In skeletal muscle, Ca²⁺ release from the sarcoplasmic reticulum (SR) is triggered when dihydropyridine sensitive voltage dependent L-type Ca²⁺ channels (DHPR) mechanically activate the ryanodine receptor (RyR) type 1 (Stephenson et al., 1998). Alternatively, DHPR-mediated Ca²⁺ current also induces Ca²⁺ release via activation of RyR type 3 (Ca²⁺ induced Ca²⁺ release, CICR; Koulen et al., 2001). The Ca²⁺ release from the SR via the RyR increases the free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) about 100-fold enabling the shortening of the myocyte myofilaments (Stephenson et al., 1998). After Ca²⁺ release, the contraction process is terminated as a result of Ca²⁺ uptake mechanisms, removing Ca²⁺ from the cytosol primarily back into the SR mediated by SR Calcium ATPase (SERCA) and secondarily extruded into the extracellular space by a sodium-calcium exchanger (NCX). This dynamic and fast Ca²⁺ movement from the SR into the myoplasm and then back into the SR is denominated Ca²⁺ transient. The time course and the amplitude of the Ca²⁺ transients determine force production (Stephenson et al., 1998; Baylor and Hollingworth, 2012; Hollingworth et al., 2012).

Although, both force production and Ca²⁺ homeostasis processes are well-described, several modulators have been identified, but their role on muscle contraction are not completely understood. Nitric Oxide (NO) is a small bioactive gas molecule, with complex arrays of message intermediation in vivo (Ignarro et al., 1987) that actively modulates force production and Ca²⁺ homeostasis. Its effect is dose dependent, where high concentrations induce an increase of force production via increase of Ca²⁺ release, whereas low concentrations induce a reduction of force. NO is synthesized endogenously from the catalytic action of NO synthase (NOS; Knowles and Moncada, 1994; Nathan and Xie, 1994), whereas a small fraction of NO can be released from nitrovasodilators such as glyceryl-trinitrate (nitroglycerin) or the redox reaction of nitrite under acidic conditions (Nakahira et al., 2010). So far four NOS isoforms have been described in mammalian cells: neuronal NOS (nNOS or NOS1) and endothelial NOS (eNOS or NOS3), are both constitutively expressed and activated by Ca²⁺/calmodulin; inducible NOS (iNOS or NOS2), whose activity is Ca²⁺ independent (Knowles and Moncada, 1994; Nathan and Xie, 1994) and mitochondrial NOS (mtNOS), as α-isoform of nNOS, is constitutively expressed at the inner membrane of mitochondria and is considered the fourth isoform (Giulivi, 1998; Giulivi et al., 1998; Tatoyan and Giulivi, 1998; Carreras et al., 2007). In skeletal muscle fibers, nNOS is located in cytoskeletal structures or plasma membranes through binding to the dystrophin-associated protein, α1-syntrophin (Wakayama et al., 1997) and is highly concentrated at the inner surface of the sarcolemma, subsarcolemmal areas near neuromuscular junctions, myotendinous junctions and costamers (Chang et al., 1996; Grozdanovic and Gossrau, 1998). Zebrafish express two types of NOS: NOS1 and NOS2, which are highly conserved in evolution, showing similar amino acid sequences, cofactor binding domains, specific domains for each isoform and intron position and phases (Andreakis et al., 2011). Although, both larvae and adult zebrafish express NOS1 in the brain and peripheral nervous system (Holmqvist et al., 2000, 2004; Shin et al., 2000; Poon et al., 2003), where NO/cGMP can modulate formation and maturation of neuromuscular junctions (Jay et al., 2014), this isoform has not yet been characterized in skeletal muscle of the zebrafish. NOS2 is widely expressed in all zebrafish tissues in two different isoforms: NOS2a, which is expressed after LPS injection in adult zebrafish and NOS2b, which presents a N-terminal myristoylation sequence and is constitutively expressed in all tested tissues, suggesting that NOS2a behaves as a classical iNOS and NOS2b is similar to the mammalian eNOS (Lepiller et al., 2009).

