Pharmacological Modulation of the Ca2+/cAMP/Adenosine Signaling in Cardiac Cells as a New Cardioprotective Strategy to Reduce Severe Arrhythmias in Myocardial Infarction

Acute myocardial infarction (AMI) is the main cause of morbidity and mortality worldwide and is characterized by severe and fatal arrhythmias induced by cardiac ischemia/reperfusion (CIR). However, the molecular mechanisms involved in these arrhythmias are still little understood. To investigate the cardioprotective role of the cardiac Ca2+/cAMP/adenosine signaling pathway in AMI, L-type Ca2+ channels (LTCC) were blocked with either nifedipine (NIF) or verapamil (VER), with or without A1-adenosine (ADO), receptors (A1R), antagonist (DPCPX), or cAMP efflux blocker probenecid (PROB), and the incidence of ventricular arrhythmias (VA), atrioventricular block (AVB), and lethality (LET) induced by CIR in rats was evaluated. VA, AVB and LET incidences were evaluated by ECG analysis and compared between control (CIR group) and intravenously treated 5 min before CIR with NIF 1, 10, and 30 mg/kg and VER 1 mg/kg in the presence or absence of PROB 100 mg/kg or DPCPX 100 µg/kg. The serum levels of cardiac injury biomarkers total creatine kinase (CK) and CK-MB were quantified. Both NIF and VER treatment were able to attenuate cardiac arrhythmias caused by CIR; however, these antiarrhythmic effects were abolished by pretreatment with PROB and DPCPX. The total serum CK and CK-MB were similar in all groups. These results indicate that the pharmacological modulation of Ca2+/cAMP/ADO in cardiac cells by means of attenuation of Ca2+ influx via LTCC and the activation of A1R by endogenous ADO could be a promising therapeutic strategy to reduce the incidence of severe and fatal arrhythmias caused by AMI in humans.


