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Eur J Cardiothorac Surg 2001;20:147-152
© 2001 Elsevier Science NL

Protamine cardiotoxicity and nitric oxide

^a Department of Cardiothoracic Surgery, Tel Aviv Sourasky Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel
^b Department of Nephrology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

Received 18 December 2000; received in revised form 20 April 2001; accepted 20 April 2001.

Corresponding author. Tel.: +972-3-6973322; fax: +972-3-6974439
e-mail: shapiraiz{at}tasmc.health.gov.il

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objectives: The purpose of this study is to assess the role of the nitric oxide (NO) pathway in protamine-induced cardiotoxicity and to formulate a possible explanation for this adverse effect. Methods: Isolated rat hearts were perfused by Krebs–Henseleit (KH) solution using a modified Langendorff model. They were randomized into three groups: A, 40 min perfusion with KH solution; B, 20 min perfusion with KH solution and 20 min with protamine; C, as B but Ng-monomethyl-L-arginine (L-NMMA), a non-selective inhibitor of the NO pathway, was added during 40 min of the perfusion period. Left ventricular (LV) function was measured every 10 min. NO and tumor necrosis factor- (TNF) were detected in the effluent from the coronary sinus (CS) and in the supernatant of the cardiac myocytes culture. Nitric oxide synthases (NOS) mRNA levels were determined in groups A and B from LV samples at baseline and after 40 min of perfusion. Results: We found that protamine at a dose of 12 µg/ml causes significant depression of LV function (decreased peak systolic pressure to 22.5±3.2% and dP/dt max to 22.9±3.1%). L-NMMA did not prevent protamine cardiotoxicity. NOS mRNA was not detected from LV samples in any group. The NO in the effluent from the CS and from the supernatant of the cardiomyocytes culture was below detectable levels. However, a significant amount of TNF was measured in the effluent from the CS (108±17 pg/min for group B and 117±13 pg/min for group C) and in the supernatant of the cardiomyocytes culture (65±21 pg/ml). Conclusions: This study suggests that direct protamine-induced cardiotoxicity does not depend on the NO pathway. Our finding that protamine induced TNF release by cardiomyocytes can shed new light on the understanding of protamine cardiotoxicity.

Key Words: Protamine • Nitric oxide • Tumor necrosis factor- • Isolated perfused heart

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The administration of protamine sulfate (protamine) for heparin reversal may cause several circulatory side effects such as systemic hypotension, pulmonary hypertension and left ventricular (LV) dysfunction [1–3]. Despite its extensive clinical application, the mechanisms of protamine-induced hemodynamic changes and direct cardiotoxic effect have not been fully determined.

One of the mechanisms that has been suggested for protamine-induced systemic vasodilatation is an increased production of nitric oxide (NO). Protamine is an arginine-rich protein, and L-arginine is the physiological substrate of NO production [4,5]. Cardiac myocytes express two types of nitric oxide synthases (NOS), endothelial (eNOS) and inducible (iNOS). eNOS activity is regulated by the contractile state of the heart, while iNOS expression is induced by cytokines [6,7]. Excessive production of NO by cardiomyocytes causes contractile dysfunction and depression of cardiac function [7,8]. Some laboratory studies found that protamine has a direct cardiotoxic effect [9]. Some of the investigators proposed that protamine cardiotoxicity was related to excessive positive charging of protamine [3,10]. On the other hand, it was shown that the protamine causes activation of leukocytes and increases in the blood level of the cardiotoxic cytokine, tumor necrosis factor- (TNF) [11]. TNF may result itself in an increased NO production [12].

The present study was undertaken to determinate the direct effect of protamine on the NO system in the isolated perfused rat heart. NO release and specific myocardial eNOS and iNOS mRNA were determined, as was the influence of NO release after protamine induced its effect on rat cardiomyocytes culture. TNF was detected in the heart perfusate effluent and in the supernatant of cardiac myocytes culture.

