Beta2-adrenergic receptor activation increases susceptibility to arrhythmogenesis in aging heart by impairing repolarization reserve via Giβγ–PLC signaling pathway

Figures

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  Fig. 1. Age-related differences in cardiac histology and biomarkers. (A) Representative H&E staining and Masson’s trichrome staining of myocardial tissues. Scale bar = 50 μm. (B) Representative ROS fluorescence plots of myocardial tissue obtained using DCFH-DA staining. Scale bar = 100 μm. (C) Representative β-galactosidase staining of myocardial tissue. Scale bar = 50 μm. (D) Relative weight ratio of the heart. HW, heart weight; BW, body weight. n ≥ 7 guinea pigs per group. Data are presented as mean ± SEM.

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  Fig. 2. Age-related differences in surface ECG parameters. (A) Representative in vivo ECG recordings. (B–F) Summary data for the quantification of the QTc interval (B), RR interval (C), PR interval (D), Tp–e interval (E), and Tp–e/QTc ratio (F). n ≥ 6 guinea pigs per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. the young group.

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  Fig. 3. Age-related differences in action potential from isolated left ventricular myocytes. (A) Representative AP traces recorded at pacing frequencies of 0.5, 1, and 2 Hz. (B) Summary data for APD90 measured at the indicated pacing frequencies. (C) Percentage change in APD90 relative to baseline (0.5 Hz). n = 15 cells from five hearts per group. Data are expressed as mean ± SEM. **P < 0.01 vs. 0.5 Hz. #P < 0.05, ##P < 0.01 vs. the young group.

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  Fig. 4. Age-related differences in susceptibility to arrhythmogenesis. (A) Representative ECG waveforms from ex vivo guinea pig hearts in the presence or absence of SAL (5 and 10 µM). Representative traces of ventricular arrhythmia shown at the bottom. (B) Percentage change in QTc intervals following SAL treatment relative to baseline. (C) Quantification of PVCs within 20 min after SAL treatment. (D) Incidence of VT/VF induced by SAL. n = 5 hearts per group. Data are expressed as mean ± SEM. *P < 0.05 vs. control within the same group. #P < 0.05 vs. the young group.

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  Fig. 5. Involvement of Giβγ–PLC signaling in ventricular arrhythmias in isolated perfused hearts. (A, C, E, G) Representative ECG waveforms from ex vivo aging guinea pig hearts pretreated with ML277 (1 µM, A), PTX (1.5 µg/mL, C), gallein (100 µM, E) or U73122 (1 µM, G), followed by SAL (5 and 10 µM). (B, D, F, H) Percentage change in QTc interval relative to baseline after SAL treatment in hearts pretreated with ML277 (B), PTX (D), gallein (F), or U73122 (H). n = 4 hearts per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. the pretreatment condition.

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  Fig. 6. Effects of Giβγ–PLC signaling inhibition on IP3 levels in myocardial homogenates from aged hearts. (A) Standard curve for the IP3 ELISA. Serial dilutions of IP3 standards (0–10,000 pg/mL) used to generate a four-parameter logistic (4-PL) curve R2 = 0.996. (B) IP3 content in myocardial tissue after treatment with SAL (10 µM) alone or in combination with PTX (1.5 µg/mL), gallein (100 µM), or U73122 (1 µM). (C) Fold change in IP3 levels normalized to the control condition. n = 5 hearts per group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01 vs. the contorl condition.

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  Fig. 7. Involvement of Giβγ–PLC signaling in cell arrhythmias in hiPSC-CMs. (A1–A5) Representative extracellular field potential (EFP) waveforms recorded from control hiPSC-CMs under baseline conditions or after pretreatment with ML277 (1 µM, A2), PTX (1.5 µg/mL, A3), gallein (100 µM, A4) and U73122 (1 µM, A5), followed by salbutamol (SAL, 10 µM). (A6) Normalized FPDc from recordings shown in A1–A5. (B1–B5) Representative EFP waveforms from D-gal–induced aging hiPSC-CMs under the same experimental conditions. (B6) Normalized FPDc from recordings shown in B1–B5. (C) Percentage change in FPDc from recordings shown in A and B. (D) Incidence of tachyarrhythmias induced by SAL. (E) Representative types of cellular arrhythmia recorded in hiPSC-CMs. (F) Heatmap of arrhythmia scores under different treatment conditions. n ≥ 3 per group. Data are expressed as mean ± SEM. **P < 0.01 vs. control within the same group. #P < 0.05 for intergroup comparisons.