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Table of Contents
ORIGINAL ARTICLE
Year : 2017  |  Volume : 6  |  Issue : 4  |  Page : 45-49

Cardiac surgery with crystalloid cardioplegia: Improved functional recovery due to molecular adaptations in adult rat hearts


1 Department of Cardiovascular Surgery, Justus Liebig University Gießen, Germany
2 Institute of Physiology, Justus Liebig University Gießen, Hesse, Germany

Date of Web Publication22-Jan-2018

Correspondence Address:
Prof. Andreas Boening
Department of Cardiovascular Surgery, Justus-Liebig-University Giessen, Hesse
Germany
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/rcm.rcm_33_17

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  Abstract 


Background: The effect of aging on functional recovery after a period of crystalloid cardioplegic arrest is still a matter of debate. We hypothesized that age-dependent differences in the polyamine metabolism may contribute to such differences. Methods: Hearts from juvenile and adult Wistar rats were placed in a perfused beating heart model and given Bretschneider's cardioplegia for an ischemia period of 60 min. During reperfusion, recovery of contractile function and coronary blood flow were measured for 90 min. In addition, adult hearts received putrescine to bypass polyamine metabolism during the 1st min of reperfusion. In comparison, the effect of putrescine was analyzed from hearts reperfused after 45-min flow arrest for 90 min. The rate-limiting enzyme of the polyamine metabolism, ornithine decarboxylase (ODC), the proapoptotic enzyme bax, and the relation between SR-calcium-ATPase (SERCA2a) and a natrium-calcium-exchanger enzyme were determined on mRNA-level through real-time polymerase chain reaction. Results: Adult hearts had lower basal performance and lower SERCA mRNA expression compared to juvenile hearts. However, after a 60-min aortic clamping period, recovery of left ventricular developed pressure (105.6 ± 39.7% of baseline) in the adult group was better than in the young group (61.3 ± 34.1% of baseline). ODC mRNA was significantly (P = 0.04228) lower in adult hearts (0.60 ± 0.09-fold vs. juvenile rats). Similar, bax mRNA was significantly (P = 0.01662) lower in adult hearts (0.22 ± 0.03-fold vs. juvenile rats). Addition of putrescine to adult hearts during reperfusion attenuated a better outcome of these hearts suggesting a detrimental effect of polyamine metabolism after cardioplegic arrest. In contrast, putrescine improved recovery in postischemic hearts without exposure to cardioplegic solution. Conclusion: Adult rat hearts tolerate cardioplegia-mitigated ischemia better than juvenile hearts because they express less ODC during resubstitution of normal calcium levels.

Keywords: Apoptosis, ischemia, polyamines, reperfusion


How to cite this article:
Boening A, Attmann T, Heep M, Niemann B, Grieshaber P, Schreckenberg R, Schlueter KD. Cardiac surgery with crystalloid cardioplegia: Improved functional recovery due to molecular adaptations in adult rat hearts. Res Cardiovasc Med 2017;6:45-9

How to cite this URL:
Boening A, Attmann T, Heep M, Niemann B, Grieshaber P, Schreckenberg R, Schlueter KD. Cardiac surgery with crystalloid cardioplegia: Improved functional recovery due to molecular adaptations in adult rat hearts. Res Cardiovasc Med [serial online] 2017 [cited 2022 Jul 2];6:45-9. Available from: https://www.rcvmonline.com/text.asp?2017/6/4/45/223786


  Introduction Top


Cardiac arrest is an essential part of heart surgery, unavoidable leading to ischemia/reperfusion changes in the heart. Cardioplegia induces diastolic cardiac arrest and attenuates ischemic injuries, not being able to avoid them totally. The degree of these changes influences the intra- and postoperative recovery of the heart and the circulation. Young and old hearts seem to behave differently regarding the ability to cope with ischemia/reperfusion injury.[1]

