Nanette H. Bishopric

Expression of Inducible Nitric Oxide Synthase in Human Heart Failure

BACKGROUND There is increasing evidence that alterations in nitric oxide synthesis are of pathophysiological importance in heart failure. A number of studies have shown altered nitric oxide production by the endothelial constitutive isoform of nitric oxide synthase (NOS), but there is very little information on the role of the inducible isoform. METHODS AND RESULTS We analyzed inducible NOS (iNOS) expression in ventricular myocardium taken from 11 control subjects (who had died suddenly from noncardiac causes), from 10 donor hearts before implantation, and from 51 patients with heart failure (24 with dilated cardiomyopathy [DCM], 17 with ischemic heart disease [IHD], and 10 with valvular heart disease [VHD]). Reverse transcription-polymerase chain reaction was used to confirm the presence of intact mRNA and to detect expression of iNOS and atrial natriuretic peptide (ANP). ANP was used as a molecular phenotypic marker of ventricular failure. iNOS was expressed in 36 of 51 biopsies (71%) from patients with heart failure and in none of the control patients (P<.0001). iNOS expression could also be detected in 50% of the donor hearts. All samples that expressed iNOS also expressed ANP. iNOS gene expression occurred in 67% of patients with DCM, 59% of patients with IHD, and 100% of patients with VHD. To determine whether iNOS protein was expressed in failing ventricles, immunohistochemistry was performed on three donor hearts and nine failing hearts with iNOS mRNA expression. Staining for iNOS was almost undetectable in the donor myocardium and in control sections, but all failing hearts showed diffuse cytoplasmic staining in cardiac myocytes. Expression of iNOS could be observed in all four chambers. Western blot analysis with the same primary antibody showed a specific positive band for iNOS protein in the heart failure specimens; minimal iNOS protein expression was seen in donor heart samples. CONCLUSIONS iNOS expression occurs in failing human cardiac myocytes and may be involved in the pathophysiology of DCM, IHD, and VHD.

Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system.

A method has been developed for culturing cardiac myocytes in a collagen matrix to produce a coherently contracting 3‐dimensional model heart tissue that allows direct measurement of isometric contractile force. Embryonic chick cardiomyocytes were mixed with collagen solution and allowed to gel between two Velcro‐coated glass tubes. During culture, the cardiomyocytes formed spontaneously beating cardiac myocyte‐populated matrices (CMPMs) anchored at opposite ends to the Velcro‐covered tubes through which they could be attached to a force measuring system. Immunohistochemistry and electron microscopy revealed a highly organized tissue‐like structure of α‐actin and α‐tropomyosin‐positive cardiac myocytes exhibiting typical cross‐striation, sarcomeric myofilaments, intercalated discs, desmosomes, and tight junctions. Force measurements of paced or unpaced CMPMs were performed in organ baths after 6–11 days of cultivation and were stable for up to 24 h. Force increased with frequency between 0.8 and 2.0 Hz (positive “staircase”), increasing rest length (Starling mechanism), and increasing extracellular calcium. The utility of this system as a test bed for genetic manipulation was demonstrated by infecting the CMPMs with a recombinant β‐galactosidase‐carrying adenovirus. Transduction efficiency increased from about 5% (MOI 0.1) to about 50% (MOI 100). CMPMs display more physiological characteristics of intact heart tissue than monolayer cultures. This approach, simpler and faster than generation of transgenic animals, should allow functional consequences of genetic or pharmacological manipulation of cardiomyocytes in vitro to be studied under highly controlled conditions.—Eschenhagen, T., Fink, C., Remmers, U., Scholz, H., Wattchow, J., Weil, J., Zimmermann, W., Dohmen, H. H., Schäfer, H., Bishopric, N., Wakatsuki, T., Elson, E. L. Three‐dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J. 11, 683–694 (1997)

Hypoxia and acidosis activate cardiac myocyte death through the Bcl-2 family protein BNIP3

