Glenn A. Langer

The myocardial cell surface, its histochemistry, and the effect of sialic acid and calcium removal on its stucture and cellular ionic exchange.

The surface of neonatal rat cells in culture, neonatal rat hearts, and adult rabbit hearts have qualitatively similar responses to lanthanum, ruthenium red, and colloidal iron stains. All demonstrate a surface coat and external lamina with abundant negatively charged sites. Cells with intact surface structure do not permit entry of lanthanum (La) intracellularly. The surface of all the myocardial cells studied contained abundant sialic acid distributed in two distinct layers, one in the surface coat next to the lipid bilayer, the other in the external lamina at the interstitial interface. The removal of sialic acid from the cellular surface increases calcium (Ca) exchangeability 5to 6-fold. Its removal also permits La to enter the cell and displace more than 80% of cellular Ca. Despite these marked alterations in Ca and La permeability, sialic acid removal has no effect on potassium <K) permeability. This indicates that the integrity of surface coat is critical in the regulation of Ca (and La) exchange but that K* permeability is controlled at the bilayer region. Exposure of the cells to [Ca]0 = 5 /»M produces a change in the surface. A peeling of the external lamina from the surface coat occurs with separation of the two sialic acid layers and the formation of a fluid-filled bleb between them. We propose that Ca removal ruptures Ca carbohydrate couplings (e.g., fucose-Ca-fucose bridges) which may anchor the external lamina to the surface coat. The effect of Ca removal on ionic permeability is not specific. The cell demonstrates increased permeability to both La and K. This indicates that Ca depletion affects both the surface structure and the bilayer region.

Effects of strophanthidin upon contraction and ionic exchange in rabbit ventricular myocardium: Relation to control of active state

Abstract The mechanical responses (active and resting tension, d P d t , TPT) and ionic exchange characteristics (Ca, K, Na) which follow upon perfusion with cardiac glycoside were studied in the arterially perfused rabbit interventricular septum. The augmentation of tension by glycoside is associated with an increase of maximum d P d t and an unchanged or decreased TPT. This inotropic response is accompanied by a net uptake of cellular Ca, a net loss of K and a net uptake of Na. The appearance of toxicity (ectopic beating and contracture) correlates with further net changes in the same direction. The component of Na and K active transport which is affected by glycoside is coupled at a ratio of approximately 2.5 Na : 1 K. Glycoside results in the retention of a fraction of Na influx in a very slowly exchangeable, kinetically inhomogeneous, cellular “compartment”. No significant inotropic response can be measured without evidence for inhibition of Na transport. The study is consistent with the hypothesis that inhibition of active cellular Na transport results in an increased Ca uptake. The latter is the basis for the augmentation of the rate of development of active state typical of glycosidic action. It is proposed that the ionic mechanisms associated with the inotropic response which follows increased frequency of contraction (Bowditch Staircase) and glycoside administration are essentially the same.

Correlation of the glycoside response, the force staircase, and the action potential configuration in the neonatal rat heart.

The rat heart demonstrates marked alterations in its responses to ouabain and increased frequencies of stimulation and in the duration of its action potential during the initial 21 days of life. At an age of 6.2 days 5 × 10−5m ouabain produced a 158.2% increase in dP/dt compared with a 17.2% increase at 21.1 days (P < 0.001). At 6.2 days dP/dt increased by 53.4% when the heart rate was accelerated from 30 to 90 beats/min compared with an increase of 12.2% at 21.1 days (P ≪ 0.005). The positive glycoside and staircase responses at the younger age were virtually eliminated when the hearts were perfused with a solution containing 50% [Na+]o and 25% [Ca2+]o ([Ca2+]o/[Na+]02 maintained constant). The duration of the ventricular action potential progressively decreased from 350–400 msec at birth to 100–150 msec at 21 days of life. This decrease was due to a shortening and a decrease in the potential level of the plateau phase. The prominent plateau typical of the early neonatal period was significantly diminished by perfusion with 50% [Na+]o. The results suggest that Na+ flux through a slow membrane channel plays a significant role in the positive staircase and glycoside responses of the early neonatal rat heart. As the heart matures and becomes functionally anomalous relative to other mammalian species, the slow channel progressively closes.

Role of Ca2+ in maintenance of rabbit myocardial cell membrane structural and functional integrity.

