Charlene M. Hohl

Response of Failing Canine and Human Heart Cells to β2-Adrenergic Stimulation

Background Failing human hearts lose β1- but not β2-adrenergic receptors. In canine hearts with tachypacing failure, the ratio of β2- to β1-adrenergic receptors is increased. The present study was designed to determine whether heart failure increases sensitivity to β2-adrenergic stimulation in isolated canine ventricular cardiomyocytes and to verify that myocytes from failing human ventricles contain functional β2-adrenergic receptors. Methods and Results Myocytes from healthy dogs, dogs with tachypacing failure, and human transplant recipients were loaded with fura 2-AM and subjected to electric field stimulation in the presence of zinterol, a highly selective β2-adrenergic agonist. Zinterol significantly increased [Ca2+]i transient amplitudes in all three groups. The failing canine myocytes were significantly more responsive than normal to β2-adrenergic stimulation. We also measured isotonic twitches, indo-1 fluorescence transients, and L-type Ca2+ currents in healthy canine myocytes. Zinterol (10−5 mol...

β2-Adrenergic Receptor Antagonists Protect Against Ventricular Fibrillation In Vivo and In Vitro Evidence for Enhanced Sensitivity to β2-Adrenergic Stimulation in Animals Susceptible to Sudden Death

BACKGROUND The ventricular myocardium contains functional beta2-adrenergic receptors that when activated increase intracellular Ca2+ transients. Because elevated Ca2+ has been implicated in the induction of ventricular fibrillation (VF), it is possible that the activation of these receptors may also provoke malignant arrhythmias. METHODS AND RESULTS To test this hypothesis, a 2-minute occlusion of the left circumflex coronary artery was made during the last minute of exercise in 28 dogs with healed anterior myocardial infarctions: 17 had VF (susceptible) and 11 did not (resistant). On a subsequent day, this test was repeated after administration of the beta2-adrenergic receptor antagonist ICI 118,551 (0.2 mg/kg). This drug did not alter the hemodynamic response to the coronary occlusion, yet it prevented VF in 10 of 11 animals tested (P<.001). However, heart rate was reduced in 6 animals. Therefore, the ICI 118,551 exercise-plus-ischemia test was repeated with heart rate held constant by ventricular pacing (n=3). ICI 118,551 still prevented VF when heart rate was maintained. Next, the effects of increasing doses of the beta2-adrenergic receptor agonist zinterol on Ca2+ transient amplitudes were examined in ventricular myocytes. Zinterol elicited significantly greater increases in Ca2+ transient amplitudes at all doses tested (10(-9) to 10(-6) mol/L) in myocytes prepared from susceptible versus resistant animals. The cardiomyocyte response to isoproterenol (10(-7) mol/L) in the presence or absence of the selective beta1- (CGP-20712A, 300 nmol/L) or beta2- (ICI 118,551, 100 nmol/L) adrenergic receptor antagonist was also examined. Isoproterenol elicited larger Ca2+ transient increases in the susceptible myocytes, which were eliminated by ICI but not by CGP. CONCLUSIONS When considered together, these data demonstrate that canine myocytes contain functional beta2-adrenergic receptors that are activated to a greater extent in the susceptible animals. The resulting cytosolic Ca2+ transient increases may lead to afterpotentials that ultimately trigger VF in these animals.

Compartmentation of cAMP in adult canine ventricular myocytes. Relation to single-cell free Ca2+ transients.

Isolated adult canine ventricular myocytes were used to study the role of compartmentation of cAMP in the diverse functional responses to various drugs that elevate cAMP. Myocytes presented with the beta-agonist isoproterenol accumulated cAMP with a half maximally effective concentration (EC50) of 3.55 x 10(-8) M. Approximately 45% of the total cAMP was recovered in the particulate fraction of digitonin-lysed myocytes under these conditions. With phosphodiesterase inhibition (10 microM isobutylmethylxanthine), isoproterenol-stimulated cAMP production was up to 3.4-fold greater, but the proportion of total cAMP residing in the particulate fraction declined to less than 20%. Similar results were obtained with forskolin, a direct activator of adenylate cyclase. Treatment with isoproterenol shortened the duration at 50% maximum peak height (T 1/2) and increased the peak fluorescence ratio of electrically triggered single-cell free Ca2+ transients in fura-2-loaded canine myocytes. Isoproterenol dose-response curves gave EC50 values of 1.7 x 10(-9) and 4.4 x 10(-9) M for effects on T 1/2 and peak height, respectively. Alterations in peak height and T 1/2 of Ca2+ transients also showed a dose dependency on isobutylmethylxanthine and forskolin. Comparison of myocyte cAMP content with the corresponding changes in free Ca2+ transients demonstrated a close correlation between particulate cAMP and the extent of shortening or increase in peak height of the fura-2 Ca2+ transients (r = 0.92 for each). However, when these two parameters were plotted as a function of total cAMP, the resulting curves were nonlinear and divergent for each agent tested. The results support the hypothesis that differences in responses to agents that augment cAMP can be explained in part by compartmentation of cAMP. Furthermore, Ca2+ mobilization seems to be most affected by cAMP located in the particulate compartment of canine cardiac myocytes.

