J Mol

Cell

Cardiol

24,

1179-l

188

Effects of Regional Release Channel Edward

(1992)

Ischemia of Canine

M. Darling,

on the Ryanodine-sensitive Ca2’ Cardiac Sarcoplasmic Reticulum

F. Anthony

Lai*,

and Gerhard

Meissner

Departments of Biochemistry and Biophysics, and Physiology, University of North Carolina, NC 2759S7260, USA

Chapel Hill,

(Received 10 January 1992, accepted in revised form 27 Ma_v 1992) E. M. DARLING, F. A. LAI AND G. MEISSNER. Effects of Regional Ischemia on the Ryanodine-sensitive Ca” Release Channel of Canine Cardiac Sarcoplasmic Reticulum. Journal of Molecular and Cellular Cardiology (1992) 24, 1179-l 188. In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca?’ ions through ryanodine-sensitive Ca*+ release channels present in the intracellular membrane compartmrnt. sarcoplasmic reticulum (SR). In this study, the effects of regional ischemia on intrinsic SR Ca” release channel and release, and [3H]ryanodine binding properties of function were determined by studying the Ca*+ transport whole muscle homogenates and SR-enriched membrane fractions from normal and ischemic myocardium. %a*+-uptake rates before and after pretreatment with 1 rnM ryanodinr. Measurement of oxalate-supported indicated that the SR Caz+ release channel retained its ability to be effectively closed by the channel-specific probe ryanodine after 15 and 60 min of ischemia. 95Ca2+ el?Iux from, and high-affinity [‘Hlryanodine binding m channel activity by Ca” +, .Mg” SR-enriched vesicle fractions indicated retention of regulation of Ca2+ release and adenine nucleotide in 15 and 60 min ischemic samples. Further, sodium dodecylsulfate polyacrylamidc gel and immunoblot analysis revealed no proteolytic degradation of the M, 565 000 SR Ca2+ release channel polypeptide after 15 and 60 min of ischemia. These results suggested a minimal, if any, loss of intrinsic SR Ca’ ’ release channel function in ischemic hearts. KEY

WORDS:

Ca”

release

channel;

Ryanodine

receptor;

Introduction A well recognized feature of myocardial ischemia is an altered calcium ion homeostasis with resultant changes in intracellular Ca2+ content and distribution [I]. A likely mediator of this response is the sarcoplasmic reticulum which plays a key role in cardiac W)‘, muscle contraction and relaxation by rapidly releasing and re-sequestering Ca*+ [2, 31. A membrane-bound Ca2+ pump and ryanodinesensitive Ca2+ release channel are believed to be the two primary transport proteins responsible for the uptake and release of Cazi‘ ions by the SR, respectively. Ca*+ uptake by the SR C:a2+ pump is driven by the hydrolysis of ATP [2], while the SR Ca2+ release channel is activated by Ca’+ and ATP, and inhibited by Mg”+ and calmodulin [4]. In ischemic hearts, function of these two transport proteins can

Ischemia;

Cardiac

sarcoplasmic

reticulum

therefore be expected to be impaired due to extrinsic mechanisms such as depletion of high-energy phosphates as well as increases in intracellular free Ca’+, Mg*+ and H’ ion concentrations [I, 5, s]. In addition, membrane modification reactions leading to loss of intrinsic Ca’+ pump and channel function have been implicated to contribute to ischemic contractile failure, although the exact nature of this damage has remained unclear [7-111. The release of Ca*+ ions from the cardiac SR occurs through a membrane-bound, highconductance (75 pS in 50m~ Ca’+j, Ca”gated Ca” -release channel [12). The cardiac Ca*+ release channel has been identified as the receptor for the plant alkaloid ryanodine and purified as a tetrameric protein complex comprised of M, 565 000 polypeptides [13-151: as

’ SR -- sarcoplasmic reticulum; EGTA - [ethylenebisoxyethylenenitrilo] tetraacetic acid; Chaps - 3-[(3-cholamidopropyl) dimethylammoniol-1-propanesulfonate; DIFP -- diisopropylfluorophosphate. Please address all correspondence to: Dr Gerhard Meissner, Department of Biochemistry and Biophysics, Univcrsit) of North Carolina, Chapel Hill, NC 27599-7260, USA. * Present address: MRC National Institute for Medical Research, The Ridgeway. .Mill Hill. London NW7 IA:\. I’nited Kingdom 0022%2828/92/101179+

10 $08.00/O

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Limited

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E. M.

