Atrial TRPM2 Channel-Mediated Ca2+ Influx Regulates ANP Secretion and Protects Against Isoproterenol-Induced Cardiac Hypertrophy and Fibrosis

Figures

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  Figure 1. Atrial TRPM2 expression and TRPM2-dependent Ca2+ signals. (A) RT-PCR of Trpm2 and Gapdh from atria in WT and TRPM2−/− (KO) mice; representative gel images are shown. Gapdh is the loading control. Right: Quantification of Trpm2 mRNA expression in WT and KO atria by real-time PCR normalized to Gapdh or to Myl7. Data are presented as mean ± SEM. Statistical comparisons were performed using Welch’s t-test (WT vs. KO: p < 0.05) (B) Whole-cell patch clamp from isolated atrial myocytes: time courses of currents at +100 mV (open symbols) and −100 mV (filled symbols) recorded with 0.5 mM ADP-ribose in the pipette (ADPr (+)) or without ADPr (ADPr (−)). The horizontal dotted line denotes the zero-current baseline. (C) Corresponding I–V relationships (ramps from +100 to −100 mV) at the indicated time points (a–h in panel (B)). (D) Summary of ADPr-activated current density (ΔI at +100 mV, pA/pF). Data are presented as mean ± SEM. Statistics evaluate within-genotype ADPr (+) vs. ADPr (−) using unpaired two-tailed Student’s t-tests. (n = 5–25) * p < 0.05 vs. ADPr (−). (E) Fura-2 ratiometric Ca2+ imaging in atrial myocytes during extracellular H2O2 (200 µM; bar) in Tyrode’s solution. (F)…

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  Figure 2. TRPM2 deficiency aggravates ISO-induced systolic dysfunction, hypertrophy, and fibrosis. (A) Representative M-mode echocardiograms (baseline, 1–3 weeks) in WT and TRPM2−/− (KO) mice. Scale bar (horizontal), 200 ms. (B) Time course of fractional shortening (FS, %) Data are mean ± SEM (n = 5–6). (C) Left-ventricular end-diastolic diameter (LVEDD, mm) over time (n = 5–6). (D) Heart-weight–to–body-weight ratio (HW/BW) at endpoint (n = 6–8). (E) Histology of whole-heart sections and mid-ventricular myocardium. Hematoxylin–eosin (HE) for myocyte morphology; Masson’s trichrome (MT) for interstitial/perivascular collagen. Scale bars: 100 µm (upper panels), 500 µm (lower panels). (F) Quantification of fibrotic area (% of tissue). Data are mean ± SEM (n = 6). Dots represent individual animals. (B,D,F) Statistical analysis: FS% and LVEDD were compared across time using two-way ANOVA with Tukey’s post hoc test; HW/BW and fibrosis% were compared by one-way ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01 WT vs. WT + ISO; ¶ p < 0.05, ¶¶ p < 0.01 KO vs. KO + ISO; # p < 0.05, ## p < 0.01 WT + ISO vs. KO + ISO.

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  Figure 3. TRPM2 deficiency blunts the atrial ANP program under ISO. (A) Focused heatmap of ANP/secretory–exocytosis genes (Nppa, Nppb, Npr1–3, Corin, Furin/Pcsk family, Vamp/Snap/Syt) across WT, KO, WT + ISO, KO + ISO. Values are row-wise z-scores of log2(TPM + 1); genes are clustered by Euclidean distance. ISO effect (WT) = log2FC (WT + ISO vs. WT); ISO effect (KO) = log2FC (KO + ISO vs. KO); ΔISO (KO − WT) = ISO effect (KO) − ISO effect (WT). (B) qPCR of Nppa (ANP) mRNA in atria (relative to WT) (mean ± SEM, n = 5). (C) Plasma ANP by ELISA (mean ± SEM, n = 6–7). (D) Atrial Npr1 (NPR-A) expression (mean ± SEM, n = 5). (E) Correlation between plasma ANP and HW/BW. Linear least-squares fits of HW/BW (mg·g−1) vs. plasma ANP (pg·mL−1): WT, y = −1.0 × 10−4 x + 5.14; WT + ISO, y = −5.0 × 10−4 x + 6.43; KO, y = −4.0 × 10−5 x + 5.36; KO + ISO, y = 1.0 × 10−3 x + 5.83. (B–D) Statistical testing: one-way ANOVA with Tukey’s post hoc test. ** p < 0.01 WT vs. WT + ISO; ¶¶ p < 0.01 KO vs. KO + ISO; ## p < 0.01 WT + ISO vs. KO + ISO.

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  Figure 4. Exogenous ANP rescues dysfunction and remodeling in ISO-treated TRPM2−/− hearts. TRPM2−/− (KO) mice received ISO (30 mg·kg−1·day−1, i.p., 7 days) and were co-treated with vehicle or ANP (400 µg·kg−1, s.c., 7 days). (A) Representative M-mode echocardiograms (Vehicle vs. ANP). Scale bar (horizontal), 200 ms. (B,C) Fractional shortening (FS, %) and ejection fraction (EF, %) for Vehicle and ANP groups (mean ± SEM; n = 6). (D,E) Left-ventricular diameters (LVEDD, LVESD, mm) for Vehicle and ANP groups. (F,G) Heart rate (HR, bpm) and heart-weight to body-weight ratio (HW/BW) for Vehicle and ANP groups (mean ± SEM; n = 6). (H) Representative histology: H&E and Masson’s trichrome (MT) sections. Scale bars: 100 µm (upper panels), 500 µm (lower panels). (I) Quantification of fibrosis area (% LV) (mean ± SEM; n = 6). Statistical testing (B–G,I): unpaired two-tailed Student’s t-test. * p < 0.05 Veh + ISO vs. ANP + ISO; ## p < 0.01 Veh + ISO vs. ANP + ISO.

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  Figure 5. ISO-induced ventricular hypertrophy and its attenuation by ANP are comparable in WT and TRPM2−/− cardiomyocytes. (A) Representative images of neonatal ventricular myocytes (phalloidin, F-actin) from WT and TRPM2−/− (KO) mice under control conditions and after ISO (10 µM, 24–72 h). Scale bar, 50 µm. (B) Quantification of cell cross-sectional area (CSA) over 24–72 h, normalized to WT control; WT and TRPM2−/− were compared at each time point (mean ± SEM, n =100–117). (C) Representative images of cells with or without ANP (0.1 µM, 24 h) in basal and ISO-treated conditions. Scale bar, 50 µm. (D) CSA quantification across the indicated conditions for both genotypes (mean ± SEM, n =101–108). (B,D): two-way ANOVA followed by Tukey’s post hoc test. ** p < 0.01 WT vs. WT + ISO ± ANP; ¶¶ p < 0.01 KO vs. KO + ISO ± ANP; ## p < 0.01 WT + ISO + Veh vs. WT + ISO + ANP; $$ p < 0.01 KO + ISO + Veh vs. KO + ISO + ANP; N.S. not significant.