(Circulation. 1995;92:433-446.)
© 1995 American Heart Association, Inc.
Articles |
Direct Effects of Protamine Sulfate on Myocyte Contractile Processes
Cellular and Molecular Mechanisms
From the Division of Cardiothoracic Surgery, Medical University of South Carolina (R.B.H., R.M., B.U.J., F.A.C., F.G.S.), Charleston, SC, and the Section of Vascular Surgery, Jobst Vascular Research Laboratory (T.W.W., J.C.S.), and the Department of Biochemistry (P.C.A.), University of Michigan, Ann Arbor.
Correspondence to Francis G. Spinale, MD, PhD, Division of Cardiothoracic Surgery, Medical University of South Carolina, 171 Ashley Ave, Charleston, SC 29425.
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
Background Administration of the arginine-rich, highly charged protamine (PROT) molecule has been associated with episodes of acute left ventricular (LV) dysfunction. The objective of the present study was to test the hypothesis that PROT has direct effects on isolated LV myocyte contractile processes and sarcolemmal transduction systems.
Methods and Results Exposure of porcine LV myocytes (n=305) to 40 µg/mL PROT (reflecting a dose of 2.5 mg/kg) decreased basal contractile function and ß-adrenergic responsiveness. For example, myocyte percent shortening was 4.3±0.1% in control myocytes and decreased to 2.8±0.2% in the presence of 40 µg/mL PROT (P<.05). Myocyte percent shortening was 9.3±0.7% after ß-adrenergic receptor stimulation (isoproterenol; 25 nmol/L) and was significantly reduced in the presence of 40 µg/mL PROT (5.7±0.7%, P<.05). PROT reduced myocyte responsiveness to forskolin (100 µmol/L), which directly activates adenylate cyclase, by >40% from forskolin. In addition, PROT abolished the inotropic effects of ouabain on myocyte contractile function. To determine contributory mechanisms for the effects of PROT on myocyte sarcolemmal systems, ß-receptor– and cardiac glycoside–binding characteristics were determined in sarcolemmal preparations. ß-receptor binding was 175±10 fmol/mg and was reduced to 140±6 fmol/mg in the presence of PROT (P<.05). Ouabain receptor binding was 7.1 pmol/mg and decreased to 2.6±0.4 pmol/mg in the presence of PROT. In addition, cAMP production after stimulation with isoproterenol and forskolin was significantly blunted in the presence of PROT. Variants of the PROT molecule were constructed by specific amino acid substitutions and deletions, which provided a means to vary charge as well as structure. Substitution of arginine with lysine in the PROT peptide sequence ameliorated the negative effects on myocyte contractile processes; despite identical overall charge (21+). However, a PROT variant with an 18+ charge but different amino acid sequence induced significant negative effects on myocyte function and inotropic responsiveness. Thus, the effects of PROT on myocyte contractile processes are not due simply to the high positive charge of the molecule. To further establish that PROT can contribute to changes in LV function in the clinical setting, fluorescein-labeled PROT was circulated in antegradely perfused rabbit hearts. Microscopic examination revealed that PROT could traverse the vascular compartment of the myocardium and come in direct contact with the myocyte.
Conclusions The unique findings from the present study suggest that a fundamental contributory mechanism for the changes in LV function observed after protamine administration may be the direct effect of unbound protamine on myocyte contractile processes.
Key Words: ventricles • proteins • surgery
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Protamine sulfate is routinely used to reverse the anticoagulant effects of heparin in patients undergoing cardiac and vascular surgical procedures. However, protamine administration has been associated historically with hemodynamic instability and changes in cardiovascular performance.1 2 3 4 5 6 7 8 9 10 11 More recently, it has been reported that adverse events may occur in >5% of vascular surgical patients after protamine administration.12 Furthermore, it has been suggested that patients with preexisting left ventricular (LV) dysfunction may be more susceptible to the negative effects of protamine on LV pump performance.10 Since increasing numbers of patients with underlying LV dysfunction are presenting for cardiovascular procedures,13 the negative effects of protamine may further compromise LV function and increase the complexity of intraoperative management of these patients. Although the effects of protamine on LV function are a clinically significant issue, the basic mechanisms by which protamine influences LV function are poorly understood. Past observations in whole-heart and multicellular preparations have suggested that protamine directly interferes with myocardial force production.14 15 16 17 However, it remains unclear from these whole-heart and multicellular preparations whether protamine directly affects myocyte contractile function or whether these negative effects are mediated through nonmyocyte cell populations and extracellular influences. To address this issue, our laboratory has demonstrated that isolated myocyte contractile function is significantly reduced in the presence of concentrations of protamine that are commonly encountered clinically.18 Protamine is made up primarily of the amino acid arginine, which results in a high net positive charge.8 We hypothesized that these highly charged arginine residues found in protamine may directly interfere with myocyte sarcolemmal properties and function.
To determine the direct effects of protamine on myocyte contractile processes and contributory mechanisms for these effects, several lines of investigation were pursued. First, the direct effects of protamine on myocyte ß-adrenergic receptor transduction were examined. To examine whether protamine interferes with sarcolemmal function in a more global manner, a second line of investigation determined the direct effects of protamine on sarcolemmal Na+,K+-ATPase function. The specific changes in the function of these sarcolemmal transduction systems in the presence of protamine were directly related to changes in myocyte contractile function. In light of the fact that maintenance of ionic potentials is dependent on an intact functioning sarcolemma and that protamine may be associated with arrhythmogenesis,3 4 19 we suspected that protamine would alter fundamental electrophysiological properties of myocytes. Accordingly, the effects of protamine on isolated myocyte action potential characteristics were determined.
The mechanism by which protamine reverses the anticoagulant effects of heparin appears to be related to the highly alkaline polycationic nature of the compound.8 20 21 22 However, the binding of heparin by protamine does not appear to be simply due to charge effects but the presence of a specific binding domain that consists of a helical structure with a high positive charge density.23 Direct evidence exists that protamine may specifically interact with the cardiac myocyte sarcolemma in an allosteric fashion and modulate receptor binding characteristics.24 To directly examine the mechanisms by which protamine interacts with the myocyte, protamine variants were constructed by substituting amino acids and by varying the charge of the molecule.25 26 The effects of these variants on myocyte contractile function and inotropic responsiveness were then examined to more carefully determine the structural attributes of the protamine molecule that contribute to alterations in myocyte contractile processes. It remains unknown whether the negative effects of protamine that have been observed in vitro can be directly translated to play a mechanistic role in the changes in hemodynamic status that occur in the whole organism after protamine administration. A final line of investigation of the present study was to determine whether protamine has the ability to egress from the vascular compartment and to enter the interstitial space of the myocardium.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
To determine the direct effects of protamine on myocyte contractile function and inotropic responsiveness to receptor agonists, myocytes were isolated from porcine LVs. To determine the direct effects of protamine on receptor binding and function of sarcolemmal transduction systems, sarcolemmal preparations were made from porcine LV myocardium. Examination of the movement of fluorescein-labeled protamine was performed by antegrade perfusion into adult rabbit hearts (New Zealand White rabbits). The localization of protamine molecule to the myocyte sarcolemma was performed by use of isolated porcine myocytes. To determine the effects of different molecular constructs of the protamine molecule on myocyte contractile processes, myocyte function and inotropic responsiveness were also evaluated using isolated porcine myocytes. All of the animals used in these studies were treated and cared for in accordance with the National Institutes of Health "Guide for the Care and Use of Laboratory Animals" (NIH publication No. 86-230, 1985).
