Background: The stress-responsive mitogen-activated protein kinases (MAPKs) (p38MAPK, c-Jun NH2-terminal kinase [JNK-1 and JNK-2], and extracellular signal regulated kinases [ERK-1 and ERK-2]) might be involved in angiotensin II (AIJ)-induced ischemiareperfusion injury. Cardioprotection induced by All type 1 (AT1) and type 2 (AT2) receptor blockade during ischemia-reperfusion is associated with protein kinase Cc (PKCe), nitric oxide, and cyclic guanosine monophosphate (cGMP) signaling. Our aim was to assess the effect of selective AT, and AT2 receptor blockade with losartan and PD123,319, respectively, on MAPK expression after ischemia-reperfusion in isolated working rat hearts. Methods: Groups of six hearts were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes) and exposed to no drug/no ischemia-reperfusion (control), ischemia-reperfusionlno drug, and ischemia-reperfusion with losartan (1 MM), or PD123,319 (0.3 ,uM) and additional groups. AT,/AT2 receptor expression, MAPKs, PKCE, and cGMP, and changes in mechanical function were measured. Western blotting was done on left ventricular tissue for AT,/AT2, p38/phosphorylated-p38 (p-p38), phosphorylated (p)-JNK-1/-2, phosphorylated (p)-ERK-1/-2, and PKCe proteins; Northern blots for AT,/AT2 mRNA; and enzyme immunoassay for cGMP. Results: Compared with controls, ischemia-reperfusion induced significant left ventricular dysfunction, decreased AT2 protein and mRNA, increased p-p38 and p-JNK-1/-2, did not change p-ERK-1/-2 or PKCe, and decreased cGMP. PD123,319 improved left ventricular recovery after ischemia-reperfusion, increased AT2 protein and mRNA, mildly increased pp38, normalized p-JNK-1, did not change p-ERK-1/-2, and increased PKCe and cGMP. Losartan did not change p-p38, increased p-JNK-1, and did not change pERK-1/-2, PKCe, or cGMP. Conclusions: The overall results suggest that the activation of p38-MAPK and JNK might be linked to All signaling and play a significant role in acute ischemia-reperfusion injury as well as in the cardioprotective effect of AT2 receptor blockade.

Stem cell therapy holds great promise for the treatment of injured myocardium, but is challenged by a limited supply of appropriate cells. Three different isoforms of transforming growth factor-β (TGF-β) -β1, -β2, and -β3 exhibit distinct regulatory effects on cell growth, differentiation, and migration during embryonic development. We compared the effects of these three different isoforms on cardiomyocyte differentiation from embryonic stem (ES) cells. In contrast to TGF-β1, or -β3, treatment of mouse ES cells with TGF-β2 isoform significantly increased embryoid body (EB) proliferation as well as the extent of the EB outgrowth that beat rhythmically. At 17 days, 49% of the EBs treated with TGF-β2 exhibited spontaneous beating compared with 15% in controls. Cardiac myocyte specific protein markers sarcomeric myosin and α-actin were demonstrated in beating EBs and cells isolated from EBs. In conclusion, TGF-β2 but not TGF-β1, or -β3 promotes cardiac myocyte differentiation from ES cells.

Many forms of heart disease are associated with the loss of cardiomyocytes both via apoptosis or necrosis, and despite the recent identification of resident cardiac stem cells, the native capacity for renewal and repair is inadequate. Cell transplantation strategies have emerged as a potential therapeutic approach for repairing injured myocardium. Many different cell types including embryonic stem cells have been transplanted in myocardial infarction (MI) models with resulting improvement in myocardial function. Here, we review the current state of knowledge with regard to the potential of embryonic stem (ES) cells to differentiate into cardiomyocytes in the embryonic stem cell derived-embryoid body (EB) in vitro system as well as for myocardial regeneration following myocardial infarction.

