,J Mol Cell Cardiol

Effects

23, 175-185

(1991)

of Neuraminidase on Cellular Cultured Cardiac Hal

F. Yee, Jr*,

John

H. Kuwata~

Calcium Myocytes and

Glenn

and Contraction

in

A. Langer$

Cardiovascular Research Laboratories, Departments of Medicine and Physiolou. University of California, Los Angeles School of Medicine, Center for the Health Sciences, Lo.7 Angeles, CA 90024, USA (Received 29 June 1990, accepted in revised jbrm

17 September 1990)

H. F. YEE, J. H. KUWATA AND G. A. LANCER. Effects of Neuraminidase on Cellular Calcium and Contraction in Cultured Cardiac Myocytes. 3oumal of Molecular and Cellular Cur&logy (1991) 23, 175-185. Mammalian plasma membranes, including the myocardial sarcolemma, are abundantly glycosylated. Sialic arid is a ubiquitous anionic sugar found at the periphery of sarcolemmal glycoconjugates. The physiological role of this sugar is not clear. but neuraminidase, which specifically hydrolyzes sialic acid from the sarcolemma, has been found to increase calcium exchange, cause electrophysiological abnormalities, and enhance the transient ‘1’ calcium current in cardiac myocytes. The purpose of this study was to better characterize the ctlecr 01 neuraminidase on cellular calcium (Ca) and contractile function. Neuraminidase removed up to 57”,, of total sialic acid from the cells. 45Ca exchange was measured and neuraminidase was found to increase cell calcium proportional to the amount of sialic acid removed (186 + 0.8 mmol/kg dry w, maximallyj. Over 80”,, of the increment in calcium remained rapidly exchangeable was inhibited by cations (La > Cd > Mn > Mg) and

(t$ < 15 s) under

non-perfusion

limited

conditions

and

nifedipine. Using a video-monitoring system, neuraminidase was observed to transiently increase cell shortening during contraction (30 _+ 9%). with progression to arrhythmias followed by cessation of contraction. These results indicate that neuraminidase. probably by removing sarcolemmal sialic acid residues, greatly augments cellular calcium in cultured cardiac myoryttas. .Most of the increment in Ca induced by neuraminidase was very rapidly exchangeable and most like])mediated by a Ca specific mechanism. Additionally, neuraminidase treatment altered contract& function in a manner consistent with elevated cellular Ca. Despite the many-fold increase in cellular Ca induced by sialic .icid removal. cells recovered and demonstrated rhythmic contractions upon return to control incubation conditions. KEY WORDS: Sialic acid; Neuraminidase; Glycocalyx; calcium exchange: Myocardial contraction.

Introduction The regulation of free ionized calcium (Ca) movement between the extracellular and intracellular compartments in the heart is critically important for myocardial viability and contractile function, and is tightly regulated by the sarcolemma. The sarcolemma is composed of glycosylated and nonglycosylated proteins and cholesterol embedded in a fluid bilayer of’ phospholipids and glycolipids [S. 401. The significance of sarcolemmal carbohydrate is not clear. The abundance. complexity and conserved nature of these sugar groups suggest important and general

Cell calcium;

Myocardial

tissue culture:

Myocardial

cellular roles. Sialic acid (&-acetyl neuraminit acid and its derivatives), an anionic I pk‘ 2.7) sugar residue found at the most peripheral positions of membrane glycoconjugates is responsible for a portion of the negative cell surface charge 18, 391. It has been demonstrated that treatment of cardiac myocytes with neuraminidase which specifically hydrosarcolemmal surface sialic acid, lyzes increases Ca exchange in cultured cardiac, myocytes [13, 23, 24, 321 led to electrophysirulogical abnormalities in cultured and acute]! isolated cardiac myocytes [4, 311 and selectively enhanced current through the ‘I‘ cypc

Present addresses: *Department of Medicine, UCSF School of Medicine, San Francisco. CA 94143. Johns Heart Institute and UCLA/Harbor Medical Center, Torrance, CA. USA. f Please address all correspondence to: Glenn A. Langer. Cardiovasculaer Research Laboratory. I.‘CLA School of Medicine, 10833 LeConte Avenue, Los Angeles, CA 90021. USA. Otr22-2828191

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H. F. Yee et al.