Crystalloid cardioplegic solutions are often void of calcium. Calcium is required for the maintenance of contractile activity, proper formation of cell–cell contacts, but it is also a ligand of calcium-sensing receptors that are expressed in cardiomyocytes and activated by positive-charged ions and polyamines.[2] Readministration of calcium to hearts after a period of calcium-free perfusion results in uncontrolled Ca 2+ influx and cell damage. Polyamines, such as putrescine, formed by the activation of ornithine decarboxylase (ODC) are involved in the mediation of abnormal calcium influx and subsequent functional impairment due to the calcium paradox.[3],[4] Cardiac ODC activity and ODC expression decline as a function of age.[5] Following these former findings about the role of polyamine metabolism at readmission of calcium after a period of calcium loss and the age-dependent decrease in the expression of the rate-limiting enzyme ODC, we hypothesized that adult rat hearts are better protected against reperfusion injury after a period of cardiac arrest with crystalloid cardioplegic solution than juvenile rat hearts. To verify this hypothesis, we determined the expression of ODC in adult and juvenile rat hearts and investigated the functional recovery of rat hearts exposed to 60-min cardioplegic arrest with a crystalloid cardioplegic solution and subsequent reperfusion for 90 min [Figure 1]. In the same setting, we investigated the effect of putrescine to establish a causal relationship between the activation of polyamine metabolism and the functional recovery.
Figure 1: Schematic drawing of the central role of Ca during electromechanical coupling in the myocardial cell

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  Methods Top


All experiments were approved by the regional authorities and conformed to the German Animal Protection Law.

Experimental model

Wistar rats aging either 3 months (juvenile group, 200–280 g) or 12 months (adult group, 470–690 g) were premedicated by flowing the inspiration air flow to 5% Isofluran or anesthetized with Pentobarbital-Natrium (0.375 mg/kg body weight i.p.). After exposure of the heart and anticoagulation with heparin (2000 U/kg, intravenous), the heart was rapidly excised and then mounted in an organ chamber on a Langendorff perfusion system (Isolated Heart IH-SR, Harvard Apparatus). The mean heart weight was 0.96 g in the juvenile group and 2.27 g in the adult group. The heart was retrogradely perfused using a constant flow of about 8 ml/g heart weight with a modified Krebs–Henseleit solution (KHS) with the following composition (mmol/l): NaCl (140), NaHCO3(24), NaH2 PO4(0.4), KCl (2.7), MgCl2(1.0), CaCl2(1.8), and glucose (5.0). The KHS was equilibrated with 95% O2 and 5% CO2 and adjusted to a pH of 7.35–7.45 at 37°C. While coronary flow was constant, aortic pressure was allowed to shift and served as a parameter for left ventricular power.

Measurements

Isovolumetric measurement of left ventricular (LV) performance was made using a compliant latex balloon connected to a pressure transducer, inserted in the LV across the mitral valve. The LV balloon was filled with saline. The volume of the balloon was left constant during the whole experiment. LV performance was assessed by measurement of LV systolic pressure (LSVP) and LV end-diastolic pressure (mmHg, LVSP – LV end-diastolic pressure = LV-developed pressure (LVDP). Positive and negative first derivatives of LSVP (+dP/dt and –dP/dt, mmHg/s) were recorded by an transducer amplifier module (TAM-A, Harvard Apparatus) and calculated by the software “Isoheart” (Harvard Apparatus). While coronary flow was constant, aortic pressure (AoP, mmHg) was measured.

