CC BY
17
2018, Т. 160, кн. 4 С.613-620
УЧЕНЫЕ ЗАПИСКИ КАЗАНСКОГО УНИВЕРСИТЕТА. СЕРИЯ ЕСТЕСТВЕННЫЕ НАУКИ
ISSN 2542-064X (Print) ISSN 2500-218X (Online)
UDC 612.17
STIMULATION OF a1-ADRENORECEPTORS INHIBITS MYOCARDIAL CONTRACTILITY IN RATS
T.L. Zefirova, I.I. Khabibrakhmanova, J.V. Valeevaa, J.T. Zefirovab'c, N.I. Ziyatdinovaa
aKazan Federal University, Kazan, 420008 Russia bSaint Mary's Hospital, Trinity Health of New England, Hartford, CT, 06105 USA cYale School of Medicine, New Haven, CT, 06510 USA
Abstract
The exact role of a1-adrenoreceptors (a1-AR) as myocardial contractility modulators is still not clear. We studied the effects of a1-AR stimulation with methoxamine hydrochloride (Sigma) on the myocardial contractility ex vivo and in vitro: on the isolated heart (LangendorfFs model) and on the atrium and ventricular myocardium strips in rats, respectively. The experiments were performed on random-bred albino 20-week-old rats (n = 28) with the average weight of 200-250 g. The contractility force (F) of myocardium strips was measured in grams (g). The contractility of isolated heart was measured as pressure in the left ventricle (mm Hg). All the studied concentrations of methoxamine (10-9-10-6 M) inhibited the contractility of the myocardium strips of rats' atria and ventricles. The stimulation of a1-AR with methoxamine (10-9 and 10-8 M) led to a decrease in the left ventricular pressure of the isolated heart in rats. The intensity of the negative inotropic effects was proportionate to the agonist concentration.
Keywords: a1-adrenoreceptors, heart, myocardial contractility, rats
Introduction
Adrenoreceptors (AR) play a key role in regulation of the cardiovascular system. There are nine known subtypes of AR: a1A-, a1B-, a1D-, a2A-, a2B-, a2C-, pi-, p2-, and P3-AR [1]. p-AR prevail in the mammalian heart, while ai-AR constitute 10% of all AR. In newborn rats' myocardium, the concentration of ai-AR increases over the first two weeks of postnatal period, while that of a2-AR decreases within first week after birth and then remains low [2]. Three subtypes of ai-AR have been described: aiA-, aiB-, and aiD-AP [3]. ai-AR participate in numerous physiological processes, such as increasing inotropic effects, genes transcription, protein synthesis, glucose metabolism, and inhibition of apoptosis [4].
aiD-AR mediate the sympathetic regulation of blood pressure through vasoconstriction [5]. ai-AR interact with Gq/11 proteins and activate phospholipase C. The latter induces the hydrolysis of phosphatidylinositol triphosphate to produce diacylglycerol (DAG) and inositoltriphosphate (IP3). DAG stimulates protein kinase C (PKC), which phosphorylates intracellular proteins, and inositoltriphosphate stimulates the release of Ca2+ from the sarcoplasmic reticulum [6]. PKC and IP3 can affect other intracellular reactions as well. Stimulation of ai-AR expression in the heart can activate both phospholipase C and phospholipase D [7]. PKC has been recently found to activate
protein kinase D (PKD) [8, 9]. PKD decreases the myocardial contractility [9, 10] by phosphorylation of troponin I and also accelerates the relaxation of cardiomyo-cytes [9]. In cardiomyocytes, PKD phosphorylates myosin-binding protein C, thereby decreasing the sensitivity of monofilaments to Ca2+ [11]. PKD activation leads to myocardial hypertrophy and possibly heart failure [8]. The regulation of L-type calcium channels (LTCC) through G proteins, as well as PKC activation, is a complex process which can lead to both stimulation and inhibition of I (Ca) [12]. Activation of a1-AR can also mediate negative inotropic effect though decreased Ca2+ current (ICa) though PKC [13]. PKC activation can also increase the permeability of L-type calcium channels (LTCC) [12]. PKC also mediates a1-AR stimulation in myocytes of the blood vessels; it also regulates immune reactions, gene transcription, cell cycle, and cell growth [14]. a1-AR stimulation with methoxamine induced a negative chronotropic effect on rats' myocardium during both in vivo and in vitro experiments [15]. Age-related changes in rats' myocardial contractility with a1-AR blockage have been described as well. The non-selective blockade of a1-AR with prazosin led to bradycardia in pups aged 3 weeks and older, but not in newborn rats [16].
