Microtubule affinity-regulating kinase 4 exacerbates diabetic cardiomyopathy by inhibiting UNC-51-like kinase 1-mediated mitochondrial autophagy

Figure 1 Microtubule affinity-regulating kinase 4 was upregulated in diabetic cardiomyopathy models. A and B: Microtubule affinity-regulating kinase 4 protein levels in diabetic cardiomyopathy model mice; C and D: Microtubule affinity-regulating kinase 4 protein levels in cardiomyocytes stimulated with 200 μM palmitic acid and 33.3 mmol/L glucose. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. Con: Control; DCM: Diabetic cardiomyopathy; MARK4: Microtubule affinity-regulating kinase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HG: High glucose.

Figure 2 Effect of microtubule affinity-regulating kinase 4 knockdown on mitophagy in mice with diabetic cardiomyopathy. A and B: Evaluation of microtubule affinity-regulating kinase 4 knockdown efficiency in diabetic cardiomyopathy model mice via western blotting quantification; C-G: Western blotting quantification of p62, Parkin, Beclin1, and microtubule-associated protein 1 light chain 3B. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. DCM: Diabetic cardiomyopathy; MARK4: Microtubule affinity-regulating kinase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; sh: Small hairpin; Con: Control; NC: Negative control; LC3B: Microtubule-associated protein 1 light chain 3B; AAV9: Adeno-associated virus 9.

Figure 3 Impact of microtubule affinity-regulating kinase 4 knockdown on myocardial injury and fibrosis, blood glucose, body weight, and cardiac function and structure in diabetic cardiomyopathy model mice. A: Hematoxylin and eosin staining of left ventricular tissue (× 200); B: Masson’s staining of left ventricular tissue (× 200); C: Blood glucose levels; D: Body weights; E: The left ventricular ejection fractions; F: The left ventricular short axis shortening rates; G: Left ventricular end-diastolic diameters; H: Left ventricular end-systolic diameters. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. HE: Hematoxylin and eosin; Con: Control; DCM: Diabetic cardiomyopathy; MARK4: Microtubule affinity-regulating kinase 4; sh: Small hairpin; NC: Negative control; LVEF: Left ventricular ejection fractions; LVFS: Left ventricular shortening fraction; LVIDd: Left ventricular end-diastolic diameters; LVIDs: Left ventricular end-systolic diameter; AAV9: Adeno-associated virus 9.

Figure 4 Effect of microtubule affinity-regulating kinase 4 knockdown on mitophagy and mitochondrial membrane potentials in cardiomyocytes stimulated with high glucose and palmitic acid. A and B: Western blotting and quantification of microtubule affinity-regulating kinase 4 knockdown efficiency; C-G: Western blotting and quantification of p62, Parkin, Beclin1, and microtubule-associated protein 1 light chain 3B proteins; H: Mitophagy in the groups (scale bar: 2 μm); I: Changes in the mitochondrial membrane potential (scale bar: 50 μm); J: Analysis of mitophagy via electron microscopy; K: Statistical analysis of the mitochondrial membrane potential. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. ShRNA: Small hairpin RNA; MARK4: Microtubule affinity-regulating kinase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; Con: Control; LC3B: Microtubule-associated protein 1 light chain 3B; sh: Small hairpin; HG: High glucose; NC: Negative control.

Figure 5 Transcriptomic results and verification of the effects of microtubule affinity-regulating kinase 4 knockdown. A: Downregulated genes between the control + small hairpin (sh)-negative control (NC) and high glucose (HG) + sh-NC groups and upregulated genes between the HG + sh-NC and HG + sh-microtubule affinity-regulating kinase 4 groups are shown as a cross-Venn diagram; B: Kyoto Encyclopedia of Genes and Genomes classification of differentially expressed genes; C: Kyoto Encyclopedia of Genes and Genomes bubble diagram of the differentially expressed genes; D: Heatmap of differentially expressed genes; E: RNA-seq results of UNC-51-like kinase 1; F-H: Consistency of the western blotting, quantitative real-time PCR, and RNA-seq results. Values are shown as mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. KEGG: Kyoto Encyclopedia of Genes and Genomes; ULK1: UNC-51-like kinase 1; Con: Control; NC: Negative control; sh: Small hairpin; MARK4: Microtubule affinity-regulating kinase 4; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; HG: High glucose.

Figure 6 Impact of UNC-51-like kinase 1 upregulation on the mitophagy-associated proteins and the mitochondrial membrane potential in diabetic cardiomyopathy. A and B: UNC-51-like kinase 1 protein levels in diabetic cardiomyopathy (DCM) mice; C-G: Western blotting quantification of Beclin1, p62, Parkin, and microtubule-associated protein 1 light chain 3B proteins in DCM mice; H-M: Western blotting quantification of mitophagy-associated proteins (UNC-51-like kinase 1, p62, Parkin, Beclin1, and microtubule-associated protein 1 light chain 3B) in DCM cell models; N: Electron micrographs of mitophagy (scale bar: 2 μm); O: Changes in the mitochondrial membrane potential (scale bar: 50 μm); P: Statistical analysis of mitophagy from electron micrographs; Q: Statistical analysis of the mitochondrial membrane potential. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. Con: Control; DCM: Diabetic cardiomyopathy; ULK1: UNC-51-like kinase 1; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; OE: Overexpress; NC: Negative control; LC3B: Microtubule-associated protein 1 light chain 3B; HG: High glucose; EV: Empty vector; EM: Electron microscopy.

Figure 7 Impact of microtubule affinity-regulating kinase 4 knockdown on mitophagy was counteracted by UNC-51-like kinase 1 knockdown. A-G: Western blotting quantification of microtubule affinity-regulating kinase 4, UNC-51-like kinase 1, p62, Parkin, Beclin1, and microtubule-associated protein 1 light chain 3B proteins. Values are mean ± SD (n = 4). ^aP < 0.05, and ^bP < 0.01. Sh: Small hairpin; NC: Negative control; HG: High glucose; MARK4: Microtubule affinity-regulating kinase 4; ULK1: UNC-51-like kinase 1; LC3B: Microtubule-associated protein 1 light chain 3B; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; NS: Not significance.

Figure 8 Interaction between microtubule affinity-regulating kinase 4 and microtubule-associated protein 1 light chain 3B. A: Several proteins that interact with microtubule affinity-regulating kinase 4; B: Co-immunoprecipitation verification of the interaction between microtubule affinity-regulating kinase 4 and microtubule-associated protein 1 light chain 3B in cardiomyocytes (n = 4). Results from triplicate experiments. MARK4: Microtubule affinity-regulating kinase 4; LC3B: Microtubule-associated protein 1 light chain 3B; IP: Immunoprecipitation.