To date, the impact of NO on the modulation of muscle systems is still under evaluation. At nanomolar levels NO activates soluble guanylyl cyclase (sGC), increasing cGMP levels, which leads to activation of a myriad of physiological processes such the activation of PKG, cGMP-cation gated channels, cGMP-hydrolyzing phosphodiesterases (PDE), and cGMP-hydrolyzing PDEs (Francis et al., 2010). NO-sGC-cGMP negatively modulates contractility, accelerates relaxation, and improves the stiffness of myocytes via direct PKG phosphorylation of proteins, including troponin I, L-type Ca²⁺ channel, phospholambam, titin, inositol triphosphate receptor, Na⁺/K⁺-APTase α-subunit, PDE5, ryanodine receptor, transient receptor potential Ca²⁺ channel isoform 3 and 6, among others (Francis et al., 2010). The second pathway is denominated S-nitrosylation (SNO), where NO interacts with free reactive cystein thiol groups to form S-nitrosothiols (Anzai et al., 2000; Eu et al., 2000; Stamler and Meissner, 2001; Sun et al., 2001). Although, zebrafish have been used as an animal model for skeletal muscle patho-physiological approaches, the precise mechanisms of NO action on the zebrafish Ca²⁺ homeostasis during muscle contraction are still unclear. Based on previous works, we hypothesized that in skeletal muscle of zebrafish larvae NO exerts its effects on Ca²⁺ homeostasis during muscle contraction mainly through negative modulation of Ca²⁺ release and Ca²⁺ uptake via the NO-sGC-PKG pathway. To address this, we used 5–7 days-post fecundation-larvae of zebrafish, a well-established animal model to study formation, development, and motor innervation as well as to comprehend molecular and cellular aspect of several myopathies (Schredelseker et al., 2010; Muto et al., 2011; Jay et al., 2014). In our study, we observed that nNOS is expressed in skeletal muscle and the Ca²⁺ transients recorded were limited to the sarcolemmal. Exogenous NO donors reduced force production via negative modulation of Ca²⁺ transients via the NO-sGC-PKG pathway, while blocking endogenous NO synthesis or the action of sGC produced the opposite effect.

Here we present evidence that in zebrafish larvae endogenous NO reduces force production by reducing Ca²⁺ release in a sGC-dependent pathway. Force production was negatively modulated by exogenous NO and enhanced in absence of NO produced by endogenous NOS. Although it is known the NO decrease myofibrillar Ca²⁺ sensitivity (Andrade et al., 1998), here we focused to further elucidate the inhibitory effect of NO on Ca²⁺-dependent force production. Different to Andrade et al. (1998), we (1) used a detailed method to describe different aspects of force and Ca²⁺ transients we collectively termed biophysical parameters, (2) we analyzed our data based on single twitch stimulation and not from submaximal tetanic stimulation, (3) we used L-NAME to evaluate the endogenous effect of NO on muscle contraction and on Ca²⁺ transients, and (4) we used zebrafish larvae as an animal model. These differences allowed us to quantify in detail each aspect of NO-dependent force and Ca²⁺ transient changes, measuring the effect of endogenous NO on force and Ca²⁺ transients in a well-established animal model for patho-physiological muscular approaches that have been extensively used due to easy and fast production of mutants, helpful model to test new therapeutic strategies and low-cost maintenance (Dou et al., 2008; Telfer et al., 2012; Asaduzzaman et al., 2013; McGown et al., 2013; Li et al., 2014; Cho et al., 2015; Li and Arner, 2015).