In this study, we evaluated the effects of a1-AR stimulation on the inotropic function of rats' myocardium.
1. Materials and Methods
The heart was isolated from random-bred albino 20-week-old rats (n = 28) with the average weight of 200-250 g. The rats were anesthetized intraperitoneally with 25% urethane (800 mg/kg body weight). Myocardial strips (2-3 mm in length and 0.8-1.0 mm in diameter) were cut from the right atrium and right ventricle. The preparation was immersed vertically into a chamber (20 mL) perfused with oxygenated car-bogen (97% O2, 3% CO2) at 37 °C. The upper end of the strip was fixed to a stainless rod connected to the strain gage, while its lower end was attached to a rubber plate. The strip was stimulated using an ESL-2 electric stimulator via two silver electrodes. The stimulation parameters were as follows: pulse amplitude - 10 mV, duration -5 msec, and frequency - 6-10 min-1. Initially, the immersed strips were let sit for 4060 min to optimize the tension. The optimal tension corresponded to the critical stretch beyond which a decrease in the contractility force (F, measured in g) was observed. After the conditioning period, the initial contraction parameters were recorded over 5 min. Methoxamine (MX, an agonist affecting all subtypes of a1-ARs, Sigma) was applied in concentrations of 10-9, 10-8, 10-7, 10-6, and 10-5 M. The data were processed using the Chart 5 and Statgraphics software (Power Lab platform; AD Instruments). Statistical analysis and evaluation of the significance of differences were performed using Student's Z-test and the Wilcoxon test (Microsoft Excel). The results were processed using the AcKnowledge 4.1 program on the MP-150 unit (BIOPAC Systems, USA) with the help of the Statgraphics software.
In the ex vivo series of experiments, the heart was rapidly isolated and completely arrested by placing it into cold physiological saline (2-5 °C). The aorta was carefully cannulated to prevent touching and injuring the aortic valve and penetration of the test solution into the left ventricle lumen. The heart was perfused in a Langendorff System (ADInstruments) with the carbogen-oxygenated Krebs-Henseleit solution (containing, mM: 118.0 NaCl, 4.7 KCl, 25.0 NaHCO3, 1.2 MgSO4, 2.5 CaCl2, 1.2 KH2
PO4, and 5.5 glucose; pH 7.3-7.4) at 37 °C. The retrograde perfusion was driven by the constant hydrostatic pressure of 60-65 mm Hg. To stimulate a1-ARs, MX was used at the concentrations of 10-9 and 10-8 M. To measure the intraventricular pressure, a latex water-filled balloon (V=0.03 mL) was placed into the lumen of the left ventricle via an orifice made posterior to the left atrial auricle. The end-systolic pressure was set at the level of 10-20 mm Hg. The left ventricular pressure was recorded with a MLT844 pressure transducer (ADInstruments). The left ventricular pressure (LVP) was measured in mm Hg. The signals were recorded in a PowerLab 8/35 system (ADInstruments) with the help of the LabChart Pro 8.0 software. The data were processed statistically using Microsoft Excel software and Student's i-test
2. Results and Discussion
We started with assessing the contractility (F) of the myocardium strips of atria and ventricles of adult rats' heart in response to the a1-AR-agonist methoxamine applied at the concentrations of 10-9-10-6 М.
Ten minutes after adding methoxamine at the concentration of 10-9 М (n = 10), F of the atria decreased from 0.1212 ± 0.0166 to 0.1138 ± 0.0173 g (p < 0.05). The maximal drop in the contractility of the atria by 13.47% was registered 19 min after adding the chemical and equaled 0.10492 ± 0.0166 g (p < 0.01). The maximal decrease (by 5.54%) in the F value of the myocardium strips of the ventricles (n = 12) from 0.1477 ± 0.0167 g to 0.1395 ± 0.0142 g was registered 15 min after adding the agonist (Fig. 1).
Adding methoxamine at the concentration of 10-8 М (n = 10) also decreased F of the myocardial strips in 20-week-old rats. The maximal change in the contractility of the atria by 19.3%, from 0.126 ± 0.0171 to 0.1017 ± 0.0162 g (p < 0.001), was registered 20 min later. Nineteen minutes after adding methoxamine at the concentration of 10-8 М (n = 12), F of the ventricular myocardium decreased from 0.2045 ± 0.0164 to 0.1723 ± 0.0124 g (p < 0.001).
