In vitro effects of amiodarone on coronary artery bypass grafts

Amiodaronun koroner arter baypas greftleri üzerine in vitro etkileri

Okay Güven Karaca¹, Turan Ege², Suat Canbaz², Özcan Gür³, Çetin Hakan Karadağ⁴, Ata Niyazi Ecevit¹, Mehmet Kalender¹

¹Department of Cardiovascular Surgery, Konya Training and Research Hospital, Konya, Turkey
²Departments of Cardiovascular Surgery, Medical Faculty of Trakya University, Edirne, Turkey
³Department of Cardiovascular Surgery, Medical Faculty of Namık Kemal University, Tekirdağ, Turkey
⁴Departments of Pharmacology, Medical Faculty of Trakya University, Edirne, Turkey

DOI : 10.5606/tgkdc.dergisi.2015.9871

Abstract

Background: This study aims to investigate the effects of amiodarone on the most commonly used grafts, internal thoracic artery (ITA), saphenous vein (SV), and radial artery (RA) in organ bath.

Methods: Twenty patients (16 males, 4 females; mean ages 58.4±9.9 years; range 38 to 80 years) who underwent isolated coronary artery bypass graft (CABG) surgery between May 2008 and October 2008 were included in this study. Internal thoracic artery, saphenous vein, and radial artery grafts were harvested. Specimens were taken to laboratory in +4 ºC Krebs solution. Specimens were suspended in 10 ml organ bath containing Krebs solution.

Results: Amiodarone caused relaxation in all grafts (ITA, RA, SV) between 10-9-10-3,5 M concentration in a dose dependent manner (p<0.01). Maximum relaxation rates (mean) induced by amiodarone were 78.9%, 74.9% and 66.5% for ITA, RA and SV, respectively.

Conclusion: Although we did not evaluate the endotheliumindependent relaxation response in this study, higher rates of relaxation response were observed with ITA grafts comparing to other grafts, and these results were compatible with literature. According to the results of this study, amiodarone-class III antiarrhythmic agent- caused vasodilation in all three grafts in vitro. Vasodilator effect of amiodarone on grafts may help to increase patency rates.

Abstract

The internal thoracic artery (ITA), saphenous vein (SV) and radial artery (RA) are the most common grafts used in coronary artery bypass grafting (CABG) operations.[1] However, structural and physiological differences in the grafts affect both short- and longterm patency, which can also be unfavorably affected when late relaxation of the vasospasm develops due to mechanical or pharmacological causes in the perioperative period.[2]

Amiodarone (Cordarone, Sanofi Aventis, Turkey) has been accepted as a prototype of the class III anti-arrhythmic drugs for a long time. However, the pharmacological effects of amiodarone are very complex. For instance, with long-term usage, the action potential time and refractory period are extended.[3,4] Additionaly, this agent inhibits rapid sodium channels and slow calcium channels,[5,6] it was known that its effects are anti-sympathetic, thyroid and lipid metabolism-converting and also it has affects on some cytokine's production metabolism.[7-9] Amiodarone can be administered intravenously or orally in the preoperative, intraoperative, or postoperative periods, and it is the drug of choice after CABG operations.[10] We found several reports in the literature that focused on the effects of several agents on specimens in an organ bath, but none of these analyzed amiodarone.[11] Evidently, in spite of its frequent use, the effects of this agent on grafts are not well understood. Therefore, in this study we aimed to analyze the effects of amiodarone on the ITA, SV, and RA in an organ bath.

Methods

Twenty patients (4 females, 16 males; median age 58.4±9.9; range 38 to 80) who underwent isolated CABG surgery between May 2008 and October 2008 were included in this study with their permission, and we also obtained the approval of the local ethics committee.

Amiodarone, potassium hydrochloric acid (Sigma- Aldrich, St. Louis, MO, USA), and phenylephrine (Sigma-Aldrich, St. Louis, MO, USA) were used in this study, all of which were dissolved in distilled water.

