Nx3 Is a Z-Disk Structural and Signaling Hub That Is Reduced in Heart Failure

BACKGROUND: Nx3 (novex-3) is an exceptionally small isoform of the giant protein titin, whose structural and functional roles within the sarcomere remain poorly understood. METHODS AND RESULTS: We used a comprehensive, multimodal approach to define the key properties of Nx3 in healthy and failing hearts, including its abundance relative to FLT (full-length titin), sarcomeric localization, protein interactions, and functional relevance in mouse and human cardiomyocytes under physiological and pathological conditions. Using Western blotting, quantitative polymerase chain reaction, total RNA sequencing, and ribosome profiling, we show that Nx3 is constitutively expressed from fetal development through adulthood. In adult mouse and human myocardium, Nx3 accounts for ≈20% to 25% of total titin protein, despite representing only ≈8% to 14% at the transcript level. Immunoelectron microscopy and binding studies reveal that Nx3 adopts a nonlinear configuration within the sarcomere: its N terminus is anchored at the Z-disk, although the proximal portion of its unique region encoded by Ttn exon 48, enriched in coiled-coil motifs, engages laterally with adjacent titin or Nx3 molecules at the Z-disk/I-band interface. Its monomeric C terminus extends toward the A-band but remains confined to the Z-/I-band region. This architecture confers enhanced stability and flexibility to the Z-disk under mechanical load. Protein interaction studies, including yeast 2-hybrid screening and coimmunoprecipitation, identified Pin1 (peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) and TBC1D4 (TBC1 domain family member 4) as binding partners of the Nx3 C terminal region, suggesting participation in signaling networks regulating cardiomyocyte metabolism. Genetic ablation of Nx3 in mouse hearts and human induced pluripotent stem cell–derived cardiomyocytes indicates that, although dispensable for sarcomere assembly, Nx3 is required for optimal Z-disk organization and mechanical performance. In end-stage dilated cardiomyopathy, human hearts exhibit dysregulated Nx3 expression, with reduced protein abundance relative to nonfailing controls and focal Z-disk disruption, likely contributing to impaired contractile function. CONCLUSIONS: Nx3 regulates Z-disk stability and modulates signaling pathways that optimize cardiac performance, and its dysregulation contributes to heart failure pathogenesis.