Upon remodeling of the ventricle after a provoking stimulus, such as hypertension, connections between adjacent myocytes may need to be "reformatted" to preserve synchronization of excitation of the remodeling heart. In the mammalian heart, the protein connexin forms the gap junctions that allow electrical and chemical signaling communication between neighboring cells. We aim to elucidate if mechanical load, in isolation, potentially changes the expression of connexin 43 (Cx43), the major isoform of connexin family in the ventricle, and its phosphorylation. Cx43 expression levels and contractile function of multicellular rabbit cardiac preparations were assessed in a newly developed in vitro system that allows for the study of the transition of healthy multicellular rabbit myocardium to hypertrophied myocardium. We found that in mechanically loaded cardiac trabeculae, Cx43 levels remained stable for about 12 hours, and then rapidly declined. Phosphorylation at ser-368 declined much faster, being almost absent after 2 hours of high load conditions. No-load conditions did not affect Cx43 levels, nor did phosphorylation at ser-368. The downregulation of Cx43 under mechanical load did not correspond with contractile changes that were observed. Furthermore, blocking paracrine activity of the muscle could only partially prevent the downregulation of Cx43. Additionally, no effect of mechanical loading on the expression of N-cadherin and zonula occludens-1 (ZO-1) was observed, indicating specificity of the connexin response. High mechanical load induced a rapid loss of Cx43 phosphorylation, followed by a decrease in Cx43 protein levels. Paracrine factors are partly responsible for the underlying mechanism of action, while no direct correlation to contractile ability was observed.