Cardiovascular “Patterns” of H2S and SSNO−-Mix Evaluated from 35 Rat Hemodynamic Parameters

This work is based on the hypothesis that it is possible to characterize the cardiovascular system just from the detailed shape of the arterial pulse waveform (APW). Since H2S, NO donor S-nitrosoglutathione (GSNO) and their H2S/GSNO products (SSNO−-mix) have numerous biological actions, we aimed to compare their effects on APW and to find characteristic “patterns” of their actions. The right jugular vein of anesthetized rats was cannulated for i.v. administration of the compounds. The left carotid artery was cannulated to detect APW. From APW, 35 hemodynamic parameters (HPs) were evaluated. H2S transiently influenced all 35 HPs and from their cross-relationships to systolic blood pressure “patterns” and direct/indirect signaling pathways of the H2S effect were proposed. The observed “patterns” were mostly different from the published ones for GSNO. Effect of SSNO−-mix (≤32 nmol kg−1) on blood pressure in the presence or absence of a nitric oxide synthase inhibitor (L-NAME) was minor in comparison to GSNO, suggesting that the formation of SSNO−-mix in blood diminished the hemodynamic effect of NO. The observed time-dependent changes of 35 HPs, their cross-relationships and non-hysteresis/hysteresis profiles may serve as “patterns” for the conditions of a transient decrease/increase of blood pressure caused by H2S.


Introduction
The information obtained from arterial pulse waveform (APW) analysis can provide insight into the function of the cardiovascular system in physiological and pathophysiological conditions. Several APW parameters were shown to be useful for the characterization of the cardiovascular system [1][2][3]. Recently, we introduced the evaluation of 35 parallel time-dependent rat hemodynamic parameters (HPs) from recorded APW at a high resolution [4][5][6]. From the cross-relationships of the 35 HPs, one can obtain 595 "patterns" characterizing cardiovascular system at the given conditions. In the cases when changes of HPs are transient, non-hysteresis/hysteresis "patterns" can be evaluated from the cross-relationships that may indicate direct or indirect connections of signaling pathways between two given HPs [4,5].
The present work is a continuation of our work based on the hypothesis that it is possible to characterize the cardiovascular system in many patho-physiological conditions from the detailed shape of APW. The HPs and their cross-relationships may provide "patterns" for particular cardiovascular conditions, as we obtained the conditions of decrease/increase of NO bioavailability [4][5][6].

Animals, APW Measurement and Data Evaluation
The method was essentially described in our previous studies [4][5][6]. We follow the principle of not implementing any more animals as is necessary in order to minimize animal use according to the principles of the National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs, London, UK). Male Wistar rats (n = 17; 330 ± 40 g) were purchased from the Department of Toxicology and Laboratory Animal Breeding at Dobra Voda, Slovak Academy of Sciences, Slovakia. The rats were housed under a 12 hlight-12 h-dark cycle, at a constant humidity (45-65%) and temperature (20)(21)(22) • C), with free access to standard laboratory rat chow and drinking water. The veterinary nursing care was provided by the Central Animal Housing Facility of Pavilion of Medical Sciences (registration number SK UCH 01017). In the experiments with 10 µmol kg −1 H 2 S, eight rats were anesthetized with Zoletil 100 (tiletamine + zolazepam, 80 mg kg −1 , i.p.) and Xylazine (5 mg kg −1 , i.p.). In the experiments, where we compared the effects of H 2 S, GSNO and SSNO − -mix on the same rat, long time (~100 min) anesthesia was necessary. Therefore, nine rats were anesthetized with Zoletil 100 (tiletamine + zolazepam, 60 mg kg −1 , i.p.) and Xylazine (5 mg kg −1 , i.p.), and just before surgical operation, the same anesthetic dose were administrated again. For notice, H 2 S, GSNO or SSNO − -mix were administered into the right jugular vein (500 µL kg −1 ) over a 15 s period, about 