Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes

Abstract

Rationale: During each beat, cardiac myocytes (CMs) generate the mechanical output necessary for heart function through contractile mechanisms that involve shortening of sarcomeres along myofibrils. Human-induced pluripotent stem cells (hiPSCs) can be differentiated into CMs (hiPSC-CMs) that model cardiac contractile mechanical output more robustly when micropatterned into physiological shapes. Quantifying the mechanical output of these cells enables us to assay cardiac activity in a dish.

Objective: We sought to develop a computational platform that integrates analytic approaches to quantify the mechanical output of single micropatterned hiPSC-CMs from microscopy videos.

Methods and results: We micropatterned single hiPSC-CMs on deformable polyacrylamide substrates containing fluorescent microbeads. We acquired videos of single beating cells, of microbead displacement during contractions, and of fluorescently labeled myofibrils. These videos were independently analyzed to obtain parameters that capture the mechanical output of the imaged single cells. We also developed novel methods to quantify sarcomere length from videos of moving myofibrils and to analyze loss of synchronicity of beating in cells with contractile defects. We tested this computational platform by detecting variations in mechanical output induced by drugs and in cells expressing low levels of myosin-binding protein C.

Conclusions: Our method can measure the cardiac function of single micropatterned hiPSC-CMs and determine contractile parameters that can be used to elucidate mechanisms that underlie variations in CM function. This platform will be amenable to future studies of the effects of mutations and drugs on cardiac function.

Keywords: cardiac myocyte; contractility; sarcomere length; single cell; stem cell.

Figure 1. Registering the contractile mechanical output of micropatterned hiPSC-CMs from microscopy videos

A, Three classes of videos of beating micropatterned hiPSC-CMs were acquired with microscopy: bright-field videos, videos of fluorescent microbeads embedded in the deformable gel substrate, and videos of moving fluorescent myofibrils. B, A region of interest (ROI) was defined around the contour of the cell; movement within this region was analyzed with cross-correlation from bright-field videos. Scale bar: 15 μm. C, Cell average displacement (d) due to the contractile activity of beating within the ROI was quantified and plotted as a function of time, yielding a d-curve. D, Average velocity of displacement (V) within the ROI was calculated from the first derivative of displacement and plotted as a function of time, yielding a V -curve. E, Average displacement (d) of microbeads embedded in the gel substrate was also quantified with cross-correlation from fluorescent videos. An ellipse calculated from the dimension of the ROI was automatically drawn to limit the calculation of displacement to this region. Scale bar: 15 μm. F, d-curve of microbeads plotted as a function of time. G, V-curve of microbeads plotted as a function of time. H, Contractile force (ΣF) estimated with traction force microscopy from the displacement map of microbeads (E) and plotted as a function of time, yielding an F-curve. I, Power (P) was calculated by multiplying ΣF by V of microbeads and plotted as a function of time, yielding a P-curve. J, The regions occupied by sarcomeres within labeled myofibrils were skeletonized. This cell is not the cell shown in B. K, d-curve of myofibrils of cell in J. L, myofibril V-curve for cell in J. Scale bar: 10 μm.

Figure 2. Parameters of contractile cycles are derived from plots

A, We determined three kinetic parameters from V-curves that represent each contractile cycle: V_C is the peak velocity of contraction, V_R is the peak velocity of relaxation, and $\widehat{t}$ (green rectangle) is the time between the peak velocity of contraction and the peak velocity of relaxation. The V-curve was calculated from the displacement (d) of microbeads. B, Variations in the d-curve derived from videos of moving microbeads were analyzed after slowly increasing the concentration of caffeine in the extracellular milieu. We determined maximum values (dashed lines) and minimum values (circles) of d. C, P was also analyzed while increasing the concentration of caffeine. P was calculated by multiplying F by V to determine the peak power of contraction (P_C; upper dashed lines), the peak power of relaxation (P_R; lower dashed lines), and $\widehat{t}$ (rectangles).

