Response to myocardial ischemia/reperfusion injury involves Bnip3 and autophagy.

Ischemia and reperfusion (I/R) injury is associated with extensive loss of cardiac myocytes. Bnip3 is a mitochondrial pro-apoptotic Bcl-2 protein which is expressed in the adult myocardium. To investigate if Bnip3 plays a role in I/R injury, we generated a TAT-fusion protein encoding the carboxyl terminal transmembrane deletion mutant of Bnip3 (TAT-Bnip3DeltaTM) which has been shown to act as a dominant negative to block Bnip3-induced cell death. Perfusion with TAT-Bnip3DeltaTM conferred protection against I/R injury, improved cardiac function, and protected mitochondrial integrity. Moreover, Bnip3 induced extensive fragmentation of the mitochondrial network and increased autophagy in HL-1 myocytes. 3D rendering of confocal images revealed fragmented mitochondria inside autophagosomes. Enhancement of autophagy by ATG5 protected against Bnip3-mediated cell death, whereas inhibition of autophagy by ATG5K130R enhanced cell death. These results suggest that Bnip3 contributes to I/R injury which triggers a protective stress response with upregulation of autophagy and removal of damaged mitochondria.

Highly efficient gene transfer into adult ventricular myocytes by recombinant adenovirus.

Molecular dissection of mechanisms that govern the differentiated cardiac phenotype has, for cogent technical reasons, largely been undertaken to date in neonatal ventricular myocytes. To circumvent expected limitations of other methods, the present study was initiated to determine whether replication-deficient adenovirus would enable efficient gene transfer to adult cardiac cells in culture. Adult rat ventricular myocytes were infected, 24 h after plating, with adenovirus type 5 containing a cytomegalovirus immediate-early promoter-driven lacZ reporter gene and were assayed for the presence of beta-galactosidase 48 h after infection. The frequency of lacZ+ rod-shaped myocytes was half-maximal at 4 x 10(5) plaque-forming units (PFU) and approached 90% at 1 x 10(8) PFU. Uninfected cells and cells infected with lacZ- virus remained colorless. Beta-galactosidase activity concurred with the proportion of lacZ+ cells and was contingent on the exogenous lacZ gene. At 10(8) PFU/dish, cell number, morphology, and viability each were comparable to uninfected cells. Thus, adult ventricular myocytes are amenable to efficient gene transfer with recombinant adenovirus. The relative uniformity for gene transfer by adenovirus should facilitate tests to determine the impact of putative regulators upon the endogenous genes and gene products of virally modified adult ventricular muscle cells.

Adenoviral delivery of E2F-1 directs cell cycle reentry and p53-independent apoptosis in postmitotic adult myocardium in vivo.

Irreversible exit from the cell cycle precludes the ability of cardiac muscle cells to increase cell number after infarction. Using adenoviral E1A, we previously demonstrated dual pocket protein- and p300-dependent pathways in neonatal rat cardiac myocytes, and have proven that E2F-1, which occupies the Rb pocket, suffices for these actions of E1A. By contrast, the susceptibility of adult ventricular cells to viral delivery of exogenous cell cycle regulators has not been tested, in vitro or in vivo. In cultured adult ventricular myocytes, adenoviral gene transfer of E2F-1 induced expression of proliferating cell nuclear antigen, cyclin-dependent protein kinase 4, cell division cycle 2 kinase, DNA synthesis, and apoptosis. In vivo, adenoviral delivery of E2F-1 by direct injection into myocardium induced DNA synthesis, shown by 5'-bromodeoxyuridine incorporation, and accumulation in G2/M, by image analysis of Feulgen-stained nuclei. In p53(-)/- mice, the prevalence of G1 exit was more than twofold greater; however, E2F-1 evoked apoptosis and rapid mortality comparably in both backgrounds. Thus, the differential effects of E2F-1 on G1 exit in wild-type versus p53-deficient mice illustrate the combinatorial power of viral gene delivery to genetically defined recipients: E2F-1 can override the G1/S checkpoint in postmitotic ventricular myocytes in vitro and in vivo, but leads to apoptosis even in p53(-)/- mice.

