Abnormal contractile function due to induction of nitric oxide synthesis in rat cardiac myocytes follows exposure to activated macrophage-conditioned medium.

Abstract

IntroductionThe mechanism by which soluble mediators of immune cell origin depress myocardial contractility, either globally as in systemic sepsis, or regionally in areas of inflammatory myocardial infiltrates, remains unclear. When freshly isolated ventricular myocytes from adult rat hearts were preincubated for at least 24 h in medium conditioned by endotoxin (LPS)-activated rat alveolar macrophages, their subsequent inotropic response to the ,-adrenergic agonist isoproterenol was reduced from 225±19% to 155±10% of the baseline amplitude of shortening (mean±SEM, P < 0.05). Neither baseline contractile function nor the contractile response to high extracellular calcium were affected. To determine whether an endogenous nitric-oxide (NO)-signaling pathway within ventricular myocytes was responsible for their decreased responsiveness to isoproterenol, the L-arginine analogue L-NMMA was added to the preincubation medium. While L-NMMA did not affect baseline contractile function or the response of control myocytes to isoproterenol, it completely restored the positive inotropic response to isoproterenol in myocytes preincubated in LPS-activated macrophage medium. Release of NO by ventricular myocytes following exposure to activated macrophage medium was detected as an increase in cGMP content in a reporter-cell Cellular elements of the immune system have long been suspected to play a role in mediating the global myocardial dysfunction characteristic of septic shock, cardiac allograft rejection, and some forms of idiopathic cardiomyopathy (1). However, recent work indicates that direct cell-mediated cytotoxicity is not required to induce myocardial depression in experimental models of sepsis in animals or contractile dysfunction in isolated ventricular myocytes exposed to inflammatory mediators, including sera from patients with septic shock (2-5). Cell-free supernatants obtained from activated lymphocyte or macrophage cultures reversibly inhibit the expected increase in cAMP and concomitant positive inotropic responses of ventricular myocytes to the f3-adrenergic agonist isoproterenol, but they have no effect on basal cAMP levels or on baseline contractile function (2, 3). This effect of activated immune-cell-conditioned medium on myocyte responsiveness to 3 agonists is not immediate, but requires hours to become apparent ( 1).Several cytokines that are known to be present in medium conditioned by activated immune cells have been shown to induce the synthesis of isoform(s) of nitric oxide (NO) in a number of cell types and tissues (6-9). NO is now recognized to be a nearly ubiquitous autocrine and paracrine chemical messenger, with several biologic activities including the activation of soluble intracellular guanylate cyclase ( 10, 11 ). Recent evidence from this laboratory has documented a role for an endogenous, constitutively present, NO-signaling pathway in regulating the physiologic responsiveness ofneonatal and adult rat ventricular myocytes to muscarinic cholinergic and ,Badrenergic agonists, respectively ( ...

Sphingosine Mediates the Immediate Negative Inotropic Effects of Tumor Necrosis Factor-α in the Adult Mammalian Cardiac Myocyte

“…The base-line values (mean Ϯ S.E.) for [ 14 gosine levels, similar to that which was observed with the wild type TNF-␣. As shown, the TNFM2 ligand, which does not affect cell motion (18), did not increase levels of free sphingosine in isolated contracting cardiac myocytes.…”

“…On the 1st day in culture the M199 medium was changed, and the cells were treated for 0, 5, 15, 30, and 60 min with diluent (endotoxin-free 0.1% human serum albumin) or with recombinant human TNF-␣ (200 units⅐ml Ϫ1 ). The cells were then lysed, and the resultant cell supernatants were incubated at 37°C with 1 l of [methyl- 14 C]sphingomyelin (25 Ci⅐ml Ϫ1 ). The reaction was stopped after 2 h by the addition of chloroform:methanol (2:1).…”

“…Similarly, it is equally clear that TNF-␣ exerts delayed effects on myocardial function that appear to be related to uncoupling of the ␤-adrenergic receptor from cyclic AMP, rather than from a direct depression in basal myocardial contractility per se; moreover, these effects occur only after prolonged TNF-␣ exposure (24 -72 h) (9,10). Given the recognition that TNF-␣ increases nitric oxide (NO) levels in myocardial tissue through increased transcription of the inducible Ca 2ϩ -independent form of nitric oxide synthase (NOS) (11,12), given that NO directly mediates myocardial depression (13,14), and given that NO is likely responsible for the uncoupling of the ␤-adrenergic receptor following TNF-␣ stimulation (15), the logical assumption has been that NO mediates the full spectrum of cytokine-induced cardiodepressant effects. However, no previous report to date has provided direct evidence that shows that TNF-␣ stimulates NO production in cardiac myocytes with a time course that is rapid enough to explain the immediate negative inotropic effects of TNF-␣ (6).…”

Impaired lymphatic contraction associated with immunosuppression

“…S2B). As NO derived from iNOS-expressing lipopolysaccharide (LPS)-activated macrophages attenuates the contraction of myocytes and carotid arteries (26,27), we hypothesized that lymphatic contraction is similarly attenuated by the overproduction of NO from iNOS-expressing inflammatory cells located in s.c. adipose tissue on OXd4 (Fig. 2C).…”

Sepsis-induced cardiomyopathy: a review of pathophysiologic mechanisms

“…Furthermore, NOS inhibitors such as N G -monomethyl-L-arginine (L-NMMA) block the negative inotropic effects of cytokines [13]. However One illustrative experiment by Balligand et al [14] showed the interplay of bacterial endotoxin, macrophage stimulation, NOS induction, cGMP production and reduced contractility. Here, rat macrophages were stimulated by exposure to lipopolysaccharide (LPS, a component of bacterial endotoxin) and then incubated with rat cardiomyocytes for 24 hours.…”