Myocyte Isolation and Contractile Function
Measurements
Twelve age- and weight-matched pigs (Yorkshire strain, 6
months, 30 kg) were anesthetized with isoflurane (0.5%/1.5 L
per minute), and a sternotomy was performed. The heart was then quickly
extirpated and placed in an oxygenated Krebs solution and
the LV free wall isolated. The region of the LV comprising the left
anterior descending artery was snap-frozen in liquid nitrogen and
used for sarcolemmal membrane preparations as described below. The
circumflex coronary artery was cannulated and used for the
myocyte isolation procedure. By use of methods described by this
laboratory previously,27 28 an oxygenated
modified Krebs solution containing aerobic substrates and
collagenase (0.5 mg/mL, Worthington, type II; 146 U/mg) was
perfused and recirculated through the cannulated circumflex artery for
20 minutes. The tissue was then minced into 2-mm sections and added to
an oxygenated solution containing 400 µmol/L
CaCl2 and collagenase (0.5 mg/mL). The tissue
and solution were gently agitated; then at 5-minute intervals, the
supernatant was removed and filtered, and the cells were allowed to
settle. The myocyte pellet was then resuspended in standard culture
medium (M199, 2 mmol/L Ca2+, Gibco
Laboratories). Isolated myocytes were placed in a thermostatically
controlled chamber (37°C) fitted with a coverslip on the bottom for
imaging on an inverted microscope (Axiovert IM35, Zeiss Inc). Myocyte
contractions were elicited by field-stimulating the tissue chamber
at 1 Hz (S11, Grass Instruments) with current pulses of 5-ms duration
and voltages 10% greater than contraction threshold. The polarity of
the platinum-stimulating electrodes was alternated to prevent the
buildup of electrochemical by-products. Myocyte
contractions were imaged with a charge-coupled device with a
noninterlaced scan rate of 240 Hz (GPCD60, Panasonic). Myocyte
contraction profiles were analyzed using techniques well
described by our laboratory previously.27 28
Stimulated
myocytes were allowed a 5-minute stabilization period, then the
following parameters were computed: percent shortening
(%), shortening velocity (µm/s), lengthening velocity (µm/s), and
total contraction duration (ms). These parameters were
calculated for each contraction, and the results were averaged for 20
contractions.
After collection of baseline contractile indexes, myocytes were incubated with either 40 or 80 µg/mL protamine (suspended in 0.1N saline; Elkins-Sinn, Inc) and measurements of contractile function repeated. The protamine concentrations of 40 and 80 µg/mL used in this portion of the study are approximately equal to serum concentrations obtained in patients dosed with protamine at 2.5 and 5 mg/kg, respectively.15 19 To determine the effects of protamine on myocyte ß-adrenergic receptor responsiveness, myocyte contractile function measurements were repeated in the presence of isoproterenol (25 nmol/L; Sigma Chemical Co) or forskolin (0.5 µmol/L; Sigma). The effects of protamine on the sarcolemmal Na+,K+-ATPase system were examined by measuring myocyte contractile responsiveness after the addition of ouabain (2 µmol/L; Sigma). For purposes of comparison, a subset of myocytes was not incubated with protamine and was exposed to either isoproterenol, forskolin, or ouabain alone. The concentration of isoproterenol used in the present study was previously shown to reflect the 100% effective concentration (EC100) in porcine myocytes.29 The ouabain and forskolin concentrations used in the present study represent the 50% effective concentrations (EC50) in this myocyte preparation.29 30 The pH and electrolyte concentrations of the myocyte media were routinely checked to ascertain whether any changes occurred after the addition of protamine. Characteristics of the cell culture medium with no protamine were PCO2, 31±2 mm Hg; PO2, 188±6 mm Hg; and pH 7.57±0.04. Medium characteristics did not significantly change with the addition of either 40 or 80 µg/mL protamine (all P>.05): PCO2, 32±3 mm Hg; PO2, 213±5 mm Hg; and pH 7.58±0.03.
Protamine is routinely administered to reverse the anticoagulant effects of heparin.1 2 3 4 5 6 7 8 9 10 11 12 In light of the fact that unbound protamine was observed to have significant effects on myocyte contractile function, separate series of experiments were performed to determine the effects of heparin alone and protamine alone and the interaction between heparin and protamine on myocyte contractile function. In the first set of experiments, myocyte contractile function was measured in the presence of 40 µg/mL heparin (beef lung, Upjohn). This concentration of heparin (40 µg/mL) was selected because it is assumed that heparin and protamine bind in a 1:1 fashion and therefore 40 µg/mL of heparin will neutralize 40 µg/mL protamine.22 In the second series of experiments, myocytes were incubated with 40 µg/mL heparin and then exposed to 40 µg/mL protamine, after which myocyte contractile function was measured. A final subset of experiments was performed to determine whether the effects of protamine on myocyte contractile function could be reversed by subsequent administration of heparin. In this series of experiments, myocytes were incubated with 40 µg/mL protamine and then subsequently exposed to 40 µg/mL heparin.
Isolated Myocyte Electrophysiological
Measurements
To determine whether protamine may influence ionic
properties of
the myocyte sarcolemma, myocyte action potential characteristics were
measured in the presence of protamine. Microelectrodes were made from
1.2-mm thin-walled glass capillary tubing (model 1B120F-4, World
Precision Instruments) by using a horizontal electrode puller (model
P-87, Sutter Instrument Inc). Microelectrodes (tip resistance, 20 to 35
M
) were filled with 3 mol/L KCl and attached to a high-input
impedance negative capacitance amplifier (Axoclamp-2A, Axon
Instruments). An Ag-AgCl reference electrode was placed in the
experimental chamber. Myocytes were then impaled by gently advancing
the microelectrodes at a 45° angle onto the sarcolemmal surface with
a micromanipulator (model MO-103, Narshige Instruments). Impalements
were considered stable if myocytes could be stimulated continuously for
10 minutes with stable resting membrane potential, action potential
duration, and contraction amplitude.31 Myocyte action
potentials and contractions were elicited by current injection through
the microelectrode using 1-ms pulses at intervals of 1000 ms.
Stimulation current was adjusted (3 to 5 nA) so that there was at least
a 1-ms separation between the stimulation pulse and the upstroke of the
action potential.
After a 15-minute stabilization period, myocyte action potential and contractile data were collected from a minimum of five consecutive contractions. Myocyte contraction profiles were imaged and converted into a voltage signal as described previously.27 28 The amplified transmembrane-membrane–potential signal and the myocyte contractile signal were digitized simultaneously at 12-bit resolution and a sampling frequency of 10 kHz/signal (model AT-MIO-16, National Instruments), and the digitized signals were stored to disk on a 486 computer (Zeos International). During off-line analysis, the first-order differentials of the myocyte contractile signal and the membrane potential signal were computed. Action potential parameters derived from the undifferentiated and differentiated membrane potential signals included resting membrane potential (mV), maximum action potential amplitude (mV), action potential durations at 50% (APD50) and 90% (APD90), repolarization (ms), and maximum action potential upstroke velocity (V/s). Maximum action potential amplitude was computed as the difference between the peak membrane potential and the resting membrane potential. APD50 and APD90 were computed as the time required for the action potential to repolarize to 50% and 10% of the maximum action potential amplitude, respectively. Maximum action potential upstroke velocity was defined as the peak positive value of the differentiated membrane potential signal.
ß-Adrenergic Receptor Binding and Adenylate Cyclase
Activity
To determine potential mechanisms for the effects of
protamine on myocyte ß-adrenergic responsiveness,
ß-adrenergic receptor binding and cAMP production were
measured in the presence and absence of protamine. Sarcolemmal
membranes were prepared from harvested porcine LVs as previously
described.29 32 ß-Adrenergic receptor
antagonist binding studies were performed in the presence
of six concentrations of [125I]iodocyanopindolol (ICYP,
74 Bq/mmol, Amersham Corp) from 0.015 to 0.75 nmol/L.29 A
standard Scatchard linear regression analysis was performed on
the amount of bound/free ligand, with an R2>.90
as the criterion for acceptability of the data. With this
analysis, the maximal number of binding sites
(Bmax), expressed as fmol/mg protein, and the
equilibrium dissociation constant (Kd, in
pmol/L) were computed. Adenylate cyclase activity was
determined by timed cAMP production in aliquots of 30 to 50
µg/100 µL of membrane preparation by use of previously described
methods.29 33 Reactions were terminated by placing
the
tubes in an ice-cold bath and then by centrifuging at
3250g for 5 minutes. Pellets were resuspended in 0.5 mL
buffer (50 mmol/L Tris-HCL, 10 mmol/L MgCl2, 10
µmol/L EGTA, 10 µmol/L PMSF, and 2.8 µmol/L EGTA), boiled for 5
minutes, and then centrifuged at 6500g for 10
minutes. Reactions were terminated by placing the tubes in boiling
water and then centrifuging at 6500g for 5 minutes. The
supernatant was assayed for cAMP content using a competitive
radiolabeled assay (RIA Kit, Advanced Magnetics Inc). Adenylate
cyclase activity was determined at baseline as well as in the
presence of either 10-3 mol/L (-)isoproterenol or 100
µmol/L forskolin. Results were expressed as pmol cAMP produced/mg
sarcolemmal protein per minute. All measurements were performed in
duplicate. A second series of experiments was designed to determine
whether protamine altered the adenylate cyclase activity of
intact cells. Isolated myocytes (12 500 cells/0.5 mL) were incubated
in medium for 10 minutes under the following conditions: (1) in the
basal state (no protamine, no isoproterenol), (2) in the presence of 25
nmol/L isoproterenol, (3) in the presence of 40 µg/mL protamine, or
(4) in the presence of both 25 nmol/L isoproterenol and 40 µg/mL
protamine. Results were expressed as pmol cAMP produced/12 500 cells
per 10 minutes.
