Mechanical stress is regarded as an important stimulus for the hypertrophic response and cardiomyocyte z-disc proteins are thought to be essential in this process. We have previously shown that syndecan-4, a transmembrane proteoglycan connecting the extracellular matrix to the cytoskeleton in the z-disc, is essential for development of concentric hypertrophy following pressure overload. We here propose that syndecan-4 is a cardiomyocyte mechanosensor regulating pro-hypertrophic calcineurin-Nuclear Factor of Activated T-cell (CnA-NFAT) signaling.
Syndecan-4 mRNA and protein levels were increased 1.9- and 1.7-fold in myocardial biopsies from aortic stenosis patients and were associated with a 3-fold increase in NFAT activation. When overexpressed or introduced as a cell-permeable peptide, syndecan-4 activated NFATc4. To examine whether mechanical stimuli activate NFAT through syndecan-4, cardiomyocytes from syndecan-4 KO-NFAT-luciferase reporter mice were subjected to cyclic mechanical stretch (10%, 1Hz). Importantly, NFAT activation was only 1.6-fold increased following stretch of syndecan-4 KO-NFAT luciferase cardiomyocytes compared to a 5.8-fold increase in NFAT-luciferase controls. Similar data were obtained in stretched syndecan-4 KO and wild-type cardiomyocytes as assessed by NFATc4 phosphorylation. Hypertrophy, measured by protein synthesis, as well as NFAT activation (NFAT-luciferase activity and phosphorylation), were reduced in syndecan-4 KO cardiomyocytes subjected to autonomous growth. Mechanical stress following aortic banding of syndecan-4 KO mice in vivo induced less activation of NFAT as assessed by NFATc4 phosphorylation and NFAT target gene expression (RCAN1-4 and BNP). In vitro pull-down showed that recombinant CnA binds directly to syndecan-4. Immunoprecipitation showed that association between endogenous CnA, its activator calmodulin and syndecan-4 was stronger in pressure-overloaded hearts. The syndecan-4 cytoplasmic region constitutes 28 amino and is composed of the syndecan isoform-specific V-region and the non-isoform-specific C1 and C2 regions. Pull-down and peptide array experiments showed that CnA binds to the V-C2-region of syndecan-4 through its autoinhibitory domain. Finally, a C1-V-C2 peptide disrupted NFAT activation in cardiomyocytes, indicating that membrane-localization of syndecan-4 is important for NFAT activation.
Conclusively, our data indicate that in cardiomyocytes of a pressure-overloaded heart, mechanical stimuli are sensed by the z-disc proteoglycan syndecan-4 which activates pro-hypertrophic CnA-NFAT signaling through a direct interaction with CnA.