Cytokine-induced nitric oxide inhibits mitochondrial energy production and induces myocardial dysfunction in endotoxin-treated rat hearts.

The mechanism responsible for cardiac depression in septic shock remains unknown. The present study examined whether nitric oxide (NO) overproduced by inducible NO synthase (iNOS) can inhibit aerobic energy metabolism and impair the myocardial function in endotoxin-treated rat hearts. Lipopolysaccharide (LPS) significantly decreased systolic blood pressure (BP) to 44% of control during the 48 h treatment. Hearts from control and LPS-treated rats were perfused in a Langendorff apparatus. After LPS injection, left ventricular (LV) developed pressure (LVDP) was significantly depressed, plasma NO2-/NO3- (NO(x)) concentration was markedly increased, and myocardial adenosine 5'-triphosphate (ATP), creatine phosphate (CrP), and the ratio of ATP/adenosine 5'-diphosphate were progressively decreased with time. Immunological examination showed a significant expression of iNOS protein in the LPS-treated myocytes. Aminoguanidine, an inhibitor of iNOS, significantly attenuated these LPS-induced functional and metabolic changes. Myocardial cyclic guanosine 3',5'-monophosphate (cGMP) content was significantly increased after LPS injection. Methylene blue, an inhibitor of soluble guanylate cyclase, blunted this increase in cGMP and significantly restored the LPS-induced contractile dysfunction 6 h after LPS injection. In addition, there was a significant negative correlation between LVDP and myocardial cGMP levels as well as a significant negative correlation between LVDP and plasma NO(x) levels. In contrast, 48 h after LPS injection, methylene blue no longer affected cardiac performance, and there was a significant positive correlation between LVDP and myocardial ATP content. Furthermore, the normalized activities (as a ratio of the citrate synthase activity) of mitochondrial NADH-CoQ reductase, succinate-CoQ reductase, and ATPase, were significantly inhibited, and the swelling or disruption of mitochondria cristae was seen in the 48 h LPS treatment. These LPS-induced functional and morphological disorders in the mitochondria were significantly improved by aminoguanidine. The findings suggest that sustained production of NO by iNOS leads to contractile dysfunction via cGMP in the early stage, but that it can directly impair the mitochondrial function, lower myocardial energy production, and contribute significantly to the myocardial dysfunction in the later stage of septic shock.

Nitric oxide-cGMP pathway is involved in endotoxin-induced contractile dysfunction in rat hearts.

The mechanisms by which endotoxemia causes cardiac depression have not been fully elucidated. The present study examined the involvement of nitric oxide (NO) in this pathology. Rats were infused with lipopolysaccharide (LPS) or saline, and the plasma and myocardial NO(2)(-) and NO(3)(-) (NOx) concentrations were measured before or 3, 6, and 24 h after treatment. The hearts were then immediately isolated and mounted in a Langendorff apparatus, and left ventricular developed pressure (LVDP) was determined before biochemical analysis of the myocardium. LPS injection effected the expression of inducible NO synthase (iNOS) in the myocardium, a marked increase in plasma and myocardial NOx levels, and a significant decline in LVDP compared with saline controls. The LPS-induced NO production and concomitant cardiac depression were most pronounced 6 h after LPS injection and were accompanied by a significant increase in myocardial cGMP content. Myocardial ATP levels were not significantly altered after LPS injection. Significant negative correlation was observed between LVDP and myocardial cGMP content, as well as between LVDP and plasma NOx levels. Aminoguanidine, an inhibitor of iNOS, significantly attenuated the LPS-induced NOx production and contractile dysfunction. Furthermore, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase, significantly decreased myocardial cGMP content and attenuated the contractile depression, although aminoguanidine or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one was not able to completely reverse myocardial dysfunction. Our data suggest that endotoxin-induced contractile dysfunction in rat hearts is associated with NO production by myocardial iNOS and a concomitant increase in myocardial cGMP.

Intracellular ATP is required for mitochondrial apoptotic pathways in isolated hypoxic rat cardiac myocytes.

OBJECTIVES: The present study examined the possibility that intracellular ATP levels dictate whether hypoxic cardiac myocytes die by apoptosis or necrosis. BACKGROUND: Although apoptosis and necrosis may appear to be distinct forms of cell death, recent studies suggest that the two may represent different outcomes of a common pathway. In ischemic myocardium, apoptosis appears early, while energy stores are presumably still available, followed only later by necrosis. METHODS: Neonatal rat cardiac myocytes were exposed to continuous hypoxia, during which the intracellular ATP concentration was modulated by varying the glucose content in the medium. The form of cell death was determined at the end of the hypoxic exposure. RESULTS: Under total glucose deprivation, ATP dropped precipitously and cell death occurred exclusively by necrosis as determined by nuclear staining with ethidium homodimer-1 and smearing on DNA agarose gels. However, with increasing glucose concentrations (10, 20, 50, 100 mg/dl) cellular ATP increased correspondingly, and apoptosis progressively replaced necrosis until it became the sole form of cell death, as determined by nuclear morphology, DNA fragmentation on agarose gels, and caspase-3 activation. The data showed a significantly positive correlation between myocyte ATP content and the percentage of apoptotic cells. Hypoxia resulted in lactate production and cellular acidification which stimulates apoptosis. However, acidification-induced apoptosis was also increased in an ATP-dependent fashion. Loss of mitochondrial membrane potential and cytochrome c release from the mitochondria was observed in both the apoptotic and necrotic cells. Furthermore, translocation of Bax from cytosol into mitochondria preceded these events associated with mitochondrial permeability transition. Increased lactate production and a lack of effect by the mitochondrial inhibitor oligomycin indicated that ATP was generated exclusively through glycolysis. CONCLUSIONS: We demonstrate that ATP, generated through glycolysis, is a critical determinant of the form of cell death in hypoxic myocytes, independently of cellular acidification. Our data suggest that necrosis and apoptosis represent different outcomes of the same pathway. In the absence of ATP, necrosis prevails. However, the presence of ATP favors and promotes apoptosis.