Experimental protocols

In the 3 cardioplegia groups (adult rat hearts, juvenile rat hearts, and adult hearts with putrescine), after 30 min of equilibration, the hearts received cold crystalloid cardioplegia (“Custodiol,” Bretschneider's solution, Köhler Chemie, Germany) for 5 min through the aortic cannula with a constant flow of about 8 ml/g heart weight, followed by a cold ischemic period of 60 min. The composition of Bretschneider's cardioplegia was (mmol/l): NaCl (15), KCl (9), MgCl2(4), Histidinhydrochlorid-Monohydrat (18), Histidin (180), Tryptophan (2), Mannitol (30), CaCl2(0.015), and Potassiumhydrogen-2-oxopentandioat (1). To imitate the clinical situation, we chose to give Bretschneider's cardioplegia in a single shot and to shorten the aortic clamping period down to 60 min. After a total of 60 min cardioplegic arrest, the heart was reperfused for 90 min with KHS. If putrescine (100 μM) was administered this was given during the first 10 min of reperfusion. The hearts were then excised and analyzed for apoptosis parameters and molecular comparisons.

In the two control groups, after an initial equilibration period, hearts were exposed to 45-min ischemia and subsequently reperfused for 90 min: Two groups of juvenile rat hearts were used under these conditions with one group receiving putrescine during the first 10 min of reperfusion (n = 8 each).

RNA isolation and real-time polymerase chain reaction

At the end of the experiments, left ventricles including the septum were separated from atria and right ventricles. Total RNA from left ventricles was extracted with Trizol (Invitrogen) as described by the manufacturer. RT reactions were performed for 1 h at 37°C in a final volume of 10 μl using 1 μg RNA, 100 ng of oligo (dT)15, 1 mmol/l dNTPs, 8 units of RNasin, and 60 units of Moloney murine leukemia virus reverse transcriptase. Aliquots were used for real-time polymerase chain reaction (RT-PCR) using the I-cycler (Biorad, Germany) and SYBR-green fluorescence for quantification. HPRT was used as a housekeeping gene to normalize sample contents. Primers used for determination had the following sequences: HPRT forward: CCA GCG TCG TGA TTA GTG AS, HPRT reverse: CAA GTC TTT CAG TCC TGT CC, ODC forward: GAA GAT GAG TCA AAC GAG CA, ODC reverse: AGT AGA TGT TTG GCC TCT GG; bax forward: ACT AAA GTG CCC GAG CTG ATC, bax reverse: CAC TGT CTG CCA TGT GGG G, bcl-2 forward ATG GCG CAA GCC GGG AGA AC, bcl-2 reverse: CTT GTG GCC CAG GTA TGC AC, SR-calcium-ATPase (SERCA) forward: CGA GTT GAA CCT TCC CAC AA; SERCA reverse: AGG AGA TGA GGT AGC GGA TGA; natrium-calcium-exchanger (NCX) forward: CCG TAA TCA GCA TTT CAG AG, NCX reverse: GCC AGG TTC GTC TTC TTA AT. The calculations of the results were carried out according to the 2−ΔΔCt methods as described.[6] After amplification reaction, products were controlled and separated on 2% agarose gels, stained with SYBR Safe, and photographed under ultraviolet illumination.

Data analysis

Statistical analysis was performed with SPSS 17.0, IBM, Munich, Germany. The data are presented as mean ± standard errors. Comparisons between groups were assessed for significance by analysis of variance. If significance was established, post hoc analysis was assessed by Student-Newman–Keuls test, which allowed for multiple comparisons. If only two groups were compared, this was performed by paired t-tests, as appropriate. Data were analyzed for equal variance by Levene's test. Statistically significant differences were assumed at a level of P < 0.05.


  Results Top


Functional recovery of rat hearts exposed to cardioplegic arrest and reperfusion

To determine the functional recovery of rat hearts exposed to cardioplegic arrest and reperfusion, we studied three different groups of rat hearts: One juvenile group (3-month-old rat hearts), two adult groups (12-month-old rat hearts) with or without putrescine intervention. Basal characteristics of the three different groups are given in [Table 1]. As expected, hearts from adult versus juvenile rats had higher heart weights but normal heart-to-body weight and lower basal function. We, then, investigated the functional recovery of these hearts after cardioplegic arrest: the functional recovery was determined at the end of the reperfusion period (90-min reperfusion) and was expressed relative to basal values determined before cardioplegic arrest. As shown in [Table 2], adult rat hearts had a significantly better functional recovery than juvenile rat hearts. This was found on the basis of LVDP and improved systolic power (+dP/dt). In contrast, relaxation parameters (−dP/dt) were not significantly altered. Of note, putrescine significantly reduced the better functional outcome in hearts from adult rats [Table 2].
Table 1: Basal characteristics of the experimental groups

