β-Arrestin–Biased AT1 Agonist TRV027 Causes a Neonatal-Specific Sustained Positive Inotropic Effect Without Increasing Heart Rate

Figures

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  Figure 1. TRV027 Causes a Long-Acting Positive Inotropic Effect Through AT1R in a Dose-Dependent Manner in the Neonatal Mouse Heart (A) Representative M-mode ultrasoundcardiogram recorded from neonatal mice treated with saline (left column) or TRV027 (right column). UCG was recorded 2 h after intraperitoneal injection of saline or TRV027 (3 mg/kg) (top row). Then, atropine (Atr) (1 mg/kg) plus propranolol (Prop) (1 mg/kg) was added, and the measurements were repeated 10 min later (middle row). Furthermore, candesartan (Cand) (3 mg/kg) was added, and the measurements were once again repeated 20 min later (bottom row). In these traces, a, b, c, and d indicate interventricular septum end-diastolic thickness, left ventricular end-diastolic diameter, posterior wall end-diastolic thickness, and end-systolic diameter, respectively. (B) The values of ejection fraction (EF) measured under each condition. Symbols indicate each data. The p value was calculated with unpaired Student’s t test. (C) Indicated time after injection of TRV027 (3 mg/kg) to neonatal mice, saline (open circles) or atropine + propranolol (1 mg/kg each) (closed circles) was added. Then, EF was measured 10 min later. Statistical…

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  Figure 2. Time-Dependent Effect of TRV027 on ECG, Plasma Concentrations of Adrenaline and Noradrenaline, and the Serum Concentration of Aldosterone After Administration of Saline or TRV027 (A) Electrocardiography (ECG) was recorded indicated time after injection of TRV027 (3 mg/kg) to neonatal mice (left column). Then, 10 min after additional injection of atropine + propranolol, ECG was once again recorded (right column). (B) Each parameter of ECG was measured different time after injection of TRV027 (3 mg/kg) (open circles). Then, 10 min after injection of atropine + propranolol, each parameter was measured again (closed circles). No significant time-dependent change in each parameter was detected with 1-way analysis of variance (ANOVA) followed by Dunnett’s test. Two-way ANOVA indicated a significant effect of drugs in heart rate (HR), P, and PQ but no significant interaction between time and drugs. †p < 0.05; ††p < 0.01; and †††p < 0.001 between saline and atropine + propranolol groups. n = 5 in each group. (C) Plasma adrenaline and noradrenaline concentrations 2 and 8 h after TRV027 (3 mg/kg) administration. The p value was calculated with 1-way ANOVA followed by Dunnett’s test. n = 5…

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  Figure 3. Effect of TRV027 on Twitch Ca2+ Transients of Isolated mNVCMs or hiPSC-CMs Representative twitch Ca2+ transients of (A) mouse neonatal ventricular cardiac myocytes (mNVCMs) detected with Fluo-4 and digitized at 144 Hz or (B) human induced pluripotent stem cell–derived cardiac myocytes (hiPSC-CMs) detected with Cal-520 and digitized at 60 Hz 2 h after in vitro treatment with saline (black line) or TRV027 (3 μmol/l) (red line). Action potential was elicited by field stimulation at 0.33 Hz. (C) Effects of saline or TRV027 on the peak ΔF/F0, time to peak (TTP), half decay time (T1/2), and the sarcoplasmic reticulum Ca2+ content in paced mNVCMs. n = 10 in each group. (D) Effects of saline or TRV027 on the peak ΔF/F0, TTP, and a half decay time of paced hiPSC-CMs, and their spontaneous beating rate in the absence of pacing. n = 8 to 11 in each group. Statistical significance was analyzed with unpaired Student’s t test.

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  Figure 4. Effect of TRV027 on the Mitochondrial OCR in mNVCMs (A) Oxygen consumption rate (OCR) in isolated mouse neonatal ventricular cardiac myocytes (mNVCMs) treated in vitro with saline or TRV027 (3 μmol/l) for 2 h. Oligomycin A (2 μmol/l), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (2 μmol/l), and antimycin A + rotenone (1 μmol/l each) were sequentially added as indicated. Letters in this panel (a, b, and c) indicate the timing at which parameters shown in B were calculated. (B) Unloaded and maximum OCRs calculated by subtracting OCR after antimycin A and rotenone (c in A) from that before oligomycin (a in A) or after FCCP (b in A), respectively. (C) Measurement of OCR in mNVCMs treated in vitro for 2 h with saline or TRV027 (3 μmol/l) and with nifedipine (10 μmol/l) for last 30 min. (D) Unloaded and maximum OCR in the presence of nifedipine. (E) Short term effect of wash-in of saline or TRV027 (3 μmol/l) on unloaded OCR in mNVCMs. Saline or TRV027 was added at indicated this time point while OCR was measured. (F) Time-dependent effect of saline or TRV027 on unloaded OCR for ∼35 min after their wash-in. Statistical significance was analyzed with unpaired Student’s t…

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  Figure 5. Effect of AngII and TRV027 on ROS in the Hearts of Neonatal Mice (A) Representative images of dihydroethidium (DHE) and DAPI staining of the heart 1 h after administration of isoproterenol (ISO) (30 mg/kg) or 2 h after administration of saline, angiotensin II (AngII) (3 mg/kg), or TRV027 (3 mg/kg) to neonatal mice. Scale bar = 50 μm. (B) Quantification of DHE fluorescence. Statistical significance was analyzed with 1-way analysis of variance followed by Dunnett's test. n = 6 to 10 in each group.

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  Figure 6. Effect of TRV027 on the Cardiac Contractility of Model Mice of Human Congenital DCM (A) The representative hearts of wild-type (WT) mouse and model mouse of human congenital dilated cardiomyopathy (cTnTΔK210) at postnatal day 6 (P6). (B) Representative M-mode ultrasound cardiogram of wild-type and cTnTΔK210 mice 2 h after administration of saline or TRV027 (3 mg/kg). (C) Ejection fraction (EF) in each condition. Statistical significance was analyzed with 1-way analysis of variance followed by Tukey’s test. n = 5 in each group.