Pharmacological preconditioning of primary rat cardiac myocytes by FK506

Pre-treatment with the immunosuppressant FK506 is shown to protect primary cardiocytes against a subsequent severe thermal or ischaemic stress. This effect is not observed with the related compounds cyclosporin A or rapamycin. It does not involve induction of the FK506 binding, heat inducible protein hsp56 or of the other heat shock proteins. In addition over-expression of hsp56 does not protect cardiac cells from severe stress in contrast to our previous results with hsp70 and hsp90. These results suggest the FK506 is acting via a novel mechanism to protect cardiac cells against cellular ischaemia which may not be related to its immunosuppressant action.     

Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes

When a total shoulder arthroplasty is performed, restoration of the anatomy to near normal is important in order to achieve a stable implant. So as not to sacrifice stability, it is not uncommon for soft tissues to be either over tightened or insufficiently released. This article analyzes the various factors to consider in order to obtain appropriate soft-tissue balancing for a successful total shoulder arthroplasty.     

Cardiotoxicity of chlorodibromomethane and trichloromethane in rats and isolated rat cardiac myocytes

The cardiovascular effects were investigated after acute and subacute treatment with chlorodibromomethane (CDBM; 0.4 to 3.2 mmol/kg p.o.), trichloromethane (TCM; 0.31 and 1.25 mmol/kg p.o.) and mixtures of CDBM and TCM (acute, 0.8 mmol CDBM/kg + 1.25 mmol TCM/kg p.o.; subacute, 0.4 mmol CDBM/kg + 0.31 mmol TCM/kg p.o.) in conscious and urethane anaesthetized male Wistar rats (n = 6-10 per treatment). Furthermore it was observed whether cardiovascular responses were modified in CDBM or TCM treated rats after administration of exogenous catecholamines (epinephrine, 1 microg/kg; norepinephrine, 2 microg/kg) and underpinned with in vitro alterations of Ca2+ dynamics in cardiac myocytes. The present findings demonstrated that single and subacute oral administration of CDBM or TCM and mixtures of CDBM and TCM resulted in arrhythmogenic and negative chronotropic and dromotropic effects in conscious and urethane anaesthetized rats. The atrioventricular conduction time and the intraventricular extension time were extended. A slight shortening of the repolarization velocity was observed. The myocardial contractility was depressed and the heart was sensitized to the arrhythmogenic effects of epinephrine. After catecholamine injection the adrenergic cardiovascular responses in urethane anesthetized rats were modified: increased hypertensive epinephrine and norepinephrine action as well as augmentation of negative chronotropic and negative dromotropic cardiac effects of catecholamines were observed. The positive inotropic adrenergic response was diminished. The present in vivo findings, myocardial depression after acute CDBM treatment, as determined by different indices of contractility, correlate well with the observed inhibitory actions of CDBM on Ca2+ dynamics in isolated cardiac myocytes. All cardiovascular alterations found after CDBM or TCM treatment were not intensified after treatment with mixtures of CDBM and TCM. The effects observed were distinctly stronger after TCM (1.25 and 0.31 mmol/kg) treatment compared to CDBM (0.8 and 0.4 mmol/kg) treatment.     

Titin extensibility in situ: entropic elasticity of permanently folded and permanently unfolded molecular segments

Titin (also known as connectin) is a giant protein that spans half of the striated muscle sarcomere. In the I-band titin extends as the sarcomere is stretched, developing what is known as passive force. The I-band region of titin contains tandem Ig segments (consisting of serially linked immunoglobulin-like domains) with the unique PEVK segment in between (Labeit, S., and B. Kolmerer. 1995. Science. 270:293-296). Although the tandem Ig and PEVK segments have been proposed to behave as stiff and compliant springs, respectively, precise experimental testing of the hypothesis is still needed. Here, sequence-specific antibodies were used to mark the ends of the tandem Ig and PEVK segments. By following the extension of the segments as a function of sarcomere length (SL), their respective contributions to titin's elastic behavior were established. In slack sarcomeres (approximately 2.0 micron) the tandem Ig and PEVK segments were contracted. Upon stretching sarcomeres from approximately 2.0 to 2.7 micron, the "contracted" tandem Ig segments straightened while their individual Ig domains remained folded. When sarcomeres were stretched beyond approximately 2.7 micron, the tandem Ig segments did not further extend, instead PEVK extension was now dominant. Modeling tandem Ig and PEVK segments as entropic springs with different bending rigidities (Kellermayer, M., S. Smith, H. Granzier, and C. Bustamante. 1997. Science. 276:1112-1116) indicated that in the physiological SL range (a) the Ig-like domains of the tandem Ig segments remain folded and (b) the PEVK segment behaves as a permanently unfolded polypeptide. Our model provides a molecular basis for the sequential extension of titin's different segments. Initially, the tandem Ig segments extend at low forces due to their high bending rigidity. Subsequently, extension of the PEVK segment occurs only upon reaching sufficiently high external forces due to its low bending rigidity. The serial linking of tandem Ig and PEVK segments with different bending rigidities provides a unique passive force-SL relation that is not achievable with a single elastic segment.     