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Table 2: Recovery data of the experimental groups 90 min after initiation of reperfusion in percentage of initial values

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Molecular characterization of rat hearts exposed to cardioplegic arrest and reperfusion

The data on the functional recovery of rat hearts exposed to cardioplegic arrest suggest that putrescine, the product of the enzymatic conversion of ornithine by ODC, impairs the functional recovery of rat hearts after cardioplegic arrest. The data also argue for the hypothesis that the better recovery of adult hearts compared to juvenile hearts is based on a lower ODC expression. We, therefore, confirmed that adult rat hearts have a lower ventricular expression of ODC compared to juvenile rat hearts: Performing real-time RT-PCR, we found an approximately 40% lower ODC expression in adult hearts [Table 3]. We have recently shown that ODC-dependent polyamine metabolism is linked to an upregulation of the proapoptotic gene bax.[7] As expected, we found a lower bax expression and a higher bcl-2/bax ratio in adult rat hearts as well [Table 3]. In contrast, adult rat hearts had a lower SERCA2a expression and lower SERCA2a-to NCX ratio [Table 3]. All these data are in agreement with the lower basal function of adult versus juvenile rat hearts but better recovery during reperfusion from cardioplegic arrest.
Table 3: mRNA expression of selected genes of interesting in the left ventricle of the hearts from juvenile and adult hearts

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Functional recovery of rat hearts exposed to ischemia and reperfusion

Finally, we investigated whether the detrimental effect of putrescine on the functional recovery of rat hearts exposed to cardioplegic arrest, and reperfusion is specific for hearts reperfused after cardioplegic arrest or a general behavior of reperfused hearts. Therefore, we exposed rat hearts to 45-min flow arrest and 90-min reperfusion and added putrescine during the initial phase of reperfusion. Under these conditions, putrescine significantly improved the functional recovery of rat hearts [Table 4].
Table 4: Effect of putrescine on functional recovery of ischemia/reperfusion without cardioplegia in juvenile rat hearts

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  Discussion Top


Our study shows that adult rat hearts recover better from ischemia-reperfusion induced by cardioplegia and 60-min aortic clamping time than juvenile hearts. The reasons for the better recovery of adult hearts include less ODC expression and possibly also less ODC activity in adult versus juvenile rat hearts. Less ODC expression would lead to less production of its product putrescine, lowering the cellular apoptosis by downregulation of the gene bax.

Our finding that adult rat hearts express less ODC than juvenile hearts is not novel and confirms earlier reports that investigated enzyme activity rather than ODC expression.[5] However, as ODC is the rate-limiting enzyme of the polyamine metabolism, it is strictly regulated by expression due to its extremely short half-life. A strong correlation between ODC mRNA expression and activity has been shown before from our group in cardiomyocytes stimulated with isoprenaline.[8] Here, we found that putrescine per se improved the functional recovery of rat hearts exposed to ischemia/reperfusion. However, a detrimental effect was observed in response to cardioplegic arrest. Crystalloid cardioplegic solutions such as the Bretschneider's solution used in this study are void of calcium or include unphysiologically low levels of calcium. It has been shown before that putrescine impairs functional recovery of rat hearts exposed to calcium readministration after a short period of calcium-free perfusion although it did not exert a similar detrimental effect under basal conditions.[3] Furthermore, in the same study, inhibition of ODC by difluoromethylornithine, an irreversible inhibitor of ODC, improved the functional recovery under these conditions indicating an activation of ODC by readministration of calcium. Our findings are in full agreement with these findings because one may consider reperfusion with KHS after a period of cardiac arrest with crystalloid cardioplegia as readministration of calcium. Thus, although neither the finding that adult hearts express less ODC than juvenile hearts nor the possible detrimental effect of putrescine on the functional recovery of hearts reperfused with calcium after a period of calcium depletion is novel. However, this scenario has never been investigated in the context of cardioplegic arrest and differences between adult and juvenile hearts under these conditions. Thus, our study established for the first time a rationale for a different outcome of reperfusion between adult and juvenile hearts after crystalloid cardioplegic arrest.