40-50 min after the first anesthetic application. L-NAME (25 mg kg −1 , prepared in 0.9% saline solution) was used to study the hemodynamic effects of H 2 S, GSNO or SSNO − -mix in the condition of low NO bioavailability and high BP. The left common carotid artery (arteria carotis communis) was cannulated to insert the fiber-optic microcatheter pressure transducer FISO LS 2F connected to the FISO Series Signal Conditioner embedded in the EVO Chassis (Harvard Apparatus, Holliston, MA, USA). The recorded analog APW signal was filtered by a lowpass filter at 1 kHz, digitalized at 10 kHz and stored on a computer. Ten points (a-j) of APW, marked in Figure S1, were analyzed, from which 35 HP parameters were calculated as in our published studies [4][5][6]. Definition and abbreviation of the 35 HP parameters are described in detail in the Supplementary Materials ( Figure S1). Since point a, resp. j fluctuated with time interval~5-10 ms and~1 mmHg BP ( Figure S1B), when it was necessary, the data were filtered to average the fluctuation. For better visual comparison, plots (a) and (aa) presenting systolic BP in Figures are the same. The plot of augmentation index relative (e.g., Figure 1jj) was not possible to determine in cases when the highest point at APW ( Figure S1A) was "c" and not "f" (for details see Reference [4]).  bolus administration of 10 µmol kg −1 Na2S (marked by dash lines). The red line starts 3 s before Na2S administration. Colors: time period corresponding to the decrease of systolic BP (red), increase of systolic BP to the control value (blue), further increase of systolic BP to the maximum (green) and decrease of systolic BP to the control value (black). For the explanation of (a), (b) … (rr) plots see the Description of the Table 1 and Figure S1. Units in plots (gg), (hh), (pp) and (qq) are informative only.

Effects of Na2S on Time-Dependent Changes of 35 HPs
The time-dependent changes of 35 HPs after i.v. bolus administration of 10 µmol kg −1 Na2S are shown in Figure 1 and for other seven rats in Figures S2-S8. Na2S affected all 35 HPs for the period of ~5-10 min and then the values of HPs returned to the control level. bolus administration of 10 µmol kg −1 Na 2 S (marked by dash lines). The red line starts 3 s before Na 2 S administration. Colors: time period corresponding to the decrease of systolic BP (red), increase of systolic BP to the control value (blue), further increase of systolic BP to the maximum (green) and decrease of systolic BP to the control value (black). For the explanation of (a), (b) . . . (rr) plots see the Description of the Table 1 and Figure S1. Units in plots (gg), (hh), (pp) and (qq) are informative only.  Figure 1 and Figures S2-S8) and 32 nmol kg −1 GSNO (data from Figure 6, Table 1 in Reference [4] . Results

Effects of Na 2 S on Time-Dependent Changes of 35 HPs
The time-dependent changes of 35 HPs after i.v. bolus administration of 10 µmol kg −1 Na 2 S are shown in Figure 1 and for other seven rats in Figures S2-S8. Na 2 S affected all 35 HPs for the period of~5-10 min and then the values of HPs returned to the control level. To compare the changes of the 35 HPs, the time dependency of all HPs was divided into four phases according to the systolic BP curve (Figure 1a): decrease of systolic BP (red), increase to the control value (blue), further increase to maximum BP (green) and final decrease to the control value (black). From the comparison of the phases, it is evident that most of the HPs did not follow the time-dependent changes of systolic BP after the administration of Na 2 S. Some of the HPs followed similar (but not identical) or reverse time-dependent changes as systolic BP (a). However, other HPs, e.g., systolic area (c), dP/dt max (d), dP/dt max -RL (e), dP/dt d (f), dP/dt d -RL (g), diastolic area (l), dP/dt min -RL (n), dP/dt d − dP/dt max (q), dP/dt d − dP/dt min (r), DiN-RD (nn), (DiN − AnN)/dP/dt min (pp), and (DiN − AnN)/dP/dt max (qq) showed a completely different course in time, suggesting that these APW changes are activated at different time points and probably by different signaling pathways than the changes in systolic BP.

