Intracellular calcium, DNase activity and myocyte apoptosis in aging Fischer 344 rats.

Myocyte apoptosis increases with age in Fischer 344 rats, but the multiple molecular events implicated in this phenomenon remain to be identified. Several defects involving Ca2+ homeostasis, pH, and the expression of p53 and genes of the Bcl-2 protein family may contribute to the activation of myocyte death. Therefore, changes in intracellular pH, cytosolic Ca2+, DNase I and DNase II were measured in myocytes isolated by enzymatic digestion from rats of different ages. Moreover, the expression of p53, Bcl-2 and Bax in these cells was determined. Measurements of intracellular pH by BCECF fluorescence at 3, 12 and 24 months showed that this parameter did not change with age: 3 months, 7.20+/-0.05; 12 months, 7.21+/-0.07; 24 months, 7.18+/-0.09. In contrast, diastolic Ca2+ determined by the Fura 2-AM method increased progressively from 99.8+/-1.9 nm at 3 months to 136.3+/-9.6 nm at 24 months (P<0.001). Concurrently, DNase I activity evaluated by plasmid digestion assay in myocytes increased 3.2-fold from 3 to 24 months (P<0.02). Conversely, pH-dependent-DNase II remained essentially constant with age. Western blotting performed on ventricular myocytes did not detect significant changes in p53, Bax and Bcl-2 proteins with age. Similarly, immunocytochemically, the fraction of myocytes labeled by p53, Bax and Bcl-2 did not change from 3 to 24 months. In conclusion, myocyte aging is characterized by an increase in diastolic calcium which may activate DNase I triggering apoptosis, independently from the expression of p53, Bax and Bcl-2 in the cells.

Medical case management after laminectomy or craniotomy: do all patients benefit from admission to the intensive care unit?

To define severity of illness to identify most effectively patients for whom admission to the intensive care unit (ICU) is unnecessary, the authors performed a retrospective cost-effectiveness analysis. The authors studied the records of 113 patients who were admitted to the ICU after undergoing laminectomy (or other spinal cord surgery) or craniotomy for removal of neoplasm; the Acute Physiology and Chronic Health Evaluation III prognostic system had identified these patients as having a 10% or less risk of requiring intervention while in the ICU. No patient required active intervention during a mean stay of 3.26 days in the ICU. Combined use of a "step-down" postoperative care unit and ICU can optimize allocation of medical resources while providing high-quality care for some neurosurgical patients who are at low risk of requiring postoperative intervention.

p53 Induces myocyte apoptosis via the activation of the renin-angiotensin system.

The mechanism by which p53 activates apoptosis in various cell systems is unknown. In the absence of an external death stimulus, p53 and p53-dependent genes, bcl-2 and bax, cannot trigger apoptosis. However, p53 may enhance not only transcription of bax and repress bcl-2, but also may upregulate the local renin-angiotensin system, inducing the formation and secretion of angiotensin II from the cells. To test this hypothesis, adult rat ventricular myocytes were infected with AdCMV.p53, which resulted in downregulation of Bcl-2, upregulation of Bax, and death of 34% of the cells. Gel retardation assays demonstrated p53 binding in the promoters of angiotensinogen and angiotensin II AT1 receptor subtype. Angiotensinogen and AT1 mRNAs increased in AdCMV.p53 cells and this phenomenon was associated with a 14-fold increase in the secretion of angiotensin II. The AT1 receptor blocker losartan and angiotensin II antibody prevented p53-induced apoptosis. Thus, p53 enhances the myocyte renin-angiotensin-system and decreases the Bcl-2/Bax ratio in the cells, triggering apoptosis. The identification of this new pathway in p53-mediated apoptosis may be critical in the alterations of myocardial function in the pathologic heart.

Apoptosis in the failing human heart.

BACKGROUND: Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. METHODS: Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. RESULTS: Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonstration of DNA-strand breaks in myocyte nuclei was coupled with the documentation of chromatin condensation and fragmentation by confocal microscopy. All these findings reflect apoptosis of myocytes. The percentage of myocytes labeled with BCL2 (which protects cells against apoptosis) was 1.8 times as high in the hearts of patients with cardiac failure as in the normal hearts, whereas labeling with BAX (which promotes apoptosis) remained constant. The near doubling of the expression of BCL2 in the cardiac tissue of patients with heart failure was confirmed by Western blotting. CONCLUSIONS: Programmed death of myocytes occurs in the decompensated human heart in spite of the enhanced expression of BCL2; this phenomenon may contribute to the progression of cardiac dysfunction.

Necrotic and apoptotic myocyte cell death in the aging heart of Fischer 344 rats.

