O. V. Nakipova

Sarcolemmal α2-adrenoceptors control protective cardiomyocyte-delimited sympathoadrenal response

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced β-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of β- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.

Store-operated Ca2+ entry supports contractile function in hearts of hibernators

Hibernators have a distinctive ability to adapt to seasonal changes of body temperature in a range between 37°C and near freezing, exhibiting, among other features, a unique reversibility of cardiac contractility. The adaptation of myocardial contractility in hibernation state relies on alterations of excitation contraction coupling, which becomes less-dependent from extracellular Ca2+ entry and is predominantly controlled by Ca2+ release from sarcoplasmic reticulum, replenished by the Ca2+-ATPase (SERCA). We found that the specific SERCA inhibitor cyclopiazonic acid (CPA), in contrast to its effect in papillary muscles (PM) from rat hearts, did not reduce but rather potentiated contractility of PM from hibernating ground squirrels (GS). In GS ventricles we identified drastically elevated, compared to rats, expression of Orai1, Stim1 and Trpc1/3/4/5/6/7 mRNAs, putative components of store operated Ca2+ channels (SOC). Trpc3 protein levels were found increased in winter compared to summer GS, yet levels of Trpc5, Trpc6 or Trpc7 remained unchanged. Under suppressed voltage-dependent K+, Na+ and Ca2+ currents, the SOC inhibitor 2-aminoethyl diphenylborinate (2-APB) diminished whole-cell membrane currents in isolated cardiomyocytes from hibernating GS, but not from rats. During cooling-reheating cycles (30°C–7°C–30°C) of ground squirrel PM, 2-APB did not affect typical CPA-sensitive elevation of contractile force at low temperatures, but precluded the contractility at 30°C before and after the cooling. Wash-out of 2-APB reversed PM contractility to control values. Thus, we suggest that SOC play a pivotal role in governing the ability of hibernator hearts to maintain their function during the transition in and out of hibernating states.

The effect of agmatine on the rhythmoinotropic properties of the cardiac papillary muscle of hibernating animals

203 Agmatine, which is produced in mammalian tiss sues during decarboxylation of LLarginine, originally attracted attention as an endogenous ligand of α 2 ARs and imidazoline receptors. Later, it has been found that, apart from binding to these receptors, agmatine has a broad spectrum of physiological effects on the nervous, endocrine, and cardiovascular systems, which showed a wide range of possibilities for its therr apeutic use and roused interest of researchers [1]. It has been shown that agmatine decreases the blood pressure in a doseedependent manner and has a carr dioprotective effect in the ischemia–reperfusion model [2]; decreases the heart rate and the amplitude of the action potential in pacemaker cells of the rabbit sinoatrial node [3]; is involved in the regulation of the activity of LLtype Ca 2+ channels and the level of intraa cellular Ca 2+ in rat cardiomyocytes [4–6]. However, the role of agmatine in the regulation of the process of the electromechanical coupling in myocardial cells, in general, remains poorly explored. One of the key elecc tromechanical coupling units is the sarcoplasmic reticulum (SR) [7]. The disturbance of its Ca 2+ accuu mulating capacity decreases the contractile reserve of myocardial cells and causes the development of heart failure. Analysis of rhythmoinotropic effects in isoo lated cardiac muscles is a convenient methodological approach to studying the mechanisms that regulate the functional activity of SR [8, 9]. The results of our studd ies have shown that agmatine has a significant influu ence on the contraction force and the character of rhythmoinotropic phenomena (relationship between the contraction force and stimulation frequency and the pause effect) in the heart of hibernating animals. The characteristics of changes induced by agmatine suggest the ability of agmatine to increase the calcium level in intracellular stores in the heart of ground squirrels during the period of hibernation, i.e., to actii vate the mechanism underlying the unique heart tolerr ance of this animal species to extreme factors [10]. The following reagents were used in the study: agmatine (Sigma), cyclopiazonic acid (CPA, Tocris), and 22aminoethoxydiphenylborate (22APB, Tocris). The research was carried out (in agreement with the requirements of the European Convention on Anii mal Protection, 1986, 86/609/EEC) on the papillary muscles of the right heart ventricle of Yakut ground squirrels (Spermophilus undulatus) during the periods of summer activity (June–July) and hibernation: sleeping (on the 5th to 6th day of a hibernation bout with a temperature of …

