Herbert A. Berkoff

Postoperative stroke in cardiac and peripheral vascular disease.

The postoperative stroke rate in 330 patients requiring coronary artery (170) or peripheral vascular (160) surgery was compared with the presence of carotid bruits and the results of noninvasive screening (Doppler imaging and spectral analysis of flow) to determine prevalence and significance of carotid lesions and their relationship to perioperative stroke. Carotid lesions were suspected because of bruits in 70 patients with peripheral vascular disease (PVD) and in 28 patients with coronary artery disease (CAD). Noninvasive tests showed high grade stenosis or occlusion in 62 patients with PVD and in 14 with CAD. Forty-four patients with PVD and 101 patients with CAD had normal Doppler studies. The rest in both groups had plaquing without major stenosis. Noninvasive tests uncovered severe, occult lesions in only 13 patients (9 PVD, 4 CAD). Postoperative neurologic complications occurred in 16 patients (13 strokes: 5 PVD, 8 CAD and 3 TIAs: 2 PVD, 1 CAD). Thirteen neurologic complications occurred in patients having nonstenotic plaques or normal carotids without bruits. Only three of the strokes and 1 TIA occurred in patients with bruits and detectable carotid stenosis. Few of the postoperative strokes or TIAs were focal (2 PVD, 1 CAD), and the rest were nonfocal. None of the postoperative strokes or TIAs were associated with postoperative carotid occlusion. Physical examination is not an accurate method of determining severity of carotid disease. Severe carotid stenosis is more common in PVD patients than in CAD patients, but there is no significant difference in postoperative stroke rate. No direct relationship has been found between a bruit, severity of disease, and incidence of perioperative stroke.

Contracture in isolated adult rat heart cells. Role of Ca2+, ATP, and compartmentation.

Isolated intact quiescent myocytes from the adult rat were used as a model system for investigating the determinants of contracture induced by metabolic deprivation. The model simulated the pattern of contracture and ATP decline seen in the intact heart during Ischemia. Three new insights into the contracture process were gained: (1) in the quiescent cell system, the rate of onset of contracture was independent of external Cn2+, supporting the view that the Ca2+ dependence of the rate of onset in the whole heart is related to beat-dependent substrate utilization; (2) the second phase of ATP decline was paralleled by a decline In the percentage of cells which had not undergone contracture, suggesting that–in any cell–contracture is immediately preceded by a total loss of ATP; and (3) oligomycin delayed the onset of contracture by 55 ± 12%, suggesting that mitochondria ATPase activity ia a significant drain on energy resources in the quiescent ischemic heart.

The isolation of Ca2+-resistant myocytes from the adult rat.

Abstract A method is described for the preparation of Ca2+-resistant myocytes from the adult rat. Ca2+-resistant cells were obtained in yields of 6.3 ± 1.5 × 106 cells/gm wet weight tissue, with a purity of 67.5 ± 8.8% cells excluding trypan blue. Of the cells which excluded trypan blue, over 90% were rod-shaped. The rodshaped cells when isolated were quiescent, but beat in response to electric field stimulation in the presence of Ca2+. Incubation of the cells at 37° in the presence of 2 m m Ca2+ caused only a slow decline in the percentage of cells able to exclude trypan blue or to beat when stimulated. The rate of decline was comparable to that of cells incubated without Ca2+. Incubation of the cells in the presence of EGTA (ethylene glycol bis (β-aminoethyl ether)-N,N′-tetraacetic acid) for 30 min had little effect on the ability of the cells to withstand Ca2+. The key step in preparing Ca2+ resistant cells was found to be the inclusion of trypsin and Ca2+ at the final stage of incubation of tissue pieces. The trypsin appears to act not only by selective removal of cells susceptible to Ca2+ but also by conferring Ca2+ resistance on cells which otherwise would be Ca2+ susceptible. The properties displayed by this preparation indicate that the cells are both mechanically and electrochemically intact. They therefore provide an excellent model system for the study of excitation-contraction coupling at the cellular level, and also provide a more realistic starting point for studies of pathology than previous Ca2+-susceptible preparations.

Cellular manganese uptake by the isolated perfused rat heart: a probe for the sarcolemma calcium channel ☆

Abstract The slow inward Ca channel with isolated working rat hearts was assayed by measuring the cellular uptake rate of 5 μ m 54Mn from the perfusate. This new method is successful because Mn taken up by the cell is not readily released and remains in the cell even after all the extracellular 54Mn is washed away. Reagents which have been shown to affect the Ca channel of isolated heart muscle using a voltageclamp technique, also affect similarly the rate of Mn uptake by the rat heart: Verapamil (2 μ m ) inhibited Mn uptake 73%, oligomycin (2 μ m ) inhibited 55% and isoproterenol (2 μ m ) stimulated 80%. In addition Mn uptake appeared to be inhibited by perfusate Ca because a drop of perfusate Ca from 2.5 to 0.2 m m resulted in a 137% stimulation of Mn uptake. The uptake of cellular Mn by mitochondria was also investigated. The uptake was found to be stimulated by over 300% when the activity of the heart was increased by either raising the level of perfusate Ca or by adding isoproterenol. It is possible that changes in the concentration of cellular Ca promoted the observed stimulation of mitochondrial Mn uptake by a mechanism of mitochondrial CaMn co-transport.

