1Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; 2Universitätsklinikum Schleswig-Holstein Innere Medizin III mit den Schwerpunkten Kardiologie, Angiologie und internistische Intensivmedizin Kiel, Deutschland
Introduction
The process of selective autophagy has been recognized as a highly conserved lysosomal degradation pathway and differential regulation of autophagic flux occurs in the failing human heart. The sarcomeric M-band is a hub for cardiomyocyte signalling and has been implicated in mechanical stress sensing, excitation-contraction coupling, and cardiac remodelling. Previously, our group identified the M-band protein Myomasp (LRRC 39) as a regulator of cardiac signal transduction and contractility, primarily acting via the SRF and Calcineurin pathway. Optineurin is a selective cargo receptor involved in autophagy via binding of ubiquitinated proteins to LC3 by its LIR motif. So far, a role of Optineurin in the pathogenesis of heart failure has not been described.
Methods
Yeast two-hybrid screens were done to discover protein-protein interactions of Optineurin and Myomasp. Adenoviral overexpression of Optineurin (AdOPTN), as well as knockdown by RNA interference (siOPTN), were performed in neonatal ventricular rat cardiomyocytes (NRVCMs). Gene (qPCR) and protein expression analyses (Western Blot) of M-Band associated proteins were done at baseline conditions, and after stimulation with phenylepinephrin (PE) and bafilomycin A1. Moreover, autophagocytosis and apoptotic read outs (e.g., LC3 II/I ratio, TUNEL staining, Caspase cleavage) were performed under these conditions. A cardiomyocyte (CM) restricted Optineurin knockout mouse line was generated by using a knockout-first conditional allele strategy.
Results
Yeast-Two hybdrid screens identified Optineurin as a direct interaction partner of Myomasp, and a high abundance of Optineurin in human heart muscle was observed. Moreover, Optineurin protein expression was significantly lower in human DCM and ICM specimen samples as compared to controls, hinting at a potential link to heart failure.
Adenoviral Optineurin overexpression in NRVCM resulted in reduced gene and protein expression of M-Band associated proteins like Myomasp, EH-Myomesin, Myomesin 1 and Myomesin 2, as compared to adenoviral LacZ overexpression. Members of the hypertrophic gene program like ANF and BNP, as well as the Calcineurin regulating factor Rcan 1.4., were induced upon adenoviral overexpression of Optineurin. This indicates a role of Optineurin in maintaining M-Band integrity and associated hypertrophic signaling.
Though Optineurin knockdown was effective at protein and gene expression level, the anticipated effect of higher expression of M-Band associated proteins was not observed. Conversely, co-stimulation with PE after overexpression of Optineurin resulted in increased cardiomyocyte death as shown by an activation of Caspase 3 and 7 as well as increased PARP cleavage. Beyond this, Optineurin overexpression resulted in increased LC3 II/I ratios, indicating increased cardiomyocyte autophagy flux, as also indicated by TUNEL stainings. This was exacerbated upon co-stimulation with PE and attenuated after co-stimulation with Bafilomycin A1. Reciprocal experiments after siRNA-mediated knockdown of Optineurin in NRVCM, as well as in-vivo cardiac pressure-volume assessments in CM-specific Optineurin deficient mice, will further explore the role of Optineurin in sarcomere homeostasis and cardiac contractility.
Conclusion
Optineurin plays a role in the regulation of sarcomere homeostasis by orchestrating cardiac autophagy and apoptosis and may be involved in the pathogenesis and the progression of heart failure.