Establishment and application of an in vitro 3D model of the human heart to facilitate discovery of new therapies for myocardial infarction

Cardiovascular disease (CVD) such as myocardial infarction (MI or heart attack) is a leading cause of death worldwide. MI accounts for 42% deaths of total deaths caused by coronary heart disease (CHD) in Australia. Current in vitro and in vivo models of cardiac injury recapitulate only a few aspects of myocardial ischaemia reperfusion (I/R) injury typical of MI. This leads to a poor translation of research findings from the bench to the bedside. Therefore, there is a need to develop novel 3D models that better mimic myocardial I/R in in vitro. In this study, we have developed a 3D in vitro model of myocardial I/R injury using murine and human cardiac spheroids (CSs) generated in hanging drop culture plates. To induce in vitro I/R injury, CSs were presented to hypoxic (0% O2) and normoxic (5% O2) conditions in a live imaging system. Then, the changes in electrophysiology, cell viability and mRNA expression levels were compared between I/R to control CSs. To further validate our results, these changes were compared with in vivo murine ventricular tissues isolated from control and I/R mice. CSs were also used to study the effects of doxorubicin (DOX) an anticancer agent and fibulin-3 (Fib-3) deficiency. Our results demonstrated that following 24 h standardization with 5% O2, CSs become hypoxic following 20 h of their incubation with 0% O2 and gets reoxygenated after additional 17 h incubation with 5% O2. Both I/R injury and DOX treatment significantly increases the cell toxicity in treated versus control CSs. Our 3D rendering analyses confirmed that I/R injury causes more cell toxicity than DOX treatment. A complete cessation of contractional frequency and fractional shortening was observed in I/R CSs compared to control and DOX CSs. mRNA expression of cardiac-damage related genes showed similar response to I/R injury in in vivo and in vitro. However, the significant differences were observed in mRNA expression between DOX and I/R CSs. On the other hand, Fib-3 deficient CSs showed significant decrease in endothelial network formation in comparison to wild type CSs. Fib-3 KO CSs retain their electrophysiological properties even after I/R injury and were found to be resistant to I/R injury. Overall, this thesis has presented for the first time a direct comparison between in vivo myocardial I/R injury and in vitro CSs. Our results demonstrated that in vitro CSs recapitulated major features typical of the in vivo MI and drug induced cardiac damages, such as adapting intracellular changes in O2 concentration and incubation with cardiotoxic drug, mimicking the contraction frequency and fractional shortening and changes in mRNA expression levels for genes. Together with this we have also demonstrated that Fib-3 deficiency in CSs showed inhibition of I/R injury, I/R-related cardiac fibrosis and promotes progression of angiogenesis.