Carnosine protects cardiac myocytes against lipid peroxidation products

Abstract

Endogenous histidyl dipeptides such as carnosine (β-alanine-L-histidine) form conjugates with lipid peroxidation products such as 4-hydroxy-trans-2-nonenal (HNE and acrolein), chelate metals, and protect against myocardial ischemic injury. Nevertheless, it is unclear whether these peptides protect against cardiac injury by directly reacting with lipid peroxidation products. Hence, to examine whether changes in the structure of carnosine could affect its aldehyde reactivity and metal chelating ability, we synthesized methylated analogs of carnosine, balenine (β-alanine-N^τ-methylhistidine) and dimethyl balenine (DMB), and measured their aldehyde reactivity and metal chelating properties. We found that methylation of N^τ residue of imidazole ring (balenine) or trimethylation of carnosine backbone at N^τ residue of imidazole ring and terminal amine group dimethyl balenine (DMB) abolishes the ability of these peptides to react with HNE. Incubation of balenine with acrolein resulted in the formation of single product (m/z 297), whereas DMB did not react with acrolein. In comparison with carnosine, balenine exhibited moderate acrolein quenching capacity. The Fe²⁺ chelating ability of balenine was higher than that of carnosine, whereas DMB lacked chelating capacity. Pretreatment of cardiac myocytes with carnosine increased the mean lifetime of myocytes superfused with HNE or acrolein compared with balenine or DMB. Collectively, these results suggest that carnosine protects cardiac myocytes against HNE and acrolein toxicity by directly reacting with these aldehydes. This reaction involves both the amino group of β-alanyl residue and the imidazole residue of L-histidine. Methylation of these sites prevents or abolishes the aldehyde reactivity of carnosine, alters its metal-chelating property, and diminishes its ability to prevent electrophilic injury.

Keywords: 4-Hydroxy-trans-2-nonenal; Acrolein; Cardiac myocytes; Histidyl dipeptides.

Fig. 1.. Preparation of balenine.

Reagents and conditions for synthesis of natural occurring histidyl dipeptide balenine steps 1–6.

Fig. 2.. Preparation of di-methyl-balenine (DMB).

Reagents and conditions for synthesis of synthetic carnosine analogue DMB steps 1–7

Fig. 3.. LC/MS spectra of different histidyl dipeptides with lipid peroxidation products.

Histidyl dipeptides: carnosine, balenine, and di-methyl-balenine (DMB) were incubated with HNE or acrolein (10:1 molar ratio) in 1mM phosphate buffer (pH 7.4). Ionic species were monitored by ESI/MS - (A) carnosine-HNE (m/z 383), (B) carnosine-propanal (m/z 265, 283, 303), and (C) balenine (m/z 241). Inset shows the chemical structure of (D) balenine-propanal (m/z 297); the Michael adduct of balenine with acrolein; and (E) di-methyl-balenine (m/z 269).

Fig. 4.. Methylation of carnosine backbone diminishes its aldehyde quenching potential.

Rate of disappearance of acrolein (100 μM) in a reaction mixture containing different concentrations of (A) carnosine, (C) balenine, and (E) dimethyl balenine (50–250 µM) in 0.15M potassium phosphate buffer, pH 7.4. (F) Rate of disappearance of HNE incubated with DMB. The reaction mixtures were incubated at 37°C and the decrease in absorbance of acrolein and HNE were monitored at 215 nm and 223 nm respectively. (B, D) Data are shown as discrete points and curves were best fit of a single exponential equation to the data [y=Ae^−kobs.t]. Concentration dependence of k_obs. Second order rate constants were calculated from the best fits of the linear dependence and are shown in Table I.

Fig.5.. Metal chelating capacity of different histidyl dipeptides.

Iron (Fe²⁺) chelating capacity of (A) EDTA (B) carnosine and (C) balenine. (D) EDTA equivalent chelating activity of different histidyl dipeptides.

Fig. 6.. Carnosine pretreatment prevents HNE-induced hypercontracture.

(A) Levels of histidyl dipeptides in myocytes non-treated (NT) or treated with 1mM each of carnosine (car), balenine (bal), and di-methyl-balenine (DMB). Inset shows representative images of myocytes at 0 min and after 55 min of superfusion with HNE (50–60 μM) with and without pretreatment with the indicated histidyl dipeptides. Hypercontracture was monitored at 5 min intervals for 60 min. (B) Mean lifetime of myocytes was calculated using the Weibull Survival distribution function (S(t)=exp-(λt)^γ). (C) Data for mean life time are presented as mean ± SD. *p<0.05 vs HNE treated myocytes, # p<0.05 vs carnosine and balenine treated myocytes n= 3–5 mice in each group.

Fig. 7.. Acrolein-induced hypercontracture is attenuated by carnosine pretreatment.

(A) Hypercontracture was monitored at 5 min interval for 60 min. Mean-lifetime was calculated using the Weibull distribution function. Inset shows the representative images of myocytes at 0 min and after 50 min of acrolein (5 μM) superfusion. (B) Data for mean lifetime are presented as mean ± SD. *p<0.05 vs acrolein treated myocytes, n=4–5 mice in each group.