Introduction
After 2023, cardiovascular diseases (CVD) will be responsible for over 26 million annual deaths worldwide, in both industrialized and underdeveloped nations; among these, ischemic heart disorders (IHD) and, particularly, acute myocardial infarction (AMI) are the leading cause of death and morbidity worldwide [1][2][3], estimated to affect about three million people worldwide and to significantly increase the incidence of sudden cardiac death [4,5].AMI results in irreversible damage to the myocardium primarily caused by a lack of oxygen in cardiac cells, which may lead to impairment in diastolic and systolic function and make the patient prone to severe and fatal cardiac arrhythmias [4][5][6].
Although AMI can lead to several serious complications for cardiac function, there are still few pharmacological resources for the treatment of AMI.
Additionally, reperfusion also produces important metabolic and functional alterations in cardiac cells.It increases free radical production and Ca 2+ influx overload through L-type Ca 2+ channels (LTCC).This Ca 2+ influx modulates ryanodine receptors (RyR) and important enzymes, such as adenylyl cyclase (AC), an enzyme that produces cAMP from ATP, in the T-tubules and intracellular medium [6].The mitochondria also play a role in maintaining Ca 2+ homeostasis during brief increases in cytosolic Ca 2+ ([Ca 2+ ]c) in cardiac cells, which is crucial in the contraction-relaxation cycle of myocardium [6,14].Ca 2+ concentration in the mitochondrial matrix ([Ca 2+ ]m) is finely controlled by Ca 2+ transporter proteins that are present in the mitochondrial membranes and control Ca 2+ influx and efflux in the mitochondrial matrix [6,7].Mitochondrial Ca 2+ influx in cardiac cells is primarily controlled by the mitochondrial Ca 2+ uniporter (MCU), while its efflux is mostly controlled by the mitochondrial Na + /Ca 2+ exchanger (mNCX) [6,16].As a result, the cardiac cycle and contraction-relaxation processes are significantly impacted by mitochondrial dysfunction in Ca 2+ homeostasis in cardiac cells [6,14].In addition to this decoupling of cardiac excitation-contraction (CECC), an important increase in free radical production during reperfusion leads to the oxidation of structural proteins, proteins involved in the respiratory chain, pyridine nucleotides, changes in permeability of the internal mitochondrial membrane, decoupling of oxidative phosphorylation, and a decrease in mitochondrial ATP production [6,14].
In addition to its role in CECC, Ca 2+ modulates 3 5 -cyclic adenosine monophosphate (cAMP) production with isoforms 5 and 6 of adenylyl cyclase (AC), and the pharmacological blockade of Ca 2+ influx via LTCC produces an increase in production and efflux of Pharmaceuticals 2023, 16, 1473 3 of 13 intracellular cAMP in cardiac cells [6,[18][19][20].In the extracellular medium, cAMP is transformed into adenosine (ADO), which then stimulates membrane A 1 -type ADO receptors (A 1 R) to finely regulate cardiac cell function [6].It is well known that stimulation of cardiac A 1 R by ADO is a common and effective strategy used to attenuate cardiac arrhythmias in various clinical situations, and especially in cardiac surgery [4,6].Thus, we have proposed that pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells could be a promising strategy in the treatment of AMI and other IHD in humans.
Based on the above proposal, in the present work, we investigated the effects of pharmacological modulation Ca 2+ /cAMP/ADO signaling in cardiac cells on the incidence of severe and fatal arrhythmias related to AMI.Thus, using an animal model of AMI, the effects of the blockade of LTCC-mediated Ca 2+ influx with nifedipine (NIF) or verapamil (VER), in the presence or absence of blocker of transporter-mediated cAMP efflux probenecid (PROB) or A 1 R-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), on the incidence of arrhythmias (VA and AVB) and lethality (LET) induced by CIR were studied (Figures 1 and 2).In addition, serum concentrations of cardiac injury biomarkers total creatine kinase (CK) and CK-MB were quantified.
efflux of intracellular cAMP in cardiac cells [6,[18][19][20].In the extracellular medium, cAMP is transformed into adenosine (ADO), which then stimulates membrane A1-type ADO re ceptors (A1R) to finely regulate cardiac cell function [6].It is well known that stimulation of cardiac A1R by ADO is a common and effective strategy used to attenuate cardiac ar rhythmias in various clinical situations, and especially in cardiac surgery [4,6].Thus, we have proposed that pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardia cells could be a promising strategy in the treatment of AMI and other IHD in humans.
Based on the above proposal, in the present work, we investigated the effects of phar macological modulation Ca 2+ /cAMP/ADO signaling in cardiac cells on the incidence o severe and fatal arrhythmias related to AMI.Thus, using an animal model of AMI, the effects of the blockade of LTCC-mediated Ca 2+ influx with nifedipine (NIF) or verapami (VER), in the presence or absence of blocker of transporter-mediated cAMP efflux pro benecid (PROB) or A1R-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) on the incidence of arrhythmias (VA and AVB) and lethality (LET) induced by CIR were studied (Figures 1 and 2).In addition, serum concentrations of cardiac injury biomarker total creatine kinase (CK) and CK-MB were quantified.

Incidence of VA, AVB and LET Induced by CIR
No arrhythmias were detected during the stabilization periods of any animal (15 min).During CIR, VA and AVB were detected and measured in different experimental groups.After CIR, the incidence of VA, AVB and LET were 90%, 80% and 70%, respectively (Figure 2).

Effects of the NIF and VER on the Incidence of VA, AVB and LET Induced by CIR
Figure 2 shows that incidences of AVB and LET induced by CIR were significantly reduced by treatment with NIF (1, 10 and 30 mg/kg, IV) and VER (1 mg/kg, IV).VA incidence was reduced from 90% to 30% in NIF10 + CIR and 30% in NIF30 + CIR groups compared to CIR group.AVB incidence was reduced from 80% to 30% in NIF1 + CIR, 20%

Incidence of VA, AVB and LET Induced by CIR
No arrhythmias were detected during the stabilization periods of any animal (15 min).During CIR, VA and AVB were detected and measured in different experimental groups.After CIR, the incidence of VA, AVB and LET were 90%, 80% and 70%, respectively (Figure 2).

