Polylactide Nanocapsules Attenuate Adverse Cardiac Cellular Effects of Lyso-7, a Pan-PPAR Agonist/Anti-Inflammatory New Thiazolidinedione

Lyso-7 is a novel synthetic thiazolidinedione, which is a receptor (pan) agonist of PPAR α,β/δ,γ with anti-inflammatory activity. We investigated the cardiotoxicity of free Lyso-7 in vitro (4.5–450 nM), and Lyso-7 loaded in polylactic acid nanocapsules (NC) in vivo (Lyso-7-NC, 1.6 mg/kg). In previous work, we characterized Lyso-7-NC. We administered intravenously Lyso-7, Lyso-7-NC, control, and blank-NC once a day for seven days in mice. We assessed cell contraction and intracellular Ca2+ transients on single mice cardiomyocytes enzymatically isolated. Lyso-7 reduced cell contraction and accelerated relaxation while lowering diastolic Ca2+ and reducing Ca2+ transient amplitude. Lyso-7 also promoted abnormal ectopic diastolic Ca2+ events, which isoproterenol dramatically enhanced. Incorporation of Lyso-7 in NC attenuated drug effects on cell contraction and prevented its impact on relaxation, diastolic Ca2+, Ca2+ transient amplitude, Ca2+ transient decay kinetics, and promotion of diastolic Ca2+ events. Acute effects of Lyso-7 on cardiomyocytes in vitro at high concentrations (450 nM) were globally similar to those observed after repeated administration in vivo. In conclusion, we show evidence for off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations and after repeated treatment in mice. Nano-encapsulation of Lyso-7 in polymeric NC attenuated the unwanted effects, particularly ectopic Ca2+ events known to support life-threatening arrhythmias favored by stress or exercise.


Introduction
Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family and function as ligand inducible transcription factors to regulate genes involved in lipid and glucose metabolism [1,2]. Thiazolidinediones (TZDs) are PPARγ agonists widely used as antihyperglycemic agents to improve the liver, muscles, and adipose tissue insulin sensitivity and treat type 2 diabetes mellitus [3]. The TZDs have additional anti-inflammatory and atheroprotective effects, yet the activation of PPARα may also be involved [4]. Despite reported favorable effects on cardiovascular risk factors, ischemic heart diseases and cardiovascular events may increase in diabetic patients [5,6]. TZDs also increase the risk of developing heart failure (HF) [7][8][9]. Treatment with TZD, such as rosiglitazone or pioglitazone, increase stroke risk in patients [10]. Lyso-7 is a new indole-thiazolidine compound ( Figure 1A) recently synthesized [11]. It is a promising hybrid molecule acting as a pan partial agonist of PPARγ, PPARα, and Polymeric nanocapsules (NC) ( Figure 1A) are oily core nanostructures surrounded by a polymeric wall [15,16]). They can encapsulate different lipophilic molecules at high payloads and are biodegradable. They have demonstrated outstanding potential to reduce the cardiotoxicity of several drug candidates such as artemether, halofantrine, and lychnopholide, as reported by our group [17][18][19][20]. These properties may reflect NC's ability to control drug release in the blood, reducing the free fraction producing potentially toxic adverse effects. Lyso-7 is a lipophilic compound (clog·P = 5.6). After encapsulation in NC, its physicochemical properties, release kinetics, and intravenous pharmacokinetics have been characterized [21]. NC significantly improves the Lyso-7 biopharmaceutical profile. Lyso-7 NC is suitable for oral or intravenous administration due to its nanometrical size dispersion. After intravenous administration in mice, compared with free Lyso-7 administered in solution, NC retained Lyso-7 in the plasma compartment, increasing 14-fold the plasma concentration and 3-fold the heart concentration. Upon intravenous administration, the accumulation of Lyso-7 in the heart was unrelated to blood flow. No apparent cardiotoxicity of Lyso-7 was observed in vivo when the NC formulation was administered intravenously at a dose of 1.6 mg/kg [21]. Polymeric nanocapsules (NC) ( Figure 1A) are oily core nanostructures surrounded by a polymeric wall [15,16]). They can encapsulate different lipophilic molecules at high payloads and are biodegradable. They have demonstrated outstanding potential to reduce the cardiotoxicity of several drug candidates such as artemether, halofantrine, and lychnopholide, as reported by our group [17][18][19][20]. These properties may reflect NC's ability to control drug release in the blood, reducing the free fraction producing potentially toxic adverse effects. Lyso-7 is a lipophilic compound (clog·P = 5.6). After encapsulation in NC, its physicochemical properties, release kinetics, and intravenous pharmacokinetics have been characterized [21]. NC significantly improves the Lyso-7 biopharmaceutical profile. Lyso-7 NC is suitable for oral or intravenous administration due to its nanometrical size dispersion. After intravenous administration in mice, compared with free Lyso-7 administered in solution, NC retained Lyso-7 in the plasma compartment, increasing 14-fold the plasma concentration and 3-fold the heart concentration. Upon intravenous administration, the accumulation of Lyso-7 in the heart was unrelated to blood flow. No apparent cardiotoxicity of Lyso-7 was observed in vivo when the NC formulation was administered intravenously at a dose of 1.6 mg/kg [21].
Due to the TZD class of drugs' clinical restrictions, our study now aimed to assess the cardiac cellular effects of intravenous repeated administrations of Lyso-7 (free form) and Lyso-7 encapsulated in polylactic acid NC in an experimental rodent model (mice). We investigated the effect of Lyso-7 on the contraction and Ca 2+ handling in single left ventricular (LV) myocytes in vitro and after treatment in vivo. Primary objectives were to: Pharmaceutics 2021, 13, 1521 3 of 13 (i) detect off-target effects potentially leading to unexpected harmful side effects, and (ii) determine if the use of NC provides cardiac protection.

