Cardiac Imaging Using Clinical 1.5 T MRI Scanners in a Murine Ischemia/Reperfusion Model

To perform cardiac imaging in mice without having to invest in expensive dedicated equipment, we adapted a clinical 1.5 Tesla (T) magnetic resonance imaging (MRI) scanner for use in a murine ischemia/reperfusion model. Phase-sensitive inversion recovery (PSIR) sequence facilitated the determination of infarct sizes in vivo by late gadolinium enhancement. Results were compared to histological infarct areas in mice after ischemia/reperfusion procedure with a good correlation (r = 0.807, P < .001). In addition, fractional area change (FAC) was assessed with single slice cine MRI and was matched to infarct size (r = −0.837) and fractional shortening (FS) measured with echocardiography (r = 0.860); both P < .001. Here, we demonstrate the use of clinical 1.5 MRI scanners as a feasible method for basic phenotyping in mice. These widely available scanners are capable of investigating in vivo infarct dimensions as well as assessment of cardiac functional parameters in mice with reasonable throughput

PI3Kγ Protects from Myocardial Ischemia and Reperfusion Injury through a Kinase-Independent Pathway

BACKGROUND: PI3Kgamma functions in the immune compartment to promote inflammation in response to G-protein-coupled receptor (GPCR) agonists and PI3Kgamma also acts within the heart itself both as a negative regulator of cardiac contractility and as a pro-survival factor. Thus, PI3Kgamma has the potential to both promote and limit M I/R injury. METHODOLOGY/PRINCIPAL FINDINGS: Complete PI3Kgamma-/- mutant mice, catalytically inactive PI3KgammaKD/KD (KD) knock-in mice, and control wild type (WT) mice were subjected to in vivo myocardial ischemia and reperfusion (M I/R) injury. Additionally, bone-marrow chimeric mice were constructed to elucidate the contribution of the inflammatory response to cardiac damage. PI3Kgamma-/- mice exhibited a significantly increased infarction size following reperfusion. Mechanistically, PI3Kgamma is required for activation of the Reperfusion Injury Salvage Kinase (RISK) pathway (AKT/ERK1/2) and regulates phospholamban phosphorylation in the acute injury response. Using bone marrow chimeras, the cardioprotective role of PI3Kgamma was mapped to non-haematopoietic cells. Importantly, this massive increase in M I/R injury in PI3Kgamma-/- mice was rescued in PI3Kgamma kinase-dead (PI3KgammaKD/KD) knock-in mice. However, PI3KgammaKD/KD mice exhibited a cardiac injury similar to wild type animals, suggesting that specific blockade of PI3Kgamma catalytic activity has no beneficial effects. CONCLUSIONS/SIGNIFICANCE: Our data show that PI3Kgamma is cardioprotective during M I/R injury independent of its catalytic kinase activity and that loss of PI3Kgamma function in the hematopoietic compartment does not affect disease outcome. Thus, clinical development of specific PI3Kgamma blockers should proceed with caution

PI3Kγ^−/− lipid kinase activity is not required for cardioprotection.