This study describes the coronary anatomy of C57BL/6 mice and the functional impact of that anatomy on myocardial infarctions induced by ligation of the left coronary artery. In contrast to humans, a distinct septal coronary artery was observed in all mice arising either from a separate ostium from the right sinus of Valsalva or as a branch of the right coronary artery. Ligation of the left coronary artery at the site of its emergence from under the left atrium resulted in reproducible large myocardial infarctions involving the anterolateral, posterior, and apical regions of the heart as evidenced by histology and echocardiography. Interestingly, the septum was spared from infarction as predicted by the presence of the distinct septal branch found in mice. Thus, the distinct mouse coronary artery anatomy results in different regionality of infarction compared with man and large laboratory animals, and this may affect on the associated pathological remodeling of the heart.Background The objective of this study was to describe the coronary anatomy of C57BL/6 mice and determine the functional impact of that anatomy on myocardial infarctions induced by ligation of the left coronary artery.Methods C57BL/6 mice were used to visualize mouse coronary anatomy and to generate myocardial infarction. Mouse coronary artery visualization was performed on isolated hearts using injection of Silastic sealant into the aortic arch. After left thoracotomy myocardial infarction was produced by ligating the left coronary artery at the site of the vessels' emergence past the tip of the left atrium. Echocardiography was performed to analyze heart function, and histology was performed to delineate myocardial infarction.Results The different septal coronary artery was observed in all mice arising either from a separate ostium from the right sinus of Valsalva or as a branch of the right coronary artery. The mouse left coronary artery passed obliquely across the left ventricular free wall similar to the ramus intermedius coronary artery variant in man. Ligation of the left coronary artery as it emerged from under the left atrium resulted in reproducible large infarctions involving the anterolateral, posterior, and apical regions of the heart as demonstrated by histology and echocardiography. Notably, the septum was spared from infarction as predicted by the presence of the distinct septal coronary artery found in mice.Conclusions Mouse coronary anatomy is distinct compared to man or large laboratory animals, and myocardial infarctions resulting from ligation of the mouse left coronary artery spare the septum, which may affect the pathological remodeling of the heart. (C) 2005 Lippincott Williams Wilkins.

To determine whether the increased concentration inblood of total tissue-typeplasminogenactivator (t-PA), accompanying increasedplasminogenactivator inhibitor type-1 (PAI-1) seenwith diabetes reflects increased free t-PA andhence activity or simply increased t-PA complexedwith PAI-1, wemeasured each in 39 people with type 2 diabetes.The increased total t-PAoccurred in the absence ofan increase in t-PA activity detectable in venous blood samples despite the increase in the concentrationof total t-PAprotein.

BACKGROUNDPatients surviving myocardial infarction (MI) are at a heightened risk for the development of congestive heart failure. This clinical syndrome has been associated with an antioxidant deficit and elevated oxidative stress in the myocardium. Effects of dietary vitamin E, a lipid-soluble antioxidant, on myocardial anti-oxidant enzyme activities, oxidative stress and hemodynamic function, were examined separately in the viable left ventricle (LV) and right ventricle (RV) of rats at 16 weeks post-MI.METHODS AND RESULTSAnimals were fed either a basal diet or a diet enriched with 1500 U of vitamin E/kg beginning two weeks before MI-inducing surgery and continued 16 weeks post-MI. In the MI animals on the basal diet, LV systolic pressure (LVSP) and RVSP were significantly depressed and LV end-diastolic pressure (LVEDP) and RVEDP were significantly elevated. These hemodynamic alterations were accompanied by clinical signs of heart failure including dyspnea, lethargy and cyanotic limbs. Supplementation of MI animals with dietary vitamin E resulted in complete normalization of RVSP and RVEDP. An increase in LVSP and a decrease in LVEDP was observed in the vitamin E-supplemented MI animals, although mild residual LV dysfunction remained. The myocardial enzymatic antioxidants catalase and glutathione peroxidase declined substantially in each of the ventricles of unsupplemented MI animals. Myocardial levels of vitamin E were reduced by 33% in the LV and no change was observed in the RV of the MI animals. Vitamin E-supplemented control animals and MI animals showed a significant increase in vitamin E levels in both ventricles. Myocardial oxidative stress, as assessed by lipid peroxidation and the ratio of reduced to oxidized glutathione, was significantly increased in each of the respective ventricles of untreated MI animals. Supplementation with dietary vitamin E resulted in a substantial increase in the myocardial activities of catalase and glutathione peroxidase in both the LV and RV. Furthermore, an increase in the ratio of reduced to oxidized glutathione concomitant with significantly less lipid peroxidation was also observed in each of the respective ventricles of MI animals supplemented with vitamin E. No overt clinical signs of heart failure were evident in these vitamin E-supplemented animals.CONCLUSIONSAn improved myocardial redox state and endogenous antioxidant reserve with vitamin E therapy, coupled with the modulation of the development of heart failure, lend strong support in favour of a pathophysiological role for increased oxidative stress in the pathogenesis of heart failure, at least in experimental animals. Association between an increase in oxidative stress and cardiac events in patients requires further examination.