Effects of the NIF and VER on the Incidence of VA, AVB and LET Induced by CIR
Figure 2 shows that incidences of AVB and LET induced by CIR were significantly reduced by treatment with NIF (1, 10 and 30 mg/kg, IV) and VER (1 mg/kg, IV).VA incidence was reduced from 90% to 30% in NIF10 + CIR and 30% in NIF30 + CIR groups, compared to CIR group.AVB incidence was reduced from 80% to 30% in NIF1 + CIR, 20% in NIF10 + CIR, and 20% in NIF30 + CIR groups, compared to CIR group.LET incidence was reduced from 70% to 30% in NIF1 + CIR, 10% in NIF10 + CIR, and 20% in NIF30 + CIR, compared to CIR group.In addition, treatment with VER was also able to reduce the incidences of VA (90% to 20%), AVB (90% to 20%), and LET (90% to 20%) induced by CIR.These results confirm previous studies [6, 7,11] that demonstrated that the blockade of Ca 2+ influx via LTCC in cardiac cells before CIR attenuates cardiac collapse and reduces the incidence of severe and fatal arrhythmias induced by CIR.

Effects of Pretreatment with PROB or DPCPX before Administration of NIF or VER on the Incidence of VA, AVB and LET Induced by CIR
To investigate whether Ca 2+ /cAMP/ADO signaling in cardiac cells is involved in the cardioprotective effect of NIF and VER (Figure 2), we pretreated rats subjected to CIR with DPCPX (100 µg/kg, IV) or PROB (100 mg/kg, IV), as well as NIF (10 mg/kg, IV) and VER (1 mg/kg, IV). Figure 3 shows that the reduction of VA, AVB and LET incidence in the PROB + NIF + CIR, PROB + VER + CIR, DPCPX + NIF + CIR, and DPCPX + VER + CIR groups was not statistically different from the CIR group, indicating that pretreatment with DPCPX and PROB completely abolished the cardioprotective effects of NIF and VER.These results indicate that an increase in extracellular levels of ADO due to cAMP transport to extracellular environments combined with an increase in activation of A 1 R receptors in cardiac cells directly participates in the cardioprotective response stimulated by NIF and VER in rats subjected to CIR.

Effects of Pretreatment with DPCPX before Administration of NIF and VER on Biochemical Markers of Cardiac Injury
Figure 4A shows that the serum concentration of biomarkers of cardiac lesion, total CK and CK-MB, were not statistically different in CIR (5487 ± 449 mg/dL, n = 3), NIF1 + CIR (5395 ± 876 mg/dL, n = 5), NIF10 + CIR (5344 ± 193 mg/dL, n = 5), NIF30 + CIR (5018 ±   Results were expressed as mean ± standard error of mean, and one-way analysis of variance (ANOVA) was applied, followed by Tukey's post-test.There was no statistical difference between the different groups.

Discussion
In the present work, we showed that pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells by means of attenuation of Ca 2+ influx via LTCC combined with an increase in the activation of A1R by ADO generated from extracellular cAMP reduced the incidence of severe and fatal arrhythmias induced by CIR (see Figures  + CIR groups.Results were expressed as mean ± standard error of mean, and one-way analysis of variance (ANOVA) was applied, followed by Tukey's post-test.There was no statistical difference between the different groups.