To the best of our knowledge, this is the first investigation of a possible participation of the NO pathway in direct protamine-induced heart dysfunction. In addition, we have now shown that protamine causes TNF production in an isolated non-blood perfused rat heart and in cardiac myocytes culture.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The investigation of the mechanism by which protamine induced cardiotoxicity was carried out by using a modified Langendorff perfusion system and employing a rat cardiac myocyte culture.

2.1. Langendorff perfusion system
Adult male Wistar rats (350–400 g) were anesthetized by an intraperitoneal injection of pentobarbital sodium (30 mg/kg). Their hearts were rapidly excised, immersed in ice-cold saline with heparin, and mounted on a stainless steel cannula of a modified Langendorff perfusion system. Retrograde aortic perfusion was initiated at a perfusion pressure of 85 mmHg with an oxygenated modified Krebs–Henseleit (KH) buffer solution: NaCl 118 mmol/l; KCl 4.7 mmol/l; CaCl₂ 2.0 mmol/l; MgSO₄ 7H₂O 1.2 mmol/l; KH₂PO₄ 1.2 mmol/l; glucose 11.1 mmol/l; and NaHCO₃ 25 mmol/l. The perfusate was bubbled continuously with 95% O₂ and 5% CO₂, maintaining a pH of 7.4–7.5. Our Langendorff system contained two separate reservoirs for retrograde aortic perfusion that provided the possibility of changing perfusion solutions during the experiment. The reservoirs were filled with a separate pump.

Cardiac temperature was measured by a thermistor implanted in the right ventricular wall and carefully maintained at 37°C by wrapping a water jacket around the perfusate reservoir and the isolated heart. The right atrium was partially removed, and the heart was paced to 300 beats/min at 4 V using an external pacemaker (type E4162; Devices Limited, Implants Division), ensuring an identical heart rate for all hearts. A water-filled latex balloon was inserted into the LV cavity via a small left atrium incision and connected to a Mennen Medical PI 32284 pressure transducer. The balloon was tied and inflated to a volume that produced 0 mmHg diastolic pressure.

2.2. Protocol
Twenty-four rats were randomized into three subgroups (A, B, and C) of eight animals each. After a 15 min period of stabilization (baseline point) LV pressure, contractility (dP/dt max, dP/dt min, pressure–time integral) and coronary flow were measured every 10 min. All the hearts were perfused for 40 min. Determination of NO and TNF in the effluent from the coronary sinus for all three groups was carried out at baseline and after 40 min of perfusion.

2.3. Experimental groups
Group A (control) hearts were perfused with KH solution alone. In group B, after 20 min of perfusion with KH solution, protamine (12 µg/ml) was added for 20 min. Group C hearts shared the same protocol as group B, but Ng-monomethyl-L-arginine (L-NMMA, 10 µmol/l), a competitive inhibitor of eNOS and iNOS [13], was added to the KH solution after the stabilization period during all stages of perfusion. This dose of L-NMMA was found to cause inhibition of NOS with no influence on the isolated perfused heart performance [14].

Ventricular fibrillation was caused by a concentration of 25–50 µg/ml of protamine in perfusate, equivalent to a clinical dose of 1.5–3.0 mg/kg body weight [15], assuming equal distribution throughout in the circulating volume used in our pilot experiments. After titration, we used a dose of 12 µg/ml, which caused cardiac depression without ventricular fibrillation.

2.4. TNF determination
TNF levels were detected in both the heart effluent and cell culture supernatant. Effluent samples from the coronary sinus for TNF measurement were drawn at the baseline point and after 40 min of perfusion and were immediately stored at -70°C until assayed. TNF activity was measured using the commercially available ELISA kit Cytoscreen^TM rat kit TNF, Immunoassay kit (Biosource, Camarillo, CA). The limit of detection was 4 pg/ml.

