Carnosinylation of Cardiac Antigens Attenuates Immunogenic Responses and Improves Function in Failing Hearts

Abstract

Objective: To investigate the effects of carnosine on heart failure and to examine whether this is associated with reduced immunogenicity of oxidatively-generated aldehyde modified proteins.

Background: Heart failure is associated with the accumulation of lipid derived aldehydes that form immunogenic protein adducts. However, the pathological impact of these aldehydes and aldehyde-modified proteins in heart failure has not been assessed. Histidyl dipeptides, such as carnosine found in the heart, bind to aldehydes, and their protein adducts. However, the effects of carnosine on heart failure or the antigenicity of aldehyde modified proteins have not been studied.

Methods: Male, wild type C57BL/6J mice were subjected to either sham or transverse aortic constriction (TAC) surgery. To increase carnosine levels, they were placed on drinking water with or without β-alanine prior to surgery, and for the remainder of the study. Cardiac function was evaluated by echocardiography, and the levels of histidyl dipeptides, immune cell populations, and CD4⁺ T cell activation were assessed via LC-MS/MS and flow cytometry, respectively.

Results: Myocardial levels of histidyl dipeptides decreased at both 3- and 8-weeks post-TAC. Supplementation with β-alanine increased myocardial histidyl dipeptide levels, attenuated adverse cardiac remodeling, and reduced aldehyde stress. Carnosine formed covalent bond with protein-bound aldehydes in the failing heart, reducing their antigenic potential and decreasing activation of dendritic cells and CD4⁺ T cells in vitro. β-alanine supplementation decreased the population of CD11b⁺CD64^-Ly6G⁺ neutrophils and CD4⁺ CD44⁺ effector T cells in the failing heart.

Conclusions: Increasing myocardial carnosine levels reduces aldehyde stress, dampens maladaptive immune responses, and preserves cardiac function during heart failure.

Keywords: Carnosine; T cells; dendritic cells; histidyl dipeptides; neoantigens; oxidative stress.

Figure 1.. Histidyl dipeptides are decreased in failing hearts.

Male wild type C57BL/6J 12-week-old mice were subjected to sham or transverse aortic constriction (TAC) surgery. Myocardial levels of carnosine and anserine were measured by LC-MS/MS using carnosine-d4 as internal standard (IS) after (A) 3 and (B) 8 weeks of sham and TAC surgery. (C) Schematic showing enzymes and transporters involved in carnosine homeostasis. Changes in the expression of carnosine synthase (CARNS1), carnosinase 2 (CNDP2), peptide transporter 2 (PEPT2) and taurine transporter (TAUT) were measured 3 weeks post-surgery at the (D) transcript and (E, F) protein level using RT-qPCR and Western blotting respectively. (G) Levels of carnosine-propanal and carnosine propanol were measured in the sham and TAC operated hearts by LC/MS/MS using carnosine-d₄ as an internal standard after (G) 3 and (H) 8 weeks of sham and TAC surgery. Data shown as mean ± SEM, p-values determined using Student’s t-test, n = 4 – 5 mice per group.

Figure 2.. β-alanine supplementation prior to transverse aortic constriction (TAC) attenuates cardiac remodeling.

(A) Male wild type male C57BL/6J were supplemented with either drinking water alone or drinking water with β-alanine (20 g/L) one week before TAC surgery. β-alanine supplementation was continued until the end of the experiment. After 4- and 8-weeks post-surgery cardiac parameters were evaluated via echocardiography. (B) Representative M-modes images, (C) left ventricular (LV) mass, (D) ejection fraction, (E) LV internal diameter diastole (LVID;d), (F) LV internal diameter systole (LVID; s), (G) end systolic volume, and (H) end diastolic volume. Data shown as mean ± SEM, p-values determined using Student’s t-test or repeated measures ANOVA as appropriate, n = 6 – 9 mice per group.

Figure 3.. β-alanine supplementation prior to transverse aortic constriction (TAC) increases myocardial histidyl dipeptide levels and improves aldehyde quenching.

Wild type (WT) C57BL/6J mice were pretreated with water alone or water supplemented with β-alanine (20 g/L) for 1 week and then subjected to TAC surgery. Treatment continued for 20 weeks. (A) Carnosine, anserine, and N-acetylcarnosine, (B) carnosine-propanal, and (C) carnosine-propanol levels were measured by LC-MS/MS using carnosine-d₄ or anserine-d₄ as an internal standard. Data shown as mean ± SEM, p-values were determined using Student’s t-test, n = 4 – 7 mice per group.