We assessed a total of 48 samples (16 RA, 16 ITA, and 16 SV), and Table 1 shows the perioperative status of the 20 patients. The ITA was harvested along with its peduncle via scissors in order to protect the endothelium from damage, and a section was taken prior to the papaverine administration. The SV was also harvested with its peduncle, and the distal portion was sampled while the RA was harvested in a skeletonized fashion using scissors. All of the specimens were taken to the laboratory in +4 ºC Krebs solution.

Table 1: Preoperative and postoperative variables

Next, the specimens were cut into 2 mm rings and suspended in a 10 ml organ bath chamber containing the Krebs solution, which was composed of the following: 122 mmol/L sodium chloride (NaCl), 5 mmol/L potassium chloride (KCl), 1.25 mmol/L calcium chloride (CaCl₂), 25 mmol/L sodium bicarbonate (NaHCO₃), 1.2 mmol/L magnesium sulfate (MgSO₄), 1.0 mmol/L monopotassium phosphate (KH₂PO₄), and 11.5 mmol/L glucose. Then the medium was gassed with a mixture of carbon dioxide (CO₂) (5%) and oxygen (O₂) (95%) and maintained at 37 °C (pH 7.4). Next, the specimens were hooked transversely and connected to an FDT10-A forced displacement transducer (COMMAT Ltd., Ankara, Turkey) which was connected to a TDA-97 transducer data acquisition system (COMMAT Ltd., Ankara, Turkey) that sent signals to a computer. The vascular responses were then recorded and analyzed with POLWIN97 acquisition software (COMMAT, Ankara, Turkey). All vascular tissues were stretched to a basal resting tension of 2 g. The preparations were allowed to equilibrate for 80 minutes, and the Krebs solution was refreshed every 20 minutes. Furthermore, smooth muscle cell contractility was measured by the addition of 90 mmol/L KCl to the bath. The vascular endothelium was also tested with epinephrine (10^-6 M) for contraction and acetylcholine (10^-6 M) for relaxation at the beginning and end of the experiments, and those that did not respond properly were excluded from the study. In order to standardize the assessment, all of the responses were calculated based on the percentage of responses in the control trace obtained at the beginning of the experiment.

Statistical analysis
GraphPad Prism 6.0a for Mac OS X 10.9 software (GraphPad Software, Inc., La Jolla, CA, USA) was used to draw concentration-response graphs and analyze the data, and a linear regression (variable slope) analysis was applied to the graphs. A p value of <0.05 was considered to be statistically significant.

Methods

Results

Human ITA, RA and SV were contracted to the submaximal level with 10^-6 M phenylephrine, and a relaxation response to the amiodarone was obtained using the cumulative method from a concentration of between 10^-9 M and 3.10^-4 M . The relaxation induced by the amiodarone was standardized to the contractions induced by the phenylephrine, and the mean minimum and maximum dose-response values of the ITA grafts were calculated as 3.5%, and 75.9% respectively (Figure 1). In addition, the values for the RA grafts were 7.9%, and 74.9%, respectively (Figure 2), and for the SV grafts, they were 3.9% and 66.5%, respectively (Figure 3).

Figure 1: The dose-dependent relaxation response curve of amiodarone showing that the in vitro ITA contracted 10^-6 M with phenylephrine.

Figure 2: The dose-dependent relaxation response curve of amiodarone showing that the in vitro radial artery contracted 10^-6 M with phenylephrine.

Figure 3: The dose-dependent relaxation response curve of amiodarone showing that the in vitro saphenous vein contracted 10^-6 M with phenylephrine.

The amiodarone produced a dose-dependent relaxation in all of the grafts in the concentration range of between 10^-9 and 3.10^-4 M, and t he a verage maximum relaxation percentages induced by the amiodarone in the ITA, RA and SV grafts were 78.9±1.44, 74.9±2.29, and 66.5±2.85, respectively. The amiodarone produced the highest relaxation in the ITA grafts followed by the RA. The SV grafts had the lowest relaxation percentage. Although the maximum relaxation rates in the ITA and RA were not statistically significantly different, the effect in the SV was statistically significantly lower than that of the ITA (p<0.01) and RA (p<0.05) (Table 2).