Discussion
In the present work, we showed that pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells by means of attenuation of Ca 2+ influx via LTCC combined with an increase in the activation of A 1 R by ADO generated from extracellular cAMP reduced the incidence of severe and fatal arrhythmias induced by CIR (see Figures 2 and 3).Similar pharmacological approaches using LTCC blockers and stimulation of cardiac A 1 R have been used for cardiac arrhythmias in various clinical situations, especially in cardiac surgery [4,6].Thus, pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells could be a promising therapeutic strategy to also reduce the incidence of severe and fatal arrhythmias caused by AMI in humans.
The dynamic equilibrium between the concentration of Ca 2+ into cytosol, sarcoplasmic reticulum and mitochondria is crucial to finely control cardiac excitation-contraction coupling (CECC) [6,8,9].Thus, deregulation of cellular Ca 2+ homeostasis causes decoupling of CECC, increasing the incidence of cardiac arrhythmias [6].ATP deficit during ischemia inhibits ATP-dependent ionic transporters, like Na + /K + -ATPase, Ca 2+ -ATPase plasmalemmal (PMCA), and sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), leading to the accumulation of Na + and Ca 2+ in the cytosol [6,14] and cytosolic Ca 2+ overload [6,14].This process induces an increase in mitochondrial Ca 2+ influx, which further reduces ATP production and, consequently, collapses the cardiac function [6].Cytosolic and mitochondrial Ca 2+ overload severely compromises CECC, favoring the development of severe and fatal arrhythmias [6,8,9,20].Thus, drugs that reduce Ca 2+ influx through L-type Ca 2+ channels (LTCC) in cardiac cells significantly reduced the incidence of severe and fatal arrhythmias induced by CIR, strengthening the idea that the attenuation of cytosolic and mitochondrial Ca 2+ overload reduces cardiac collapse caused by CIR [6].
In addition to its role in CECC, LTCC-mediated Ca 2+ influx in cardiac cells modulates cAMP production by AC isoforms 5 (AC5) and 6 (AC6) [6], the activation of adrenergic receptors in the heart, and pharmacological block of Ca 2+ via LTCC increases production and efflux of intracellular cAMP [6,21].In the extracellular medium, cAMP is transformed into ADO that can stimulate A 1 R located in the plasma membrane of cardiac cells to finely regulate cardiac function [6,22].Biochemical analyses of membrane preparations in overexpression systems have been used to establish the paradigm for Ca 2+ -mediated inhibition of AC5 and AC6 in the submicromolar range [6].In fact, the crystal structure of an AC5-catalytic domain-containing high affinity Ca 2+ -pyrophosphate (PPi) complex was just recently published [20].Although there are many papers reporting Ca 2+ -mediated modulation of AC6 activity in an endogenous setting, whole cell overexpression experiments provide most of the evidence for Ca 2+ inhibition of AC6 [6,19].Although less thorough, the evidence for Ca 2+ -mediated inhibition of the extremely comparable AC5 is consistent in its assertion.As a result, there is strong support for the idea that Ca 2+ inhibits both AC5 and AC6 and that this inhibition occurs both in vitro and in vivo [6,19].
The physiological effects of knocking down AC5 and AC6 have been seen in several investigations, although none of these can be clearly linked to the enzymes' Ca 2+susceptibility to inhibition by Ca 2+ .Mice lacking the AC5 gene have impaired pain perception, diminished motor activity, and altered heart function [6, 19,20].The preponderance of AC5 and AC6 in cardiac tissue has been hypothesized to have a substantial role in the rhythmicity of sympathetic regulation of inotropy [20].AC5 null animals exhibit a lower left ventricular ejection fraction, attenuated baroreflexes, and a lack of acetylcholine-mediated Gi inhibition of AC activity [20].They also have reduced Ca 2+ -mediated inhibition of cAMP.Reduced left ventricular function is shown in AC6 knockout mice, as well as a diminished Ca 2+ -mediated suppression of cAMP [20].
In stress conditions, such as hypoxia or ischemia, increased extracellular ADO levels are responsible for cardioprotective effects, which involve, at least in part, the activation of Gi-coupled A 1 R [23 -25].The activation of A 1 R and A 3 R has been shown to decrease cardiac infarction lesion size, as well as to consistently improve functional recovery in isolated hearts [22][23][24][25][26][27][28].A 1 R mediates the direct negative chronotropic and dromotropic actions of ADO, as well as indirect anti-β 1 AR actions [21,[29][30][31][32].It is significant to note that pharmacological stimulation of cardiac A 1 R lowers cardiac cell excitability [33][34][35][36], perhaps reducing the likelihood of fatal AVB.There is substantial evidence that the activation of all four AR (A 1 , A 2A , A 2B and A 3 ) is importantly involved in cardioprotective response in different pathological conditions, including the CIR [33,34].
The results obtained in this study (see Figures 2 and 3) also demonstrate that there is a positive correlation between Ca 2+ influx via LTCC and activity of the purinergic pathway through A 1 R activation in cardiac cells.This cross-communication between Ca 2+ influx and the purinergic signaling mediated by A 1 R is importantly involved in the regulation of the electrophysiology and contractile activity of cardiac cells, attenuating the severe and fatal arrhythmias induced by CIR.It was shown that the positive chronotropic response induced by the activation of cardiac β 1 AR is attenuated by an increase in extracellular levels of ADO produced by the enzymatic degradation of ATP released from intracardiac sympathetic neurons combined with the transport of cAMP to the extracellular medium from cardiac cells during stimulation [6, 21,22].According to several lines of evidence, the adrenergic-purinergic communication that is critical for controlling cardiac chronotropism also plays a significant role in cardioprotective responses under various pathological circumstances [6, 22,35].However, like other xanthine derivatives, DPCPX also functions as a phosphodiesterase (PDE) inhibitor and is virtually as powerful as rolipram at inhibiting PDE [6, 22,35].DPCPX exhibits a high selectivity for A 1 R over other AR subtypes [35,36].Figure 3 shows that DPCPX inhibited the cardioprotective effects of NIF and VER, indicating that A 1 R is involved in these effects (see Figure 3).
We have proposed that this pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells by means the attenuation of Ca 2+ influx via LTCC combined with an increase in the activation of A 1 R by ADO generated by the increment of extracellular transport of cAMP may be effective to prevent sudden mortality in individuals with AMI due to severe arrhythmias brought on by cardiac collapse.Bringing together all the results obtained in the present study and the existing data in the literature, we built a theoretical model of cardioprotective response stimulated by pharmacological modulation of the Ca 2+ /cAMP/ADO signaling in cardiac cells (see Figure 5).
ing in cardiac cells by means the attenuation of Ca 2+ influx via LTCC combined with an increase in the activation of A1R by ADO generated by the increment of extracellular transport of cAMP may be effective to prevent sudden mortality in individuals with AMI due to severe arrhythmias brought on by cardiac collapse.Bringing together all the results obtained in the present study and the existing data in the literature, we built a theoretical model of cardioprotective response stimulated by pharmacological modulation of the Ca 2+ /cAMP/ADO signaling in cardiac cells (see Figure 5).5) activates the contractile machinery generating contraction of cardiac cells.At the same time, (6) the Ca 2+ concentration in the cytosol is restored by Ca 2+ sequestration by SR via SERCA; Ca 2+ influx via LTCC also (7) inhibits the isoforms 5 and 6 of adenylyl cyclase (AC), producing reduction in the intracellular production of cAMP and phosphorylation of RYR 2 by cAMP-dependent kinases (PKA), and then reducing cardiac contraction frequency and strength; in addition (8), efflux of cAMP through membrane transporters MRP4 and (9) extracellular degradation of cAMP to adenosine (ADO) by ectonucleotides (ENCT) and ectophosphodiesterases (EPDE) increases extracellular ADO levels that lead to (10) activation of A 1 R, culminating in the cardioprotection resulting from prevention of CECC collapse and consequent reduction of incidence of severe and fatal cardiac arrhythmias induced by CIR.