Preparation of Lyso-7 Solution and Lyso-7-Loaded Nanocapsules
Lyso-7 was dissolved in absolute ethanol further diluted in buffers used in each experiment with ethanol concentration no higher than 2% v/v. We used this solution without Lyso-7 as a control in the acute experiments performed in vitro with isolated cardiomyocytes. We prepared the free Lyso-7 solution for intravenous (iv) treatment of mice as follows: we dissolved Lyso-7 in 20 µL of DMSO, 20 µL of polysobate 80, 380 µL of DMA, and 580 µL of PEG 300. This solution was mixed for 10 min and then appropriately diluted in isotonic glucose to obtain 0.5 mg/mL of free Lyso-7 solution used in vivo. We used the same solution without Lyso-7 as a control in vivo.
We used the nanoprecipitation method to make monodispersed nanocapsules loading Lyso-7 (Lyso-7-NC) or without Lyso-7 (blank-NC) as previously described [21]. We prepared Lyso-7-NC with 0.6% wt/v of PLA, 0.75% v/v of Miglyol 810N), and 0.75% wt/v of soybean lecithin dissolved in 10 mL of acetone. We added this organic solution into 0.75% wt/v of Poloxamer 188 solution (20 mL) via a syringe. We maintained dispersion under magnetic stirring for 10 min, and then all organic solvents and part of the water were evaporated under reduced pressure (Heidolph Rotary Evaporator, Schwabach, Germany) to render 5 mL of the aqueous suspension of NC.
The mean hydrodynamic diameters were 296 ± 4.2 nm and 265 ± 1.7 nm, respectively, with dispersion indexes less than 0.3 indicating the monodispersed population of particles in size. NC encapsulated Lyso-7 with high efficiency (83%) at a 0.5 mg/mL Lyso-7 concentration, attributed to Lyso-7 high lipophilicity. We have already characterized these NC in detail [21]. We used blank-NC as controls vs. Lyso-7-NC. All the formulations of Lyso-7, the 0.5 mg/mL iv solution (Lyso-7) and the 0.5 mg/mL Lyso-7 NC (Lyso-7-NC), as well as blank-NC, were passed through a 0.8 µm sterile filter before appropriate dilution in isotonic glucose to allow intravenous injection in mice of the doses established.

