Opposing effects of endothelin-1 on C-type natriuretic peptide actions in rat cardiomyocytes.

C-type natriuretic peptide (CNP) and Endothelin-1 are paracrine peptides with opposing vascular and mitogenic actions. In cardiac myocytes, CNP reduced contractility and induced accumulation of cyclic guanosine monophosphate (cGMP). Endothelin-1 caused an increase in contractile amplitude, abolished the negative inotropic effect of CNP, reduced the negative inotropic effect of a membrane permeable cGMP, and inhibited cGMP accumulation induced by CNP. We conclude that endothelin-1 abolishes the negative inotropic effect of CNP. This effect may be mediated by inhibition of the negative inotropic actions of cGMP as well as by reduction of cGMP levels.

C-type natriuretic peptide has a negative inotropic effect on cardiac myocytes.

C-type natriuretic peptide (CNP) has vasodilatory and antimitogenic actions, but its role in the control of cardiac function is unclear. We studied the effect of CNP on cultured, beating neonatal rat cardiac myocytes. CNP caused a significant reduction in the amplitude of contraction and a significant accumulation of intracellular cyclic GMP. The effect of a membrane permeable cyclic GMP on cell contraction was similar to that of CNP. CNP caused no change in Ca2+ transients. Blockade of natriuretic peptide receptors abolished the effects of CNP on contraction and accumulation of intracellular cyclic GMP. Blockade of cyclic GMP-dependent protein kinase abolished the effect of CNP on myocyte contraction. We conclude that CNP has a negative inotropic effect on neonatal rat cardiac myocytes. The effect of CNP is mediated via natriuretic peptide receptor(s) causing elevation of intracellular cyclic GMP which possibly activates protein kinase and causes attenuation of myofilament sensitivity to Ca2+.

Cytotoxic T lymphocytes are activated following myocardial infarction and can recognize and kill healthy myocytes in vitro.

The damage of myocardial infarction (MI) is often progressive. A possible mechanism for subsequent myocardial damage and heart failure after MI is immune response against cardiac self-antigens. The purpose of our study was to test the hypothesis that cytotoxic T lymphocytes are activated following acute MI and may have a role in producing further myocardial damage. Rats were allocated into three experimental groups: acute MI, Sham MI and non-operated control. One, two and three weeks after surgery, lymphocytes were obtained from rat spleens and incubated with neonatal cardiac myocytes. Lymphocyte proliferation was assessed by a thymidine incorporation assay and calculated as proliferation index (PI). Myocyte destruction was measured by a crystal-violet staining assay and expressed as percentage of cell destruction. Proliferation index was significantly higher among lymphocytes obtained from MI animals (44. 3+/-5.8 and 44.9+/-5.1, at 2 and 3 weeks after MI, respectively) than sham MI (29.3+/-5.3, 27.1+/-4.7) (P<0.05) or control animals (17.1+/-2.5, 16.2+/-2.8) (P=0.03). Cytotoxic activity of the MI lymphocytes against the cultured cardiomyocytes was significantly higher 2 and 3 weeks after MI, (36.4+/-7.3%, 69.3+/-4.9%) compared to sham MI (17.9+/-3.14%, 36.6+/-5.3%) (P<0.001) and control animals respectively (13.3+/-5.4%, 17.4+/-6.1%) (P<0.001). The cytotoxic activity against healthy cardiomyocytes was myocyte-specific, induced by CD8 lymphocytes and major-histocompatibility complex (MHC) restricted. Cytotoxic T lymphocytes (CD8) are activated following MI and can recognize and kill normal cardiomyocytes in vitro. The newly described pathophysiological insights may provide novel oportunities to prevent death of non-ischemic cardiomyocytes and heart failure following myocardial infarction.

The effect of cytotoxic lymphocytes on contraction, action potential and calcium handling in cultured myocardial cells.

Cytotoxic T lymphocytes are important in the pathogenesis of several disease states, yet the pathophysiology of the lymphocyte-myocyte interaction is not well known. We have developed in vitro viral and autoimmune models to study the physiological phenomena associated with this interaction. To produce these models, lymphocytes were obtained from adult rats injected either with mengo virus or autologous cardiac myocytes. Cardiac myocytes from neonatal rats were then exposed to these lymphocytes. In both models, reversible physiologic changes in myocytes preceded irreversible cell damage. The physiologic changes included reduced amplitude of myocyte contraction, impairment of relaxation and prolongation of the duration of contraction and action potential. In addition, oscillations were noted in the plateau phase of the action potentials. These physiologic changes were accompanied by an early elevation in the cytosolic free calcium concentration, a late elevation in the total exchangeable calcium pool, and attenuation of the [Ca2+]i transient signals. Verapamil inhibited the late elevation in the total exchangeable calcium pool, but failed to inhibit the early elevation in the cytosolic free calcium concentration. These phenomena may explain transient cardiac functional abnormalities that may appear during myocarditis prior to cell destruction.