Heterogeneity and underlying mechanism for inotropic action of endothelin-1 in rat ventricular myocytes

1. To clarify the mechanisms underlying the positive inotropic action of endothelin-1 (ET-1), we investigated the effect of ET-1 on twitch cell shortening and the Ca2+ transient in rat isolated ventricular myocytes loaded with a fluorescent Ca2+ indicator indo-1. 2. There was a cell-to-cell heterogeneity in response to ET-1. ET-1 (100 nM) increased twitch cell shortening in only 6 of 14 cells (44%) and the increase in twitch cell shortening was always accompanied by an increase in the amplitude of the Ca2+ transient. 3. The ET(A)- and ET(B)-receptors antagonist TAK-044 (100 nM) almost reversed both the ET-1-induced increases in twitch cell shortening and in the Ca2+ transient. In the ET-1 non-responding cells, the amplitude of the Ca2+ transient never increased. 4. Intracellular pH slightly increased (approximately 0.08 unit) after 30 min perfusion of ET-1 in rat ventricular myocytes. However, ET-1 did not change the myofilament responsiveness to Ca2+, which was assessed by (1) the relationship between the Ca2+ transient amplitude and twitch cell shortening, and by (2) the Ca2+ transient-cell shortening phase plane diagram during negative staircase. 5. We concluded that there was a cell-to-cell heterogeneity in the positive inotropic effect of ET-1, and that the ET-receptor-mediated positive inotropic effect was mainly due to an increase in the Ca2+ transient amplitude rather than to an increase in myofilament responsiveness to Ca2+.     

PPADS inhibits P2Y1 purinoceptors in rat brain capillary endothelial cells and in rat ileal myocytes by an indirect mechanism

P2Y1 receptor-like responses were analyzed in rat ileal myocytes and in rat brain capillary endothelial cells. In endothelial cells, pyridoxal phosphate-6-azophenyl-2',4'disulfonic acid (PPADS) inhibits ADP induced intracellular Ca2+ transients with a half maximum effect at 3 microM. PPADS shifts ADP dose response curves to larger concentrations. Yet PPADS is inactive when added at the same time as ADP. A preequilibration of the cells with PPADS is necessary to observe its inhibitory action. Similarly in ileal myocytes, PPADS has no action on ADP responses when it is applied at the same time as ADP. Actions of PPADS require a preequilibration with the cells and are fully reversible. These results suggest that PPADS is not a competitive antagonist of P2Y1 receptors and caution about its usefulness to distinguish subtypes of P2Y1 receptors.     

Rapid transition of cardiac myocytes from hyperplasia to hypertrophy during postnatal development

The switch from myocyte hyperplasia to hypertrophy occurs during the early postnatal period. The exact temporal sequence when cardiac myocytes cease dividing and become terminally differentiated is not certain, although it is currently believed that the transition takes place gradually over a 1-2-week period. The present investigation has characterized the growth pattern of cardiac myocytes during the early postnatal period. Cardiac myocytes were enzymatically isolated from the hearts of 1, 2, 3, 4, 6, 8, 10, and 12-day-old rats for the measurements of binucleation, cell volume and myocyte number. Almost all myocytes were mononucleated and cell volume remained relatively constant during the first 3 days of age. Increases in cell volume and binucleation of myocytes were first detected at day 4. Myocyte volume increased 2.5-fold from day 3 to day 12 (1416 +/- 320 compared to 3533 +/- 339 microns 3). The percentage of binucleated myocytes began to increase at day 4 and proceeded at a high rate, reaching the adult level of approximately 90% at day 12. Myocyte number increased 68% during the first 3 days (from 13.6 +/- 3.5 x 10(6) at day 1 to 22.9 +/- 5.6 x 10(10) at day 3) and remained constant thereafter. To confirm that no further myocyte division exists after 4 days, bromodeoxyuridine (Brdu) was administered to 4-day-old rats and the fate of DNA-synthesizing myocytes was examined 2 h and 2, 4, 6 and 8 days after Brdu injection. About 12% of myocytes were labeled with Brdu at 2 h and all were mononucleated at that time. Gradually, these Brdu-labeled myocytes became binucleated. However, the percentage of labeled myocytes in all groups was identical, indicating that DNA-synthesizing myocytes were becoming binucleated without further cell division after 4 days of age. Within 8 days after injection, approximately 82% of total labeled myocytes were binucleated, while the others remained mononucleated. Sarcomeric alpha-actinin was fully disassembled in dividing myocytes of 2-day-old rats, while typical alpha-actinin striations were present in dividing myocytes of 4-day-old rats. The results from this study suggest that a rapid switch from myocyte hyperplasia to hypertrophy occurs between postnatal day 3 and 4 in rat hearts.     