Since it was not possible to calculate the cross sectional area for normalization of force production as performed in other muscle models, we found that in our model single twitch force at 1 Hz prior to the force-frequency protocol was suitable for normalization to standardize the results obtained from different sizes of zebrafish larvae. The increase of force production across the entire force-frequency protocol revealed that larvae did not produce a sigmoidal force increase in zebrafish (Martin et al., 2015) as observed in mammalian skeletal muscle (Kobzik et al., 1994; Reid, 1998; Reid et al., 1998). Although we followed the force production protocol published by Dou et al. (2008), our force was substantially smaller than expected. The procedure to hold the zebrafish larvae in the aluminum foil was extremely difficult and our rate of success was very low. Therefore, we adapted the method using cyanoacrylate to better fix the larvae in the aluminum foil. Although, being very cautious we were not able to record from the entire fish as shown in Figure 1 by Dou et al. Having less muscle tissue, we expected to measure smaller forces in comparison to Dou et al. In fact, our data showed small variation and validated that our modified procedure described above did not cause major differences among the samples from the same group. Nevertheless, the maximal force production was ca. 67% higher compared to single twitch stimulation, which is in line with previous reports for zebrafish force-frequency (Dou et al., 2008). Additionally, the zebrafish larvae adapted to our method was also able to respond to extreme high frequencies of stimulation (120 Hz) without showing the clear characteristics of a fused tetanus, only at 200 Hz. This result is in line with the previous report by others (Dou et al., 2008; Martin et al., 2015), whose protocols reported that complete fused tetanus was obtained at high frequency stimulation of ~180 Hz. Attili and Hughes (2014) also field-stimulated zebrafish larvae at 200 Hz in order to evoke muscle contractions to evaluate the effect of tricaine measuring rapid escape movements. As described by Attili and Hughes, zebrafish larvae would not be able to move upon stimulation at 200 Hz (Attili and Hughes, 2014), but did upon single twitch stimulation (Attili and Hughes, 2014; Tabor et al., 2014). The physiological relevance of the force-frequency data relies on the frequency of the tail used by the zebrafish for swimming. Zebrafish larvae swim in discrete bouts with multiple oscillations of the tail, inter-spaced by quiescent periods (Budick and O'Malley, 2000) and the swimming movement of the tail is a synchronized process where motor neurons activated the correct amount of muscles fibers to produce the necessary force for a precise response, i.e., swimming for escaping, mating or preying. The measured frequency of tail's muscles during spontaneous or induced activity ranged between 15 and 100 Hz, depending on age, experimental set-up and genotype (Budick and O'Malley, 2000; Buss and Drapeau, 2001; Muller and van Leeuwen, 2004; McDearmid and Drapeau, 2006; Portugues and Engert, 2011). From this range, we observed that 60 and 90 Hz are the frequencies of stimulations able to produce the maximal force that the zebrafish larvae could generate for swimming. However, we cannot extrapolate the single twitch data analyses (that it is discussed below) to higher frequency of stimulation due to: (1) tetanic stimulation requires higher frequency and longer time compared to single twitch that modifies the EC-coupling, such as a presence of slow calcium entry during tetani stimulation that is refractory to ryanodine, but it is IP3-dependent (Eltit et al., 2004). Moreover, it is difficult to predict the role of NO under sub tetanic or tetanic conditions; and (2) we observed from the force-frequency data that the force produced at any sub-tetanic or tetanic frequency of stimulation is not a factor of the 1 Hz data (it is not lineal). Despite these limitations, we considered that our aim in this study was achieved using classical single twitch EC-coupling methods to reveal in detail the effect of NO on zebrafish larvae.

The requirement to produce force before fusion into a tetanus begins can be explained with an extreme fast action potential and fast Ca²⁺ homeostasis (Buckingham and Ali, 2004; Coutts et al., 2006). This interesting characteristic was previously reported, where the half-duration of recorded action potentials was as fast as <0.6 ms (Coutts et al., 2006). Moreover, the kinetics and the steady-state characteristics of the K⁺ and Na⁺ currents of inner skeletal muscles allow high frequency firing expected during fast swimming (Buckingham and Ali, 2004; Coutts et al., 2006). Although, both zebrafish's force production and Ca²⁺ homeostasis have been characterized, there is no information regarding the effect of NO on force and Ca²⁺ transients. We observed that exogenous NO produced by 100 μM SNAP was enough to reduce the force production at low frequencies of stimulation. We used SNAP at the concentration of 100 μM, which according to the manufacturer and our previous experience would produce NO at ca. 1.4 μM (Iturriaga et al., 2000a,b). Furthermore, the reduction of force production at low frequencies was confirmed using DEA- NONOate, a different NO donor. Conversely, blocking NOS activity with L-NAME induced an increase of force production only at very high stimulation frequency. A similar effect was extensively reported in various mammalian skeletal muscles (Kobzik et al., 1994; Andrade et al., 1998; Reid, 1998).