Adenosine, Inosine, and Guanosine Protect Glial Cells During Glucose Deprivation and Mitochondrial Inhibition: Correlation Between Protection and ATP Preservation

Abstract: The purpose of this study was to determine the mechanism by which adenosine, inosine, and guanosine delay cell death in glial cells (ROC‐1) that are subjected to glucose deprivation and mitochondrial respiratory chain inhibition with amobarbital (GDMI). ROC‐1 cells are hybrid cells formed by fusion of a rat oligodendrocyte and a rat C6 glioma cell. Under GDMI, ATP was depleted rapidly from ROC‐1 cells, followed on a much larger time scale by a loss of cell viability. Restoration of ATP synthesis during this interlude between ATP depletion and cell death prevented further loss of viability. Moreover, the addition of adenosine, inosine, or guanosine immediately before the amobarbital retarded the decline in ATP and preserved cell viability. The protective effects on ATP and viability were dependent on nucleoside concentration between 50 and 1,500 µM. Furthermore, protection required nucleoside transport into the cell and the continued presence of nucleoside during GDMI. A significant positive correlation between ATP content at 16 min and cell viability at 350 min after the onset of GDMI was established (r = 0.98). Modest increases in cellular lactate levels were observed during GDMI (1.2 nmol/mg/min lactate produced); however, incubation with 1,500 µM inosine or guanosine increased lactate accumulation sixfold. The protective effects of inosine and guanosine on cell viability and ATP were >90% blocked after treatment with 50 µM BCX‐34, a nucleoside phosphorylase inhibitor. Accordingly, lactate levels also were lower in BCX‐34‐treated cells incubated with inosine or guanosine. We conclude that under GDMI, the ribose moiety of inosine and guanosine is converted to phosphorylated glycolytic intermediates via the pentose phosphate pathway, and its subsequent catabolism in glycolysis provides the ATP necessary for maintaining plasmalemmal integrity.

An in vitro model of myocardial ischemia utilizing isolated adult rat myocytes

Isolated adult rat myocytes were used to develop an in vitro model of myocardial ischemia. Freshly isolated myocytes were spun into a cell pellet to limit extracellular volume. Excess supernatant was removed and the pellet was covered with mineral oil and incubated in a temperature controlled water bath. After various periods of incubation, cells were analyzed for adenine nucleotide levels, lactate accumulation, rate of cell death, and cell morphology. Adenine nucleotide profiles after 60 min incubation at 37 degrees C showed marked depletion of adenosine triphosphate (ATP) and large increases in adenosine monophosphate (AMP), adenosine, inosine, and lactate and no significant difference in levels of inosine monophosphate. These results are consistent with ischemic conditions. Reduction of the incubation temperature to 34 and 30 degrees C slowed the rate of cell squaring and the onset of cell death. Resuspension of ischemic cells after 30, 45, 60 and 90 min incubation in hypotonic buffer (170 mosmol) to induce acute cell swelling caused an increase in the number of non-viable cells at each time point. Control cells and ischemic cells incubated less than 30 min did not show increases in non-viable cells when subjected to hypotonic swelling. Morphological analysis revealed that isolated myocytes respond to ischemia in a heterogeneous fashion and exhibit changes at both light and electron microscopic levels similar to those seen in other ischemic models. These results indicate that pelleted isolated adult rat myocytes may be a useful in vitro model to study myocardial ischemic cells injury.

Inosine and guanosine preserve neuronal and glial cell viability in mouse spinal cord cultures during chemical hypoxia

Murine spinal cord primary mixed cultures were treated with the respiratory inhibitor, rotenone, to mimic hypoxic conditions. Under these conditions neurons rapidly underwent oncosis (necrosis) with a complete loss in viability occurring within 260 min; however, astrocytes, which accounted for most of the cell population, died more slowly with 50% viability occurring at 565 min. Inosine preserved both total cell and neuronal viability in a concentration-dependent manner. The time of inosine addition relative to hypoxic insult was critical with the most effective protection occurring when inosine was added just prior to or within 5 min after insult. Inosine was ineffective when added 30 min after hypoxic insult. The effect of guanosine was similar to that of inosine. Treatment of cultures with BCX-34, a purine nucleoside phosphorylase inhibitor, prevented protection by inosine or guanosine, suggesting involvement of a purine nucleoside phosphorylase in the nucleoside protective effect.