176

Ca channel in guinea-pig ventriculocytes [451. These findings suggest a role for sarcolemma1surface sialic acid in the control of Ca exchange and cellular function in the heart. This study further characterizes the role that sialic acid plays in regulating cellular Ca and myocardial contractile function. We report that neuraminidase, probably through its removal of sarcolemmal sialic acid, selectively augmented cellular Ca. This increment in Ca was very large and very rapidly exchangeable. In addition, neuraminidase altered contractile function in a manner consistent with changes in cellular Ca regulation.

acid content was determined by the thiobarbituric acid assay which corrects for 2deoxyribose, the major contaminating compound [44]. Sialic acid removal

Sialic acid was removed from the sarcolemma1 surface of cardiac myocytes by exposure to neuraminidase. Neuraminidase was prepared from Clostridium perfringens using afinity chromatography [17] and obtained in a lyophilized form (Worthington Biochemicals) and stored frozen until used ( t3 months). This preparation of neuraminidase had no detectable protease activity but did contain small amounts of phospholipase C, c. 0.01 U Methods phospholipase C per 1.0 U neuraminidase [I, 241. When needed the enzyme was disCardiac cell culture solved in physiological buffer, stored at 3°C Neonatal rat cardiac myocyte cultures were and used within 60 h. Cultured cells were prepared using a standard method [15] modi- incubated at room temperature (23 to 25°C) fied as described previously [Zq. Cultures with various concentrations of neuraminidase were grown in confluent monolayers on (0.0, 0.06, 0.12, 0.25, 0.37 U/ml physiological either Primaria petri dishes (Falcon 3802) or buffer; one unit of enzyme releases1.0 pmol unmodified petri dishes containing special of sialic acidlmin from bovine submaxillary discs composed of scintillator material with mucin at 37°C and pH 5.0). Myocytes were Primaria coating. The Primaria coating also treated with neuraminidase in Laplaces a positive charge on the plastic which containing (0.5 mM) or nominally Mg-free enablesmyocytes to adhere while minimizing solutions. Ca binding to the plastic. Dishes were allowed to incubate at 37°C for 3 to 5 days Exogenous sialic acid until utilized for experiments. Addition of cytosine arabinoside to the culture medium to Cells were incubated in physiological buffer containing exogenous sialic acid (N-acetyl inhibit fibroblast growth produced virtually neuraminic acid 0.35 mM) for 30 min at 23 to pure cardiac myocyte cultures. 25°C. The incubation buffer was exchanged every 10 min during this intervention. Biochemical

analysis

Cardiac myocyte cultures were analyzed after 3 to 5 days of incubation. Cultured cells were washedwith 5 ml of physiological buffer (mM: NaCl 133, KC1 3.6, JV-Z-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) 3.0, glucose 16.0, CaCl, 1.0, MgCl, 0.3, pH 7.3) three times. Fluid was decanted and the cellular material was scraped off the dish or disc with a rubber scraper. Cellular material was suspended in physiological buffer and frozen in liquid nitrogen. Vials of cellular material were stored frozen until analyzed ( < 2 weeks). Protein content was determined using the folin phenol method 1301. Sialic

Analysis of contractile function

Myocytes were visualized using an inverted Nikon microscope ( x 40)-video camera system attached to a television monitor [21]. A photocell mounted on the monitor allowed cell motion to be recorded. Contractile function as measured by magnitude of cell shortening was studied in electricalIy paced myocytes before, during and after treatment with neuraminidase (0.25 U/ml). ion exchange experiments

unique on-line method for monitoring cellular Ca instantaneously and continuously

A

Calcium

and Contraction

was utilized [13, 20, 231.Briefly cardiac myocytes were cultured on scintillator discs. Insertion of the flow cell, of which the discs formed the walls, into a spectrophotometer allowed continuous measurement of /?emission if radioisotope was present. 45Ca was used as a tracer for monitoring cellassociatedCa. Thus, myocytes could be perfused and treated within the flow cell with continuous measurement of cell-associated Ca. All phasesof theseexperiments were conducted at pH 7.3 and 23 to 25°C unless otherwise noted. Uptake experiments

Discs within the flow cell were perfused with physiological buffer for 10 min at 10 ml/min, then erfused with radiolabelled solution (c. 1 ,uCi ‘5Cajml buffer) at 10 ml/min until a steady-state of counts per minute was obtained (uptake). This was followed by perfusion of the flow cell with non-radioisotope labelled buffer for 30 to 40 min at 26 ml/min (washout). Cells were then exposed to various controls and interventions (e.g. treatment with neuraminidase). The discs were then subjected to 45Ca uptake and washout again. Figure 2 demonstrates such a experiment. At the end of the second washout, the cells were scraped from the discs onto a pre-weighed dried piece of filter paper. The cells were dried at 1OO“Cfor > 24 h. The cells and filter paper were then weighed and the cellular dry weight was determined. Aliquots of the 4sCalabelled solution were taken, weighed, suspended in scintillation fluid, and counted in the /I-scintillation counter. Specific activity of the 45Ca-labelled solution was then determined. The scraped discswere washed in 1.0 N HCl to remove bound 45Ca and subjected to an uptake and washout to obtain steadystate 45Ca activity in the absenceof cells; this was termed the blank. The interventions studied included: (1) neuraminidase treatment (0.0, 0.06, 0.12, 0.25, 0.37 U/ml for 30 min) and (2) exogenoussialic acid exposure (0.35 mM JV-acetyl neuraminic acid for 30 min). In addition, the effects of various agents on the responseof cell associatedCa to neuraminidase treatment (0.25 U/ml) were evaluated. In these experiments nifedipine (0.01, 0.001 mM), LaCI,, CdCI,, MnCI,, MgCl,