Kumar, Dinender; LeWinter, Martin M.Editor(s): Kumar, Dinender; LeWinter, Martin M. Author Information

Initial studies have suggested that transplantation of embryonic stem (ES) cells following myocardial infarction (MI) in animal models is beneficial; however, the mechanism of benefit is largely unknown. The present study investigated the fate of mouse ES cells transplanted post-MI to determine if the ES cells give rise to the range of major cell types present in the native myocardium. MI was produced by coronary artery ligation in C57BL/6 mice. Two different mouse ES cell lines, expressing eGFP and β-galactosidase, respectively, were tested. Post-MI intramyocardial injection of 3 × 104 ES cells was compared to injection of media alone. Histochemistry and immunofluorescence were used to track the transplanted ES cells and identify the resulting cell types. Echocardiography assessed the cardiac size and function in a blinded fashion. Two weeks post-MI, engraftment of the transplanted ES cells was demonstrated by eGFP or β-galactosidase-positive cells in the infarct region without evidence for tumor formation. Co-immunolabeling demonstrated that the transplanted ES cells had become cardiomyocytes, vascular smooth muscle, and endothelial cells. Echocardiographic analysis showed that ES cell transplantation resulted in reduced post-MI remodeling of the heart and improved cardiac function. In conclusion, transplanted mouse ES cells can regenerate infarcted myocardium in part by becoming cardiomyocytes, vascular smooth muscle, and endothelial cells that result in an improvement in cardiac structure and function. Therefore, ES cells hold promise for myocardial cellular therapy.

Background/Aims: Angiotensin II (Ang II) mediates progressive nephron loss in diabetes and stimulates apoptotic cell death in several tissues. We studied the extent of apoptosis in streptozotocin (STZ) induced diabetic nephropathy in the rat and the effects of insulin and type 1 (AT(1)) or type 2 (AT(2)) Ang II receptor blockade with losartan or PD123319, respectively. Methods: Three groups of rats were studied after 2 and 12 weeks: (1) controls; (2) STZ-diabetic rats (STZ rats), and (3) STZ-diabetic rats with insulin implants. Additional rats were treated with losartan (25 mg/kg/day) and/or PD123319 (10 mg/kg/day) for 2 weeks. Kidneys were examined for apoptosis, using the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, DNA laddering, and electron microscopy. Immunoblotting determined expression of the proapoptotic protein Bax and of the antiapoptotic protein Bcl-2 in proximal tubules. Results: Diabetes caused a significant increase in apoptosis, involving tubular and interstitial cells of cortex and medulla, but not glomerular cells (2 weeks: controls 264+/-94 vs. STZ rats 1,501+/-471 apoptotic nuclei/kidney section; p<0.02; n=6-8), an effect reversed by insulin. In STZ rats, ultrastructural examination revealed chromatin condensation and nuclear fragmentation in tubular and interstitial cells. At 2 and 12 weeks, a significant decrease in the expression of the antiapoptotic protein Bcl-2 occurred in STZ rat proximal tubules, with restoration by insulin. In STZ rats, treatment for 2 weeks with losartan or PD123319 inhibited apoptosis in the kidneys, with no additive effect of the combination therapy. Conclusions: Apoptosis occurs in diabetic nephropathy, involving tubular and interstitial cells, an effect reversed by insulin therapy. Furthermore, the effects of AT(1) or AT(2) receptor blockade suggest that Ang II is involved in mediating apoptosis in the diabetic kidney. Copyright (C) 2004 S. Karger AG, Basel.