2.5. NO measurement
Effluent nitrite (NO₂) and nitrate (NO₃), stable metabolites of NO representing NO production, were detected in the heart perfusate samples and cardiac myocytes culture supernatant, as previously described [16]. NO₃ was reduced to NO₂ by nitrate reductase prepared in our laboratory from Escherichia coli incubated in anaerobic conditions with KNO₃ [17]. After incubation of samples with E. coli preparate, the concentration of total NO₂ (formed from NO₃ and preexisted) was determined using Griess reagent (1% sulfanilamide and 0.1% N-(1-naphtyl)-ethylenediamine dihydrochloride in 2.5% phosphoric acid). Reagent was added to the sample in the proportion of 2:1, absorbance was measured at 546 nm and the NO₂ concentration was calculated from the calibration curve obtained by spectrophotometry of similarly treated standard solutions (2–50 µmol/l Na NO₂). The limit of detection was 2 µmol/l.

2.6. Myocardial tissue eNOS and iNOS determination
Immediately after a stabilization period (baseline, four additional hearts) and after perfusion with protamine (n=5), the myocardium of the LV was excised and placed in cold Hank's balanced solution. The LV of hearts perfused for 40 min with KH alone served as a control for hearts perfused with protamine (n=5). Total RNA was extracted from myocardial samples using the guanidinium thiocyanate method [18]. RNA pellets were kept at -20°C with 75% ethanol until assay. Dried sediments were dissolved in sterile RNAse-free water and quantified spectrophotometrically at =260 nm.

Total RNA (2 µg) was subjected to reverse transcription reaction in 20 µl of reaction mixture using a transcription system (Promega, USA). After completion of the reaction, 5 µl of this reaction mixture was used for eNOS and iNOS cDNA polymerase chain reaction (PCR) amplification, and 5 µl of 1:10 diluted reaction mixture was used for GAPDH cDNA amplification. Our PCR negative control contained H₂O instead of cDNA, and the cDNA negative control contained H₂O instead of RNA. Primer sequences, annealing temperature, number of cycles and PCR product size [19] are reported in Table 1.

2.8. Rat cardiac myocyte cultures
Myocyte cultures were derived from the excised hearts of neonatal Wistar rats, which were finely minced. The pieces were immersed in dissociation solution (calcium- and magnesium-free Hank's balanced salt solution, Gibco, Bassel, Switzerland) containing a 1:200 dilution of RDB enzyme (Institute of Biology, Nes-Ziona, Israel). One million cells were placed in 0.1% gelatin pre-coated 35 mm tissue culture dishes. The medium was replaced on the following day. Cardiac myocyte cultures were treated with cytosine arabinoside (3 mmol/l) 1 day after seeding for 2 days to abort the multiplication of fibroblasts and other dividing cells, but there was no effect on cardiac myocytes since they were essentially post-mitotic. Thus, experiments were done on a highly rich cardiac myocytes population [20]. Using the periodic acid Schiff procedure [21], the amount of fibroblasts in the cardiac myocytes culture never exceeded 10%.

Cardiac myocytes were treated with protamine on the fourth day after seeding, when the cultures were confluent. Fresh medium was introduced and protamine (150 µg/ml) was added to the cardiac myocyte cultures. Measurement of NO was performed after 24 h of storage with protamine, and TNF assessment was performed after 1 h. For the purpose of comparison, NO and TNF were measured in the myocytes culture without protamine after 24 h and after 1 h of incubation, respectively.

2.9. Drugs
Protamine sulfate and heparin sodium were purchased from Kamada (Israel). L-NMMA was purchased from Sigma.

2.10. Ethics
Animal care complied with the ‘Principles of Laboratory Animal Care’ and the ‘Guide for the Care and Use of Laboratory Animals’ (NIH No. 85-23, Revised 1985).

2.11. Statistical analysis
The results are presented as the mean±SE. All measurements were subjected to one-way analysis of variance (ANOVA) with repeated measures. NO measurements in the coronary effluent were normalized to a 1 min volume of coronary flow. Significance was established at a level of P<0.05. All statistical analyses were performed by the Statistical Department of our Medical Center using an SPSS computer program.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. Hemodynamic changes
The baseline values for different LV hemodynamic parameters are given in Table 2. No significant differences were found between the experimental groups. Protamine was found to cause significant depression of LV function and to decrease coronary flow (for all variables of group B after protamine addition in comparison to group A: P<0.05, Fig. 1) . Thus, protamine caused a decrease of peak systolic pressure (to 22.5±3.2%, P<0.005), dP/dt max (to 22.9±3.1%, P<0.005), pressure–time integral (to 25.1±3.7, P<0.005), and coronary flow (to 59±4%, P<0.05), and an elevation of end-diastolic pressure (to 22±6 mmHg, P<0.05). The L-NMMA competitive inhibitor of eNOS and iNOS (group C) did not prevent protamine cardiotoxicity. No significant differences were found between groups B and C (Fig. 1).

