Citrullination of myofilament proteins in heart failure

Abstract

Aims: Citrullination, the post-translational conversion of arginine to citrulline by the enzyme family of peptidylarginine deiminases (PADs), is associated with several diseases, and specific citrullinated proteins have been shown to alter function while others act as auto-antigens. In this study, we identified citrullinated proteins in human myocardial samples, from healthy and heart failure patients, and determined several potential functional consequences. Further we investigated PAD isoform cell-specific expression in the heart.

Methods and results: A citrullination-targeted proteomic strategy using data-independent (SWATH) acquisition method was used to identify the modified cardiac proteins. Citrullinated-induced sarcomeric proteins were validated using two-dimensional gel electrophoresis and investigated using biochemical and functional assays. Myocardial PAD isoforms were confirmed by RT-PCR with PAD2 being the major isoform in myocytes. In total, 304 citrullinated sites were identified that map to 145 proteins among the three study groups: normal, ischaemia, and dilated cardiomyopathy. Citrullination of myosin (using HMM fragment) decreased its intrinsic ATPase activity and inhibited the acto-HMM-ATPase activity. Citrullinated TM resulted in stronger F-actin binding and inhibited the acto-HMM-ATPase activity. Citrullinated TnI did not alter the binding to F-actin or acto-HMM-ATPase activity. Overall, citrullination of sarcomeric proteins caused a decrease in Ca(2+) sensitivity in skinned cardiomyocytes, with no change in maximal calcium-activated force or hill coefficient.

Conclusion: Citrullination unique to the cardiac proteome was identified. Our data indicate important structural and functional alterations to the cardiac sarcomere and the contribution of protein citrullination to this process.

Keywords: Citrullination; Heart failure; Myofilament; Peptidylarginine deiminases; Sarcomere.

Figure 1

The cardiac citrullinated proteome. Diagrams show citrullinated proteins with significant P-value group by (A) cellular component and (B) molecular function. Details can be found in Table 1.

Figure 2

2DE DIGE analysis with samples treated with PAD2. Samples labelled with Cy2 (internal control), Cy3 (untreated), and Cy5 (treated) as described in Methods section. Details can be found in insert table.

Figure 3

Actin-binding studies. Increasing concentrations of (A) TM (0.5–2 µM) were incubated with F-actin or (B) actin-HMM and (C) TnI (0.5–2 µM) in buffer containing 40 mM Tris–HCl (pH 7.6), 100 mM NaCl, 5 mM MgCl₂, and 1 mM DTT. Binding of TM to F-actin was carried out at 25°C for 30 min and ultracentrifuged at 156 565 g for 25 min, 20°C, in a Beckman model TL-100.2. Both pellet and supernatant (unbound protein) were analysed. Representative silver-stained gels show proteins composition of the supernatants and pellets. A triplicate set of gels was analysed by densitometry. Each data point is an average (and range) of the values obtained from the three sets of gels.

Figure 4

Citrullination of sarcomeric proteins, biochemical and physiological effects. (A) Regulation of the actomyosin HMM-ATPase activity by citrullinated F-actin and/or citrullinated HMM. (B) Inhibition of actomyosin HMM-ATPase activity by TM. ATPase activity was measured as a function of TM concentration. The results are the average of four independent experiments for each protein at each TM concentration. Assay conditions: 0.2 mg/mL F-actin, 0.02 mg/mL HMM, 0–2.0 µM TM in 10 mM Hepes, pH 7.5, 30 mM NaCl, 5 mM MgCl₂, 4 mM ATP. PAD2 treatment reduced myofilament calcium sensitivity. (C) Force–calcium relationships for untreated membrane-permeabilized myocytes from untreated control (n = 8 myocytes from three mice, grey circles) and PAD2-treated (n = 8 myocytes from three mice, open circles) groups. (D) There was no difference in maximal calcium-activated force (F_max) between the two groups. (E) PAD2 treatment caused a significant (P = 0.009) increase in EC₅₀ (calcium required to generate 50% F_max), indicating a decrease in calcium sensitivity. (F) While the hill coefficient (nH) trended to be decreased by PAD2 treatment (see steepness of curve in C), the difference was not significant (P = 0.34).

Figure 5

RT-PCR analysis of expression level of PAD isoforms in (A and B) heart from control mouse, (C) mouse keratinocytes, and (D) mouse macrophage cell line activated by lipopolysaccharide. The PCR product PAD2 is seen in all types of samples; PAD4 and PAD1 are seen in cardiac fibroblast, keratinocytes, and macrophage cell line. PAD3 has not been detected. (MW: PAD1, 285 bp; PAD2, 390 bp; PAD3, 200 bp; PAD4, 550 bp).

Figure 6

Citrullinated peptides up-/down-regulated between heart failure groups, ischaemia, and IDCM.

Figure 7

Citrullination of the contractile proteins could affect different aspects of regulatory function. It could either trigger a structural change or stabilize a conformation that is necessary for actin-activated release of Pi and completion of the ATPase cycle.