To determine the effects of aging on myocyte cell death, Fischer 344 rats at 3, 7, 12, 16, and 24 mo of age were injected with myosin monoclonal antibody for the localization and quantification of necrotic myocyte cell death in the left ventricle, interventricular septum, and right ventricle. Conversely, the presence of DNA strand breaks in myocyte nuclei, indicative of programmed cell death, was evaluated by the terminal deoxynucleotidyl transferase assay and confirmed by DNA laddering. Myocyte necrosis, which involved nearly 1,000 myocytes in the left ventricular free wall at 3 mo, progressively increased with aging, reaching a value of 13,600 myocytes at 24 mo. Corre- sponding values in the interventricular septum were 300 and 9,400 myocytes. In the right ventricle, there were 270 necrotic myocytes at 3 mo and 9,000 at 24 mo. Programmed myocyte cell death was restricted to the left ventricular free wall and included 140 cells at 3 mo. This form of myocyte cell death increased at the subsequent age intervals, resulting in the involvement of 874 cells at 24 mo. The combination of necrosis and apoptosis in the left ventricular free wall was associated with 1,150 cells dying at 3 mo and 14,500 at 24 mo. In conclusion, myocyte cell death, apoptotic and necrotic in nature, constitutes an important determinant of the aging process, possibly mediating the occurrence of ventricular dysfunction and failure in the old heart.

Programmed myocyte cell death affects the viable myocardium after infarction in rats.

To determine whether apoptotic and necrotic myocyte cell death occur acutely and chronically after infarction, the formation of DNA strand breaks and the localization of myosin monoclonal antibody labeling were analyzed in the surviving myocardium from 20 min to 1 month. DNA strand breaks in myocyte nuclei were detected as early as 3 h following coronary artery occlusion and were still present at 1 month. This cellular process was characterized biochemically by internucleosomal DNA fragmentation which produced DNA laddering on agarose gel electrophoresis. Quantitatively, 155 myocyte nuclei per 10(6) cells exhibited DNA strand breaks in the portion adjacent to the infarcted tissue at 3-12 h. This parameter increased to 704 at 1-2 days and subsequently decreased to 364 at 7 days, 188 at 14 days, and 204 at 1 month. In the remote myocardium, the number of myocyte nuclei with DNA strand breaks was 84 per 10(6) at 3-12 h and remained essentially constant up to 1 month. Programmed myocyte cell death was accompanied by a decrease in the expression of bcl-2 and an increase in the expression of bax. The changes in the expression of these genes were present at 1 and 7 days after coronary artery occlusion. In conclusion, the mechanical load produced by myocardial infarction and ventricular failure may affect the regulation of bcl-2 and bax in the viable myocytes, triggering programmed cell death and the remodeling of the ventricular wall.

Acute aortic occlusion--factors that influence outcome.

PURPOSE: The purpose of this study was to report our experience in the management of acute aortic occlusion and to analyze factors that influenced the outcome. METHODS: This was a retrospective analysis of 48 patients with acute aortic occlusion treated over a 19-year period. Presentation included limb ischemia in 34, acute abdomen in four, spinal cord compression-like symptoms in eight, and sudden onset of hypertension in two patients. Thrombosis was the cause of acute aortic occlusion in 44, and embolus in four patients. Acute thrombosis was associated with underlying atherosclerotic occlusive disease in 36 patients. In these, thrombosis was due to low-flow state caused by cardiac dysfunction or severe volume depletion. Thrombosed aneurysms caused aortic occlusion in two patients. Hypercoagulable state caused thrombosis of relatively normal aorta in six patients. Angiography in 39 patients revealed occlusion to be juxtarenal or infrarenal in 37 and suprarenal in two. Left ventricular function (LVF) was assessed in 42 patients. Circulation was restored in 45 (aortofemoral bypass in 22, axillofemoral bypass in 12, and thromboembolectomy in 11). This was not feasible in three patients. Additional surgical procedures were required in 29 patients (64%). RESULTS: The overall mortality rate was 52% (25 of 48). Of the 20 patients with severely compromised LVF, 17 died (85%). In contrast, only five (23%) deaths occurred among 22 with good LVF. Among 29 patients who required additional operations, 18 died (62%). All four patients with embolic occlusion survived. Patients with normal LVF but hypercoagulable state had dismal outcome--only one of the six survived. CONCLUSIONS: Acute aortic occlusion is infrequent. Presentation may be varied, thus delaying diagnosis. Poor LVF, thrombosis of arteries below the inguinal ligament or of visceral arteries, and "hypercoagulable state" portend ominous prognosis.

Nicotine increases expression of tyrosine hydroxylase gene. Involvement of protein kinase A-mediated pathway.

Nicotine, a major component of tobacco smoke, stimulates catecholamine secretion and activates catecholamine biosynthetic enzymes such as tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) in adrenal medullary cells. We investigated the effect of long term treatment with nicotine on TH and DBH gene expression in rat PC12 pheochromocytoma cells. Nicotine treatment for 1-2 days increased both the TH and DBH mRNA levels. The effect of nicotine on TH mRNA seems to be transcriptionally mediated. Deletion analysis of the 5' promoter region of the TH gene showed that the region containing a cyclic AMP/calcium regulatory element is sufficient for the nicotinic induction of TH. Nicotine did not induce TH mRNA or chloramphenicol acetyltransferase reporter activity in mutant PC12 cells deficient in protein kinase A activity. However, the deficiency in protein kinase A activity did not affect the elevation in intracellular calcium concentration caused by nicotine, indicating normal receptor function. These results suggest that a cAMP-mediated pathway plays a crucial role in the long term nicotine-induced activation of the TH gene.