Animals and Cardiomyocytes
We used 7-week-old male C57BL/6J mice (Janvier Labs, Le Genest-Saint-Isle, France) bred and housed (4 mice/cage) under pathogen-free conditions (22 ± 2 • C; 12-h day/12-h night cycle) with standard rodent chow diet, ad libitum access to water, and behavioral needs (wood bedding material, cardboard tunnel, nesting material, and wooden gnawing sticks). For repeated-dose intravenous administration (via tail vein), mice were blindly randomized to the four different groups: vehicle (intravenous solution diluted in isotonic glucose), Lyso-7 (1.6 mg/kg/day), blank-NC (equivalent dose of NC excipients related to 1.6 mg/kg/day), and Lyso-7-NC (1.6 mg/kg/day). We administered mice vehicle and blank-NC as control groups for Lyso-7 and Lyso-7-NC, respectively, for comparisons. Repeated doses of both Lyso-7 and Lyso-7-NC for seven days produced no death in mice. We isolated myocytes freshly from mice of all experimental groups after the seven days of repeated administrations, as shown in Figure 1B.
We assessed the acute effects of Lyso-7 in vitro following the application of 4.5 nM, 45 nM, and 450 nM of Lyso-7 for 15 min on single cardiomyocytes isolated from untreated animals. The hearts were excised after euthanasia of mice by cervical dislocation to ensure rapid death without injecting any substance interfering with cellular physiology. Whole hearts were submitted to the liberase action using a Langendorf perfusion system to obtain individual LV myocytes as described [18,20,[22][23][24]. We used only quiescent cardiomyocytes with precise edges. Otherwise, we investigated cells randomly. We carried out the experiments 1 to 4 h after cells enzymatic isolation. Figure 1B shows the general schema of animal experiments and protocols.

Sparks Confocal
We visualized Ca 2+ sparks in quiescent myocytes incubated with the Ca 2+ indicator Fluo-4AM (4 µM) (Molecular Probes Inc., Eugene, OR, USA), visualized by confocal imaging with a Zeiss LSM510 microscope (Carl Zeiss Inc., Oberkochen, Germany) equipped with 63×/1.2 N.A. water immersion objective, at 25 • C in line-scan mode (1.5 ms/line, 512 pixels × 3000 lines) as described [18,22,23]. The dye was excited at 488 nm, and the fluorescence emission was collected through a 505-nm long-pass filter. Myocytes were field-stimulated at 1 Hz with 1-ms current pulses delivered via two platinum electrodes, one on each side of the perfusion chamber. During the rest period that followed stimulation, myocytes were repetitively scanned along the entire length of the cell at 1.5-ms intervals, for a maximum of 6 s. We reduced the laser intensity to 5% maximum to decrease cell damage and dye bleaching. Ca 2+ sparks were determined by averaging the intensity of each sequential scan line and plotting the mean intensity as a function of time. We used the SparkMaster plugin for ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) to detect and analyze Ca 2+ sparks.

Statistical Analysis
We performed statistical analyses using GraphPad Prism ® Software (Version 6.0, GraphPad Software Inc., San Diego, CA, USA). We expressed all data as mean ± SEM. We used D'Agostino and Pearson omnibus and Shapiro-Wilk normality tests, then a Oneway ANOVA test for multiple comparisons. We performed post hoc tests where F was significant (p < 0.05) and no variance inhomogeneity. We used a Tukey post-test to compare all pairs of columns. We used a t-test for Ca 2+ waves and Ca 2+ sparks. p < 0.05 indicated a significant difference. N indicates the number of mice, and n indicates the number of independent cardiomyocytes isolated from these mice.

Differential Effects of Lyso-7 and Lyso-7-NC on Cellular Contraction and Ca 2+
We investigated the effects of the repeated administration of Lyso-7 (free form) on contraction and Ca 2+ transient evoked by electrical stimulation of LV myocytes at 1 Hz. No mice died under treatment with the formulations. First, it is worth noting that blank-NC did not affect any parameter relative to the control group ( Figure 2, white bars). In contrast, Lyso-7 decreased cell contraction (−64%, SL shortening) and accelerated relaxation (25%) during systolic activity while not affecting resting SL (Figure 2A-D). In parallel, Lyso-7 lowered diastolic Ca 2+ (−5%), reduced the Ca 2+ transient amplitude (−35%) and accelerated the Ca 2+ transient decay kinetics (16%) ( Figure 2E-H). However, when Lyso-7 was incorporated in NC, its effect on contraction was less pronounced, by a factor of two (−33%, Lyso-7-NC vs. NC; Figure 2C, ! p < 0.05), and the impact of Lyso-7 on cell relaxation was prevented ( Figure 2D) as well as its effect on the diastolic Ca 2+ , Ca 2+ transient amplitude and Ca 2+ transient decay kinetics ( Figure 2F-H, ! p < 0.05).
used D'Agostino and Pearson omnibus and Shapiro-Wilk normality tests, then a Oneway ANOVA test for multiple comparisons. We performed post hoc tests where F was significant (p < 0.05) and no variance inhomogeneity. We used a Tukey post-test to compare all pairs of columns. We used a t-test for Ca 2+ waves and Ca 2+ sparks. p < 0.05 indicated a significant difference. N indicates the number of mice, and n indicates the number of independent cardiomyocytes isolated from these mice.

