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. 2018 Mar;22(3):1517-1526.
doi: 10.1111/jcmm.13419. Epub 2017 Dec 20.

Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats

Li Jin  1   2 , Zhe Hao Piao  3 , Chun Ping Liu  2 , Simei Sun  1 , Bin Liu  3 , Gwi Ran Kim  1 , Sin Young Choi  1 , Yuhee Ryu  1 , Hae Jin Kee  1 , Myung Ho Jeong  1
Affiliations

Gallic acid attenuates calcium calmodulin-dependent kinase II-induced apoptosis in spontaneously hypertensive rats

Li Jin et al. J Cell Mol Med. 2018 Mar.

Abstract

Hypertension causes cardiac hypertrophy and leads to heart failure. Apoptotic cells are common in hypertensive hearts. Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is associated with apoptosis. We recently demonstrated that gallic acid reduces nitric oxide synthase inhibition-induced hypertension. Gallic acid is a trihydroxybenzoic acid and has been shown to have beneficial effects, such as anti-cancer, anti-calcification and anti-oxidant activity. The purpose of this study was to determine whether gallic acid regulates cardiac hypertrophy and apoptosis in essential hypertension. Gallic acid significantly lowered systolic and diastolic blood pressure in spontaneously hypertensive rats (SHRs). Wheat germ agglutinin (WGA) and H&E staining revealed that gallic acid reduced cardiac enlargement in SHRs. Gallic acid treatment decreased cardiac hypertrophy marker genes, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), in SHRs. The four isoforms, α, β, δ and γ, of CaMKII were increased in SHRs and were significantly reduced by gallic acid administration. Gallic acid reduced cleaved caspase-3 protein as well as bax, p53 and p300 mRNA levels in SHRs. CaMKII δ overexpression induced bax and p53 expression, which was attenuated by gallic acid treatment in H9c2 cells. Gallic acid treatment reduced DNA fragmentation and the TUNEL positive cells induced by angiotensin II. Taken together, gallic acid could be a novel therapeutic for the treatment of hypertension through suppression of CaMKII δ-induced apoptosis.

Keywords: Ca2+/calmodulin-dependent protein kinase II; apoptosis; cardiac hypertrophy; gallic acid; spontaneously hypertensive rats (SHR).

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Figures

Figure 1
Figure 1
Gallic acid lowers blood pressure in spontaneously hypertensive rats. (A, B) Systolic and diastolic blood pressures were measured in three groups: WKY rats, SHRs and SHRs plus gallic acid. ***P < 0.001 versus WKY rats. ### P < 0.001 versus SHRs.
Figure 2
Figure 2
Gallic acid attenuates left ventricular hypertrophy in spontaneously hypertensive rats (A) WGA (top panel) and H&E (bottom panel) staining performed to evaluate the increased size in myocytes. Scale bar = 50 μm. (B) Cross‐sectional area of the left ventricle was evaluated (n = 8 per group). ***P < 0.001 compared with WKY rats. ### P < 0.001 versus SHRs. (C) The ratio of the left ventricular weight to tibia length (mg/mm) in WKY, SHR, SHR+GA (n = 8 per group). ***P < 0.001 compared with WKY rats. ### P < 0.001 versus SHRs. (D, E) The mRNA levels of ANP and BNP were evaluated by real‐time RT‐PCR from three groups (n = 14). The transcript levels were normalized to those for 18S and presented as relative values. ***P < 0.001 versus WKY rats. # P < 0.05 versus SHRs.
Figure 3
Figure 3
Gallic acid down‐regulates expression of Ca2+/calmodulin‐dependent protein kinase II in spontaneously hypertensive rats. (A–D) Real‐time RT‐PCR was performed in LV tissues from WKY rats, SHRs and SHRs plus gallic acid. The mRNA levels of CaMKII α, β, δ and γ were determined. The transcript levels were normalized to those for 18S. ***P < 0.001 versus WKY rats. # P < 0.05 versus SHRs. (E) Representative immunoblots. Western blot analysis for pan‐CaMKII, CaMKII δ and CaMKII γ in LV tissues from WKY rats, SHRs and SHRs plus gallic acid (n = 8 per group). Antibodies for pan‐CaMKII detected CaMKII β (60 kD) and CaMKII α (55 kD).
Figure 4
Figure 4
Gallic acid reduces apoptosis in spontaneously hypertensive rats. (A) Representative immunoblots. Western blot analysis for the expression of cleaved caspase‐3 performed in LV tissues from WKY rats, SHRs and SHRs plus gallic acid (n = 8 per group). Caspase‐3 and cleaved caspase‐3 have sizes of 35 kD and 23 kD, respectively. GAPDH was used as a loading control. (B) Western blot analysis for the expression of bax protein was performed in LV tissues from WKY rats, SHRs and SHRs plus gallic acid. (C–E) The mRNA levels of bax, p53 and p300 were evaluated by real‐time RT‐PCR. The transcript levels were normalized to those for 18S. ***P < 0.001 versus WKY rats. # P < 0.05 versus SHRs.
Figure 5
Figure 5
Gallic acid suppresses apoptosis induced by CaMKII δ overexpression or angiotensin II stimulus in H9c2 cells. (A–C) H9c2 cells were transfected with empty vector or pcDNA3‐CaMKIIδ and then were treated with gallic acid for 6 hrs. The expression of CaMKII δ (A), bax (B) and p53 (C) was assayed by real‐time RT‐PCR. The transcript levels were normalized to those for GAPDH. ***P < 0.001 versus empty vector. ## P < 0.01 and ### P < 0.001 versus CaMKII δ transfection. (D–F) H9c2 cells were incubated with angiotensin II (100 μM) and then treated with gallic acid (100 μM) for 6–9 hrs. CaMKII δ (D), bax (E) and p53 (F) transcript levels were determined by real‐time RT‐PCR. **P < 0.01 and ***P < 0.001 versus empty vector. # P < 0.05 and ### P < 0.001 versus angiotensin II‐treated group. Data represent the means ± standard error (S.E.) for at least four independent experiments.
Figure 6
Figure 6
Gallic acid reduces angiotensin II‐induced apoptosis as determined by TUNEL assay and DNA fragmentation. (A) Representative images of TUNEL staining. H9c2 cells were serum starved for 24 hrs and were treated with gallic acid (50 μM) in the presence or absence of angiotensin II (10 μM) for 36 hrs. Green and blue colours indicate apoptotic cells and nuclei in H9c2 cells. (B) Quantification of positive TUNEL staining. **P < 0.01 versus vehicle group. ### P < 0.001 versus the angiotensin II‐treated group. Data represent the means ± S.E. of at least three independent experiments. (C) Representative DNA fragmentation of angiotensin II (100 μM)‐treated H9c2 cells in the presence or absence of gallic acid (25 μM). H9c2 cells lysates were treated with RNase A and proteinase K before DNA extraction. Apoptosis was determined by 1.7% agarose gel electrophoresis; 100 bp DNA ladder was used as a loading control.
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