Dapagliflozin reduces the vulnerability of rats with pulmonary arterial hypertension-induced right heart failure to ventricular arrhythmia by restoring calcium handling

Figures

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  Fig. 1. Measurement and analysis of PAP and RVP in the 4 groups of rats. a Representative PAP waves and RVP waves in the 4 groups of rats from haemodynamic experiments. b mPAP in the 4 groups of rats. c mRVP in the 4 groups of rats. d The NT-pro-BNP level in the 4 groups of rats. mPAP mean pulmonary arterial pressure, mRVP mean right ventricular pressure, NT-pro-BNP the serum N-terminal pro-Brain natriuretic peptide. N = 8 per group. The horizontal lines show the mean ± SEM. One-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 2. Histological analysis of RV tissue in the 4 groups of rats. a Representative image of the RV cryosections stained with H&E (× 400). Nuclei were stained dark purple, the cytoplasm was stained red, the scale bar is 50 µm. b Representative images of the RV WGA immunofluorescence detection (× 400), the scale bar is 50 µm. c Representative images of the RV cryosections stained with Masson’s trichrome (× 200). Collagen fibres are stained blue, nuclei are stained dark purple, and cytoplasm is stained red/pink, the scale bar is 100 µm. d Quantitative analysis of the RV cardiomyocyte cross-sectional area that was WGA-stained (µm2). e Quantitative analysis of the average CVF from the Masson’s trichrome-stained section in the RV (%). WGA wheat germ agglutinin, CVF collagen volume fraction. N = 5 per group. The horizontal lines show the mean ± SEM. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 3. Western blot analysis. Total RyR2 (t-RyR2), phosphorylated RyR2 (p-RyR2-Ser2814), SERCA2a, total CaMKII (t-CaMKII), p-CaMKII (P-CaMKII-Thr287), phospholamban (PLB), Cav1.2, and Na+–Ca2+ exchanger protein (NCX) levels in the RV tissue from the 4 groups of rats were analysed and normalised to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). N = 5 per group. The horizontal lines show the mean ± SEM. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 4. Ca2+ microfluorometry. a Recordings of CaTs from RVCMs in each group. b The diastolic [Ca2+]i level, c CaT amplitude, and d CaT decay time constant (Tau) of a single RVCM were recorded and quantitatively analysed for RVCMs from the 4 groups of rats. Each point represents the result from a single RVCM. RVCM right ventricular cardiomyocyte, CaT Ca2+ transient. n/N = 15/5 = cells/rats per group. The horizontal lines show the mean ± SEM. One-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 5. Assessment of CaT alternans and SCaEs. a Representative image of signals at progressively greater frequencies with and without the record of the alternans. The results from one cell in each group are shown in a column. CaT alternans is defined as 4 or more consecutive CaTs with more than a 5% change in amplitude. b Recording of SCaEs by 4 s at 5-Hz pacing and post-pacing under each condition from a single RVCM for the 4 groups of rats. c Threshold for cellular CaT alternans. d Calculation of the number of SCaEs per cell. Each point represents the result from a single RVCM. RVCM right ventricular cardiomyocyte, CaT Ca2+ transient, SCaEs spontaneous calcium events, PF pacing frequency. n/N = 15/5 = cells/rats per group. One-way ANOVA or the nonparametric Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 6. Assessment of [Ca2+]SR by measurement of caffeine-induced CaTs. a Representative recording of caffeine-induced CaTs from a single RVCM for the 4 groups (caffeine = 10 mmol/L), which was used to estimate the total [Ca2+]SR content. b CaT amplitude. c CaT decay time constant. Each point represents the result from a single RVCM. RVCM right ventricular cardiomyocyte, CaT Ca2+ transient, [Ca2+]SR sarcoplasmic reticulum Ca2+ content. n/N = 15/5 = cells/rats per group. The horizontal lines show the mean ± SEM. One-way ANOVA or the nonparametric Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 7. Assessment of the SCaS properties. a Representative image of the SCaS. b Ca2+ spark frequency. c Ca2+ spark amplitude. d Ca2+ spark width. e Ca2+ spark duration. f Ca2+ spark mass. g Ca2+ spark-mediated leakage. All parameters were recorded and quantitatively analysed from the RVCMs of the 4 groups of rats. Each point represents the result from a single RVCM. RVCM right ventricular cardiomyocyte, SCaS spontaneous Ca2+ sparks. n/N = 15/5 = cells/rats per group. The horizontal lines show the mean ± SEM. One-way ANOVA. *p < 0.05, **p < 0.001, ***p < 0.001

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  Fig. 8. Optical mapping study of the RV. a Representative results of the activation time map at BCL 5 Hz (200 ms) in the RV free wall for the 4 groups. The arrow indicates the conduction direction. Right bar: colour-coded activation times across a representative RV surface. b Local CV in the four groups (cm/s). BCL basic cycle length, CV conduction velocity. n = 10 per group. The horizontal lines show the mean ± SEM. One-way ANOVA

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  Fig. 9. Assessment of APD alternans. a Representative recording of SCA-APD from a CTL heart. ΔAPD map and an AP recording from a single pixel at 80 ms BCL (position shown with a triangle on the map). b Example of the SDA-APD from an MCT heart. ΔAPD map and AP recordings from two pixels at 100 ms BCL. c The BCL threshold for SCA-APD alternans in each group. Each point represents the result from one heart. The horizontal lines show the median values for BCL that induce the SCA-APD alternans. d Inducibility of SDA-APD in each group. SCA-APD spatially concordant alternans action potential duration, SDA-APD spatially discordant alternans action potential duration, AP action potential, BCL basic cycle length. n = 10 per group. One-way ANOVA or χ2 tests. *p < 0.05, **p < 0.01, ***p < 0.001

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  Fig. 10. Detection and analysis of VA in the four groups of rats. a Pseudo-ECG before and during VA initiated by pacing at 100-ms BCL. T-wave alternans (shown with arrows) was found prior to VA initiation. b The panels show eight snapshots of the phase maps, which correspond to the time points indicated in red points of the amplified pseudo-ECG episode. The positions of phase singularities (PSs) are indicated by the white circle. The formation of the rotor, a hallmark of re-entrant VA, was observed in MCT-treated RVs. c Inducibility of VA in each group. VA ventricular arrhythmia, BCL basic cycle length, n = 10 per group. χ2 tests. *p < 0.05, **p < 0.01