Na+,K+-ATPase Glycoside Binding and
Hydrolytic Activity
To determine whether protamine affected other
sarcolemmal
receptor systems, the present study examined the effects of
protamine on ouabain binding and
Na+,K+-ATPase activity. Sarcolemmal
membranes were prepared as described in the previous section, and
binding of [3H]ouabain (31.5 Ci/mmol; Amersham Corp) was
performed using a displacement assay as previously
described.30 34 The binding assays were performed in
a
reaction volume of 900 µL (pH 7.4, 37°C) containing the following:
5 mmol/L Tris-PO4, 5 mmol/L
MgCl2, 10 mmol/L MOPS, 2 µmol/L
[3H]ouabain, and 0 to 1.0 µmol/L unlabeled ouabain. A
100-µL aliquot of homogenate was added to the prewarmed
reaction mixture, yielding a final protein concentration of 0.5 to 1.0
mg/mL. The mixture was incubated for 45 minutes, the reaction
terminated by the addition of 0.5 mmol/L ice-cold unlabeled
ouabain, and the mixture rapidly filtered through a membrane filter
(HAWP, Millipore Corp). The filters were dissolved in 20 mL of liquid
scintillation cocktail (Scintiverse, Fisher Scientific) and the
radioactivity determined using a liquid scintillation spectrometer.
Specific binding was computed as the difference in values observed in
the absence and presence of 50 µmol/L unlabeled ouabain. The maximum
ouabain bound (Ba) and affinity (Kd)
were estimated as described previously.34 Briefly, the
[3H]ouabain bound at each unlabeled ouabain concentration
was transformed into a probit scale.34 All assays were run
in duplicate, and the Ba computations were expressed as
pmol/mg protein.
The effects of protamine on Na+,K+-ATPase hydrolytic capacity were examined by assessing p-nitrophenol-phosphatase (p-NPPase) activity.30 Previously frozen LV myocardial samples were homogenized in 30 mmol/L histidine buffer (pH 7.4) at a 4:1 mixture (vol/wt). The assay was run at 37°C by using a 215-µL aliquot of tissue homogenate in a reaction medium containing (in final concentration): 150 mmol/L KCl, 20 mmol/L MgCl2, 30 mmol/L histidine (pH 7.4), 2 mmol/L EGTA, 10 mmol/L p-NPP (pH 7.4), and 3.3% BSA. A standard curve was established using serial concentrations of p-nitrophenol read at 410 nm with a digital spectrophotometer (Spectronic 21D, Milton Roy). Ouabain-sensitive p-NPPase activity was determined by adding 10 mmol/L ouabain to reaction mixtures and subtracting these results from those obtained in the reaction mixtures without ouabain. The reaction was terminated after 30 minutes by addition of 0.1 mL 50% trichloroacetic acid. Assays were run in duplicate, and results were expressed as µg of p-nitrophenol released/mg protein per hour.
Total protein concentrations of tissue homogenates and sarcolemmal preparations were determined using the Bradford assay.35
Protamine Variants: Effect of Molecular Constructs on Myocyte
Function
These series of experiments were performed to determine the
potential structural mechanisms for the toxic effects of the protamine
molecule on myocyte contractile processes. Variations of the original
protamine molecule were constructed to modify overall charge and amino
acid composition. The construct proteins were synthesized with an
Applied Biosystems model 431 peptide synthesizer as previously
described.25 26 A total of five different variations
on
the protamine molecule were constructed. Since protamine is comprised
of approximately 67% arginine, the first construct examined whether
the arginine moieties of the protamine molecule play a contributory
role in its effects on myocyte contractile function. Accordingly, the
first variant was constructed so that arginine was replaced by lysine.
This amino acid substitution maintained both the number of amino acids
and charge (21+) of the native protamine molecule. The second protamine
construct was designed to reduce the total protein charge number by
three and to change the sequence of amino acids. The structure of this
second construct was a lysine/alanine copolymer with spacing optimized
for formation of an amphipathic helix. This construct was
acetylated and amidated to deter in vitro degradation and to
enhance 
-helix formation.26 To more carefully
determine the effects of secondary conformation of the protamine
molecule, the third construct was similar to the second construct with
the exception that a proline was replaced by a glutamic acid with the
addition of an extra alanine for spacing considerations. This construct
was identical to the second construct with respect to overall charge
(18+). The final two constructs were synthesized to more carefully
determine the charge effects of the protamine molecule. Each had a
different number of positive charges. One construct was 29 amino acids
long with a charge of 16+. This construct was acetylated and
amidated at the terminal residues with the same proline replaced by
glutamic acid. The final construct was a compound with 29 amino acids
and a 14+ charge in which alanine was inserted between each lysine. The
efficiency of these variants in reversing the anticoagulant effects of
standard heparin or low-molecular-weight heparin with respect
to thrombin clotting time and activated clotting time was
reported previously.25 26 Characterization of each of
these constructs as well as the protamine molecule with respect to
charge, pH, and polarity were determined by computer modeling software
(LASERGENE, DNASTAR, Inc), and the
two-dimensional space filling models for each of these molecules
are shown in Fig 1
. A summary of the characteristics of
protamine and each of the protamine variants is presented in
Table 1. Myocyte contractile function and
ß-adrenergic responsiveness for each of these protamine variants
were examined at a final concentration of 80 µg/mL protamine.
|
|
Protamine Localization Studies
A final series of experiments
was performed to address whether
the protamine molecule could enter the interstitial space
of intact hearts and thereby potentially interact with the myocyte. In
addition, the specific interaction of protamine with the myocyte
sarcolemma was more carefully characterized.
Fluorescein-labeled protamine was produced by
conjugating fluorescein (Molecular Probes, Inc) to the
serine residues of the protamine molecule with well-established
methods.36 37 Briefly, DTAF was prepared with the
aminofluorescein and cyanuric chloride
reaction.36 37 DTAF was then incubated with
protamine, and
the conjugated product was purified by filtration using Sephadex
G-25. The purified fluorescein-labeled protamine was
maintained in the dark at 4°C before use. For the intact heart
experiments, adult rabbits (New Zealand White rabbits, 3.5 to 4.5 kg;
n=3) were sedated with 10 mg diazepam IM and subsequently
anesthetized using isoflurane (2%/800 mL per minute) and
nitrous oxide (200 mL/min). A median sternotomy was performed, and the
hearts quickly extirpated and placed in oxygenated Krebs
solution. The hearts were then perfused in an antegrade fashion with
Krebs buffer and fluorescein-labeled protamine (80
µg/mL) for 20 minutes at 37°C, maintaining a perfusion pressure of
50 mm Hg. The LV was then dissected free, rinsed in chilled Krebs
solution, and snap-frozen in liquid nitrogen. The LV sections were
mounted in cryoprotectant embedding compound (OTC, Miles Scientific)
and serially sectioned at 5-µm thickness using a microtome cryostat
(Microm HM 500M, Microm GmbH). The sections were fixed in a 3.7%
buffered formalin solution for 10 minutes, air dried for 15 minutes at
room temperature, and rinsed two consecutive times in PBS, pH 7.4. The
sections were then counterstained with 1% toluidine blue for the
purposes of section orientation and mounted on slides with a
glycerol/PBS solution. For the isolated myocyte experiments, porcine
myocytes (10 000/0.5 mL) were incubated for 20 minutes at 37°C with
standard cell medium containing 80 µg/mL of
fluorescein-labeled protamine. The myocytes were then
passed through three washes of cell medium and placed in a buffered
sodium cacodylate solution containing 2%
paraformaldehyde and 2% glutaraldehyde
(pH 7.4, 325 mOsm). After fixation, myocytes were washed three times in
PBS to remove any residual background staining and mounted on slides
with a glycerol/PBS solution. Fluorescein-stained
tissue was examined on an Axiovert 35 (Zeiss Inc) equipped with
epifluorescent illumination and a 50-W mercury light
source. The LV sections and myocytes were imaged using 40x and 60x
Plan-Neoflar objectives. Photomicrographs were recorded using Kodak
Tmax 400 film (Eastman Kodak Co) at a fixed exposure time
of 15 seconds.
