Margaret P. Moffat

Role of protein kinase C in mediating effects of hydrogen peroxide in guinea-pig ventricular myocytes

The present study examined the effects of hydrogen peroxide (H2O2) on intracellular calcium transients and unloaded cell shortening in the presence of the protein kinase C (PKC) inhibitors 1-(5-isoquinolinesulfonyl-2-methylpiperazine (H7) or chelerythrine chloride (CHC) or the PKC activator phorbol 12-myristate 13-acetate (PMA). Calcium transient amplitudes and cell shortening were measured simultaneously in single, enzymatically dissociated ventricular myocytes loaded with fura2-AM. Exposure of myocytes to H2O2, 25 microM or 75 microM, for 15 min caused a time- and concentration-dependent increase in calcium transient amplitude, cell shortening and the diastolic 340/380 fluorescence ratio. Significant increases in calcium transient amplitude were observed from 7 to 15 min of superfusion with 25 microM H2O2 and the transient amplitude remained elevated throughout the 10 min washout period. In the presence of 75 microM H2O2, transient amplitude was elevated following 2 min and remained elevated for the remainder of the experiment. Significant increases in cell shortening were also observed from 7 to 15 min in the presence of either 25 or 75 microM H2O2. This effect was reversed upon washout of the lower concentration of H2O2 but persisted during the initial 5 min of washout at the higher concentration. The diastolic 340/380 fluorescence ratio was unaltered in the presence of 25 microM of H2O2, however this parameter was significantly increased from 7 to 15 min following exposure to 75 microM H2O2 and remained elevated throughout the washout period. The H2O2-induced increases in calcium transient amplitude and cell shortening were significantly attenuated in myocytes which were pretreated with either H7 or CHC.(ABSTRACT TRUNCATED AT 250 WORDS)

Positive and negative inotropic effects of phorbol 12-myristate 13-acetate: Relationship to PKC-dependence and changes in [Ca2+]i

The present study examined the concentration-dependent effects of phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, on contractile force and [Ca2+]i in guinea-pig hearts and isolated cardiac myocytes, respectively. Contractile force was measured using isolated Langendorff-perfused hearts while [Ca2+]i was measured independently in isolated cardiac myocytes loaded with fura2-AM. Phorbol 12-myristate 13-acetate, as well as another PKC-activating phorbol, phorbol dibutyrate (PDBu), and two non-PKC-activating phorbols, alpha-phorbol didecanoate (alpha PDD) and 4 alpha-phorbol, exerted time- and concentration-dependent effects on contractility. A significant positive inotropic response was observed with either PMA (10(-12) M; 5-15 min of perfusion) or PDBu (10(-12) M; 5 min of perfusion). In contrast, 10(-10) M PMA caused a significant negative inotropic effect following 30 min of perfusion while 10(-8) M PMA produced a significant negative inotropic effect which occurred earlier (10 min) and was sustained throughout the 30 min perfusion period. A similar negative inotropic effect was seen with 10(-8) M of either PDBu or alpha PDD. In addition, 4 alpha-phorbol (10(-8) M) exerted a modest, but significant negative inotropic effect following 25 and 30 min of perfusion. Both concentration-dependent increases and decreases of +dF/dt and -dF/dt were observed in the presence of PMA. In addition, both PMA and PDBu caused a concentration-dependent increase in coronary perfusion pressure. The positive inotropic responses and coronary perfusion pressure effects elicited by PMA and PDBu were largely prevented by the addition of the PKC inhibitors H7 (6 nM) or HAG (10 nM); however, these drugs were without effect on the negative inotropic response to higher concentrations of both PKC-activating (PMA, PDBu) and non-PKC-activating (alpha PDD, 4 alpha-phorbol) phorbol compounds. The lowest concentration of either PMA or PDBu (10(-12) M) increased the 340/380 fluorescence ratio of isolated cardiac myocytes loaded with fura2-AM on a time scale similar to that at which the positive inotropic response was seen in the whole heart. However, in contrast to results in the isolated heart, PDBu elicited a greater and sustained increase in the fluorescence ratio measured in isolated cardiac myocytes. The higher concentration of either PMA or PDBu (10(-8) M), resulted in a decrease in the 340/380 ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts

