Mst1 silencing alleviates hypertensive myocardial injury associated with the augmentation of microvascular endothelial cell autophagy

Abstract

The activation of mammalian ste20‑like kinase1 (Mst1) is a crucial event in cardiac disease development. The inhibition of Mst1 has been recently suggested as a potential therapeutic strategy for the treatment of diabetic cardiomyopathy. However, whether silencing Mst1 also protects against hypertensive (HP) myocardial injury, or the mechanisms through which this protection is conferred are not yet fully understood. The present study aimed to explore the role of Mst1 in HP myocardial injury using in vivo and in vitro hypertension (HP) models. Angiotensin II (Ang II) was used to establish HP mouse and cardiac microvascular endothelial cell (CMEC) models. CRISPR/adenovirus vector transfection was used to silence Mst1 in these models. Using echocardiography, hematoxylin and eosin staining, Masson's trichrome staining, the enzyme‑linked immunosorbent assay detection of inflammatory factors, the enzyme immunoassay detection of oxidative stress markers, terminal deoxynucleotidyl transferase dUTP nick‑end labeling staining, scanning electron microscopy, transmission electron microscopy, as well as immunofluorescence and western blot analysis of the autophagy markers, p62, microtubule‑associated proteins 1A/1B light chain 3B and Beclin‑1, it was found that Ang II induced HP myocardial injury with impaired cardiac function, increased the expression of inflammatory factors, and elevated oxidative stress in mice. In addition, it was found that Ang II reduced autophagy, enhanced apoptosis, and disrupted endothelial integrity and mitochondrial membrane potential in cultured CMECs. The silencing of Mst1 in both in vivo and in vitro HP models attenuated the HP myocardial injury. On the whole, these findings suggest that Mst1 is a key contributor to HP myocardial injury through the regulation of cardiomyocyte autophagy.

Keywords: CMEC; Mst1; autophagy; hypertension; myocardium.

Figure 1

Cardiac morphology and function in mice. (A) Systolic blood pressure of the mice. ^*P<0.05 vs. WT or Mst1^−/−. (B) Representative M-mode echocardiography images of WT, Mst1^−/−, HP and HP Mst1^−/− mice. (C) Quantitative assessment of M-mode images describing left ventricular function; n=6. (D) Representative hematoxylin and eosin staining of mouse cardiac sections. (E) Representative Masson's trichrome staining of mouse cardiac sections. (F) Quantification of cardiomyocyte hypertrophy. (G) Quantification of fibrosis. ^*P<0.05 and ^**P<0.01. WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension.

Figure 2

Cardiac levels of inflammatory factors and oxidative stress markers in mice. (A and B) Expression level of IL-6 and TNF-α in mouse myocardium; n=6. (C-E) Protein levels of MDA, SOD and GSH-PX in mouse myocardium; n=6. ^*P<0.05. IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-PX, glutathione peroxidase; WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension.

Figure 3

Cardiomyocyte apoptosis in mice. (A) Representative TUNEL staining of mouse cardiac sections. (B) Quantitative assessment of TUNEL staining; n=6. ^*P<0.05. WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension.

Figure 4

Expression of apoptosis-related proteins. (A) representative western blots. Quantification of the expression level of (B) Bcl-2, (C) BAX, (D) PARP-1, (E) cleaved caspase-3, and (F) cleaved caspase-9 in WT, Mst1^−/−, HP and HP Mst1^−/− mice. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension.

Figure 5

Microvascular endothelial cell integrity and mitochondrial membrane potential of Ang II-treated endothelial cells. (A) Representative scanning electron microscopy images of mouse myocardial microvascular endothelial cells. (B) FACS measurement of mitochondrial membrane potential of CMECs. (C) Quantitative assessment of mitochondrial membrane potential of CMECs; n=3. ^*P<0.05 and ^**P<0.01. CMECs, cardiac microvascular endothelial cells; WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension; KD, knockdown; NC, negative control.

Figure 6

Increased autophagy in Mst1-deficient endothelial cells. (A) TEM images of CMECs. Scale bar, 2 µm. (B) Representative immunofluorescence images of p62 and LC3B in mouse myocardium; magnification, ×200. (C) Quantitative assessment of immunofluorescence images; n=3. (D) Representative immunofluorescence images of p62 and LC3B in CMECs; magnification, ×200. (E) Quantitative assessment of immunofluorescence images; n=3. ^*P<0.05. WT, wild-type; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension; LC3B, microtubule-associated proteins 1A/1B light chain 3B; CMECs, cardiac microvascular endothelial cells; KD, knockdown; NC, negative control.

Figure 7

Modulation of core autophagy protein expression by Mst1. (A) Representative western blots of Mst1, p-Mst1, Beclin-1, p62, LC3B and β-actin proteins in CMECs and the quantitative assessment of the western blots; n=4. (B) Representative western blots of Mst1, p-Mst1, Beclin-1, p62, LC3B and β-actin proteins in mouse myocardium and the quantitative assessment of the western blots; n=3. ^*P<0.05, ^**P<0.01, ^***P<0.001 and ^****P<0.0001. Mst1, mammalian ste20-like kinase 1; LC3B, microtubule-associated proteins 1A/1B light chain 3B; CMECs, cardiac microvascular endothelial cells; WT, wild-type; HP, hypertensive/hypertension; KD, knockdown; NC, negative control.

Figure 8

Autophagy-related protein expression with autophagy inhibitors. (A) Representative western blots. Quantification of the expression of (B) LC3B and (C) p62 in CMECs treated with or without bafilomycin A1. ^**P<0.01, ^***P<0.001 and ^****P<0.0001. LC3B, microtubule-associated proteins 1A/1B light chain 3B; CMECs, cardiac microvascular endothelial cells; Mst1, mammalian ste20-like kinase 1; HP, hypertensive/hypertension; KD, knockdown; NC, negative control.