Data Analysis
Analyses of myocyte function data were
performed using
ANOVA. If the ANOVA revealed significant differences, pairwise tests of
individual group means were compared using Bonferroni
probabilities.38 Changes in ß-adrenergic binding and
cAMP production in the presence and absence of protamine
were tested by using a Student's t test. Changes in ouabain
binding and Na+,K+-ATPase activity in the
presence and absence of protamine were examined in similar fashion.
Changes in myocyte electrophysiological
parameters were examined in the presence and absence of
protamine with the Mann-Whitney U test.38
Results are presented as mean±SEM. Values of P<.05
were considered statistically significant.
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
Effects of Protamine on Myocyte Contractile Function
Indexes of contractile function from isolated myocytes in the baseline (no protamine) state and after the addition of protamine to achieve a final bath concentration of 40 or 80 µg/mL are summarized in Fig 2
. Baseline indexes of myocyte contractile
function were very similar to values reported previously by this
laboratory.27 28 29 30 In the
presence of 40 µg/mL protamine,
indexes of myocyte contractile function decreased significantly from
baseline values. For example, 40 µg/mL protamine caused a 35%
decrease in both myocyte percent shortening and velocity of shortening.
A further decline in myocyte contractile function was observed in the
presence of 80 µg/mL protamine. For example, myocyte percent
shortening fell by 50% in the presence of 80 µg/mL protamine.
Thus, as is consistent with a recent report from our
laboratory,18 protamine had a direct effect on myocyte
contractile function.
|
In the next portion of the study, the interactive
effects of heparin
and protamine were examined. The results from the different series of
experiments are summarized in Table 2. Myocyte
contractile function was unaffected in the presence of 40 µg/mL
heparin. Furthermore, the addition of heparin followed by an equivalent
concentration of protamine had no significant effect on myocyte
contractile function. In contrast, in myocytes exposed to protamine
before the addition of heparin, contractile function was significantly
decreased compared with baseline values. Thus, heparin administration
after previous exposure to protamine could not reverse the negative
effects of protamine on myocyte contractile function.
|
Effects of Protamine on Myocyte Contractile Response to
ß-Adrenergic Simulation and Cardiac Glycosides
To determine
whether protamine influences myocyte
ß-adrenergic responsiveness, myocyte contractile function was
examined by using the ß-adrenergic receptor agonist
isoproterenol. The results from this series of studies are summarized
in Table 3. Consistent with past
reports,29 25 nmol/L isoproterenol significantly increased
myocyte contractile function compared with baseline values. For
example, myocyte velocity of shortening increased by 177% after the
addition of isoproterenol. In the presence of protamine, myocyte
ß-adrenergic responsiveness was significantly reduced. For
example, in the presence of 40 µg/mL protamine, myocyte percent
shortening and velocity of shortening decreased by >35% from
isoproterenol-alone values. The presence of 80 µg/mL protamine
caused a >50% decrease from isoproterenol-alone values in both
myocyte percent shortening and velocity of shortening. To more
carefully determine the basis for the effects of protamine on myocyte
ß-adrenergic responsiveness, a series of experiments was
performed in which adenylate cyclase was directly
activated by forskolin. In the presence of forskolin, myocyte
contractile function significantly increased from baseline values. In
myocytes preincubated with 40 µg/mL protamine, all indexes of myocyte
contractile function decreased significantly from forskolin-alone
values. For example, myocyte velocity of shortening decreased by 44%
from forskolin-alone values in the presence of 40 µg/mL
protamine. In the presence of 80 µg/mL protamine, myocyte
responsiveness to forskolin was further reduced from
forskolin-alone values.
|
To determine whether protamine influences
myocyte sarcolemmal
transduction systems in a more global manner, the effects of protamine
on myocyte contractile responsiveness to ouabain were examined. The
results from this series of experiments are summarized in Table
4. In the presence of the cardiac glycoside ouabain,
myocyte contractile function significantly increased from baseline
values. For example, myocyte velocity of shortening increased by >35%
from baseline values in the presence of 2 µmol/L ouabain. In
contrast, preincubation with either 40 or 80 µg/mL protamine
completely abolished the positive inotropic effects of ouabain.
|
Effects of Protamine on Myocyte Electrophysiology
Action
potential parameters at baseline and in the
presence of either 40 or 80 µg/mL protamine are summarized in Table
5. Baseline (no protamine) resting membrane potential
and action potential duration obtained in the present study were
similar to previously reported values.39 In the presence
of either 40 or 80 µg/mL protamine, myocyte resting membrane
potential was significantly decreased from baseline (no protamine)
values, ie, became less negative. The time required for
APD50 to occur was significantly prolonged in the presence
of both 40 and 80 µg/mL protamine when compared with baseline values.
In contrast to 40 µg/mL protamine, maximum upstroke velocity of the
action potential was significantly reduced in the presence of 80
µg/mL protamine.
|
Effects of Protamine on the ß-Adrenergic–Receptor
System
To determine the contributory mechanism for the effects of
protamine on myocyte ß-adrenergic responsiveness,
ß-adrenergic receptor binding characteristics and
adenylate cyclase activity were measured under control
conditions (no protamine) and after the addition of 40 µg/mL
protamine. The results from these studies are shown in Table 6.
The ß-adrenergic receptor density and affinity
computed for LV sarcolemmal preparations that were obtained in the
present study were consistent with past
reports.29 In the presence of protamine,
ß-adrenergic receptor binding was decreased by >20% from
control values with no apparent change in receptor affinity. In the
presence of protamine, a 50% reduction in basal adenylate
cyclase activity was observed. In the presence of protamine,
isoproterenol-stimulated cAMP production was reduced by
52%, and forskolin-stimulated cAMP production was reduced
by 41%. These experiments were performed using purified sarcolemmal
preparations to directly examine the fundamental effects of protamine
on ß-adrenergic receptor binding and adenylate
cyclase activity. However, these studies failed to address whether
protamine could directly influence adenylate cyclase
activity in viable myocytes with an intact sarcolemma. Accordingly,
cAMP production was examined using isolated myocyte
preparations in the presence and absence of 40 µg/mL protamine. The
results from this series of experiments are summarized in Fig
3. Consistent with the observations made using
sarcolemmal preparations, myocyte cAMP production was
diminished at basal states as well as after ß-adrenergic receptor
stimulation with isoproterenol.
|
|
Effects of Protamine on the
Na+,K+-ATPase System
Results from the
myocyte function studies suggest that protamine
may significantly influence the sarcolemmal
Na+,K+-ATPase system. The
Na+,K+-ATPase system plays a fundamental role
in maintaining myocyte homeostatic processes, such as
maintenance of resting membrane potentials.40
Accordingly, ouabain-binding characteristics and
Na+,K+-ATPase hydrolytic activity were
examined
in the presence and absence of protamine. The results from this series
of experiments are summarized in Table 6
. In the
presence of 40 µg/mL protamine, maximal ouabain binding was reduced
by >60% from control values. In addition, receptor affinity for
ouabain was significantly reduced in the presence of protamine.
Interestingly, Na+,K+-ATPase hydrolytic
activity, expressed as K+-dependent p-NPP ase
activity, was not significantly altered in the presence of
protamine.
Protamine Variants and Myocyte Contractile Function
As
outlined in the previous sections, the native protamine
molecule caused significant alterations in myocyte contractile
processes and inotropic responsiveness. This portion of the study
attempted to elucidate whether these effects were due to the charge
effects of protamine and/or the secondary structure of the protamine
molecule. Five variants of the native protamine molecule were
constructed, and the effects of these constructs on myocyte contractile
function and ß-adrenergic responsiveness were examined.
Two-dimensional space-filling models for the native protamine
molecule and each of the protamine variants were constructed on the
basis of the amino acid sequence (LASERGENE) and are
shown in Fig 1. The effects of these protamine variants on
myocyte
contractile function and ß-adrenergic responsiveness are
summarized in Table 7. When lysine was substituted for
arginine in the protamine construct, myocyte contractile function was
significantly increased from native protamine values. Specifically,
myocyte velocity of shortening increased by 63% from native
protamine values with this amino acid substitution. Myocyte
ß-adrenergic responsiveness to isoproterenol was significantly
improved in the presence of the lysine-substituted variant compared
with the native protamine molecule. In contrast to these findings,
myocyte contractile function was significantly affected when the number
of charges was reduced to 18+ by altering the arrangement of lysine
residues (18B, Fig 1, Table 7). Specifically,
this 18+-charged
protamine variant caused a similar reduction in myocyte velocity of
shortening when compared with the native protamine molecule.
