Effect of α2-Adrenoreceptors stimulation after blockade of the current activated by hyperpolarization, if, in 6-week-old rat’s isolated heart

Efecto de la estimulación de los receptores adrenérgicos-α2 después del bloqueo de la corriente activada por la hiperpolarización, if, en el corazón aislado la rata de 6 semanas de edad

Anna Mikhailovna Kuptsova anuta0285@mail.ru

Associate Professor (Department of Human Health Protection) Institute of Fundamental Medicine and Biology, Rusia

Alina Maratovna Galieva galieva_alina94@mail.ru

Рostgraduate, Department of Human Health Protection) Institute of Fundamental Medicine and Biology,, Rusia

Arnold Geinrikhovich Miller, arnold_17@mail.ru

Рostgraduate, Department of Human Health Protection) Institute of Fundamental Medicine and Biology, Rusia

Nafisa Ilgizovna Ziyatdinova nafisaz@mail.ru

Professor (Department of Human Health Protection) Institute of Fundamental Medicine and Biology, Rusia

Timur Lvovich Zefirov, zefirovtl@mail.ru

Professor (Department of Human Health Protection) Institute of Fundamental Medicine and Biology, Rusia

This work is licensed under Creative Commons Attribution-NoDerivs 4.0 International.

The α2-adrenergic receptor (α2-AR) agonists are used to treat cardiovascular, psychiatric, and neurological diseases, asthma, glaucoma¹, as well as for anesthesia, sedation, hypotension, and hypothermia². Using DNA microarrays, the expression of three α2-AR isoforms (α_2A/D, α_2B, α_2C-AR) was detected in all mammalian species, including humans, as well as at different stages of cardiogenesis².

In mammals, α_2A- and α_2C-AR isoforms are significantly present in the central nervous system, while all three α2-AR isoforms are widespread in such structures as epithelial tissue of the intestine, kidneys, vascular smooth muscle cells, membranes of presynaptic adrenergic endings, cell membranes myocardium^3,4. All 3 subtypes of α2-AR have been detected in the human heart using the polymerase chain reaction method⁵. Radioligand analysis and immunoblotting revealed the presence of all three subtypes of α2-AR in the myocardium of normotensive as well as spontaneously hypertensive rats⁶.

In the late 1990s, studies were focused on the current activated by hyperpolarization (Ih, If). The research interest in this type of current is due to the biophysical properties and features of regulation of isoforms of HCN channels conducting this current, including their distribution in tissues^7,8. In the heart, isoforms of HCN channels are distributed in the vascular system⁹, as well as in working cardiomyocytes¹⁰.

The evidence indicates the existence of the interaction between α₂-AR and If to reduce mechanical and thermal pain¹¹. The α₂-AR agonist, clonidine, by inhibiting HCN channels causes a strong bradycardic effect¹². It should be noted that the inhibition of HCN channels with clonidine in humans can be applied with rapid intravenous administration during a hypertensive crisis or opioid detoxification¹³.

The findings in regard to the ontogenetic features of the formation and development of the cardiovascular system indicate that the age of 6 weeks is of absolute interest, since at this age rats undergo significant changes in the activity of the heart, the minimum heart rate, the completion of adrenergic innervation formation of the heart, and the initiation rat body’s work as adult animals^14,15.

The objective of this work is to study the parameters of the isolated rat heart after the formation of adrenergic regulation of the heart after activation of α2-AR in the presence of If blockade.

Experiments and procedures on laboratory animals were performed following the principles of good laboratory practice and animal ethical guidelines.

A 25% urethane solution (800 mg/kg) administered intraperitoneally was used as anesthesia. A transverse incision was made at the level of the diaphragm, the chest cavity was opened, and the heart was removed. The isolated heart specimen was washed in the temperature related to the Krebs-Henselite solution (around 2-5^oC). Then, an isolated heart specimen was hooked onto the cannula behind the aorta, fixed, and perfused with Krebs-Henselite solution. The solution was pre-saturated with carbogen (95% O₂, 5% CO₂) for 30 minutes, and then continuously during the experiment. The pH was maintained in the range of 7.3-7.4, the temperature of the perfused solution was maintained at 37°C. Throughout the experiment, the pressure of the perfused solution was maintained at a constant level of 55-60 mmHg. Mechanical activity of the left ventricle was determined using a latex balloon connected through a catheter to a pressure transducer. Signals of pressure developed in the left ventricular cavity were recorded on a computer using LabChart Pro V8.