View larger version (21K): [in this window] [in a new window]   Fig. 1. Hemodynamic performance of isolated rat hearts during 1 h of perfusion: control group (white bars), hearts treated with protamine (dashed bars), and hearts treated with L-NMMA before protamine action (black bars). No significant differences were found between the group treated with protamine and the group treated with L-NMMA+protamine. Results are presented as each heart's percentage of baseline measurements.

After 24 h of cardiomyocytes culture incubation, the control NO supernatant levels were 3.85±2.6 µmol/l. There was no significant (P=0.34) increase of NO supernatant levels after 24 h of storage with protamine (4.19±2.3 µmol/l).

3.3. Effect of protamine on eNOS and iNOS mRNA expression
Basal eNOS and iNOS mRNA expression (group A) was detected in LV samples after the stabilization period and did not change following 40 min of perfusion (Figs. 2A and 3A) . The intensities of the bands at baseline and at the end of 40 min of perfusion were 0.41±0.03 and 0.39±0.03 (P=0.157, Fig. 2B), respectively, for eNOS and 0.60±0.02 and 0.67±0.02 (P=0.136, Fig. 3B), respectively, for iNOS.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Effect of protamine on myocardial eNOS mRNA expression in isolated perfused hearts: representative PCR analysis of RNA samples (in duplicates) (A), and relative optical density of eNOS PCR signal. Data were normalized to the relative GAPDH PCR signal. (B) Samples withdrawn at 15 min after onset of perfusion (1 or baseline), 20 min after protamine addition (40 min of perfusion since baseline, 2 or 20' Protamine) and after 40 min of perfusion with KH solution (3 or 40' KH, controls). eNOS mRNA expression is the same in all groups, and no significant changes were found in any band intensity.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effect of protamine on myocardial iNOS mRNA expression in isolated perfused hearts: representative PCR analysis of RNA samples (in duplicates) (A), and relative optical density of iNOS PCR signal. Data were normalized to the relative GAPDH PCR signal. (B) Samples withdrawn at 15 min after onset of perfusion (1 or baseline), 20 min after protamine addition (40 min of perfusion since baseline, 2 or 20' Protamine) and after 40 min of perfusion with KH solution (3 or 40' KH, controls). iNOS mRNA expression is the same in all groups and no significant changes were founded in any band intensity.

3.4. Effect of protamine on TNF release
Significant amounts of TNF, i.e. 108.3±16.7 pg/min (group B) and 117±12.8 pg/min (group C), were detected in the effluent after a 20 min perfusion (i.e. 40 min of perfusion from baseline) with protamine and with L-NMMA+protamine, respectively. The TNF was below detectable levels in samples taken before the addition of protamine and in the control group after the 40 min perfusion.

Significant amounts of TNF (65.4±21.3 pg/ml) were detected in the supernatant of the cardiomyocytes culture after 1 h of incubation with protamine. The TNF in the supernatant was below detectable levels in the non-protamine-treated cardiomyocytes culture.

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
In an excellent review of the heparin protamine interaction, Carr and Silverman [3] summarize the multiple effects of protamine neutralization of heparin. It was suggested that the numerous cardiovascular effects of protamine are mediated by complement activation, histamine, thromboxane antibody formation and by increased NO production [3,22].

The possible effects of protamine on the vascular smooth muscle relaxation by NO have already been suggested [22]. Protamine causes stimulation of NO production by endothelial cells in tissue culture [23].