Interestingly, although the 18+-charged protamine variant (18B)
produced negative effects on myocyte ß-adrenergic responsiveness,
these negative effects were significantly less when compared with the
native protamine molecule. To more carefully determine whether the
negative effects of the 18+-charged protamine variant on myocyte
contractile processes were due to secondary structural effects,
glutamic acid was substituted for proline at the initial amino acid
site (18BE, Fig 1). With this amino acid substitution, the
negative
effects of the original 18+-charged protamine construct (18B) were
completely abolished. Specifically, this modified 18+-charged
protamine variant (18BE) exhibited no effects on baseline myocyte
contractile function or ß-adrenergic responsiveness. The final
two constructs were used to more carefully determine the effects of
amino acid sequence and charge on myocyte contractile function. A
16+-charged variant constructed by deletion of four amino acids
(16BE, Fig 1) exhibited no significant effects on myocyte
contractile
function and ß-adrenergic responsiveness. Finally, a 14+ molecule
was constructed in which positions of alanine and lysine were
alternated (14ALA, Fig 1). This 14+-charged molecule had
no significant
effects on myocyte contractile function or ß-adrenergic
responsiveness.
|
Protamine Localization Studies
In light of the significant
negative effects of unbound protamine
on myocyte contractile processes and inotropic responsiveness, these
final series of studies were undertaken to examine whether the
protamine molecule can enter the interstitial space in the
intact ventricle and interact with the myocyte sarcolemma. In the first
set of experiments, fluorescein-labeled protamine was
circulated in antegradely perfused rabbit hearts, the LV was flushed,
and sections were prepared for microscopy.
Representative photomicrographs of LV
myocardium taken from these experiments are shown in Fig 4.
Strong fluorescein staining was readily
visible along all of the myocardial vasculature.
Fluorescein staining was particularly evident along the
luminal and abluminal surfaces of small venules. More importantly,
strong fluorescein staining could be readily appreciated in
the interstitial space within the myocardium
(Fig 4A). The fluorescein staining appeared to form a
linear pattern and surrounded individual myocytes as well as fascicles
of myocytes. At higher magnification, the
fluorescein-labeled protamine could be readily detected
on both sides of the endothelial cell border of the
myocardial vasculature (Fig 4B). The fluorescein staining
appeared to form a linear array and to stream away from the abluminal
surface of the vasculature into the myocardial interstitium. In larger
blood vessels, the fluorescein-labeled protamine formed
a highly linear pattern between the luminal and abluminal surfaces and
appeared to cascade into the surrounding interstitial space
in a linear fashion (Fig 4C). In a final series of experiments,
isolated myocytes were incubated with
fluorescein-labeled protamine, vigorously washed,
and examined. Representative photomicrographs of
isolated myocytes after incubation with
fluorescein-labeled protamine are shown in Fig 5. Strong
fluorescein staining was observed
in the isolated myocyte preparations, and this staining appeared to be
in a focal pattern of distribution. In all of the myocytes examined
(n=125), a similar patchy distribution of fluorescein
labeling was observed.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
In 1938, Chargraff and Olson41 described the use of protamine as a means by which to reverse the anticoagulant effects of heparin. In 1949, Jaques1 reported potential toxic effects of protamine sulfate when administered to conscious dogs. Since that time, there have been a number of clinical reports on the detrimental and sometimes fatal effects seen after protamine administration in patients.2 3 4 5 6 7 8 9 10 11 12 13 Despite numerous reports regarding the toxicity of the protamine molecule with respect to effects on the systemic vasculature,7 20 42 43 44 45 coagulation system,6 46 47 48 and ventricular pump function,2 3 4 5 10 11 14 15 16 it remains the clinical cornerstone for the reversal of the anticoagulant effects of heparin in cardiovascular procedures. The overall goal of the present project was to determine fundamental mechanisms responsible for the effects of protamine on LV myocyte contractile processes. There were several important and unique findings that were obtained from the present study. First, protamine exposure to isolated LV myocytes decreased basal state contractile function, diminished ß-adrenergic responsiveness, and eliminated the inotropic effects of the cardiac glycoside ouabain. Second, protamine induced significant changes in myocyte sarcolemmal transduction systems that included a reduction in ß-adrenergic receptor and glycoside receptor binding and diminished cAMP production. Third, the fundamental mechanisms for the negative effects of protamine on myocyte contractile processes are not simply due to a high positive charge but also are due to the fundamental structure of the protamine molecule itself. Fourth, in the intact heart, protamine can egress from the myocardial vasculature, enter the extracellular space, and directly come in contact with the myocyte sarcolemma. Thus, findings from the present study suggest that fundamental contributory mechanisms for the observed changes in LV function after protamine administration2 3 4 5 10 11 may be the direct interactive effects of the protamine molecule on myocyte contractile processes and sarcolemmal transduction systems.
Significant changes in LV loading conditions and neurohormonal systems occur in the immediate post–cardiopulmonary bypass period.49 Thus, it remained unclear from these past studies whether protamine had a direct effect on myocyte contractile function. Results from the present study provide direct evidence that unbound protamine has a direct and negative effect on myocyte contractile function and inotropic responsiveness. Although the present study demonstrated that unbound protamine had a direct and negative effect on myocyte contractile function, protamine is routinely administered in the presence of heparin. Accordingly, the present study examined the interactive effects of heparin and protamine on myocyte contractile function. In myocytes exposed to an equivalent concentration of heparin before protamine administration, there was no effect on myocyte contractile function. In contrast, when protamine was placed in the myocyte chamber before the addition of heparin, a significant reduction in myocyte contractile function was observed. These findings suggest that the formation of the heparin-protamine complex has no significant effect on myocyte contractile function. However, if unbound protamine is allowed to interact with the myocyte, subsequent heparin administration cannot reverse the negative effects on myocyte contractile function. The present study demonstrated that unbound protamine adheres in a focal distribution to the myocyte sarcolemma. Taken together, these results suggest that unbound protamine directly interacts with the myocyte and that subsequent heparin administration cannot "rescue" the unbound protamine from the sarcolemmal surface.
ß-Adrenergic receptor agonists are commonly used to improve LV pump function in the early postoperative setting.50 The intracellular effect of ß-adrenergic receptor stimulation is the activation of adenylate cyclase with subsequent cAMP generation. In the present study, protamine caused a significant reduction in myocyte ß-adrenergic responsiveness. The present study also demonstrated that contributory mechanisms for the effects of protamine on myocyte ß-adrenergic responsiveness included diminished ß-adrenergic receptor binding and subsequent cAMP production. Specifically, in the presence of 40 µg/mL protamine, both basal and isoproterenol-stimulated adenylate cyclase activity were reduced. Moreover, in the presence of protamine, cAMP production was diminished despite direct activation of adenylate cyclase using forskolin. Finally, the present study demonstrated that the inhibitory effects of protamine on cAMP production persist in myocytes with an intact sarcolemma. These findings suggest that unbound protamine directly modulates the ß-adrenergic receptor transduction system with subsequent blunting of the contractile response to ß-adrenergic agonists. The clinical implications of the findings from this portion of the study are threefold. First, administration of protamine and ß-adrenergic agonists has a close temporal relationship in the immediate post–cardiopulmonary bypass period. Thus, unbound protamine may cause diminished ß-adrenergic responsiveness in this important postoperative period. Second, in patients with chronic LV dysfunction, blunted ß-adrenergic responsiveness and changes within the ß-adrenergic receptor system have been reported.31 32 51 Findings from the present study suggest that protamine may further exacerbate abnormalities in the ß-adrenergic receptor transduction system in patients with underlying chronic LV dysfunction. Third, results from the present study suggest that administration of ß-adrenergic receptor agonists to ameliorate or reverse the negative effects of protamine on myocyte contractile function may be less than optimal. The present study demonstrated that adenylate cyclase remains active (albeit reduced) in the presence of protamine. Thus, alternative pharmacological approaches such as phosphodiesterase inhibitors that prevent the deactivation of cAMP33 may improve contractile function after protamine administration.
The Na+,K+-ATPase system is located
within the myocyte sarcolemma and is composed of two subunits: and
ß.39 The -subunit folds across the sarcolemma
approximately eight times, and the catalytic portion of this subunit is
located on the intracellular face of the membrane.52 The
Na+,K+-ATPase system is responsible for
maintenance of the myocyte resting membrane potential by
extrusion of Na+ during the late repolarization phase of
the myocyte action potential.53 Thus,
Na+,K+-ATPase function is an important
determinant of myocyte contractile function and ionic homeostasis. The
present study demonstrated that in the presence of protamine,
myocyte inotropic responsiveness to the cardiac glycoside ouabain was
abolished. In addition, the present study demonstrated that a
fundamental contributory mechanism for the effects of protamine on
myocyte responsiveness to ouabain was significant alterations in the
Na+,K+-ATPase glycoside receptor.
