Metformin's effects beyond ischemia: Evaluating cardioprotection in nonischemic myocardium in a large animal model of coronary and metabolic disease

FIGURE 1.. Flowchart illustrating the experimental design, timeline of surgical procedures, treatments, and group assignments. Surgical procedures and treatments are displayed in the blue/gray boxes within the Procedures section, aligned horizontally with their corresponding time points on the timeline. Group assignments are shown in colored boxes under the Groups section, along with the number of animals in each group.

FIGURE 2.. Immunoblot images for apoptotic markers in control (CON) (n = 5; 1 excluded due to sample loss during storage) versus metformin-administered (MET) (n = 6) nonischemic tissue. Compared with the relative expression in the CON group, the MET group showed a significantly decreased average expression of caspase 9, cleaved caspase 9, BCL2-associated agonist of cell death (BAD), and apoptosis-inducing factor (AIF). In addition, the MET group showed a significantly increased average phosphorylated BCL2-associated agonist of cell death (p-BAD)/BAD ratio. The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.

FIGURE 3.. Immunofluorescence analysis in control (CON) (n = 5; 1 excluded due to sample loss during storage) versus metformin-treated (MET) (n = 6) animals. Transferase dUTP nick end labeling (TUNEL) staining was performed to assess apoptosis in myocardial tissue collected at harvest. The proportion of positive staining was significantly reduced in the MET group compared with the CON group (P = .0064). Representative images display nuclei stained with 4′, 6-diamidino-2-phenylindole (blue) and apoptotic cells labeled with fluorescein isothiocyanate (green) in control (middle) and MET-treated (right) samples, respectively. The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.

FIGURE 4.. Immunoblot images for prosurvival markers in control (CON) (n = 5; 1 excluded due to sample loss during storage) versus metformin-administered (MET) (n = 6) nonischemic tissue. Compared with the relative expression in the CON group, the MET group showed a significantly increased average expression of phosphorylated protein kinase B (p-AKT), phosphorylated mammalian target of rapamycin (p-mTOR), and phosphorylated AMP-activated protein kinase (p-AMPK). In addition, the MET group showed a significantly increased average p-AKT/AKT, p-mTOR/mTOR, and p-AMPK/AMPK ratio. Lastly, the MET group showed a significantly decreased average expression of AKT (protein kinase B) and a significantly increased average expression of mTOR and AMP-activated protein kinase (AMPK). The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.

FIGURE 5.. Blood flow (mL/min/g tissue) for control (CON) (n = 6) versus metformin-administered (MET) (n = 6) nonischemic animals. As shown in the graphs, there appears to be no statistically significant difference between CON and MET animals both at rest and during rapid myocardial pacing. The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.

FIGURE 6.. Cardiac functional parameters and associated correlations in control (CON) (n = 6) versus metformin (MET) treated (n = 6) animals. Top row, MET treatment significantly improved cardiac output (CO), stroke volume (SV), and maximal rate of volume change (dV/dt max) in nonischemic tissue. Although end-systolic volume (Ves) trended higher in the MET group, the difference was not statistically significant. Bottom row, Correlation analyses, performed using Spearman rank-order correlation, revealed that cardiac output trended toward a negative correlation with p-AKT, whereas Ves inversely correlated with phosphorylated BCL2-associated agonist of cell death (p-BAD), and dV/dt max trended toward a negative correlation with phosphorylated AMP-activated protein kinase (p-AMPK), indicating that improved ventricular function is associated with reduced proapoptotic signaling and modulation of AMPK-mediated prosurvival pathways. The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.

FIGURE 7.. Immunofluorescence analysis in control (CON) (n = 5; 1 excluded due to sample loss during storage) versus metformin (MET) treated (n = 6) animals. Isolectin B4 staining was used to label endothelial cell structures. Although the difference between the groups did not reach statistical significance (P = .0748), a downward trend in signal was observed in MET-treated tissue. Representative images show 4′, 6-diamidino-2–stained nuclei (blue) and isolectin B4 labeled with Cy5 (magenta) in control (middle) and MET-treated (right) samples, respectively. The upper and lower borders of the boxes represent the upper and lower quartiles, the middle horizontal line represents the median, and the upper and lower whiskers represent the maximum and minimum values of nonoutliers.