Abstract
IntroductionCaspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. We have recently demonstrated that β1-adrenoceptor (AR) activation by endogenous norepinephrine contributes to cardiomyocyte apoptosis in endotoxemic mice. Here, we further investigated the molecular mechanisms for the enhancing effect of β1-AR activation on LPS-induced cardiomyocyte apoptosis.MethodsThe adult mouse ventricular myocytes were exposed to LPS, dobutamine, protein kinase A (PKA) inhibitor or/and nifedipine, an L-type Ca2+ channel blocker. Male BALB/c mice were treated with LPS or/ and β1-AR antagonist, atenolol. Cardiomyocyte apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) assay and apoptosis-associated molecules were detected.ResultsLPS induced apoptosis in adult mouse ventricular myocytes, dobutamine (DOB), a β1-AR agonist, promoted apoptosis, caspase-8, 9 and 3 activation and increased cytosolic Ca2+ concentration in LPS-challenged cardiomyocytes. DOB also up-regulated TNF-α expression, decreased Bcl-2 levels, promoted Bax translocation to mitochondria, mitochondrial membrane potential loss and cytochrome c release as well as IκBα, p38 MAPK, JNK and Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation in LPS-treated cardiomyocytes. PKA inhibitor abolished the effects of DOB on caspase-9 activation, Bcl-2 levels as well as JNK and p38 MAPK phosphorylation, but not on IκBα phosphorylation, TNF-α expression and caspase-8 activation in LPS-stimulated cardiomyocytes. Pretreatment with nifedipine not only significantly blocked the enhancing effects of DOB on LPS-induced elevation in cytosolic Ca2+ concentration and CaMKII phosphorylation in cardiomyocytes, but also partly reversed the effects of DOB on caspase-9 and caspase-3/7 activities in LPS-treated cardiomyocytes. Furthermore, atenolol suppressed TNF-α expression, JNK, p38 MAPK and CaMKII phosphorylation, increased Bcl-2 expression, and inhibited cytochrome c release and cardiomyocyte apoptosis in the myocardium of endotoxemic mice.Conclusionsβ1-AR activation promotes LPS-induced apoptosis through activating PKA, increasing CaMKII phosphorylation as well as enhancing IκBα phosphorylation and TNF-α expression in cardiomyocytes.
Highlights
Caspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction
Dobutamine promotes LPS-induced caspase 3/7 activation and nuclear apoptosis in adult mouse cardiomyocytes To assess the direct effect of cardiomyocyte β1-AR activation on LPS-stimulated cardiomyocyte apoptosis, we performed transferase-mediated dUTP nick-endlabeling (TUNEL) assay and examined the caspase 3/7 activity in isolated adult mouse ventricular myocytes treated with DOB, a β1-AR agonist, or/and LPS at various concentrations for 24 hours
At various concentrations for 24 hours. (E) Representative confocal images of TUNEL assay of adult mouse ventricular myocytes treated with 0.02 μM DOB or/and 10 ng/ml LPS for 24 hours; all cardiomyocytes were stained with anti-cardiac troponin I antibody and total nuclei with 4', 6-diamidino-2-phenylindole (DAPI, blue)
Summary
Caspase activation and cardiomyocyte apoptosis have been implicated in lipopolysaccharide (LPS)-induced cardiac contractile dysfunction. To date LPS-induced cardiomyocyte apoptosis has been attributed to increased production of TNF-α, macrophage migration inhibitory factor and reactive oxygen species [7,12,13,14,15]. It is well-documented that plasma norepinephrine levels are significantly elevated in the setting of sepsis [16] and norepinephrine alone can directly stimulate cardiomyocyte apoptosis by activating the β-adrenoceptor (AR) [17,18], little is known about the causative contribution of endogenous norepinephrine in sepsis-induced cardiomyocyte apoptosis. Stimulation of β1-AR activates PKA, increases CaMKII and IκBα phosphorylation as well as TNF-α expression, in turn enhances caspase-9 and 8 activities, and promotes LPS-induced cardiomyocyte apoptosis
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