Mechanical response of cardiac microtissues to acute localized injury

Abstract

After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury.NEW & NOTEWORTHY We report an in vitro cardiac injury model that uses a high-powered laser to induce regional cell death and a focal fibrotic response within a human-engineered cardiac microtissue. The model captures the effects of acute injury on tissue response, remodeling, and electromechanical recovery in both the damaged region and surrounding healthy tissue, modeling similar changes to contractile function observed in vivo following myocardial infarction.

Keywords: cardiac fibrosis; cardiac mechanics; cardiac tissue engineering; iPSC-derived cardiomyocytes; organ-on-chip.

Figure 1.

Focal laser injury to engineered cardiac tissues leads to regional cell death. A: brightfield images of CMTs 7 days postseeding before (left) and after (right) laser ablation. White dashed box indicates region of targeted laser injury. Scale bar = 200 μm. B: maximum projections of fluorescence images for phosphatidylserine (apoptosis, magenta) and 7-aminoactinomycin D (7-AAD) (necrosis, cyan) of control (top) and injury (bottom) conditions 2 h postinjury. Scale bar = 200 μm. C: mean intensity of phosphatidylserine of z-stacks in center region of tissues. ****P < 0.0001; n = 18 (control) and 19 (injury). D: mean intensity of 7-AAD of z-stacks in center region of tissues. **P < 0.01; n = 18 (control) and 19 (injury). E: maximum projections of fluorescence imaging of DAPI (blue) and TTN-GFP (green) of control (top) and injury (bottom) tissues 2 h after injury. Insets: white dashed boxes are 25 μm × 25 μm. Scale bar = 100 μm. F: median intensity of DAPI of z-stacks in center region of tissues. **P < 0.01; n = 14 (control) and 15 (injury). G: average of sum of myofibril length over slices from z-stacks of center region of tissues. ****P < 0.0001; n = 13 (control) and 15 (injury). Bar plots represent means ± SE. CMT, cardiac microtissue; GFP, green fluorescent protein.

Figure 2.

Injured tissues demonstrate increased ECM production over time. A: maximum projections of CMT on day 7 postinjury with TTN-GFP (green), vimentin (magenta), and fibronectin (cyan); low-magnification (left), scale bar = 200 μm, black dashed boxes indicate corresponding high-magnification images (right), scale bar = 50 μm. B: mean intensity of vimentin for sum of z-stacks in center region of tissues. ***P < 0.001; n = 6 (injury) and 4 (control). Bar plots represent means ± SD. C: mean intensity of fibronectin for sum of z-stacks in center region of tissues. ***P < 0.001; n = 4 (injury) and 3 (control). D: change in resting tension from before injury over time for control (black) and injured (gray) tissues. ****P < 0.0001, *P < 0.05, n = 26 (injury) and 25 (control). E: Young’s modulus measured in the center region of the tissue at day 0 after injury and day 7 after injury. ***P < 0.001, ****P < 0.0001; n = 9–11. Bar plots represent means ± SE. Brackets above bars indicate significance or nonsignificance in indicated pairwise comparisons. CMT, cardiac microtissue; ECM, extracellular matrix; GFP, green fluorescent protein.

Figure 3.

Electrical function is regained in adjacent tissue but not in the region of injury. A: widefield fluorescence images of control (top) and injured (bottom) CMTs stained with Rhod-3 calcium dye 2 h postinjury showing baseline (left) and peak (right) signal. Dashed boxes indicate regions of interest for traces and analysis in B–E. Center region (cyan) and edge region (magenta). Scale bar = 200 μm. B: example traces for a control and an injury tissue at day 0 after injury in center (cyan) and edge (magenta) regions indicated in A. All traces are normalized to control center region trace. C: normalized intensity amplitudes for traces at day 0 after injury. Normalized to control center region. **P < 0.01, ***P < 0.001; n = 9–10. D: example traces for a control and an injury tissue at day 7 after injury in center (cyan) and edge (magenta) regions indicated in A. All traces are normalized to control center region trace. E: normalized intensity amplitudes for traces at day 7 after injury. Normalized to control center region. **P < 0.01; n = 10. Bar plots represent means ± SE. CMT, cardiac microtissue.

Figure 4.

Force recovery over time corresponds with increased contraction in regions adjacent to injury. A: normalized twitch force before injury and over time after injury for control (black) and injured (gray) tissues normalized to before injury force for each tissue. ***P < 0.001, **P < 0.01; n = 26 (injury) and 25 (control). B: brightfield images of control (top) and injured (bottom) CMT postinjury. Dashed boxes indicate regions of interest for traces and analysis in C–F. Center region (cyan) and adjacent region (magenta). Scale bar = 200 μm. C: example traces of E_xx for the same control (top) and injury (bottom) tissues over time in center region indicated in B (cyan) at day 0 before injury, day 0 after injury, and day 7 after injury. D: E_xx in center regions as defined in B (cyan) for control and injury tissues at day 0 before injury, day 0 after injury, and day 7 after injury. **P < 0.01, ****P < 0.0001; n = 6–13. E: example traces of E_xx for the same control (top) and injury (bottom) tissues over time in adjacent region indicated in B (magenta) at day 0 before injury, day 0 after injury, and day 7 after injury. F: E_xx in adjacent regions as defined in B (magenta) for control and injury tissues at day 0 before injury, day 0 after injury, and day 7 after injury. *P < 0.05, n = 6–13. Bar plots represent means ± SE. CMT, cardiac microtissue.