Pulsatile stretch stimulates vascular endothelial growth factor (VEGF) secretion by cultured rat cardiac myocytes

Evidence has accumulated that vascular endothelial growth factor (VEGF) is expressed in the heart, and its expression is markedly increased in response to hypoxia. Recently, it was shown that pulsatile myocardial stretch in vivo markedly enhanced VEGF mRNA level in the heart. To investigate whether pulsatile mechanical stretch really stimulates VEGF expression by cardiac myocytes, using an in vitro preparation, we examined the secretion of VEGF into the culture media from cardiac myocytes subjected to pulsatile stretch. We found that pulsatile mechanical stretch induced rapid secretion of VEGF by cultured rat cardiac myocytes and mRNA expression of VEGF and VEGF receptors in the cardiac myocytes. We also found that the stretch-induced secretion of VEGF was at least in part mediated by TGF-beta. These data provide the direct evidence that mechanical overload itself can induce VEGF secretion by cardiac myocytes, which may play a role in ameliorating the relative myocardial hypoxia.     

Insulin-like growth factor-I rapidly activates multiple signal transduction pathways in cultured rat cardiac myocytes

In response to insulin-like growth factor-I (IGF-I), neonatal rat cardiac myocytes exhibit a hypertrophic response. The elucidation of the IGF-I signal transduction system in these cells remains unknown. We show here that cardiac myocytes present a single class of high affinity receptors (12,446 +/- 3,669 binding sites/cell) with a dissociation constant of 0.36 +/- 0.10 nM. Two different beta-subunits of IGF-I receptor were detected, and their autophosphorylation was followed by increases in the phosphotyrosine content of extracellular signal-regulated kinases (ERKs), insulin receptor substrate 1, phospholipase C-gamma1, and phosphatidylinositol 3-kinase. IGF-I transiently activates c-Raf in cultured neonatal cardiac myocytes, whereas A-raf is activated much less than c-Raf. Two peaks of ERK activity (ERK1 and ERK2) were resolved in cardiac myocytes treated with IGF-I by fast protein liquid chromatography, both being stimulated by IGF-I (with EC50 values for the stimulation of ERK1 and ERK2 by IGF-I of 0.10 and 0. 12 nM, respectively). Maximal activation of ERK2 (12-fold) and ERK1 (8.3-fold) activities was attained after a 5-min exposure to IGF-I. Maximal activation of p90 S6 kinase by IGF-I was achieved after 10 min, and then the activity decreased slowly. Interestingly, IGF-I stimulates incorporation of [3H]phenylalanine (1.6-fold) without any effect on [3H]thymidine incorporation. These data suggest that IGF-I activates multiple signal transduction pathways in cardiac myocytes some of which may be relevant to the hypertrophic response of the heart.     

Highly selective sigma receptor ligands elevate inositol 1,4,5-trisphosphate production in rat cardiac myocytes

Exposure of cardiac myocytes from adult rat ventricles to the highly selective, high affinity sigma receptor ligands 1S,2 R-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1-pyrrolidinyl)-cycloh exylamine (BD-737) (0.1-100 nM) and N-[2-(3,4-dichlorophenyl)ethyl]-N,N',N'-trimethylethylenediamine (BD-1047) (0.01-10 nM), caused potentiation of electrically-evoked amplitudes of contraction and Ca2+ transients, while exposure to 100 nM BD-1047 caused attenuation of these amplitudes. In addition, BD-737 (1-100 nM) and BD-1047 (10-100 nM) caused an increase in the incidence of spontaneous twitches. These effects were inhibited when the incubation with BD-737 was done in the presence of the phospholipase C inhibitor, neomycin, or after pre-incubation with thapsigargin or caffeine which deplete the sarcoplasmic reticulum Ca2+ stores. Inositol 1,4,5-trisphosphate (IP3) production in cardiac myocytes was determined by the IP3 binding protein assay. Both substances caused an increase in the intracellular concentration of IP3. BD-737 caused a rapid transient increase to 3.2-fold in 1 min and stabilization at 2.1-fold of control thereafter. BD-1047 caused a gradual increase reaching 4.4-fold after 5 min. The results suggest that the effects of these sigma receptor ligands on contractility and spontaneous contractions are mediated by activation of phospholipase C and elevation of intracellular IP3 level.     

