Charles E. Ganote

Potassium channels and preconditioning of isolated rabbit cardiomyocytes: effects of glyburide and pinacidil.

Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37 degrees C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOSM media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 microM) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 microM) added with pinacidil and calphostin C (200nM) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 microM) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 microM) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidils protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3,dipropylxanthine (DPCPX) (10 microM) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.

In vitro ischaemic preconditioning of isolated rabbit cardiomyocytes: effects of selective adenosine receptor blockade and calphostin C

OBJECTIVEnThe aim was to determine if in vitro ischaemic preincubation can precondition cardiomyocytes and if the responses to adenosine receptor antagonists are similar to those previously determined during metabolic preconditioning with glucose deprivation or adenosine agonists.nnnMETHODSnIsolated rabbit cardiomyocytes were preconditioned with 10 min of ischaemic preincubation, followed by a 30 min postincubation before the final sustained ischaemic period. The protein kinase C inhibitor calphostin C or the adenosine receptor antagonists 8-sulphophenyltheophylline (SPT), BW 1433U, and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were added either during the preincubation or into the final ischaemic pellet. Adenosine deaminase (10 U.ml-1) was added during ischaemic preincubation. Rates of contracture and extent of injury were determined by sequential sampling and assessment of trypan blue permeability following 85 mOsM swelling.nnnRESULTSnMyocytes were preconditioned by a 10 min in vitro ischaemic preincubation. Preincubation with 100 microM SPT or with adenosine deaminase, or addition of 200 nM calphostin C into the final ischaemic pellet did not alter rates of rigor contracture but nearly abolished protection. A significant degree of protection was maintained following ischaemic preincubation with the highly selective adenosine A1 receptor blocker DPCPX (10 microM), while the A1/A3 antagonist BW 1433U (1 microM) severely limited protection. SPT and BW 1433U added only into the final ischaemic pellet of preconditioned cells significantly blocked protection, while protection was maintained in the presence of DPCPX.nnnCONCLUSIONSnIschaemic preconditioning of cardiomyocytes is blocked by adenosine receptor antagonists known to bind to A3 receptors but not by DPCPX which has high affinity for A1 receptors, but little affinity for A3 receptors. Maintenance of protection during the final ischaemic phase has a similar receptor specificity. Blockade of protein kinase C activity abolishes protection. Ischaemic and metabolic preconditioning in vitro appear to occur through similar pathways.

Effects of 2,3-butanedione monoxime (BDM) on contracture and injury of isolated rat myocytes following metabolic inhibition and ischemia

The relationship between myocardial cell contracture and injury during total metabolic inhibition (amylobarbital and iodoacetic acid) and ischemia was examined, using 5-50 mM butanedione monoxime (BDM) as an inhibitor of contracture. BDM had no apparent effect on control myocytes during 180 min incubations, but inhibited contracture following anoxia or ischemia in a dose-dependent fashion, as directly quantitated by length/width ratios. Cellular ATP levels decreased at a similar rate in the absence or presence of BDM, following metabolic inhibition. BDM-mediated inhibition of contracture was associated with accelerated cell injury, as defined by: the uptake of an extracellular marker (trypan blue) by the cardiomyocytes, by direct analysis of myoglobin released into the supernatant and by ultrastructural demonstration of defects in sarcolemmal membrane integrity. Calcium was not required for BDMs enhancement of injury, in that cells incubated in calcium free-EGTA buffer showed a similar BDM-mediated acceleration of injury. In the presence or absence of calcium, enhancement of injury was more marked in cells osmotically stressed with a brief incubation in hypotonic buffer, than in cells resuspended in isotonic media. It is concluded that BDM enhances development of osmotic fragility of inhibited or ischemic cardiomyocytes and that contracture is not a necessary contributing factor to myocardial cell death.

Enhanced cell volume regulation: a key protective mechanism of ischemic preconditioning in rabbit ventricular myocytes

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ~60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 microM calyculin A, or 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl(-) channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

Effects of the protein phosphatase inhibitors okadaic acid and calyculin A on metabolically inhibited and ischaemic isolated myocytes.

Isolated adult rat myocytes were subjected to 180 min of metabolic inhibition or incubated in ischaemic pellets, in the presence and absence of 10 microM okadaic acid (OA) or calyculin A (CL-A). Contracture and viability was determined by light microscopic analysis of trypan blue-stained preparations and ATP levels by HPLC. Osmotic fragility was assessed by brief hypotonic swelling of cells in 170 or 85 mOsm media prior to determination of viability. Neither drug significantly affected the relatively rapid rates of contracture of myocytes during metabolic inhibition, and both afforded significant protection from development of trypan blue permeability and osmotic fragility. Both OA and CL-A significantly accelerated the rates of contracture and ATP depletion of myocytes during ischaemic incubations. Despite an enhanced rate of ATP depletion, which would be expected to accelerate development of injury, neither drug accelerated development of loss of viability or development of osmotic fragility as measured by 170 mOsm swelling. Mathematical compensation for different rates of ATP depletion confirmed that a protective effect of the drugs, during ischaemic incubation, was masked by their enhancement of the rate of injury, following swelling at 170 mOsm. When the effects of CL-A on ischaemic cells were examined at 85 mOsm, a more stringent test for osmotic fragility, protection was found without compensation for differing rates of ATP depletion. A dose/response curve for CL-A showed some effect at 100 nM and a nearly full effect during metabolic inhibition at 1 microM concentrations. It is concluded that protein phosphatase inhibitors reduce the rates of development of osmotic fragility of metabolically inhibited cells and reduces the rate of injury relative to the rate of ATP depletion of ischaemic cardiomyocytes. Phosphorylation mechanisms may be important to development of irreversible myocardial cell injury.