Differential Effects of Lyso-7 and Lyso-7-NC on Cellular Contraction and Ca 2+
We investigated the effects of the repeated administration of Lyso-7 (free form) on contraction and Ca 2+ transient evoked by electrical stimulation of LV myocytes at 1 Hz. No mice died under treatment with the formulations. First, it is worth noting that blank-NC did not affect any parameter relative to the control group ( Figure 2, white bars). In contrast, Lyso-7 decreased cell contraction (−64%, SL shortening) and accelerated relaxation (25%) during systolic activity while not affecting resting SL (Figure 2A-D). In parallel, Lyso-7 lowered diastolic Ca 2+ (−5%), reduced the Ca 2+ transient amplitude (−35%) and accelerated the Ca 2+ transient decay kinetics (16%) ( Figure 2E-H). However, when Lyso-7 was incorporated in NC, its effect on contraction was less pronounced, by a factor of two (−33%, Lyso-7-NC vs. NC; Figure 2C, ! p < 0.05), and the impact of Lyso-7 on cell relaxation was prevented ( Figure 2D) as well as its effect on the diastolic Ca 2+ , Ca 2+ transient amplitude and Ca 2+ transient decay kinetics ( Figure 2F-H, ! p < 0.05).  We next evaluated the effects of Lyso-7 under the challenge of a maximally active concentration of the β-adrenergic receptor agonist isoproterenol (ISO,10 nM) to mimic the effects of physical exercise and stress [25]. We exposed myocytes to ISO for 5 min before recordings. As expected, in all experimental groups (compare averaged data in Figure 3 with corresponding values in Figure 2), acute exposure of cells to ISO enhanced cell contraction, accelerated relaxation, and, in parallel, increased Ca 2+ transient amplitude and accelerated Ca 2+ transient decay kinetics in line with enhanced reuptake of Ca 2+ in the sarcoplasmic reticulum (SR) via the SERCA2a pump [26]. For example, in the control group, ISO increased cell contraction by 43%, accelerated relaxation by 29%, increased Ca 2+ transient amplitude by 81%, and accelerated Ca 2+ transient decay kinetics by 60%. Overall, these effects were consistent with those reported in the literature [25]. However, ISO had a two-fold more positive inotropic effect on contraction, compensating for the decreasing impact of Lyso-7 as found in basal conditions while also enhancing the lusitropic impact ( Figure 3B,C). In the same line, free Lyso-7 still lowered diastolic Ca 2+ and accelerated the Ca 2+ transient decay kinetics ( Figure 3D,F), similarly to the effects observed in the  Figure 2G with Figure 3E). The different effects of Lyso-7 in the presence of ISO were absent or attenuated by nano-encapsulation (Lyso-7-NC formulation). In basal conditions and with ISO, these data showed that Lyso-7 impacts cellular contraction and intracellular Ca 2+ , but its effects could be mostly prevented or significantly attenuated (e.g., contraction) by encapsulation in NC.
ISO increased cell contraction by 43%, accelerated relaxation by 29%, increased Ca 2+ transient amplitude by 81%, and accelerated Ca 2+ transient decay kinetics by 60%. Overall, these effects were consistent with those reported in the literature [25]. However, ISO had a two-fold more positive inotropic effect on contraction, compensating for the decreasing impact of Lyso-7 as found in basal conditions while also enhancing the lusitropic impact ( Figure 3B,C). In the same line, free Lyso-7 still lowered diastolic Ca 2+ and accelerated the Ca 2+ transient decay kinetics (Figure 3D,F), similarly to the effects observed in the absence of ISO ( Figure 2F,H) except for the Ca 2+ transient where the decreasing impact of ISO was partly compensated (compare Figure 2G with Figure 3E). The different effects of Lyso-7 in the presence of ISO were absent or attenuated by nano-encapsulation (Lyso-7-NC formulation). In basal conditions and with ISO, these data showed that Lyso-7 impacts cellular contraction and intracellular Ca 2+ , but its effects could be mostly prevented or significantly attenuated (e.g., contraction) by encapsulation in NC.