Tongue protrusion: a simple test for neurological recovery in rats following focal cerebral ischemia.

A simple tongue protrusion (TP) test is described for rats following focal ischemia induced by middle cerebral artery occlusion (MCAO). MCAO resulted in a dramatic decrease in TP that correlated with a concomitant decline in neurological performance in standard 5- and 20-point tests and deficits in performance in the Morris water maze and the accelerating rotarod. TP values also correlated with infarct size at 7 and 24 days following MCAO. This simple and inexpensive test, that monitors the ability of rats to lick food out of a glass tube, is easily administered, can be administered frequently without changing baseline performance, is not susceptible to behavioral compensation and should not interfere with other tests used concurrently to evaluate neurological deficit. The TP test may, therefore, serve as a useful addition to the battery of tests commonly used to assess neurological damage in rats, particularly in models of stroke.

Amlodipine inhibits doxorubicin-induced apoptosis in neonatal rat cardiac myocytes.

OBJECTIVES: We examined whether amlodipine, a calcium channel antagonist with potent antioxidant activity, inhibits doxorubicin-induced apoptosis in cultured neonatal rat cardiac myocytes. BACKGROUND: Recent studies have shown that doxorubicin induces apoptosis as well as necrosis in myocytes through generation of reactive oxygen species. METHODS: The effects of amlodipine and several other antioxidants on doxorubicin-induced oxidative stress and mitochondria-mediated apoptosis were examined. RESULTS: Treatment of myocytes with doxorubicin (10(-6) mol/l) for 14 h increased the number of cells with elevated peroxides, as histochemically estimated by 2',7'-dichlorofluorescin (DCF) diacetate, and the percentage of apoptotic myocytes, as estimated by Hoechst 33258 nuclear staining, compared with control myocytes (25.0 +/- 1.6% vs. 5.2 +/- 1.2%). Moreover, doxorubicin-induced myocyte apoptosis was also confirmed by annexin V-fluorescein isothiocyanate binding assay. Doxorubicin induced a reduction in myocyte adenosine 5'-triphosphate content, a loss of mitochondrial membrane potential, cytochrome c release from the mitochondria into the cytosol, and caspase-3 activation to 1.9-fold of control. Amlodipine significantly attenuated increased DCF fluorescence, inhibited the mitochondria-mediated apoptotic responses described earlier, and decreased apoptosis in the doxorubicin-treated myocytes in a dose-dependent fashion. Amlodipine at 10(-6) mol/l significantly decreased apoptosis to 15.4 +/- 0.7%, and this antiapoptotic action was more effective than that seen with other antioxidants, including probucol, ascorbic acid, and alpha-tocopherol. In contrast, the calcium channel antagonist nifedipine (10(-6) mol/l) did not inhibit apoptosis. Catalase, glutathione, and N-acetylcysteine, but not mannitol or superoxide dismutase, significantly decreased DCF fluorescence and attenuated myocyte apoptosis induced by doxorubicin to 18.7 +/- 1.2%, 19.1 +/- 1.7%, and 18.7 +/- 0.6%, respectively. CONCLUSIONS: Amlodipine significantly inhibits doxorubicin-induced myocyte apoptosis by suppressing the mitochondrial apoptotic pathway. This effect is attributed to the antioxidant properties of amlodipine, affecting mainly hydrogen peroxide.

Lethal effect of cytokine-induced nitric oxide and peroxynitrite on cultured rat cardiac myocytes.

We examined the cytotoxic effect of iNOS-generated NO in cultured cardiac myocytes treated with IL-1 beta, IFN- gamma and LPS. Treatment of the myocytes with cytokines for 48 h resulted in a marked NO production, a significant decline in cellular ATP content, and a significant increase in myocyte death with morphological characteristics of necrosis. Moreover, immunohistochemical examination showed that the cytokines caused nitrotyrosine formation in the injured myocytes. Uric acid and L-cysteine which have the ability to quench peroxynitrite significantly attenuated these cytokine-induced effects, although they did not alter NO production or the decline in cellular ATP. These data suggest that NO production induced by cytokines can not only cause deleterious effects in the myocardial energy balance but also induce myocytes necrosis, through the formation of peroxynitrite.