Citrullination is linked to reduced Ca²⁺ sensitivity in hearts of a murine model of rheumatoid arthritis

Figures

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  FIGURE 1. Development of the collagen‐induced arthritis (CIA) mouse model of Rheumatoid Arthritis. DBA mice were subcutaneously (s.c.) injected with 100 μg of type II collagen (CII) and 300 μg of M tuberculosis in 0.1 ml of the emulsion into the base of the tail. On day 5, the animals were boosted with a subcutaneous (s.c.) injection of 100 μg of CII in Freund's incomplete adjuvant. Control mice were injected with 0.1 ml saline on both occasions. The CIA mice were sacrificed when they had developed overt arthritis in at least 2 paws (day 10–15) and the hearts were collected for: cardiomyocytes isolation ([Ca2+]i transients), myofibrils (force‐Ca2+ relationship) and biochemistry (western blot).

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  FIGURE 2. [Ca2+]i and fractional shortening measured in isolated cardiomyocytes, Fluo‐3 AM was used to measure [Ca2+]i transients in cardiomyocytes that were electrically paced (1 Hz) to elicit repeated contractions. Representative [Ca2+]i transient records (A). Mean data of [Ca2+]i transient amplitudes (B), Tau, [Ca2+]i decay time‐constant (C). Caffeine (10 mM) was used to trigger the opening of the RyR2 and cause the maximum release of SR Ca2+ (D). Fractional shortening (E) of cardiomyocytes stimulated at 1 Hz under control conditions (n = 19–33). (F) XY graph of Ca2+ fluorescence on the X axis and contractility (FS%) on the Y axis for each cardiomyocyte. Cardiomyocytes of the CIA group showed higher Ca2+ transients as they distributed on the right part of the graph and right shifting of the linear regression curve of the CIA group. Control r 2 0.069, p = 0.29, slope 0.731 ± 0.7, CIA r 2 0.183, p < 0.05, slope 0.139 ± 0.5. Data are presented as box and whiskers min and max including all points. *p < 0.05, †p < 0.01, ‡p < 0.001 with unpaired t test.

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  FIGURE 3. Mitochondrial Ca2+ uptake and ROS production in cardiomyocytes from CIA and control mice. (A) Mean (±SEM) of mitochondrial Ca2+ in cardiomyocytes electrically paced at 0.5–3 Hz by brief current pulses, n = 21–30. Rhod‐2 fluorescence is normalized to the resting value in each cardiomyocyte. (B) Representative images of Rhod‐2 fluorescence in CIA (i and ii) and control (iii and iv) cardiomyocytes at the end of 0.5 Hz (i and iii) and 3 Hz (ii and iv) pacing. (C) Mitochondrial ROS in cardiomyocytes was measured as MitoSOX Red fluorescence (mean data ± SEM, n = 12–22) at 0.5–3 Hz electrical stimulation. Hydrogen peroxide (H2O2; 1 μM) was used as a positive control at the end of the experiment. (D) Representative images of MitoSOX Red fluorescence in CIA (i and ii) and control (iii and iv) cardiomyocytes at the end of 0.5 Hz (i and iii) and 3 Hz (ii and iv) pacing. Data are presented as mean ± SEM. *p < 0.05, †p < 0.01, ‡p < 0.001 with Mann–Whitney non‐parametric test.

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  FIGURE 4. Ca2+ handling protein expression in hearts of CIA and control mice. (A) Representative immunoblots of, ryanodine receptor 2 (RyR2), dihydropyridin receptor (DHPR), calsequestrin 2 (CSQ2), SERCA2a, phospholamban (PLN, pentameric and monomeric form) and housekeeping protein glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). (B) Mean band densities normalized to GAPDH and the mean value in control hearts set to 1.0 (n = 6–8). Data are presented as box and whiskers min and max including all points. *p < 0.05, †p < 0.01, ‡p < 0.001 with unpaired t test. Data were analyzed with Mann–Whitney non‐parametric test.

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  FIGURE 5. Reduced Ca2+ sensitivity in myofibrils isolated from CIA hearts. (A) The submaximal Ca2+ activated force was lower in isolated myofibrils from CIA hearts than in controls, whereas maximal Ca2+ activated force was not different between the groups. (B) The [Ca2+] needed to achieve 50% of maximal myofibrillar force (Ca50) was significantly increased in the CIA group, showing that myofibrillar Ca2+ sensitivity was lower in the CIA hearts. (C,D) The rates of force development at the onset of maximum contractions (KAct) and redevelopment after a shortening step (KTr) were both reduced in CIA than in control myofibrils. (E) The rate of relaxation following deactivation (KRel) did not differ between CIA and control myofibrils. Average data in (A–E) is displayed as box and whiskers min and max including all points., n = 9. *p < 0.05, †p < 0.01, ‡p < 0.001, repeated measurements anova (A), t test (B–E).

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  FIGURE 6. Expression of PAD2 and post‐translational modification of α‐Actin and RyR2 in hearts of CIA and control mice. (A) (left) Expression of PAD2 in hearts of CIA and control mice; (right) mean band densities normalized to GAPDH and the mean value in control hearts set to 1.0 (n = 6). (B) (left) Representative immunoblots from immunoprecipitated α‐Actin; (right) mean citrulline band densities normalized to total α‐Actin and the mean value in control hearts set to 1.0 (n = 6). (C) (left) Representative immunoblots from immunoprecipitated RyR2; (right) mean phosphorylated (p‐RyR2; PKA phosphorylation site RyR2‐S2808) and citrullinated RyR2 bands densities normalized to total RyR2 and the mean value in control hearts set to 1.0 (n = 7). Data are presented as box and whiskers min and max including all points. *p < 0.05, †p < 0.01, ‡p < 0.001 with Mann–Whitney non‐parametric test.

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  FIGURE 7. TNF‐α triggered PAD‐dependent citrullination in cardiomyocytes. (A) Cartoon depicting the in vitro model to mimic the inflammatory phase of RA. We used cardiomyocytes isolated from DBA mice (N = 4 mice) treated for 20 h with TNF‐α (50 ng/ml), pretreated with or without 2‐chloroacetamidine (2‐CA, 200 μM) a small molecule that inhibits PADs active‐sites. Then, PAD‐dependent citrullination was measured and normalized to baseline levels (e.g. cells untreated, CTR). (B) Representative immunoblots from cardiomyocytes lysates blotted for PAD2, citrullination, and GAPDH. (C) Mean band density of PAD2 normalized to GAPDH. (D) The mean band density of protein citrullination normalized to GAPDH, which appeared to be significantly increased in cardiomyocytes treated with TNF‐α. Data are presented as box and whiskers min and max including all points. *p < 0.05, †p < 0.01, ‡p < 0.001 with one‐way anova test, multiple comparisons.

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  FIGURE 8. Graphical abstract. In the acute inflammation phase of rheumatoid arthritis, the expression and activity of peptidyl arginine deaminase 2 (PAD2) in the heart increases. This links to aggravated PAD‐dependent citrullination of proteins involved in contractile regulation, which causes reduced myofilament Ca2+ sensitivity (e.g. increased amount of Ca2+ needed to elicit 50% of maximal myofibrillar force, Ca50). This reduction is counteracted by increased [Ca2+]i transient amplitudes, yet it decreases the contractile reserve. A declined contractile reserve that sensitizes the heart to insults is a factor that could explain the high prevalence of heart failure amongst patients with RA and shows the importance of monitoring RA patients for the development of HF.