Physiological changes induced in cardiac myocytes by cytotoxic lymphocytes: an autoimmune model.

BACKGROUND: cytotoxic lymphocytes are important in the pathogenesis of several disease states, yet, the pathophysiology of lymphocyte-myocyte interaction is not well known. METHODS AND RESULTS: We have developed a model for the in vitro evaluation of autoimmune cytotoxic myocardial damage. Cardiac myocytes were repeatedly injected to adult autologous rats. Following 3 months, histological evidence of myocarditis was seen in 20% of the hearts. Cultured myocytes obtained from newborn rats were exposed to lymphocytes isolated from the immunized animals. Cytotoxic activity was measured using crystal violet staining test. The percentage of killing was increased as the ratio of lymphocytes/myocytes was increased. Verapamil did not block this cytotoxic effect. No killing was seen when myocytes were exposed to non-sensitized lymphocytes. Physiological changes induced in myocytes by cytotoxic lymphocytes were studied. Cell wall motion was measured by an optical method and action potentials with intracellular microelectrodes. Physiological changes observed in myocytes following exposure to cytotoxic lymphocytes included: Impaired relaxation with prolonged contractions, oscillations and prolongation of the plateau of the action potential. Cellular contraction was prolonged up to 4 s before total arrest of spontaneous activity. Verapamil but not tetrodotoxin restored action potentials and contractions to normal. Supernatant collected from cultures of myocytes and lymphocytes had the same effect on myocytes contractility as observed following exposure of myocytes to cytotoxic lymphocytes. CONCLUSIONS: This supports our hypothesis that these physiological alterations observed in myocytes are mediated by a soluble factor secreted by cytotoxic lymphocytes.

Role of the Na+/K+/Cl- transporter in the positive inotropic effect of ouabain in cardiac myocytes.

In this study we have characterized the bumetanide-sensitive K+/Na+/Cl- cotransport in cultured rat cardiac myocytes. 1) It carries about 10% of the total K+ influx. 2) It is sensitive to furosemide (Ki0.5 = 10(-6)M) and bumetanide (Ki0.5 = 10(-7)M). 3) It is strongly dependent on the extracellular concentrations of Na+ and Cl-. 4) It carries out influx of both ions, K+ and Na+. A therapeutic concentration of ouabain (10(-7) M) stimulated the bumetanide-sensitive K+ influx (as measured by 86Rb+), in the cultured myocytes, with no effect on the bumetanide-resistant K+ influx, which was mediated mostly by the Na+/K+ pump. Stimulation of the bumetanide-sensitive Rb+ influx by a low ouabain concentration was strongly dependent on Na+ and Cl- in the extracellular medium. A low concentration of ouabain (10(-7) M) was found to increase the steady-state level of cytosolic Na+ by 15%. This increase was abolished by the addition of bumetanide or furosemide. These findings suggest that ouabain, at a low (10(-7) M) concentration, induced its positive inotropic effect in rat cardiac myocytes by increasing Na+ influx into the cells through the bumetanide-sensitive Na+/K+/Cl- cotransporter. In order to examine this hypothesis, we measured the effect of bumetanide on the increased amplitude of systolic cell motion induced by ouabain. Bumetanide or furosemide, added to cultured cardiac myocytes, inhibited the increased amplitude of systolic cell motion induced by ouabain. Neither bumetanide nor furosemide alone has any significant effect on the basal amplitude of systolic cell motion. We propose that stimulation of bumetanide-sensitive Na+ influx plays an essential role in the positive inotropic effect in rat cardiac myocytes induced by low concentration of ouabain.

Effect of ouabain on the concentration of free cytosolic Ca++ and on contractility in cultured rat cardiac myocytes.

Effects of ouabain on [Ca++]i and on contractility was measured in quin2 and fura2 loaded cultured neonatal rat cardiac myocytes. Addition of ouabain (5 x 10(-8) to 5 x 10(-6) M) to cultured myocytes exposed to balanced buffered salt solution (BSS) caused a transient increase in [Ca++]i, followed by slow oscillations for about 10 min, and by an elevated steady state level of [Ca++]i thereafter. Concentrations of ouabain between 10(-7) and 5 x 10(-7) M caused an increase in the amplitude of systolic motion (ASM) whereas concentrations above 10(-6) caused a decrease in the ASM, an increase in the beating frequency and an upward shift of the base line, indicating impaired relaxation. When ouabain was added to cardiac myocytes exposed to Ca++-free BSS the increase in [Ca++]i was not observed, but only a transient decrease. To investigate the effect of [K+]o on the ouabain-induced changes in [Ca++]i, ouabain was added to cells exposed to BSS containing low K+ concentration (1 mM instead of 5 mM in balanced BSS). In this medium the increase in ASM by ouabain was similar to that in balanced BSS. Addition of ouabain caused a transient decrease in [Ca++]i. There was no initial increase in [Ca++]i and the steady state level of [Ca++]i was not elevated as compared with the same cells before the addition of ouabain. Similar results were observed in cells loaded with quin2 or with fura2. In view of these results the mechanism of action of ouabain on cardiac myocytes is discussed.

