MST1 coordinately regulates autophagy and apoptosis in diabetic cardiomyopathy in mice

Abstract

Aims/hypothesis Diabetic cardiomyopathy (DCM) is associated with suppressed autophagy and augmented apoptosis in the heart although the interplay between the two remains elusive. The ability of mammalian sterile 20-like kinase 1 to regulate both autophagy and apoptosis prompted us to investigate it as a possible candidate in the progression of DCM. Methods Wild-type, Mst1 (also known as Stk4) transgenic and Mst1-knockout mice were challenged with streptozotocin to induce experimental diabetes. In addition, cultured neonatal mouse cardiomyocytes were subjected to simulated diabetes to probe mechanisms. Results Mst1 knockout alleviated while Mst1 overexpression aggravated cardiac dysfunction in diabetes. Diabetic Mst1 transgenic mice exhibited decreased LC3 expression and enhanced protein aggregation. In contrast, typical autophagosomes were observed in diabetic Mst1-knockout mice with increased LC3 expression and reduced protein aggregation. Mst1 downregulation promoted autophagic flux as demonstrated by increased LC3-II and decreased p62 expression in the presence of bafilomycin A1. Furthermore, Mst1 overexpression increased, while Mst1 knockout decreased, cardiomyocyte apoptosis both in vivo and in vitro. Coimmunoprecipitation assays showed that Mst1 overexpression promoted Beclin1 binding to B cell lymphoma 2 (Bcl-2) and induced dissociation of Bcl-2 from Bax in diabetic mice. Conversely, Mst1 knockout disrupted the Beclin1-Bcl-2 complex and enhanced the interaction between Bcl-2 and Bax. Conclusions/interpretation Mst1 knockout restores autophagy and protects against apoptosis in cardiomyocytes, en route to the rescue against DCM.

Melatonin activates Parkin translocation and rescues the impaired mitophagy activity of diabetic cardiomyopathy through Mst1 inhibition

“…In agreement with a previous report,39 electron microscopy revealed that the mitochondrial number and volume density per equivalent fields were obviously increased in diabetic mouse hearts compared with non‐diabetic controls. Melatonin markedly reduced the amount and cross section area of mitochondria in diabetic hearts (Figure 1A‐C).…”

“…In its most severe state, diabetic cardiomyopathy is manifested as a ventricular dilation and decreased cardiac output 3, 4, 39. Consistent with our previous results, diabetic mice exhibited left ventricular enlargement and a drop in LVEF.…”

“…Previously, we showed that mitochondrial injury plays a primary role in the development of diabetic cardiomyopathy. The mitochondrial ATP content, diabetic myocardium citrate synthase activity, and activities of complexes I/II/III/IV/V in isolated mitochondria were significantly reduced in the diabetic and high‐glucose‐treated groups 38, 39. Recent studies also indicated that dysfunctional mitochondria accumulation can be detrimental to the function of cardiomyocytes, necessitating removal of damaged organelles for cardiomyocyte survival.…”

“…Cardiomyocytes were adhered to dishes and grown for 48 hours and then transduced with recombinant adenoviruses harbouring the control vector (Ad‐LacZ), Parkin shRNA (Ad‐sh‐Parkin), Mst1 (Ad‐Mst1) or Mst1 shRNA (Ad‐sh‐Mst1) for 48 hours with a MOI of approximately 80‐100:1. Next, cells were cultured in 100 μmol/L melatonin for 4 hours and subjected to high‐glucose culture for 48 hours 39. Finally, cells were incubated for 4 hours with 3‐methyladenine (3‐MA) or chloroquine (CHQ) to inhibit autophagy when necessary 42…”

“…Cells were dyed with MitoTracker Red CMXRos (MTR, 50 nmol/L) for 25 minutes at 37°C to visualize mitochondria and stained with LysoTracker Green DND‐26 (LTG, 100 nmol/L) for 60 minutes to detect lysosomes. Following staining by MTR and fixation with 4% paraformaldehyde, anti‐Parkin and FITC‐conjugated secondary antibodies were used to visualize the Parkin distribution according to previously described protocols 39. Images were visualized under an Olympus FV1000 laser confocal microscope and analysed using ImageJ software.…”