Serpin protein CrmA suppresses hypoxia-mediated apoptosis of ventricular myocytes.

BACKGROUND: In this study, we ascertain whether caspase 8 activation and mitochondrial defects underlie apoptosis of ventricular myocytes during hypoxia. As an approach to circumvent the potential shortcomings surrounding the limited permeability and short half-life of the synthetic peptide inhibitors designed to block caspase activation, we constructed a replication-defective adenovirus encoding the serpin caspase inhibitor protein CrmA to ensure efficient and continual inhibition of caspase 8 activity during chronic hypoxia. METHODS AND RESULTS: In contrast to normoxic cells, oxygen deprivation of postnatal ventricular myocytes for 24 hours resulted in a 9-fold increase (P<0.05) in apoptosis as determined by Hoechst 33258 staining and nucleosomal DNA laddering. Moreover, hypoxia provoked a 1.5-fold increase (P<0.01) in caspase 8-like activity. Furthermore, hypoxia provoked perturbations to mitochondria consistent with the mitochondrial death pathway, including permeability transition pore (PT) opening, loss of mitochondrial membrane potential ((m)), and cytochrome c release. Importantly, CrmA suppressed caspase 8 activity, PT pore changes, loss of (m), and apoptosis but had no effect on hypoxia-mediated cytochrome c release. Furthermore, Bongkrekic acid, an inhibitor of PT pore, prevented hypoxia-induced PT pore changes, loss of (m), and apoptosis but had no effect on hypoxia-mediated cytochrome c release. CONCLUSIONS: To our knowledge, we provide the first direct evidence for the operation of CrmA as an antiapoptotic factor in ventricular myocytes during prolonged durations of hypoxia. Furthermore, our data suggest that perturbations to mitochondria including PT pore changes and (m) loss are caspase-regulated events that appear to be separable from cytochrome c release.

Does multiple-dose charcoal therapy enhance salicylate excretion?

Multiple-dose charcoal therapy has been shown to increase the excretion of some drugs. This study assesses the effects of this intervention on salicylate excretion in the postabsorptive phase. Ten human volunteers participated in this randomized, controlled, crossover, two-limbed protocol. On two occasions each volunteer ingested 2880 mg of aspirin. During the experimental limb, 25 g of activated charcoal was ingested at 4, 6, 8, and 10 hours after drug ingestion. Pharmacokinetic data were derived from serial serum salicylate concentrations, and urinary salicylate excretion was quantified. Treatment effects were 9% and 18%, respectively. Although both are significant, they are clinically modest, making multiple-dose charcoal therapy of questionable value for acute salicylate poisoning. Controlled data demonstrating the clinical efficacy of this therapy are required to validate it as an intervention for this condition.

The cardiac cell cycle, pocket proteins, and p300.

Identification of cell cycle regulators-tumor suppressor "pocket" proteins, cyclins, cyclin-dependent protein kinases (cdks), and an emerging family of cdk inhibitors-has fueled fundamental research into mechanisms that regulate cell proliferation, as well as clinical investigation in the settings, especially, of cancer and restenosis. The failure of ventricular myocytes to regenerate through cell proliferation following infarction might arguably best be viewed as the ultimate problem in growth control, though until recently, only sporadic studies were available that addressed the identity or function of proteins governing the cell cycle in ventricular muscle. From this perspective, it may be less fruitful to debate whether mitoses never occur in adult ventricular muscle or merely do so with such rarity as to be inconsequential, than to define the repertoire of molecules that hold the cardiac cell cycle in check. To substantiate their operation in cardiac cells is the prerequisite step toward establishing what role such pathways might play in cardiac myogenesis, organogenesis, and pathophysiology.

Whole-bowel irrigation versus activated charcoal in sorbitol for the ingestion of modified-release pharmaceuticals.