Physiological changes induced in cardiac myocytes by cytotoxic T lymphocytes.

The "lethal hit" induced by viral specific, sensitized, cytotoxic T lymphocytes (CTL) attacking virus-infected heart cells is important in the pathogenesis of viral myocarditis and reflects the key role of CTL in this immune response. The mechanisms involved are incompletely understood. Studies of the physiological changes induced in mengovirus-infected, cultured, neonatal, rat heart cells by CTL that had been previously sensitized by the same virus are presented. The CTL were obtained from spleens of mengovirus-infected, major histocompatibility complex (MHC) matched adult rats. Cell wall motion was measured by an optical method, action potentials with intracellular microelectrodes, and total exchangeable calcium content by 45Ca tracer measurements after loading the myocytes with 45Ca and then exposing them to CTL. After 50 min (mean time) of exposing mengovirus-infected myocytes to the CTL, the mechanical relaxation of the myocyte was slowed, with a subsequent slowing of beating rate and a reduced amplitude of contraction. Impaired relaxation progressed, and prolonged oscillatory contractions lasting up to several seconds appeared, with accompanying oscillations in the prolonged plateau phase of the action potentials. Arrest of the myocyte contractions appeared 98 min (mean time) after exposure to CTL. These changes in action potentials and contractions were reversible either by washout with the normal medium or by the addition of verapamil. The amount of total exchangeable calcium in the cultured myocytes, 1 h after exposure to CTL, was significantly increased. This increase was prevented by pretreatment with verapamil. (ABSTRACT TRUNCATED AT 250 WORDS)

T-2 toxin effect on cultured myocardial cells.

Beat rate, contractility and viability of cultured myocardial cells perfused with solutions containing various concentrations of T-2 toxin were studied. While doses below 50 micrograms/ml had no immediate effect, those above 250 micrograms/ml decreased beat rate and amplitude. After 10-30 min of perfusion most cells stopped beating and did not restart after withdrawal of toxin. Nevertheless, most cells remained viable as judged by morphology and trypan blue exclusion. A 24-h exposure to doses of 5 or 2.5 micrograms/ml of toxin decreased the beat rate and inotropic responses of the myocytes. After 48 h cell death ensued. Thus T-2 toxin has some direct toxicity to myocardial cells but the lethal dose seems too high to make this the cause of cardiovascular failure.

Cytotoxic T lymphocytes and natural killer cell activity in the course of mengo virus infection of mice.

Inbred C57BL/6 mice were inoculated intraperitoneally (i.p.) with mengo virus. The activity of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells were measured during the first 22 days following infection. The CTL response began 7 days after virus inoculation, persisted for at least 22 days and was related to the dose of the virus inoculated. NK cell activity was elevated within 24 hr, reached its peak level on the fourth day and declined to normal levels on the eleventh day after exposure to the virus. These results suggest that NK cells represent the first cellular immune response to restrict mengo virus spread while specific CTL appear later and are probably responsible for further restriction, elimination and prevention of the viral disease.

Antiviral activity of a thymic factor in experimental viral infections. I. Thymic hormonal effect on survival, interferon production and NK cell activity in Mengo virus-infected mice.

A partially purified thymic factor, thymostimulin (TS), significantly increased the survival rate of adult, immune-intact mice infected with the neurotropic Mengo virus. TS treatment was begun after virus inoculation by daily i.p. injections. In untreated C57BL/6 mice, LD50 was reached with 1 X 10(4) PFU, but 10-fold more virus (i.e., 1 X 10(5) PFU) was needed to reach LD50 in TS-treated animals. TS effect on survival, though, could be observed with several virus doses (1 X 10(3) to 1 X 10(6) PFU) (p less than 0.001). A significant effect on survival was also observed with outbred ICR mice (p less than 0.005). Serum interferon (IFN) levels in the Mengo virus-infected mice were relatively low (average peak 300 U/ml), but were significantly increased (two- to ninefold) in the TS-treated mice. Peak serum levels were reached earlier in TS than in control animals (24 hr and 72 hr, respectively). Both acid-labile and acid-stable type I IFN production were augmented by TS in the Mengo virus-infected mice. Natural killer activity was also enhanced by TS, in particular on the second day after virus inoculation. In addition, MP-virus was used as a second, unrelated virus challenge. This virus caused a nonlethal infection, with relatively high levels of serum IFN (average peak 10,000 U/ml). TS increased IFN levels (two- to eight-fold) also in this challenge system. In conclusion, TS causes a nonspecific enhancement of endogenous production of IFN and has a significant effect on the survival of lethally infected mice. The data indicate a potential application of thymic factors for the treatment of viral infections.