An Explorative Approach to Examining the Role of Ischemia and Inflammation on the Function of Autoantibodies Against G Protein–Coupled Receptors and Their Corresponding Agonists

Figures

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  Figure 1. Influence of ω-6 fatty acid (AA) and eicosapentaenoic acid (EPA) on the dose response of cardiomyocytes to noradrenaline (NA). Dose–response curves for NA in spontaneously beating cardiomyocytes in the presence of AA (1 µM) (A), AA with the β2-adrenoceptor antagonist ICI118.551 (0.3 µM) (B), and AA with the β1-adrenoceptor inhibitor bisoprolol (1 µM) (C) and EPA (D). An unusually elongated dose–response curve for NA is presented with a maximal response at 10 µM and a threshold concentration at 1 pM (A). The overall chronotropic response reflects contributions from both β1 adrenoceptor and β2 adrenoceptor receptor–mediated signaling. The first sensitive part of the dose–response curve was strongly attenuated by the β2-adrenoreceptor antagonist ICI118.551 (B). The β1-adrenoceptor antagonist bisoprolol (1 µM) reduced the maximal chronotropic response and left the more sensitive portion of the curve largely unchanged (C). In the presence of EPA, the NA-induced chronotropic response was markedly attenuated, resulting in a dose–response profile resembling that observed in the presence of the β2-adrenoceptor antagonist (D).

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  Figure 2. Influence of AA and EPA on cardiomyocyte responses induced by G protein–coupled receptors (GPCRs) to agonists or functional autoantibodies (fAAbs). AA modulated the responses of β2-fAAb (A), α1-fAAb (B), angiotensin II (AT1)-fAAb (C), endothelin A (ETA)-fAAb (D), and angiotensin 1–7 MAS-fAAb (E), whereas responses induced by muscarinic M2-fAAb remained largely unchanged (F). EPA also influenced cardiomyocyte responses to several fAAbs. In particular, EPA significantly reduced the stimulatory responses induced by fAAbs against the β2 adrenoceptor (A), α1 adrenoceptor (B), and AT1 receptor (C), whereas the negative chronotropic response induced by ETA-fAAbs was markedly attenuated in the presence of EPA (D). In contrast, EPA did not significantly alter the responses associated with angiotensin 1–7 MAS-fAAbs (E). Similarly, for fAAbs against the muscarinic M2 receptor, neither AA nor EPA resulted in statistically significant differences between conditions (F). Receptor-specific antagonists were used to block the respective GPCRs, resulting in attenuation or disappearance of the corresponding fAAb-induced chronotropic responses. The following inhibitors were applied: 1 µM ICI118.551…

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  Figure 3. Ciliary neurotrophic factor (CNTF) induces a time-dependent increase in cardiomyocyte beating rate. No effect was observed after 5 min, whereas a moderate increase occurred after 120 min of incubation. Δ beats/15 s values represent the mean response of 10 clusters per flask. n = 4 independent experimental preparations. Normality of paired differences was assessed using the Shapiro–Wilk test and was confirmed. Therefore, paired comparisons were performed using the two-tailed paired t-test (complete paired observations); p = 0.0029. Statistical significance is indicated as ns, not significant; ** p < 0.01. Data are presented as means ± SEM.

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  Figure 4. Effect of CNTF on negative chronotropic responses in cardiomyocytes. Cardiomyocyte responses to angiotensin 1–7 (Ang 1–7) (A), 17,18-epoxyeicosatetraenoic acid (17,18-EETeTr) (B), and carbachol (C) following CNTF incubation. CNTF altered the chronotropic responses of cardiomyocytes to Ang 1–7 and 17,18-EETeTr, whereas the muscarinic receptor–mediated response to carbachol remained largely unchanged (A–C). For both Ang 1–7 and 17,18-EETeTr, a reduction in Δ beats was already observable after 5 min of CNTF incubation and became more pronounced after 60 min. Δ beats/15 s values represent the mean response of 10 clusters per flask. Cardiomyocytes originated from at least three independent isolations across the study. Statistical analysis was performed as described in the Methods section. Statistical significance is indicated as ns, not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are presented as means ± SEM.

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  Figure 5. Explorative effects of CNTF on positive chronotropic response in cardiomyocytes. CNTF (20 pM) modestly enhanced the chronotropic responses induced by angiotensin II (A) and isoprenaline (B), with a modest time-dependent trend. Δ beats/15 s values represent the mean response of 10 clusters per flask. Cardiomyocytes originated from at least three independent isolations across the study. Statistical analysis was performed as described in the Methods section. Statistical significance is indicated as ns, not significant; * p < 0.05, ** p < 0.01. Data are presented as means ± SEM.