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determined by cDNA cloning and sequencing [16, 171. Morphological studies have suggested that the mammalian cardiac Cazf release channel is identical with large protein bridging structures (“feet”) present at specialized areas where the SR comes in close contact with the surface membrane and its tubular infoldings (T-tubule) [13, II]. In the present report, we have compared the Ca*+ transport and release properties of whole muscle homogenates and SR vesicle fractions which were obtained from normal (non-ischemic) and ischemic canine cardiac tissue. In these studies, significant changes in intrinsic SR Ca2+ release channel function were not detectable after 15 and 60 min of ischemia.

Materials

and Methods Chemicals

Ryanodine was obtained from AgriSystems International (Wind Gap, PA), [3H]ryanodine (54.7 Ci/mmol) from DuPont-New England Nuclear, and 45Ca2+ from ICN Radiochemical (Irvine, CA). Protease inhibitors and SDS gel molecular weight standards were purchased from Sigma, and ruthenium red from Fluka. All other reagents were of analytical grade.

et al. the same manner as the non-ischemic sample. Non-ischemic and ischemic ventricular tissues were minced and homogenized at 4°C for 60 s in an ultra torrex homogenizer in 7 volumes of 0.3 M sucrose, 0.5 mM EDTA and 20 mM Tris/Hepes, pH 7.4, containing various protease inhibitors (1 mM diisopropyl Buorophosphate (DIFP), 100 nM aprotinin, 1 ,UM leupeptin, 1 ,UM pepstatin, 1 mM benzamide, and 1 mM iodacetamide). An aliquot of the homogenate was quickly frozen and stored at - 135°C for subsequent studies. The remaining homogenate was centrifuged for 25 min at 4500 rpm (9000 x g) at 4°C in a GSA rotor in a Sorvall RC-2 centrifuge, and the resulting supernatant was passed through two layers of cheesecloth and then re-centrifuged for 30 min at 33 000 rpm (90 000 x g) in a Beckman type 35 rotor. After resuspension of pellets in - 30 ml of 20 mM K Pipes, pH 7.0 buffer containing 0.6 M KCl, 0.1 mM MgCl,, 0.1 mM EGTA, 5OpM CaCI,, and the above protease inhibitors, membranes were placed at the top of a 20% (w/w) sucrose solution containing 0.6 M KCI, 0.1 mM MgCl,, 0.1 mM EGTA, 50 PM CaCl,, 20 mM K Pipes, pH 7.0, and centrifuged for 60 min at 33 000 rpm (90 000 x g) in a Beckman type 35 rotor. The resulting pellet fraction was resuspended at a protein concentration of 10-15 mg/ml in 0.3 M sucrose, 5 mM K Pipes, pH 7.0, quickly frozen, and stored at - 135°C.

Preparation of non-ischemic and ischemic muscle homogenates and microsomal fractions

45Ca2+ -uptake measurements

Whole muscle homogenates and SR enriched membrane fractions were obtained from canine left ventricular wall of normal (nonischemic) and ischemic hearts. Mongrel dogs weighing 15 to 25 kg were anesthetized with sodium pentobarbital (30 mg/kg), and following a midsternal sternotomy, the hearts were rapidly excised and immediately trimmed of fat and atria1 tissue. A 5-10 g portion of left ventricular tissue, designated as non-ischemic sample, was removed, placed into ice-cold 0.3 M sucrose solution, and processed as described below. The remainder was placed into a beaker, flushed with a H,O-saturated stream of argon gas and maintained at 37°C. At 15 and 60 min, 5-log segments of left ventricular tissue were removed, placed into ice-cold 0.3 M sucrose and then processed in

Unless otherwise indicated, homogenates (25 mg wet muscle/ml) or microsomes (1.s 2.5 mg protein/ml), were incubated at 37°C for 15 min in the presence or absence of 1 PM or 1 mM ryanodine in 20 mM K Pipes, pH 7.0, 0.1 M KCI, 5mM AMP, 150~~ Ca2+, 100,~~~ EGTA, 1 mM DIFP and 5p~ leupeptin. Incubation of homogenates with 1 FM ryanodine locked all channels into an open configuration, whereas incubation with 1 mM ryanodine completely closed all cardiac Ca2+ release channels, and thereby allowed determination of uptake rates by all intact SR vesicles, i.e. vesicles which contained or lacked the SR Ca2+ release channel [II, 18, 191. Incubated samples were placed on ice and kept there for less than 5 min before their use in ‘%a2+ uptake measurements. Sample aliquots of

