Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes

Abstract

In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1-80 microm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13 microm, and the average depth in the range from 140 to 700 nm (AFM). The surfaces with abrasions 13 microm wide and 700 nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1 ms duration, 1 Hz, 2.3 or 4.6 V/cm) was initiated 24 h after cell seeding and maintained for additional 72 h. The cover slips were positioned between the carbon rod electrodes such that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability. The presence of a well-developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that (i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation, (ii) electrical field stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular to the field lines and (iii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. The orientation and elongation response of cardiomyocytes was completely abolished by inhibition of actin polymerization (Cytochalasin D) and only partially by inhibition of phosphatidyl-inositol 3 kinase (PI3K) pathway (LY294002).

Figure 1. Abraded surfaces

A) SEM images of the abraded surfaces B) AFM images of the abraded surfaces C) Abrasion width as estimated by AFM. D) Abrasion depth as estimated by AFM. E) Abrasion density as estimated by AFM. The letters a, b, c, d and e represent abrasions made by lapping paper of grain size 1, 9, 12, 40 and 80μm respectively. (p<0.05, Dunn's test).

Figure 2. Elongation and orientation of fibroblasts (A-C) and cardiomyocytes (D-E) cultivated on abraded and non-abraded surfaces

Abraded surfaces were created by lapping paper with grain sizes of 9μm, 40μm and 80μm respectively (b, d, e). Aspect ratio is defined as the ratio between the long and the short axis of the cell. Orientation angle is defined as the angle between the long axis of the cell and the direction of abrasion. Arrows indicate direction of abrasion. For cells cultured on non-abraded surfaces orientation angle is measured with respect to the horizontal axis of the image. A) Giemsa staining of fibroblasts. (Scale bar: 100μm for b, d, e and 50μm for non-abraded). B) Elongation of fibroblasts as measured by the aspect ratio. C) Alignment of fibroblasts as measured by the orientation angle (box plots). D) Live/dead (green/red) staining of cardiomyocytes. (Scale bar: 100μm) E) Elongation of cardiomyocytes as measured by the aspect ratio. F) Alignment of cardiomyocytes as measured by the orientation angle (box plots).

Figure 3. Interactive effects of topographical cues and electrical field stimulation on fibroblasts (A,B) and cardiomyocytes (C,D)

Electrical field stimulation using square pulses 1ms duration, 1Hz and 2.3V/cm or 4.6V/cm was initiated 24hr after cell seeding and maintained for additional 72hr. Abraded surfaces were placed between the electrodes so that the abrasions were either perpendicular or parallel to the field lines. A) Elongation of fibroblasts as measured by the aspect ratio. B) Alignment of fibroblasts as measured by the orientation angle (box plots). C) Elongation of cardiomyocytes as measured by the aspect ratio. D) Alignment of cardiomyocytes as measured by the orientation angle (box plots). Total N=2-3 independent samples (cover slips) per group; 30-90 cells were analysed per group. (p<0.05 was considered significant). *significantly different than non-abraded surface at identical stimulation voltage.

Figure 4. Immunostaining for cardiac Troponin I of cardiomyocytes cultivated in the presence of electrical field stimulation on abraded surfaces

A) Orientation and morphology of cells expressing cardiac troponin I. Scale bar 100μm. B) Higher magnification images indicate the presence of contractile apparatus (cross-striations). Scale bar 20μm.

Figure 5. Actin cytoskeleton in cardiomyocytes cultivated in the presence of electrical field stimulation on abraded surfaces

A) Orientation and morphology of cells stained with phalloidin-TRITC. Scale bar 100μm. B) Higher magnification images indicate orientation of actin microfilaments. Scale bar 10μm.

Figure 6. Effects of pharmacological agents on elongation (A, C) and orientation (B, D) of cardiomyocytes cultivated on non-abraded (A, B) and abraded (C, D) surfaces

A) Elongation of cardiomyocytes on non-abraded surfaces B) Alignment of cardiomyocytes on non-abraded surfaces C) Elongation of cardiomyocytes on abrasions placed parallel to the field lines D) Alignment of cardiomyocytes on abrasions placed parallel to the field lines N=2 -3 independent samples per treatment; 30 cells were analyzed per treatment. (p<0.05 was considered significant). *significantly different than non-abraded surface at identical stimulation voltage.

Figure 7. Immunostaining for cardiac Troponin I of cardiomyocytes treated with pharmacologic agents

Main panels indicate the orientation and morphology of cells expressing cardiac troponin I. Scale bar 100μm. Insets: Higher magnification images indicate the presence of contractile apparatus (cross-striations) in No-drug and LY294002 group (arrows). Scale bar 20μm.

Figure 8. Actin cytoskeleton in cardiomyocytes treated with pharmacologic agents

Main panels indicate the orientation and morphology of cells expressing stained with phalloidin-TRITC. Scale bar 100μm. Insets: Higher magnification images indicate orientation of actin microfilaments. Scale bar 20μm.