INTRODUCTION

Cardioplegic solution administration in open-heart surgeries has been the main strategy enable to antagonize the effects of global ischemia and reperfusion (IR). Cardioplegic arrest in combination with hypothermia corresponds to a standard method to reduce cardiac metabolism during the period of absence of oxygen and substrate, avoiding the collapse of contractile function after reperfusion^[1,2].

Despite being essential, this scientific field faces a major challenge in determining a single type of cardioplegic solution protocol amidst a variety of surgical procedures. A pharmacological approach to drugs that act as cardioplegic agents makes this puzzle even more difficult to understand. In the presence of cardioplegic agents, these solutions can block or open ion channels present in the sarcolemma, influencing on the ion dynamic between extra and intracellular means.

In the present study, two cardioplegic agents with distinct actions on ion channels were evaluated, followed by (I) pinacidil, a potassium channel opener, and (II) lidocaine, a sodium channel blocker. Rationally, two different types of solution, very eminent in clinical practice, were elected to drive these drugs on myocardium during global ischemia^[3].

Firstly, Custodiol solution (Custodiol^®-HTK), commonly known as HTK solution (histidine/tryptophan/alpha-ketoglutarate) and which has been used primarily in organ preservation, was conceived as an alternative to crystalloid hyperkalemic cardioplegic solutions^[4,5]. Custodiol solution has been used in some countries as the first choice for conventional cardiac surgeries, mainly in myocardial preservation^[6]. It is considered a type of microplegia or cardioplegia solution with intracellular action due to reduction of potassium, sodium, and calcium concentration, resulting in cardiac arrest by polarization and avoiding damages from hypercontractures related to the depolarized arrest. In addition, Custodiol solution has an important buffering effect attributed to the amino acid histidine, which is important in controlling the balance of hydrogen ion levels released during a long period of ischemia^[7,8].

Secondly, del Nido cardioplegia, initially developed to be used in a single dose in neonatal surgeries, is classified as a modified hyperkalemic type due to high potassium concentration, although the lidocaine in its composition avoids the ionic imbalance by blocking sodium current flow^[9-11]. This sodium channel blockage prevents intracellular calcium overload, avoiding contractile dysfunction after reperfusion^[11]. Lidocaine is a selective and reversible blocker frequently used in clinical practice for the treatment of ventricular tachycardia and also has been assisted recovery hearts submitted to IR in experimental models^[12,13].

This study aimed to evaluate cardioplegic solutions under pharmacological modification. Pinacidil was added to both solutions and lidocaine only to the Custodiol solution, since del Nido cardioplegia original formulation already has this agent. Cardioplegic solutions were termed as Custodiol-LP and del Nido-LP. The main goal was to evaluate functional recovery after 3 hours of ischemia and 90 minutes of reperfusion. Moreover, cardiomyocyte integrity by alpha-fodrin degradation was also evaluated from heart samples collected at the end of the protocol.

METHODS

Cardioplegic Solutions Preparation

The Custodiol solution was acquired from a commercial vendor (Table 1). Blood cardioplegia del Nido was prepared with autologous blood collected directly from the thoracic cavity after heart explantation 1:4 (blood:solution) (Table 1).

Table 1 - Composition of cardioplegic solutions.

Components (mM)	Custodiol-LP	del Nido-LP
Carrier	Sterile water	Plasma-Lyte A®
Blood:cardioplegia	__	1:4
Calcium chloride	0.02	0.24
Histidine	180	__
Histidine-HCl	18	__
Magnesium chloride	4	1.13
Magnesium sulfate	__	6.18
Mannitol	30	13.72
Potassium chloride	9	24.3
Potassium hydrogen 2-ketoglutarate	1	__
Sodium acetate trihydrate	__	20.34
Sodium bicarbonate	__	13.79
Sodium chloride	15	91.66
Sodium gluconate	__	17.33
Tryptophan	2	__
Drugs concentration (del Nido-LP without added blood)
Lidocaine	0.45	0.45
Pinacidil	0.5	0.5

Table 1 - Composition of cardioplegic solutions.

RESULTS

Functional Left Ventricle Analysis

Two hearts from the control group were excluded due to ventricular fibrillation and no beating recovery after ischemia. Lower levels of developed pressure (Figure 1A) and RPP (Figure 1B) recovery were found in the control group than in Custodiol-LP and del Nido-LP groups, respectively. However, no relevant differences among the cardioplegic solution groups were found with these variables. Relevant lower levels of maximum dP/dt (Figure 1C) and minimum dP/dt (Figure 1D) were found in the control group than in Custodiol-LP and del Nido-LP groups, respectively. However, at 90 minutes of reperfusion, higher levels of maximum dP/dt (Figure 1C) and minimum dP/dt (Figure 1D) were found in del Nido-LP group than in Custodiol-LP.

Fig. 1 - Left ventricular function data expressed as percentage of recovery after 3 hours. (A) Developed pressure. (B) Rate pressure product. (C) Maximum dP/dt. (D) Minimum dP/dt. All values are expressed as mean and standard error of mean (SEM) (error bars). Analyses were performed by 2-way ANOVA with Sidak's post-hoc test for multiple comparisons (*P < 0.05 compared to del Nido group). Six animals per group.