Comparison of Transient Effects of Na 2 S with Published Data of GSNO on 35 HPs
In our previous studies, we evaluated time-dependent changes of 35 HPs in the condition of increased NO bioavailability by GSNO and compared these effects with the changes observed in the condition of decreased NO biovailability by L-NAME [4,5]. Since GSNO transiently decreased systolic BP, similarly to H 2 S, we compared trends of their effects on 35 HPs during the decrease of systolic BP ( Table 1). The comparison revealed that the time-dependent trends of 25 HPs were similar for both compounds. The diastolic area (l) was affected in the opposite direction, GSNO decreased and Na 2 S increased this HP (red). The remaining 9 HPs showed different correlations, e.g., increase/decrease vs no effect for heart rate (b), dP/dt max (d), dP/dt min -RL (n), dP/dt d − dP/dt min (r), AnN-RD (ee), (DiN − AnN)/dP/dt max (hh), DiN-RD (nn) (blue); biphasic effect vs. no effect for dP/dt d (f) (green) and biphasic effect vs increase for dP/dt d − dP/dt min (i) (brown). Reproducibility of the effects of Na 2 S on HPs was significantly lower than the reproducibility of the effects for NO-synthase inhibitor L-NAME and NO-donor GSNO [4,6].

Non-Hysteresis/Hysteresis Relationships between HPs to Systolic BP after Na 2 S Administration
In order to describe "patterns" of effects of Na 2 S on the cardiovascular system in more detail, time-dependent changes between systolic BP and other HPs, described as their cross-relationships, were evaluated. For the full description of the cross-relationships of the 34 HPs to systolic BP (mmHg) each plot in Figure 2 should be three-dimensional, which we found visually confusing. Therefore, we omitted the time dimension and presented two-dimensional cross-relationships only.
From the maximum sum of 595 derived relationships between each HP, the crossrelationships between 34 HPs and the systolic BP are shown only (Figure 2 and Figures S9-S15). The "patterns" were different for each relationship and their reproducibility for the eight rats was low. These cross-relationships showed hysteresis or non-hysteresis "patterns". We evaluated the hysteresis/non-hysteresis pattern during the time period corresponding to the decrease ( Figure 2; red lines) and increase of systolic BP ( Figure 2; blue lines) after Na 2 S administration. The non-hysteresis cross-relationships mean that the changes of HP during the decrease ( Figure 2; red lines) and increase of systolic BP ( Figure 2; blue lines) followed approximately the same line in opposite directions (e.g., Figure 2; plots (dd) or (ee)). However, the hysteresis cross-relationships mean that the changes of HP during the decrease and increase of systolic BP did not follow the same line in opposite directions and form a loop > 5 mmHg of systolic BP (e.g., Figure 2; plots (p) or (q)). Similarly, one can reconsider the non-hysteresis/hysteresis cross-relationships of the response of the cardiovascular system to the administration of Na 2 S during the increase of systolic BP to maximum ( Figure 2; green lines) and decrease of systolic BP to the control value ( Figure 2; black lines). Figure 3 shows the number of rats, which represent the non-hysteresis or hysteresis cross-relationship of 34 HPs to the decrease/increase of systolic BP (data were taken from Figure 2 and Figures S9-S15, red and blue lines). There was a low reproducibility of the non-hysteresis/hysteresis cross-relationships in eight rats. The non-hysteresis crossrelationships (at least in 6 of 8 rats) were observed in plots: (e), (o), (cc), (dd) and (mm). The hysteresis cross-relationships (at least in 6 of 8 rats) were observed in plots: (c), (d), (j), (k), (q), (hh) and (rr).  Table 1 and Figure S1. Units in plots (gg), (hh), (pp) and (qq) are informative only.
From the maximum sum of 595 derived relationships between each HP, the crossrelationships between 34 HPs and the systolic BP are shown only (Figure 2 and Figures  S9-S15). The ''patterns'' were different for each relationship and their reproducibility for the eight rats was low. These cross-relationships showed hysteresis or non-hysteresis ''patterns". We evaluated the hysteresis/non-hysteresis pattern during the time period  Table 1 and Figure S1. Units in plots (gg), (hh), (pp) and (qq) are informative only. Figure 3 shows the number of rats, which represent the non-hysteresis or hysteresis cross-relationship of 34 HPs to the decrease/increase of systolic BP (data were taken from Figure 2 and Figures S9-S15, red and blue lines). There was a low reproducibility of the non-hysteresis/hysteresis cross-relationships in eight rats. The non-hysteresis crossrelationships (at least in 6 of 8 rats) were observed in plots: (e), (o), (cc), (dd) and (mm). The hysteresis cross-relationships (at least in 6 of 8 rats) were observed in plots: (c), (d), (j), (k), (q), (hh) and (rr). Since H2S transiently decreased systolic BP, similarly to NO-donor GSNO, we compared the H2S data from Figure 3 to the published GSNO data (Figure 3c in Reference [4]). From the comparison of the H2S and GSNO non-hysteresis/hysteresis cross- Figure 3. Number of rats, that represent the non-hysteresis or hysteresis "patterns" of the relationships of HPs to the systolic BP after the administrations of 10 µmol kg −1 Na 2 S during decrease (red line in Figures 1 and 2) and increase (blue line in Figures 1 and 2) of BP. Data were taken from Figure 2 and Figures S9-S15. The total number of rats in which non-hysteresis (blue) or hysteresis (red) "patterns" were evaluated is n = 8. The hysteresis was arbitrary defined as HP − systolic BP loop >5 mmHg of systolic BP. For the explanation of (a), (b) . . . (rr) plots see the Description of the Table 1 and Figure S1.
