To study the ameliorating effects of curcumin in lipopolysaccharide (LPS) induced cardiac hypertrophy, mice were assigned to 4 groups (3 males and 3 females in each group): (A) control, (B) curcumin: 100
In the United States, foodborne pathogens cause 76 million illnesses, 325,000 hospitalizations, and 5,000 deaths annually [
HAT activity in cardiac muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression [
Curcumin which is derived from turmeric (
This study was designed to determine the potential for curcumin to attenuate LPS induced cardiac hypertrophy in rodents. Additionally, the mechanism of action for this attenuation was investigated.
Curcumin C3 complex (R) was a kind gift from Sabinsa Corporation, Hyderabad, India. The patented C3 complex (R) contains curcumin and its derivates demethoxycurcumin and bisdemethoxycurcumin, also known as curcuminoids. Curcumin is 1–5% of the turmeric. LPS sc-3535 was obtained from Santa Cruz Biotechnology as a white to yellow lyophilized powder. For the live and dead experiment, LPS was used as 1.5 mg/mice (dissolved in distilled water), and in curcumin attenuation of LPS induced cardiac hypertrophy, LPS was used as 60 mg/kg body weight dose (dissolved in distilled water). Histone H3 antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA. It is a purified antibody. Histone H3 (N-20), was produced against a peptide mapping at the N-terminus of histone H3 of human origin. Histone H4 was obtained from Santa Cruz Biotechnology, Santa Cruz, CA. histone H4 (N-18), purified goat polyclonal antibody, was produced against a peptide mapping at the N-terminus of histone H4 of human origin. p300 (C-20) was obtained from Santa Cruz Biotechnology, Santa Cruz, CA. p300 (C-20), affinity purified rabbit polyclonal antibody, was raised against a peptide mapping at the C-terminus of p300 of human origin. Acetyl-histone H3 (Lys9) antibody was obtained from cell signaling Technology, Danvers, MA. Endogenous levels of Histone H3 were detected by acetyl-histone H3 (Lys9) antibody only when acetylated at lysine 9. Acetyl-histone H4 (Lys12) antibody was obtained from cell signaling Technology, Danvers, MA. Endogenous levels of histone H4 were detected by acetyl-histone H4 (Lys12) antibody only when acetylated at Lys12.
Acetyl-CBP (Lys1535)/p300 (Lys1499) antibody was obtained from Cell Signaling Technology, Danvers, MA. Acetyl-CBP (Lys1535)/p300 (Lys1499) antibody detects endogenous levels of CBP or p300 only when acetylated at lysine 1535 or lysine 1499, respectively.
The Animal Care and Use Committee of Tuskegee University approved the experimental protocol (protocol number R0804-12-1). Eight breeding pairs of mice (
The hearts were collected and photographed for cardiomegaly. Hearts and lungs of the sacrificed mice were dissected and weighed to compare heart weight/brain weight (mg/g) and lung weight/brain weight (mg/g) ratios in LPS and curcumin treated mice. The collected organs were then preserved in 10% regular formalin in 50 mL tubes.
Immunohistochemistry is the process of detecting antigens in cells of a tissue section by exploiting the principle of antigen-antibody binding specificity. The antibody is conjugated to peroxidase that catalyses a color-producing reaction. The sections of murine tissue were placed on the heated water bath and picked up, placed on the slides, and placed in a rack. After all slides had been cut and placed in a rack they were put into a 60 degrees Celsius oven over night. The slides were deparaffinized by going through 3 changes of xylene 2 minutes each, followed by 3 changes of 100% alcohol at 1 minute and one change of 95% alcohol at 1 minute. Slides were washed in running tap water for 1 minute and stored in distilled water slides. The slides were then subjected to antibody pretreatment.
Comparisons between 2 groups were performed by paired Student’s
A
Mice in the LPS treated group showed significant cardiac hypertrophy as measured by heart weight/brain weight (HW/BW) ratio. Figure
Heart wt./brain wt. ratios of rats at 12 hrs and 24 hrs after respective treatment. The LPS treated group was significantly different from all other groups (
Curcumin protects mice against LPS induced cardiomegaly. The heart collected 24 hrs after LPS treated mice group showed significant cardiomegaly, and the heart from the LPS + Curcumin treated group showed that curcumin reverses LPS induced cardiomegaly.
Chromatin structure modulation has critical role in the regulation of transcription in eukaryotes. DNA wound around eight core histone proteins (two each of H2A, H2B, H3, and H4) form the nucleosome which is the primary building block of chromatin [
In order to evaluate the mechanism of action of curcumin against LPS induced cardiac hypertrophy, we determined the condition of histone H3 acetylation by using acetylation specific antibodies. In this study, curcumin repressed the LPS induced acetylation of histone-3 in mice (Figure
Histone proteins were detected by histone H3 antibody in control (a), curcumin (b), LPS (c), and curcumin + LPS (d) groups.
