Abstract Body: Introduction: Extracellular matrix (ECM) collagen levels accumulate during development and confer rigidity to tissues while paralleling increases in nuclear Lamin-A levels. However, the impact of physical stress on turnover of fibrous collagen and filamentous Lamin-A remains understudied in living tissues including heart despite its fundamental applications in tissue maturation, regeneration as well as clinical contractility modulators. We hypothesize that degradation of collagen-fibers and Lamin-A filaments is strain-suppressed in live mature and developing tissues.
Materials and Methods: To track kinetics of collagen-matrix over ~hours timescale, live imaging by label-free second harmonic generation (SHG) is applied to fresh heart tissue from adult humans (Fig.1-i), normal or diseased mice, and chick embryos under acute perturbations and combined with paired measurements of tissue rigidity. Our acute perturbations target cellular-contractility and collagen-fiber integrity (Fig.1-ii) with or without inhibitors of endogenous matrix metalloproteinases (MMPs). Immunostaining assessed ECM collagen-I, cytoskeletal Actin and nuclear Lamin-A while mass-spectrometry (MS) complemented some of the SHG measurements. Additionally, a custom-designed mechano-bioreactor used cell-free mouse tendon model under external strain plus exogenous purified MMP-1 or collagenases to show connective-tissue changes while micropipette aspiration measured tissue rigidity (i.e., elasticity, viscosity, and plasticity).
Results and Discussion: Parsing backward (bSHG) and forward (fSHG) signals show myosin-II inhibition in human myocardium drives losses in cardiomyocyte-striation and collagen-fiber density, with nuclear wrinkling via Lamin-A staining (Fig.1-iii). Exogenous collagenase decreases collagen-fibers and tissue rigidity, and eventually, cardiomyocyte-striation and Lamin-A filaments. Developing hearts yield similar results based on MS, with rescue of collagen-fibers and Lamin-A upon inhibiting MMPs. Lamin-A depleted hearts confirm a bidirectional coupling with collagen-fibers and tissue rigidity. Contractile strains of ~5% are relaxed by human and chick heart perturbations, and applying ~5% strain to tendon showed strain-suppressed collagen-fiber degradation despite a >1000-fold higher collagen-fiber level and stiffness than heart.
Conclusion: Our results support generality of the hypothesis for collagen-fiber and Lamin-A filament degradation by MMPs is suppressed by ~5% strain.