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

Figures

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  Figure 1. Chemical structure of Lyso-7 and schematic representation of polymeric nanocapsules (A). Presentation of the experimental protocol used to treat mice and isolate the cardiomyocytes to test cardiotoxicity of the formulations (B) directly in vitro (acute effects) or after in vivo treatment followed by cardiomyocyte isolation.

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  Figure 2. Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on contraction (A–D) and Ca2+ transient (E–H) in freshly isolated cardiomyocytes. Typical recordings of the effects of Lyso-7 (free form) and Lyso-7-NC on sarcomere length (SL) shortening (A) and Ca2+ transient (E), both under field stimulation at 1 Hz. (B–D): averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, all for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control; ! Lyso-7-NC vs. Lyso-7. n = 4; n = 12–18.

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  Figure 3. Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on contraction (A–D) and Ca2+ transient (E–H) of freshly isolated cardiomyocytes exposed in vitro to Isoproterenol (ISO, 10 nM). (A–C): averaged data of resting sarcomere length (SL), SL shortening and relaxation, respectively, and (D–F): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, all for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control; ! Lyso-7-NC vs. Lyso-7. n = 4; n = 12–18.

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  Figure 4. Effect of in vivo administration of Lyso-7 and Lyso-7-NC (1.6 mg/kg, once a day for seven days) on cardiomyocytes’ ectopic diastolic Ca2+ waves. (A) Representative recordings of Ca2+ waves during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes from the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC); (B) Representative recordings of Ca2+ waves during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes from the four the same experimental groups treated in vitro with Isoproterenol (ISO, 10 nM); (C,D) percentage of cardiomyocytes developing at least one spontaneous Ca2+ wave in absence and presence of ISO (10 nM) in vitro, respectively, for the four experimental groups (Control, Lyso-7, NC, and Lyso-7-NC). We used a t-test. p < 0.05,* Lyso-7 vs. control; ! Lyso-7-NC vs. Lyso-7. a.u.: arbitrary units. n = 4; n = 12–18.

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  Figure 5. Acute effect of Lyso-7 on contraction (A–D) and Ca2+ transient (E–H) in cardiomyocytes of untreated mice. Typical recordings of Lyso-7 (450 nM) effects on sarcomere length (SL) shortening (A) and Ca2+ transient (E), both under field stimulation at 1 Hz. (B–D) averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H): averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, for Control, and increasing concentration of Lyso-7 (4.5, 45, and 450 nM). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control. Control refers to the absence of Lyso-7. n = 12–18.

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  Figure 6. Acute in vitro effect of Lyso-7 on contraction (A–D) and Ca2+ transient (E–H) of cardiomyocytes under IsoproTable 10. nM). Typical recordings of Lyso-7 (450 nM) effects on (A) sarcomere length (SL) shortening and (E) Ca2+ transient, both under field stimulation at 1 Hz. (B–D) averaged data of resting SL, SL shortening and relaxation, respectively, and (F–H) averaged data of diastolic Ca2+, Ca2+ transient and decay of Ca2+ transient (Tau), respectively, for Control, and increasing concentration of Lyso-7 (4.5, 45, and 450 nM). ANOVA followed by Tukey post-test (p < 0.05). * Lyso-7 vs. Control. Control refers to the absence of Lyso-7. n = 12–18.

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  Figure 7. Acute in vitro effect of Lyso-7 on cardiomyocytes’ abnormal spontaneous diastolic Ca2+ events under IsoproTable 10. nM) challenge. (A) percentage of cardiomyocytes developing at least one spontaneous Ca2+ wave in the absence (left panel) and presence (right panel) of Isoproterenol (ISO, 10 nM) during resting periods after a train of field stimulation at 1 Hz in cardiomyocytes. (B) Representative recordings of Ca2+ waves under ISO. n = 12–18 cells. (C) Typical line-scan confocal images of Ca2+ sparks from Fluo-4-AM loaded cardiomyocytes; n = 7–10. (D) Mean of frequency of Ca2+ sparks measured in the different conditions indicated. C = Control refers to the absence of Lyso-7 (450 nM). We used a t-test. p < 0.05, * Lyso-7 vs. Control in the absence of ISO (left part of the panel); # Lyso-7 vs. Control in the presence of ISO (right part of the panel); ! with ISO vs. without ISO.