Calcium tolerance of isolated rat heart cells.

Abstract Freshly isolated adult rat heart cells, which initially show the elongated, rod-shaped morphology typical of heart cells in situ , are almost quantitatively converted to rounded contracture forms by exposure to 1 m m Ca 2+ . These Ca 2+ -sensitive cells became Ca 2+ -tolerant following a short period of metabolic activity in a low-Ca 2+ medium, in that they retain their rod-shaped configuration when challenged with Ca 2+ after this preincubation step. Tolerance to Ca 2+ develops in parallel with the establishment of low Na + /K + ratios in these cells and both processes are sensitive to ouabain. The initial net uptake of Ca 2+ is greater in Ca 2+ -sensitive than in Ca 2+ -tolerant cells. These results suggest that contracture in the Ca 2+ -sensitive cells is a consequence of the rapid entry of excessive amounts of Ca 2+ in exchange for internal Na + .

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligo‐dendroglial‐glioma hybrid cells (ROC‐1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to <5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehy‐drogenase release) which began at ∼20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K+‐ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to ∼10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at ∼55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracel‐lular calcium ([Ca2+]i) was measured using fura‐2. In calcium‐containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 ± 17 nM) followed by a low steady‐state level preceding plasma membrane disruption. Following deenergization in calcium‐free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.

MECHANICAL ALTERNANS AND THE FORCE-FREQUENCY RELATIONSHIP IN FAILING RAT HEARTS

We examined contractile performance in perfused ventricles from normal rats and from SHHF/Mccfacp rats with end-stage heart failure. Changes in pacing frequency from 3 to 5 Hz evoked a complex response in normal rat myocardium. The first beat after a switch to 5 Hz was extremely weak, but each successive beat was stronger until force exceeded the 3 Hz steady state value by approximately 30%. Force then gradually declined to a new steady state where developed pressure was depressed but rate-pressure product was slightly greater than that at 3 Hz. By contrast, in failing SHHF/Mcc-facp hearts, an increase in pacing frequency from 3 to 5 Hz did not increase force development. Instead, the isovolumic left ventricles exhibited mechanical alternans. This alternation between weak and strong beats was abolished by 1 mM caffeine but restored by its washout. Inhibition of SR Ca2+ accumulation by 50-500 nM thapsigargin in normal ventricles did not evoke alternans when pacing frequencies were increased. The results indicate that mechanical alternans in failing rat hearts is due to altered reactions of the sarcoplasmic reticulum, but a decreased rate of Ca2+ accumulation is not the primary cause.

Cellular glutathione and the response of adult rat heart myocytes to oxidant stress

Freshly isolated adult rat heart myocytes contain total glutathione and reduced glutathione (GSH) at levels quite comparable to those in intact rat heart. Total glutathione can be depleted from 11 to 1 nmol/mg protein or less by treatment with cyclohex-2-ene-1-one without effect on either cellular ATP, rod-cell morphology or the integrity of the sarcolemma. Glutathione levels and redox state are not altered significantly when the Ca-tolerant, quiescent cells are subjected to a period of anoxia followed by reoxygenation. This oxygen paradox protocol results in irreversible hypercontracture of the contractile elements into an amorphous mass in the bulk of the cells, but little loss of sarcolemmal integrity. When the myocytes are subjected to an externally applied oxidant stress by the addition of either diamide or t-butylhydroperoxide, GSH is rapidly depleted with accumulation of oxidized glutathione (GSSG. On continued aerobic incubation both of these reagents promote a slower depletion of cellular ATP and a parallel hypercontracture. Cells treated with t-butylhydroperoxide, but not those with diamide, also generate increasing amounts of thiobarbituric acid reactive species as an indication of lipid peroxidation and show a parallel loss of sarcolemmal integrity. It is concluded that respiring myocytes and those subjected to the oxygen paradox do not produce oxygen radicals in sufficient amounts to displace the GSH/GSSG redox poise and depletion of myocyte glutathione per se is not detrimental to the short term survival of the cells. In addition, aerobic myocytes subjected to external oxidant stress can be damaged irreversibly by two pathways, a hypercontracture that correlates with depletion of ATP and a loss of sarcolemmal integrity that correlates with lipid peroxidation.