Force-frequency relationship and rest potentiation in papillary muscles of Siberian ground squirrel in the period of preparation to hibernation

activity to hibernation. To date, any published data on the nature of rhythm-inotropic relationship in the myocardium of hibernating animals in this period are completely absent. Experiments were performed on the right ventricular papillary muscle isolated from ground squirrels ( C. undulatus ). The isolation, stimulation, and contraction amplitude ( A ) measurement were performed as described previously [11] at 30 ± 1°e . The steady-state force‐frequency relationships were studied in the frequency range from 0.1 to 1.0 Hz. The contraction amplitude of papillary muscles at a frequency of 0.1 Hz was taken as 100%. The maximum rest potentiation of contractility (in the range of 1 to 60 s), sometimes up to 600 s, was determined as an increment in the amplitude of the first post-rest contraction ( A 1 ) relative to the amplitude of the preceding steady-state contraction ( A 0 ) at a stimulation frequency of 0.8 Hz and calculated by the formula (( A 1 – A 0 )/ A 0 ) × 100, %) [14]. The mechanical restitution curves were obtained by plotting the increment in A 1 versus pause duration. The data are expressed as the mean and square error of the mean ( p < 0.05).

Seasonal specificity of the frequency-force dependence in the myocardium of ground squirrel, Citellus undulatus.

The heart of hibernators is capable of functioning without arrhythmia and calcium overload within the body temperature range from 0 to 37 ° C. In contrast, the body temperature decrease to 32 ° C induces atrial fibrillation in nonhibernating mammals. Further decrease in body temperature of nonhibernating mammals below 20 ° C exerts extrasystole and ventricular fibrillation [1, 2]. The mechanisms of the unique resistance of the heart of hibernators are not sufficiently understood. The rhythmoinotropic relations (dependence of the contraction force on the stimulation frequency) is an important characteristic of myocardium contractility [3]. It was found that the type of the frequency–force relationship (FFR) in the myocardium of hibernators varies in accordance with the animal’s state (hibernating or active) [4–6]. However, the mechanisms of this variability are obscure, whereas the literature on this subject is scarce and controversial. Seasonal Specificity of the Frequency–Force Dependence in the Myocardium of Ground Squirrel, Citellus undulatus L. A. Andreeva*, O. V. Nakipova*, N. A. Chumaeva*, L. S. Kosarskii*, S. G. Kolaeva*, N. I. Kukushkin*, and Corresponding Member of the RAS N. G. Solomonov**

The effects of KB-R7943, an inhibitor of reverse Na+/Ca2+ exchange, on the force of contraction of papillary muscles in the heart of the ground squirrel Spermophilus undulatus

We investigated the effect of KB-R7943, an inhibitor of the reverse mode of Na+/Ca2+ exchanger, on the force of isometric contractions, the contractile force–frequency relationship and post-rest potentiation (a qualitative parameter of Ca2+ levels in sarcoplasmic reticulum) in the right ventricle papillary muscles isolated from ground squirrel hearts during summer (June, n = 4) and autumn (October, n = 4) activities. In the presence of 1.8 mM Ca2+at 36°C, 1–1.5 hours-long treatment of the summer papillary muscles with KB-R7943 produced no significant effects on the contractile indices at the majority of stimulation frequencies. In the autumn papillary muscles KB-R7943 induced a 40–50% decrease in the force of contraction (negative inotropic effect) at low stimulation frequencies (0.1–0.3 Hz) without any significant effect at higher stimulation frequencies (0.4–3.0 Hz). Furthermore, in this group, KB-R7943 suppressed the post-rest potentiation of contractility by 50 ± 21% at pause durations exceeding 120 s. These observations indicate that KB-R7943 can affect Ca2+ levels in sarcoplasmic reticulum and that Na+/Ca2+ exchange may contribute to the physiological remodeling of intracellular Ca2+ homeostasis in myocardium of hibernating animals prior their transition to a hypometabolic torpid state.

