and (B and E) improved CM relaxation as shown by increased speed of relaxation (return velocity) and (C and F) shortened time constant of relaxation (tau). (A and D) Pre-treatment of CMECs with TNF-α (EC+TNF) abolished the beneficial effect of CMECs on CM contraction and (B, C, E, and F) relaxation. (A to C) Graphs representing single CMs isolated from 1 individual rat, distributed into the 3 corresponding experimental conditions; 40 to 45 CMs were measured per condition. (D to F) Data are representative of 8 independent experiments. (D to F) Graphs of combined average values obtained from 8 independent experiments corresponding to 8 individual rats. All data are represented as mean ± SD. Other abbreviations as in Figure 1.

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  Figure 3. Inhibition of Endothelial NOS Eliminates the Beneficial Effect of CMEC on CM Contraction and Relaxation (A) Inhibition of CMEC nitric oxide (NO) generation with N(ω)-nitro-L-arginine methyl ester (L-NAME) abrogated the effect of CMECs on CM sarcomere shortening, (B) return velocity, and (C) tau. (A to C) Graphs representing single CMs isolated from 1 individual rat, distributed into the 3 corresponding experimental conditions; 40 to 45 CMs were measured per condition. Data are representative of 3 independent experiments. Data are represented as mean ± SD. NOS = nitric oxide synthase; other abbreviations as in Figures 1 and 2.

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  Figure 4. TNF-α Reduces NO Bioavailability in CMECs (A and B) NO levels in CMEC. Acetylcholine (Ach)−induced upregulation of NO production in CMECs. Similar to L-NAME treatment, TNF-α reduced CMEC NO levels. (n = 4 to 50, refers to number of cells per group from 3 independent experiments). Data represented as mean ± SD. Other abbreviations as in Figures 1, 2, and 3.

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  Figure 5. Empa Restores CMEC-Mediated CM Function During TNF-α Exposure (A) Co-culture of CMEC with CMs increased CM shortening and (B) speed of relaxation, and (C) shortened tau. (A) Pre-treatment of CMECs with empagliflozin (EC+Empa) slightly increased CM contraction compared with CMEC alone (EC). (B) Empa pre-treatment increased CM return velocity but did not affect relaxation time, in comparison to CMEC alone. (D to F) Pre-incubation of CMEC with either TNF-α or a combination of TNF-α and Empa and subsequent co-culture of CMEC with CMs. (D) Co-treatment of TNF-α-stimulated CMEC with Empa prevented reduction of CM contraction in comparison to TNF-α stimulation alone. Co-treatment with Empa also maintained CMEC beneficial effect on (E) CM relaxation velocity and (F) tau. (A to C) Graphs representing single CMs isolated from 1 individual rat, distributed into the 3 corresponding experimental conditions; 30 to 40 CMs were measured per condition. Data are representative of 3 independent experiments. (D to F) Graphs representing single CMs isolated from 1 individual rat, distributed into the 4 corresponding experimental conditions; 30 to 40 CMs were measured per condition. Data are…

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  Figure 6. NO Mediates the Beneficial Effect of Empa on CMEC-CM Interaction (A) Co-treatment with Empa maintained the beneficial effects of CMEC on CM contraction and (B and C) relaxation after TNF-α stimulation. (A to C) The beneficial effect of Empa was abolished when CMECs were co-treated with L-NAME. (A to C) Graphs representing single CMs isolated from 1 individual rat, distributed into the 4 corresponding experimental conditions; 40 to 45 CMs were measured per condition. Data are representative of 3 independent experiments; data are represented as mean ± SD. Abbreviations as in Figures 1, 2, and 3 and 5.

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  Figure 7. Empagliflozin Reduced Mitochondrial and Cytoplasmic ROS Level and Restores NO Bioavailability in CMECs After TNF-α Stimulation (A and B) TNF-α treatment reduced CMEC NO levels. Co-treatment of CMEC with TNF-α and Empa resulted in higher CMEC NO bioavailability in comparison to TNF-α treatment alone (n = 40 to 50, refers to number of cells per group from 3 independent experiments; data represented as mean ± SD). (C and D) TNF-α stimulation led to increased VCAM and E-selectin expression on the cell surface of CMECs, as assayed by a cell-bound enzyme linked immunosorbent assay, and co-treatment with Empa did not change the level of both proteins (n = 4, refers to number of independent experiments; data represented as mean ± SD). (E and F) TNF-α treatment increased cytoplasmic reactive oxygen species (ROS) levels in CMECs, which were attenuated by simultaneous treatment of Empa (n = 40 to 50, refers to number of cells per group from 3 independent experiments; data represented as mean ± SD). (G) Empa did not possess direct antioxidant activity at 1 and 10 μM concentrations, whereas 2 potent antioxidants, butylated hydroxytoluene (BHT) and ascorbic acid, showed a strong radical…