Differentially regulated genes in wildtype compared to <i>Per2^−/−</i> mice during myocardial ischemia and reperfusion.

<p>Shown are the number of genes that were differentially regulated in wildtype or <i>Per2^−/−</i> mice using different ischemia and reperfusion protocols: wildtype or <i>Per2^−/−</i> mice were exposed to 1.) Ischemia of 30 minutes without reperfusion (I30), 2.) Ischemic preconditioning (consisting of 4 times 5 minutes of ischemia and 5 minutes of reperfusion, IP0), and 3.) 30 minutes of ischemia followed by 60 minutes of reperfusion (I30R60).</p

Microarray design comparing wildtype and <i>Per2</i><i>⁻</i>^{<i>/</i><i>−</i>} mice.

<p>(<b>A</b>) Different ischemia and reperfusion protocols used on one 24 multi-plate array. 1.) 30 minutes of ischemia without reperfusion, I30. 2.) Ischemic preconditioning consisting of 4×5 minutes of ischemia followed by 5 minutes of reperfusion each, IP0. 3.) 30 minutes of ischemia and 60 minutes of reperfusion, I30R60. (<b>B</b>) Box plots for each of the samples with the intensity (arbitrary units) of the probes graphed on the X-axis to identify outliers in the data set.</p

Ingenuity pathway analysis in wildtype and <i>Per2</i><i>⁻</i>^{<i>/</i><i>−</i>} after IP or IR treatment.

<p>(<b>A</b>) Top networks or canonical pathways from differentially regulated genes after ischemic preconditioning (4×5 minutes of ischemia and reperfusion, IP0) treatment. Analysis is based on genes regulated in wildtype mice only. (<b>B</b>) Top networks or canonical pathways from differentially regulated genes after 30 minutes of ischemia and 60 minutes of reperfusion (I30R60) treatment, comparing wildtype and <i>Per2^−/−</i> mice. Analysis is based on genes regulated in wildtype mice only. (<b>C</b>) Top networks or canonical pathways from differentially regulated genes after ischemia and reperfusion (I30R60) treatment comparing <i>Per2^−/−</i> and wildtype mice. Analysis is based on genes regulated in <i>Per2^−/−</i> mice only.</p

Lactate measurements from whole blood in wildtype, <i>Per1</i><i>⁻</i>^{<i>/</i><i>−</i>} and <i>Per2</i><i>⁻</i>^{<i>/</i><i>−</i>} mice.

<p>(<b>A</b>) Murine model of in situ myocardial ischemia and reperfusion. After 60 minutes of ischemia and indicated time points of reperfusion whole blood samples were obtained by left ventricular puncture. (<b>B</b>) Lactate measurements in wildtype (WT), Period 1 deficient (<i>Per1^−/−</i>) and Period 2 deficient (<i>Per2^−/−</i>) mice after 60 minutes of ischemia and 5 minutes of reperfusion. (C) Time course of lactate levels in whole blood after 60 minutes of ischemia and indicated time points of reperfusion (0, 5, 10, 15, 30, 45 and 60 minutes) in wildtype and <i>Per2^−/−</i> mice; n = 3 mice in all groups.</p

Disrupted fatty acid metabolism in <i>Per2</i><i>⁻</i>^{<i>/</i><i>−</i>} mice during myocardial ischemia.

<p>(<b>A, B</b>) Pathway analysis comparing wildtype and <i>Per2^−/−</i> mice after 30 minutes of ischemia without reperfusion. Differentially regulated genes and pathways were analyzed using Partek and Ingenuity software, respectively. (<b>C</b>) Fatty acid subpopulation analysis in wildtype and <i>Per2^−/−</i> hearts at baseline using nuclear magnet resonance (NMR) technique. Representative NMR spectra for total lipids or monounsaturated fatty acids (MUFAs) at baseline are displayed. To calculate an absolute monounsaturated fatty acid (MUFA) concentration, the concentration of polyunsaturated fatty acids, triacylglycerides and glycerides are subtracted from the total for this peak. (<b>D</b>) WT or <i>Per2^−/−</i> mice were exposed to 30 minutes of ischemia without reperfusion. Shock frozen hearts were analyzed for total lipid and MUFA content at baseline (B) and Ischemia (I) using NMR, n = 3 mice in all groups.</p

‘Principal Components Analysis’ (PCA) of a 24 multi-plate microarray.

<p>Each point represents a chip (sample) and corresponds to a row on the top-level spreadsheet. The color of the dot represents the type of the sample. Points that are close together within the plots have similar intensity values across the probe sets on the whole chip (genome), and points that are far apart within the plots are dissimilar. (<b>A</b>) PCA of the genetic background. (<b>B, C</b>) PCA of the different treatment conditions. WT =  wildtype, <i>Per2^−/−</i>  =  Period 2 deficient mice, I30  = 30 minutes of ischemia without reperfusion, IP0  =  ischemic preconditioning (4×5 minutes of ischemia and reperfusion), I30R60  = 30 minutes of ischemia followed by 60 minutes of reperfusion. The units on the axes represent the different measurement points of all arrays where the percentage for one axis indicates how many of these measurement points are representable by this axis. <i>NOTE:</i> Due to the rotation of the 3-D graph using <i>Partek Genomics Suite 6.6</i> not all values are visible.</p

Metabolism under the control of Per2.

<p>Shown are the top metabolic genes accounting for the identification of carbohydrate or fatty acid metabolism as top networks or canonical pathways when analyzing genes that are only regulated in wildtype but not in <i>Per2^−/−</i> mice using different ischemia and reperfusion protocols. WT  =  wildtype, I30  = 30 minutes of ischemia, IP0  =  ischemic preconditioning (4 times 5 minutes of ischemia and reperfusion), I30R60  = 30 minutes of ischemia and 60 minutes of reperfusion. Given are the expression values (fold change) obtained by Ingenuity pathway analysis. <b>Bold genes</b> appear in more than one treatment group, indicating a robust differentially regulated gene.</p

Initiation of a pro- inflammatory program in <i>Per2</i><i>⁻</i>^{<i>/</i><i>−</i>} mice during ischemia and reperfusion.

<p>(<b>A,B</b>) Pattern recognition analysis (heat map of biological functions) from genes only regulated in <i>Per2^−/−</i> (<b>A</b>) or WT (<b>B</b>) mice after 30 minutes of ischemia and 60 minutes of reperfusion. (<b>C, D</b>) Wildtype or <i>Per2^−/−</i> mice were exposed to 60 minutes of ischemia and 5 (I60/R5) or 60 (I60/R60) minutes of reperfusion. The area at risk was excised and analyzed for IL-6 (<b>C</b>) or TNF-α (<b>D</b>) cardiac tissue concentration; n = 3 mice in all groups.</p