Hydrostatic stress regulates tissue compaction, polarity, and matrix stiffness in the developing atrioventricular valve

The morphogenesis of the amorphous cardiac cushions into thin fibrous leaflets requires controlled cellular compaction and elongation in an intensifying mechanical environment. We have previously reported that as atrioventricular (AV) valve maturation progresses, interstitial cells respond differently to cyclic mechanical loading through signaling of small GTPases RhoA and Rac1. RhoA activation was found natively in early (HH25) AV cushions and promotes a myofibroblastic phenotype though up regulation of alpha smooth muscle actin (αSMA). As the AV valves mature (up to HH40), we found a transition to Rac1 signaling and a higher degree of compaction. However, it is not established how increasing cellular compaction behavior is balanced with increasing matrix collagen density to achieve a compacted and correctly sized valve. To examine the effect of both compressive and tensile hydrostatic pressure on tissue explants that are natively dense, we cultured atrioventricular (AV) cushions from HH25, HH30 and HH40 chick embryos in hypotonic (tensile) and hypertonic (compressive) medias. Cushions isolated from the AV canal spontaneously round and compact to near 50% of their original size over 24 hours in hanging drop culture ( Fig 1A). Interestingly, as cushion maturity progresses, the inherently greater matrix density is not represented in the compaction behavior, and is instead similar across stages. On the other hand, mature cushions maintain their shape fidelity, shrinking in thickness while maintaining planar morphology ( Fig 2). Tensile stress significantly increased compaction compared to normotensive conditions, while compressive stress decreased compaction. In the hypertonic condition, the 18hr compaction rate is 2‐fold less than the control, indicating that the coherent cellular compaction response at this time was abolished. Further, through mechanical testing with micropipette aspiration, we determined that tissue cultured under compressive stress is stiffer compared to controls ( Fig 1B). Reduced compaction combined with higher stiffness supports tissue growth in the presence of compressive stress. Compaction was found to be ROCK independent, suggesting a greater role for Rac1 in cushion remodeling. Increased compaction was associated with an up regulation of αSMAs and Cyclin b, supporting to increased contractility and proliferation. Compressive stress leads to a reduction in compaction, with an associated increase in TGFb3 expression. This reduction in compaction suggests that compressive stress may promote growth, demonstrating how a mechanical parameter can influence the balance of contractility and structural maintenance. Together, our studies demonstrate that varying the directionality of mechanical stimuli from a tensile to a compressive load can predictably direct tissue morphogenesis. Support or Funding Information NIH RO1 HL110328, HL1287845,NSF CBET 0955‐172,NSF Graduate Research Fellowship(A) A greater extent of compaction (lower final area to original area ratio) is observed for tensile loading, while a lesser extent of compaction is seen for compressive loading. (B) The resulting mechanical stiffness (strain energy density) increases based on the stress treatment, increasing from tensile to compressive. The inset shows the calculation of strain energy density.(A) HH25 cushion, imaged during pipette aspiration, is rounded on all sides, forming a spherical shape (B) HH30 cushion shows preservation of thinned axis. Insets show general rounded shapes in both cases when looking from the top.