SR Ca’+

Release

Channel

90 ~1 were diluted into 3.5 ml of a solution kept at 23°C and containing 20 mM K Pipes, pH 7.0, 0.1 M KCl, loo,UM EGTA, 150,UM 45CaCl,, 5 mM MgCl,, 0.1 mM DIFP, 1 PM leupeptin, 5 mM NaN, (to inhibit mitochondrial Ca2+ accumulation), and 5 mM K oxalate (a Ca2+ precipitating agent that increases the Ca’+ storing capacity of SR vesicles). 4jca2 + -uptake by SR vesicles was started by the addition of 5 mM ATP. At timed intervals, a 0.4 ml aliquot was placed on a 0.45pm Millipore filter (under vacuum) and rinsed with 3 volumes of a quench solution containing 0.1 M KCI, 5 mM Mg*+, 20 pM ruthenium red, 0.1 mM EGTA, and 20 mM K Pipes, pH 7.0. Radioactivity retained on the filters was determined by liquid scintillation counting. “‘Ca”

in Ischemic

Heart

1181

1 mM DIFP, 5 PM leupeptin, 100 PM EGTA, 200 p CaCl,, 5 mM AMP and i-50 nM [3H]ryanodine (specific activity 54.7 Ci/ mmol) Non-specific binding was assessed using a lOOO-fold excess of unlabeled ryanodine. After 4 h, an aliquot of vesicles was placed into a scintillation vial to determine total radioactivity. ,4nother aliquot was sedimented by centrifugation for 30 min at 90000 x g in a Beckman Airfuge. Free [3H]ryanodine in the supernatant was determined by liquid scintillation counting. A third aliquot was diluted 20-fold in ice-cold water and vacuum filtered through a Whatman GF/ B filter soaked in 1% polyethylenimine. hfter rinsing with three 5 ml volumes of ice-cold water, bound [“Hlryanodine remaining on the filter was determined by liquid scintillation counting.

E$ux

SR-enriched vesicle fractions (2.5 mg protein) were diluted into 10 ml of ice-cold buffer containing 20 mM K Mes, pH 6.15, 0.1 M KCI, 100~~ EGTA, 50~~ CaCl,, 1 mM DIFP, and 5 pM leupeptin, and centrifuged for 30 min at 35 000 rpm in a Beckman type 42.1 rotor. Vesicles were resuspended at a protein concentration of N 10 mg/ml in the above medium but containing 1 mM 45Ca2+ instead of 50 PM Ca* +, and then passively loaded with I mM “Ca”+ by incubation for 60 min at 23°C. .‘%a’+ efflux was initiated as described [ZO] by diluting vesicles loo-fold into isoosmolar release medium containing varying c.oncentrations of Ca”, Mg2+, and adenine nucleotide. ‘5Ca’+ efflux was stopped by placing an aliquot on a 0.45,~m Millipore filter ! under vacuum) and washing of vesicles with a quench solution containing 20 mM K Mes, pH 6.15, 0.1 M KCl, 2OpM ruthenium red, 5rnM Mg’+, and 0.1 mM EGTA. Radioactivity remaining with the vesicles on the filters was determined by liquid scintillation counting. ! ‘H]Ryanodine

binding

[‘HJRyanodine binding to SR-enriched membrane fractions (-0.5 mg of protein/ml) was determined similarly as described [21]. Membranes were incubated at 37°C in a medium containing 20 mM Na Pipes, pH 7.0, 1 M NaCl,

