STK35 Gene Therapy Attenuates Endothelial Dysfunction and Improves Cardiac Function in Diabetes

Abstract

Diabetic cardiomyopathy (DCM) is characterized by microvascular pathology and interstitial fibrosis that leads to progressive heart failure. The mechanisms underlying DCM pathogenesis remain obscure, and no effective treatments for the disease have been available. In the present study, we observed that STK35, a novel kinase, is decreased in the diabetic human heart. High glucose treatment, mimicking hyperglycemia in diabetes, downregulated STK35 expression in mouse cardiac endothelial cells (MCEC). Knockdown of STK35 attenuated MCEC proliferation, migration, and tube formation, whereas STK35 overexpression restored the high glucose-suppressed MCEC migration and tube formation. Angiogenesis gene PCR array analysis revealed that HG downregulated the expression of several angiogenic genes, and this suppression was fully restored by STK35 overexpression. Intravenous injection of AAV9-STK35 viral particles successfully overexpressed STK35 in diabetic mouse hearts, leading to increased vascular density, suppression of fibrosis in the heart, and amelioration of left ventricular function. Altogether, our results suggest that hyperglycemia downregulates endothelial STK35 expression, leading to microvascular dysfunction in diabetic hearts, representing a novel mechanism underlying DCM pathogenesis. Our study also emerges STK35 is a novel gene therapeutic target for preventing and treating DCM.

Keywords: STK35; angiogenesis; cardiac function; diabetes; gene therapy; serine threonine kinase.

Figure 1

STK35 expression is decreased in the diabetic human heart. (A) STK35 expression in diabetic human heart from publicly available RNA sequence data. STK35 expression in the GSE26887 datasheet includes 5 non-diabetic human hearts and 7 diabetic human heart tissues. (B) Immunohistochemistry staining showed a decrease in STK35 expression in the diabetic human heart compared to the non-diabetic human heart. Scale bar: 100 μm. (C) Western blotting and quantification analyses of STK35 expression in both non-diabetic and diabetic human hearts. (D) qRT-PCR data showing a decrease in STK35 mRNA expression in the diabetic human heart compared to the non-diabetic human heart. Data were normalized against GAPDH mRNA. n = 4, non-diabetic human heart; n = 5, diabetic human heart. **P < 0.01, and ***P < 0.001.

Figure 2

High glucose downregulates STK35 expression in mouse cardiac endothelial cells. (A) Cell type-specific STK35 mRNA expression in the adult human heart. The STK35 mRNA levels in different human heart cell types were determined by mining single-cell RNAseq data deposited in THE HUMAN PROTEIN ATLAS database (https://www.proteinatlas.org/ENSG00000125834-STK35/celltype/heart+muscle). The different heart cell types are colored according to clusters and are represented using Uniform manifold approximation and projection (UMPA). The cell-type expressions of STK35 mRNA were represented by the mean protein-coding transcripts per million (pTPM). (B) Immunofluorescence staining observed decreased STK35 expression in high glucose treated MCEC compared to MCEC treated with normal glucose. Scale bars: 50 μm. (C) Western blotting analysis of STK35 expression in MCEC treated with high glucose. (D) qRT-PCR data showing decreased STK35 mRNA expression in high glucose-treated MCEC compared to MCEC treated with normal glucose. Data were normalized to GAPDH mRNA. n = 3, *P < 0.05 and **P < 0.01.

Figure 3

STK35 knockdown impairs MCEC proliferation, migration, and tube formation. (A) Western blotting and (B) qRT-PCR analyses showing STK35 was knocked down in MCEC transduced with lentiviral STK35 shRNA. Data were normalized to β-actin and GAPDH, respectively. n = 3. (C) MCEC proliferation (n = 8). (D) Boyden chamber migration assay determining the migratory responses of lentiviral STK35 shRNA or scrambled control shRNA transduced MCEC toward VEGF gradient (n = 6). (E) Matrigel tube formation assay. Tubes formed by MCEC transduced with lentiviral STK35 shRNA or scrambled control shRNA were imaged, and tube length was quantified. n = 6. ****P < 0.0001.

Figure 4

STK35 overexpression restores high glucose-induced impairment of MCEC migration and vascular tube formation. (A,B) Western blotting and qRT-PCR analyses showing transfection with pcDNA3.4-TOPO-STK35 over-expressed STK35 in MCEC. Data normalized to β-actin and GAPDH, respectively. n = 3. (C) Boyden chamber migration assay determining the migratory responses of STK35 shRNA or scrambled control shRNA transduced MCEC toward VEGF gradient in high glucose culture condition. (D) Matrigel tube formation assay. Tubes formed by MCEC transfected with pcDNA3.4-TOPO-STK35 or scramble control plasmid in high glucose culture were imaged, and tube length was quantified. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.001.

Figure 5

Overexpression of STK35 restores high glucose-induced downregulation of pro-angiogenic gene expression. (A) Mouse PCR angiogenesis array showing high glucose downregulates several angiogenic factor expressions in MCEC, including Ctgf and Vegfa, whereas the downregulations were restored by STK35 overexpression. The gene expression data were normalized to normal glucose control and are presented as fold change. (B) Angiogenic gene expression in diabetic human hearts from publicly available RNA sequence data (GSE26887).

Figure 6

AAV9-mediated overexpression of STK35 enhances neovascularization, reduces fibrosis, and improves left ventricular function in diabetic mice. (A) Immunohistochemistry staining shows AAV-9-STK35 expression in diabetic mouse hearts. STK35. (B) Representative images of the capillary vasculature in diabetic mouse heart treated with saline or i.v. injected with scramble AAV9 or AAV9-STK35. Capillary vessels were stained with CD31+ (red), and nuclei were counterstained with DAPI (blue). Graph depicting capillary density as the percentage of CD31+ cells. (C) Trichrome staining and percentage of fibrosis in the diabetic heart from the mice treated with saline or i.v. injected with scramble AAV9 or AAV9-STK35. Scale bar 100 μm. (D) Quantitative analyses of percent ejection fraction (%EF) and percent fractional shortening (%FS). (n = 4); *P < 0.05 and **P < 0.01.