Inhibition of calcium influx in isolated adult rat heart cells by ATP depletion.

Using 45Ca, indo1, and quin2, calcium uptake was measured in isolated quiescent adult rat heart cells under different metabolic conditions. Exposure of cells in a medium containing 1 mM CaCl2 to rotenone and uncoupler resulted in adenosine triphosphate (ATP) depletion from 17.08 +/- 2.26 to 0.63 +/- 0.11 nmol/mg within 8 minutes, and the cells went into contracture. In this time, the cells lost 1.65 +/- 0.1 nmol Ca/mg of total rapidly exchangeable cellular calcium, and the level of free cytosolic calcium as measured by indo1 rose from 47.4 +/- 16.3 nM to 79.8 +/- 27.6 nM. The subsequent rate of rise of intracellular free calcium concentration was just 4 nM/min for at least 40 minutes. Therefore, we investigated the effect of ATP depletion on the rate of calcium entry. In cells loaded with sodium by ouabain treatment without calcium, the initial rate of calcium influx on calcium addition was inhibited by 82-84% when cellular ATP was depleted, as measured by 45Ca or indo1. Quin2 also showed a strong inhibition of calcium influx by ATP depletion, but itself also caused a strong inhibition of calcium influx. The rate of calcium influx declined even further in ATP-depleted cells after the initial influx: Between 1 and 12 minutes after calcium addition, the residual 45Ca uptake rate of the first minute was inhibited by an additional 90%. We conclude that ATP depletion per se does not quickly elevate cytoplasmic free calcium and that such an elevation is prevented by a very strong inhibition of the rate of calcium entry.

Inhibition of ATP-sensitive potassium channels of adult rat heart cells by antiarrhythmic drugs.

We have investigated the effect of antiarrhythmic drugs on the increased potassium conductance induced in isolated adult rat heart cells by ATP depletion. The rate of 86Rb uptake in the presence of ouabain was used as a measure of potassium conductance. Treatment of cells with rotenone plus p-trinuoromethoxyphenylhydrazone (FCCP) rapidly depleted ATP levels and strongly stimulated the rate of 86Rb uptake. The stimulated uptake and the ATP depletion were inhibited by oligomycin; thus, the uptake was not induced by roteuone plus FCCP directly. The stimulated uptake, but not the ATP depletion, was inhibited potently by glyburide (ICJO, 38.3 nM), quinidine (IC50, 2.7 μM), verapamil (IC50, 4.5 μM), and amiodarone (IC50, 19.1 μM). The stimulated uptake was also inhibited by tetraethylammonium ion and by 4-aminopyridine but not by tetrodotoxiii or manganese. We conclude that 1) the stimulated 86Rb uptake is measuring ATP-sensitive potassium channel activity, 2) the ATP-sensitive potassium channel is strongly inhibited by quinidine, verapamil, and amiodarone, and 3) this inhibition may contribute to the antiarrhythmic action of these drugs.

Na+ releases Ca2+ from liver, kidney and lung mitochondria.

Intracelhrlar CaZ’ has assumed increasing importance as a regulator of a variety of cellular processes, including muscle contraction, secretion and cell division [l]. A prime candidate for a controller of intracellular Cazf levels, by virtue of its high affinity Ca2* uptake mechanism, is the mitochondrion. Such a role for the mitochondrion has gamed support from the demonstration that mitochondri~ Ca*” release is under separate control from uptake [2]. Two systems capable of releasing mitochondria Ca”’ are the Na+induced release system [3] and the Ca2+ -induced transition in permeability of the inner membrane 243. In elucidating the role of these release systems in Ca’* homeostasis it is important to know the extent of their occurence in mitochondria from different tissues. Such a study for the Na+ system has concluded that this system was most active in brain, heart and adrenal cortex, present in striated muscle, but absent in kidney, liver, lung, smooth muscie and hepatoma [5]. We report that the Na+ system is indeed present in mitochondria from liver, kidney and lung. We have not studied smooth muscle or hepatoma.

Modulation of cellular calcium stores in the perfused rat heart by isoproterenol and ryanodine.