Specifically,
in the presence of 40 µg/mL protamine, glycoside-binding capacity
and affinity were significantly reduced. In contrast, protamine did not
significantly affect the catalytic activity of the
Na+,K+-ATPase. One potential explanation for
the differential effects of protamine on the glycoside receptor and
catalytic activity is the location of these two components on the
-subunit of
Na+,K+-ATPase.39
Na+,K+-ATPase ouabain binding has been shown
to
be influenced by alterations in surrounding
phospholipids.54 The present study provided evidence
to suggest that protamine has the capacity to interact with the myocyte
sarcolemmal surface. Thus, the interaction of protamine with
sarcolemmal phospholipids may diminish glycoside receptor binding
characteristics, whereas the catalytic portion of the
Na+,K+-ATPase located on the cytoplasmic
face of the molecule may be unaffected. However, it must be recognized
that the present study examined the hydrolytic capacity of
Na+,K+-ATPase in the presence of
protamine, not actual ionic pumping capacity. Thus, Na+
pumping capacity of Na+,K+-ATPase may be
significantly compromised in the presence of unbound protamine. As
discussed in the following paragraph, the evidence to support this
possibility is the significant changes in myocyte resting membrane
potential that were observed in the presence of protamine.
Several past studies have suggested that protamine may influence electrophysiological characteristics of the myocardium.3 4 19 In the present study, protamine decreased myocyte resting membrane potential (ie, made it less negative). In addition, a higher concentration of protamine (80 µg/mL) reduced action potential upstroke velocity. Important determinants of myocyte action potential upstroke velocity include the electrochemical gradient of Na+ across the sarcolemma,53 the number of channels that are available for Na+ conduction,53 and the resting membrane potential. The more positive resting membrane potential induced by protamine may cause a voltage-dependent inactivation of sarcolemmal Na+ channels and thereby reduce action potential upstroke velocity. During rapid depolarization, Na+ moves into the myocyte, which in turn increases cytosolic Ca2+ and subsequent myocyte contraction. The changes in the upstroke velocity of the myocyte action potential with protamine may cause diminished Na+ influx into the myocyte and thereby cause a reduction in myocyte contractile processes. The present study also demonstrated a significant prolongation of myocyte action potential repolarization in the presence of protamine. These findings suggest that an additional contributory mechanism for the abnormalities in myocyte contractile function in the presence of unbound protamine is the alteration of the normalization of ionic homeostatic processes. Finally, protamine induced changes in the myocyte action potential that may favor the genesis of arrhythmias. There are three important determinants of the myocyte action potential that may contribute to a proarrhythmic substrate: (1) a less negative resting membrane potential, (2) decreased maximum upstroke velocity, and (3) prolongation of action potential duration.55 Lin et al19 demonstrated action potential afterdepolarizations in human atrial tissue after protamine administration. Thus, results from the present study as well as their report suggest that protamine alters myocyte action potential morphology, which may be a potential contributory factor for the genesis of arrhythmias. However, more focused electrophysiological studies performed in the presence of protamine will be necessary to more carefully examine this possibility.
Findings from the present study clearly demonstrate that the highly charged, arginine-rich protamine molecule directly modulates myocyte contractile function and sarcolemmal receptor systems. To more carefully determine fundamental mechanisms for these effects, the present study examined myocyte contractile function and inotropic responsiveness using equivalent concentrations of protamine-like molecular constructs. The effects of these protamine variants on coagulation profiles have been the subject of recent reports.25 26 When the amino acid lysine was substituted for arginine in the amino acid sequence of the native protamine molecule, the overall charge of 21+ was maintained. However, in direct contrast to native protamine, this lysine variant had little effect on myocyte contractile function and inotropic responsiveness. In contrast, when a protamine-like construct containing an overall charge of 18+ was exposed to the myocyte, contractile function and inotropic responsiveness were significantly reduced. When a simple amino acid substitution on this 18+-charge protamine variant (18BE) was performed, the negative effects on myocyte contractile function and inotropic responsiveness were abolished. Thus, the results from this portion of the study clearly suggest that the negative effects of protamine on myocyte contractile processes are not simply due to the overall charge effects of the molecule. Rather, these results suggest that the specific primary and secondary conformations of the protamine molecule play a significant contributory role in the changes in myocyte contractile function and inotropic responsiveness. Several past studies have demonstrated that protamine has a toxic effect on blood cell elements (platelets) by direct interaction with phospholipid moieties on the cell membrane.47 48 The present study demonstrated that protamine altered the ß-adrenergic and cardiac glycoside receptors. A past report demonstrated that protamine had direct effects on cardiac muscarinic receptor binding and suggested that these receptors were allosterically modulated by the protamine molecule.24 Thus, a fundamental mechanism for the negative effects of protamine on myocyte contractile processes may be the direct interaction between a specific region of the protamine molecule and specific phospholipids in the myocyte sarcolemma. Future studies using more sensitive in vitro assay systems will be necessary to test this hypothesis.
Several past experimental studies provide indirect evidence to suggest that protamine has the capacity to exit the vascular compartment and enter the extracellular space, thereby exposing the myocyte to significant concentrations of protamine.56 57 Specifically, it has been demonstrated that the negative charges localized to the endothelium and interstitium result in an interface for ionic exchange.56 Thus, on the basis of these electrochemical conditions, it was suspected that cationic molecules such as protamine would freely enter the interstitium. Results from the present study directly demonstrate that protamine can traverse the myocardial vasculature and directly interface with the myocyte. To the best of our knowledge, this is the first study to directly demonstrate that protamine can directly enter the myocardial interstitium and bind to the myocyte. Delucia and colleagues58 reported that radiolabeled protamine was preferentially distributed to the kidneys, lung, and heart. The results from the present study suggest that a proportion of the radiolabeled protamine quantitated within the myocardium obtained in this past report was located within the myocardial interstitium. These findings coupled with the myocyte contractile function studies provide evidence to suggest that a contributory factor for the changes in LV function after administration of protamine2 3 4 5 6 7 8 9 10 11 may be that the unbound protamine directly interacts with the myocyte. Past reports have suggested that hypothermic cardioplegic arrest and cardiopulmonary bypass cause increased permeability of endothelial cells comprising the myocardial vasculature.59 Since protamine is commonly administered after hypothermic cardioplegic arrest and cardiopulmonary bypass, then findings of the present study that demonstrated an influx of protamine into the extracellular space may be significant in this particular clinical situation.
There have been several clinical and experimental studies that have examined the effects of protamine on LV pump function.2 3 4 5 6 7 8 9 10 11 14 15 16 17 18 19 20 For example, Wakefield and colleagues15 demonstrated that protamine caused a dose-dependent decline in LV peak developed pressure in isolated rabbit hearts. In contrast, there have been past reports that failed to demonstrate a significant change in LV function after protamine administration.60 61 The different findings from these past reports are probably caused by several factors, including experimental design and systemic influences. It has been established that protamine causes the elaboration of a wide variety of cytokines and bioactive peptides.42 43 44 45 For example, Pearson et al44 reported that protamine induces the release of endothelium-derived relaxing factor. Several reports have demonstrated that protamine causes increased plasma thromboxane concentrations.42 43 45 Thus, measurement of the direct effects of protamine on contractile function in the intact LV may be confounded by these extracellular factors. The present study examined the direct effects of protamine on contractile processes in over 300 isolated myocytes and clearly demonstrated that protamine had a direct and negative effect on myocyte contractile function and inotropic responsiveness.
