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.
W. Linke, Lisa Kümper, A. Fomin et al.· Circulation Research· 0 citations
Sodium–glucose cotransporter 2 inhibitors (SGLT2i) are now widely applied in treatment plans for heart failure with reduced ejection fraction (HFrEF) patients, regardless of the presence of diabetes. SGLT2i, such as empagliflozin (EMP), reduce the risk of cardiovascular death and heart failure hospitalizations in HFrEF patients. However, the underlying molecular mechanisms of action, by which SGLT2i benefit cardiomyocytes (CMs) in HFrEF hearts, are not well understood. This study investigated the role of the SGLT2i, EMP, in a patient-specific human model of dilated cardiomyopathy (DCM), a primary cause of HFrEF, which is frequently caused by inherited mutations in sarcomere proteins. We employed DCM patient-specific induced pluripotent stem cell-derived CMs (iPSC-CMs) carrying the inherited sarcomere protein mutation tropomyosin (TPM1)-L185F together with high-speed motion traction and optical action potential mapping analysis, as well as biochemical methods to dissect molecular signaling dysfunctions. Our findings indicate that in DCM patient-specific iPSC-CMs, SGLT2i may act via different mechanisms. This includes the regulation of signaling pathways, to modulate sarcomere function via improving mitochondrial respiration and ATP production. As our results show, the SGLT2i, EMP, ameliorates primary sarcomere dysfunctions in DCM TPM1-L185F iPSC-CMs, such as disrupted sarcomere organization, reduced contractile function, and prolonged action potential duration. Moreover, we found that a subcellular signaling pathway dysregulated in DCM CMs, clathrin-mediated endocytosis (CME)-dependent signaling, may be recovered by EMP treatment in DCM TPM1-L185F iPSC-CMs. This pathway is essential for the uptake and distribution of critical cargo, such as transferrin-bound Fe, in CMs. In the presence of the DCM-causing mutation, EMP recovered mitochondrial Fe levels and mitochondrial functional output, which are reduced in DCM iPSC-CMs as part of pathologically defective CME-dependent signaling. These findings support that EMP modulates aspects of CME-signaling, and contributes to restored sarcomere functions. To further elucidate the effects of SGLT2i in DCM CMs, we investigated the molecular functions of a state-of-the-art combinatorial therapy for DCM (HFrEF) patients, the drug combination referred to as “fantastic four” (F4), including an SGLT2i, in DCM iPSC-CMs. Our in-vitro results point to a significant benefit of F4 to be attributed to the SGLT2i, EMP, regarding sarcomere functions as well as aspects of CME-dependent recovery of molecular signaling. Moreover, studying the recovery of subcellular dysfunctions by EMP treatment, findings from an in-vivo porcine model of HF support improvements observed in the presence of EMP regarding contractile and electrophysiological parameters. Together, our findings provide molecular insights into the subcellular function of SGLT2i and their role as part of the F4 state-of-the-art combination therapy for HFrEF, via targeting different molecular signaling pathways in a human patient-specific 2D model of HFrEF.
Daria Plota, H. N. Saleem, K. Lin et al.· Journal of Translational Med...· 0 citations
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