Mitogen-activated protein kinases (MAPKs) have been implicated during ischemia-reperfusion (IR) and angiotensin II (AngII) type 2 receptor (AT2R) blockade has been shown to induce cardioprotection involving protein kinase Cε-(PKCε) signaling after IR. We examined whether the 3 major MAPKs, p38, c-Jun NH2-terminal kinase (JNK-1 and JNK-2), and extracellular signal regulated kinases (ERK-1 and ERK-2) are activated after IR and whether treatment with the AT2R antagonist PD123,319 (PD) alters their expression. Isolated rat hearts were randomized to control (aerobic perfusion, 80 min), IR (no drug; 50 min of perfusion, 30 min global ischemia and 30 min reperfusion; working mode), and IR + PD (0.3 μmol/l) and left ventricular (LV) work was measured. We measured LV tissue content of p38, p-p38, p-JNK-1 (54 kDa), p-JNK-2 (46 kDa), p-ERK-1 (44 kDa), p-ERK-2 (42 kDa) and PKCε proteins by immunoblotting and cGMP by enzyme immunoassay. IR resulted in significant LV dysfunction, increase in p-p38 and p-JNK-1/-2, no change in p-ERK-1/-2 or PKCε, and decrease in cGMP. PD improved LV recovery after IR, induced a slight increase in p-p38 (p < 0.01 vs. control), normalized p-JNK-1, did not change p-ERK-1/-2, and increased PKCε and cGMP. The overall results suggest that p38 and JNK might play a significant role in acute IR injury and the cardioprotective effect of AT2R blockade independent of ERK. The activation of p38 and JNKs during IR may be linked, in part, to AT2R stimulation.

Diabetic nephropathy is characterized by an early period of renal growth with glomerular and tubular cell hypertrophy, but this is followed by progressive glomerulosclerosis and tubulointerstitial fibrosis, associated with loss of renal tissue. We studied whether apoptotic cell death occurs in human diabetic nephropathy. Percutaneous renal biopsy samples were obtained from five patients with diabetic nephropathy who were receiving insulin and/or angiotensin-converting enzyme inhibitor therapy. Apoptosis was determined by the presence of DNA fragmentation, detected by in situ TUNEL staining, and by characteristic features on electron microscopy, such as chromatin condensation. Apoptosis was present in all five biopsy specimens, either in epithelial cells of the proximal or distal tubules, or in endothelial cells or interstitial cells. No apoptosis was detected in cells of the glomeruli. The present study provides evidence for apoptosis in human diabetic kidney, and suggests a role for apoptosis in the gradual loss of renal mass.

Cardiomyocyte (CM) apoptosis has been reported in a variety of cardiovascular diseases, including myocardial infarction, ischemia/reperfusion, end-stage heart failure, arrhythmogenic right ventricular dysplasia, and adriamycin-induced cardiomyopathy. The role of CM apoptosis in the development and progression of cardiac diseases merits further investigation. Cumulative evidence suggests that reactive oxygen species (ROS), which have been implicated in cardiac pathophysiology, can trigger myocyte apoptosis by up-regulating proapoptotic proteins, such as Bax and caspases, and the mitochondria-dependent pathway. These apoptotic proteins and pathways are inhibited by various antioxidants, as well as by overexpression of the antiapoptotic protein Bcl-2 by way of the antioxidant pathway. Detection of CM apoptosis with the terminal transferase-mediated DNA nick-end labeling assay alone has recently been questioned because of technical concerns regarding its sensitivity and specificity. Because CMs are mononuclear or binuclear, if only one nucleus or a certain percentage of fragmented nuclei is stained with TUNEL assay at the early stage of apoptotic cell death, it remains unknown whether this particular early apoptotic CM is still functionally active. The issue of TUNEL specificity further questions reports of high percentages of apoptotic CM nuclei (0.02%-35%) in the heart. Nevertheless, oxidative stress is a major apoptotic stimulus in many cardiovascular diseases and the process can be inhibited by antioxidants both in vitro and in vivo.