Abstract The structure and function of the sarcolemmas superficial layers and the sarcolemmas dependence upon [Ca] 0 was studied in the isolated arterially perfused rabbit interventricular septum. Perfusion of tissue for 20 min with 0 Ca ( m ) solution produced a distinctive separation of membrane superficial laminae. These separations were found to be irreversible and associated with an increased cellular 45 Ca content and irreversible contracture when normal [Ca] 0 was reperfused. Perfusion with 50 μ m Ca produced no alteration of membrane structure, contractile function or 45 Ca uptake. 0 Ca perfusion resulted in a 14% mean cellular 42 K loss and this appeared to be independent of superficial membrane separations. Mg supplements in 0 Ca perfusate resulted in membrane separations and contracture intermediate to those of 0 Ca without supplements and 50 μ m Ca; Mg supplements completely protected against cellular K loss. Zero [Ca]-induced separation of superficial laminae correlates with increased Ca permeability and suggests that this membrane region is involved in the control of contractile Ca. Ca ion is also essential for control of K permeability, probably in the lipid bilayer. The importance of the presence of [Ca] 0 to the normal structure and function of these membrane sites is emphasized.

Calcium depletion in rabbit myocardium. Calcium paradox protection by hypothermia and cation substitution.

The purpose of this study was to define further the basis of control of myocardial membrane permeability by further examination of the “calcium paradox.” To this end, the protective effect of hypothermia and addition of micromolar amounts of divalent cations during the Ca-free perfusion period were studied. Damage during Ca++ repletion to the isolated arterially perfused, interventricular rabbit septum was assessed by contracture development, loss of developed tension, and loss of 42K and creatine kinase. Progressive hypothermia prolongs the time of Ca-free perfusion needed to cause similar 42K, creatine kinase and developed tension losses upon Ca++ repletion. Complete protection against the Ca-paradox after 30–60 minutes Ca-free perfusion is seen at 18°C. The inclusion of 50 JIIM Ca++ during 30 minutes “Ca-free” perfusion also provides complete protection during Ca++ repletion, i.e., there was full mechanical recovery with no 42K or creatine kinase loss. Other divalent cations perfused in 50 JUM concentrations during the Ca-free period exhibited variable ability to protect when Ca++ was reperfused. The order of effectiveness (Ca++ > Cd++ > Mn++ > Co++ > Mg++) was related to the crystal ionic radius, with those cations whose radii are closest to that of Ca++ (0.99 A) exerting the greatest protective effect. The cation sequence for effectiveness in Ca-paradox protection is the same sequence for potency of excitation-contraction uncoupling. The mechanism of hypothermic protection is likely a phase transition in the membrane lipids (from a more liquid to a less liquid state) which stabilizes membrane structure and preserves Ca++ permeability characteristics during the Ca-free period. The mechanism of protection via cation addition is perhaps a cations ability to substitute for Ca++ (dependent on unhydrated crystal ionic radius) at critical sarcolemmal binding sites to preserve control of Ca++ permability during the Cafree period.

Activation-dependent cumulative depletions of extracellular free calcium in guinea pig atrium measured with antipyrylazo III and tetramethylmurexide.

We have used a spectrophotometric method to monitor mean free extracellular calcium concentrations in isolated left atria of guinea pigs via extracellular application of the calcium-sensitive absorption dyes, antipyrylazo III and tetramethylmurexide. Exchange of extracellular free calcium with the bathing medium takes several minutes and closely parallels contractile response in this preparation. Under conditions favoring a rapid positive force staircase response during repetitive stimulation after a long rest period (2-10 minutes), cumulative depletions of extracellular calcium can clearly be differentiated from motion artifact due to muscle movement by multiple-wavelength spectrophotometry. Responses of similar magnitude and characteristics are obtained with both dyes employed. In the presence of 10(-7) M isoproterenol, the mean extracellular calcium concentration falls by at least 5% (0.25-0.8 mM total calcium concentration) in four beats at 0.5 Hz; extracellular calcium replenishes during rest with an apparent t1/2 of 25-60 seconds. A 10-minute pretreatment with 10(-8) M ryanodine greatly reduces the contraction force and motion artifact of the first beat after a rest period, whereby the magnitude of depletion response to one post-rest stimulation is increased 2- to 3-fold. With further ryanodine treatment, the magnitude of depletion responses remains stable, and the rate of calcium replenishment during rest increases many-fold. After ryanodine treatment and 10(-7) M isoproterenol, at least 10% of total dye accessible calcium (0.25-1.0 mM) is lost during two to five rapid stimulations, and returns to the extracellular space within 20 seconds of rest. Cumulative extracellular calcium depletion responses are strongly suppressed by 10(-6) M nifedipine. Cumulative depletion responses are also inhibited by 10 mM caffeine, whereby contraction and corresponding motion artifacts are increased at post-rest stimulation.