The obtained curves were used to calculate the coronary flow (CF), heart rate (HR), pressure wave amplitude (PWA), maximum wave rise rate (dP/dt_max), and maximum wave drop rate (dP/dt_min).

In the experiments, the α2-AR agonist, clonidine hydrochloride, and the hyperpolarization-activated current blocker ZD7288 were used. There were one group with clonidine alone, one with the blocker alone and a third group with the combination of both. Besides, the used substances are manufactured by Sigma and Tocris.

Paired and unpaired Student's t-test was used for statistical processing of experimental data. Values were considered significant at *p <0.05; **p <0.01 relative to the initial values.

To evaluate the interaction of α₂-AR and If, experiments were carried out on with the administration of clonidine hydrochloride (10^-6M) in 6-week-old rats pretreated with ZD7288, If blocker (10^-9M and 10^-5M). The If blocker and the α₂-AR agonist at these concentrations caused significant changes in the functioning of an isolated heart^16,17. In effect, the application of a α₂-AR agonist in the presence of blockade of If-currents at a concentration of 10^-9 M led to a significant change in all studied parameters.

Over the course of the experiment it could be observed he PWA gradually increased during the experiment. By the 5th minute of the experiment, its value increased from 23.2±2.2 mm Hg, up to 29.6±2.4 mm Hg (p <0.05). Further, by the 10th minute of observation, the PWA increased to 37.5±2.7 mm Hg. (p<0.01). Maximum PWA 41.1±3 mm Hg (p <0.01) was recorded during the 13th minute of the experiment. At the final minute of observation, the PWA decreased slightly and amounted to 39.6±4.7 mm Hg (p<0.01).

An increase in dP/dt_max from 585.2±61 mm Hg/sec to 595±70.3 mm Hg/sec was observed during the first minute. By the 5th minute, dP/dt_max continued to increase to 794.5±87.9 mm Hg/sec (p <0.05). During the 10th minute it was 861.2 ± 92.2 mm Hg/sec (p <0.01). Then, by the 15th minute, dP/dt_max increased to 954.7±126.2 mm Hg/sec (p <0.01). At the last minute of the experiment, dP/dtmax was 983±142.6 mm Hg/sec (p <0.01).

Application of an α₂-AR agonist secondary to If blockade led to the increase in dP/dtmin from 480±44.2 mm Hg/sec to 492.51.8 mm Hg/sec. By the 5th minute of the experiment, dP/dt_min was 699.9 ± 74.3 mm Hg/sec (p <0.01). During the tenth minute of observation, dP/dt_min increased to 783.6±77.7 mm Hg/sec (p <0.01). Further, during observation, dP/dt_min continued to increase to 813.1±89.5 mm Hg/sec (p<0.01) and 824±115 mm Hg/sec (p <0.01) during the 15th and 20th minutes, respectively.

Figure 1.

Figure 1. Dynamics of changes in the indices of the isolated heart of 6-week-old rats upon application of a α.-AR agonist secondary to the blockade of If-currents at a concentration of 10^-9M. The ordinate axis is the change in values (%), the abscissa is the performance of an isolated heart - the pressure wave amplitude (PWA), heart rate (HR), coronary flow (CF), the pressure wave rise rate (dP/dt_max), the pressure wave drop rate (dP/dt_min). *p <0.05; **p <0.01 relative to the initial values.

Heart rate in one group of animals at 1 minute of the experiment showed over time a decrease from 180.2±25.3 bpm to 175.6±23 bpm. By the 5th minute, the heart rate dynamics continued to decrease to 141.6±20.2 bpm (p<0.05). By the 10th minute, the heart rate decreased to 124.6±19 bpm (p<0.05), by the 15th minute the heart rate was 130.8±18.3 bpm (p<0.05). At the final minute of observation, the heart rate decreased to 125.6±18.2 bpm (p<0.05). In the other group, an increase in heart rate was observed already by the 5th minute of the experiment from 179.5±21.6 bpm to 186.4±20 bpm. The maximum increase in heart rate was recorded during the 9th minute of observation - 224.3±67.5 bpm. Further, the heart rate decreased slightly to 217.6±62.4 bpm during the 15th minute and to 207.1±37 bpm during the 20th minute of experimental observation.

