Investigating a universal stress response of muscle to mechanical stress: CARP-induced cross-linking of titin/actin filaments in the sarcomere

• Applicant: Jennifer Fleming, Ph.D.
• Subject Area: Structural Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 507930106

The sarcomere is the contractile unit of skeletal and cardiac striated muscle. Numerous stressors including mechanical overload, metabolic stress and chronic disease trigger an adaptive regulation of its mechanical performance. To date, the molecular mechanisms mediating such an adaptive response remain poorly understood. In this regard, the protein CARP is a universal response factor of striated muscle to stress. Upon tissue insult, CARP is rapidly and robustly upregulated and targets the sarcomere, interacting with the N2A element of the titin myofilament in the elastic I-band. Only very recently (2021), our laboratory revealed that sarcomere targeting by CARP induces the cross-link of the titin and actin filaments and that this increases myofibrillar stiffness, predictably by shortening the effective length of titin’s spring regions. We concluded that CARP is a regulator of sarcomere mechanics that provides rapid protection to muscle against mechanical overload in the early stages of tissue insult, protecting skeletal muscle against injury and preserving beating force in the heart. In this proposal, I aim to characterise at the molecular level the CARP/titin/F-actin interaction and its mechanisms of regulation by applying structural and biochemical methodologies in combination. Specifically, I will build on materials, protocols and preliminary data now available to elucidate the 3D-structure of the CARP/titin-N2A complex using X-ray crystallography and the 3D-structure of the CARP/titin-N2A/F-actin assembly using cryo-electron microscopy. Structures will advance the understanding of the mode of action of this cross-link in the sarcomere and guide the identification of its molecular determinants. The latter will enable the future development of cell probes that can help to dissect the functional pathways of CARP in the myofibril and clarify the biomedical benefit of their manipulation to treat muscle pathology. In addition, I will aim to reveal how the CARP response mechanism is affected by the presence in the myofibril of the conserved family member, MARP, which also binds sarcomeric titin-N2A but that is constitutively expressed. Finally, I will explore the mechanisms of regulation and removal of the CARP-induced sarcomere cross-link. Specifically, I will study the effect of the post-translational modifications of phosphorylation, ubiquitination and methylation using myoblast co-transfections, in vitro enzyme-linked immunosorbent assays (ELISA) to monitor protein interactions, enzymatic assays and actin co-sedimentation. Emphasis will be placed in revealing whether cytosolic methylation and ubiquitination at the N2A locus act to counter-regulate the life time of CARP in the sarcomere. Taken together, the investigation of this newly discovered regulatory element will aid the understanding of its function in muscle stress adaptation and its link to clinical pathological outcomes.

• DFG Programme: Research Grants