Influence of NO and [Ca2+]o on [Ca2+]i homeostasis in rat ventricular cardiomyocytes

Abstract In this study, we explored the effect of NO-induced changes in [Ca2+]i homeostasis in rat ventricular cardiomyocytes through variation in extracellular Ca2+ and application of SNAP (S-nitroso-N-acetyl-d,l-penicillamine) as an NO-donor. The Fura-2 signal dynamics and phalloidin intensity profile of z-disks in rat cardiomyocytes served as endpoints to describe the mechanisms involved in the control of the intracellular Ca2+ levels, depending on the kinetics of distribution of the SNAP-produced NO. The results demonstrated that SNAP caused small phalloidin intensity profile changes between the z-lines in the presence of [Ca2+]o. However, in the absence of [Ca2+]o, SNAP in a concentration of 300 μmol/L induced a significant decrease in the distance between z-lines. This SNAP-induced decrease was reflected as a decrease in the phalloidin fluorescence intensity in the middle of the sarcomere, due to the preferential imaging of greater fluorescence in the bulk of the thin filaments. Based on the Ca2+ fluorescence intensity profile, we could suggest that intracellular Ca2+ is mainly affected by the mechanisms of Ca2+ outflow, rather than the mechanisms of Ca2+ inflow in the presence of NO. Actually, through variation in the electrochemical gradient of Ca2+, we induced mechanisms of faster/slower cytosolic Ca2+ emptying. The obtained data showed that the delay in the cytosolic Ca2+ emptying in the presence of [Ca2+]o is due to the increased electrochemical gradient of Ca2+.


Introduction
L-type Ca 2þ channels (LTCCs) are the major mediator of Ca 2þ influx in cardiomyocytes, regulating both excitation-contraction coupling and the activation of several signaling cascades. The Ca 2þ current (I Ca ) is itself regulated by several pathways, including b-adrenergic, Ca 2þ /calmodulin-dependent protein kinase and calcineurin [1]. Calcineurin is linked to the z-disk via calsarcin but also directly binds to and dephosphorylates the LTCCs [2,3]. Published data suggest that described patterns are clearly dependent on intracellular Ca 2þ ([Ca 2þ ] i ) homeostasis, and their proper functioning is dependent on [Ca 2þ ] i regulatory mechanisms as well [4].
Nitric oxide (NO) markedly influences the [Ca 2þ ] i homeostasis [5,6] by affecting the influx of Ca 2þ through the plasma membrane and its release from the intracellular stores. Many reports have discussed the inhibitory effects of NO on the heart's I Ca . Sodium nitroprusside (SNP, 100 lmol/L) inhibits I Ca in neonatal rat cardiac ventricular myocytes [7]. NO-synthase inhibitors, L-N G -monomethyl arginine citrate (L-NMMA, 1 mmol/L) and L-N G -Nitroarginine (L-NNA, 1 mmol/L) induce rapidly pronounced stimulation of I Ca in isolated guinea-pig ventricular myocytes [8,9]. Other authors have shown that rat ventricular myocytes [10] and rabbit sinoatrial cells pre-stimulated with b-adrenergic agonist [11][12][13] show inhibited I Ca in the presence of different NO-donors. There are also reports of biphasic effects of NO on I Ca in cardiomyocytes [14]. Kirstein et al. [15] showed that the NO-donor 3-morpholino-sydnonymine (SIN-1) has a pronounced stimulatory effect on I Ca in isolated human atrial myocytes, starting from 1 pmol/L concentration. Moreover, it attains a maximal stimulatory effect (doubling of current amplitude) at 1 nmol/L, while increasing its concentration to 100 mmol/L abruptly reduces its stimulatory effect on I Ca .
Several authors report dual effects of NO on I Ca . Campbell et al. [16] showed that in isolated ferret ventricular myocytes, SIN-1 (1 mmol/L) has a stimulatory effect on I Ca in $40% of cells, an inhibitory effect in 40% of cells, and a biphasic effect in 20% of cells. Furthermore, Whaler and Dollinger (1995) used SIN-1 (10 lmol/L) in isolated guinea-pig ventricular myocytes to create a inhibitory effect, while in myocytes prestimulated with b-adrenergic agonist, the SIN-1 created a stimulatory effect on LTCCs [17].
There are conflicting data about the influence of NO on [Ca 2þ ] i homeostasis. The few studies performed with NO donors indicated different dose-dependent effects. In order to determine the NO-induced changes in intracellular Ca 2þ homeostasis in rat ventricular cardiomyocytes, we employed a novel approach using fluorescent dyes. Through variation in extracellular Ca 2þ ([Ca 2þ ] o ), and application of the NO-donor, S-nitroso-N-acetyl-D,L-penicillamine (SNAP), based on the Fura-2 signal dynamics and phalloidin intensity profile between z-disks in rat ventricular cardiomyocytes, we explored the mechanisms involved in the control of [Ca 2þ ] i , depending on the kinetics of distribution of the SNAP-produced NO.