Based on results described above, which confirm those previously obtained in mammalian models that NO negatively modulates force production, we evaluated whether the effect of NO on force production could be due to modification of Ca²⁺ transients. To test this hypothesis, we isolated the skeletal muscle from zebrafish larvae and loaded them with Fluo4 Ca²⁺ indicator. Although, we used a different method, we obtained similar size and shape of isolated skeletal muscles as described by Robin and Allard (2015). However, we observed a difference in sarcomere length compared to reported by Dou et al. where they measured the change of sarcomere length during electrical stimulation using a laser beam method in alive zebrafish tissue while is still fixed in the force transducer. In our case, the sarcomere length was measured in single cells after the immunostaining protocol using the fluorescence signal from three different proteins (DHPR, RyR, and SERCA). If it assumed that the single cell is relaxed (1.9 μm) as opposed to the electrically stimulated mounted muscle in the force transducer, the sarcomere lengths seem comparable. Next, we evaluated whether nNOS would have a similar periodic distance as DHPR, RyR, and SERCA. The double immuno-fluorescence images suggested that the nNOS signal would be distributed similarly to DHPR, RyR1, and SERCA. However, our confocal microscope allowed us to have a maximal resolution of 440 nm at lateral direction (xy dimension) and 1.62 μm at axial direction (z dimension) for both 488 and 650 nm wavelengths necessary to excite the second fluorescence antibodies (Georgiev et al., 2015). Therefore, interpretation of co-localization of nNOs with the other tested proteins has to be done with care. In order to avoid misinterpretation of our immune-fluorescence data, we used a mathematical approach using TTorg plugin for ImageJ, which measures the periodic distance of the peak amplitude in the Fourier spectrum of the image, determining whether nNOS would have the same pattern of distribution (periodic distance) of DHPR, RyR, and SERCA. The analysis from the quantification obtained by the TTorg plugin showed only a significant difference in the periodic distance of nNOS respect to RyR. This result suggested that nNOS at 5–7 dpf larvae zebrafish is not distributed randomly in the sarcolemma and presents a similar periodic distance measured for DHPR and SERCA. It is interesting to note that we observed the Ca²⁺ transients were not uniformly distributed across the entire myocyte. They were rather more intense at the subsarcolemmal region (Figure S1). Therefore, we further investigated measuring the Ca²⁺ transient using line-scan recordings on a confocal microscope and to clarify whether the Ca²⁺ transients were limited to the salcolemmal or it also happened into the myocyte. We observed that in 5–7 dpf, the Ca²⁺ transients occurred starting in the subsarcolemmal region until approximately the center of the myocyte. Previous electron microscopy data showed that t-tubules seem to be well formed in 7 dpf (Zhang et al., 2009; Gupta et al., 2011; Housley et al., 2016). However, Gupta et al. (2011), showed that both RyR and DHPR are present at the outermost edge of the cell (Figure 7K, p. 1,720) and only the RyR is present at the sarcolemma, supporting our observations regarding Ca²⁺ transients described above.

The kinetics of Fluo4 Ca²⁺ transients were slower compared to the previously described Ca²⁺ transients obtained using whole cell patch clamp technique. However, this trait, of Fluo4 Ca²⁺ transients was useful for evaluation of the individual biophysical parameters of the modulatory effect of NO in zebrafish larvae from a selected region of the isolated myocyte. In addition, we successfully used the same method described here were in isolated zebrafish cardiomyocytes (Scheid et al., 2016). Moreover, the possibility to record several cells per experiment allowed us to statistically analyze a large number of cells compared to the patch clamp technique obtaining data. Our analyses of the biophysical parameters of single twitch-evoked Ca²⁺ transients showed that exogenous NO significantly reduced the Peak and Peak Area relative to control solution, which supports the finding that NO significantly reduced force production. In detail, the analyses of other parameters such as Time to Peak and Slope suggested that exogenous NO significantly prolonged the time of Ca²⁺ release with a decreasing rate of release, which results in a smaller Ca²⁺ transients, which in turn is translated to reduced force production. Conversely, blocking NOS activity enhanced the Peak of Ca²⁺ transients and significantly increased Peak Area compared to control. Moreover, Time to Peak was significantly reduced and the Slope was increased, but this was not statistically significant. These results suggest that NO modulates crucial events of Ca²⁺ transients such as Ca²⁺ release and Ca²⁺ uptake. To further answer this question, we tested the effect of NO on Ca²⁺ release and uptake in isolated myocytes using traditional pharmacological tools. The analyses from blocking DHPR with 20 μM verapamil showed that in combination with either exogenous NO or blocking NOS the Ca²⁺ transients displayed similar behavior regarding Peak, Time to Peak and Slope as observed from addition of SNAP or L-NAME without verapamil (Figure 4). These results support previous reports showing that the DHPR is not responsible for Ca²⁺ conductance but rather for the activation of the RyR1 in order to activate the EC-coupling via voltage-dependent conformational change of the β_1a subunit (Schredelseker et al., 2009, 2010).

