Structural changes in heart and kidney tissue in response to modification of substrate availability

Thesis

Introduction

Diabetes is a disease of impaired metabolism caused by a disparity between the insulin secretion necessary to maintain normoglycaemia and the ability of β-cells to secrete insulin. In all types of diabetes this results in hyperglycaemia which may lead to tissue damage. This project investigates the structural changes in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) as a result of maturation and insulin resistance.

Separately, this project also briefly investigates the effects of hyperglycaemia on the structure of glomeruli in a mouse model of diabetic nephropathy (βV59M).

Methods

hiPSC-CM in monolayer and in 3D as engineered heart tissue (EHT) were treated with oleic acid to increase maturity, and with palmitic acid and high insulin to induce insulin resistance. Previous work in the Carr group had shown that both treatment with oleic acid and culture as EHT induced a switch from glycolysis to oxidative metabolism and induction of insulin resistance impaired glucose metabolism. Transmission Electron Microscopy (TEM) was used to produce high magnification images which were assessed for quantitative analysis of muscle, nuclear area and mitochondrial area and structure. Statistical analysis was performed using R.

Sections of diabetic and control mouse kidney were stained with periodic acid Schiff’s assay (PAS), and confocal microscopy was used to observe mesangial matrix expansion. The glomeruli in these images were graded to produce quantitative data.

Results

Nuclear area, muscle area, as well as mitochondrial size, number and total area, were all increased by culturing hiPSC-CM in EHT. However only mitochondrial size and muscle area were further increased by oleic acid treatment. Insulin resistance had a significant effect only on average muscle area and on the distribution of the data for mitochondrial size and nuclear area.

There was a significant increase in mesangial matrix expansion in 12-week diabetic mice compared to non-diabetic mice.

Conclusions

Transmission electron microscopy images of hiPSC-CM showed changes in muscle area and mitochondrial morphology in hiPSC-CM cultured in 3D as EHT. Induction of insulin resistance caused increased disorder of mitochondria. Image analysis using FIJI and statistical analysis using R enabled some conclusions to be supported quantitatively. Orientation of slicing and separating mitochondrial populations would be important aspects of future work.

The βV59M mouse model shows significant indications of mesangial matrix expansion. Further work should include identifying the cause of the PAS staining using glycogen anti-bodies.

Authors: Brisk, K

• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: electron microscopy; engineered heart tissue; cardomyocytes; mitochondria