Figure 4.. β-alanine supplementation prior to transverse aortic constriction (TAC) decreases carbonyl stress during heart failure.

Male wild type C57BL/6J mice were pretreated with drinking water alone or drinking water supplemented with β-alanine (20 g/L) one week prior to TAC surgery, which was continued for 4 weeks. (A) Ratio of heart weight:tibia length (TL) and (B) carbonyl content of cardiac proteins determined by using a spectrophotometric dinitrophenylhydrazine-based assay. Data shown as mean ± SEM, p-values were determined using one-way ANOVA, n = 5 – 7 mice per group.

Figure 5.. Carnosine binds to the aldehyde moiety of aldehyde-protein adducts.

(A) Schematic demonstrating binding of reactive aldehydes with proteins either through Micheal adduct or Schiff base formation. (B) Hen egg lysozyme (HEL) was incubated with 1 mM 4-hydroxynonenal (HNE) or 1mM acrolein for 1 h at 37°C. The aldehyde-modified proteins were further incubated with 10 mM carnosine, reduced with 10 mM NaBH₄, or reduced with NaBH₄ followed by carnosine incubation (both 10 mM). (C) Modified HEL was immunoblotted and developed with anti-carnosine, anti-HNE and anti-acrolein antibodies. (D) Heart lysates from mice subjected to sham, TAC, and TAC + β-alanine supplementation were immunoblotted and developed using anti-carnosine antibody. (E) Intensity of bands were quantified and normalized to total protein signal using amidoblack (AB) stain. Data shown as mean ± SEM, p-values were determined using one-way ANOVA, n=3 mice in each group.

Figure 6.. Carnosine abrogates immunogenicity of aldehyde modified cardiac proteins.

(A-D) Bone marrow derived dendritic cells (BMDCs) were incubated with 100 μg/mL hen lysozyme (HEL), HEL-malonaldehyde, HEL-MDA incubated with carnosine, or HEL-MDA reduced by NaBH₄ for 6 h. BMDC activation was assessed by MHCII and CD80 expression levels in CD45⁺CD11b⁺CD11c⁺ dendritic cells by flow cytometry. (E) Splenocytes from Nur77-GFP mice were treated with 200 μg/mL naïve cardiac lysate, lysate incubated with an aldehyde (F, MDA; G, acrolein; H, HNE), aldehyde-lysate incubated with carnosine, or lysate-MDA reduced with NaBH₄. Extent of CD4⁺ T cell activation was determined by measuring GFP fluorescence in CD45⁺CD3⁺CD4⁺ cells using flow cytometry. (I, J) Splenocytes from Nur77-GFP mice were treated with 200 μg/mL lysate from sham mice, lysate from TAC mice, TAC lysate incubated with carnosine, or TAC lysate reduced with NaBH₄. Extent of CD4⁺ T cell activation was then determined by measuring GFP fluorescence in CD45⁺CD3⁺CD4⁺ cells using flow cytometry. Data shown as mean ± SEM, p-values were determined using one-way ANOVA.

Figure 7.. β-alanine supplementation reduces cardiac immune cell infiltration and CD4⁺ T cell activation in mice subjected to transverse aortic constriction (TAC).

Male wild type C57BL/6J mice were subjected to sham or TAC surgery and given either plain drinking water or drinking water supplemented with 20 g/L β-alanine 1 week prior to surgery and continued until the end of the experiment. (A) 4 weeks after surgery, infiltration of (B) CD45⁺ leukocytes, (C) CD45⁺CD11b⁻CD3⁺CD4⁺ T cells, (D) CD45⁺CD11b⁻CD3⁺CD4⁺CD44⁺ T cells, (E) CD45⁺CD11b⁻CD3⁺CD8⁺ T cells, (F) CD45⁺CD11b⁺CD64⁻Ly6G⁺ neutrophils, and (G) CD45⁺CD11b⁺Ly6G⁻CD64⁺ monocytes and macrophages were determined using flow cytometry. Data shown as mean ± SEM, p-values were determined using one-way ANOVA, n = 7 – 11 mice per group.