Overdose with modified-release pharmaceuticals is an increasing phenomenon. This study examines whole-bowel irrigation as a potential decontamination strategy after overdose with enteric-coated acetylsalicylic acid and compares it with administration of activated charcoal in sorbitol, which is currently the recommended intervention. A three-phase randomized crossover protocol was used in 10 adult volunteers. Each volunteer ingested nine 325 mg doses of enteric-coated acetylsalicylic acid on three occasions, with at least 1 week between each administration period. Serum samples were analyzed for salicylic acid concentration by HPLC. Both interventions decreased peak salicylic acid concentration, time-to-zero salicylic acid concentration, and AUC when compared with control (p less than 0.01). Whole-bowel irrigation was superior to activated charcoal in sorbitol by all three criteria (p less than 0.05). Adverse effects were qualitatively and quantitatively greater during activated charcoal in sorbitol, and the volunteers preferred whole-bowel irrigation over charcoal in sorbitol. Our data suggest that whole-bowel irrigation should be considered for overdose of other modified-release pharmaceuticals.

Apoptosis in adriamycin cardiomyopathy and its modulation by probucol.

The dose-dependent cardiomyopathy and heart failure due to adriamycin have been shown to be due to increased oxidative stress and loss of myocytes. We examined the incidence of myocardial apoptosis as well as changes in the expression of apoptotic regulatory gene products in an established animal model of adriamycin cardiomyopathy. Rats were treated with adriamycin (cumulative dose, 15 mg/kg), and the hearts were examined for apoptosis as well as expression of Bax, caspase 3, and Bcl-2 at 0, 4, 10, 16, and 21 days after the treatment. A significant increase in the incidence of apoptosis was seen at 4 days, followed by a decline at 10 and 16 days of posttreatment. At 21 days, the number of apoptotic cells increased again and included cells of the conducting system. Expression of Bax corresponded to these biphasic changes, whereas the converse was true for the expression of Bcl-2. The latter peaked at 10 days followed by a decline at 16 and 21 days. The Bax/Bcl-2 ratio also correlated with the incidence of apoptosis. Expression of caspase 3 correlated with increased apoptosis, but only at early time points. Probucol (cumulative dose, 120 mg/kg), a known antioxidant as well as promoter of endogenous antioxidants, significantly reduced the incidence of apoptosis as well as expression of Bax. Adriamycin-induced hemodynamic changes were also prevented by probucol. These data suggest that adriamycin-induced apoptosis is mediated by oxidative stress and may play a role in the development of heart failure.

Regulators of apoptosis in the heart: a matter of life and death.

Programmed cell death or apoptosis is an active physiological process that permits the removal of unwanted or damaged cells from the body through an intrinsic cell-suicide program. Apoptosis is characterized by condensation of the nucleus and cytoplasm without loss of membrane integrity. The occurrence of apoptosis in the vasculature and myocardium has recently been described. Inappropriate loss of myocardial cells is suggested to contribute to conduction defects and ventricular remodelling after injury. The molecular mechanisms that regulate programmed cell death in cardiac muscle cells are poorly defined. However, recent evidence has suggested that specific genes can either provoke or prevent apoptosis. In this regard, the tumour suppressor protein p53 has been proposed to mediate apoptosis, while the Bcl-2 protein prevents it. Prevention of apoptosis in the heart is potentially of significant therapeutic value given the limited capacity of the heart to repair itself after injury. This study determined that the expression of p53 in ventricular myocytes is sufficient to trigger apoptosis. Moreover, p53 results in a significant increase in the expression of the death-promoting protein Bax. Importantly, the antiapoptotic factor Bcl-2 is sufficient to prevent p53-mediated apoptosis and p53-dependent transcription of Bax in ventricular myocytes. The data substantiate a role for p53 and Bcl-2 as crucial regulators of apoptosis in the heart.

A relative deficit in antioxidant reserve may contribute in cardiac failure.

Antioxidant enzyme activities, including superoxide dismutase, glutathione peroxidase and catalase, are known to be altered under various physiological and pathophysiological conditions. There is a significant increase in some of these activities in the myocardium during stable hyperfunctional heart hypertrophy subsequent to pressure overload, as well as after exercise training in rats. Hearts with increased antioxidant capacity have been reported to be more resistant to in vivo and in vitro oxidative stress. On the other hand, cardiomyopathy and heart failure under a variety of conditions are accompanied by increased free radicals and lipid peroxidation. These data lead to the hypothesis that maintained or improved function during compensated heart hypertrophy may be supported by an increased antioxidant capacity, and a relative deficit in this 'antioxidant reserve' may contribute in the decompensated state.