<p>(<b>A</b>) TroponinT levels measured after 30 minutes of ischemia and 3 hours of reperfusion are comparable between PI3Kγ^KD/KD (KD) and control wild type (WT) mice. In total PI3Kγ knock-out (KO) mice TroponinT levels are significantly increased (*p<0.05). n = 13 for WT and KO, n = 11 for KD mice. (<b>B</b>) No significant difference in the area at risk (AAR)/left ventricle (LV) in PI3Kγ^−/− (KO), PI3Kγ^KD/KD (KD), and control WT mice. Data are at 24 hours after M/I injury. (<b>C</b>) PI3Kγ KO, but not PI3Kγ^KD/KD (KD) mice display a markedly increased area of infarction/area at risk (AAR) 24 hours after reperfusion evaluated by TTCstaining. n = 6 per group. *p<0.05. NS = not significant. (<b>D</b>) Representative left ventricular Trichrome-stained sections from the basis (left) to the apex (right) of hearts from wild type (WT), PI3Kγ^KD/KD knock-in (KD), and PI3Kγ^−/− (KO) mice challenged with 30 minutes of ischemia plus 1 week of reperfusion. Arrows represent infarcted regions. Bars indicate 1 mm. (<b>E</b>) PI3Kγ^KD/KD (KD) mice display similar infarctions/left ventricle (LV) compared to wild type controls after 30 minutes of ischemia and 1 week of reperfusion. Data from PI3Kγ^−/− (KO) are also shown. n = 6 per group. (<b>F</b>) PI3Kγ^−/− (KO), but not PI3Kγ^KD/KD knock-in (KD) mice exhibit a significant greater impairment in percentage fractional shortening (FS) relative to WT controls after 30 minutes of ischemia and 1 week of reperfusion. n = 6 per group. Note that the data are calculated to baseline FS of the respective genotype. All bar graphs show mean values +/− SEM. *p<0.05. (<b>G</b>) Representative M-mode echocardiography of wild type (WT), PI3Kγ^KD/KD knock-in (KD), and PI3Kγ^−/− (KO) mice one week after M I/R injury. Note, that baseline contractility is enhanced in PI3Kγ^−/− (KO), but not PI3Kγ^KD/KD knock-in (KD) mice as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009350#pone.0009350-Crackower1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009350#pone.0009350-Patrucco1" target="_blank">[12]</a>. Bars indicate 50 ms (horizontal) and 1 mm (vertical).</p

Loss of PI3Kγ results in long term myocardial ischemia/reperfusion induced heart damage.

<p>(<b>A</b>) Areas of infarction (arrows) in representative left mid-ventricular sections 1 week after M I/R injury. (H&E staining). Bars indicate 1 mm. (<b>B</b>) PI3Kγ^−/− (KO) hearts show a markedly greater infarction size in the left ventricle (LV) at 1 week after M I/R. n = 7 per group. (<b>C</b>) No significant difference between PI3Kγ^−/− and WT mice in the numbers of infiltrating inflammatory cells 1 week after M I/R. n = 7 per group. (<b>D</b>) Representative left mid-ventricular sections stained with Trichrome and Aniline blue to visualize collagen deposits (arrows) in hearts of PI3Kγ^−/− (KO) and wild type (WT) mice 3 weeks after M I/R. Magnifications ×20. (<b>E</b>) Markedly increased scar tissue in PI3Kγ^−/− 3 weeks after M I/R injury. n = 7 per group. (<b>F</b>) PI3Kγ^−/− mice exhibit a significant greater loss in percentage fractional shortening (FS) relative to their respective baseline FS after 3 weeks of M I/R compared to WT controls. n = 9 per group. In all bar graphs mean values +/− SEM are shown. *p<0.05. NS = not significant.</p

PI3Kγ functions in non-haematopoietic cells in myocardial ischemia/reperfusion injury.

<p>(<b>A</b>) Representative left mid-ventricular histological sections (H&E staining) from WT mice receiving WT bone marrow (WT→WT) and WT mice receiving PI3Kγ^−/− bone marrow (KO→WT). Hearts were isolated and analysed from mice 1 week after M I/R injury. Arrows indicate areas of infarction. (<b>B</b>) Representative left mid-ventricular histological sections (H&E staining) from PI3Kγ^−/− mice receiving WT bone marrow (WT→KO) and PI3Kγ^−/− mice receiving PI3Kγ^−/− bone marrow (KO→KO) after 1 week of M I/R injury. Arrows point at areas of infarction. (<b>C</b>) No significant difference between WT→WT and KO→WT chimeras and no difference between WT→KO and KO→KO chimeras in the size of infarction determined 1 week after M I/R injury. n = 5 per group. (<b>D</b>) No significant difference between WT→WT and KO→WT chimeras and no difference between WT→KO and KO→KO chimeras in TroponinT levels assayed 3 hours after reperfusion. n = 7 per group. (<b>E</b>) No significant difference between all 4 groups of chimeric mice in the numbers of infiltrating inflammatory cells 1 week after M I/R. n = 5 per group. In all histological pictures the bar indicates 1 mm. All bar graphs show mean values +/− SEM. *p<0.05. NS = no significant difference.</p