β-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of IL6R-Signaling in Cardiomyocytes upon Increased Workload

Figures

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  Figure 1. β-adrenergic signaling prevents NCX upregulation under increased workload in neonatal rat ventricular cardiomyocytes (NRVCMs). (A) Representative fluorescent images of NRVCMs immunostained against NCX exposed to 3 h of low pacing (1 Hz) or tachycardic pacing (8 Hz) frequency under control conditions (CTL), β-adrenergic stimulation with isoprenaline (ISO) or β-adrenergic stimulation following 1 h preincubation with β-blocker propranolol (ISO + BB). Scale bar indicates 50 µm. (B) Mean fluorescent values of CTL under low pacing frequency (1 Hz) or tachycardia (8 Hz) normalized to the 1Hz condition. (C) Mean fluorescent values of CTL, ISO and ISO + BB at 8 Hz normalized to the CTL at 1 Hz. Data are shown as mean ± SEM (n = 760–1171 cells from N = 4 isolations). Indicated p-values were calculated using (B) Mann–Whitney test comparing 8 Hz to 1 Hz under CTL conditions or (C) Kruskal–Wallis test comparing normalized CTL, ISO or ISO + BB levels at 8 Hz.

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  Figure 2. β-adrenergic signaling potentiates CaMKII activation on subcellular Ca2+ stores in neonatal rat ventricular cardiomyocytes (NRVCMs) under conditions of tachypacing. (A) Representative fluorescent images of NRVCMs immunostained against phospho-CaMKII (pCaMKII) exposed to 3 h of either low pacing (1 Hz) or tachycardic pacing (8 Hz) frequency under control conditions (CTL), β-adrenergic stimulation with isoprenaline (ISO) or β-adrenergic stimulation following 1 h preincubation with β-blocker propranolol (ISO + BB). Scale bar indicates 50 µm. (B) Magnified nuclei of indicated areas in (A). Scale bar indicates 10 µm. (C) Mean fluorescent values of CTL under low pacing frequency (1 Hz) or tachycardia (8 Hz) in cytoplasm, in nucleoplasm and on the nuclear envelope normalized to the 1 Hz CTL signal of the respective compartment. (D) Mean fluorescent values of CTL, ISO and ISO + BB conditions at 8 Hz normalized to the CTL at 1 Hz in cytoplasm, in nucleoplasm and on the nuclear envelope. Data are shown as mean ± SEM (n = 308–415 cells from N = 4 isolations). Indicated p-values were calculated using (C) Mann–Whitney test comparing 8 Hz to 1 Hz in the respective compartments or (D)…

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  Figure 3. Effects of tachycardic pacing and β-adrenergic stimulation/inhibition on the expression of hypertrophy- and cardiac-remodeling-relevant genes in neonatal rat ventricular cardiomyocytes (NRVCMs). Relative expression of (A) interleukin-6 receptor (IL6R), (B) regulator of calcineurin 1 (RCAN1) and (C) transforming growth factor β 1 (TGFB1) exposed to 3 h of tachycardia (8 Hz) under control conditions or combined with β-adrenergic stimulation with isoprenaline (ISO), β-adrenergic stimulation following 1 h preincubation with β-blocker propranolol (ISO + BB) or treatment with NCX inhibitor ORM-10962 (ORM). All 8 Hz conditions were normalized to the respective low pacing frequency (1 Hz) condition (using the 2−ΔΔCt method). Data are shown as mean ± SEM (N = 5 isolations). Indicated p-values were calculated using a one-sample t-test.

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  Figure 4. Hypertension is associated with blunted β-adrenergic signaling and increased NCX expression in Dahl salt-sensitive rats. (A) Representative bands of immunoblotted Serca2a and housekeeping protein GAPDH in cardiac tissue of Dahl salt-sensitive rats kept on low-salt diet (LSD) or high-salt diet (HSD) for 10 weeks. (B) Corresponding quantification of Serca2a expression, normalized to GAPDH. (C) Representative bands of immunoblotted NCX and housekeeping protein GAPDH in cardiac tissue of Dahl salt-sensitive rats kept on LSD or HSD for 10 weeks. (D) Corresponding mean values of NCX expression, normalized to GAPDH. (E) Representative fluorescent images of isolated cardiomyocytes immunostained against β-adrenergic receptor 1 (ADRB1). Scale bar indicates 50 µm. (F) Mean fluorescent values for ADRB1 normalized to LSD. Mean values for (G) noradrenaline and (H) adrenaline plasma levels in Dahl salt-sensitive rats kept on LSD or HSD for 10 weeks. Data are shown as mean ± SEM (N = 7/3 rats for LSD/HSD for B and D, N = 2/2 rats and n = 76/79 cells for LSD/HSD and N = 4–5/4 rats for LSD/HSD). Indicated p-values were calculated using unpaired t-test (B,D,G,H) or Mann–Whitney test (F) comparing…

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  Figure 5. Effects of hypertension on the expression of disease-relevant mRNAs in Dahl salt-sensitive rats.Dahl salt-sensitive rats were kept on low-salt diet (LSD) or high-salt diet (HSD) for 10 weeks to induce hypertension. mRNA levels of (A) SLC8A1 (gene encoding for NCX), (B) ATP2A2 (gene encoding for Serca2a), (C) brain natriuretic peptide (NPPB), (D) β-adrenergic receptor 1 (ADRB1), (E) interleukin-6 (IL6) and (F) interleukin-6 receptor (IL6R) were quantified by microarray analysis and normalized to the mean expression level of LSD. Data are shown as mean ± SEM (N = 5/6 rats for LSD/HSD). Indicated p-values were calculated using unpaired t-test (except for B, where a Mann–Whitney test was applied).

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  Figure 6. Graphical representation of effects of tachycardia alone, which are abolished by tachycardia combined withβ-adrenergic stimulation, on expression of NCX, subcellular CaMKII activation profile and IL6(R) in cardiomyocytes. Abbreviations: ADRB1—β-adrenergic receptor 1, CaMKII—calcium/calmodulin-dependent kinase II, IL6(R)—interleukin 6 (receptor), ISO—isoprenaline, LTCC—L-type calcium channel, NCX—sodium/calcium exchanger, NE—nuclear envelope, PLB—phospholamban, RyR—ryanodine receptor, Serca2a—sarcoplasmic reticulum calcium ATPase 2a, SR—sarcoplasmic reticulum.