Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, C010

Oral Communications

Intra-tissue specific gene and miRNA changes in response to myocardial infarction in fetal compared to adolescent sheep

M. C. Lock1, J. R. Darby1, J. Soo1, R. L. Tellam1, D. A. Brooks1, E. R. Porrello2, M. Seed3, J. Selvanayagam4, C. K. Macgowan3, M. Keller-Wood5, J. L. Morrison1

1. School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia, Australia. 2. Murdoch Childrens Research Institute, Melbourne, Victoria, Australia. 3. The Hospital for Sick Children, Toronto, Ontario, Canada. 4. Flinders University, Adelaide, South Australia, Australia. 5. School of Pharmacodynamics, University of Florida, Gainesville, Florida, United States.

  • Figure 1. MTS assay for H9c2 cardiomyoblast proliferation after treatment with miRNA inhibitors. Nx, normoxia; LG, low glucose; Hx, hypoxia.

Animal models indicate that there are critical molecular mechanisms that can be activated to induce myocardial repair at specific points in development. Neonatal mice and fetal sheep have the capacity to regenerate heart tissue after myocardial infarction (MI). We investigated the immediate response to injury within specific regions of the infarcted cardiac tissue in fetal and postnatal hearts to characterise the differences between proliferative and quiescent hearts in a large animal model. We used sheep, which like humans complete most cardiomyocyte maturation before birth, to model the effect of age on the molecular response to cardiac damage by ligating the second diagonal of the left anterior descending coronary artery. Surgery was performed on fetuses (at ~102 days gestation when all cardiomyocytes are mononucleated and proliferative) and postnatal sheep (at 6 months of age when all cardiomyocytes contribute to heart growth by hypertrophy). Ewes underwent surgery using general anesthesia induced by the intravenous injection of diazepam (0.3mg/kg) and ketamine (7mg/kg) and maintained with inhalation of isoflurane (1-2%) in oxygen and received antibiotics and analgesia post-operatively. Total RNA was extracted for qRT-PCR, gene array and miRNA array analyses and immunohistochemistry was performed on fixed sections. Specific gene and miRNA targets were filtered from significantly upregulated or downregulated probes in the infract area compared with sham tissue and remote-zone tissue. Identified target miRNAs were tested in vitro in H9c2 cardiomyoblasts under hypoxic and low substrate conditions to simulate MI. Proliferation of H9c2 cells was assessed using MTS assay and Ki67 staining. Gene ontology analysis revealed several enriched pathways that were differentially regulated in the fetal compared to adolescent sheep that appear to be important in the immediate response to infarction. These included; E2F Targets, Cardiac Muscle Hypertrophy, WNT and MTORC1 signaling. Specific miRNAs that were oppositely regulated in the fetal compared to adolescent sheep were identified based on their expression profile across the infarcted tissue regions. miR-150 and miR-1538 were upregulated in the adolescent infarct but downregulated in the fetal infarct compared with remote-zone tissue. In H9c2 cell culture, miR-558 and miR-1538 inhibitors significantly increased cell proliferation in normoxia and hypoxia (Figure 1). These results indicate that fetal hearts have a ‘resistance' to cardiac damage. This tissue-wide response indicates a prioritisation within the fetal heart to increase proliferation/replacement of cardiomyocytes within the border and remote-zones, and retain a normal developmental pattern in the infarct area. Further investigation of specific gene pathways and miRNAs that are uniquely modulated in the fetal heart after infarction may allow for development of new targeted therapies of human heart disease.

Where applicable, experiments conform with Society ethical requirements