Diabetes Metab J.  2023 Jul;47(4):487-499. 10.4093/dmj.2022.0125.

Pharmacologic Activation of Angiotensin-Converting Enzyme II Alleviates Diabetic Cardiomyopathy in db/db Mice by Reducing Reactive Oxidative Stress

• Kim D
• Jeong W
• Kim Y
• Lee J
• Cho SW
• Oh CM
• Park R

• KMID: 2544728
• DOI: http://doi.org/10.4093/dmj.2022.0125

Abstract

Background
Diabetes mellitus is one of the most common chronic diseases worldwide, and cardiovascular disease is the leading cause of morbidity and mortality in diabetic patients. Diabetic cardiomyopathy (DCM) is a phenomenon characterized by a deterioration in cardiac function and structure, independent of vascular complications. Among many possible causes, the renin-angiotensin-aldosterone system and angiotensin II have been proposed as major drivers of DCM development. In the current study, we aimed to investigate the effects of pharmacological activation of angiotensin-converting enzyme 2 (ACE2) on DCM.
Methods
The ACE2 activator diminazene aceturate (DIZE) was administered intraperitoneally to male db/db mice (8 weeks old) for 8 weeks. Transthoracic echocardiography was used to assess cardiac mass and function in mice. Cardiac structure and fibrotic changes were examined using histology and immunohistochemistry. Gene and protein expression levels were examined using quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Additionally, RNA sequencing was performed to investigate the underlying mechanisms of the effects of DIZE and identify novel potential therapeutic targets for DCM.
Results
Echocardiography revealed that in DCM, the administration of DIZE significantly improved cardiac function as well as reduced cardiac hypertrophy and fibrosis. Transcriptome analysis revealed that DIZE treatment suppresses oxidative stress and several pathways related to cardiac hypertrophy.
Conclusion
DIZE prevented the diabetes mellitus-mediated structural and functional deterioration of mouse hearts. Our findings suggest that the pharmacological activation of ACE2 could be a novel treatment strategy for DCM.

Keyword

Angiotensin-converting enzyme 2; Diabetic cardiomyopathies; Diminazene aceturate; Heart failure

Figure

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  Fig. 1. Angiotensin-converting enzyme 2 (ACE2) activator has a beneficial effect on the diabetic heart. (A) The bodyweights of the four groups (db/+ with the vehicle [CTL_VEH], db/+ with diminazene aceturate [DIZE] [CTL_DIZE], db/db with the vehicle [DB_VEH], and db/db with DIZE [DB_DIZE]) for 8 weeks. Comparisons between db/db groups were statistically significant, following Sidak’s multiple comparisons test and one-way analysis of variance (ANOVA). (B) Representative left ventricular (LV) short axis and M-mode echocardiographic tracings. (C) LV function between the db/db groups. DIZE groups showed reduced LV mass and wall thickness, decreased LV internal diameter (LVIDd) end systole, improved left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) percentages, and increased LV internal diameter end diastole compared with the vehicle groups (n=5 mice per group). Each bar represents the mean±standard deviation. Two-tailed unpaired Student’s t-test. aP<0.05, bP<0.005 compared to the DB_VEH groups.