1 The effects of the phorbol ester, phorbol myristate acetate (PMA) were examined on function and energy metabolism in the isolated working heart of the rat. 2 At a concentration of 10−9 m. PMA produced a rapid loss in cardiac function in terms of aortic flow rate (AFR) and coronary flow rates (CFR) whereas a similar concentration of 4α‐phorbol 12,13‐didecanoate was ineffective. At a concentration of 10−10 m., the PMA‐induced depression was more gradual but nevertheless very pronounced with an almost total loss in AFR after 30 min perfusion. The reduction in CFR was more moderate than that observed with respect to AFR. 3 The protein kinase C (PKC) inhibitor (±)‐l‐O‐hexadecyl‐2‐O‐acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. 4 Two inhibitors of Na+/H+ exchange, amiloride and quinacrine, totally prevented the reduction in AFR. Although the PMA‐induced depression in CFR was also attenuated by both amiloride and quinacrine, these effects were not significant, probably reflecting the less pronounced effect of PMA on this parameter. 5 Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/H+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. 6 Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. 7 We conclude that PKC activation is necessary for phorbol ester‐induced cardiac dysfunction. The consequence of PKC stimulation includes (1) Na+/H+ exchange activation and a subsequent elevation in intracellular calcium [Ca2+]i via Na+/Ca2+ exchange and (2) PKC‐dependent phosphorylation of the calcium channel, both of which would produce toxicity by elevation of [Ca2+]i. Pharmacological manipulation of any of these steps prevents PMA toxicity by virtue of a reduction in the accumulation of [Ca2+]i. PMA effects or their prevention are unrelated to any changes in energy metabolism.

Adenosine-sensitive α1-adrenoceptor effects on reperfused ischaemic hearts : comparison with phorbol ester

1 We have examined the effects of the α1‐adrenoceptor agonists, phenylephrine or methoxamine, on contractility in rat and rabbit isolated hearts as well as their effects on postischaemic ventricular recovery. We compared these effects to those of 12‐phorbol 13‐myristate acetate (PMA), a direct activator of protein kinase C (PKC). 2 The positive inotropic effect of α1‐receptor agonists was significantly attenuated in the presence of the Na/H exchange inhibitor, methylisobutyl amiloride (MIA, 1 μm), whereas the positive inotropic effect of PMA was unaffected. 3 Reperfusion of rat hearts subjected to either 30 or 60 min of zero‐flow ischaemia, resulted in recovery of contractility to 91 ± 2% and 57 ± 7% of the preischaemic values, respectively which was unaffected by phenylephrine. In contrast, PMA at a concentration (10 pM) devoid of direct depressant effects, significantly decreased recovery following 60 min of ischaemia to 31 ± 4% of pre‐ischaemic value (P < 0.05 from control); an effect which was completely prevented by the PKC inhibitor, bisindolyl‐maleimide. A similar inhibitory effect of PMA and lack of effect of phenylephrine were seen in reperfused rabbit hearts. 4 As α1‐receptor activation has been shown previously to stimulate cardiac adenosine production, we assessed whether blockade of adenosine A1 receptors with the specific antagonist, 1,3‐dipropyl‐8‐cyclopentylxanthine (DPCPX, 0.5 μm) would unmask the actions of phenylephrine in hearts subjected to 30 min ischaemia and reperfusion. In the presence of DPCPX, phenylephrine reduced recovery to 44 ± 9% compared to 82 ± 10% recovery in the absence of phenylephrine (P < 0.05). Identical results were observed in rabbit hearts treated with DPCPX in which recovery was reduced from 57.1 ± 11.2% to 17.8 ± 6.8% by phenylephrine (P < 0.05). Another A1 receptor antagonist, (±)‐N6‐endonorbornan‐2‐yl‐9‐methyladenine (N‐0861, 0.5 μm) produced virtually identical results to those observed with DPCPX. 5 MIA failed to modulate the inhibition of postischaemic recovery by phenylephrine. Bisindolyl‐maleimide, on the other hand, partially prevented the effects of phenylephrine on postischaemic contractile dysfunction. The inhibitory effect of either PMA or phenylephrine on postischaemic recovery of both rat and rabbit hearts was generally dissociated from alterations in energy metabolism, although in the case of rat hearts, inhibition by phenylephrine was associated with diminished high energy phosphate content. 6 Our results demonstrate that both α1‐receptor activation as well as direct activation of PKC with phorbol ester can attenuate post‐ischaemic ventricular recovery. Moreover, our results strongly suggest that endogenous adenosine protects the heart against the deleterious effects of α1‐receptor activation during ischaemia and reperfusion.