The present study also examined the direct effects of 40 and 80 µg/mL protamine on myocyte contractile function and inotropic responsiveness. These concentrations reflect estimated serum levels that would be found with the clinically administered doses of 2.5 and 5 mg/kg protamine, respectively.8 However, the concentrations used in the present study are dependent on several assumptions. First, the concentrations of protamine used in the current experimental design assume that unbound protamine has an equivalent and predictable volume of distribution. However, it remains unclear whether protamine is equally distributed within the extracellular compartment. Second, protamine is administered in the presence of heparin with subsequent formation of a heparin-protamine complex.20 Thus, it remains speculative as to whether the concentration of protamine used in the present in vitro study reflects actual protamine concentrations to which myocytes would be exposed in vivo. Another limitation of the present study is that the effects of unbound protamine were examined in isolated myocytes and therefore were independent of extracellular influences or nonmyocyte cell populations; the effects of protamine that may be modulated by these extracellular or nonmyocyte cell influences could not be addressed in the present study. Furthermore, in vivo there are several determinants that can influence the amount and duration of protamine to which the myocytes are exposed, including myocardial blood flow, capillary permeability, and extracellular matrix buffering and diffusion. These limitations notwithstanding, the present study demonstrated for the first time that protamine has a direct and negative effect on myocyte contractile function and sarcolemmal transduction systems. In addition, the present study provides evidence that the mechanism for these effects is not simply the high positive charge of the protamine molecule but also the amino acid structure and conformation. On the basis of these findings, future studies addressing the limitations outlined above would be appropriate.
|
Acknowledgments |
|---|
This study was supported by NIH grant R29-HL-45024 (Dr Spinale) and Veterans Administration merit review grant 331 (Dr Wakefield). Dr Spinale is an Established Investigator of the American Heart Association.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1. Jaques LB. A study of the toxicity of the protamine, salmine. Br J Pharmacol. 1949;4:135-144. [Medline] [Order article via Infotrieve]
2.
Jastrzebski J, Sykes MK, Woods DG.
Cardiorespiratory effects of protamine after
cardiopulmonary bypass in man. Thorax. 1974;29:534-538.
3. Olinger GN, Becker RM, Bonchek LI. Noncardiogenic pulmonary edema and peripheral vascular collapse following cardiopulmonary bypass: rare protamine reaction? Ann Thorac Surg. 1980;29:20-25. [Abstract]
4. Gourin A, Streisand RL, Greineder JK, Stuckey JH. Protamine administration and the cardiovascular system. J Thorac Cardiovasc Surg. 1971;62:193-204. [Medline] [Order article via Infotrieve]
5. Shapira N, Schaff HV, Piehler JM, White RD, Still JC, Pluth JR. Cardiovascular effects of protamine sulfate in man. J Thorac Cardiovasc Surg. 1982;84:505-514. [Abstract]
6. Kirklin JK, Chenoweth DE, Naftel DC, Blackstone EH, Kirklin JW, Bitran DD, Curd JG, Reves G, Samuelson PN. Effects of protamine administration after cardiopulmonary bypass on complement, blood elements, and the hemodynamic state. Ann Thorac Surg. 1986;41:193-199.[Abstract]
7. Lowenstein E, Zapoli WM. Protamine reactions, explosive mediator release, and pulmonary vasoconstriction. Anesthesiology. 1990;73:373-375. [Medline] [Order article via Infotrieve]
8.
Horrow JC. Protamine: a review of its
toxicity. Anesth Analg. 1985;64:348-361.
9. Fadali MA, Ledbetter M, Papacostas C, Duke LJ, Lemole GM. Mechanism responsible for the cardiovascular depressant effect of protamine sulfate. Ann Surg. 1974;180:232-235. [Medline] [Order article via Infotrieve]
10.
Del Re MR, Ayd JD, Schultheis LW, Heitmiller ES.
Protamine and left ventricular function: a
transesophageal echocardiography
study. Anesth Analg. 1993;77:1098-1103.
11.
Michaels IA, Barash PG.
Hemodynamic changes during protamine
administration. Anesth Analg. 1983;62:831-835.
12. Wakefield TW, Lindblad B, Stanley TJ, Nichol BJ, Stanley JC, Bergqvist D, Greenfield LJ, Bergentz SE. Heparin and protamine use in peripheral vascular surgery: a comparison between surgeons of the Society for Vascular Surgery and the European Society for Vascular Surgery. Eur J Vasc Surg. 1994;8:193-198. [Medline] [Order article via Infotrieve]
13. Weintraub WS, Wenger NK, Jones EL, Craver JM, Guyton RA. Changing clinical characteristics of coronary surgery patients: differences between men and women. Circulation. 1993;88(suppl II):II-79-II-86.
14. Wakefield TW, Wrobleski SK, Nichol BJ, Kadell AM, Stanley JC. Heparin-mediated reductions of the toxic effects of protamine sulfate on rabbit myocardium. J Vasc Surg. 1992;16:47-53. [Medline] [Order article via Infotrieve]
15. Wakefield TW, Bies LE, Wrobleski SK, Bolling SF, Stanley JC. Impaired myocardial function and oxygen utilization due to protamine sulfate in an isolated rabbit heart preparation. Ann Surg. 1990;212:387-393.[Medline] [Order article via Infotrieve]
16. Hendry PJ, Taichman GC, Keon WJ. The myocardial contractile responses to protamine sulfate and heparin. Ann Thorac Surg. 1987;44:263-268. [Abstract]
17.
Caplan RA, Su JY. Differences in threshold for
protamine toxicity in isolated atrial and ventricular
tissue. Anesth Analg. 1984;63:1111-1115.
18. Hird BR, Crawford FA, Mukherjee R, Zile MR, Spinale FG. The direct effects of protamine sulfate on myocyte contractile function and beta-adrenergic responsiveness. Ann Thor Surg. 1994;57:1066-1075. [Abstract]
19.
Lin CI, Luk HN, Wei J, Tsao SJ.
Electromechanical effects of protamine in isolated human atrial
and canine ventricular tissues. Anesth
Analg. 1989;68:479-485.
20. Hurt R, Perkins HA, Osborn JJ, Gerbode F. The neutralization of heparin by protamine in extracorporeal circulation. J Thorac Surg. 1956;32:612-619.
21. Wakefield TW, Hinshaw DB, Burger JM, Burkel WE, Stanley JC. Protamine-induced reductions of endothelial cell ATP. Surgery. 1989;106:378-385. [Medline] [Order article via Infotrieve]
22. Jaques LB. Protamine-antagonist to heparin. Can Med Assoc J. 1973;108:1291-1297. [Medline] [Order article via Infotrieve]
23. Ferran DS, Sobel M, Harris RB. Design and synthesis of a helix heparin-binding peptide. Biochem. 1992;31:5010-5016. [Medline] [Order article via Infotrieve]
24. Hu J, Wang SZ, Forray C, El-Fakahany EE. Complex allosteric modulation of cardiac muscarinic receptors by protamine: potential model for putative endogenous ligands. Mol Pharmacol. 1992;42:311-321. [Abstract]
25. Wakefield TW, Andrews PC, Wrobleski SK, Kadell AM, Fazzalari A, Nichol BJ, Vanderkooi T, Stanley JC. Reversal of low-molecular-weight heparin anticoagulation by synthetic protamine analogues. J Surg Res. 1994;56:586-593. [Medline] [Order article via Infotrieve]
26. DeLucia A, Wakefield TW, Andrews PC, Nichol BJ, Kadell AM, Wrobleski SK, Downing LJ, Stanley JC. Efficacy and toxicity of differently charged polycationic protamine-like peptides for heparin anticoagulation reversal. J Vasc Surg. 1993;18:49-60. [Medline] [Order article via Infotrieve]
27.
Spinale FG, Mukherjee R, Fulbright BM, Hi J, Crawford
FA, Zile MR. Contractile properties of isolated porcine
ventricular myocytes. Cardiovasc Res. 1993;27:304-311.
28.
Mukherjee R, Crawford FA, Hewett KW, Spinale FG.
Cell and sarcomere contractile performance from the same
cardiocyte using video microscopy. J
Appl Physiol. 1993;74:2023-2033.
29. Tanaka R, Fulbright BM, Mukherjee R, Burchell SA, Crawford FA, Zile MR, Spinale FG. The cellular basis for the blunted response to ß-adrenergic stimulation in supraventricular tachycardia-induced cardiomyopathy. J Mol Cell Cardiol. 1993;25:101-119.
30. Spinale FG, Clayton C, Tanaka R, Fulbright BM, Mukherjee R, Schulte BA, Crawford FA, Zile MR. Myocardial Na+,K+-ATPase in tachycardia induced cardiomyopathy. J Mol Cell Cardiol. 1992;24:277-294. [Medline] [Order article via Infotrieve]
31. Terrar DA, Mitchell MR. Current and voltage-clamp with a single microelectrode: electrical activity associated with contraction in rat and guinea-pig ventricular muscle cells. In: Noble D, Powell T, eds. Electrophysiology of Single Cardiac Cells. Orlando, Fla: Academic Press; 1987:5-24.
32.
Bristow MR, Ginsburg R, Umans V, Fowler M, Minobe W,
Rasmussen R, Zera P, Menlove R, Shah P, Jamieson S, Stinson EB.
