Abstract:Muscle specific signaling has been shown to originate from myofilaments and their associated cellular structures, including the sarcomeres, costameres or the cardiac intercalated disc. Two signaling hubs that play important biomechanical roles for cardiac and/or skeletal muscle physiology are the N2B and N2A regions in the giant protein titin. Prominent proteins associated with these regions in titin are chaperones Hsp90 and αB-crystallin, members of the four-and-a-half LIM (FHL) and muscle ankyrin repeat prot… Show more
“…Second, this study focuses primarily on the transcriptional role of FHL5 in SMCs. Other FHL family members, FHL1 and FHL3, are known to function as scaffolds to regulate sarcomere formation in myoblasts 14,81,82 . We speculate that FHL5, due to the presence of similar conserved LIM domains, may mediate similar interactions with the actin cytoskeleton 83,84 or focal adhesions 85,86 .…”
Background: Genome-wide association studies (GWAS) have identified hundreds of loci associated with common vascular diseases such as coronary artery disease (CAD), myocardial infarction (MI), and hypertension. However, the lack of mechanistic insights for a majority of these loci limits translation of these findings into the clinic. Among these loci with unknown functions is UFL1-FHL5 (chr6q16.1), a locus that reached genome-wide significance in a recent CAD/MI GWAS meta-analysis. In addition to CAD/MI, UFL1-FHL5 is also implicated to coronary calcium, intracranial aneurysm, and migraine risk, consistent with the widespread pleiotropy observed among other GWAS loci. Methods: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and imaging of human coronary artery tissues to implicate Four-and-a-half LIM domain 5 (FHL5) as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments. Results: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through eQTL colocalization methods. FHL5 gene expression was enriched in the SMC and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating SMC contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream CAD/MI loci, such as FOXL1 and FN1 that have roles in vascular remodeling. Conclusion: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling loci. These trans-acting mechanisms may account for a portion of the heritable risk for complex vascular diseases.
“…Second, this study focuses primarily on the transcriptional role of FHL5 in SMCs. Other FHL family members, FHL1 and FHL3, are known to function as scaffolds to regulate sarcomere formation in myoblasts 14,81,82 . We speculate that FHL5, due to the presence of similar conserved LIM domains, may mediate similar interactions with the actin cytoskeleton 83,84 or focal adhesions 85,86 .…”
Background: Genome-wide association studies (GWAS) have identified hundreds of loci associated with common vascular diseases such as coronary artery disease (CAD), myocardial infarction (MI), and hypertension. However, the lack of mechanistic insights for a majority of these loci limits translation of these findings into the clinic. Among these loci with unknown functions is UFL1-FHL5 (chr6q16.1), a locus that reached genome-wide significance in a recent CAD/MI GWAS meta-analysis. In addition to CAD/MI, UFL1-FHL5 is also implicated to coronary calcium, intracranial aneurysm, and migraine risk, consistent with the widespread pleiotropy observed among other GWAS loci. Methods: We apply a multimodal approach leveraging statistical fine-mapping, epigenomic profiling, and imaging of human coronary artery tissues to implicate Four-and-a-half LIM domain 5 (FHL5) as the top candidate causal gene. We unravel the molecular mechanisms of the cross-phenotype genetic associations through in vitro functional analyses and epigenomic profiling experiments. Results: We prioritized FHL5 as the top candidate causal gene at the UFL1-FHL5 locus through eQTL colocalization methods. FHL5 gene expression was enriched in the SMC and pericyte population in human artery tissues with coexpression network analyses supporting a functional role in regulating SMC contraction. Unexpectedly, under procalcifying conditions, FHL5 overexpression promoted vascular calcification and dysregulated processes related to extracellular matrix organization and calcium handling. Lastly, by mapping FHL5 binding sites and inferring FHL5 target gene function using artery tissue gene regulatory network analyses, we highlight regulatory interactions between FHL5 and downstream CAD/MI loci, such as FOXL1 and FN1 that have roles in vascular remodeling. Conclusion: Taken together, these studies provide mechanistic insights into the pleiotropic genetic associations of UFL1-FHL5. We show that FHL5 mediates vascular disease risk through transcriptional regulation of downstream vascular remodeling loci. These trans-acting mechanisms may account for a portion of the heritable risk for complex vascular diseases.
