Embryonic stem cells overexpressing Pitx2c engraft in infarcted myocardium and improve cardiac function

Abstract

This study investigated the effects on cardiomyocyte differentiation of embryonic stem cells by the overexpression of the transcription factor, Pitx2c, and examined the effects of transplantation of these differentiated cells on cardiac function in a mouse model of myocardial infarction. Pitx2c overexpressing embryonic stem cells were characterized for cardiac differentiation by immunocytochemistry, RNA analysis, and electrophysiology. Differentiated cells were transplanted by directed injection into the infarcted murine myocardium and functional measurements of blood pressure, contractility, and relaxation were performed. Histochemistry and FISH analysis performed on these mice confirmed the engraftment and cardiac nature of the transplanted cells. Pitx2c overexpressing embryonic stem cells robustly differentiated into spontaneously contracting cells which acquired cardiac protein markers and exhibited action potentials resembling that of cardiomyocytes. These cells could also be synchronized to an external pacemaker. Significant improvements (P < 0.01) in blood pressure (56%), contractility (57%), and relaxation (59%) were observed in infarcted mice with transplants of these differentiated cells but not in mice which were transplanted with control cells. The Pitx2c overexpressing cells secrete paracrine factors which when adsorbed onto a heparinated gel and injected into the infarcted myocardium produce a comparable and significant (P < 0.01) functional recovery. Pitx2c overexpression is a valuable method for producing cardiomyocytes from embryonic stem cells, and transplantation of these cardiomyocytes into infracted myocardium restores cardiac function through multiple mechanisms.

Figure 1. Expression of pitx2c induces cardiomyocyte differentiation in ES cells

ES cells stably transfected with either empty vector or pitx2c were seeded for EB formation at 2-5 × 10⁵ cells/ml and cultured for 8 days. EBs were dissociated and cultured for an additional 5 days. Spontaneously beating clusters were observed 2 days after plating the pitx2 transfected cells, but only rarely in the empty vector transfected cells. Empty vector transfected cells (A) or pitx2c transfected cells (B) were fixed and stained for cardiac troponin-T (green) and counterstained with Hoechst to visualize nuclei. Virtually every partially dissociated EB contained clusters of cardiac Troponin-T positive cells. Cells in the pitx2c transfected cells were positive for the transcription factors GATA-4 (C) and Nkx2.5 (D). High magnification of a cardiac cluster in the pitx2c transfected cells showing nuclear localization of Nkx2.5 (E). Cardiomyocyte like cells tended to appear in clusters of cells intimately associated with one another. Quantification of cardiac Troponin T positive EBs in different clones of pitx2 transfected cells shows a significant (* and ** p<0.05 compared to control group by ANOVA) increase compared to control empty vector transfected cells (F).

Figure 2. Cardiac specific transcripts are upregulated in pitx2 transfected cells

Real time quantitative PCR shows an increase in the expression of the mRNA of transcription factors like Nkx2.5, Mef2c,GATA4 and Tbx5. mRNA of structural proteins like MLC2a, MLC2v and Cardiac Troponin I are also upregulated (A). RT-PCR of various cardiac specific transcripts shows a similar trend in the pitx2 transfected cells compared to control cells.Oct3/4 transcripts decrease with differentiation (B).

Figure 3. Ca2⁺ Transients and Ventricular-like Action Potentials

Panel A shows a 2-D image of pitx2 expressing cells in an 8 day EB after loading with fluo-4AM. The red line shows the site of repetitive scanning used to record linescan images in subsequent panels. The scale of the image is 340μ square. Panel B shows a linescan image recorded by repetitive scanning at site indicated by the red line in Panel A at a rate of 2msec/line. Spontaneous Ca2+ transients were activated simultaneously at a number of cells throughout the EB. The integrated profile across the entire image is shown above. When field stimulation (2Hz) was initiated (Panel C), Ca2+ transients were activated in response to each stimulus, indicating the ability of these same cells within the EB to react to external stimulation and the likely presence of sarcolemmal ion channels. Panel D shows a ventricular-like action potential in a cell from an EB induced by pitx2. The action potential closely resembles that reported by others to be characteristic of left ventricular myocytes from neonatal mouse [29]. Action potentials were recorded by delivery of a current pulse through the microelectrode for 2msec at 1.2X threshold at 2Hz.

Figure 4. Pitx2 and Notch Signaling are Antagonistic

Cross-talk between notch and pitx2 signaling was tested by observing the regulation of two notch regulated promoters, HES-1 and myoD. (A) HES-1 promoter activity was found to be activated several fold by expression of Nicd (** p<0.05) Pitx2c expression had no effect on this promoter. However, cotransfection of Nicd with pitx2c blocked the transactivation of the HES1 promoter by Nicd. (B) Regulation of the myoD promoter by pitx2c and Nicd. Pitx2c significantly stimulated the myoD promoter. Transfection of Nicd had no significant effect. Cotransfection of pitx2c with Nicd significantly inhibited the activation of the myoD promoter by pitx2c (* p<0.05). (C-F) L cells were transfected with the Nkx2.5 heart specific enhancer driving β-galactosidase expression with empty vector (C), HES6 (D), pitx2c (E), or HES6 and pitx2c (F). L-cells were incubated overnight after the transfection, fixed with glutaraldehyde and then stained for β-galactosidase. Both pitx2c and HES6 transfected cells showed elevated β-galactosidase staining. However, transfection of both pitx2c and HES6 resulted in a synergistic increase in β-galactosidase staining.

Figure 5. Pitx2 overexpressing ES cells contribute to the functional recovery of infarcted mice

ES cells overexpressing pitx2 were differentiated into embryoid bodies and transplanted into infarcted mice. Functional measurements were done to assess Blood pressure, Contractility and Relaxation (A,B,C) one month after cell transplantation (* p<0.05 compared to all other groups by ANOVA, R1-ES cell group and MI control group do not significantly differ from each other). (D) The relatively acellular nature of the infarct is evident at the junction of the infarct (black arrowhead) in an H&E stain, and the transplanted cells can be seen as in laminar arrangement (white arrow) or as rosettes (black arrow). (E) The same heart was stained for cardiac differentiation of transplanted cells with cardiac troponin T in the scar (20x). (F) The presence of transplanted cells was confirmed by FISH analysis of the Y chromosome with an FITC conjugated probe (white arrows) and cardiac differentiation by cardiac troponin T immunoflourescence (red) (40x).

Figure 6. Proposed model of Pitx2 induced cardiomyocyte differentiation

Notch signaling induces intramembrane proteolysis to release its intracellular domain (Nicd) which activates transcription factors such as Hes1 that have inhibitory activity on Nkx2.5’s and other cardiac specific genes. Pitx2 expression inhibits the ability of Nicd to activate the HES1 promoter. In addition to inhibiting notch signaling, pitx2 also induces the expression of the early cardiac transcription factor Nkx2.5.