Removal of the endothelium or the administration of L-NMMA prevented protamine-induced vasodilatation in mammalian arteries. In our laboratory protamine induces endothelial NO-dependent relaxation of the human internal thoracic artery and this was prevented by NOS inhibitors. Recent studies showed that an excessive production of NO by myocytes causes contractile dysfunction and depression of cardiac function [6–8]. Cardiac myocytes express two types of NO synthase: eNOS and iNOS [6,8]. iNOS is not a constitutive form, and needs to be activated. However, in some cells there is low basal-specific iNOS activity. iNOS can be activated by cytokines leading to NO overproduction and causes inappropriate vasodilatation and negative inotropic effects [8,9]. The NO produced endogenously in cardiac myocytes is reversed by NOS inhibition [8]. All these favor a possible protamine–NO system mechanism in the protamine cardiotoxicity. We hypothesized that protamine, which is rich in L-arginine, a physiological precursor of NO [4], augmented NO formation by supplying exogenous L-arginine as a metabolic substrate for NO production.

In the present study, we could not find any evidence that the protamine-induced cardiotoxic effects on isolated perfused rat hearts are related to increased NO production. Neither the NO level in effluent of perfusate nor the LV myocardium-specific eNOS or iNOS mRNA changed after the addition of protamine. Furthermore, the cardiotoxic effect caused by protamine was not reversed by the non-specific NOS inhibitor, L-NMMA.

In the present study TNF increased in both perfusate effluent and cardiomyocytes cell culture supernatants after protamine exposure. However, this did not result in NO changes. TNF is a proinflammatory cytokine with potent negative inotropic properties [24]. Our study has several limitations. The study was performed in vitro. Non-blood perfusion excluded the possibility for protamine to bind to blood protein and excluded an influence of blood immunological factors.

The mechanisms of protamine-induced TNF release are unknown. The possible causes of TNF production were described by Dr Meldrum in his excellent review [25]. Free radicals or cytokines cause activation of the intracellular signaling cascade, leading to activation of nuclear transcription factor kappa B and translocation of this factor to the nucleus for activation of TNF promoters. At least two TNF promoter sites must be occupied by nuclear transcription factor kappa B in order for TNF gene transcription to occur. Protamine probably induces the formation of free radicals or directly binds to the cell membrane and causes activation of the intracellular signaling cascade. Our findings of protamine-induced local TNF synthesis by the rat's myocytes can shed new light on the understanding of the cardiotoxic effect of protamine.

4.1. Conclusions
In contrast to the findings of many investigators who showed that protamine causes its adverse effects via excessive NO production in isolated vessels, our results demonstrated that its direct cardiotoxic effect is not associated with the NO pathway. We found that protamine induces the release of the cardiotoxic cytokine TNF from cardiomyocytes. This TNF release may be one possible mechanism of protamine-induced cardiotoxicity.

	Acknowledgments

 
We thank Esther Eshkol for editorial assistance.