We have recently linked the effect of putrescine to the expression of the proapoptotic gene bax.[7] Mechanistically, this has been linked to a putrescine-dependent activation of the calcium-sensing receptor of cardiomyocytes. Following these assumptions, we must expect less bax expression in adult rat hearts compared to juvenile rat hearts in our new study because adult hearts express less ODC and are expected to form less putrescine. Indeed, we found less bax and an improved bcl-2-to-bax ratio in the hearts analyzed here. Interestingly, the effect of calcium-sensing receptor stimulation on bax expression is induced by ischemia itself.[9] This indicates that signal transduction through calcium-sensing receptors in cardiomyocytes can be rapidly modified. Our data argue for a similar modification under calcium depletion and readministration. However, calcium-containing blood cardioplegia has similar bcl-2 and bax expression than crystalloid cardioplegia.[10] The future studies are needed to characterize the effect of calcium depletion on calcium-sensing receptor coupling in cardiomyocytes in more detail. Of note, in the study cited above, putrescine preferentially impaired cardiac calcium handling in cardiomyocytes exposed to a calcium paradox protocol but not under basal conditions.[3]

Despite an improved recovery of adult versus juvenile hearts described in this study, older hearts have more impaired ventricular function that leads to an increased surgical risk. This is independent of the improved recovery and was confirmed in our study as well. Older hearts had lower basal function and lower SERCA2a expression and subsequently a lower SERCA2a/NCX ratio. SERCA has a prominent role in excitation/contraction coupling of myocytes.[11] SERCA downregulation has been shown in models of cardiac hypertrophy and failure, together with an increased expression of the NCX. Possibly, the overexpression of NCX may facilitate Ca 2+ reuptake by SERCA und compensate for the impaired SR Ca 2+ load.[12] The antagonistic effects of SERCA and NCX led us to evaluate the ratio between both enzymes rather than the mRNA expression of the single enzyme alone. SERCA upregulation was associated with improvement in cardiac function and in heart-failure-associated inflammatory markers.[13] Of note, we have previously shown that there is no general age-dependent impairment in classical ischemia/reperfusion protocols.[14] Therefore, despite the lower basal function in older hearts used in this study, the observed difference in the outcome of reperfusion after cardioplegic arrest is specific for the cardioplegic conditions.

The lifespan of rats is approximately 24 months. Therefore, 12-month-old rats represent a middle-aged population rather than a geriatric population. However, our new finding that already at this stage significant differences between young adolescent and elder rats occur suggest an even more dramatic difference in geriatric rats that might mimic a clinical situation even better.


  Conclusion Top


Adult rat hearts tolerate cardioplegia-mitigated ischemia better than juvenile hearts. The mechanisms of improved ischemia tolerance could include a lower ODC expression and therefore also a lower formation of putrescine, an agonist of cardiac calcium-sensing receptors. This would lead to better functional recovery after cardioplegia and protection against apoptosis. As mentioned before, adult and senescent hearts have an increased risk profile compared to young hearts. This may also be relevant in light of our findings of this study: spontaneously hypertensive rat hearts express more ODC, and the future studies are needed to investigate the effect of cardioplegic arrest, reperfusion, ODC expression, and functional recovery in these settings.[15] On the other hand, a quantitative analysis of cardiac-specific ODC expression may predict the functional recovery in patients undergoing cardiac surgery and identify patients at high risk.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