Animals
Male Wistar rats (14-to 16-week-old) weighing between 290 and 320 g, were kept at 21 ± 2 • C with a 12:12 h light/dark cycle and were given food and water ad libitum.All experimental protocols used in this study were approved by the Ethics Committee of the Escola Paulista de Medicina-Universidade Federal de São Paulo (UNIFESP #1130/11 and #0065/12).

Cardiac Ischemia and Reperfusion (CIR) Induction
To produce an animal model of AMI, rats were subjected to surgical procedures in accordance with the approach previously published by our laboratory [7][8][9].Initially, the rats were anesthetized with ketamine (75 mg/kg, intraperitoneally) and xylazine (8 mg/kg, intraperitoneally).After anesthesia, rats were intubated using a Jelco 14G catheter (New York, NY, USA), and mechanically ventilated using an Insight model EFF 312 mechanical ventilator (Insight Equipamentos Cientificos, Ribeirão Preto, SP, Brazil) [7][8][9].A thoracotomy was carried out to insert a vascular tourniquet (4/0 braided silk suture linked to a 10 mm micropoint reverse cutting needle, Ethicon K-890H, Cincinnati, OH, USA) around the left anterior descending coronary artery to induce ischemia after the animal had been stabilized for 15 min.The tourniquet was removed after 10 min of myocardial ischemia to allow 75 min of coronary recirculation (cardiac reperfusion) [7][8][9].