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Fig. 1 - Left ventricular function data expressed as percentage of recovery after 3 hours. (A) Developed pressure. (B) Rate pressure product. (C) Maximum dP/dt. (D) Minimum dP/dt. All values are expressed as mean and standard error of mean (SEM) (error bars). Analyses were performed by 2-way ANOVA with Sidak's post-hoc test for multiple comparisons (*P < 0.05 compared to del Nido group). Six animals per group.

Coronary Resistance

CR levels found in the control group were higher than in Custodiol-LP group up to 60 minutes of reperfusion (Figure 2). Compared to del Nido-LP group, the control group showed high levels of CR throughout reperfusion. However, del Nido-LP group showed lower levels of CR than Custodiol-LP group at 90 minutes of reperfusion (Figure 2).

Fig. 2 - Coronary resistance data expressed as percentage of recovery after 3 hours. All values are expressed as mean and standard error of mean (SEM) (error bars). Analyses were performed by 2-way ANOVA with Sidak's post-hoc test for multiple comparisons (*P<0.05 compared to del Nido group). Six animals per group.

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Fig. 2 - Coronary resistance data expressed as percentage of recovery after 3 hours. All values are expressed as mean and standard error of mean (SEM) (error bars). Analyses were performed by 2-way ANOVA with Sidak's post-hoc test for multiple comparisons (*P<0.05 compared to del Nido group). Six animals per group.

Protein Expression by Western Blot Technique

Higher degradation of alpha-fodrin expression was found in Custodiol-LP group than in del Nido group in samples collected after 3 hours of ischemia and 90 minutes of reperfusion (Figure 3).

Fig. 3 - Representative Western blot results for alpha-fodrin. All values are expressed as mean, standard error of mean (SEM) (error bars), and arbitrary units. Analyses were performed by t-test (*P<0.05). Four animals per group.

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Fig. 3 - Representative Western blot results for alpha-fodrin. All values are expressed as mean, standard error of mean (SEM) (error bars), and arbitrary units. Analyses were performed by t-test (*P<0.05). Four animals per group.

DISCUSSION

Myocardial global IR insult is well known to promote multifactorial effects on cellular metabolism resulting in irreversible damage to the heart function. Cardioplegic solutions with pharmacological combination as an alternative to provide further myocardial protection have encouraged researchers to carry out different combinations^[15]. Therefore, the present study evaluated pharmacological modifications with sodium inhibitor channels and potassium channel openers added to clinical relevant cardioplegic solutions, Custodiol and del Nido.

The potassium channel opener pinacidil has two main effects: vasodilatation and contractility reduction, due to its effect on potassium efflux avoiding calcium overload into the cardiomyocytes^[16]. In experimental models, pinacidil induced cardiac arrest by polarization, consequently reducing the adenosine triphosphate (ATP) consumption, compared to the cardiac arrest induced by depolarization^[17,18]. Pinacidil has a non-selective pharmacological effect regarding the potassium channels' location, being able to activate them in the sarcolemma and mitochondria, since both have an important role in myocardial protection^[17,19]. Differently, lidocaine is a reversible sodium channel blocker which has a pharmacological effect on calcium overload, preventing sodium-calcium exchanger reverse-mode action during IR^[9,10]. In combination, these cardioplegic agents might improve cardiac resistance against long-term ischemia.

Custodiol-LP showed detrimental effects on cardiac function by the maximum and minimum dP/dt at 90 minutes of reperfusion compared to del Nido-LP. However, developed pressure and RPP parameters were similar throughout reperfusion time. CR analysis showed Custodiol-LP group with higher levels than del Nido-LP and marked reduction of coronary flow after 90 minutes of reperfusion.

The superiority of del Nido-LP solution may be associated with its greater ability to protect the myocardium over prolonged ischemia. The immunoblot assay performed by Western blot technique showed alpha-fodrin significant degradation in Custodiol-LP group compared to del Nido-LP. Alpha-fodrin present in extracellular matrix is consumed as a substrate for calpains after IR. The increase of calpain activity is correlated with myocardial injury and loss of contractile function^[20-22]. Therefore, alpha-fodrin degradation is an indirect marker for cell integrity.

According to our findings, the addition of pharmacological agents to cardioplegic solutions showed greater myocardial protection in del Nido-LP group than in Custodiol-LP. Other components present in this solution may have possibly collaborated for these results. A single dose of the del Nido original formulation per se has shown great efficacy in pediatric and adult surgeries; perhaps, new solution proposals to induce long periods of ischemia should be based on its composition.

CONCLUSION

The addition of cardioplegic agents pinacidil and lidocaine provided greater protective capacity to del Nido-LP solution than to Custodiol-LP in hearts submitted to 3 hours of ischemia and 90 minutes of reperfusion. Contractile function data, according to variables of maximum dP/dt, minimum dP/dt and CR, showed a higher percentage of recovery in del Nido-LP group. Also, lower levels of alpha-fodrin degradation in del Nido-LP can support these findings showing higher myocardial protection against IR injury.