Huoxue Qianyang Qutan recipe attenuates Ang II-induced cardiomyocyte hypertrophy by regulating reactive oxygen species production

Abstract

Continuous and irreversible cardiac hypertrophy can induce cardiac maladaptation and cardiac remodeling, resulting in increased risk of developing cardiovascular diseases. The present study was conducted to investigate the therapeutic effect of Huoxue Qianyang Qutan recipe (HQQR) on angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. Primary cardiomyocytes were isolated from the cardiac tissue of neonatal rats, followed by flow cytometry detection to confirm the proportion of primary cardiomyocytes. Cell Counting Kit-8 assay and immunofluorescence detection were performed to examine the effect of Ang II and HQQR on cardiomyocyte hypertrophy. Reactive oxygen species (ROS) and a series of metabolic indicators were quantified to investigate the effect of HQQR on Ang II-induced cardiomyocyte hypertrophy. Mitochondrial electron transport chain complex activity and related coding gene expression were determined to explore the effect of HQQR on mitochondrial function. HQQR significantly inhibited Ang II-induced cardiomyocyte hypertrophy and restored Ang II-induced ROS accumulation, metabolic indicators, and membrane potential levels. HQQR also regulated the mitochondrial function related to the sirtuin 1 pathway in Ang II-induced cardiomyocytes by increasing the activity of the mitochondrial electron transport chain complex and affecting the expression of genes encoding mitochondrial electron transport chain complex subunits. HQQR could alleviate Ang II-induced cardiomyocyte hypertrophy by modulating oxidative stress, accumulating ROS and increasing mitochondrial electron transport chain activity.

Keywords: Huoxue Qianyang Qutan recipe; cardiomyocyte hypertrophy; mitochondrial dysfunction; mitochondrial electron transport chain.

Figure 1

HQQR attenuates the inhibitory effect of Ang II on rat cardiomyocyte proliferation. (A) TnI was detected using flow cytometry analysis to identify the percentage of primary cardiomyocytes. (B) Image of isolated primary rat cardiomyocytes (magnification, x200; scale bar, 50 µm). (C) Cell Counting Kit-8 assays were used to examine the proliferation of primary cardiomyocytes at 0, 12, 24 and 48 h after treatment with Ang II (1 µmol/l) and HQQR powder (0, 0.05, 0.1, 0.2, 0.5 and 1 mg/ml). Vehicle and valsartan were used as negative and positive controls, respectively. ^***P<0.001 vs. the vehicle group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the Ang II + vehicle group. HQQR, Huoxue Qianyang Qutan recipe; Ang II, angiotensin II; TnI, troponin I; OD, optical density.

Figure 2

HQQR improves Ang II-induced cardiomyocyte hypertrophy. (A) Immunofluorescence experiments were performed using anti-α-actinin antibodies to analyze cardiomyocyte hypertrophy (magnification, x200; scale bar, 50 µm). (B) mRNA expression levels of the myocardial hypertrophy markers ANP, BNP and β-MHC were examined using reverse transcription-quantitative PCR. (C) Protein expression levels of the myocardial hypertrophy markers ANP, BNP and β-MHC were examined using western blotting. ^**P<0.01 and ^***P<0.001 vs. the vehicle group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the Ang II + vehicle group. HQQR, Huoxue Qianyang Qutan recipe; Ang II, angiotensin II; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; β-MHC, β-major histocompatibility complex.

Figure 3

HQQR alleviates AngII-induced cardiomyocyte hypertrophy by modulating ROS accumulation. (A) Flow cytometry analysis to detect the changes in ROS accumulation. Levels of (B) ATP and (C) protein carbonyl content, and activities of (D) MDA, (E) SOD, (F) CAT and (G) GSH-Px were quantified. (H) Immunofluorescence experiments were performed using anti-α-SMA antibodies to analyze cardiomyocyte hypertrophy (magnification, x200; scale bar, 50 µm). ^**P<0.01 and ^***P<0.001 vs. the vehicle group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the Ang II + vehicle group. HQQR, Huoxue Qianyang Qutan recipe; Ang II, angiotensin II; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GSH-PX, glutathione peroxidase; α-SMA, α-smooth muscle actin.

Figure 4

HQQR reverts cardiomyocyte hypertrophy-induced membrane potential reduction and apoptosis increase. (A) Flow cytometry analysis to detect the changes in membrane potential. (B) Flow cytometry analysis to detect the changes in cell apoptosis. ^**P<0.01 and ^***P<0.001 vs. the vehicle group; ^#P<0.05, ^##P<0.01 and ^###P<0.001 vs. the Ang II + vehicle group. HQQR, Huoxue Qianyang Qutan recipe; Ang II, angiotensin II.

Figure 5

HQQR increases the activity of mitochondrial electron transport chain complexes in Ang II-treated rat cardiomyocytes. (A) The activities of the mitochondrial complexes I-IV and V were determined. (B) The level of mtDNA was quantified using RT-qPCR. (C) Protein levels of SIRT1, PGC-1α, NRF1, Tfam, NDUFA13, SDHB, COX IV, COX1 and ATPase 6 in primary cardiomyocytes were examined by western blotting. ^**P<0.01 and ^***P<0.001 vs. the vehicle group; ^#P<0.05 and ^##P<0.01 vs. the Ang II + vehicle group. HQQR, Huoxue Qianyang Qutan recipe; Ang II, angiotensin II; SIRT1, sirtuin 1; mtDNA, mitochondrial DNA; RT-qPCR, reverse transcription-quantitative PCR; PGC-1α, peroxisome proliferator-activated receptor-gamma coactivator-1α; NRF1, nuclear respiratory factor 1; Tfam, mitochondrial transcription factor A; NDUFA13, NADH:ubiquinone oxidoreductase subunit A13; SDHB, succinate dehydrogenase complex iron sulfur subunit B; COX IV, cytochrome c oxidase subunit IV; COX1, anti-cyclooxygenase 1; RT-qPCR, reverse transcription-quantitative PCR.