SDS Gel Electrophoresis

and immunoblot ana&i.r

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in the Laemmli buffer system [Z?] using a 512% linear polyacrylamide gradient gel and 3% stacking gel. Samples were denatured for 3 min at 95°C in 0.1 M Tris/HCl, pH 6.8, containing 2% SDS, 3% /I-mercaptoethanol, and 10% glycerol. Electrophoresis was at 15°C with a constant current (30 mA/gelj and was monitored with 0.004% bromphenol blur as a tracking dye. Gels were stained with 0.1% Coomassie Brilliant Blue R-250 in 50% methanol, 10% acetic acid and destained with 10% methanol and 15% acetic acid. For immunoblots. the separated proteins from SDS-PAGE were electrophoretically transfered onto Immobilon PVDF membranes (Millipore Corp.) for 1 h at 400 mA, then 1500 mA for 12-15 h. Transfer mcmbranes were blocked with PBS/5% non-fat dried milk for 1 h at 37°C and washed twicr for 20 min in PBS/0.5% Tween 20, bcfirrf overnight incubation at 4°C with a monoclonal antibody (C22997) raised against the purified canine cardiac muscle ryanodinc receptor. Following 1 h room temperature incubation and washing, blots were developed with a peroxidase-conjugated secondary antibody ( I :5000), using 3,3’-diaminohenzidine and H,O,.

1182

E. M.

Darling

Other biochemical assays “Basic” and Mg2+-dependent, Ca’+-stimulated ATPase activities were determined in the presence of the Ca2+ ionophore A23187 (2pg/ml) at 32°C as the difference of total (with 50,~ free Ca*+) and “basic” (with 1 mM EGTA) ATPase activities [20]. Free Ca*+ concentrations were calculated according to a computer program using binding constants published by Fabiato [23]. Results

‘%a’+

uptake by whole muscle homogenates

The time course of ATP-dependent, oxalatesupported *Ca*+ uptake by non-ischemic and 60 min ischemic whole muscle homogenates was determined following their prior incubation with 0, 1 PM or 1 mM ryanodine. In the absence of ryanodine, homogenates from normal hearts sequestered ‘Ca*+ with an about two-fold higher initial rate than those obtained from 60 min ischemic hearts (Fig. 1). After 30 min of loading, amounts of 45Ca2+ accumulated by the two homogenates differed by a factor of about 3. Prior incubation with 1 mM ryanodine increased “Ca*+ uptake rates and the extent of “Ca’+ loading in both the ischemic and non-ischemic homogenates. The uptake rates were increased to a greater extent in the ischemic than non-ischemic homogenates, so that after ryanodine treat-

I

/ 40

Time

(mm1

FIGURE I. ‘5Ca’f uptake by non-ischemic and 60 min ischemic canine cardiac homogenates. +TW+ uptake by non-ischemic (N) and 60 min ischemic (60’-I) samples was measured following preincubation for 15 min at 37°C in the presence or absence of 1 mx ryanodine as described in Materials and Methods.

et al.

ment the difference in 45Ca2’ uptake by the two samples was only -20%. Incubation of homogenates with 1 ,u~ ryanodine had no noticeable effect on 45Ca2f uptake rates or the extent of loading (not shown). This suggested that in the absence of ryanodine, the Ca*+ release channel was sufficiently activated by 50,~~ free Cazf (Materials and Methods; ref. 2 1 ), to cause rapid Ca*+ release and thereby to prevent accumulation of transported “Ca’+ by those SR vesicles that contained the channel. Table 1 compares the 45Ca’+ uptake rates for homogenates obtained from non-ischemic, 15 min and 60 min ischemic tissues following pretreatment with and without 1 mM ryanodine. In the absence of ryanodine, initial 45Ca2+ uptake rates were 1.2f0.2, l.Of0.2, and 0.5 f 0.1 nmol 45Ca2+/mg wet muscle/min for non-ischemic and 15 and 60 min ischemic samples, respectively. This corresponded to a decrease of uptake rates of 17% and 58% in 15 and 60 min samples, respectively. Prior incubation with I mM ryanodine permitted measurement of 45Ca2’ uptake by all intact SR vesicles and indicated an only modest loss of SR Ca*+ pump function in the ischemic samples (8% and 20% for the 15 min and 60 min ischemic samples, respectively). Student’s t test showed that the differences in 45Ca2’ uptake rates for the non-ischemic and 60 min ischemic samples were statistically significant (P

Effects of regional ischemia on the ryanodine-sensitive Ca2+ release channel of canine cardiac sarcoplasmic reticulum.

In mammalian myocardium, muscle contraction is regulated by the rapid release of Ca2+ ions through ryanodine-sensitive Ca2+ release channels present i...
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