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

1. Horrow J.C. Protamine: a review of its toxicity. Anesth Analg 1985;64:348-361.[Free Full Text]
2. Porsche R., Brenner Z.R. Allergy to protamine sulfate. Heart Lung 1999;28:418-428.[Medline]
3. Carr J.A., Silverman N. The heparin protamine interaction. J Cardiovasc Surg (Torino) 1999;40:659-666.[Medline]
4. Palmer R.M.J., Rees D.D., Ashton D.S., Moncada S. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-1256.[Medline]
5. Fan B., Wang J., Stuehr D.S., Rousseau D.L. NO synthase isoenzymes have distinct substrate binding sites. Biochemistry 1997;21:12660-12665.
6. Balligand J.L., Cannon P.J. Nitric oxide synthases and cardiac muscle. Autocrine and paracrine influences. Arterioscler Thromb Vasc Biol 1997;17:1846-1858.[Abstract/Free Full Text]
7. Ikeda U., Shimada K. Nitric oxide and cardiac failure. Clin Cardiol 1997;20:837-841.[Medline]
8. Joe E.K., Schussheim A.E., Longrois D., Maki T., Kelly R.A., Smith T.W., Balligand J.L. Regulation of cardiac myocytes contractile function by inducible nitric oxide synthase (iNOS): mechanisms of contractile depression by nitric oxide. J Mol Cell Cardiol 1998;30:303-315.[Medline]
9. Hird R.B., Crawford F.A., Mukherjee R., Zile M.R., Spinale F.G. Effect of protamine on myocyte contractile function and ß adrenergic responsiveness. Ann Thorac Surg 1994;57:1066-1074.[Abstract]
10. Wakefield T.W., Wrobleski S.K., Nichol B.J., Radell A.M., Stanley J.C. Heparin-mediated redaction of the toxic effect of protamine sulfate on rabbit myocardium. J Vasc Surg 1992;1:47-53.
11. Katircioglu S.F., Ulus T., Yamak B., Saritas Z., Yildiz U. Experimental inhibition of protamine cardiotoxicity by prostacyclin. Angiology 1999;11:929-935.
12. Ferdinandy P., Danial H., Ambrus I., Rothery R.A., Shulz R. Peroxynitrite is a major contributor to cytokine induced myocardial contractile failure. Circ Res 2000;87:241-247.[Abstract/Free Full Text]
13. Wolff D.J., Lubeskie A. Inactivation of nitric oxide synthase isoforms by diaminoguanidine and N^G-amino-L-argenine. Arch Biochem Biophys 1996;325:227-234.[Medline]
14. Finkel M.S., Oddis C.V., Jacob T.D., Watkins S.C., Halter B.G., Simmons R.L. Negative inotropic effect of cytokines on the heart mediated by nitric oxide. Science 1992;257:387-389.[Abstract/Free Full Text]
15. Halpern P., Hochhauser E., Puzhevsky A., Rudick V., Sorkine P., Vidne B. The isolated post ischemic heart is more vulnerable to protamine sulphate than the non-ischemic heart. Cardiovasc Surg 1997;5:235-240.[Medline]
16. Goor Y., Peer G., Laina A., Blum M., Wollman Y., Chernihovsky T., Silverberg D., Cabili S. Nitric oxide in ischemic acute renal failure of streptozocin diabetic rats. Diabetologia 1996;39:1036-1040.[Medline]
17. Cole J.A., Wimpenny J.W. Metabolic pathways for nitrate reduction in Escherichia coli. Biochem Biophys Acta 1968;162:39-48.[Medline]
18. Terada Y., Tomita K., Nonoguchi H., Marumo F. Polymerase chain reaction localization of constitutive nitric oxide synthase and soluble guanylate cyclase messenger RNAs in microdissected rat nephron segment. J Clin Invest 1992;90:659-665.
19. Morrissey J.J., McCracken R., Kaneto H., Vehaskari M., Montani D., Klahr S. Location of an inducible nitric oxide synthase mRNA in the normal kidney. Kidney Int 1994;45:998-1005.[Medline]
20. Zangen A., Shainberg A. Thiamine deficiency in cardiac cells in culture. Biochem Pharmacol 1997;54:575-582.[Medline]
21. Wasserman L., Kessler-Icekson G. A simple method for the preparation of permanent slides from cell cultures. Stain Technol 1984;59:353-354.[Medline]
22. Pearson P.J., Evora P.R., Ayrancioglu K., Shaff H.V. Protamine releases endothelium-derived relaxing factor from systemic arteries. Circulation 1992;86:289-294.[Abstract/Free Full Text]
23. Li J.M., Hajarizdeh H., La Rosa C.A., Rohrer M.J., VanderSalm T.J., Cutler B.S. Heparin and protamine stimulate the production of nitric oxide. J Cardiovasc Surg (Torino) 1996;37:445-452.[Medline]
24. Yokoyama T., Vaca L., Rossen R.D., Durante W., Hazarica P., Mann D.L. Cellular basis for the negative inotropic effect of tumor necrosis factor in the adult mammalian heart. J Clin Invest 1993;92:2303-2312.
25. Meldrum D.R. Tumor necrosis factor in the heart. Am J Physiol 1998;274:R577-R595.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Pevni, D. Articles by Mohr, R. Search for Related Content PubMed PubMed Citation Articles by Pevni, D. Articles by Mohr, R. Related Collections Cardiac - pharmacology Cardiac - physiology