Differential Effects of Lyso-7 and Lyso-7-NC on Ectopic Ca 2+ Events at Rest
The repeated administration of Lyso-7 promoted ectopic diastolic Ca 2+ events, not observed in the control and NC groups, during resting periods following a train of stim-

Differential Effects of Lyso-7 and Lyso-7-NC on Ectopic Ca 2+ Events at Rest
The repeated administration of Lyso-7 promoted ectopic diastolic Ca 2+ events, not observed in the control and NC groups, during resting periods following a train of stimulations ( Figure 4A,C). When we incorporated Lyso-7 in NC (Lyso-7-NC group), we detected no significant abnormal activity. We also observed no irregular Ca 2+ transient during pacing in any of the four groups. We next tested the effect of an ISO challenge on the promotion of spontaneous Ca 2+ events. First of all, ISO (10 nM), as such, favored their occurrence ( Figure 4B,D) in line with reports by others [27][28][29]. Nevertheless, Lyso-7 severely enhanced the number of cells exhibiting ectopic Ca 2+ events (80% vs. 10% in the absence of ISO), which was abolished in the Lyso-7-NC group ( Figure 4D). Therefore, Lyso-7 induced aberrant diastolic Ca 2+ events, severely increased by ISO but controlled by NC.
ing pacing in any of the four groups. We next tested the effect of an ISO challenge on the promotion of spontaneous Ca 2+ events. First of all, ISO (10 nM), as such, favored their occurrence ( Figure 4B,D) in line with reports by others [27][28][29]. Nevertheless, Lyso-7 severely enhanced the number of cells exhibiting ectopic Ca 2+ events (80% vs. 10% in the absence of ISO), which was abolished in the Lyso-7-NC group ( Figure 4D). Therefore, Lyso-7 induced aberrant diastolic Ca 2+ events, severely increased by ISO but controlled by NC.

Lyso-7 Promotes Abnormal Ca 2+ Events at Rest
We detected no abnormal activity, either during pacing or rest, in control cells for low concentrations of Lyso-7. However, Lyso-7 at 450 nM promoted diastolic ectopic Ca 2+ events in nearly 20% of cells ( Figure 7A) though they were small and infrequent. The presence of abnormal macroscopic Ca 2+ events at rest often results from a ryanodine receptor 2 (RyR2) leakage, generating microscopic Ca 2+ events called Ca 2+ sparks [27]. Consistent with this possibility, Lyso-7 at 450 nM increased the onset of Ca 2+ sparks (+125%; Figure 7C,D). We next tested the effect of Lyso-7 in cells subjected to an ISO challenge. Once again, ISO (10 nM), as such, promoted the occurrence of ectopic Ca 2+ waves (in 30% of cells, Figure 7A) and Ca 2+ sparks (approximately two-fold, Figure 7C,D). Lyso-7 had no further impact at 4.5 nM and 45 nM in the presence of ISO, but it was worth noting that Lyso-7 severely enhanced the number of cells exhibiting ectopic Ca 2+ events (80% vs. 10% in the absence of ISO). In the presence of ISO, all cells exhibited spontaneous diastolic Ca 2+ events at 450 nM ( Figure 7A). Lyso-7 also further increased Ca 2+ sparks frequency (+28%; Figure 7C,D). Therefore, Lyso-7 promoted unwanted spontaneous Ca 2+ events, both microscopically and macroscopically, particularly during the β-adrenergic challenge of ISO in line with this pathway's additive or synergic effect.