Background: The stress-responsive mitogen-activated protein kinases (MAPKs) (p38-MAPK, c-Jun NH 2 -terminal kinase [JNK-1 and JNK-2], and extracellular signal regulated kinases [ERK-1 and ERK-2]) might be involved in angiotensin II (AIJ)-induced ischemiareperfusion injury. Cardioprotection induced by All type 1 (AT 1 ) and type 2 (AT 2 ) receptor blockade during ischemia-reperfusion is associated with protein kinase Cc (PKCe), nitric oxide, and cyclic guanosine monophosphate (cGMP) signaling. Our aim was to assess the effect of selective AT, and AT 2 receptor blockade with losartan and PD123,319, respectively, on MAPK expression after ischemia-reperfusion in isolated working rat hearts. Methods: Groups of six hearts were subjected to global ischemia (30 minutes) followed by reperfusion (30 minutes) and exposed to no drug/no ischemia-reperfusion (control), ischemia-reperfusionlno drug, and ischemia-reperfusion with losartan (1 MM), or PD123,319 (0.3,uM) and additional groups. AT,/AT 2 receptor expression, MAPKs, PKCE, and cGMP, and changes in mechanical function were measured. Western blotting was done on left ventricular tissue for AT,/AT 2 , p38/phosphorylated-p38 (p-p38), phosphorylated (p)-JNK-1/-2, phosphorylated (p)-ERK-1/-2, and PKCe proteins; Northern blots for AT,/AT 2 mRNA; and enzyme immunoassay for cGMP. Results: Compared with controls, ischemia-reperfusion induced significant left ventricular dysfunction, decreased AT 2 protein and mRNA, increased p-p38 and p-JNK-1/-2, did not change p-ERK-1/-2 or PKCe, and decreased cGMP. PD123,319 improved left ventricular recovery after ischemia-reperfusion, increased AT 2 protein and mRNA, mildly increased p-p38, normalized p-JNK-1, did not change p-ERK-1/-2, and increased PKCe and cGMP. Losartan did not change p-p38, increased p-JNK-1, and did not change pERK- 1/-2, PKCe, or cGMP. Conclusions: The overall results suggest that the activation of p38-MAPK and JNK might be linked to All signaling and play a significant role in acute ischemia-reperfusion injury as well as in the cardioprotective effect of AT 2 receptor blockade.

OBJECTIVES We sought to determine whether reperfusion and the calcium channel blocker amlodipine or the angiotensin-converting enzyme inhibitor enalapril, during healing over six weeks after myocardial infarction (MI), limit structural vascular remodeling in the noninfarct zone (NIZ),BACKGROUND The effect of reperfusion and amlodipine or enalapril on structural vascular remodeling during healing of MI has not been determined.METHODS We randomly, assigned 54 dogs to reperfused or nonreperfused MI, followed by, twice-daily doses of oral placebo, amlodipine (5 mg) or enalapril (5 mg) for six weeks and three days off treatment, or to three matching sham groups. We measured in vivo hemodynamic data and left ventricular (LV) function and remodeling (by echocardiography) over the six weeks, as well as ex vivo structural vascular, ventricular and collagen remodeling in the hearts after six weeks.RESULTS Compared with placebo and sham groups, both amlodipine arid enalapril with or without reperfusion produced LV unloading and limited structural LV remodeling arid dysfunction over six weeks in vivo, and also decreased the NIZ resistance vessel media/lumen area ratio at six weeks ex vivo. In addition, amlodipine, but not enalapril, preserved infarct scar collagen and increased the border zone collagen volume fraction and perivascular fibrosis, its well as NIZ resistance vessel media thickness. Enalapril, but not amlodipine, decreased transforming growth factor-beta in the border zone and NIZ.CONCLUSIONS The results indicate that therapy with amlodipine and enalapril during healing after reperfused MI Emits structural vascular remodeling in the NIZ, probably by different mechanisms. (C) 2002 by the American College of Cardiology Foundation.