Coronary artery disease leads to injury and loss of myocardial tissue by deprivation of blood flow (ischemia) and is a major underlying cause of heart failure. Prolonged ischemia causes necrosis and apoptosis of cardiac myocytes and vascular cells; however, the mechanisms of ischemia-mediated cell death are poorly understood. Ischemia is associated with both hypoxia and acidosis due to increased glycolysis and lactic acid production. We recently reported that hypoxia does not induce cardiac myocyte apoptosis in the absence of acidosis. We now report that hypoxia-acidosis-associated cell death is mediated by BNIP3, a member of the Bcl-2 family of apoptosis-regulating proteins. Chronic hypoxia induced the expression and accumulation of BNIP3 mRNA and protein in cardiac myocytes, but acidosis was required to activate the death pathway. Acidosis stabilized BNIP3 protein and increased the association with mitochondria. Cell death by hypoxia-acidosis was blocked by pretreatment with antisense BNIP3 oligonucleotides. The pathway included extensive DNA fragmentation and opening of the mitochondrial permeability transition pore, but no apparent caspase activation. Overexpression of wild-type BNIP3, but not a translocation-defective mutant, activated cardiac myocyte death only when the myocytes were acidic. This pathway may figure significantly in muscle loss during myocardial ischemia.

Modulation of Cytokine-Induced Cardiac Myocyte Apoptosis by Nitric Oxide, Bak, and Bcl-x

-Cytokine-induced NO production depresses myocardial contractility and has been shown to be cytotoxic to cardiac myocytes. However, the mechanisms of cytokine-induced cardiac myocyte cell death are unclear. To analyze these mechanisms in detail, we treated neonatal cardiac myocytes in serum-free culture with a combination of the macrophage-derived cytokines interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma. These cytokines caused a time-dependent induction of cardiac myocyte apoptosis, but not necrosis, beginning 72 hours after treatment, as determined by nuclear morphology, DNA internucleosomal cleavage, and cleavage of poly(ADP-ribose) polymerase, reflecting caspase activation. Apoptosis was preceded by a >50-fold induction of inducible NO synthase mRNA and the release of large amounts (5 to 8 nmol/ microgram protein) of NO metabolites (NOx) into the medium. Cell death was completely blocked by an NO synthase inhibitor and attenuated by antioxidants (N-acetylcysteine and DTT) and the caspase inhibitor ZVAD-fmk. Cytokines also mediated an NO-dependent, sustained increase in myocyte expression of the Bcl-2 homologs Bak and Bcl-x(L). The NO donor S-nitrosoglutathione also induced apoptosis and cell levels of Bak, but not of Bcl-x(L). All effects of cytokines, including poly(ADP-ribose) polymerase cleavage, could be attributed to interleukin-1beta; interferon-gamma and tumor necrosis factor-alpha had no independent effects on apoptosis or on NOx production. We conclude that cytokine toxicity to neonatal cardiac myocytes results from the induction of NO and subsequent activation of apoptosis, at least in part through the generation of oxygen free radicals. The rate and extent of this apoptosis is modulated by alterations in the cellular balance of Bak and Bcl-x(L), which respond differentially to cytokine-induced and exogenous NO and by the availability of oxidant species.

Hypoxia-activated apoptosis of cardiac myocytes requires reoxygenation or a pH shift and is independent of p53.

Ischemia and reperfusion activate cardiac myocyte apoptosis, which may be an important feature in the progression of ischemic heart disease. The relative contributions of ischemia and reperfusion to apoptotic signal transduction have not been established. We report here that severe chronic hypoxia alone does not cause apoptosis of cardiac myocytes in culture. When rapidly contracting cardiac myocytes were exposed to chronic hypoxia, apoptosis occurred only when there was a decrease in extracellular pH ([pH](o)). Apoptosis did not occur when [pH](o) was neutralized. Addition of acidic medium from hypoxic cultures or exogenous lactic acid stimulated apoptosis in aerobic myocytes. Hypoxia-acidosis-mediated cell death was independent of p53: equivalent apoptosis occurred in cardiac myocytes isolated from wild-type and p53 knockout mice, and hypoxia caused no detectable change in p53 abundance or p53-dependent transcription. Reoxygenation of hypoxic cardiac myocytes induced apoptosis in 25-30% of the cells and was also independent of p53 by the same criteria. Finally, equivalent levels of apoptosis, as demonstrated by DNA fragmentation, were induced by ischemia-reperfusion, but not by ischemia alone, of Langendorff-perfused hearts from wild-type and p53 knockout mice. We conclude that acidosis, reoxygenation, and reperfusion, but not hypoxia (or ischemia) alone, are strong stimuli for programmed cell death that is substantially independent of p53.