Antioxidant protection against adrenaline-induced arrhythmias in rats with chronic heart hypertrophy.

Effects of vitamin E on adrenaline-induced arrhythmias were examined in rats with chronic heart hypertrophy subsequent to narrowing of the abdominal aorta. After 60 weeks of pressure overload, the rats showed an increase of about 21% in heart/body weight ratio and a small but significant rise in left ventricular end diastolic pressure (LVEDP) (sham control 1.7 +/- 0.67 mmHg; hypertrophy 7.1 +/- 2.7 mmHg) without any change in left ventricular peak systolic pressure (LVSP). Intravenous infusion of adrenaline caused rhythm disorders in a dose-dependent manner and pathological arrhythmias (occurrence of six premature ventricular complexes/min) were observed at doses of 2.9 +/- 0.6 and 3.8 +/- 1.0 micrograms/kg of the drug in control and hypertrophy animals, respectively. Administration of two doses of vitamin E (50 mg/kg intraperitoneally), given 24 h and 1 h before adrenaline infusion, significantly increased the amount of adrenaline required to produce pathological arrhythmias (control 8.0 +/- 3.0; hypertrophy 7.7 +/- 2.0 micrograms/kg). Vitamin E pretreatment did not have any detrimental effect on the pressure readings nor did it have any influence on adrenaline-induced pressure changes. The data suggest that a combination therapy with vitamin E may allow therapeutic use of higher concentrations of adrenaline required to improve function in failing hearts with a reduced risk of arrhythmias.

Cardiac reanimation: targeting cardiomyocyte death by BNIP3 and NIX/BNIP3L.

Programmed cardiac myocyte death contributes to pathological ventricular remodeling and the progression of myocardial infarction or pressure overload hypertrophy to dilated cardiomyopathy. Recent work has identified importance of stress-mediated transcriptional induction of BNIP3 (BCL2 and 19-kDa interacting protein-3) and NIX/BNIP3L in cardiac remodeling. Here, the regulatory mechanisms for these two factors in the heart and their effects on programmed cardiomyocyte death are reviewed, with a focus on information derived from studies using mouse models of cardiac BNIP3 and NIX/BNIP3L overexpression and gene ablation.

Adenovirus mediated-gene transfer into cardiomyocytes.

To circumvent limitations imposed by conventional gene transfer techniques into cardiac muscle cells, we studied whether replication defective adenovirus would obviate this limitation to basic studies of signal transduction and transcriptional control processes in the heart. We demonstrate here the utility of adenovirus mediated gene transfer to introduce foreign DNA into post-mitotic terminally differentiated ventricular myocytes with uniformity and high efficiency. We also provide evidence for the genetic modification of neonatal ventricular myocytes by adenovirus early region 1 (E1) proteins and their impact on cardiac gene transcription and DNA synthesis respectively. Thus, for studies of transcriptional control processes in the heart, which until now have been restricted to neonatal ventricular myocytes; adenovirus mediated gene transfer provides a means to genetically manipulate adult cardiac muscle cells. The advent of adenovirus gene transfer will extend our understanding of the molecular mechanisms that mediate basic cardiac disease and may ultimately provide a means to therapeutically mitigate the disease process.

Bcl-2 intersects the NFkappaB signalling pathway and suppresses apoptosis in ventricular myocytes.