Animals
All animal experiments were carried out in accordance with the Guide for Care and Use of Laboratory Animals published by the US National Institute of Health (8th edition, 2011). The experimental protocol was approved by the Bioethics Committee of the Moscow State University. Male outbred white rats weighing 200-250 g (n ¼ 28) were held in the animal house for 4 weeks under 12:12-h light:dark period in standard T4 cages prior to the experiment. The animals were fed ad libitum.

Isolated cardiomyocyte preparation
The previously described cell isolation procedure was used [17] (with slight modifications). Also, the rat hearts were isolated as described previously [17]. The hearts were attached to the Langendorff apparatus for retrograde perfusion with a Ca 2þ -free solution containing: 120 mmol/L NaCl, 5.4 mmol/L KCl, 5 mmol/L MgSO 4 Á7H 2 O, 5 mmol/L Na-pyruvate, 20 mmol/L glucose, 20 mmol/L taurine and 10 mmol/L Hepes at pH 7.4, adjusted with NaOH [18][19][20][21]. After an initial perfusion period of 5 min with a Ca 2þ -free solution, the hearts were perfused for 18-20 min with the same solution and supplemented with type II collagenase (0.5 mg/mL), type XIV protease (0.08 mg/mL) and 20 lmol/L CaCl 2 . The perfusate was continuously bubbled with carbogen, and the temperature was equilibrated at 37 C. Finally, the ventricles were separated, chopped and gently triturated to release the cells into a standard Kraftbr€ uhe medium [17]. The suspension of the cells obtained from each of the hearts was stored in a normal Kraftbr€ uhe medium.
Images were collected using an inverted confocal laser scanning microscope, LSM-700 (Zeiss, Germany). The fluorescence of Alexa Fluor Phalloidin was excited at 488 nm and monitored using an LP505 emission filter; DAPI fluorescence was excited at 405 nm and registered through a 420-480 emission filter.
The images were analyzed using the Fiji/ImageJ software. The laser power and the detector settings were kept constant to maintain consistency in the data collection system.

Calcium imaging
The cardiomyocytes were loaded with the acetoxymethyl ester form of Fura-2 (Fura-2 AM; 1 lmol/L) for 20-30 min at room temperature. After loading, the cells were incubated in a dye-free solution for 30 min to allow conversion of the colour to its Ca 2þ -sensitive form. The cells were plated on glass-bottom dishes and mounted on the stage of an inverted Olympus IX81 microscope with a 20Â objective. Only wellattached cells (as assessed by whether a brief test pulse of shear fluid would move or blow the cell away) were used. Fura-2 fluorescence was excited at 340 nm and focused on the cells via a 20Â objective (LUC Plan FL N 20x/0.45 Ph1, Olympus, Tokyo, Japan). The 510 nm emitted fluorescence was collected by a high-speed cooled CCD camera (Olympus, Tokyo, Japan) and recorded with cellM&cellR software. A quantitative analysis of fluorescent images was performed using Fiji/ImageJ software.