Modulation of sodium-hydrogen exchange activity in cardiac myocytes during acidosis and realkalinisation: effects on calcium, pHi, and cell shortening

OBJECTIVEnThe aim was to examine the effects of the Na+/H+ exchange inhibitor methylisobutylamiloride (MIA) as well as protein kinase C, a putative regulator of Na+/H+ exchange, on intracellular calcium, intracellular pH, and unloaded cell shortening in isolated guinea pig cardiac myocytes subjected to lactic acid induced acidosis followed by realkalinisation.nnnMETHODSnCalcium transient amplitude and cell shortening were measured simultaneously in single isolated myocytes loaded with fura2-AM. Intracellular pH was measured in cells loaded with BCECF-AM.nnnRESULTSnExposure of cells to 5 min of lactate (20 mM) acidosis (pH 6.8) caused an increase in calcium transient amplitude and a decrease in cell shortening and intracellular pH. During realkalinisation (pH 7.3), the calcium transient gradually decreased while intracellular pH became more alkaline than pre-acidosis values. The cells underwent transient hypercontractility as evidenced by a marked increase in systolic cell shortening and a decrease in diastolic cell length. Inhibition of sodium/hydrogen exchange with MIA (1 microM) caused a significant attenuation of the increase in calcium transient amplitude during acidosis and further depressed cell shortening as well as intracellular pH. In addition, MIA significantly attenuated hypercontractility and abolished cell contracture upon realkalinisation. In contrast, phorbol 12-myristate 13-acetate (10(-12) M) exerted no effects on the response to acidosis; however, this treatment exacerbated cell hypercontractility and reduced functional recovery upon realkalinisation.nnnCONCLUSIONSnInhibition of Na+/H+ exchange activity during acidosis/realkalinisation enhances recovery of cell function.

Activated neutrophils impair rabbit heart recovery after hypothermic global ischemia

Cardiopulmonary bypass is known to cause neutrophil activation, and activated neutrophils appear to be of importance in myocardial reperfusion injury. This study examined the effect of a preischemic infusion of activated neutrophils on the recovery of myocardial function after 40 minutes of hypothermic global ischemia. Studies were carried out in three groups of Langendorff-perfused rabbit hearts: control, control (unactivated) neutrophil infusion, and phorbol myristate acetate-activated neutrophil infusion. The activated neutrophil group showed significant deterioration in function during the activated neutrophil infusion. All three groups demonstrated significant depression of function initially after reperfusion, but the two control groups subsequently recovered to baseline levels. The activated neutrophil group, however, showed a persistent significant depression in ventricular force, rate of ventricular tension development, and rate of ventricular relaxation as well as a significant increase in coronary vascular resistance. It is concluded that activated neutrophils depress myocardial function and contribute to impaired recovery of function after global hypothermic ischemia.

Protective effects of D,L-carnitine against arrhythmias induced by lysophosphatidylcholine or reperfusion

Electrophysiological effects of lysophosphatidylcholine (50 or 100 microM) and D,L-carnitine (100 microM) were studied under control conditions and in response to simulated ischaemia and reperfusion using the superfused right ventricular free wall preparation from the guinea pig heart. Lysophosphatidylcholine, 100 microM, induced a significant depolarization of the maximum diastolic potential (MDP) in the epicardium, as well as the development of ventricular premature beats, salvos and ventricular tachycardia. Both coupled beats and abnormal automaticity were observed in lysophosphatidylcholine (100 microM)-treated preparations. Carnitine (100 microM) alone had no effect on preparations superfused with normal Tyrode solution. However, it delayed the time to onset and reduced the cumulative duration of lysophosphatidylcholine-induced arrhythmias (P less than 0.05). The incidence of lysophosphatidylcholine-induced abnormal automaticity and salvos was also significantly decreased in the presence of carnitine. Twenty minutes of simulated ischaemia caused depolarization of MDP as well as prolongation followed by block of transmural conduction. Lysophosphatidylcholine (100 microM) did not alter this response however, carnitine significantly reduced ischaemia-induced depolarization in the epicardium. All control preparations developed arrhythmic activity during 30 min of reperfusion. Carnitine accelerated recovery of MDP in the epicardium upon reperfusion, prolonged the time to onset of arrhythmic activity and reduced both its cumulative duration and incidence. In contrast, reperfusion in the presence of lysophosphatidylcholine (100 microM) significantly increased the incidence of arrhythmic activity. Carnitine exerted only minimal antiarrhythmic action when preparations were exposed to reperfusion in the presence of lysophosphatidylcholine. In conclusion, this study demonstrates that carnitine can modify various cellular mechanisms of arrhythmia induced by lysophosphatidylcholine or by reperfusion but is much less effective when lysophosphatidylcholine and reperfusion are combined.