As a first step toward identifying putative regulators of apoptosis in the heart, the impact of the anti-apoptosis protein Bcl-2 (B-cell lymphoma gene) on the NFkappaB (nuclear factor kappa beta) signalling pathway in suppressing apoptosis in ventricular myocytes was studied. The data indicate that adenovirus-mediated delivery of Bcl-2 resulted in a significant increase in NFkappaB-dependent DNA binding and NFkappaB-directed gene transcription. No change in NFkappaB protein content was observed in myocytes expressing Bcl-2. Moreover, the Bcl-2-mediated NFkappaB activation was found to be related to changes in the activity of the NFkappaB regulatory protein IkappaBalpha (inhibitor of kappa beta). In this regard, a marked reduction in IkappaBalpha protein content was observed in ventricular myocytes expressing Bcl-2. The mode by which Bcl-2 regulates IkappaBalpha was related to the N-terminal phosphorylation and degradation of IkappaBalpha by the proteasome since an N-terminal deletion mutant of IkappaBalpha or the proteasome inhibitor lactacystin abrogated Bcl-2's inhibitory effects on IkappaBalpha and prevented NFkappaB activation. Furthermore, adenovirus-mediated delivery of a phosphorylation defective form of IkappaBalpha rendered ventricular myocytes incapable of NFkappaB activation and susceptible to tumour necrosis factor alpha-mediated apoptosis. Moreover, Bcl-2's anti-apoptotic function was lost in cells defective for NFkappaB activation. The data provide evidence for a link between Bcl-2 and the NFkappaB signalling pathway for the suppression of apoptosis in ventricular myocytes.

Increase in endogenous antioxidant enzymes protects hearts against reperfusion injury.

Coarctation of the abdominal aorta in rats for 10 wk increased the heart weight-to-body weight ratio by 36% and peak left ventricular systolic pressure by 75%; there was no apparent change in the end-diastolic pressure, and animals did not show any clinical signs of heart failure. These hypertrophied (H) hearts showed increased activities of superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) with no change in catalase. Lipid peroxide content as indicated by the malondialdehyde (MDA) level was lower in H hearts. There was no apparent difference in either Na+ and Ca2+ content or high-energy phosphates between sham (S) and H hearts. Control and H hearts were subjected to 10 min of ischemia (I) and 15 min of reperfusion (R). Contractile failure and rise in resting tension due to I, in both S and H hearts, were comparable. On reperfusion, H hearts showed better recovery of the developed force and resting tension as well as reduced incidence of arrhythmias when compared with corresponding S hearts. Both SOD and GSHPx activities were depressed due to I-R, but these activities were significantly higher in reperfused H hearts. Reperfused H hearts also showed a better maintenance of the ultrastructure and Na+ and Ca2+ contents, recovery of high-energy phosphates, and reduced MDA levels compared with S hearts. Supplementation of the perfusion medium with SOD (120 U/ml) and catalase (80 U/ml) significantly attenuated the I-R injury in S hearts, and the response in many ways was comparable to H hearts. The study documents the therapeutic potential of increased myocardial endogenous antioxidants against oxidative stress.

Adenovirus E1A represses cardiac gene transcription and reactivates DNA synthesis in ventricular myocytes, via alternative pocket protein- and p300-binding domains.

To examine the potential impact of disrupting "pocket" protein function on cardiac differentiation and growth, we introduced 12 S E1A genes into neonatal ventricular myocytes, by adenoviral gene transfer. In the absence of E1B, E1A was cytotoxic, with features typical of apoptosis. In the presence of E1B, E1A preferentially inhibited transcription of cardiac-restricted alpha-actin promoters, and reactivated DNA synthesis in cardiac myocytes, without cell death. Mutations that abrogate known activities of the amino terminus of E1A, versus conserved region 2, demonstrate that the "pocket" protein- and p300-binding domains each suffice, in the absence of the other, for transcriptional repression and re-entry into S phase.

Apoptosis in ventricular myocytes: the role of tumor suppressor proteins.

Apoptosis or programmed cell death is an important physiologic event crucial for the selective removal of damaged or unwanted cells from body tissues. In the cardiovascular system, apoptosis has been observed in the vasculature and myocardium. Untimely or inappropriate myocardial cell loss through an apoptotic process may contribute to ventricular remodeling and the ultimate demise of ventricular function following injury. Therapeutic interventions designed to modulate or prevent myocardial apoptotic cell loss may therefore prove beneficial in maintaining cardiac function. Incite into the molecular mechanisms that govern apoptosis in mammalian cells has led to the identification of several key factors that promote or prevent the apoptotic process. In this report, we discuss putative regulators of cardiac cell apoptosis with specific reference to the tumor suppressor proteins, p53 and Rb. The interplay between these factors, as well as the anti-apoptotic molecules related to the Bcl-2 the family are discussed in the context of the heart under normal and disease conditions.