Since H 2 S transiently decreased systolic BP, similarly to NO-donor GSNO, we compared the H 2 S data from Figure 3 to the published GSNO data (Figure 3c in Reference [4]). From the comparison of the H 2 S and GSNO non-hysteresis/hysteresis cross-relationship "patterns" of the 34 HPs to systolic BP, it is evident that most of the "patterns" are not the same-actually, some of them are opposite. For example, in the dP/dt max to the systolic BP (plot (d)), hysteresis was observed in the case of H 2 S, but non-hysteresis in the case of GSNO response. Similarly, opposite non-hysteresis/hysteresis relationships were observed for diastolic BP (j), pulse BP (k) or dP/dt d − dP/dt max (q). However, similar non-hysteresis/hysteresis relationships for H 2 S and GSNO were observed for dP/dt max -RL (e), (DiN − AnN)/dP/dt max (hh) or AnN − 1Max (rr). Figure S16 represents the non-hysteresis/hysteresis "patterns" of the relationships of HPs to systolic BP, which were evaluated during the increase and decrease of BP ( Figure 2 and Figures S9-S15, green and black lines, respectively) after the i.v. administration of 10 µmol kg −1 Na 2 S. The non-hysteresis cross-relationships (at least in 6 of 8 rats) were observed in plots: dP/dt max -RL (e), diastolic area (l), dP/dt min (m), dP/dt min delay (o), AnN-RL (cc), AnN delay (dd), AnN-RD (ee), (DiN − AnN)/dP/dt min (gg), DiN delay (mm) and DiN-RD (nn). The hysteresis cross-relationships (at least in 6 of 8 rats) were observed in: systolic area (c), dP/dt max (d), diastolic BP (j), pulse BP (k), dP/dt d − dP/dt max (q) and AnN − 1Max (rr).

Effect of H2S on Distinct Fluctuation of Diastolic BP
The bolus administration of H2S also influenced a minor fluctuation of diastolic BP ( Figure 5 and Figures S17-S23) that is clearly seen from the measurement of the time interval between AnN (point d in Figure S1A) and diastolic BP (point a1 or a2 in Figure  S1B). This time interval (d-a1) or (d-a2) can be depicted as the time-dependent plot of

Effect of H 2 S on Distinct Fluctuation of Diastolic BP
The bolus administration of H 2 S also influenced a minor fluctuation of diastolic BP ( Figure 5 and Figures S17-S23) that is clearly seen from the measurement of the time interval between AnN (point d in Figure S1A) and diastolic BP (point a1 or a2 in Figure S1B). This time interval (d-a1) or (d-a2) can be depicted as the time-dependent plot of AnN delay ( Figure 5A, plot (dd)) and Figure 5B,C at a higher time resolution. The higher value of AnN delay in ms ( Figure 5A(dd),B,C) indicates that diastolic BP (in mmHg) at point a1 was lower than at point a2. The time dependence of AnN delay more or less reflected the time-dependent changes of systolic BP ( Figure 5A, plot (aa)) and the position of AnN in mmHg ( Figure 5A, plot (bb)). The comparison of the fluctuation between a1 and a2 values at single pulses, expressed as the ratio of a1/(a1 + a2) pulses, increased~5-12 s after the H 2 S administration, when systolic BP reached the minimum and decreased after 120 s ( Figure 5D). of AnN in mmHg ( Figure 5A, plot (bb)). The comparison of the fluctuation between a1 and a2 values at single pulses, expressed as the ratio of a1/(a1 + a2) pulses, increased ~5-12 s after the H2S administration, when systolic BP reached the minimum and decreased after 120 s ( Figure 5D).  Figure S1B). The higher value of AnN delay in ms indicates that diastolic BP at point a1 was lower than at point a2. (C) The fluctuation between a1 and a2 points in pulses (black circles) recorded at high resolution at 120 s after the 10 µmol kg −1 Na2S administration. Data were taken from Figure S8. (D) Comparison of the ratio of a1/(a1 + a2) pulses in 6 s long interval: Starting 6 s before the 10 µmol kg −1 Na2S administration; from 3 s before to 3 s after the minimum of systolic BP, and 120 s after the 10 µmol kg −1 Na2S administration. Data represent means ± SEM. Statistically significant differences between group means were determined by one-way repeated-measures ANOVA (p < 0.0001) with Dunnett's post-hoc test (*p < 0.05, **p < 0.01, n = 8). For the explanation of (aa), (bb) and (dd) plots see the Description of the Table 1 and Figure S1.  