Christopher J. French

A profibrotic effect of plasminogen activator inhibitor type-1 (PAI-1) in the heart.

Increased expression of PAI-1 is profibrotic in several organs. However, its potentially profibrotic effects in the heart subjected to infarction have not been elucidated. Accordingly, we induced coronary occlusion in 10-week-old mice congenic on a C57BL6 background and in mice overexpressing PAI-1 (PTG) in multiple tissues. Compared with C57BL6 control mice without myocardial infarction (MI), PTG mice exhibited consistently elevated PAI-1 in plasma at 16 weeks of age but virtually identical PAI-1 content in left ventricular (LV) myocardium. However, they exhibited a 2-fold increase in LV PAI-1 content 6 weeks after induction of MI (4.21 ± 1.0 ng/ml tissue protein) compared with that in C57BL6 mice (2.04 ± 0.5, P < 0.05). In 16-week-old mice, ultrasonically delineated LV fractional shortening (FS) was comparable in normal PTG and normal C57BL6 controls. However, 6 weeks after MI, PTG (n = 21) compared with C57BL6 (n = 14) mice exhibited markedly thinner LV posterior walls in both diastole (C57BL6 0.79 ± 0.05 mm, PTG 0.55 ± 0.06, P < 0.05) and systole (0.97 ± 0.05 mm, 0.75 ± 0.06, P < 0.05); increased end systolic LV dimensions (4.54 ± 0.2 mm, 5.17 ± 0.2, P < 0.05); and significantly depressed FS, more impaired LV segmental function, and greater mitral E wave amplitude. Compared with fibrosis assessed by Masson staining of sections from apex to base in C57BL6 mice (10.85 ± 0.43% LV area), PTG mice exhibited 33% more LV fibrosis after MI (P < 0.05). Thus, PAI-1 is profibrotic in the heart subjected to infarction. Accordingly, overexpression of PAI-1 is a promising target for attenuation of heart failure after MI that may be exacerbated by fibrosis.

The magnitude and temporal dependence of apoptosis early after myocardial ischemia with or without reperfusion

In view of the conventional wisdom in the cardiology literature that apoptosis is extensive early after myocardial ischemia, predicated largely from results with the TUNEL assay known to be nonspecific, this study was performed to delineate its extent with multiple assays and at multiple intervals. Coronary occlusion with and without subsequent revascularization was induced in 10‐wk‐old C57BL6 mice subjected to 1 or4hof transient ligation followed by 24 h of reperfusion, or 24 h persistent ligation. Apoptosis was quantified throughout the left ventricle immu‐nohistochemically by assay of TUNEL, single‐stranded DNA (ssDNA), and cleaved caspase 3;electron microscopy (EM); and activity assays of caspase 3 and 8. TUNEL staining was marked, but ssDNA and cleaved caspase 3 staining were significantly less (P< 0.001 compared with TUNEL), and apoptosis defined by EM was virtually absent in all groups. Caspase 3 and caspase 8 activities per milligram protein were not significantly different from those in normal hearts. Only rare, potentially apoptotic cells were seen by EM in hearts from any group. Thus, the results with TUNEL were not specific, and the extent of apoptosis was markedly less than that predicated on the results with the TUNEL procedure. Apoptosis is de minimus early after transitory or persistent ischemia, though it is overestimated by TUNEL assays. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of myocardium early after ischemic insults.—French, C. J., Spees, J. L., Tarikuz Zaman, A. K. M., Taatjes, D. J., Sobel, B. E. The magnitude and temporal dependence of apoptosis early after myocardial ischemia with or without reperfusion. FASEB J. 23, 1177–1185 (2009)

Failure of erythropoietin to render jeopardized ischemic myocardium amenable to incremental salvage by early reperfusion.

ObjectivesErythropoietin (EPO) has been thought to be capable of potentiating protection of jeopardized myocardium by reperfusion in evolving myocardial infarction. However, diversity in study design and measurements of infarct size in studies evaluating EPO has led to inconsistent results. We sought to characterize the effect of EPO on infarct size after myocardial ischemia and reperfusion with the use of assessment of left-ventricular (LV) creatine kinase (CK) depletion and echocardiography. MethodsAcute coronary occlusion was induced in 10-week-old C57BL6 mice by left anterior descending coronary artery ligation for 3 h followed by 72 h of reperfusion. EPO (10 000 U/kg) or an equivalent amount of saline vehicle alone was injected intraperitoneally before ligation or immediately after the onset of reperfusion. Assays of residual LV CK activity and calculation of LV CK depletion were performed on LV homogenates harvested 72 h after onset of reperfusion for measurement of infarct size, and echocardiography was performed immediately before harvest of tissue for measurement of function. ResultsMice administered EPO before ligation had similar infarct size (37.1±4.1%) and echo scores (22.9±0.4) compared with those in corresponding control mice administered saline (35.29±1.9 and 21.3±1.1%, respectively). Mice administered EPO after reperfusion had similar infarct size (39.1±4.8%) and echo scores (19.5±1.0) compared with those in corresponding control mice administered saline (40.3±4.9 and 21.5±1.9%, respectively). ConclusionEPO does not protect ischemic myocardium such that reperfusion after 3 h can yield additional salvage.