Physical and functional sensitivity of zinc finger transcription factors to redox change.

Redox regulation of DNA-binding proteins through the reversible oxidation of key cysteine sulfhydryl groups has been demonstrated to occur in vitro for a range of transcription factors. The direct redox regulation of DNA binding has not been described in vivo, possibly because most protein thiol groups are strongly buffered against oxidation by the highly reduced intracellular environment mediated by glutathione, thioredoxin, and associated pathways. For this reason, only accessible protein thiol groups with high thiol-disulfide oxidation potentials are likely to be responsive to intracellular redox changes. In this article, we demonstrate that zinc finger DNA-binding proteins, in particular members of the Sp-1 family, appear to contain such redox-sensitive -SH groups. These proteins displayed a higher sensitivity to redox regulation than other redox-responsive factors both in vitro and in vivo. This effect was reflected in the hyperoxidative repression of transcription from promoters with essential Sp-1 binding sites, including the simian virus 40 early region, glycolytic enzyme, and dihydrofolate reductase genes. Promoter analyses implicated the Sp-1 sites in this repression. Non-Sp-1-dependent redox-regulated genes including metallothionein and heme oxygenase were induced by the same hyperoxic stress. The studies demonstrate that cellular redox changes can directly regulate gene expression in vivo by determining the level of occupancy of strategically positioned GC-binding sites.

Activation of c-Jun N-terminal kinase promotes survival of cardiac myocytes after oxidative stress

Reperfusion injury occurs when ischaemic tissue is reperfused. It involves the generation and release of reactive oxygen that activates numerous signalling pathways and initiates cell death. Exposure of isolated cardiac myocytes to chronic hypoxia followed by reoxygenation results in the early activation of c-Jun N-terminal kinase (JNK) and death by apoptosis of approx. 30% of the myocytes. Although JNK activation has been described in a number of models of ischaemia/reperfusion, the contribution of JNK activation to cell fate has not been established. Here we report that the activation of JNK by reoxygenation correlates with myocyte survival. Transfection of myocytes with JNK pathway interfering plasmid vectors or infection with adenoviral vectors support the hypothesis that JNK is protective. Transfection or infection with JNK inhibitory mutants increased the rates of apoptosis by almost 2-fold compared with control cultures grown aerobically or subjected to hypoxia and reoxygenation. Caspase 9 activity, measured by LEHD cleavage, increased >3-fold during reoxygenation and this activity was enhanced significantly at all times in cultures infected with dominant negative JNK adenovirus. Hypoxia-reoxygenation mediated a biphasic (2.6- and 2.9-fold) activation of p38 mitogen-activated protein kinase, as well as a small increase of tumour necrosis factor alpha (TNFalpha) secretion, but treatments with the p38 MAPK-specific inhibitor SB203580 or saturating levels of a TNFalpha-1 blocking antibody provided only partial protection against apoptosis. The results suggest that JNK activation is protective and that the pathway is largely independent of p38 MAPK or secreted TNFalpha.

