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Intramyocardial delivery of miR-29a improves cardiac function and prevents pathological remodeling following myocardial infarction

Z.B. Ma,1 G.A. Quaife-Ryan,1 J.J. Cooper-White,2 J.E. Hudson1 and E.R. Porrello,1 1School of Biomedical Sciences, University of Queensland, St. Lucia, QLD 4072, Australia and 2Australian Institute for Bioengineering and Nanotechnology, University of Queensland, St. Lucia, QLD 4072, Australia.

MicroRNAs are endogenous, small, noncoding RNA molecules that regulate the expression of protein-coding genes. They have been reported to impact on numerous biological processes and diseases including cardiovascular physiology and diseases (Hudson & Porrello, 2013). Following myocardial infarction (MI), members of the miR-29 family have been shown to be down-regulated in the region of the heart adjacent to the infarct border and repress the expression of extracellular matrix proteins associated with fibrosis (van Rooij et al., 2008). It has been demonstrated that inhibition of miR-29 in vivo induces collagen mRNA expression, and also that over-expression of miR-29 in cultured cardiac fibroblasts reduces collagen expression in vitro (van Rooij et al., 2008). Together these studies indicate that miR-29 could be an anti-fibrotic therapy for heart disease, however, the therapeutic potential of miR-29 in the setting of acute myocardial infarction remains unclear as systemic delivery of microRNA mimics has proven challenging. In order to verify whether miR-29 can improve cardiac function and exert anti-fibrotic effects in vivo, we developed an intramyocardial injection protocol for microRNA mimic delivery to the infarct border zone following permanent occlusion of the left anterior descending (LAD) coronary artery. Briefly, 8-week-old CD-1 male mice were anaesthetized with a combination of ketamine (50mg/kg, i.p.) and metetomidine (10mg/kg i.p.). Following anaesthesia, mice were intubated and ventilated (Harvard Mini-Vent, Type 845, USA) prior to thoracotomy. An incision was made between the 3rd and 4th intercostal space and the LAD was permanently ligated. Synthetic microRNA mimics (QIAGEN) for either miR-29a or a scrambled control sequence were injected into the infarct border zone at a low dose (0.1mg/kg) using a Hamilton syringe (30 gauge). Mice were euthanized by CO2 asphyxiation followed by cervical dislocation and infarcted cardiac tissue samples were separated from non-infarcted regions for further analysis. All protocols were approved by the UQ Animal Ethics Committee. Quantitative PCR (qPCR) analysis showed that our intra-cardiac delivery strategy leads to miR-29a up-regulation specifically in the infarct border zone at day 3 post-MI. Additionally, the miR-29 was functional as it down-regulated its target genes, collagen 1 (Col1a1) and collagen 3 (Col3a1).. We next assessed whether these anti-fibrotic affects could improve cardiac function following MI. Cardiac function was assessed by echocardiography in mice injected with miR-29a or the scrambled sequence control mimic at baseline and at 3, 7, 14 and 21 days post-MI. Several cardiac functional parameters including ejection fraction, fractional shortening and left ventricular internal diameter were significantly improved in the miR-29a treatment group. Furthermore, histological sectioning and staining with Masson’s trichrome demonstrated a marked reduction in infarct size at day 21 post-MI. Moreover, injection with miR-29a prevented the development of cardiac hypertrophy and associated increases in cell size of cardiomyocytes following MI. Interestingly, isolation of different cell types from adult hearts at Day 3 following intramyocardial delivery of microRNA mimics revealed that miR-29a was specifically increased in cardiomyocytes. The present findings suggest that the anti-fibrotic and cardioprotective effects of miR-29a following myocardial infarction may be mediated through an as yet unidentified mechanism involving cardiomyocytes in the infarct border zone.

Hudson JE, Porrello ER. (2013). The non-coding road towards cardiac regeneration. J Cardiovasc Transl Res 6, 909-923.

van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS, Hill JA, Olson EN. (2008). Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci USA 105, 13027-13032.