Microtubules are needed for dispersal of alpha-myosin heavy chain mRNA in rat neonatal cardiac myocytes

In some cell types, microtubules are used for transport of mRNA through the cytoplasm to the translation site. The number of microtubules increases during growth of cardiac myocytes, suggesting a functional role exists. Here, we test the need for microtubules to transport alpha-myosin heavy chain (alpha-MyHC) mRNA through the cytoplasm of neonatal cardiac myocytes. The alpha-MyHC mRNA concentration was assessed by non-radioactive in situ hybridization. The relative mRNA distributions were expressed as slopes (m=OD/micrometer), since optical density declined linearly from the nucleus to the cell periphery. Spontaneously-contracting myocytes displayed a gradual decrease in alpha-MyHC mRNA away from the nucleus (m=-1.27+/-0.12 OD/micrometer). To test whether microtubules were necessary for alpha-MyHC mRNA dispersal, contraction was first arrested with the Ca2+-channel blocker verapamil (10 micrometer) for 18 h, which aggregated the mRNA perinuclearly. Contractile activity was then resumed by washing out verapamil and using isoproterenol (10 micrometer) in the presence or absence of a microtubule depolymerizing drug, colchicine (3 micrometer). Within 6 h, the alpha-MyHC mRNA distribution in myocytes with microtubules returned to normal values (m=-1.11+/-0.14 OD/micrometer), while cells lacking microtubules maintained a perinuclear mRNA distribution (m-1.50+/-0.16 OD/micrometer; P<0.05 from control). Despite this perinuclear pattern of mRNA distribution, the myocytes still produced new myofibrils. These data indicate that microtubules are necessary for dispersal of alpha-MyHC mRNA outward from the nucleus. Furthermore, myofibrillogenesis may occur independently of mRNA localization and microtubule organization.     

Expression of calcium channels in adult cardiac myocytes is regulated by calcium

In addition to playing a significant role in cardiac excitation-contraction coupling, intracellular Ca2+ ([Ca2+]i) can regulate gene expression. While the mechanisms regulating expression of Ca2+ channels are not entirely defined, some evidence exists for Ca2+-dependent regulation. Using an adult ventricular myocyte culture system, we determined the effects of Ca2+ on: (1) abundance of mRNA for L-type Ca2+ channel alpha1 subunit (DHP receptor); (2) amount of DHP receptors; and (3) whole-cell Ca2+ current (ICa). Rat ventricular myocytes were cultured for 1-3 days in serum-free medium containing either normal (1.8 mM) or high (4.8 mM) Ca2+. Exposing myocytes to high Ca2+ rapidly elevated [Ca2+]i as determined by fura-2. Northern blot analysis revealed that culturing cells in high Ca2+ produced 1.5-fold increase in mRNA levels for the DHP receptor. The abundance of DHP receptors, determined by ligand binding, was two-fold greater in myocytes after 3 days in high Ca2+. Moreover, peak ICa was larger in myocytes cultured for 3 days in high Ca2+ (-17.8+/-1.5 pA/pF, n=26) than in control cells (-11.0+/-1.0 pA/pF, n=23). Voltage-dependent activation and inactivation, rates of current decay, as well as percent increases in ICa elicited by Bay K8644 were similar in all groups. Therefore, larger ICa is likely to represent a greater number of functional channels with unchanged kinetics. Our data support the conclusion that transient changes in [Ca2+]i can modulate DHP receptor mRNA and protein abundance, producing a corresponding change in functional Ca2+ channels in adult ventricular myocytes.     

Adrenergic stimulation of Na/K pump current in adult rat cardiac myocytes in short-term culture

This research examined self-perception and the perception of age groups by young and elderly adults from the perspective of social identity theory and social categorization theory. Respondents rated either themselves or unfamiliar stimulus persons from three age categories in adulthood: young, middle-aged, and elderly. As expected, an ingroup bias was found in the evaluation of elderly adults. Specifically, compared to ratings made by younger adults, older adults evaluated elderly persons more favorably. Moreover, as predicted, elderly adults' self-evaluations and those of young adults asked to imagine themselves as elderly were more positive than the ratings made by respondents who evaluated an unfamiliar older adult (e.g., elderly woman, elderly man). Both cognitive and motivational processes were discussed as contributing to the phenomenon of self-other discrepancy in beliefs about and attitudes toward older adults.     