Contractile and Electrophysiologic Effects of Realkalization in Cardiac Tissues: Role of Na/H Exchange and Increased [CA]i

The effects of lactate acidosis followed by realkalization were studied in fast and slow response ventricular tissues and isolated cardiac myocytes. Both electrophysiological and contractile responses were altered by these conditions in isolated ventricular tissues and [Ca]i was elevated in isolated myocytes.

Signal transduction mechanisms in the ischemic and reperfused myocardium

The cellular mechanisms regulating myocardial dysfunction during ischemia and subsequent reperfusion are complex. As can be determined from this review, it is clear that signal transduction pathways are altered during these conditions, which may explain, in part, the pathophysiology of ischemia and reperfusion. With respect to beta-adrenoceptor signal transduction, adaptive changes during ischemia and reperfusion ensure that this critical pathway for the regulation of cardiac function remains intact. Additionally, although the relative contribution of alpha 1-adrenoceptors to the regulation of cardiac function is minimal in normal myocardium, these receptors clearly exacerbate conditions associated with the generation of arrhythmias during reperfusion. It is likely that this enhancement of arrhythmogenesis is related to the activation of NHE by a PKC-dependent mechanisms. The importance of non-receptor-mediated signal transduction as a mediator of ischemia and reperfusion injury has long been established with respect to products of membrane lipid breakdown. As discussed, recent evidence now suggests that other compounds formed during ischemia and reperfusion, such as reactive oxygen species and NO, are also linked to cellular second messenger systems. In conclusion, as signal transduction is critical for normal myocardial function, signal transduction pathways are of even more importance during ischemia and reperfusion. There is an increasing interest in the role of non-receptor-mediated signal transduction as a mediator of ischemia and reperfusion injury and it is hoped that these pathways may represent new levels for therapeutic intervention.

Role of Na/H Exchange and [Ca2+]i in Electrophysiological Responses to Acidosis and Realkalization in Isolated Guinea Pig Ventricular Myocytes

It is well known that reperfusion of the ischemic heart results in a rapid increase in the intracellular calcium concentration ([Ca2+]i) (1). However, the mechanisms responsible for the increase in [Ca2+]i remain unclear (1). In this regard, since the intracellular pH is much lower than that of the perfusate during early reperfusion (2), a pH gradient develops instantaneously across the cardiac cell membrane. This pH gradient activates the sodium/hydrogen (Na+/H+) exchange, leading to a rapid accumulation of intracellular sodium ([Na+]i). The increase in [Na+]i may, in turn, result in an increase in [Ca2+]i via sodium/calcium (Na+/Ca2+) exchange (reviewed in 1,3,4). Therefore, the realkalization-induced activation of Na+/H+ exchange, and subsequent accumulation of [Na+]i may explain, at least in part, the reperfusion-induced increase in [Ca2+]i (reviewed in 1, 3, 4). This hypothesis has been supported recently by several lines of evidence. Firstly, it has been reported that the increase in [Na+]i occurs prior to and is closely correlated to the subsequent increase in [Ca2+]i (5). Secondly, the increases in [Na+]i and [Ca2+]i associated with reperfusion can be attenuated either by reperfusion with an acidotic buffer (6), or by inhibitors of the Na+/H+ exchanger (5, 7). Finally, improved ventricular recovery and a reduction in reperfusion associated contracture has been demonstrated using amiloride or amiloride analogues in rat and guinea pig hearts (8, 9, 10). Conversely, administration of lactate to isolated rat hearts prior to the induction of ischemia significantly reduces recovery of function upon reperfusion; an effect which can be reversed by inhibitors of Na+/H+ exchange (11).