The effect of insulin on the heart rate and temperature of the ground squirrel Spermofilus undulatus during arousal from hibernation

The effect of insulin on the heart rate and body temperature, measured per rectum, of ground squirrels (Spermophilus undulatus) during triggered arousal from winter hibernation was studied. We found that the outcomes of insulin injection to hibernating ground squirrels varied in the course of arousal. During the first stage, while body temperatures were less than 10°C, the heart rates and rectal temperatures in both control and insulin-treated groups changed in the same manner. During the next stage of arousal, when the body temperature rose above 12°C, elevation of the heart rate and rectal temperature in the insulin-treated animals was significantly retarded and lasted 110 min compared to 80 min in the control group. Conversely, in the final stage of arousal at body temperatures above 20°C, the heart rate and body temperature increased more rapidly in the insulin-treated animals that reached normal body temperature within 40 min compared to 60 min in the control group. Suggested mechanisms of bidirectional effects of insulin on the heart rates and body temperatures in ground squirrels at the particular stages of arousal, with regard to the progression of endogenous insulin and glucose levels in the blood serum, are discussed.

Possible reasons for the variability of the inotropic insulin effect in papillary muscles of ground squirrel myocardium

The effects of insulin (0.1–50 nM) on isometric twitch force (0.1 to 1.0 Hz; 30 ± 1°C; 1.8 mM Ca2+) were studied in right ventricular papillary muscles from active ground squirrels of different seasons (summer, n = 14; autumn, n = 16 and winter interbout, n = 16) in control conditions and after one-hour pretreatment of PM with 2 μM nifedipine (an L-type Ca2+-channel inhibitor) and 1.0 mM orthovanadate (a tyrosine phosphatase inhibitor). In active animals of different seasonal periods insulin causes both positive and negative inotropic effects. At low frequencies (0.1–0.5 Hz), insulin of low concentrations (0.1–1.0 nM) induces a transient (within the first 20 min after application) positive effect (about 15–25%). Application of high hormone concentration (10 nM) in a low range of stimulation frequencies causes a biphasic effect (a small initial positive inotropic effect followed by a marked negative one). At frequencies above 0.5-Hz stimulation, insulin of 10 nM concentration causes presumably a negative inotropic effect. It was proposed that ICaL is possibly involved in the insulin-induced negative inotropy in ground squirrels hearts. Alteration of protein phosphorylation in tyrosine residues is known to be a major link in the mechanism of insulin action. We performed a study on sodium orthovanadate action (a known inhibitor of tyrosine phosphatase) on the inotropic insulin effect. In the group of summer animals the pretreatment of papillary muscles with sodium orthovanadate (100 μM) does not change the negative inotropic effect of insulin in a low range of stimulation frequencies but almost completely removes this effect at stimulation frequencies above 0.3 Hz (n = 4). Nifedipine (1–1.5 h pretreatment), a blocker of L-type calcium channel, reduces the inhibitory effect of insulin in autumn and winter animals, and on the contrary intensifies it in summer animals. This fact indicates that different mechanisms must be involved in insulin actions in animals of summer and winter periods. The main findings of the present study are that insulin induces positive, negative or no inotropic effects in papillary muscles of ground squirrels myocardium. The character of the effects of insulin depends on the physiological state of animals; time and concentrations of the hormone applied; affected by conditions that alter cellular Ca2+ loading and the ratio of protein-tyrosine kinases/phosphatases activity.

Frequency-dependent effect of insulin on myocardial contractility in active ground squirrel Citellus undulatus in different seasons.

Insulin plays a key role in the regulation of myocardial contractility in normal and pathological states. However, the mechanisms of its inotropic effect are still unknown, and the published data are contradictory [1–4]. It is known that insulin increases the contractility of the myocardium in different animal species [1, 2]. However, in some cases, insulin and insulin-like agents either have no effect on the contractility [1, 3, 4] or suppress it [5, 6]. It was shown that the pattern of insulin effects may change in pathologies [4] and depends on the age of the animals [3, 5] and the experimental conditions [6]. Earlier, we demonstrated a pronounced multidirectional effect of insulin in the myocardium of the Yakutian ground squirrel [7, 8]. The discovered seasonal variability of the sensitivity of the myocardium to insulin and a pronounced dependence of its effects on the animal’s state (active, hibernating, or awakening) make the myocardium of hibernators a convenient model for studying the mechanisms of the insulin effects on the myocardium.