ß1- and ß2-adrenergic-receptor
subpopulations in nonfailing and failing human ventricular
myocardium: coupling of both receptor subtypes to muscle
contraction and selective ß1-receptor downregulation in
heart failure. Circ Res. 1986;59:297-309.
33.
Feldman MD, Copelas L, Gwathmey JK, Phillips P, Warren
SE, Schoen FJ, Grossman W, Morgan JP. Deficient
production of cyclic AMP: pharmacologic evidence of an
important cause of contractile dysfunction in patients with
end-stage heart failure. Circulation. 1987;75:331-339.
34. Akera T, Cheng VK. A simple method for the determination of affinity and binding site concentration in receptor binding studies. Biochim Biophys Acta. 1977;470:412-423. [Medline] [Order article via Infotrieve]
35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. [Medline] [Order article via Infotrieve]
36. Blakeslee D, Baines MG. Immunofluorescence using dichlorotriazinylaminofluorescein (DTAF): preparation and fractionation of labelled IgG. J Immunol Methods. 1976;13:305-320. [Medline] [Order article via Infotrieve]
37. Blakeslee D. Immunofluorescence using dichlorotriazinylaminoflourescein (DTAF): preparation, purity and stability of the compound. J Immunol Methods. 1977;17:361-364. [Medline] [Order article via Infotrieve]
38. Steel RG, Torrie JH. Principles and Procedures of Statistics: A Biomedical Approach. New York, NY: McGraw-Hill; 1980:172-553.
39. Eisner DA, Smith TW. The Na-K pump and its effectors in cardiac muscle. In: Fozzard HA, et al, eds. The Heart and Cardiovascular System. New York, NY: Raven Press; 1992:863-902.
40.
Litovsky SH, Antzelevitch C. Transient outward
current prominent in canine ventricular epicardium but not
endocardium. Circ Res. 1988;62:116-126.
41. Chargraff E, Olson KB. Studies on the chemistry of blood coagulation, VI: studies on the action of heparin and other anticoagulants-the influence of protamine on the anticoagulant effect in vivo. J Biol Chem. 1938;122:153-167.
42.
Conzen PR, Habazettl H, Gutmann R, Hobbhahn J, Goetz
AE, Peter K, Brendel W. Thromboxane mediation of
pulmonary hemodynamic responses after
neutralization of heparin by protamine in pigs. Anesth
Analg. 1989;68:25-31.
43.
Morel DR, Lowenstein E, Nguyenduy T, Robinson DR,
Repine JE, Chenoweth DE, Zapol WM. Acute pulmonary
vasoconstriction and thromboxane release during protamine
reversal of heparin anticoagulation in awake sheep: evidence for the
role of reactive oxygen metabolites following nonimmunological
complement activation. Circ Res. 1988;62:905-915.
44.
Pearson PJ, Evora PR, Ayrancioglu K, Schaff HV.
Protamine releases endothelium-derived
relaxing factor from systemic arteries: a possible mechanism of
hypotension during heparin neutralization.
Circulation. 1992;86:289-294.
45. Morel DR, Costabella PM, Pittet JF. Adverse cardiopulmonary effects and increased plasma thromboxane concentrations following the neutralization of heparin with protamine in awake sheep are infusion rate-dependent. Anesthesiology. 1990;73:415-424. [Medline] [Order article via Infotrieve]
46. Lindblad B, Wakefield TW, Whitehouse WM, Stanley JC. The effect of protamine sulfate on platelet function. Scand J Thorac Cardiovasc Surg. 1988;22:55-59. [Medline] [Order article via Infotrieve]
47. Geard-Cobel RJ, Hassouna HI. Interaction of protamine sulfate with thrombin. Am J Hematol. 1983;14:227-233. [Medline] [Order article via Infotrieve]
48. Eika C. On the mechanism of platelet aggregation induced by heparin, protamine and polybrene. Scand J Haematol. 1972;9:248-257. [Medline] [Order article via Infotrieve]
49. Edmunds LH. Systemic inflammatory responses secondary to cardiopulmonary bypass. In: Wechsler AS, ed. Systemic Effects of Cardiopulmonary Bypass. New York, NY: Cahners Publishing Co; 1993:4-98.
50.
Steen PA, Tinker JH, Pluth JR, Barnhorst DA, Tarhan S.
Efficacy of dopamine, dobutamine, and
epinephrine during emergence from cardiopulmonary
bypass in man. Circulation. 1978;57:378-384.
51.
Eschenhagen T, Mende U, Nose M, Schmitz W, Scholz H,
Haverich A, Hirt S, Doring V, Kalmar P, Hoppner W, Seitz HJ.
Increased messenger RNA level of the inhibitory G
protein subunit Gi-2 in human end-stage heart
failure. Circ Res. 1992;70:688-696.
52. Ovchinnikov YA, Modyanov NN, Broude NE, Petrukhin KE, Grishin AV, Arzamazova NM, Aldanova NA, Monastryrskaya GS, Sverdlov ED. Pig kidney Na+,K+-ATPase: primary structure and spatial organization. FEBS Lett. 1986;201:237-245. [Medline] [Order article via Infotrieve]
53. Katz AM. Physiology of the Heart. 2nd ed. New York, NY: Raven Press; 1992:415-472.
54. Yingst DR. Modulation of the Na+,K+-ATPase by Ca and intracellular proteins. Annu Rev Physiol. 1988;50:291-303. [Medline] [Order article via Infotrieve]
55.
Hoffman BF, Rosen MR. Cellular mechanisms for
cardiac arrhythmias. Circ Res. 1981;49:1-15.
56.
Sunnergren KP, Fairman RP, Deblois GG, Glauser FL.
Effects of protamine, heparinase, and hyaluronidase on
endothelial permeability and surface charge.
J Appl Physiol. 1987;63:1987-1992.
57.
Peterson MW, Gruenhaupt D. Protamine interaction
with the epithelial cell surface. J Appl
Physiol. 1992;72:236-241.
58. Delucia A, Wakefield TW, Kadell AM, Wrobleski SK, Vandort M, Stanley JC. Tissue distribution, circulation half-life, and excretion of intravenously administered protamine sulfate. ASAIO J. 1993;39:M715-M718. [Medline] [Order article via Infotrieve]
59. Harjula A, Maattila S, Harkonen M, Myllarniemi H, Nickels J, Merikallio E. Coronary endothelial damage after crystalloid cardioplegia. J Cardiovasc Surg. 1984;25:147-152. [Medline] [Order article via Infotrieve]
60. Pauca AL, Graham JE, Hudspeth AS. Hemodynamic effects of intraaortic administration of protamine. Ann Thorac Surg. 1983;35:637-642. [Abstract]
61. Taylor RL, Little WC, Freeman GL, Avery DM, Norris SE, Trinkle JK, Grover FL. Comparison of the cardiovascular effects of intravenous and intraaortic protamine in the concious and anesthetized dog. Ann Thorac Surg. 1986;42:22-26.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  S. J. Khundmiri, M. A. Metzler, M. Ameen, V. Amin, M. J. Rane, and N. A. Delamere Ouabain induces cell proliferation through calcium-dependent phosphorylation of Akt (protein kinase B) in opossum kidney proximal tubule cells Am J Physiol Cell Physiol, December 1, 2006; 291(6): C1247 - C1257. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Khundmiri, E. J. Weinman, D. Steplock, J. Cole, A. Ahmad, P. D. Baumann, M. Barati, M. J. Rane, and E. Lederer Parathyroid Hormone Regulation of Na+,K+-ATPase Requires the PDZ 1 Domain of Sodium Hydrogen Exchanger Regulatory Factor-1 in Opossum Kidney Cells J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2598 - 2607. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. J. Khundmiri, W. L. Dean, K. R. McLeish, and E. D. Lederer Parathyroid Hormone-mediated Regulation of Na+-K+-ATPase Requires ERK-dependent Translocation of Protein Kinase C{alpha} J. Biol. Chem., March 11, 2005; 280(10): 8705 - 8713. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. D. Lederer, S. J. Khundmiri, and E. J. Weinman Role of NHERF-1 in Regulation of the Activity of Na-K ATPase and Sodium-Phosphate Co-transport in Epithelial Cells J. Am. Soc. Nephrol., July 1, 2003; 14(7): 1711 - 1719. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-J. O, M. H. Cox, R. Mukherjee, M. J. Clair, F. A. Crawford Jr, and F. G. Spinale Direct and Interactive Effects of Cardioplegic Arrest and Protamine on Myocyte Contractility Ann. Thorac. Surg., August 1, 1996; 62(2): 489 - 494. [Abstract] [Full Text]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||