Figure S1B). The higher value of AnN delay in ms indicates that diastolic BP at point a1 was lower than at point a2. (C) The fluctuation between a1 and a2 points in pulses (black circles) recorded at high resolution at 120 s after the 10 µmol kg −1 Na 2 S administration. Data were taken from Figure S8. (D) Comparison of the ratio of a1/(a1 + a2) pulses in 6 s long interval: Starting 6 s before the 10 µmol kg −1 Na 2 S administration; from 3 s before to 3 s after the minimum of systolic BP, and 120 s after the 10 µmol kg −1 Na 2 S administration. Data represent means ± SEM. Statistically significant differences between group means were determined by one-way repeated-measures ANOVA (p < 0.0001) with Dunnett's post-hoc test (*p < 0.05, **p < 0.01, n = 8). For the explanation of (aa), (bb) and (dd) plots see the Description of the Table 1 and Figure S1.

Comparison of Na 2 S, GSNO and SSNO − -Mix Effects on Rat BP
It is known that GSNO and H 2 S influence BP and interact together to form a mixture of several bioactive products. Nitrosopersulfide (SSNO − ) is one of the major products of this interaction. Therefore, we termed the mixture as the SSNO − -mix. Here, we compared the effects of Na 2 S, GSNO and SSNO − -mix on rat BP. Bolus administration of 40, 80, 160 and 320 nmol kg −1 Na 2 S did not affect BP, whereas the administration of 4, 8, 16 and 32 nmol kg −1 of NO-donor GSNO transiently decreased BP in a concentration-dependent manner ( Figure 6). SSNO − -mix applied at a 10:1 molar ratio (by dilution of stock solution prepared by the reaction of 10 mmol L −1 Na 2 S with 1 mmol L −1 GSNO to final bolus administrations of Na 2 S:GSNO at a ratio of 40:4; 80:8; 160:16; and 320:32 in nmol kg −1 ) had a minor effect on BP. It is known that GSNO and H2S influence BP and interact together to form a mixture of several bioactive products. Nitrosopersulfide (SSNO − ) is one of the major products of this interaction. Therefore, we termed the mixture as the SSNO − -mix. Here, we compared the effects of Na2S, GSNO and SSNO − -mix on rat BP. Bolus administration of 40, 80, 160 and 320 nmol kg −1 Na2S did not affect BP, whereas the administration of 4, 8, 16 and 32 nmol kg −1 of NO-donor GSNO transiently decreased BP in a concentration-dependent manner ( Figure 6). SSNO − -mix applied at a 10:1 molar ratio (by dilution of stock solution prepared by the reaction of 10 mmol L −1 Na2S with 1 mmol L −1 GSNO to final bolus administrations of Na2S:GSNO at a ratio of 40:4; 80:8; 160:16; and 320:32 in nmol kg −1 ) had a minor effect on BP. The comparison of Na2S, GSNO and SSNO − -mix effects on rat BP was also studied in the condition of a decreased NO bioavailability. It has been shown that L-NAME, an inhibitor of nitric oxide synthase (NOS), decreased NO bioavailability and significantly influenced HPs [6]. Particularly, L-NAME (25 mg kg −1 ) significantly increased BP and influenced shape of APW (Figure 7). Bolus administration of Na2S at concentrations 40-320 nmol kg −1 did not significantly affect BP. However, GSNO at the concentrations 4-32 nmol kg −1 transiently and pronouncedly decreased BP in a concentration-dependent manner in the presence of L-NAME (Figure 7). However, SSNO − -mix (Na2S:GSNO; 80:8; 160:16; and 320:32 in nmol kg −1 ) had only a minor effect on BP. The comparison of Na 2 S, GSNO and SSNO − -mix effects on rat BP was also