Shi J. Liu

Stimulation of different phospholipase A2 isoforms by TNF-α and IL-1β in adult rat ventricular myocytes

We previously showed that in adult rat ventricular myocytes interleukin (IL)-1β activates a membrane-associated, Ca2+-independent phospholipase A2(iPLA2). In this study, we examined the possible existence of different PLA2 isoforms and effects of tumor necrosis factor (TNF)-α on iPLA2activities. Western blot analysis identified iPLA2 in both membrane (∼82 kDa) and cytosolic (∼40 kDa) fractions and identified Ca2+-dependent PLA2(cPLA2) only in cytosolic fractions. With plasmenylcholine or alkylacyl glycerophosphorylcholine as substrate, TNF-α elicited a twofold transient increase in cytosolic iPLA2 activity accompanied by an increase in arachidonic acid release and decreased membrane-associated iPLA2 activity with plasmenylcholine. With phosphatidylcholine as substrate, TNF-α decreased both cytosolic and membrane-associated iPLA2 activities. TNF-α-induced increases in cytosolic iPLA2activity and arachidonic acid release were completely blocked by methyl arachidonyl fluorophosphonate (MAFP) but not by bromoenol lactone (BEL). TNF-α and IL-1β together enhanced synergistically cytosolic and membrane PLA2 activities and arachidonic acid release that were blocked differentially by MAFP and BEL, respectively, and inhibited completely by MAFP plus BEL. These results suggest that TNF-α and IL-1β act on different PLA2 isoforms in ventricular myocytes.

Suppression of β-adrenergic responsiveness of L-type Ca2+ current by IL-1β in rat ventricular myocytes

The possible mechanism by which interleukin-1β (IL-1β) affects β-adrenergic responsiveness of L-type Ca2+ current ( I Ca,L) was examined in adult rat ventricular myocytes by use of whole cell patch-clamp techniques. In the presence of isoproterenol (Iso), exposure for 3 min to IL-1β suppressed the Iso-activated I Ca,L. In the presence of IL-1β, the response of I Ca,L to Iso was decreased, and the EC50 for Iso stimulation was increased. However, IL-1β had no effect on [3H]CGP-12177 binding, displacement of [3H]CGP-12177 binding by Iso, or on basal and Iso-enhanced cAMP content. When I Ca,L was activated by extracellular application of forskolin or 8-(4-chlorophenylthio)-cAMP, a membrane-permeable cAMP analog, or by intracellular dialysis with cAMP, IL-1β had little effect on I Ca,L. In contrast, in the presence of cAMP, IL-1β still suppressed the Iso-enhanced I Ca,L. These results show that the IL-1β-induced decrease in β-adrenergic responsiveness of I Ca,L does not result from inhibition of β-adrenoceptor binding, adenylyl cyclase activity, or cAMP-mediated pathways, suggesting a cAMP-independent mechanism.

Inhibition of sarcoplasmic reticular function by chronic interleukin‐6 exposure via iNOS in adult ventricular myocytes

Interleukin (IL)‐6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)‐dependent pathway during acute exposure. We previously reported that IL‐6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL‐6‐induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml−1 IL‐6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from 0 to 2 mm) in ARVM. We first examined whether IL‐6 reduced Ca2+ influx via L‐type Ca2+‐channel current (ICa,L). Whole‐cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL‐6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL‐6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL‐6, PRP and responsiveness to 10 mm caffeine were also reduced. Meanwhile, the IL‐6‐induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG‐monomethyl‐l‐arginine (l‐NMMA) and 2‐amino‐5,6‐dihydro‐6‐methyl‐4H‐1,3‐thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL‐6‐elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L‐NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL‐6‐induced inhibition of SR function in ARVM during chronic exposure.

α1-Adrenergic activation of L-type Ca current in rat ventricular myocytes: perforated patch-clamp recordings

α1-Adrenergic stimulation has little effect on L-type Ca2+channel current ( I Ca,L) in adult cardiac myocytes measured using conventional whole cell voltage-clamp techniques. In this study using perforated-patch techniques, we reevaluated the effect of α1-adrenergic stimulation on I Ca,L in adult rat ventricular myocytes. Action potentials and I Ca,L were examined in the presence of 1 μM nadolol, a β-adrenergic antagonist, in myocytes internally dialyzed with Na+- and K+-free solutions (Cs+ and tetraethylammonium as substitutes). Phenylephrine (PE; 30 μM) increased the action potential duration measured at 25 and 70% of repolarization by 104 and 86%, respectively. In the perforated-patch configuration, PE elicited a transient decrease followed by a ∼60% increase in I Ca,L, whereas only the transient decrease in I Ca,L was observed in myocytes when the conventional whole cell configuration was used. The PE-induced increase in I Ca,L was reversibly blocked by 1 μM prazosin, an α1-adrenergic antagonist. These results suggest that α1-adrenergic stimulation enhances cardiac I Ca,L and that obligatory intracellular mediators for this action are lost during whole cell recordings.

Both cGMP and peroxynitrite mediate chronic interleukin‐6‐induced negative inotropy in adult rat ventricular myocytes

We previously showed that chronic exposure to interleukin (IL)‐6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)‐dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO‐associated negative inotropic effects of IL‐6 during acute exposure; however, its role in chronic cardiac effects of IL‐6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO−) in chronic IL‐6‐induced negative inotropy in ARVM. After ARVM were exposed to IL‐6 for 2–24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp‐8‐Br‐cGMP, an inhibitor of cGMP‐dependent protein kinase G (PKG). Meanwhile, the IL‐6‐induced decrease in PRP at 2 h was blocked by ODQ or Rp‐8‐Br‐cGMP. By contrast, ODQ or Rp‐8‐Br‐cGMP only attenuated the inhibition of PRP induced by IL‐6 after 24 h exposure. Furthermore, IL‐6 time dependently increased superoxide anion production and ONOO− formation; the latter was abolished by 5,10,15,20‐tetrakis‐(4‐sulphonatophenyl)‐porphyrinato iron (III) (FeTPPS), an ONOO− decomposition catalyst. Interestingly, FeTPPS had no effect on the IL‐6‐elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO− formation, abolished the IL‐6‐induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL‐6‐induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO−, at least in part, mediate the IL‐6‐induced inhibition of SR function after 24 h exposure.

Sex differences in L-type calcium current after chronic ethanol consumption in rats.

Chronic ethanol consumption elicits a progressive cardiac contractile dysfunction, and studies in rats suggest that this alcoholic heart muscle disease is more pronounced in males than females. Cellular changes associated with the ethanol-induced cardiotoxicity remain largely undefined; however, it is possible that L-type Ca(2+) channel current (I(Ca,L)) is affected. Using whole-cell patch-clamp techniques, this study examined I(Ca,L) in adult ventricular myocytes isolated from male and female P-rats that had consumed drinking water (controls) or a 25% ethanol/water mixture for 14 months. The peak amplitude and maximum conductance of I(Ca,L) were 32 and 26% greater, respectively, in cardiomyocytes isolated from ethanol-consuming compared to control male rats. In contrast, no differences in the amplitude or conductance of I(Ca,L) were observed when comparing myocytes isolated from control and ethanol-consuming females. Ethanol treatment had no significant effects on the kinetics I(Ca,L) inactivation or on steady-state activation and inactivation in either gender. In conclusion, male but not female cardiomyocytes respond to chronic ethanol consumption with an increased I(Ca,L) that may represent a compensatory response to the depressed contractility.