Vascular rhexis in mice subjected to non-sustained myocardial ischemia and its therapeutic implications

We previously described the death of vascular cells (vascular rhexis) following persistent coronary occlusion. The present study was designed to determine whether non-sustained ischemia can initiate vascular rhexis and if so, whether relatively brief ischemic insults are sufficient. C57BL6 mice were subjected to coronary ligation for 15 min or 3 h followed by reperfusion. Soluble fractions of left ventricular (LV) homogenates were obtained 48 h after the onset of transitory coronary occlusion. They were assayed by Western blotting for quantification of alpha smooth muscle actin (α-SMA) and smooth muscle myosin heavy chain (SM-MHC) that we have shown reflect vascular rhexis delineated immunohistochemically. Non-sustained coronary occlusion for 3 h initiated vascular rhexis evident 45 h after reperfusion, but not earlier, as judged from Western blotting of α-SMA and SM-MHC. The number of small- and medium-sized vessels in the previously ischemic zones was reduced at 45 h after reperfusion as well. Thus, vascular rhexis occurs after ischemia as brief as 3 h but evolves slowly and is not evident for 45 h. The delayed disintegration of the vasculature makes it likely that it can be ameliorated by interventions initiated after non-sustained ischemia, rendering it an attractive target for diminution of phenomena such as late negative LV remodeling, and ‘no reflow.’

Vascular rhexis: loss of integrity of coronary vasculature in mice subjected to myocardial infarction

We previously observed gross hemorrhage in plasminogen activator inhibitor type-1 (PAI-1) knockout (PKO) mice with induced myocardial infarction (MI). We hypothesized that it reflected degradation of vessels – a phenomenon we termed vascular rhexis. Accordingly, in the present study we characterized vascular rhexis in C57BL6 mice. MI was induced in 10- to 12-week-old mice by coronary artery ligation for 24, 48, 72 or 96 h. Hemorrhage was quantified by non-cross-reacting enzyme-linked immunosorbent assay of left ventricular (LV) hemoglobin corrected for myoglobin. Degradation of vasculature was quantified by the appearance of alpha smooth muscle actin (αSMA) in low salt soluble fractions of LV homogenates (Western blotting) and by immunohistochemistry (residual αSMA). Co-staining for CD31 (endothelial cells) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) (a marker of cell death) was used to identify capillary rhexis. PKO mice (n = 9) had marked hemorrhage in infarct zones (432 ± 27 standard error of mean µL blood/g). Hemorrhage was evident in C57BL6 mice as well (n = 6): 51 ± 8 μL/g LV 96 h after coronary occlusion compared with 10 ± 5 μL /g, n = 13 in normal LVs. Residual intact vasculature was reduced 48 h after infarction. Thus, an average of 16 ± 1.6 small- and medium-sized vessels (n = 5 hearts) were seen compared with 84 ± 4.8 in normal LVs (n = 3, P ≤ 0.05). An approximately three-fold increase in soluble αSMA 48 h after MI (2.68 ± 0.28, n = 6) was seen relative to that in normal LVs defined as 1.0 ± 0.04, n = 10, P ≤ 0.05. Capillary degradation was evident as well, as judged from CD31 and TUNEL co-localization. Vascular rhexis occurs within 48 h after the onset of MI. It may contribute to the early no-reflow phenomenon and to late negative LV remodeling.

Cardiac fibrosis and diastolic dysfunction after myocardial infarction in apolipoprotein E knockout mice.

Apolipoprotein E knockout mice (ApoE – / – ) have been of inestimable value in atherosclerotic research. They lack apolipoprotein E, a ligand for receptors that clear remnants of chylomicrons and very low-density lipoproteins [1,2]. As a result, they develop severe hypercholesterolemia with concentrations of cholesterol in plasma five times higher than those in C57BL6 (C57) mice. The ApoE – / – mice develop foam cell-rich depositions in their proximal aortas by 3 months of age [1,2], and these lesions progress to cause severe occlusion of the coronary artery ostium by 8 months of age [3–6]. Despite differences in atherosclersis in humans and mice, as a result of their predisposition to develop atheromas, the ApoE – / – mice are widely utilized to study the progression of atherosclerosis and factors that modify its evolution. They may be useful as well in the study of coronary insults and their consequences. However, to the best of our knowledge, the effect of the ApoE – / – genotype on the response of the heart to induced myocardial infarction (MI) has not been elucidated. We sought to determine whether derangements in lipid metabolism or the ApoE / – genotype itself lead to adverse effects on the heart after experimentally induced MI.

BRIEF MYOCARDIAL ISCHEMIA FOLLOWED BY REPERFUSION INITIATES MICROVASCULAR CELL DEATH THAT CAN CONTRIBUTE TO THE “NO-REFLOW” PHENOMENON AND POTENTIALLY BE AMELIORATED

Introduction: The “no-reflow” phenomenon refers to diminution of perfusion in the microcirculation despite restitution of perfusion in the macrovascular supply by relief of epicardial coronary artery occlusion. We have previously described death of vascular cells (vascular rhexis) following persistent coronary occlusion. The present study was designed to determine whether transitory ischemia can initiate vascular rhexis in the coronary microcirculation that can contribute to “no-reflow” and late negative left ventricular (LV) remodeling.