The inhibitory action of procaine on cellular calcium release was utilized to define a new cellular calcium pool which, under physiological conditions, is present only during catecholamine stimulation. Rat hearts labeled with 45Ca++ were perfused with medium containing procaine and EGTA at 23 degrees C to remove extracellular calcium, and then cellular calcium was released by removal of procaine and restoration of calcium. By this method we have identified a cellular calcium pool (pool C) whose release is inhibited by procaine, but which does not require extracellular calcium for its release. Release of pool C can also be triggered by caffeine. [We have previously identified a cellular calcium pool (pool A) whose release is triggered by caffeine, inhibited by procaine, and which does require extracellular calcium for its release.] When hearts were labeled for 3 minutes with perfusate containing 1 mM 45Ca++, 48 +/- 6 nmol Ca++/g wet weight was found in pool A, but only 3 +/- 1 nmol Ca++/g in pool C. However, if isoproterenol was present during labeling, the hearts contained 72 +/- 5 nmol Ca++/g in pool A and 42 +/- 6 nmol Ca++/g in pool C. When calcium concentration in the labeling perfusate was varied, with and without isoproterenol, it was found that pool C does not begin to fill until pool A is almost full. The same effect was seen when excess cellular calcium uptake was induced by removing sodium from the perfusate. Ryanodine (0.2 microM) induced contractile failure (t1/2 = 3.4 +/- 0.4 min) and depleted pool A in control hearts by 85%. Ryanodine also similarly depleted pools A and C in isoproterenol-treated hearts. When contractility was monitored at the same time as the hearts were labeled, a linear relationship between dP/dt and the sum of pools A and C was observed over a wide range of conditions. Pools A and C both selected strongly for calcium over barium. These observations suggest that both pools A and C are located in the sarcoplasmic reticulum and are intimately involved in the regulation of contractility.

Comparison of Ca2+, Sr2+, and Mn2+ fluxes in mitochondria of the perfused rat heart.

The amount of readily exchangeable Ca2+ in mitochondria of an isolated working rat heart is less than 10 ng-ions/g heart. We therefore conclude that either no Ca2+ enters mitochondria or that the Ca+ which does enter is removed continuously. Using Sr2+ and Mn2+, we obtained evidence that the mitochondrial Na+-Ca2+ exchanger was indeed operational in releasing metal from mitochondria of the heart. When Ca2+ in the perfusate was replaced by Sr2+, we found that a significant amount of Sr2+ (approximately 100 ng-ions/g heart) entered mitochondria. When the heart then was returned to a Ca2+-containing perfusate, over 80% of the Sr2+ was washed out of mitochondria within 30 seconds. When low levels of Mn2+ were added to the perfusate, we found that Mn2+ accumulated in mitochondria irreversibly. This is evidence for the operation of the Na+-Ca2+ exchanger because Na+ was found to release Ca2+ and Sr2+ but not Mn2+ from isolated rat heart mitochondria. Our estimates indicate that when the Na+-Ca2+ exchanger is maximally operative, as in the Sr2+-perfused heart, the flux of Sr2+ through mitochondria is at most 10% of the total flux needed for the activation of contraction. The low level of Ca2+ in the mitochondria of Ca2+-perfused hearts suggests a much smaller flux of Ca2+ through the mitochondria in this case. We therefore conclude that mitochondria play little if any role in the beat-to-beat regulation of normal Ca2+ fluxes in the rat heart.

Metabolic cost of the stimulated beating of isolated adult rat heart cells in suspension.

Heart cells from adult rats were induced to beat in suspension by electric field stimulation. We have gained evidence that all the rod-shaped cells in suspension were indeed beating, and that the beat had dynamic characteristics similar to those of intact heart muscle contracting under zero load. The cells were undamaged in the process, as judged by maintenance of ATP levels, morphology, and ability to beat. In gaining such evidence, we also measured the metabolic cost to the cells of beating under zero load. In cells with oxidative phosphorylation inhibited by rotenone plus oligomycin (termed anaerobic), the rate of beat-dependent lactate production suggested an equivalent rate of ATP utilization of 0.126 ± 0.013 nmol ATP/beat per mg protein (plus isoproterenol), and 0.058 ± 0.005 nmol ATP/beat per mg protein (minus isoproterenol). In respiring cells, the rate of beat-dependent oligomycin-sensitive oxygen consumption gave an equivalent rate of ATP utilization of 0.198 ± 0.009 nmol ATP/beat per mg protein (plus isoproter-enol), and 0.126 ± 0.013 nmol ATP/beat per mg protein (minus isoproterenol). The cells beat with the same approximate maximum velocity whether isoproterenol was present or not. We calculate that-in the case of anaerobic cells without isoproterenol-this rate of ATP utilization can account for only about a 15% degree of activation of the contractile proteins. In addition, we have found an oligomycin-insensitive beat-dependent mitochondrial respiration of 0.023 ± 0.006 nanoatom O/beat per mg. The cause of this respiration is not known. The total rate of oxygen consumption of cells and also the rate per beat was comparable to that measured in nonworking whole hearts.