Cell contractility effects on fibrillar collagen in developing and mature hearts

Introduction: Tissue fibrosis causes almost half the deaths in the developed world, and cardiac fibrosis alone leads to heart failure in >6-million U.S. adults. Collagen-rich extracellular matrix (ECM) accumulates in fibrosis and both stiffens hearts and compromises beating. Current treatment efforts for progressive cardiac fibrosis primarily focus on ECM rather than the cell forces that conceivably regulate ECM levels. Cardiac myosin-II is one possible target based on recent studies, but the Rho kinases (ROCK) expressed in varying amounts by many cell-types including fibroblasts seem to also promote expression of ECM genes possibly via acto-myosin contractility. A new ROCK inhibitor (ROCKi) was FDA-approved recently for chronic Graft-versus-host disease (cGVHD) that affects many organs in recipients of stem-cell graft transplants. To visualize any effects of such contractility inhibitors on heart function and ECM collagen levels, we studied beating hearts from chick embryos. To track real-time kinetics of fibrillar collagen during perturbations with various myosin-II inhibitors, we used second harmonic imaging (SHG).

Materials and

Methods: For studying cellular contractility-based ECM effects and heart functions, we selected isolated embryonic chick hearts that beat autonomously (without any electrical stimulation) in contrast to mature hearts and comprises of different cell-types namely cardiomyocytes, fibroblasts as revealed by single-cell RNA sequencing. Acto-myosin based cellular contractility was perturbed by several small-molecule inhibitors namely ROCK-2 (Belumosudil) and Blebbistatin (a pan myosin-II inhibitor) whereas a pan matrix metalloproteinase inhibitor (MMPi) and collagenases were used to perturb ECM protein levels. The heart function, defined by beating strain magnitude, was accessed by means of wide-field time-lapse imaging during above mentioned perturbations in cellular-contractility and ECM protein levels (Fig.1) whereas second harmonic imaging (SHG) was used to assess the real-time kinetics of fibrillar collagen levels in the tissues.

Results, Conclusions, and Discussions: Exposure of the heart tissues to ROCKi resulted in heart beating strain suppression similar to that of Blebbistatin treated hearts (Fig.2). Given that ROCK-2 is primarily expressed by fibroblastic cells in the heart and cardiomyocytes express extremely low levels, the slower rate of beating strain inhibition during ROCKi treatments (~30 mins) compared to blebbistatin treated hearts ( < 15 mins) likely results from cell-type specific distribution of ROCK-2 within the heart tissue while blebbistatin targets several myosin types abundant in most cell-types present in the heart. Considering the propagation of contractile wave among sparse immature cardiomyocytes within the heart relies on robust cell-ECM interactions, ROCK-2 inhibition likely promotes ECM catabolism and thus, disables contractile wave propagation in the heart. Such effects of altered ECM turnover on beating strain are evident during the presence of pan-MMPi when tissue function recovery is observed following drug-washouts (Fig.2). For various mature mice tissues and beating embryonic chick hearts, we find collagen-sensitive second harmonic generation (SHG) image intensity scales non-linearly versus tissue stiffness, aligning well with the results from cellularized gels of collagen. Chick hearts beating at ~5% strain maintain collagen levels until their contractile strain is suppressed by myosin-II inhibition and endogenous matrix metalloproteinases then degrade collagens within ~30-60 minutes based on SHG (Fig.3). Our results have potential to deepen the understanding of cell-based contractile forces on ECM in development, adaptation, and disease. The modulation of cellular forces during pathologies via targeting ROCK-pathway or other means might help target fibroblastic cells over other cell-types. Given that fibroblasts are primarily responsible for synthesis and degradation of a tissue ECM, such targeting of fibroblasts might help restore “normal" ECM and function of fibrotic hearts and other tissues.

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