studied in the condition of a decreased NO bioavailability. It has been shown that L-NAME, an inhibitor of nitric oxide synthase (NOS), decreased NO bioavailability and significantly influenced HPs [6]. Particularly, L-NAME (25 mg kg −1 ) significantly increased BP and influenced shape of APW (Figure 7). Bolus administration of Na 2 S at concentrations 40-320 nmol kg −1 did not significantly affect BP. However, GSNO at the concentrations 4-32 nmol kg −1 transiently and pronouncedly decreased BP in a concentration-dependent manner in the presence of L-NAME (Figure 7). However, SSNO − -mix (Na 2 S:GSNO; 80:8; 160:16; and 320:32 in nmol kg −1 ) had only a minor effect on BP.

Discussion
This work is a continuation of studies based on the hypothesis that it is possible to significantly characterize the cardiovascular system in physiological and pathophysiological conditions just from the shape of APW [4][5][6]. To characterize the cardiovascular system from APW, the connections between ''unique patterns'' of APW parameters and specific physiological and patho-physiological conditions must be known. Effects of drugs on APW, for example specific inhibitors/activators of ion channels or regulation enzymes, can help to find these ''patterns'' of APW parameters specific for

Discussion
This work is a continuation of studies based on the hypothesis that it is possible to significantly characterize the cardiovascular system in physiological and patho-physiological conditions just from the shape of APW [4][5][6]. To characterize the cardiovascular system from APW, the connections between "unique patterns" of APW parameters and specific physiological and patho-physiological conditions must be known. Effects of drugs on APW, for example specific inhibitors/activators of ion channels or regulation enzymes, can help to find these "patterns" of APW parameters specific for particular patho-physiological conditions of the cardiovascular system. Our previous studies indicate that the recording system used in our work detects subtle changes of APW with sufficient reproducibility under equal experimental conditions [4]. Using this approach, we characterized timedependent changes of APW caused by i.v. administration of H 2 S, and compared these changes with the effects of NO-donor GSNO and with the effects of H 2 S/GSNO interaction mixture (SSNO − -mix). It is noticed that the results were obtained in rats under anesthesia.
Reproducibility of the effects of H 2 S on HPs, their cross-relationships and nonhysteresis/hysteresis "patterns" were significantly lower than the reproducibility of the effects for the NO-synthase inhibitor L-NAME and NO-donor GSNO [4][5][6]. It might result from numerous interactions of H 2 S with different components of the cardiovascular system, which were reported previously [21]. Additionally, the interaction of H 2 S with biological disulfides (RSSR) results in the formation of equilibrium of various sulfur species, e.g., H 2 S, RSH, RSSR, RSSH and RSSSR, which may be responsible for its diverse activity [22]. These may contribute to the explanation of dissimilar effects of H 2 S on single HPs, as discussed below.
From the time-dependent H 2 S effects, it is evident that most of the 34 HPs did not follow the time-dependent changes of systolic BP and that nearly each HP showed a different time-dependent response to H 2 S (Figure 1 and Figures S2-S8). These results indicate that H 2 S influences each of the 35 HPs differently, confirming specific "patterns" of responses included in the shape of APW. H 2 S transiently decreased systolic BP similarly to GSNO, but 10 out of 35 HPs showed diverse trends for H 2 S and GSNO, indicating different "patterns" of their effects on hemodynamics, based on different signaling pathways.