We next evaluated the role of RyRs on Ca²⁺ transients under the effect of NO using 100 nM of ryanodine or 250 μM caffeine. The observed effect of exogenous NO (SNAP) on the biophysical parameters of Ca²⁺ transients were absent once SNAP was simultaneously applied with 100 nM ryanodine. At high ryanodine concentrations, the RyRs were completely blocked and it was no possible to elicit Ca²⁺ transients even at higher or longer electrical stimulation. However, at 100 nM ryanodine, the RyRs are locked at a subconductance state and do not open to a full conductance state (Koulen et al., 2001). Taken together, this suggests that exogenous NO released by SNAP would not modulate RyR activity. Conversely, the results of L-NAME (blockage of endogenous NO production) in combination with ryanodine was similar to those obtained without ryanodine, suggesting that endogenous NO would modulate the activity of RyRs. Nevertheless, these results should be treated with caution due to the high variability caused by the constant open subconductance of the RyRs (Koulen et al., 2001). Similar experiments were carried out using 250 μM caffeine as an activator of RyRs obtaining Ca²⁺ transient results comparable to the experiments without caffeine (Table S1). This suggested that under the effect of caffeine, only exogenous NO negatively modulates RyR activity. Zebrafish skeletal muscles express almost equal amounts of both RyR-α and RyR-β types (Ottini et al., 1996; Koulen et al., 2001), which are homologs to the mammalian RyR type 1 and 3, respectively (O'Brien et al., 1995; Ottini et al., 1996; Sonnleitner et al., 1998). Anatomically RyR-α is expressed in the junctional feet where it interacts with the DHPR, forming the triad in skeletal muscle, whereas the RyR-β is expressed in the parajunctional feet, which is located next to the triad facing toward the myofibrils rather than toward the t-tubule (Perni et al., 2015). Both RyR-α and RyR-β show different conductance and Ca²⁺ mediated inhibition: although both RyR-α and RyR-β are activated by physiological low Ca²⁺ concentration, RyR-α has higher conductance and Ca²⁺ inhibition at submillimolar free cytosolic Ca²⁺, whereas RyR-β has lower conductance and is active at millimolar Ca²⁺ concentration (Koulen et al., 2001). Another functional difference between RyR-α and RyR-β is that the first does not respond with Ca²⁺ sparks upon caffeine stimulation, while the second does (Perni et al., 2015). While the role of RyR-α is well-established in zebrafish skeletal muscle, the relevance of RyR-β is still being discussed, where it has been suggested to be part of the calcium-induced-calcium release mechanism and that it is not essential for normal movement of the tail in zebrafish morfants for RyR-β (Perni et al., 2015). In the 1B5 myogenic cell line both RyR1 and RyR3 have the same affinity to ryanodine, but electrical stimulation and 4-chloro-m-cresol are able to induce Ca²⁺ transients in RyR1-expressing cells, while the same protocol does not produce a response in RyR3-expressing cells. Moreover, the application of caffeine induces Ca²⁺ oscillations in RyR1-expressing 1B5 myotubes whereas the same concentration generates a small, localized and sustained Ca²⁺ increase that does not propagate and resolves rapidly in RyR3-expresing cells. Here we were interested in the general response of the RyR to NO and thus, we did not differentiate the contribution of RyR-α and RyR-β on the Ca²⁺ transients, neither whether the effect of NO was equal or not on both RyRs.

