Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease

Figures

• 

  Fig. 1.. In vitro exposure of cTnI-G203S myocytes to AID-TAT peptide restores ICa-L kinetics, Ψm and flavoprotein oxidation in response to activation of ICa-L. (A–E) ICa-L kinetics and intracellular calcium handling recorded in wt and cTnI-G203S cardiac myocytes in the presence of 1 μM AID(S)-TAT or AID-TAT. (A) Representative ICa-L current traces. (Inset) Pulse protocol. (B and C) Mean ± SEM of rate of inactivation (tau) (B) and current density (C) for all myocytes. (D) Current/voltage (I-V) relationships (P = NS, not significant). (Inset) Pulse protocol. (E) Calcium transients recorded in cTnI-G203S cardiac myocytes loaded with Fura-2, in the presence of 1 μM AID(S)-TAT or AID-TAT, presented as mean ± SEM (dashed line indicates SEM) (P = NS). Statistical significance determined by the Kruskal–Wallis test (B and C), two-way ANOVA with Geisser–Greenhouse correction (D), or the two-stage linear step-up method of Benjamini, et al. (68) (E). (F–K) Representative ratiometric JC-1 (F–H) and flavoprotein (I–K) fluorescence recorded from wt and cTnI-G203S myocytes before and after exposure to 10 μM BayK(−) in the presence of either 1 μM AID(S)-TAT, 1 μM AID-TAT, or 10 μM AID-TAT. JC-1 studies…

• 

  Fig. 2.. Cardiac uptake of AID-TAT peptide is significantly greater than AID(S)-TAT when administered in vivo. (A) Representative whole animal Cy7 fluorescent images from adult BALB/c nude mice administered a single 10-μM bolus dose of AID(S)-TAT-Cy7 or AID-TAT-Cy7, 4 h posttreatment. (B) Mean ± SEM of cardiac Cy7 fluorescence for all mice (n) administered AID(S)-TAT-Cy7 or AID-TAT-Cy7 acquired 20 and 40 min postinjection, and every hour after for up to 4 h. *P < 0.05 compared with AID(S)-TAT at matched time-points as determined by a Kruskal–Wallis test. (C) Representative ex vivo cardiac Cy7 fluorescent images from adult BALB/c nude mice administered AID(S)-TAT-Cy7 or AID-TAT-Cy7, 4 h posttreatment. (D) Mean ± SEM of cardiac Cy7 fluorescence for all ex vivo hearts (n), 4 h following administration of AID(S)-TAT-Cy7 or AID-TAT-Cy7. P < 0.05 as determined by a Mann–Whitney U test.

• 

  Fig. 3.. AID-TAT peptide is not retained by the kidneys or liver when administered in vivo. Clearance of AID-TAT and AID(S)-TAT via the left kidney (A), right kidney (B), and liver (C) for all mice administered AID(S)-TAT-Cy7 (n = 9) or AID-TAT-Cy7 (n = 11) over time (1 to 4 h postinjection). Insets, Right: Mean ± SEM of decay rates (exponential decrease) in Cy7 for all mice (n) over time (t = 1 to 4 h). Statistical significance determined by Mann–Whitney U tests (A and B) or unpaired t test with Welch’s correction (C). NS, not significant.

• 

  Fig. 4.. In vivo treatment of precardiomyopathic cTnI-G203S mice to AID-TAT peptide restores cellular Ψm and mitochondrial metabolic activity in response to activation of ICa-L. Representative ratiometric JC-1 (A) and flavoprotein (C) fluorescence recorded from myocytes isolated from wt and cTnI-G203S mice treated with AID(S)-TAT or AID-TAT (10 μM, 3×/wk/5 wk), recorded before and after exposure to 10 μM BayK(−). JC-1 studies were performed under calcium-free conditions (0 mM calcium). Arrows indicate addition of drugs. NaCN: 40 mM, FCCP: 50 μM. Mean ± SEM of JC-1 (B) and flavoprotein (D) fluorescence for all myocytes (n) treated with AID(S)-TAT or AID-TAT, before or after the onset of HCM (Pre HCM Rx and Post HCM). Myocytes were exposed to BayK(+), BayK(−) or 15 μM nisoldipine (Nisol) as indicated. *P < 0.05 compared with wt AID(S)-TAT [BayK(− )], **P < 0.05 compared with cTnI-G203S AID(S)-TAT [BayK(−)], ***P < 0.05 compared with cTnI-G203S AID-TAT [BayK(− )], ****P < 0.05 compared with cTnI-G203S AID-TAT [BayK(−)] (Post-HCM Rx). (E) Formation of formazan measured as change in absorbance in myocytes from wt and cTnI-G203S mice recorded after addition of BayK(+) or BayK(−). Italicized…

• 

  Fig. 5.. Metabolomics assessment of whole heart tissue reveals alterations in key metabolic pathways in the cTnI-G203S heart. Mean ± SEM of biochemical intermediates found to be significantly altered between wt and cTnI-G203S murine heart extracts by one-way ANOVA (false-discovery rate-adjusted P < 0.05). Between-group Fisher’s least significant difference post hoc significant differences are denoted with a bracketed line (adjusted P < 0.05) for citrulline (A), histidine (B), glutamine (C), adenylosuccinic acid (D), AMP (E), and IMP (F). n = 16 treated mice (four per treatment group).

• 

  Fig. 6.. In vivo treatment of precardiomyopathic cTnI-G203S mice to AID-TAT peptide prevents development of cardiac hypertrophy. (A, i–iv) Representative images of echocardiographic measurements from wt and cTnI-G203S mice treated with AID(S)-TAT or AID-TAT (10 μM, 3×/wk/5 wk), before or after the onset of HCM (Pre HCM Rx and Post HCM Rx, respectively). (B) Mean ± SEM of cell size from myocytes isolated from wt and cTnI-G203S mice treated with AID(S)-TAT or AID-TAT (10 μM, 3×/wk/5 wk), before or after the onset of HCM (Pre HCM Rx and Post HCM Rx, respectively). P < 0.05 as determined by a Kruskal–Wallis test. (C) Representative confocal images of intact cardiac myocytes isolated from wt mice treated with 10 μM AID(S)-TAT (i, n = 10) or AID-TAT (ii, n = 8), and cTnI-G203S mice treated with 10 μM AID(S)-TAT (iii, n = 17) or AID-TAT (iv, n = 12), prior to the onset of HCM (Pre HCM Rx) as indicated. F-actin shown in green (phalloidin), nuclei shown in blue (DAPI).

• 

  Fig. 7.. In vivo treatment of precardiomyopathic cTnI-G203S mice to AID-TAT peptide is not toxic. Mean ± SEM of urea (A), creatinine (B), ALT (C), and AST (D) concentrations from terminal serum from wt and cTnI-G203S mice treated with AID(S)-TAT or AID-TAT (10 μM, 3×/wk/5 wk) as indicated. n = number of mice. P = NS (not significant) determined by the Brown-Forsythe and Welch ANOVA test (A) or Kruskal–Wallis tests (B–D). (E) Mean ± SEM of body weight recorded from wt and cTnI-G203S mice treated with AID(S)-TAT or AID-TAT (10 μM, 3×/wk/5 wk, equaling 15 doses: 1 to 15) reported as a percentage (%) of day 1 BW. n = number of mice. Red line indicates 5% weight-loss threshold.