Assessment of Cardiac Activity during CIR
All animals underwent ECG analysis to evaluate cardiac activity during CIR, in accordance to previously described methodology [7][8][9][10][11][12].This ECG analysis was performed to evaluate the effects of NIF and VER, in the presence or absence of blocker ABC transportermediated cAMP efflux PROB or A 1 R-selective antagonist DPCPX, on the incidence of arrhythmias (VA and AVB) and lethality (LET) induced by CIR.Initially, ECG was recorded for 15 min before ischemia protocol (stabilization period) and during ischemia (10 min) and reperfusion (75 min) protocol [7][8][9][10][11][12].A biopotential amplifier was used to record the ECG using needle electrodes inserted subcutaneously on the limbs.ECG changes (increase in R wave and ST segment) brought on by CIR were utilized to confirm that the coronary artery had successfully been blocked by surgery [7][8][9][10].A heated operating table and the proper heating lamps were used to keep body temperature at 37.5 • C, and a rectal thermometer was regularly used to check the temperature [7][8][9][10].ECG data were captured using the AqDados 7.02 collection equipment from Lynx Tecnologia Ltda.(São Paulo, Brazil) and examined using AqDAnalysis 7 software.We assessed heart rates and the incidence of VA, AVB, and LET brought on by CIR using this program.All three conditions were regarded as VA: ventricular fibrillation, torsades de pointes, and ventricular tachycardia [8,9].

Biochemical Assessment of Biomarkers of Cardiac Lesion
Blood samples (3-4 mL) were taken from the abdominal aorta and placed in siliconized tubes to determine the serum levels of biomarkers of cardiac lesion, total creatine kinase (CK), and creatine kinase-MB fraction (CK-MB).These samples were taken from rats that survived the entire 75 min CIR protocol.Centrifugation of blood samples (2500 rpm for 40 min at 5 • C) was performed and the supernatant was removed and kept at −20 • C for enzymatic analysis.A commercial kit from Vida Biotecnologia, Belo Horizonte, Brazil, was used to perform a kinetic UV test, measuring at 340 nm the enzymatic activity of CK and CK-MB in serum [8].
CIR group (n = 40): animals were treated with 0.9% saline solution (IV) one minute before they were subjected to a surgical procedure to induce cardiac ischemia (10 min), followed by coronary reperfusion (75 min), and subsequent ECG monitoring (100 min) for determination of VA, AVB and LET incidence; 2.
PROB + CIR group (n = 20): animals were treated with ABC transporter-mediated cAMP efflux blocker probenecid (PROB, 100 mg/kg, IV), five minutes before they were subjected to a surgical procedure to induce cardiac ischemia (10 min), followed by coronary reperfusion (75 min), and subsequent ECG monitoring (100 min) for determination of VA, AVB and LET incidence; 3.
NIF1 + CIR group (n = 20): animals were treated with NIF (1 mg/kg, IV) one minute before they were subjected to a surgical procedure to induce cardiac ischemia (10 min), followed by coronary reperfusion (75 min), and subsequent ECG monitoring (100 min) for determination of VA, AVB and LET incidence; 4.
NIF10 + CIR group (n = 20): animals were treated with NIF (10 mg/kg, IV) one minute before they were subjected to a surgical procedure to induce cardiac ischemia (10 min), followed by coronary reperfusion (75 min), and subsequent ECG monitoring (100 min) for determination of VA, AVB and LET incidence; 5.
NIF30 + CIR group (n = 20): animals were treated with NIF (30 mg/kg, IV) one minute before they were subjected to a surgical procedure to induce cardiac ischemia (10 min), followed by coronary reperfusion (75 min), and subsequent ECG monitoring (100 min) for determination of VA, AVB and LET incidence; 6.

Data Analysis
Data corresponding to VA, AVB, and LET incidences were expressed as percentages and statistically compared using Fisher's exact test with the Prism 5.0 software (GraphPad, San Diego, CA, USA) [7][8][9][10][11][12].Data corresponding to the serum concentration (mg/dL) of biomarkers of cardiac lesion (total CK and CK-MB) were expressed as the mean ± the standard error of the mean (SEM) and statistically analyzed with an analysis of variance test using Prism [8].The results were considered statistically significant when p < 0.05.