Methoxamine at the concentration of 10-7 М (n = 8) caused a decrease in F of the atria from 0.1058 ± 0.015 to 0.0841 ± 0.0137 g (p < 0.01). The contractility of ventricular myocardium strips after adding methoxamine at the same concentration (10-7 М (n = 8)) decreased from 0.1856 ± 0.0157 to 0.1457 ± 0.0116 g (p < 0.001), i.e., by 21.5% in total.
F of the isolated myocardium strips of the right atria after adding methoxamine at the concentration of 10-6 М (n = 8) decreased from 0.0883 ± 0.0137 to 0.0744 ± 0.0138 g (p < 0.05). The ventricular myocardium contractility after adding methoxamine at the same concentration decreased by 20.8%, from 0.1544 ± 0.0135 to 0.1221 ± 0.0118 g (p < 0.001).
A considerable limitation for accurate assessment of the contractility of the myocardium strip is its fixed position and predetermined parameters of stimulation.
In the second part of the study, the perfusion with methoxamine (10-9 and 10-8 M) without electric stimulation was used to reveal the effect of a1 -AR stimulation on the isolated heart of adult rats. Here again, the Langendorff s technique was applied.
One minute after adding methoxamine at the concentration of 10-9 М (n = 7), LVP decreased from 56.2 ± 3.8 to 46.2 ± 3.2 mm Hg (p < 0.01). In the second minute, LVP decreased to 44.2 ± 2.8 mm Hg (p < 0.01). Seven minutes after the methoxamine
Fig. 1. The influence of a1-AR methoxamine stimulation on the myocardial contractility in 20-week-old rats. Y-axis is the effect, % of the original values; X-axis is the concentration (mol). Note: * p < 0.05, ** p < 0.01, and *** p < 0.001 in comparison with the initial level. Original values are the values of myocardial contractility before methoxamine administration
Ph" J
Vv
\ \I - * h
\ ; r "1
\ * «
■-1 * *
initial 2 mill
—O— 10-9 mol(n=7)
"mill 12 mill
— 10-8 iiiol(ii=9)
Fig. 2. The effect of methoxamine on LVP of the hearts isolated from 20-week-old rats. Y-axis is LVP (%); X-axis is time (min). Note: ** is the reliability in comparison with the original values: p < 0.01. Original values are the values of LVP before methoxamine administration. ** -p < 0.01
administration, LVP equaled 44.4 ± 3.38 mm Hg (p < 0.01). The maximal decrease in the left ventricular contractility (43.7 ± 3.88 mm Hg (p < 0.01) was registered after 12 min; this value was 22.2% lower than the initial pressure (Fig. 2).
Two minutes after the perfusion of the isolated heart with 10-8 M (n = 9) of methoxamine, a decrease in the ventricular pressure from 64 ± 5.2 to 41.16 ± 4.4 mm Hg (p < 0.01) was observed. In the fifth minute, the pressure went down to 34.6 ± 5.8 mm Hg (p < 0.01). The maximal decrease of the LVP value by 37%, 33.8 ± 6.6 mm Hg (p < 0.01), was registered in the seventh minute. Further observations were not possible due to the absence of contractile activity.
Conclusions
The exact role of a1-AR as modulators of the myocardium contractility is not fully understood. One of the reasons may be that chatecholamines activate numerous intracellular targets which can affect the myocardium in opposite ways [12, 13].
Although a1-AR contribute to merely 10% of all adrenergic receptors in the mammalian heart, they still play an important role in its regulation. It is widely believed that a1-AR normally enhance the myocardium contractility, but the opposite effect -a decrease in the contractility force - is possible [17]. In our experiments, all the studied concentrations of methoxamine induced a negative effect on the atria and ventricular contractility in 20-week-old rats. The non-selective stimulation of a1-AR with methoxamine caused a negative inotropic reaction of the isolated left ventricle in adult rats. The intensity of the negative inotropic effect was determined by the agonist concentration. In our prior experiments, we discovered that a1-AR stimulation with methoxamine decreases the contractility in the isolated heart [5]. a1-AR in the heart are stimulated by postganglionic sympathetic neurons and further mediate the signaling pathways activated by Gq/11 proteins. A decrease in the myocardium contractility with a1-AR activation may be secondary to that of the Са2+ current (ICa) due the activation of PKC [13]. It is possible that a1-AR participate in a more delicate regulation of the cardiac function and most likely effects of the stimulation depend on the activity of other receptors and various intracellular systems.
Acknowledgments. The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University and supported by the Russian Foundation for Basic Research and the Government of the Republic of Tatarstan (project no. 18-44-160022).