atIer

Neuraminidase

177

(all 0.5 mM), or no blocking agent, was present throughout the experiment. Experiments with nifedipine, which is photosensitive, were carried out in the dark.

Washout experiments

Very rapid washout experiments were carried out [20]. In these experiments, cultured myocytes in the flow cell were treated with neuraminidase (0.25 U/ml for 30 minj or not treated, and then ex osed for 20 min to 45Ca solution (c. 30 &i 4PCalm1 buffer). This was followed by perfusion of the flow cell with physiological buffer (no 45Ca) at a flow rate of 500 ml/min. This flow rate is well tolerated by the cells and approaches the level at which 4 Ca exchange is not perfusion-limited. Due to the very high flow and pressure(c. 9 p.s.i.) these experiments were not paired. This is in contrast to the uptake experiments where cells served as their own controls. The effects of sialic acid removal on potassium (K) exchange were also studied using washout experiments. Due to the high emission energy of 42K (3.52 meV), #?-emission from 42K in the bulk fluid was not quenched (maximum range 2000 pm) so that background emission from radioisotope labelled perfusate during an uptake obscuresthe cellassociated fl-emission. Thus, only washout data was meaningful. Discs were placed in the flow cell and equilibrated for 10 min with physiological buffer. The flow cell was then incubated for 1 h with 42K solution (0.3 to 0.6 ,uCi 42K/ml buffer). This solution was exchanged every 15 min. The flow cell was then placed in the spectrophotometer and the 42K washout pattern establishedby perfusion with non-isotopic buffer solution for 20 min at 26 ml/min. The washout was then interrupted by neuraminidase treatment (0.25 U/ml) for 30 min. Washout was then reinitiated and recorded for 20 min in order to ascertain the effect of neuraminidase treatment on 42K exchange.

Statistical analysis

Exchange data were analyzed as has been previously described [13, 20, 231. Results are reported as mean ) S.E.M. Student’s paired

Ii. F. Yee et al.

178 70

45Ca exchange

,

+

+

n=4

n=6 0

I I 0. I 0.2 [Neuraminidase]

n=3

I 0.3 U/ml

1 0.4

45Ca uptake experiments were carried out in order to examine the effect of neuraminidase on steady-state, cell-associated Ca. In these experiments on cultured rat cardiac myocytes, cell-associated Ca was determined before and after treatment in the same cardiac myocytes, so that the cells served as their own control group. Initial control experiments revealed that cell-associated Ca was not different after a sham treatment (exposure to physiological buffer for 30 min). The change in cell-associatedCa after sham treatment was 0.09 f 0.07 mmol Ca/kg dry wt (n = 4).