Conclusions
The results obtained in the present study indicate that pharmacological modulation of Ca 2+ /cAMP/ADO signaling in cardiac cells by means of the attenuation of Ca 2+ influx via LTCC and the activation of A 1 R by endogenous ADO could be a promising therapeutic strategy to reduce the incidence of severe and fatal arrhythmias caused by AMI in humans.

Figure 1 .
Figure 1.(Blue) ECG recording of control rats submitted to cardiac ischemia and reperfusion (CIR protocol; (Yellow) ECG recording of rats treated with nifedipine or/and verapamil before of CIR (Purple) ECG recording of rats treated with probenecid and nifedipine before of CIR; (Red) ECG recording of rats treated with probenecid and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) befor of CIR.

Figure 1 .
Figure 1.(Blue) ECG recording of control rats submitted to cardiac ischemia and reperfusion (CIR) protocol; (Yellow) ECG recording of rats treated with nifedipine or/and verapamil before of CIR; (Purple) ECG recording of rats treated with probenecid and nifedipine before of CIR; (Red) ECG recording of rats treated with probenecid and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) before of CIR.

Pharmaceuticals 2023 ,
16,  x FOR PEER REVIEW 5 of 13 transport to extracellular environments combined with an increase in activation of A1R receptors in cardiac cells directly participates in the cardioprotective response stimulated by NIF and VER in rats subjected to CIR.

Figure 4 .
Figure 4. Histograms representing the serum concentrations of total CK (A) and CK-MB (B) in the CIR, NIF + CIR, NIF10 + CIR, NIF30 + CIR, DPCPX + NIF10 + CIR, VER 1 + CIR, and DPCPX + VER1+ CIR groups.Results were expressed as mean ± standard error of mean, and one-way analysis of variance (ANOVA) was applied, followed by Tukey's post-test.There was no statistical difference between the different groups.
Figure 4. Histograms representing the serum concentrations of total CK (A) and CK-MB (B) in the CIR, NIF + CIR, NIF10 + CIR, NIF30 + CIR, DPCPX + NIF10 + CIR, VER 1 + CIR, and DPCPX + VER1+ CIR groups.Results were expressed as mean ± standard error of mean, and one-way analysis of variance (ANOVA) was applied, followed by Tukey's post-test.There was no statistical difference between the different groups.

Figure 5 .
Figure 5. Theoretical model of cardioprotective response stimulated by pharmacological modulation of the Ca 2+ /cAMP/ADO signaling in cardiac cells.The membrane depolarization of cardiac cells generates the (1) Na + influx via voltage-operated Na + channels (VOC Na + ) that induces (2) opening of the L-type Ca 2+ channels (LTCC) containing dihydropyridine receptors (DHPR) located in the T-tubules and depolarization of the sarcolemma that lead to Ca 2+ influx that (3) stimulates the release of Ca 2+ from sarcoplasmic reticulum (SR) mediated by activation by Ca 2+ of the R 2 -type ryanodine receptors (RYR 2 ); this Ca 2+ released from SR (4) binds to troponin C and (5) activates the contractile machinery generating contraction of cardiac cells.At the same time, (6) the Ca 2+ concentration in the cytosol is restored by Ca 2+ sequestration by SR via SERCA; Ca 2+ influx via LTCC also (7) inhibits the isoforms 5 and 6 of adenylyl cyclase (AC), producing reduction in the intracellular production of cAMP and phosphorylation of RYR 2 by cAMP-dependent kinases (PKA), and then reducing cardiac contraction frequency and strength; in addition (8), efflux of cAMP through membrane transporters MRP4 and (9) extracellular degradation of cAMP to adenosine (ADO) by ectonucleotides (ENCT) and ectophosphodiesterases (EPDE) increases extracellular ADO levels that lead to (10) activation of A 1 R, culminating in the cardioprotection resulting from prevention of CECC collapse and consequent reduction of incidence of severe and fatal cardiac arrhythmias induced by CIR.