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The mechanisms by which apoptotic myocytes are removed by macrophages have not been fully elucidated. This study examined whether apoptotic myocytes actively recruit macrophages by generating monocyte chemoattractant protein-i (MCP-1) in experiments in vitro and in vivo. Neonatal rat cardiac myocytes were incubated for 4 h in the presence or absence of staurosporine (STS, 0.2-1 μmol/l), an apoptosis inducer. Nuclear staining with DAPI showed that STS induced apoptosis in a dose-dependent fashion. STS (1 μmol/l) caused extensive DNA fragmentation and increased caspase-3 activity compared with a serum-deprived control. MCP-l mRNA and protein levels in myocytes increased twofold and fourfold, respectively, on STS treatment, and immunochemical staining revealed that apoptotic myocytes expressed MCP-l. To elucidate the role of MCP-1 expressed in apoptotic myocytes to recruit macrophages/monocytes, rat monocytes were incubated in the supernatant of STS-treated myocytes using a transwell system. The culture medium of STS-treated myocytes recruited monocytes in a MCP- I-dependent fashion. In addition, experiments were performed in vivo using ischemia-reperfused rat hearts. Rats were subjected to 30 mm of ligation of the left coronary artery followed by 24 h of reperfusion. After the reperfusion, in the ischemic border myocardium, 17.1 ± 1.1% of myocytes were terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) positive. Moreover, double staining using the TUNEL technique and immunohistochemistry with MCP- I antibody showed that 69.8 ± 3.9% of TUNEL-positive myocytes expressed MCP-l protein. Concomitantly, activated macrophages infiltrated the areas of apoptosis remarkably. These results suggest that apoptotic myocytes produce MCP-l, which have a critical role in the active recruitment of macrophages. [ABSTRACT FROM AUTHOR]

Examines the involvement of nitric oxide (NO) in the mechanisms by which endotoxemia causes cardiac depression. Infusion of rats with lipopolysaccharide (LPS) or saline; Isolation of hearts and mounting in a Langendorff apparatus; Determination of the left ventricular developed pressure before biochemical analysis of the myocardium; Finding that the myocardial adenosine triphosphate levels were not significantly altered after LPS injection.

Examines the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Effect of glucose concentration on adenosine triphosphate content; Effect of staurosporine treatment on cell viability; Mitochondrial transmembrane potential; Effect of glucose concentration on caspase-3 activity.

The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial NO(2)(-) and NO(3)(-) (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.