“…The binding to the exposed binding sites in unfolded structural domain kinetically inhibits refolding of the structural domain when forces were decreased to below 5 pN. The numerous FHL2 binding sites make the N2B-us a hot spot for FHL2 binding, which could scaffold downstream binding and potential activation of several important kinases such MAPK and PFK(van der Pijl et al ., 2021).…”
The 572 amino acids unique sequence on titin N2B element (N2B-us) is known to regulate the passive elasticity of muscle as an elastic spring. It also serves as a hub for cardiac hypertrophic signaling by interacting with multiple proteins such as FHL1(Sheikh et al, 2008), FHL2(Lange et al, 2002), and Erk2(Perkin et al, 2015). N2B-us is thought to be an intrinsically disordered region. In addition, N2B-us bears force; therefore, the functions of N2B-us are likely regulated by mechanical stretching. In the work, we investigated the conformation of N2B-us as well as its force-dependent interaction with FHL2 using a combination of AlphaFold2 predictions and single-molecule experimental validation. Surprisingly, a stable alpha/beta structural domain (~115 a.a.) was predicted and confirmed in N2B-us, which can be mechanically unfolded at forces greater than 5 pN. More than twenty FHL2 LIM domain binding sites were predicted to spread throughout N2B-us including the regions cryptic in the structural domain. Mechanosensitive binding of FHL2 to N2B-us is revealed in single-molecule manipulation experiments. Together, the results unveil several previously unknown aspects of the N2B-us conformations and its force-dependent interactions with FHL2, which provides new insights into the physiological functions of the force-bearing N2B-us region.
“…Several additional proteins, localized in the M-band and Z-disc of the sarcomere, share similar domain layout to titin however, they have much smaller size. These include the myomesin family in the M-band, the filamins and the myopalladin in the Z-disc, the myosin binding protein C (MyBP-C) in the A-band and the obscurin proteins which are localized both in the Z-disc and the M-band [ 8 , 9 ]. Fig.…”
Transversal structural elements in cross-striated muscles, such as the M-band or the Z-disc, anchor and mechanically stabilize the contractile apparatus and its minimal unit—the sarcomere. The ability of proteins to target and interact with these structural sarcomeric elements is an inevitable necessity for the correct assembly and functionality of the myofibrillar apparatus. Specifically, the M-band is a well-recognized mechanical and signaling hub dealing with active forces during contraction, while impairment of its function leads to disease and death. Research on the M-band architecture is focusing on the assembly and interactions of the three major filamentous proteins in the region, mainly the three myomesin proteins including their embryonic heart (EH) isoform, titin and obscurin. These proteins form the basic filamentous network of the M-band, interacting with each other as also with additional proteins in the region that are involved in signaling, energetic or mechanosensitive processes. While myomesin-1, titin and obscurin are found in every muscle, the expression levels of myomesin-2 (also known as M-protein) and myomesin-3 are tissue specific: myomesin-2 is mainly expressed in the cardiac and fast skeletal muscles, while myomesin-3 is mainly expressed in intermediate muscles and specific regions of the cardiac muscle. Furthermore, EH-myomesin apart from its role during embryonic stages, is present in adults with specific cardiac diseases. The current work in structural, molecular, and cellular biology as well as in animal models, provides important details about the assembly of myomesin-1, obscurin and titin, the information however about the myomesin-2 and -3, such as their interactions, localization and structural details remain very limited. Remarkably, an increasing number of reports is linking all three myomesin proteins and particularly myomesin-2 to serious cardiovascular diseases suggesting that this protein family could be more important than originally thought. In this review we will focus on the myomesin protein family, the myomesin interactions and structural differences between isoforms and we will provide the most recent evidence why the structurally and biophysically unexplored myomesin-2 and myomesin-3 are emerging as hot targets for understanding muscle function and disease.
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