Table 2: Comparison of maximum relaxation with amiodarone in the internal thoracic artery, saphenous vein, and radial artery

Results

Discussion

Amiodarone is a well known, effective anti-arrhythmic agent that is also beneficial for preventing sudden cardiac death in hypertrophic cardiomyopathy.[12] Following CABG operations, atrial fibrillation (AF) occurs in 5-65% of patients,[13,14] and amiodarone, which is highly tolerated, can be used before, during or after CABG operations to prevent and treat arrhythmias via peri-oral or intravenous administration.[15] Preoperative amiodarone prophylaxis reduces by 25% the incidence of postoperative AF, but postoperative adverse events, such bradyarrhythmia, acute lung injury, and infusionrelated hypotension, have been reported with this agent.[16]

Using ITA, RA, and SV grafts during CABG operations may affect the short- and long-term patency rates because of their different characteristics with regard to contraction and endothelial function,[17] and these differences depend on the physiological and anatomical properties of the grafts. Thus, it is accepted that postoperative graft function and patency are affected by the susceptibility to vasospasm, which can occur due to either physical factors, such as surgical manipulations, or biochemical, molecular and pharmacological factors, for example an increase in smooth muscle hyperactivity.[17] As a result, increased perioperative morbidity and postoperative myocardial failure, which can lead to death, may be seen.

Initially, the endothelial layer was thought to be made up of a simple array of cells that lined up between the vessel lumen and intima. However, with the discovery of endothelium-derived vasoactive substances, the complex functions of the vascular endothelium are now better understood. The endothelium-dependent hyperpolarization factor (EDHF) plays an important role in ensuring the appropriate vascular tone. In addition to variations in the components of EDHF, there may be dissimilarities because it includes at least three components [endothelium-dependent nitric oxide (NO), prostaglandin I2 (PGI2), and the EDHF itself).[18] Endothelium-dependent relaxation occurs as a result of the effect of various factors caused by the vasodilatation of different mechanisms. Endothelium NO causes vasodilatation by using cGMP pathway and PGI2 uses the cAMP pathway. The structure of EDHF is not fully understood, however it is through that it creates hyperpolarization by affecting the potassium channels of smooth muscle cells.[18] Endotheliumdependent relaxation also plays an important role in providing appropriate vascular tonus. In this study, the arterial graft samples had an increase in basal production as well as a higher NO release than the SV grafts. Furthermore, Luscher et al.[19] found a significant difference in favor of ITA in terms of the NO release versus the SV, and Sellke et al.[20] attributed the higher patency rate of ITA to the higher basal NO release.

In clinical studies, the most common side effect of intravenous amiodarone administration was hypotension, with incidence rates ranging between 15 and 26%.[12] Additionally, in experimental studies that involved that use of dog hearts, the vasodilatory effects of amiodarone were only partially endotheliumdependent.[21] Moreover, according to G essner e t al.,[22] the amiodarone derivate KB130015 induced endothelium-independent relaxation in porcine pulmonary arteries, and Traupe et al.[23] reported that amiodarone acutely increased endothelium-dependent vasodilation and phasic contractions in atherosclerotic arteries in a gender-dependent fashion.

Despite the frequent use of commercial amiodarone after CABG operations, to our knowledge, no one has addressed the impact that this agent has on graft function in the literature. In this in vitro study, amiodarone resulted in relaxation for all three graft samples, with the relaxation responses in both arterial grafts being higher that in the SV. Furthermore, although this study did not evaluate the endotheliumindependent relaxation response, the higher rates in the ITA grafts were compatible with the literature; thus, we concluded that these were associated with the anatomical and physiological properties of the graft. At this point, we think that the differences in the NO synthesis and release rates from the ITA grafts and the differences in the level of the receptors on ITA grafts may affect this condition. Furthermore, the variations in the relaxation response between the arterial grafts and the SV may be explain with the same reasons. However, further studies are needed to investigate our suppositions.

Discussion

Conclusion

According to the results of this study, amiodarone, a class III anti-arrhythmic agent, dilated the ITA, RA, and SV grafts used in CABG in vitro, and based on the vasodilator effect of amiodarone on these grafts, the use of this agent may help reduce the short-term complications associated with this type of surgery.

Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The authors received no financial support for the research and/or authorship of this article.

Conclusion