Keywords

Apoptosis, Embryonic stem cells, Fibrosis, Myocardial infarction

Abstract

Transplanted embryonic stem (ES) cells following myocardial infarction (MI) contribute to limited cardiac repair and regeneration with improved function. Therefore novel strategies are still needed to enhance the efficacy by which ES cells differentiate into cardiac cell types and inhibit adverse remodeling in the infarcted myocardium. Our studies evaluate whether genetic manipulation of transplanted ES cells employing miR- 1, a pro-cardiac microRNA, and TIMP-1, an anti-apoptotic and anti-fibrotic protein, will enhance cardiac myocyte differentiation, inhibit native cardiac apoptosis, and reduce fibrosis in the infarcted myocardium. Furthermore, we assess levels of associated pro- (caspase-3, PTEN) and anti-(Akt) apoptotic proteins as well as a pro-fibrotic protein (MMP-9) in the post-MI and cell transplanted heart. microRNAs (miRs) have emerged as critical regulators of various physiological processes including development, differentiation, metabolism, and death. Indeed, miR- 1 plays an integral role in early cardiac development in Drosophila and mice as well as mediates differentiation of cardiac myocytes in vitro. To that end, we generated ES cells overexpressing miR-1 (miR-1-ES cells), transplanted them into the infarcted myocardium, and evaluated their impact on cardiac myocyte differentiation, myocardial repair, and left ventricular dysfunction post-MI. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared to ES cell and culture media transplanted hearts. Assessment of apoptosis revealed overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-Akt and inhibition of caspase-3, PTEN, and superoxide anion production. A significant reduction iv in interstitial and vascular fibrosis was quantified in miR-1-ES and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1- ES cell and ES cell groups was observed. Finally mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls. Our data suggests miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI ultimately giving rise to enhanced cardiac repair, regeneration, and function. Next, we assessed the role of miR-1-ES cells in a chronic model of MI as research has shown that apoptosis occurs not only hours but months following ischemia. 4 weeks following transplantation into the infarcted myocardium, we provide evidence demonstrating reduced cardiac apoptosis in miR-1-ES cell transplanted hearts compared to respective controls. Moreover, we show significant elevation of p-Akt levels and diminished PTEN levels in hearts transplanted with miR-1-ES cells as determined by enzyme-linked immunoassays. Finally, using echocardiography, we reveal mice receiving miR-1-ES cell transplantation post-MI had significantly improved cardiac function compared with animals transplanted with ES cell and culture media. Our data suggests that miR-1, when overexpressed in transplanted ES cells, has the capacity to inhibit apoptosis long term while attenuating contractility loss. In addition to enhancing cardiac-specific donor cell differentiation, improving the efficacy by which stem cells promote cell survival and repair in the host myocardium is imperative in the pursuit of refining and optimizing stem cell therapy. To that end, we overexpressed TIMP-1, an endogenous inhibitor of apoptosis and fibrosis, in ES cells (TIMP-1-ES cells), transplanted them into infarcted myocardium, and evaluated their v impact on adverse cardiac remodeling. Immunofluorescence, TUNEL staining, caspase-3 activity, ELISAs, histology, and echocardiography were used to assess apoptosis, fibrosis, and heart function. Hearts transplanted with TIMP-1-ES cells demonstrated a reduction in apoptosis as well as an increase in p-Akt activity compared with ES cells or culture media controls. Interstitial and vascular fibrosis was significantly decreased in the TIMP-1-ES cell group compared to controls. Furthermore, MMP-9, a key pro-fibrotic protein, was significantly reduced following TIMP-1-ES cell transplantation. Echocardiography data showed fractional shortening and ejection fraction were significantly improved in the TIMP-1-ES cell group compared with respective controls. Our data suggest that transplanted ES cells overexpressing TIMP- 1 attenuate adverse myocardial remodeling and improve cardiac function compared with ES cells. Overall, our data suggest that genetic manipulation of ES cells following transplantation in the infarcted heart enhances cardiac myocyte differentiation, inhibits apoptosis and fibrosis as well as improves cardiac function.

Notes

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Graduation Date

2011

Semester

Summer

Advisor

Singla, Dinender K.

Degree

Doctor of Philosophy (Ph.D.)

College

College of Graduate Studies

Degree Program

Biomedical Sciences

Format

application/pdf

Identifier

CFE0003936

URL

http://purl.fcla.edu/fcla/etd/CFE0003936

Language

English

Release Date

July 2014

Length of Campus-only Access

3 years

Access Status

Doctoral Dissertation (Open Access)

Subjects

Dissertations, Academic -- Graduate Studies, Graduate Studies -- Dissertations, Academic

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