BACKGROUND: The angiotensin 11 type 2 receptor (AT(2)R) is considered to be antigrowth and to mediate apoptosis in several cell types. Whether AT(2)R upregulation, associated with angiotensin II type 1 receptor (AT(1)R) blockade and cardioprotection after ischemia-reperfusion (IR), might not result in increased cardiomyocyte (CM) apoptosis has not been documented. OBJECTIVES: To determine whether increased AT 2 R protein expression, during AT(1)R blockade after acute IR, is associated with no increase in CM apoptosis. MATERIALS AND METHODS: The recovery of left ventricular (LV) mechanical function after acute IR (30 min of ischernia, 40 min of reperfusion) was measured in isolated Langendorff rat hearts following pretreatment with the AT(1)R antagonist candesartan (CN) (CN 10 nmol/L) for 40 min before ischemia. The authors established with an initial dose-response curve using escalating concentrations of CN that 10 nmol/L abrogated vasoconstriction induced by angiotensin 11 (0.1 mumol/L). AT(1)R and AT(2)R protein expression (Western immunoblot), CM apoptosis (terminal deoxynucleotidyl transferase-mediated 2',deoxyuridine 5'-triphosphate nick end-labelling assay and nuclear morphology) and apoptotic markers (Bax, Bcl-2, caspase-3, p53) were assessed in LV tissue. RESULTS: Compared with IR controls, CN improved peak systolic pressure, IV developed pressure and positive dp/dt, and increased AT(2)R (not AT(1)R) protein, but did not change the level of apoptosis or the expression of Bax, Bcl-2, caspase-3 or p53. CN also increased AT(2)R protein after ischernia alone but did not change CM apoptosis or expression of the markers. CONCLUSIONS: Increased AT(2)R protein expression during AT(1)R blockade after IR in the isolated Langendorff rat heart is associated with cardioprotection but no increase in CM apoptosis.

Heart disease is a major cause of morbidity and mortality due to its complex pathogenesis. Myocyte cell loss through apoptosis has been reported in a variety of cardiovascular disease conditions including myocardial infarction (MI), ischemia-reperfusion injury, end-stage heart failure and adriamycin cardiomyopathy.

Significant progress has been made in developing an understanding of the cell biology of the apoptotic process. It is also clear that myocyte apoptosis occurs in a variety of cardiovascular disease states such as myocardial infarction, ischemia-reperfusion injury, end stage heart failure, arrhythmogenic right ventricular dysfunction, adriamycin cardiomyopathy and atherosclerosis. There are still many gaps in our knowledge concerning the biology as well as the role of apoptosis in cardiac pathophysiologies. Another aspect that needs resolution is an enhanced sensitivity and specificity of techniques used for the analysis of myocardium apoptosis. The true picture of the number of myocyte apoptotic cell deaths is also far from clear. Among different stimuli as the cause of apoptosis, oxidative stress as the instigator and/or mediator of apoptosis merits a serious consideration.

BACKGROUND:The angiotensin II type 2 receptor (AT2R) is considered to be antigrowth and to mediate apoptosis in several cell types. Whether AT2R upregulation, associated with angiotensin II type 1 receptor (AT1R) blockade and cardioprotection after ischemia-reperfusion (IR), might not result in increased cardiomyocyte (CM) apoptosis has not been documented.OBJECTIVES:To determine whether increased AT2R protein expression, during AT1R blockade after acute IR, is associated with no increase in CM apoptosis.MATERIALS AND METHODS:The recovery of left ventricular (LV) mechanical function after acute IR (30 min of ischemia, 40 min of reperfusion) was measured in isolated Langendorff rat hearts following pretreatment with the AT1R antagonist candesartan (CN) (CN 10 nmol/L) for 40 min before ischemia. The authors established with an initial dose-response curve using escalating concentrations of CN that 10 nmol/L abrogated vasoconstriction induced by angiotensin II (0.1 mol/L). AT1R and AT2R protein expression (Western immunoblot), CM apoptosis (terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end-labelling assay and nuclear morphology) and apoptotic markers (Bax, Bcl-2, caspase-3, p53) were assessed in LV tissue.RESULTS:Compared with IR controls, CN improved peak systolic pressure, LV developed pressure and positive dp/dt, and increased AT2R (not AT1R) protein, but did not change the level of apoptosis or the expression of Bax, Bcl-2, caspase-3 or p53. CN also increased AT2R protein after ischemia alone but did not change CM apoptosis or expression of the markers.CONCLUSIONS:Increased AT2R protein expression during AT1R blockade after IR in the isolated Langendorff rat heart is associated with cardioprotection but no increase in CM apoptosis.