The CF change was also multidirectional. In one group, CF decreased during the 1st minute of the experiment from 5.9 ± 1.9 ml/min to 5.7±1.3 ml/min (p <0.05). Then, until 10 minutes, CF remained unchanged. By the 15th minute of experimental observation, CF decreased to 5.3±1.3 ml/min (p <0.05). At the end of the experimental observation, CF decreased to 5.1±1.2 ml/min (p <0.05). In the second group, CF increased from 10.8±2.9 ml/min to 11.4±3 ml/min (p <0.05) by the 5th minute of the experiment. Further, CF did not change. During the 20th minute of observation, CF was 12±2.6 ml/min (p <0.05).

The addition of clonidine hydrochloride against the background of a high concentration (10^-5M) of the If blocker caused multidirectional effects of all studied parameters.

The PWA in the first group gradually increased during the experiment. By the 1st minute of the experiment, its value increased from 23.9±5.1 mm Hg up to 24.8 ± 4.9 mm Hg. By the 5th minute, the PWA was 31.6±4.1 mm Hg (p <0.01). The dynamics of the increase in PWA were also observed during the 10th minute - up to 36.3±3.9 mm Hg (p <0.01), and during the 15th minute the value changed to 36.6±3.7 mm Hg (p <0.01). In the final minute, the PWA increased to 37.7±4.6 mm Hg (p <0.01).

There was also the second experimental group, which showed a decrease in PWA. During the 5th minute of the experiment, its value decreased from 23.3±4.7 mm Hg up to 20.9±5.9 mm Hg. Further, until the 10th minute, the values did not change. During the 15th minute of observation, the PWA decreased to 12.7±2 mm Hg. The maximum decrease in PWA was recorded at the 19th minute of the experiment - 11.6±1.7 mm Hg.

Figure 2.

Figure 2. Dynamics of changes in the indices of the isolated heart of 6-week-old rats upon application of a α.-AR agonist in the presence of If-currents blockade with ZD7288, at a concentration of 10^-5M. The ordinate axis is the change in values (%), the abscissa is the performance of an isolated heart - the pressure wave amplitude (PWA), heart rate (HR), coronary flow (CF), the pressure wave rise rate (dP/dt_max), the pressure wave drop rate (dP/dt_min).

In the first experimental group, dP/dt_max during the 1st minute of the experiment increased from 505.6±51.8 mm Hg/sec to 519.3±54 mm Hg/sec. During the 5th minute of observation, dP/dt_max increased to 669.2±50.7 mm Hg/sec (p <0.01). By the 10th minute after adding clonidine hydrochloride to the solution secondary to If-blockade, dP/dt_max increased to 774.6 ± 53.5 mm Hg /sec (p <0.01). The maximum increase in dP/dt_max was observed during the 17th minute of the experiment, 890.7±84.8 mm Hg/sec (p <0.01). In the second experimental group, a decrease in dP/dt_max was from 777.7±266.4 mm Hg/sec to 759.8 ± 363.7 mm Hg/sec, which did not change until the 15th minute. During the 15th-minute dP/dt_max was 626.9±416.6 mm Hg/sec. During the last 20th minute of the experiment, dP/dt_max decreased to 579.1±372.9 mm Hg/sec.

dP/dt_min in one experimental group increased from 380.9±30.7 mm Hg/sec to 399.2±39.4 mm Hg/sec during the 1st minute of observation. By the 5th minute of the experiment, dP/dt_min increased to 541.6±22.2 mm Hg/sec (p <0.01). During the 10th and 15th minutes, dP/dtmin increased to 611.1±46 mm Hg/sec (p <0.01) and 656.9±50.6 mm Hg/sec (p <0.01), respectively. During the 20th minute of the experiment, dP/dt_min was 634.2±54.3 mm Hg/sec (p <0.01). In another experimental group, dP/dt_min decreased from 692.9±319.9 mm Hg/sec to 644.8±275.4 mm Hg/sec. By the 10th minute of observation, dP/dt_min decreased to -625.7±355.8 mm Hg/sec. At the 17th minute of the experiment, the α2-AR agonist administered after preliminary If blockade led to the decrease in dP/dt_min to 520.4±382.3 mm Hg/sec. During the 20th minute of observation, dP/dt_min decreased to 479.2±330.1 mm Hg/sec.