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Recieved June 25, 2018
Zefirov Timur L'vovich, Doctor of Medical Sciences, Professor, Department of Human Health Protection Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: zefirovtl@mail.ru
Khabibrakhmanov Insaf Ilkhamovich, Candidate of Biological Sciences, Senior Lecturer, Department of Human Health Protection Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: insaf1201@mail.ru
Valeeva Julia Vladimirovna, Candidate of Medical Sciences, Associate Professor, Department of Emergency Aid and Simulation Medicine Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: JVValeeva@kpfu.ru
Zefirova Julia Timurovna, Candidate of Medical Sciences Saint Mary's Hospital; Trinity Health of New England Hartford, CT, 061505 USA
Yale School of Medicine
New Haven, CT, 06510 USA
Ziyatdinova Nafisa Il'gizovna, Doctor of Biological Sciences, Professor, Department of Human Health Protection
Kazan Federal University
ul. Kremlevskaya, 18, Kazan, 420008 Russia E-mail: nafisaz@mail.ru
УДК 612.17
Стимуляция а1-адренорецепторов ингибирует сократимость миокарда у крыс
Т.Л. Зефиров\ И.И. Хабибрахманов1, Ю.В. Валеева1, Ю.Т.Зефирова2,ъ, Н.И. Зиятдинова1
1 Казанский (Приволжский) федеральный университет, г. Казань, 420008, Россия 2Госпиталь Св. Марии, «Тринити Хэлз» Новой Англии, г. Хартфорд, CT 061505 США 3Йельская школа медицины, г. Нью-Хейвен, CT 06510 США
Аннотация
Влияние стимуляции al-адренорецепторов на сократимость миокарда до сих пор не изучено в полной мере. В данной работе обсуждаются результаты исследования воздействия метоксамина гидрохлорида (Sigma) на сократимость миокарда у крыс, полученные в ходе экспериментов ex vivo (изолированное сердце, модель Лангендорфа) и in vitro (изолированные полоски миокарда предсердий и желудочков). Эксперименты проводились на белых беспородных крысах 20-недельного возраста (n = 28), средняя масса тела которых составляла 200-250 г. Силу сокращения (F) полосок миокарда выражали в граммах (г), a сократимость изолированного сердца понимали как кровяное давление в левом желудочке (мм рт. ст.). Метоксамин во всех концентрациях (10-9-10-6 М) ингибировал сократимость полосок миокарда предсердий и желудочков. Стимуляция al-АР меток-самином (10-9 и 10-8 М) приводила к снижению кровяного давления в левом желудочке изолированного сердца. Интенсивность негативных инотропных реакций миокарда была пропорциональна концентрации метоксамина.
Ключевые слова: а1-адренорецепторы, сердце, сократимость миокарда, крысы
Поступила в редакцию 25.06.18
Зефиров Тимур Львович, доктор медицинских наук, профессор кафедры охраны здоровья человека
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: zefirovtl@mail.ru
Хабибрахманов Инсаф Илхамович, кандидат биологических наук, старший преподаватель кафедры охраны здоровья человека
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: insaf1201@mail.ru
Валеева Юлия Владимировна, кандидат медицинских наук, доцент кафедры неотложной медицинской помощи и симуляционной медицины
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: JVValeeva@kpfu.ru
Зефирова Юлия Тимуровна, кандидат медицинских наук
Госпиталь Св. Марии, «Тринити Хэлз» Новой Англии
г. Хартфорд, CT, 061505 США Йельская школа медицины
г. Нью-Хейвен, CT, 06510 США
Зиятдинова Нафиса Ильгизовна, доктор биологических наук, профессор кафедры охраны здоровья человека
Казанский (Приволжский) федеральный университет ул. Кремлевская, д. 18, г. Казань, 420008, Россия E-mail: nafisaz@mail.ru
<For citation: Zefirov T.L., Khabibrakhmanov I.I., Valeeva J.V., Zefirova J.T., Ziyatdinova N.I. Stimulation of a1-adrenoreceptors inhibits myocardial contractility in rats. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2018, vol. 160, no. 4, pp. 613-620.
<Для цитирования: Zefirov T.L., Khabibrakhmanov I.I., Valeeva J.V., Zefirova J.T., Ziyatdinova N.I. Stimulation of a1-adrenoreceptors inhibits myocardial contractility in rats |/ Учен. зап. Казан. ун-та. Сер. Естеств. науки. - 2018. - Т. 160, кн. 4. - С. 613-620.
