*2.3. Protocols*

The experimental protocols are illustrated in Figure 1. The baseline readings were taken after 20 min stabilization.

**Figure 1.** Shows the experimental protocols. The hearts were isolated from eight-week-old male Brown Norway rats and mounted in a Langendorff setup. After 20 min equilibration and a baseline (bl) reading, the hearts in the first set of experiments (n = 12; Panel (**A**)) were given rosiglitazone (Rosi, 50 μM) or a vehicle (Con) for two times, 5 min each with a 5 min washout period interspersed and followed by 15 min washout before 30 min of global no-flow ischemia and 120 min of reperfusion (IR) and subsequent infarct size (IS) determination. In a second set of experiments (Panel (**B**)), the hearts were given increasing concentrations (2, 10, and 50 μM) of either rosiglitazone (Rosi, blue colors; n = 6) or pioglitazone (Pio, red colors; n = 6) for 5 min each followed by 30 min washout. This series was repeated in the presence of 10 μM of the PPARγ antagonist GW9662 (GW, white). The hearts were not subject to IR or to IS determination.

## 2.3.1. IR Experiments

In this first set of experiments (n = 12; Figure 1, Panel A), rosiglitazone (Rosi, 50 μM) as a clinically used TZD was given as a preconditioning agen<sup>t</sup> for two times, 5 min each with a 5 min washout period interspersed and followed by 15 min washout before 30 min of acute global no-flow ischemia and 120 min of reperfusion. This protocol was chosen to mimic our ischemic and other pharmacological preconditioning protocols [30,31,35,36] shown to be more effective than single exposure [31,37]. The control hearts (Con) received 0.1% DMSO as a vehicle only.

After removal of the hearts at the end of the experiments, the atria were discarded and the ventricles were cut into 2-mm transverse slices and incubated for 10 min in 1% 2,3,5-triphenyltetrazolium chloride in a 0.1 M KH2PO4 buffer (pH 7.4, 38◦C) [38,39] which stains viable tissue red. The slices were digitally imaged on a green background, and the infarcted areas of each slice were measured automatically by planimetry using Image J 1.44i software (NIH, Bethesda, MD). The individual slice infarctions were weight-averaged to calculate the total ventricular infarct size (IS) per heart [32,40].

## 2.3.2. Dose-Response Experiments

In a second set of experiments (n = 12; Figure 1, Panel B), the hearts were given increasing concentrations (2, 10 and 50 μM) of either rosiglitazone (Rosi) or pioglitazone (Pio) for 5 min each, without intervening IR or IS determination. After a 30 min washout, this series was repeated in the presence of the PPARγ antagonist GW9662 [12] at a concentration of 10 μM [11,41,42].

#### *2.4. Fluorescence Measurement of Mitochondrial Redox State*

Autofluorescence is widely used to measure mitochondrial electron transport in myocardial tissue [30,43,44]. Thus, the experiments were conducted in a light-blocking Faraday cage to assess the online autofluorescence of reduced NADH and oxidized FAD. The distal end of a trifurcated fiberoptic cable was placed gently against the left anterior ventricular wall while the proximal ends were connected to a modified spectrophotometer (Horiba, Piscataway, NJ, USA). At selected times, the shutter for excitation was opened for 2.5 sec intervals. The NADH fluorescence was excited at 350 nm followed by FAD fluorescence excitation at 488 nm. The NADH emissions were filtered at 460 ± 10 nm (Chroma Technology Corp., Brattleboro, VT, USA), FAD emissions at 540 ± 10 nm, and their respective fluorescence intensities were measured by photomultipliers.