Neuraminidase (0.25 U/ml, 30 min) treatment of cultured rat cardiac myocytes led to a large steady-state increase (18.6 + 0.8 mmol Ca/kg dry wt, R = 12) in cell-associated Ca (Fig. 2). Increases in cellassociated Ca were determined after treatment with different concentrations of neuraminidase, and these increases were directly and unpaired t-tests were used to determine proportional to the amount of sialic acid statistical significance. The level of signifi- removed from the cardiac myocytes (see cance was defined as P < O-05. In experi- Fig. 3). ments utilizing the microscope-video system, Rapid washout experiments (Fig. 4) were n refers to the number of cells; in experiments done to investigate the compartmentation of utilizing ion exchange method or biochemical the increment in cell-associatedCa related to assays,n refers to the number of batches of neuraminidase treatment [20]. Cultured rat cells. cardiac myocytes were treated with neuraminidase (0.25 U/ml for 30 min) or not treated, then exposed to 45Ca solution for 20 min, by Results which time 45Ca exchange had reached a Biochemistry steady-state. The cells were then perfused The sialic acid content of the cultured myo- with physiological buffer (no 45Ca) at a rate cytes was determined to be 61 + 5.1 nmol of 500 ml/min (Fig. 4). The washout curve sialic acid/mg protein (n = 6). The cells were for the increment in cellular Ca associated exposed to various concentrations of neura- with neuraminidase treatment (the difference minidase (0.06, 0.12, 0.25, 0.37 U/ml) for 30 between the average of the neuraminidase min at 23 to 25°C and pH 7.3, and their treated and untreated curves in Fig. 4) is sialic acid contents were assayed (Fig. 1). presented in Figure 5. The increment in cellIncreasing concentrations of neuraminidase associated Ca following neuraminidase was removed an increasing proportion of the total rapidly exchangable. Note that half of the cellular sialic acid until a maximum of c. 57% neuraminidase associatedCa was washed out was removed (0.25 and 0.37 U/ml neurami- in Cd > Mn > Mg (Fig. nous with stimulation, and eventual cessation 6). La completely blocked the increase in cell of measurable cell shortening within 5 min. associatedCa following neuraminidase treat- Non-electrically paced, but spontaneously ment. The neuraminidase induced increment contracting myocytes similarly ceased conin cell-associated Ca was partially inhibited tracting within 5 min of exposure to 0.25 by nifedipine in a dose-related manner (Fig. U/ml neuraminidase (n = 20 dishes). When neuraminidase treated (0.25 U/ml for 10 6). Cell-associated Ca was not significantly altered by exposure of the cells to 0.35 mM min) cultured myocytes were washed and exogenous jV-acetyl neuraminic acid (change reincubated synchronous and spontaneous in cell-associated Ca was -0.50 + 0.58, rhythmic contractions returned within 8 h n = 2). (n = 4 dishes). 42K exchange

Discussion

In cultured cardiac myocytes, 42K exchange during washout experiments (n = 5) was monoexponential and rate constants were not significantly different before (0.021 f O.OOS/min) and after (0.023 f O.O04/min)

The plasma membranes of all mammalian cells, including cardiac myocytes, are heavily glycosylated. Virtually all integral membrane proteins and some of the bilayer lipid have carbohydrate bound extracellularly [5, 40/o].

16.0

*

0 x

g c s5

10.0

6.0 6.0

Time

FIGURE neuraminidase

~INeurominidose (min)

2. 45Ca exchange in a representative culture of cultured rat cardiac myocytes before and after treatment (0.25 U/ml for 30 min) indicated by the vertical arrow. See Methods for experiment details. Steady-state, cell-associated 45Ca activity prior to neuraminidase treatment was significantly greater than the blank steady-state value indicated by the horizontal arrow. Note the very large increase in steady-state, cell-associated 45Ca activity following neuraminidase treatment.

180

Ii. F. Yee et al.

f

! e 0

L 0

n=4

n=4 I IO

I I 20 30 % Sialic acid

I 40 released

I 50

1 60

FIGURE 3. The change in cell-associated Ca after removal of different amounts of sialic acid from cultured rat cardiac myocytes. The increase in cell-associated Ca was proportional to the amount of sialic acid removed from the cells.

Sialic acid, n-acetyl neuraminic acid and its derivatives, is an anionic sugar found in abundance at the periphery of membrane glycoconjugates [38]. It is responsible for a portion of the negative cell surface charge and is found on important integral membrane proteins, including ion channels and surface receptors [Z, 34, 431. The physiologic role of surface sugars, including sialic acid, is not completely clear. The location, abundance and negative charge of sialic acid suggest

Time

IO Time

(s)

FIGURE 5. Washout of the increment in calcium induced by neuraminidase treatment calculated as the difference between the average untreated and average neuraminidase-treated cell washout curves (see Fig. 4). Note that the bulk of the Ca was rapidly exchangeable with a fi of Cd > Mn > Mg. This sequencewas the same as that for displacing sarcolemmal bound Ca, blocking Ca channels and inhibiting the Na-Ca exchanger, evidence that the neuraminidase induced increment in cellular Ca was mediated by some Ca-specific mechanism. Furthermore, nifedipine, a Ca channel antagonist, reduced the increase in Ca induced by neuraminidase treatment by 20 to 30% in a dose-related manner. This is attributed to blockage of Ca flux through the “L” channel, unrelated to the action of neuraminidase. This would, however, diminish total flux (normal plus neuraminidase-activated), and thereby produce a modest inhibition as shown in Figure 6. Preliminary experiments