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  Fig. 2. Angiotensin-converting enzyme 2 (ACE2) activator attenuates fibrosis in the diabetic heart. (A) Representative gross view of heart (vehicle=db/db with vehicle, diminazene aceturate [DIZE]=db/db with DIZE). (B) Representative images of H&E stained left ventricular (LV) sections (scale bar=500 μМ). The right images showed the different myofibril thickness in transverse cross-sectioned cells between the vehicle and DIZE groups in the diabetic heart (scale bar=20 μМ). (C) Measured diameters of myofibrils at 7 points of transverse cross-sectioning (B) in each diabetic group. The DIZE group’s diameters significantly decreased compared to the vehicle group. Each bar represents the mean±standard deviation. (D) Representative images of LV sections stained with Picrosirius red (scale bar=500 μМ). (E) Quantification of interstitial fibrosis (scale bar=20 μМ). (F) LV mRNA expression of collagen type I alpha 1 chain (Col1a1), Col3a1, connective tissue growth factor (Ctgf), and transforming growth factor-beta 1 (Tgfβ1) is related to fibrosis in LV tissues of all mouse groups (n≥3 mice per group). (G) LV mRNA expression of natriuretic peptide A (Nppa), natriuretic peptide B (Nppb), natriuretic peptide receptor type A (Npra), and natriuretic peptide receptor type B (Nprb) that is related to cardiac hypertrophy in LV tissues of all mice groups (n≥3 mice per group). Two-tailed unpaired Student’s t-test or sidak’s multiple comparisons test and one-way analysis of variance (ANOVA). CTL_VEH, db/+ with vehicle; CTL_DIZE, db/+ with DIZE; DB_VEH, db/db with vehicle; DB_DIZE, db/db with DIZE. aP<0.05, bP<0.005, and cP<0. 0005 compared to the DB_VEH groups.

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  Fig. 3. Angiotensin-converting enzyme 2 (ACE2) activator reduces oxidative stress in the diabetic heart. (A) Left ventricular (LV) mRNA expression of Ace2 in db/+ with the vehicle and db/db groups. Plasma concentration of angiotensin II (Ang II) and serum concentration of Ang-(1-7) in three different groups (n≥3 mice per group). (B) LV mRNA expression of superoxide dismutase 1 (Sod1), Sod2, and glutathione peroxidase (Gpx) in db/+ with the vehicle and db/db groups (n≥5 mice per group). (C) Representative western blotting images of Sod1, Sod2, and α-tubulin in three different groups. (D) Densitometry analysis of immunoreactive bands of Sod1 and Sod2. Diminazene aceturate (DIZE) reduced Sod1 and Sod2 expression in the diabetic heart. (E) Representative confocal images of dichlorodihydrofluorescein diacetate (DCFDA) assay. Two-tailed unpaired Student’s t-test or sidak’s multiple comparisons test and one-way analysis of variance (ANOVA). CTL_VEH, db/+ with vehicle; DB_VEH, db/db with vehicle; DB_DIZE, db/db with DIZE. aP<0.05.

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  Fig. 4. Angiotensin-converting enzyme 2 (ACE2) activator’s beneficial effect on the transcriptome level in the diabetic heart. (A) A volcano plot showing the differentially expressed genes between db/db with diminazene aceturate (DIZE) (DB_DIZE) and db/db with vehicle (DB_VEH). The threshold of the y-axis is the cutoff of the overall P value (0.05) and the x-axis is the cutoff of log2 fold change (±0.4625). (B) A heatmap representing the functional enrichment analysis between the two groups as mentioned above. Color bars indicate the gradients of the Z-score. (C) Gene Set Enrichment Analysis (GSEA) enrichment plots showed a negative association with Ras protein signaling and a positive association with adenosine triphosphate (ATP) generation in the DB_DIZE group. (D) Upstream regulatory analysis (URA) showed potential upstream molecules in db/db with the DIZE group. The threshold for the x-axis is the cutoff of the activation Z-score (±2) and that for the y-axis is the cutoff of the P value (0.05) (up). Gene regulatory network of Rock1. Each node represents gene colored according to whether it is differentially expressed gene (DEG) or not (down). (E) The heatmap represents the functional enrichment analysis between the two datasets. In the right-hand section (No. 1) are our db/db mice groups and in the opposite section (No. 2) are the db/db and wild-type mice from the public transcriptome data (GSE161931). ERK, extracellular signal-regulated kinase; ERBB, erb-b2 receptor tyrosine kinase 2; MAPK, mitogen-activted protein kinase 1; PI3K, phosphatidylinositol 3-kinase; ECM, extracellular matrix; NES, normalized enrichment score; ADP, adenosine diphosphate; WT, wild type.

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