Lyso-7 Promotes Abnormal Ca 2+ Events at Rest
We detected no abnormal activity, either during pacing or rest, in control cells for low concentrations of Lyso-7. However, Lyso-7 at 450 nM promoted diastolic ectopic Ca 2+ events in nearly 20% of cells ( Figure 7A) though they were small and infrequent. The presence of abnormal macroscopic Ca 2+ events at rest often results from a ryanodine receptor 2 (RyR2) leakage, generating microscopic Ca 2+ events called Ca 2+ sparks [27]. Consistent with this possibility, Lyso-7 at 450 nM increased the onset of Ca 2+ sparks (+125%; Figure  7C,D). We next tested the effect of Lyso-7 in cells subjected to an ISO challenge. Once again, ISO (10 nM), as such, promoted the occurrence of ectopic Ca 2+ waves (in 30% of cells, Figure 7A) and Ca 2+ sparks (approximately two-fold, Figure 7C,D). Lyso-7 had no further impact at 4.5 nM and 45 nM in the presence of ISO, but it was worth noting that Lyso-7 severely enhanced the number of cells exhibiting ectopic Ca 2+ events (80% vs. 10% in the absence of ISO). In the presence of ISO, all cells exhibited spontaneous diastolic Ca 2+ events at 450 nM ( Figure 7A). Lyso-7 also further increased Ca 2+ sparks frequency (+28%; Figure 7C,D). Therefore, Lyso-7 promoted unwanted spontaneous Ca 2+ events, both microscopically and macroscopically, particularly during the β-adrenergic challenge of ISO in line with this pathway's additive or synergic effect.