Statistical analysis
One-way analysis of variance (ANOVA), followed by the Newman-Keuls multiple comparison tests, was used to analyze the tested groups. The statistical significance was used for testing two-tailed probabilities. The statistical significance was set at p < .05. All the analyses were performed with Graph Pad Prism 4.0 (San Diego, CA).  (Figure 1(B)). However, in the absence of [Ca 2þ ] o , SNAP in a concentration of 300 lmol/L, induced a significant reduction in the distance between the z-lines (p < .05) (Figure 1(A)). We believe that such changes are a reflection of NO-induced changes in intracellular Ca 2þ . It was demonstrated that exogenous NO triggers classic ischemic preconditioning by preventing intracellular Ca 2þ overload in cardiomyocytes [22]. In fact, we believe that NO released by SNAP inhibits LTTCs and stimulates mechanisms of extracellular Ca 2þ release. It appears that SNAP-induced Ca 2þ release is reflected as a decrease in phalloidin fluorescence intensity in the middle of the sarcomere, due to preferential imaging of greater fluorescence in the bulk of thin filaments (Figure 2(B,C)). However, in the presence of high [Ca 2þ ] o (1.8 mmol/L), the effect of NO was not pronounced, probably due to the high concentration gradient of Ca 2þ . This high [Ca 2þ ] o is the reason for replenished intracellular Ca 2þ stores [23], and stable intracellular Ca 2þ homeostasis (Figure 1(B)). More evidence that the entire process is Ca 2þ -dependent comes from the comparison between the date in Figure 1(A,B). As can be seen, 300 lmol/L SNAP did not show the same effect in the presence or absence of [Ca 2þ ] o . This indicates some involvement of NO in the regulation of intracellular Ca 2þ homeostasis through Ca 2þ regulatory players [24].    gradient for Ca 2þ , the low NO level at the very beginning (t 1/2 for SNAP decomposition is $37 h) initiates mechanisms that lead to an increase in cytosolic Ca 2þ concentration. As intracellular Ca 2þ grows, intracellular stores are replenished, and at some point, Ca 2þ overload is achieved. Meanwhile, the partial pressure of NO increases and induces inhibition of LTCCs, and at the same time, fosters the restoration of intracellular Ca 2þ homeostasis by induction of mechanisms for extracellular Ca 2þ transport [25]. Actually, in the absence of [Ca 2þ ] o , the increased outflow of Ca 2þ along the gradient and the Na þ /Ca 2þ exchanger can change the mode and stoichiometry of the Ca 2þ transfer, which was not the purpose of this study. Taking into account the NO-driven parallel dynamics in the Ca 2þ outflow in the presence or absence of [Ca 2þ ] o , we assume that [Ca 2þ ] i is mainly affected by the mechanisms of Ca 2þ outflow rather than the mechanisms of Ca 2þ inflow in the presence of NO. This is in line with previous reports that submicromolar concentration changes in the cytoplasmic Ca 2þ concentration increase the peak amplitude of the outward Na þ /Ca 2þ exchange current, even in the condition of reduced electrochemical gradient of Ca 2þ [26,27]. This reduced electrochemical gradient of Ca 2þ in our case (empty circles in Figure 3), was the reason for the faster cytosolic Ca 2þ emptying. Therefore, it seems that the time delay in the presence of [Ca 2þ ] o was due to the increased electrochemical gradient of Ca 2þ .

Results and discussion
In all experiments from this setting, we obtained high standard errors when [Ca 2þ ] i decreasing began, which may be an indicator of a partially dys-synchronous Ca 2þ release. A similarly dys-synchronous Ca 2þ release has been previously demonstrated in atrial cells [28], in model systems with high background Ca 2þ influxes [29], in dedifferentiated myocytes [25,30] and in detubulated myocytes subjected to osmotic shock [28]. Consistent with the important role of high Ca 2þ influxes [29]

Limitations
One of the basic limitations of this study is that we did not clarify whether calmodulin is involved in the examined mechanisms. Also, additional experiments with Ca 2þ -troponin C should be performed to determine any additional signaling pathways affected by SNAP and/or [Ca 2þ ] o .

Conclusions
This report is among the firsts that, based on the Fura-2 signal dynamics and phalloidin intensity profile between z-lines, examined [Ca 2þ ] i changes in the presence of 300 lmol/L SNAP and/or 1.8 mmol/L [Ca 2þ ] o . The results, addressed a possible role of the Na þ /Ca 2þ exchanger in the regulation of [Ca 2þ ] i homeostasis during high NO production. This finding does not minimize the role of SR Ca 2þ uptake, but could help fill the gap in the literature regarding the role of the Na þ /Ca 2þ exchanger, suggesting that in conditions of higher NO production, this transporter can play an important role in the regulation of the [Ca 2þ ] i homeostasis in cardiomyocytes. Additional investigations should be done in order to be able to give full clarification of the role of the Na þ /Ca 2þ exchanger in conditions of increased NO production in cardiomyocytes.

Disclosure statement
No potential conflict of interests was reported by the author(s).

Funding
This work was supported by the Russian Science Foundation under grant number 16-14-10372.