Adenosine and preconditioning in the rat heart.

The report by Cave et al. [21] was a landmark paper which demonstrated that functional recovery protection by ischemic preconditioning (IPC) is not mediated by adenosine in the globally ischemic rat heart. This report raised several issues in the field of preconditioning. (1) It was enigmatic in that recent studies had demonstrated a predominant role of adenosine-receptors in preconditioning in the rabbit and dog so that the inability of adenosine to precondition in the rat implied that species differences might exist in the receptor pathways that lead to initiation of preconditioning. (2) Since rat cardiomyocyte metabolism preferentially relies upon exogenous glucose to support glycolysis, whereas the rabbit, dog, pig and man are more dependent upon endogenous glycogen, this report opened the possibility that species-dependent metabolic differences could also be involved in the mechanisms of IPC. (3) Since adenosine did not appear to precondition the rat, emphasis was placed on multiple G-protein coupled receptors that can induce preconditioning, e.g., α1-adrenergic-receptors in the rat [1], and on focused research on postreceptor, downstream events in the search for the ultimate mediators of protection. (4) Finally, the manuscript focused debate on the relationships between the mechanisms of preconditioning as assessed by functional recovery (stunning), reductions in arrythmias or ischemic cell death (infarction).nnAortic flow during reperfusion is a sensitive end-point commonly used to assess postischemic functional recovery. Postischemic functional recovery is influenced by multiple parameters including: (1) degree of stunning during ischemia; (2) rate of anaerobic glycolysis; (3) ability to recover oxidative respiration, intracellular pH and ionic equilibrium; and (4) degree of diastolic contracture and vascular integrity at onset of reperfusion. Increased diastolic pressures subsequent to ischemic contracture reduce subendocardial perfusion such that zones of no-reflow may occur upon reperfusion. These subendocardial zones of no-reflow are a perfused heart artifact due …

Flow cytometric analysis of isolated adult cardiomyocytes: Vinculin and tubulin fluorescence during metabolic inhibition and ischemia

Immunofluorescence and quantitative flow cytometry was used to determine if alterations in cytoskeletal proteins (vinculin and tubulin) occur during metabolic inhibition and ischemic incubation of isolated adult rat cardiomyocytes. Effects of cell shape changes on fluorescence, were controlled for by the contractile inhibitor, butanedione monoxime (BDM) and gated analysis. Flow cytometry differentiated rod- and round-shaped myocytes on the basis of forward and side scattering. Severe contracture of metabolically inhibited (iodoacetic acid and amytal) myocytes caused an artefactual increase in fluorescence intensity and a redistribution of tubulin into microblebs on the cell surface, which tended to mask specific losses of fluorescence. Fluorescence microscopy showed that round cells stained intensely for vinculin, but not for tubulin and that vinculin redistributed into coarse patches between 60 and 90 min, times which corresponded to small rebounds of fluorescence. With gated analysis, to exclude severely contracted round and squared cells, and with BDM inhibition of contracture, both metabolically inhibited and ischemic pelleted myocytes showed an early decrease in specific immunofluorescence staining for tubulin and vinculin, which preceded loss of cell viability, as determined by trypan blue staining. In both ischemic and metabolically inhibited cells, decreases of vinculin fluorescence preceded or coincided with increasing osmotic fragility. It is concluded that early cytoskeletal alterations of vinculin in ischemic and anoxic injury correlate with the development of osmotic fragility and irreversible myocyte injury.

Effects of calcium depletion and loading on injury during metabolic inhibition of isolated adult rat myocytes

The hypothesis that calcium influxes from the extracellular space play an important role in the pathogenesis of irreversible anoxic injury was tested using isolated adult rat myocytes. Myocytes treated with 6 mM amytal and 3 mM iodoacetate and subsequently incubated in either calcium-containing (1.12 mM) or calcium-free media (with or without 1 mM EGTA) developed rigor contracture (cell squaring) and cell death (trypan blue permeability) at the same rate. The rates of cell death in both calcium-containing and calcium-free media were increased by incubation in hypotonic media even though the rates of contracture development remained unaltered. Cells developed osmotic fragility prior to membrane permeability increases. The calcium ionophore, A23187 (10 microM), induced rapid rounding of rod-shaped cells subjected only to mitochondrial inhibition in calcium containing media, confirming its ability to cause an increase in cellular permeability to calcium. However, A23187 did not alter the rates of cell death of totally metabolically inhibited myocytes in either calcium-containing or calcium-free media with EGTA. The results indicate that influxes of calcium are not necessary for the development of irreversible injury in metabolically inhibited, isolated myocytes.

Ischemic Preconditioning in a Model of Isolated Cardiomyocytes

An intact heart is an integrated complex of tissues including the cardiomyocyte and interstitial elements (e.g. the extracellular collagen matrix, fibroblasts, mast cells, lymphatics, blood vascular elements and nerves). The complexity of the intact heart often excludes the reliable experimental isolation of single variables which influence cardiomyocyte physiology. The need for a simpler model led to development of methods to isolate cardiomyocytes. Initial attempts produced viable cells from neonatal and fetal hearts, but adult preparations were intolerant of calcium. Powell and Twist (1) first isolated of pure preparations of calcium tolerant adult myocytes. Cardiomyocytes have morphologic, metabolic, electrical and mechanical properties similar to those established for myocytes in intact hearts (2). In addition to the study of normal physiological processes, isolated cardiomyocytes have contributed significantly to understanding of the oxygen paradox, ischemia and reperfusion injury and preconditioning. An understanding of how in vitro cardiomyocyte models contribute to our understanding of cardiac disease might be facilitated by a brief review of these entities.