Sarcolemmal-bound calcium and contractility in the mammalian myocardium

Abstract Using the perfused rabbit interventricular septum we have studied the kinetics of changes in contractility when the calcium concentration of the perfusate is either raised or lowered. We find that the rate of decline of d T d t in low calcium is first-order ( t 1 2 ∼45 s ) and independent of initial perfusate calcium levels. The redevelopment of d T d t when the calcium level is raised is 3 to 5 times faster. Lanthanum inhibits contraction with a t 1 2 of about 6 s. Analysis of these kinetics in the light of morphological considerations leads us to conclude that there are specific sarcolemmal calcium receptors which are rapidly accessible ( t 1 2 ∼6 s ) to the perfusate and which control the kinetics of changes in contractility. Based on this model we propose that the contractile state of the septum is proportional to the fraction of these receptors which are occupied. Although one type of receptor appears to dominate, the presence of a second receptor is indicated and we are able to estimate the association binding constant of each.

The effect of calcium and high-energy phosphate compounds on myocardial contracture in the newborn and adult rabbit.

Abstract Anoxic myocardial contracture was studied in the arterially perfused septal preparation of the newborn and adult rabbit. Myocardial energy requirement was varied by changing the pacing rate, and myocardial energy supplies were varied by altering the perfusion rate or adding iodoacetate to the perfusate. Myocardial contracture began at an ATP concentration of 16μmol/g dry weight. Full myocardial contracture was observed at a higher ATP concentration in the newborn as compared with the adult. In the absence of external calcium the onset of myocardial contracture was delayed and the degree of contracture was attenuated. At similar ATP concentrations myocardial contracture was significantly (P

ORGANIZATION AND FUNCTION OF SARCOLEMMAL PHOSPHOLIPIDS IN CONTROL AND ISCHEMIC/REPERFUSED CARDIOMYOCYTES

The topic of this review is the lipidic part of the sarcolemma, the plasma membrane of the myocardial cell, and its role in (dis)function of the cardiomyocyte. First the isolation of the sarcolemma and its lipid composition are discussed. These phospholipids are not randomly distributed over the two monolayers of the lipid bilayer and negatively charged phospholipids are exclusively present in the cytoplasmic leaflet of the sarcolemma, which also contains the majority of phosphatidylethanolamine. This distribution is most likely caused by an active transport of these lipids and by an interaction of the headgroup of these lipids with the cytoskeleton. Subsequently the physicochemical properties of sarcolemmal phospholipids are discussed, where it is shown that certain phospholipids prefer non-bilayer phases, and the effects of sarcolemmal phospholipids on trans-sarcolemmal ion fluxes and calcium compartmentation are discussed. In the second part the effect of ischemia on sarcolemmal phospholipids is discussed with regard to: transbilayer distribution, hydrolysis, lateral distribution and sarcolemmal bilayer stability. In our view, onset of ischemia initiates a sequence of events leading to a loss of normal sarcolemmal phospholipid distribution with an outward migration of phosphatidylethanolamine. There follows, as ischemia progresses, loss of sarcolemmal bilayer stability due to the expression of the non-bilayer behavior of phosphatidylethanolamine, leading to irreversible disruption of the sarcolemma and cell death.

Caffeine effects upon contraction and calcium exchange in rabbit myocardium

Abstract Caffeine was employed to study the movements of contractile calcium in the isolated blood-perfused rabbit interventricular septum. Caffeine (1 to 20 m m ) induced a rise in resting tension with four functional concomitants: an initial transient positive inotropy, followed by a transient decrease in tension and d P d t , a second positive inotropic interval and then progressive contracture. The second inotropic interval was characterized by a striking decline in the rate of relaxation while the rate of tension development climbed. These effects were associated with a not increase in tissue 45Ca, and with a decrease in 45Ca efflux which was rapidly reversible upon removal of caffeine. Caffeine did not affect mechanical function after exposure to zero calcium. Caffeine (20 m m ) significantly shortened time to peak tension and the action potential. This concentration caused vacuolation and “rounding up” of the mitochondria but had no other obvious morphologic effect. Caffeine primarily inhibits calcium sequestration. Its action suggests that a portion of calcium influx associated with contraction coupling will accumulate within the cell unless a specific effluent pathway remains open.