The heart rate changes upon stimulation of α.-AR with preliminary If blockade were multidirectional. A decrease in heart rate was observed from 128.3±23.1 bpm to 122.5±26.1 bpm in the first minute of the experiment. 5 minutes after the start of α.-AR stimulation against the background of preliminary If blockade, HR was 108.3±23.2 bpm (p <0.05). By the 10th minute, HR smoothly decreased to 105.3±22.8 bpm (p <0.05) and did not change until the end of the experiment. Also, some animals showed an increase in heart rate from 111.5±18.2 bpm to 160.5±90.4 bpm during the 4th minute of the experiment and a further decrease in heart rate to 152.8±66 bpm during the 15th minute of observation, up to 135.1±46.1 bpm during the 20th minute of observation.

CF in one experimental group increased from 8±3.1 ml/min to 8.54±3.2 ml/min (p <0.05) during the 4th minute of observation. Then CF decreased to 8.36±3.2 ml/min (p <0.05) by the 10th minute of the experiment. During the 15th minute, CF was 8.3±3.1 ml/min. At the end of the experiment, CF was up to 8.4±3.1 ml/min. In the second experimental group of animals, during the 1st minute of the experiment, we recorded a decrease in CF from 5.4±2 ml/min to 5.2±2 ml/min. 5 minutes after the injection of the substance, CF decreased and amounted to 4.8 ± 1.9 ml/min (p <0.01); by the 15th minute of the experiment, CF decreased to 4.4±2 ml/min (p <0.01). By the 20th minute of experimental observation, CF was lower than the initial value and amounted to 4.5±2 ml/min (p <0.01).

Analysis of the results revealed that stimulation of α₂-AR in the control group led to an increase in contractility by 18%, and administration of an α₂-AR agonist after the If blocker (10^-9M) led to the increase in the pressure wave amplitude by 70%. The α2-AR agonist against the background of the If-blockade (10^-5M) led to an increase in the contraction force by 58% and the decrease in the contraction force by 45%. In the control group, clonidine hydrochloride caused bradycardia, reducing the heart rate by 34%. However, stimulation of α₂-AR against the background of If blockade had multidirectional effects on heart rate. The decrease in heart rate during the application of clonidine hydrochloride against the background of ZD7288 blockade at 10^-9 M and 10^-5 M was less pronounced - 21% and 18%, respectively. The increase in heart rate was 15% and 21%. There was a change in CF indicators. In the control group, CF decreased by 9%. The introduction of an agonist against the background of If blockade (10^-9M and 10^-5M) caused a more pronounced negative CF dynamic by 14% and 16%, respectively. But there was also an increase in CF by 12% and 4%.

The development and formation of adrenergic innervation of the heart play an important role in the regulation of ionic currents of cardiomyocytes, the development of the autonomic reaction of the heart^14,15. Undoubtedly, the study of the mechanism of action of the α₂-AR agonist in the presence of If blockade during the development of adrenergic innervation of the heart is of absolute interest. If-current plays a key role in the autonomic regulation of the heart rate, as well as rhythmic activity in the nervous system. Reviewing the literature data, mechanisms of the possible role of If in the realization of the signaling pathways of α₂-AR are suggested. The activation of α2-adrenergic receptors on the cell membrane can lead to the activation of G-protein-bound internal K+ rectification channels and inhibition of HCN, which leads to membrane hyperpolarization^16,17. The evidence showed that such α2-AR agonists as clonidine and UK14304 led to the decrease in the amplitude of the current activated by hyperpolarization and slowed down the rate of its activation¹⁸. A direct inhibitory effect of the α₂-AR agonist on If in the mouse heart was also shown^19,20. Thus, we investigated the age-related mechanisms of adrenergic regulation of the heart of 6-week-old rats. As a result, If-blockade and subsequent stimulation of α₂-AR showed to cause an increase in heart inotropy, as well as multidirectional effects on the rhythm and blood supply of an isolated rat heart at the stage of completion of the formation of adrenergic innervation, in contrast to adult animals with a formed innervation of the heart in which unidirectional increase in inotropy and decrease in heart rate and CF.

Conflict of Interests

The author declares that the provided information has no conflicts of interest.

Acknowledgments

This study was prepared in accordance with the Russian state program of competitive growth of Kazan Federal University and supported by the RFBR and Government of the Republic of № 18-44-160022.