In order to look for more detailed "patterns" of effects of H 2 S on the cardiovascular system, the time-dependent changes between HPs, described as their cross-relationships, were evaluated. Each of the cross-relationship between 34 HPs and the systolic BP (Figure 2 and Figures S9-S15) showed a specific "pattern", indicating that various signaling pathways influence the shape of APW after the administration of H 2 S. Some of the cross-relationships showed non-hysteresis or hysteresis loops (Figure 3). Similarly, as in our previous studies [4,5], it is suggested that the non-hysteresis of the cross-relationship between two particular HPs indicates a direct connection between the signaling pathways regulating these two HPs, whereas hysteresis indicates that the signaling pathways are connected indirectly. The direct or indirect connections of signaling pathways regulating the connection between two HPs are also considered as a "pattern" for H 2 S action. However, more studies are needed to conclude which of the time-dependent and cross-relationships "patterns" are unique for the H 2 S action.
The comparison of the non-hysteresis or hysteresis cross-relationships of HPs between H 2 S ( Figure 3) and GSNO ( Figure 3C in [4]) showed similar, but also opposite "patterns" for particular HPs (Figure 4). Therefore, we may suggest that some of the HPs are regulated by the activation of the same pathways for both compounds, however others were modulated by different mechanisms.
NO and H 2 S coupled signaling pathways in the cardiovascular system are complex and not fully understood [9,21,[23][24][25][26][27][28]. The involvement of NO in the regulation of BP is associated with the modulation of activity of various ion channels. For instance, NO signaling leads to an increased activity of large conductance Ca 2+ -dependent K + channels (BK Ca ), resulting in K + efflux, causing hyperpolarization of cell membrane and consequent deactivation of L-Type Ca 2+ channels. In addition, NO may decrease intracellular Ca 2+ levels by direct inhibition of L-Type Ca 2+ channels, thus reducing Ca 2+ influx, leading to smooth muscle relaxation [21,[23][24][25][26]. Similarly, the effects of H 2 S on BP are mediated through the modulation of the activity of membrane channels. Several reports show that the H 2 S induced vasorelaxation is caused by the activation of potassium channels, particularly ATP-sensitive potassium channels (K ATP ) or small to medium conductance K Ca channels [21,23,29]. Furthermore, vascular effects of NO and H 2 S are mediated through cyclic guanosine monophosphate (cGMP) signaling. NO increases the cGMP levels by activating guanylyl cyclase and H 2 S inhibits phosphodiesterase activity that catalyzes the degradation of cGMP [30]. In turn, cGMP stimulates the activity of protein kinase G (PKG), leading to the dephosphorylation of myosin light chains (MLC) and smooth muscle relaxation. Alternatively, H 2 S activates PKG by oxidation and disulfide formation, independently of cGMP signaling [31].
The transient decrease of BP induced by H 2 S and NO may at least partially result from hyperpolarization, closing of voltage-dependent calcium channels and reduction of intracellular Ca 2+ or reduction of MLC phosphorylation. We may suggest that the similar responses of the cross-relationships of HP S to systolic BP for H 2 S and NO (Table 1) and resembling non-hysteresis/hysteresis "patterns" (Figure 4) reflect the "ion channel signaling pathways" or the "PKG signaling pathway" by both compounds. The different responses of the cross-relationships of HP S to H 2 S and NO and dissimilar non-hysteresis/hysteresis "patterns" may reflect other pathways that are specific for single compounds. For instance, Nier et al. suggested that the changes in the relative levels of DiN are specific for NO signaling and negatively correlate with NO bioavailability [32]. Our study showed that H 2 S decreased the relative level of DiN, indicating that the NO-dependent signaling pathway was activated. Moreover, the relationships between DiN-RL (ll) and systolic BP showed mostly hysteresis "patterns" after GSNO and Na 2 S administration, implicating that the changes in DiN-RL (ll) are activated at different time points and probably by different signaling pathways than the changes in systolic BP ( Figure 4). While having a similar effect on BP, GSNO and H 2 S affected HPs, which reflect heart functions differently. GSNO had a negative inotropic (dP/dt max -RL) and lusitropic (dP/dt min -RL) effect, however H 2 S had a minor effect on heart contraction and relaxation. At the same time, H 2 S decreased heart rate, but GSNO did not affect the heart chronotropy. This is in line with a negative inotropic effect of H 2 S mediated through the opening of K ATP channels and no effect on heart contractile function reported by others [33,34]. For the depressive actions of NO on contraction and relaxation, desensitization of cardiac myofilaments through the cGMP/PKG pathway was described [35,36]. Interestingly, the relationships between dP/dt max -RL (e) and BP showed mostly non-hysteresis "patterns", however the dP/dt min -RL (n) to BP showed mostly hysteresis "patterns" for both compounds (Figure 4). Therefore, we suppose that the effects on heart contraction may be mediated by the same signaling pathways as the effects of the compounds on BP. However, the changes in heart relaxation are postponed in comparison to BP changes and probably mediated by different signaling pathways.