A unique pathway of cardiac myocyte death caused by hypoxia–acidosis

SUMMARY Chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiac myocytes in culture. The condition parallels myocardial ischemia in vivo, where ischemic tissue becomes rapidly hypoxic and acidotic. Cardiac myocytes are resistant to chronic hypoxia at neutral pH but undergo extensive death when the extracellular pH (pH[o]) drops below 6.5. A microarray analysis of 20 000 genes (cDNAs and expressed sequence tags) screened with cDNAs from aerobic and hypoxic cardiac myocytes identified> 100 genes that were induced by >2-fold and ∼20 genes that were induced by >5-fold. One of the most strongly induced transcripts was identified as the gene encoding the pro-apoptotic Bcl-2 family member BNIP3. Northern and western blot analyses confirmed that BNIP3 was induced by 12-fold (mRNA) and 6-fold (protein) during 24 h of hypoxia. BNIP3 protein, but not the mRNA, accumulated 3.5-fold more rapidly under hypoxia–acidosis. Cell fractionation experiments indicated that BNIP3 was loosely bound to mitochondria under conditions of neutral hypoxia but was translocated into the membrane when the myocytes were acidotic. Translocation of BNIP3 coincided with opening of the mitochondrial permeability pore (MPTP). Paradoxically, mitochondrial pore opening did not promote caspase activation, and broad-range caspase inhibitors do not block this cell death pathway. The pathway was blocked by antisense BNIP3 oligonucleotides and MPTP inhibitors. Therefore, cardiac myocyte death during hypoxia–acidosis involves two distinct steps: (1) hypoxia activates transcription of the death-promoting BNIP3 gene through a hypoxia-inducible factor-1 (HIF-1) site in the promoter and (2) acidosis activates BNIP3 by promoting membrane translocation. This is an atypical programmed death pathway involving a combination of the features of apoptosis and necrosis. In this article, we will review the evidence for this unique pathway of cell death and discuss its relevance to ischemic heart disease. The article also contains new evidence that chronic hypoxia at neutral pH does not promote apoptosis or activate caspases in neonatal cardiac myocytes.

Induction of the skeletal alpha-actin gene in alpha 1-adrenoceptor-mediated hypertrophy of rat cardiac myocytes.

Myocardial hypertrophy in vivo is associated with reexpression of contractile protein isogenes characteristic of fetal and neonatal development. The molecular signals for hypertrophy and isogene switching are unknown. We studied alpha (sarcomeric)-actin messenger RNA (mRNA) expression in cultured cardiac myocytes from the neonatal rat. In the cultured cells, as in the adult heart in vivo, expression of cardiac alpha-actin (cACT) predominated over that of skeletal alpha-actin (sACT) mRNA, the fetal/neonatal isoform. alpha 1-Adrenergic receptor stimulation induced hypertrophy of these cells, increasing total RNA and cytoskeletal actin mRNA by 1.8-fold over control, and total alpha-actin mRNA by 4.3 fold. This disproportionate increase in total alpha-actin mRNA was produced by a preferential induction of sACT mRNA, which increased by 10.6-fold over control versus only 2.6-fold for cACT mRNA. The alpha 1-adrenoceptor is the first identified molecular mediator of early developmental isogene reexpression in cardiac myocyte hypertrophy.

Genome-wide association study identifies a susceptibility locus at 21q21 for ventricular fibrillation in acute myocardial infarction

Sudden cardiac death from ventricular fibrillation during acute myocardial infarction is a leading cause of total and cardiovascular mortality. To our knowledge, we here report the first genome-wide association study for this trait, conducted in a set of 972 individuals with a first acute myocardial infarction, 515 of whom had ventricular fibrillation and 457 of whom did not, from the Arrhythmia Genetics in The Netherlands (AGNES) study. The most significant association to ventricular fibrillation was found at 21q21 (rs2824292, odds ratio = 1.78, 95% CI 1.47–2.13, P = 3.3 × 10−10). The association of rs2824292 with ventricular fibrillation was replicated in an independent case-control set consisting of 146 out-of-hospital cardiac arrest individuals with myocardial infarction complicated by ventricular fibrillation and 391 individuals who survived a myocardial infarction (controls) (odds ratio = 1.49, 95% CI 1.14–1.95, P = 0.004). The closest gene to this SNP is CXADR, which encodes a viral receptor previously implicated in myocarditis and dilated cardiomyopathy and which has recently been identified as a modulator of cardiac conduction. This locus has not previously been implicated in arrhythmia susceptibility.