Discussion
The main result of our study is that the nano-encapsulation of Lyso-7 in PLA-NC protects cardiac cells from the deleterious off-target effects of this drug. Indeed, Lyso-7 free impacted several parameters of the cellular excitation-contraction coupling and, in particular, promoted abnormal spontaneous firing of undesirable ectopic diastolic Ca 2+ events known to increase the arrhythmogenic risk in vivo. Nano-encapsulation of Lyso-7 abolished or significantly attenuated the unwanted effects.
A significant result of our study was evidence for some off-target effects of Lyso-7, seen during acute exposure of cardiomyocytes to high concentrations or after repeated treatment in vivo. Indeed, most of the adverse effects, namely on contraction, intracellular Ca 2+ handling, and pro-arrhythmogenic Ca 2+ events, were retrieved after repeated intravenous administration of Lyso-7 at 1.6 mg/kg/day in mice for seven days. Of note, a counterintuitively divergent effect appeared on cell contraction (i.e., a decrease instead of an increase following acute exposure), suggesting different mechanisms or that cell physiology has been impacted during the repeated administration. Nevertheless, Lyso-7 had many similar effects and profoundly modified the Ca 2+ homeostasis of the myocytes when administered acutely and directly on the cardiomyocytes or repeatedly to mice. Lyso-7 lowered the resting Ca 2+ , decreased the amplitude of the Ca 2+ transient, and accelerated its decay kinetics. Finally, Lyso-7 promoted abnormal firing of Ca 2+ events during diastolic periods in both experimental conditions. Ca 2+ sparks reflect discrete, elementary Ca 2+ signaling events arising from abnormally leaky RyR2 channels [27,28]. They participate critically in initiating and propagating proarrhythmogenic spontaneous Ca 2+ waves. From a mechanistic point of view, the processes involved here are unclear. RyR2 leakage may participate in depleting SR Ca 2+ content, therefore explaining the decrease of Ca 2+ transient amplitude. However, since this depletion should increase intracellular Ca 2+ , it was counterintuitive to find that Lyso-7 decreases diastolic Ca 2+ . The Ca 2+ levels are controlled mainly by the balance between uptake of Ca 2+ via SERCA2a and Ca 2+ release of the RyR2 receptors. In case of imbalance, abnormal diastolic Ca 2+ discharge activates the electrogenic Na + /Ca 2+ exchanger (NCX), producing delayed afterdepolarization and potentially ectopic Ca 2+ transients [26,27,30]. Therefore, Lyso-7 may enhance the Na + /Ca 2+ exchanger (NCX) activity, essential for removing intracellular Ca 2+ during relaxation [31], to account for the reduced diastolic Ca 2+ .
The β-adrenergic challenge did not change much the overall effect of Lyso-7 on the basic Ca 2+ handling parameters, but it dramatically enhanced its impact on the firing of macroscopic Ca 2+ events as well as Ca 2+ sparks at rest. Low RyR2 leakage is insufficient per se to produce arrhythmogenic diastolic Ca 2+ release, particularly with enhanced NCXmediated Ca 2+ extrusion, because of the depletion in the SR Ca 2+ content [32]. However, under β-adrenergic stimulation, increased SR-Ca 2+ content, consistent with Ca 2+ transient decay acceleration found here, is known to enhance RyR2 open probability and the associated arrhythmia risks [32] in line with our findings on Ca 2+ waves and Ca 2+ sparks (Figures 4 and 7). Overall, this result points out a genuine cumulative pro-arrhythmogenic risk of Lyso-7 under stress or physical exercise.
The effects of Lyso-7 were complex, probably involving several mechanisms. In contrast with its impact on Ca 2+ handling, Lyso-7 had divergent acute and long-term effects on cell contraction (increase vs. decrease, respectively). The amount of Ca 2+ released depends on the amount of Ca 2+ stored in the SR [33]. The positive inotropic effect of Lyso-7 on contraction was uncorrelated with the concomitant reduction in the Ca 2+ transient amplitude, which was paradoxical. Since this latter parameter is a crucial determinant of contraction, another mechanism must be responsible for the positive inotropy. An increase in the sensitivity of the contractile proteins to Ca 2+ is a likely candidate. We did not investigate the underlying mechanism because that was not the main objective of our study. ISO could not prevent, or compensate for, the impact of Lyso-7 on Ca 2+ handling parameters such as the decrease in diastolic Ca 2+ and Ca 2+ transient amplitude. Overall, our data suggest that Lyso-7 has multiple molecular off-targets, direct targeting of contractile protein activity being a likely possibility in addition to Ca 2+ handling. Moreover, the divergent effect on contraction, particularly the decrease resulting from repeated exposure of cells to Lyso-7 in vivo, may reflect a long-term impact of the molecule on this parameter. This possibility warrants further thorough investigation on the effect of more prolonged, repeated administration to investigate whether morphological and functional changes occur during chronic exposure at the whole organ level and whether NC ensures long-term cardioprotection.
The most important result of our study was the safety provided by the administration of Lyso-7 in the NC dosage form for seven days. Indeed, Lyso-7 in NC was devoid of cardiotoxicity for most parameters investigated at the cellular level. Lyso-7-NC had only little effect on Ca 2+ handling, spontaneous Ca 2+ events, and contraction of cardiomyocytes. The overall result was somewhat unexpected because the administration of Lyso-7 encapsu-lated in polymeric NC increases Lyso-7 body exposure by 14-fold and heart concentration by 3.5-fold, respectively, according to the pharmacokinetics and biodistribution studies [21]. The lipophilic character of free Lyso-7 is also likely to direct the molecule preferentially to the cell membrane, contributing to the increase in the whole heart. Cell membranes would then act as a storage site, a reservoir, gradually releasing the active principle towards its targets over time. The prolonged release of Lyso-7 from the NC in vivo may significantly reduce the fraction of the free molecule and minimize the interaction with the cardiomyocytes and the off-target effects. This is consistent with the absence of the impact of free Lyso-7 when applied at low concentrations (<45 nM). In vitro, the time required to release 50% Lyso-7 from NC is 420 min compared to 30 min dissolution of free Lyso-7, indicating that NC is a sustained release device for this molecule [21]. From our previous pharmacokinetic data, the intravenous dose of 1.6 mg/kg is large enough to induce the effects on COX-1, COX-2, PPAR-α, PPAR-β/δ or PPAR-γ [12,13].

Conclusions
The association of Lyso-7 with PLA-NC is promising to provide a potential therapeutic option devoid of significant adverse cardiac effects in line with previous findings from our group showing that NC can attenuate the toxicity of artemether and lychnopholide both at the heart and cardiomyocytes levels [18][19][20]. The NCs were efficient to prevent aberrant Ca 2+ events known to support life-threatening Ca 2+ -dependent ventricular arrhythmias potentially favored during stress or exercise. The interest of this concept can extend to all TZD to treat type 2 diabetes because of risks of increased HF and mortality incidence [34] and all the potential uses of Lyso-7. Although more investigation is warranted, our data may pave the way for NC in future applications.