Identifying cardiac actinin interactomes reveals sarcomere crosstalk with RNA-binding proteins

Abstract

1 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in 2 heritable diseases. We deployed proximity-dependent biotinylation to identify actinin proximity 3 proteins and RNA transcripts in human cardiomyocytes to reveal new functions of the actin 4 cytoskeleton. We identified 285 proximity protein partners including unexpected effectors of 5 RNA-binding and metabolism, and interrogated dynamic partners using a sarcomere assembly 6 model. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein 7 associated with type 2 diabetes, interacted with the rod domain of actinin through its K 8 Homology domain. This interaction was necessary for actinin localization and translation of 9 electron transport chain transcripts, and oxidative phosphorylation. Diminished IGF2BP2 in 10 cardiac microtissues impaired contractile function in accord with diminished metabolic functions. 11 This actinin proximity protein and RNA study expands our functional knowledge of the actin 12 cytoskeleton, and uncovers new modes of metabolic regulation through interactions with RNA-13 binding proteins. 14 15 Main 16 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are important 17 for a myriad of cellular functions including cell adhesion, migration, contraction, and signaling 1 . 18 The four human actinin isoforms have distinct expression profiles (non-muscle ACTN1 and 19 ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share homologous amino 20 acid sequences that are organized into three structural domains-an actin-binding (AB) domain 21 composed of two calponin-homology domains, a central rod domain containing four spectrin-like 22 repeats (SR), and a calmodulin-like domain (CaM) containing two EF hand-like motifs 2 . While it 23is thought that actinin evolved initially to regulate the early eukaryotic actin-based cytoskeleton 3 , 24 it has acquired more elaborate functions in vertebrates, including a mechanical role in the 25 sarcomere, a specialized contractile system required for striated muscle function 3, 4 . Moreover, 21 microtissues with heart failure-associated mutations, twitch force was unaltered by Actinin-BirA* 22 expression (extended data Fig. 1b). We confirmed appropriate localization of Actinin-BirA* to the 23 Z-disk by both co-localization immunofluorescence (Fig.1d) and HA-immunoprecipitation assays 24 (Fig.1e). Actinin-BirA* interacted with the Z-disk protein titin-cap (TCAP), but not the sarcomere 25 M-line protein myomesin [22][23][24][25] , which supported appropriate localization. With the knowledge that 26 Confidential Manuscript 6 Actinin-BirA* functions similarly to unmodified actinin, we next optimized biotin supplementation

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes

“…A deeper analysis of proteomic data revealed that the protein levels of ACTN2, several other sarcomere-associated proteins and desmosomal proteins were markedly lower in ACTN2mut hiPSC-CMs ( Table S1 ). Some of these proteins (FLNC, MYOZ2, NEBL, SYNPO2, SYNPO2L, TTN) are known to interact directly with ACTN2 [ 22 , 23 , 24 ]. On the other hand, FHL1 and FHL2, located at the Z-disk of the sarcomere [ 25 , 26 ] were more abundant in ACTN2mut.…”

“…This is in line with the low abundance of several sarcomeric proteins, including ACTN2 in ACTN2mut hiPSC-CMs, leading to a poorly developed sarcomere and possibly to a more immature cardiomyocyte state. In addition, SYNPO2 and SYNPO2L, which contribute to early assembly and stabilization of the Z-disk via interaction with filamin and ACTN2 [ 22 , 27 ], were also less abundant, supporting disruption of the ACTN2 interactome and deficient sarcomere development in ACTN2mut hiPSC-CMs. The reduced Z-disk integration of exogenous MUT-ACTN2 in ACTN2wt by live cell imaging supports the susceptibility of mutant ACTN2 to aggregate.…”

Cardiomyopathies in Children: Genetics, Pathomechanisms and Therapeutic Strategies

“…As a result, the mutations may alter the very sensitive interplay between the components of the sarcomere and/or protein dynamics and thereby induce contractile dysfunction (for review, see [4]). Furthermore, interactions of sarcomeric proteins with associated proteins might be altered, affecting signaling and thereby protein transcription, metabolism, inflammation, oxidation, cell death, protein degradation, fibrosis, cell-cell communication, Ca 2þ homeostasis, and so on [95][96][97].…”

Structural and signaling proteins in the Z-disk and their role in cardiomyopathies

“…Global inactivation of alpha-actinin two in mice is not compatible with life, but a cardiac specific mosaic inactivation suggests a crucial role of the protein for cardimyocyte maturation via serum response factor signaling (Guo et al, 2021). Moreover, a role of alpha-actinin two in regulating cardiac mitochondrial function has been proposed recently (Zech et al, 2022), in part by acting as a scaffold for mitochondrial messenger RNAs via binding to RNA-binding proteins (Ladha et al, 2020).…”