Figure 3. Detection of isoproterenol (ISO)-induced variations in the mechanical output of a single micropatterned hiPSC-CM

We used two different concentrations of isoproterenol (0.1 μM and 1 μM) to test the ability of our image analysis platform to detect contractile variations. A, Heat map in which cell-generated traction stresses on the surface of the gel substrate were estimated with traction force microscopy; F was calculated within the region delimited by an ellipse around the cell. D, Myofibrils were fluorescently labeled in the analyzed micropatterned hiPSC-CM (Online Movie V) and imaged for quantification of myofibril movement. B, F-curves and C, P-curves were estimated from videos of moving microbeads acquired before and after the cell was exposed to isoproterenol. D, Myofibrils were fluorescently labeled in the micropatterned hiPSC-CM (Online Movie V) and imaged to quantify myofibril movement. E, d-curves and F, V-curves calculated from videos of moving myofibrils before and after adding isoproterenol. G, Bright-field video of the analyzed single cell. H, d-curves and I, V-curves obtained from brighfield videos of the cell at different isoproterenol concentrations. Scale bar: 10 μm.

Figure 4. Validating the ability of our integrated platform to detect changes in the mechanical output of a single hiPSC-CM induced by omecamtiv mecarbil (OM)

OM was added to the extracellular milieu of a beating micropatterned hiPSC-CM at a concentration of 0.1 μM, and we acquired videos of microbeads in the substrate, of moving myofibrils, and of the cell before and after adding OM. A, Fluorescently labeled myofibrils before adding OM (Online Movie VIII). B, Acute tightening of sarcomeres detected within 10 s after adding OM (Online Movie IX). C, Chronic damage of myofibrils imaged 2 minutes after adding OM (Online Movie X). Red arrows point at the locations of myofibril damages. D, F-curves and E, P-curves were estimated from videos of moving microbeads acquired before adding OM and after acute and chronic exposure. F, d-curves and G, V-curves calculated from videos of moving myofibrils. H, d-curves and I, V-curves obtained from brighfield videos of the cell. Scale bar: 10 μm.

Figure 5. Detection of changes in sarcomere length and sarcomere shortening induced by isoproterenol (ISO) and omecamtiv mecarbil (OM)

We measured sarcomere length (sl) and sarcomere shortening (ss) from videos of myofibrils labeled in the beating micropatterned hiPSC-CMs in Figure 4 and Figure 6. A and E, Box plots of average sarcomere length values calculated for all frames of the analyzed videos (n=53 frames for the cell exposed to ISO (Online Movies V–VII); n=50 frames for the cell exposed to OM (Online Movies VIII–X). B and F, Maximum values of sarcomere length. C and G, minimum values of sarcomere length. D and H, sarcomere shortening calculated by subtracting the minimum values of sarcomere length from the maximum values of sarcomere length. Each point represents a value in the contractile curve of moving sarcomeres. *P<0.05, **P<0.01, and ***P<0.005 by the unpaired Wilcoxon-Mann-Whitney rank-sum test and by Bonferroni’s all-pairs comparison test; n.s., not significant with any test. ANOVA P<0.001 (A–E) and ANOVA P<0.02 (F and G).

Figure 6. Measuring parameters of spatial (a_θ) or temporal (a_δ) asynchronicity in micropatterned hiPSC-CMs harboring homozygous and heterozygous knockout of the gene encoding MYBPC3

a_δ was calculated from the offset times (δ) of intracellular displacement. A, δ was determined for each pixel i within an ROI delimited by the borders of the cell by subtracting the time of each displacement peak for each pixel i by the time of the displacement peak for the average of displacement in the ROI. B, Representative ROI in a bright-field video of a beating micropatterned hiPSC-CM. C, Heat map of δ within the pixels of the ROI. D-G, parameters calculated from micropatterned hiPSC-CMs that lack both copies of the gene encoding MYBPC3 (−/−) or that lack one copy of the gene (+/−). MYBPC3 +/+ cells contain both copies of the gene. D, a_θ. E, a_δ. F, $\widehat{t}$. *P < 0.05, **P<0.01, and ***P<0.005 by the unpaired Wilcoxon-Mann-Whitney rank-sum test and by Bonferroni’s all-pairs comparison test; n.s., not significant with any test. ANOVA P<0.01 (D–F) and ANOVA P<0.04 (G). Scale bar: 15 μm.