Calcium

aud Contraction

with relatively specific “T” channel blockade indicate an inhibition of greater than 90%, consistent with the demonstrated effect of neuraminidase on the “T” channel. These results support the belief that the neuraminidase-induced increase in cellular Ca was mediated by a Ca-specific mechanism and not related to nonspecific changes in sarcolemmal permeability. The specific effect of neuraminidase on Ca flux is also supported by the finding of Fermini and Nathan [rl] that its application to sinusnode cells did not affect inward rectifying current in these cells. The maintenance of selective sarcolemmal permeability may relate to the ability of the cell to survive such large Ca overload. Presumably neuraminidase treatment leads to an increase in the free sialic acid concentration in the vicinity of the sarcolemma. To test whether an increase in free sialic acid could explain the increase in cellular Ca, cultured cardiac myocytes were exposed to an amount of exogenous n-acetyl neuraminic acid greater than 10 times that which would be expected to be released from the sarcolemma by neuraminidase. Exogenous n-acetyl neuraminic acid had no effect on cellular Ca, again supporting the hypothesis that the effects of neuraminidase are related specifically to the removal of the sarcolemmal sialic acid. The effects of neuraminidase on contractile function were studied in cultured cardiac myocytes. Spontaneously contracting cultured rat cardiac myocytes ceased contracting within minutes of treatment with neuraminidase. When cultured cardiac myocytes were electrically paced they exhibited transient increases in cell shortening during exposure to neuraminidase, followed by a decline in cell shortening, contraction asynchronous with stimulation, and eventually cessationof contraction. These findings are in contrast to two previous studies which did not find changes in inotropy following neuraminidase treatment of superfusedintact atria1 preparations [X,18]. It is not, however, clear that sialic acid moieties were removed from sarcolemmal glycoconjugates by neuraminidasesuperfusion of intact tissue preparations. The question arisesas to whether the contractile changes induced by neuraminidase

after

Neumminidase

183

could be related to the augmentation of cellular Ca. Contractile function can be divided into those processesinvolved in initiation of contraction (pacemaking) and those related to the force of contraction (inotropy). Ca influx via the slow inward current contributes to the action potential [3.7J. Recently, Ca influx through the transient (T) Ca channel has been postulated to play a role in pacemaking in sinoatrial node cells [14]. The force of contraction is also dependent on Ca entry into the cardiac myocyte [IZ, 221. Furthermore, Ca influx across the sarcolemma is necessary for the coupling of excitation and contraction in cardiac myocytes. It, therefore, seemsreasonable that an increase in cellular Ca could cause arrhythmic contractions, increased cell shortening which presumably correlates with positive inotropy, and cessation of contraction. Supporting the association between the increasein cellular Ca and the contractile changes induced by neuraminidase tretment, was the fact that the Ca in contraction important was rapidly exchangeable as was the increment in Ca induced by neuraminidase. It was, however, curious that the increase in cell shortening with neuraminidase treatment of cultured cardiac myocytes was only 3096 while the increment in Ca was many times greater than that necessaryfor maximal activation of the myofilaments. Two explanations for this discrepancy are possible. (1) cardiac myocytes, after the initial positive inotropy, ceasedcontracting within 5 min of neuraminidase treatment. This could be due to Ca overload and onset of contracture which is difficult to discern in the tightly adherent cells. (2) The increment of Ca induced by neuraminidase may have been compartmentalized in a location that was distinct from the Ca responsible for contraction. In summary, the contractile changes following neuraminidase treatment were not inconsistent with the increases in cellular Ca. Interestingly, cultured rat cardiac myocytes which had ceased contracting due to neuraminidase treatment, began to spontaneously beat again within 8 h after being washed and reincubated despite cellular levels usually associatedwith cell death. This indicates that the cells are able to reverse the

184

H. F. Yee es al.

effects of neuraminidase treatment, either by adapting to the increased sarcolemmal permeability to Ca or by synthesizing and inserting sarcolemmal glycoconjugates critical for Ca regulation to replace those that had sialic acid removed. In conclusion, neuraminidase, most likely by removing sarcolemmal sialic acid residues, induced a very large increment in cellular Ca. The increment was rapidly exchangeable and probably mediated by a Ca-specific mechanism such as selective augmentation of the T Ca channel current as in guinea-pig

ventricular myocytes [44. In addition, neuraminidase altered contractile function in a manner consistent with alterations in cellular Ca. This study suggests that sialic acid may play a significant role in the regulation of cellular Ca and contractile function. Acknowledgement This work was supported by USPHS grants HL 28539-08, the Laubisch Fund and the Castera Endowment; H. F. Yee was the recipient of a USPHS Medical Scientist Training Program Fellowship (GM08042).

References 1 2

3 4 5 6 7 8 9 10 11 12 13

14 15 16 17 18 19 20 21 22

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myocardial

24

and Contraction