Control
Curcumin
LPS
LPS + curcumin
Curcumin attenuates histone H3 acetylation. LPS group showed that lipopolysaccharide (LPS) induced a significant increase in histone H3 acetylation, and curcumin group showed curcumin blocked LPS induced histone H3 acetylation.
Control
Curcumin
LPS
LPS + curcumin
Histone proteins were detected by histone H3 antibody in control (Figure
Figure
These findings indicate that acetylation of histone tails by histone specific antibodies, mediated by histone acetyltransferases, is involved in activation of gene expression in the myocardium. Conversely, repression of gene expression can be induced by histone deacetylation, which is mediated by histone deacetylases (HDACs). Pretreatment with curcumin, which has a HAT inhibitory activity, attenuates histone modifications.
Modulation of chromatin structure has a central role in the regulation of transcription in eukaryotes [
Histone H4 acetylation at the position of lysine 16 (H4-K16Ac) is a posttranslational chromatin modification. In addition to histone H3 we also measured histone H4 acetylation by measuring the [4H] acetate incorporation into histones using H4 acetylation specific antibody.
Figure
Histone proteins were detected by histone H4 antibody in control (a), curcumin (b), LPS (c), and curcumin + LPS (d) treated groups.
Control
Curcumin
LPS
LPS + curcumin
The histone (H4) acetylation results are illustrated in Figure
Curcumin attenuates histone H4 acetylation. LPS treated group (c) showed marked histone acetylation and curcumin + LPS treated group (d) showed curcumin reverses histone H4 acetylation.
Control
Curcumin
LPS
Curcumin + LPS
Our findings also suggested that histone acetylation is associated with pathological cardiac hypertrophy. HATs (histone acetyl-transferase) acetylate histone proteins, relax chromatin, and expose genes for activation by cardiogenic transcription factors. Inhibition of histone acetylation has a central role for the antihypertrophy activity of curcumin.
Two highly related and widely expressed molecules, cAMP regulated enhancer binding protein (CBP) and p300, have emerged as important cofactors for a broad number of transcription factors [
p300, high molecular weight transcriptional factor, has three cysteine and histidine-rich regions. p300 knockout mice showed the defects of cardiac muscle differentiation and trabeculation, indicating the importance of p300 for early cardiac morphogenesis and heart development. However, p300 is also involved in the pathological process of cardiac hypertrophy [
p300 levels were markedly increased by stimulation with lipopolysaccharide. To further check the mechanism of action of curcumin, we analyzed p300 protein acetylation levels. The results are illustrated in Figure
Curcumin attenuates p300 acetylation. LPS treated group (c) showed marked p300 acetylation, and curcumin + LPS treated group (d) showed curcumin reverses LPS induced p300 acetylation.
Control
Curcumin
LPS
Curcumin + LPS
p300 is involved in the pathological process of cardiac hypertrophy. The results of the present study indicate that inhibition of histone acetylation is a key mechanism for the anti-cardiac hypertrophy activity of curcumin and that p300-HAT serves as its molecular target. p300 may maintain basal function in the normal heart but it also promotes cardiac hypertrophy under LPS infusion. Our data also indicate that curcumin inhibits p300-HAT activity.
We can conclude from our study that curcumin attenuated LPS induced cardiac hypertrophy in vivo. Cardiac hypertrophy can be monitored by increased protein synthesis and induction of gene expression. Curcumin plays its anti-cardiac hypertrophy role by the involvement of chromatin remodeling, especially histone acetylation. Acetylation of lysine residues upon a single histone tail (hyperacetylation) would produce a stronger effect. Histone acetyltransferases have a critical role to acetylate histone proteins, relax chromatin, and expose cardiac hypertrophy genes for activation by cardiogenic transcription factors. Cardiac hypertrophy is also responsible for involvement of transcriptional factor p300. HAT activity in muscle is determined by p300 that causes modification of chromatin and associated transcription factors and gene expression. LPS induced cardiac hypertrophy accentuates p300 transcriptional activity, and LPS mediated cardiac hypertrophy is reversed by blocking of p300-HAT activity. Therefore, blocking p300-HAT activity may be a viable method for the treatment or prevention of myocardial hypertrophy.
The results showed that curcumin attenuated LPS induced cardiac hypertrophy in rodents and that the most probable mechanism of action involves inhibiting the p300-HAT activity.
The authors would like to thank all the faculty members of the College of Veterinary Medicine, Nursing and Allied Health and Department of Biology, at Tuskegee University.