1.
Karimi M, Wang LX, Hammel JM, Mascio CE, Abdulhamid M, Barner EW, et al. Neonatal vulnerability to ischemia and reperfusion: Cardioplegic arrest causes greater myocardial apoptosis in neonatal lambs than in mature lambs. J Thorac Cardiovasc Surg 2004;127:490-7.  Back to cited text no. 1
    
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Smajilovic S, Chattopadhyay N, Tfelt-Hansen J. Calcium-sensing receptor in cardiac physiology. Open Heart Fail J 2010;3:11-5.  Back to cited text no. 2
    
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Flamigni F, Rossoni C, Stefanelli C, Caldarera CM. Polyamine metabolism and function in the heart. J Mol Cell Cardiol 1986;18:3-11.  Back to cited text no. 3
    
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Koenig H, Goldstone AD, Trout JJ, Lu CY. Polyamines mediate uncontrolled calcium entry and cell damage in rat heart in the calcium paradox. J Clin Invest 1987;80:1322-31.  Back to cited text no. 4
    
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Das R, Kanungo MS. Activity and modulation of ornithine decarboxylase and concentrations of polyamines in various tissues of rats as a function of age. Exp Gerontol 1982;17:95-103.  Back to cited text no. 5
    
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Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402-8.  Back to cited text no. 6
    
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Mörlein C, Schreckenberg R, Schlüter KD. Basal ornithine decarboxylase activity modifies apoptotic and hypertrophic marker expression in post-ischemic hearts. Open Heart Fail J 2010;3:31-7.  Back to cited text no. 7
    
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Schlüter KD, Frischkopf K, Flesch M, Rosenkranz S, Taimor G, Piper HM, et al. Central role for ornithine decarboxylase in beta-adrenoceptor mediated hypertrophy. Cardiovasc Res 2000;45:410-7.  Back to cited text no. 8
    
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Tantini B, Fiumana E, Cetrullo S, Pignatti C, Bonavita F, Shantz LM, et al. Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia. J Mol Cell Cardiol 2006;40:775-82.  Back to cited text no. 9
    
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Feng J, Bianchi C, Sandmeyer JL, Li J, Sellke FW. Molecular indices of apoptosis after intermittent blood and crystalloid cardioplegia. Circulation 2005;112:I184-9.  Back to cited text no. 10
    
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Inesi G, Prasad AM, Pilankatta R. The ca2+ATPase of cardiac sarcoplasmic reticulum: Physiological role and relevance to diseases. Biochem Biophys Res Commun 2008;369:182-7.  Back to cited text no. 11
    
12.
Isenberg G. How can overexpression of Na+, Ca2+-exchanger compensate the negative inotropic effects of downregulated SERCA? Cardiovasc Res 2001;49:1-6.  Back to cited text no. 12
    
13.
Gupta D, Palma J, Molina E, Gaughan JP, Long W, Houser S, et al. Improved exercise capacity and reduced systemic inflammation after adenoviral-mediated SERCA-2a gene transfer. J Surg Res 2008;145:257-65.  Back to cited text no. 13
    
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Ross G, Schlüter KD. Cardiac-specific effects of parathyroid hormone-related peptide: Modification by aging and hypertension. Cardiovasc Res 2005;66:334-44.  Back to cited text no. 14
    
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Ruskoaho H, Raunio H. Cardiac polyamine metabolism in spontaneously hypertensive rats: Effect of antihypertensive treatment. J Hypertens Suppl 1986;4:S71-4.  Back to cited text no. 15
    


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


This article has been cited by
1 Cardiac surgery in acute myocardial infarction: crystalloid versus blood cardioplegia an experimental study
Andreas Boening,Maximilian Hinke,Martina Heep,Kerstin Boengler,Bernd Niemann,Philippe Grieshaber
Journal of Cardiothoracic Surgery. 2020; 15(1)
[Pubmed] | [DOI]



 

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