Several reports propose that the products of H 2 S-NO interaction, particularly SSNO − , play an important role in the regulation of BP [9,17,18,37]. Therefore, we have studied the effect of SSNO − -mix on BP. SSNO − -mix in the presence or absence of L-NAME had minor effects on BP in comparison to GSNO (Figures 6 and 7). Effects of bolus i.v. administration of SSNO − -mix on BP have been previously studied at concentrations ≥30 nmol kg −1 and decreased BP in a concentration-dependent manner [12,14]. The observed minor effects of i.v. administration of SSNO − -mix at low concentrations (Figures 6 and 7) with comparison to GSNO on HPs is in contrast to its published potency to relax isolated aortic rings exvivo, in which the effects of SSNO − -mix was several times more potent and faster than GSNO [37]. It has been suggested that NO and HNO, among other species formed in the SSNO − -mix, were involved in the vessels relaxation [14,37]. However, in blood a fast inactivation of NO and HNO occurs after the interaction with hemoglobin [38,39]. While the membrane of red blood cells is permeable to NO, the consumption of NO by red blood cells is reduced by an intrinsic barrier and an extracellular unstirred layer of solvent [40,41]. Our data suggest that the products of the SSNO − -mix are decomposed or metabolized faster in the blood than GSNO. From this, it is suggested that, at low concentrations (4-32 nmol kg −1 ), the SSNO − -mix and/or the products of SSNO − -mix decomposition are almost all quenched by blood components before reaching the surface of vascular endothelial cells, leading to minor effects on BP. We may speculate that the mechanisms limiting the consumption of NO by red blood cells are less effective in the case of the products formed in the SSNO − -mix due to different physical and chemical properties of these species. While in the case of isolated aortic rings, SSNO − -mix and its decomposition products directly interact with the membrane and/or intracellular targets [37]. These results suggest that the formation of SSNO − -mix in blood, but not in the cells, diminishes the effects of NO on BP.
As discussed in our previous studies [4,6], the physiological origin of anacrotic notch (point d in Figure S1A) and the minor time/pressure fluctuation of diastolic BP ( Figure S1A,B, point a-a1 or a2) are not yet fully understood. Effect of H 2 S on the distinct fluctuation of a1 or a2 ( Figure 5) was similar (but not identical) to the effect reported for GSNO [4]. The observations that the points fluctuated between two distinct positions and that the fluctuation was influenced by H 2 S and GSNO during decreasing/increasing of BP indicate that it might be a physiological, yet unknown, important cardiovascular phenomenon.

Conclusions
Our work characterizes novel details of the cardiovascular responses and presents numerous original data characterizing APW by 35 HPs and by their cross-relationships after i.v. administration of 10 µmol kg −1 H 2 S. Since the effect of H 2 S on BP was transient, we showed non-hysteresis/hysteresis time-dependent changes of 35 HPs, and from their cross-relationships to systolic BP, direct/indirect signaling pathways of the H 2 S effects were proposed. We assume that the similar responses of HP S to H 2 S and NO, their resembling cross-relationships and non-hysteresis/hysteresis "patterns" reflect the activation of "channels or PKG signaling pathways" by both compounds. In addition, the H 2 S/GSNO product (SSNO − -mix) at a concentration ≤32 nmol kg −1 showed a minor effect on hemodynamics in comparison to GSNO, suggesting that the formation of SSNO − -mix in blood diminished the effect of NO on BP. The observed time-dependent changes of 35 HPs, their cross and non-hysteresis/hysteresis relationships may serve as "patterns" for the conditions of a transient decrease/increase of BP caused by H 2 S.
In summary, the analysis of the hemodynamic effects of H 2 S is a starting point for the characterization of the cardiovascular system by 35 HPs under physiological conditions. However, further validation of the results in animal models of diseases will be useful and represents a challenge for future studies.