Based on the results obtained above (Figure 3), we investigated whether the presence of SNAP or L-NAME would modulate cytosolic Ca²⁺ removal (Figure 5). In mammalian skeletal muscle NO inhibits SERCA activity in a dose-dependent manner in both fast and slow muscles (Kobzik et al., 1994; Ishii et al., 1998) and it has been suggested that NO inhibit SERCA via GMP-PKG pathway (Bredt et al., 1990) or via direct action of NO on SERCA (Ishii et al., 1998); however, the mechanism of action is not entirely clear. Another discussed possibility is that NO inhibits transient receptor potential Ca²⁺ channel isoforms 3 and 6, decreasing Ca²⁺ influx and thus decreasing or depleting Ca²⁺ stores in the SR and consequently inhibit muscle contraction (Ishii et al., 1998; Francis et al., 2010); we did not evaluate this hypothesis due to its complexity and extension of the experiments. Here we pharmacologically evaluated the role of SERCA and NCX, only the most prominent Ca²⁺ extruders of skeletal muscle (Gehlert et al., 2015). We used CPA, a SERCA blocker, in order to evaluate the contribution of NCX to Ca²⁺ uptake. As expected, time durations of Ca²⁺ transients were extremely long under CPA treatment, without a change in the amplitude of the Peak. Under the effect of CPA, the evaluated Ca²⁺ uptake measuring uptake time and tau parameters did not show a significant change with either SNAP or L-NAME. When the myocytes were incubated with the NCX-blocker NiCl₂ (Gonzalez-Serratos et al., 1996; Deval et al., 2000, 2002a,b; Torok, 2007) the measured SERCA-mediated Ca²⁺ removal showed no significant reduction in uptake time parameter with SNAP (due to a non-significant reduction on the total duration of Ca²⁺ transient). Tau parameter was not different from NiCl₂ solution with either SNAP or L-NAME. It is interesting to note that higher NiCl₂ concentrations inhibit calcium transients, possibly because it blocked calcium release. We observed that only at 2 mM there was no such interference of NiCl₂. These results suggest that SERCA is the main cytosolic Ca²⁺ removal protein in zebrafish myocytes, while NCX showed no measurable effect. Regarding the effect of NO on cytosolic Ca²⁺ removal, there were no significant differences in the tau parameter, suggesting that neither endogenous nor exogenous NO would modulate either NCX- or SERCA-dependent Ca²⁺ uptake.

The NO action on Ca²⁺ transients described above could be mediated by NO-sGC pathway or via S-nitrosylation mechanism. In order to clarify this question, we analyzed the alterations of our proposed biophysical parameters under the effect of the sGC blocker, ODQ (10 μM). Moreover, we analyzed the effect of the S-nitrosylation blocker NEM (100 μM) on the same biophysical parameters. We observed that the sGC blockage with ODQ produced a similar effect as observed with L-NAME, suggesting that the effect of endogenous NO could be via the NO-sGC pathway. We wanted to exclude that this effect could also be related to NO-sGC-independent mechanisms evaluating whether the S-nitrosylation could contribute to the NO-mediated alterations of the biophysical parameters of the calcium transient. We observed that NEM, an S-nitrosylation blocker did not differ from control solution, except for Duration and Uptake time suggesting that S-nitrosylation via endogenous NO was not required for normal Ca²⁺ homeostasis. These two sets of data allowed us to propose that the main effect of NO on Ca²⁺ transients of 5–7 days old zebrafish skeletal muscle is via the NO-sGC pathway.

In summary, our results support previous reports of the NO effect in mammalian skeletal muscle (Kobzik et al., 1994; Andrade et al., 1998; Ishii et al., 1998) and extend them to 5–7 dpf zebrafish skeletal muscle. Our data suggested that NO negatively modulates force production reducing Ca²⁺ release from the SR via NO-sGC pathway, an effect probably mediated by protein kinase G (PKG) since this effect was antagonized by the action of NOS inhibitor or blocker of sGC. The next step would be to evaluate the targets and how PKG activation is able to decrease Ca²⁺ transient and thus force production in zebrafish skeletal muscle. Furthermore, in light of our results it would be of future interest to address the role of NO in other muscle pathways such as PKG activation as seen in skeletal muscle myofilaments (Beckendorf and Linke, 2015) and cardiac muscles (Francis et al., 2010), in the inhibition of the RyR (Aghdasi et al., 1997). The implication of understanding the NO-sGC-PKG pathway would help to design new therapeutic strategies in muscle diseases, such as Duchenne muscular dystrophy, where the correct localization and expression of nNOS is impaired, being replaced by the expression of iNOS hindering correct function of NO (Mosqueira et al., 2013).

ZX and MM: Contributed with design and performing all experiments, acquisition, analysis, and interpretation of data presented in the manuscript. He also drafted the manuscript and gave his final approval of the version to be published, agreeing with all aspects of the manuscript. RF: Contributed with interpretation of data for the work, drafting the manuscript and final approval of the version to be published and agreed with all aspects of the manuscript.