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery

OBJECTIVE: To assess whether hypertension is a risk factor for hysterectomy performed for benign diseases. METHODS: Self-report questionnaires were collected from 77% of 2301 Danish women aged 30, 40, 50, or 60 years selected at random in 1982 for a prevalence study. Information about cardiovascular diseases, hypertension, use of medicine, weight and dieting history, life-styles, psychologic factors, gynecologic history (including history of hysterectomy), and social background were recorded. Weight, height, and blood pressure were measured. In an incidence study, the cohort was followed during 1982-1990 via central registers to assess the incidence of hysterectomy. Logistic and Cox regressions were used to analyze data. RESULTS: In the prevalence study, history of hypertension partly explained the relation between hysterectomy and cardiovascular diseases. In the incidence study, history of hypertension and use of diuretics were significant risk factors for hysterectomy. After confounder control, use of diuretics was explained by weight-related variables, and hypertension was a risk factor for hysterectomy in educated women (adjusted relative risk [RR] 2.88, 95% confidence interval [CI] 1.07, 7.76) and in women with weight fluctuations (adjusted RR 3.31, 95% CI 1.35, 8.14). Weight cycling and lack of education remained significant risk factors for hysterectomy in women with and without hypertension, respectively. CONCLUSION: History of hypertension, weight cycling, and lack of education are closely related risk factors for premenopausal hysterectomy. These three risk factors contribute to women undergoing hysterectomy having an increased risk for cardiovascular diseases. We proposed that hypertension might be a plausible biological cause of menorrhagia and an indication for hysterectomy.     

Calcium transients in single myocytes and membranous ultrastructures during the development of cardiac hypertrophy and heart failure in rats

1. We examined changes in intracellular calcium transients of separated single myocytes from the right ventricle (RV) of the rat heart during the change from adaptation to maladaptation in response to a pressure overload. 2. Right ventricular hypertrophy (RVH) secondary to pulmonary hypertension was induced by a subcutaneous injection of monocrotaline. Developed tensions of the RV-free wall were decreased as RVH progressed. Single myocytes were separated from the RV during different stages of RVH. Fura-2/AM-loaded cells were field stimulated, and changes in calcium transients were measured by Olympus OSP-3 system. We also examined membranous ultrastructures (sarcoplasmic reticulum, mitochondria, surface caveolae) involved in calcium metabolism in the hearts using scanning electron microscopy. 3. We observed characteristic changes in calcium transients during the change from adaptation to maladaptation, and also found that one parameter (amplitude) of calcium transients appeared to be correlated with the changes in the number of sarcoplasmic reticulum. 4. These results provided some insights into the mechanism of calcium handling of hypertrophied heart in response to a pressure overload from adaptation to maladaptation especially when stimulatory frequency was high, and suggested that heart rate control is a very important factor for the treatment of patients with congestive heart failure.     

Apoptosis of cardiac myocytes in Gsalpha transgenic mice

We examined the potential involvement of two CC chemokine receptors (CCRs), CCR-1 and CCR-3, in the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (DCs). Flow cytometric analysis showed that CCR-1, CCR-3, CCR-5, and CXC chemokine receptor (CXCR)-4 were expressed on the cell surface of monocyte-derived DCs. Treatment with a monoclonal antibody (MoAb) to either CCR-1 or CCR-3 but not MoAbs to CCR-5 and CXCR-4 abolished chemotactic migration of monocyte-derived DCs. The DCs treated with either the anti-CCR-1 MoAb or anti-CCR-3 MoAb were less efficient than untreated DCs in proliferation of allogeneic T cells (TCs) and TC-derived secretion of interferon-gamma (IFN-gamma). The homotypic aggregation of DCs and heterotypic aggregation of DCs with TCs were suppressed by the anti-CCR-1 MoAb or anti-CCR-3 MoAb. These results indicate that CCR-1 and CCR-3 specifically regulate interaction of TCs and DCs in the process of antigen presentation.     

Hydrogen peroxide activates mitogen-activated protein kinases and Na+-H+ exchange in neonatal rat cardiac myocytes

Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na+-H+ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 micromol/L hydrogen peroxide (H2O2) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H2O2 (100 micromol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H2O2. Pretreatment with chelerythrine (10 micromol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 micromol/L, 45 minutes) or herbimycin A (3 micromol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H2O2-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H2O2 also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 micromol/L, 60 minutes). These results suggest that low doses of H2O2 induce MAP kinase-dependent pathways that regulate NHE activity during reperfusion injury.     

Cardioprotective effect of angiotensin-converting enzyme inhibition against hypoxia/reoxygenation injury in cultured rat cardiac myocytes

BACKGROUND: Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS: Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS: The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.