Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions

Chopra, Anant; Kutys, Matthew L.; Zhang, Kehan; Polacheck, William J.; Sheng, Calvin C.; Luu, Rebeccah J.; Eyckmans, Jeroen; Hinson, J. Travis; Seidman, Jonathan G.; Seidman, Christine E.; Chen, Christopher

doi:10.1016/j.devcel.2017.12.012

Cited by 141 publications

(205 citation statements)

References 56 publications

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“…Compared to the WT hPSC-CMs, ΔK210 hPSC-CMs display more disorganized sarcomeres with patches of punctate staining (Figure 5B). Similar disorganization and punctate sarcomere structure has been seen with other hPSC-CM models of DCM cultured on stiff substrates (6, 18, 54), suggesting that it might be a common feature of some forms of DCM.…”

Section: Resultssupporting

confidence: 72%

“…While healthy cardiomyocytes adapt their contractility and structure to changes in their mechanical environment, such as age or disease-related stiffening of the heart tissue (21), ΔK210 cells do not adapt in the same way as the WT cells. Similar to other hPSC-CM models of DCM grown on stiff substrates, ΔK210 hPSC-CMs cultured on glass show impaired sarcomeric organization (18, 54, 76); however, ΔK210 hPSC-CMs can organize normally when given mechanobiological cues that mimic the healthy heart (Figures 5 and 6). Therefore, the ΔK210 mutation affects not only contractility, but also the ability of the hPSC-CMs to sense and respond to their environment.…”

Section: Discussionsupporting

confidence: 74%

“…We used human hPSC-CMs to study how the initial insult of a molecular-based change leads to the early disease pathogenesis in human cells. For these studies, hPSC-CMs are excellent models, since they can capture cellular changes that occur before major compensatory mechanisms (e.g., fibrosis, activation of neurohormonal pathways, changes in gene expression) mask the initial molecular phenotype (18–20, 54, 72). Contractile changes associated with the disease have been observed in cardiomyopathy patients before adverse remodeling of the heart (73–75).…”

Section: Discussionmentioning

confidence: 99%

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Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

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27Familial dilated cardiomyopathy (DCM) is a leading cause of sudden cardiac death and a 28 major indicator for heart transplant. The disease is frequently caused by mutations of 29 sarcomeric proteins; however, it is not well understood how these molecular mutations 30 lead to alterations in cellular organization and contractility. To address this critical gap in 31 our knowledge, we studied the molecular and cellular consequences of a DCM mutation 32 in troponin-T, DK210. We determined the molecular mechanism of DK210 and used 33 computational modeling to predict that the mutation should reduce the force per 34 sarcomere. In mutant cardiomyocytes, we found that DK210 not only reduces contractility, 35 but also causes cellular hypertrophy and impairs cardiomyocytes' ability to adapt to 36 changes in substrate stiffness (e.g., heart tissue fibrosis that occurs with aging and 37 disease). These results link the molecular and cellular phenotypes and implicate 38 alterations in mechanosensing as an important factor in the development of DCM. 39Results 100 DK210 decreases calcium sensitivity in an in vitro motility assay 101We set out to decipher the molecular mechanism of the DK210 mutation in vitro. 102The molecular effects of cardiomyopathy mutations depend on the myosin isoform (7-9, 103 35-37) and therefore, we used porcine cardiac ventricular myosin (38). Porcine ventricular 104 cardiac myosin (MYH7) is 97% identical to human, while murine cardiac myosin (MYH6) 105 is only 92% identical. Porcine cardiac myosin has very similar biophysical properties to 106 human cardiac myosin, including the kinetics of the ATPase cycle, step size, and 107 sensitivity to load (38-41), making it an ideal myosin for biophysical studies. 108Given the role of troponin-T in thin filament regulation, we first determined whether 109 the DK210 mutation affects calcium-based regulation of myosin binding to thin filaments 110 using an in vitro motility assay (42). Reconstituted thin filaments, consisting of porcine 111 cardiac actin and recombinantly expressed human troponin and tropomyosin, were added 112 to a flow cell coated with porcine cardiac myosin in the presence of ATP. The speed of 113 filament translocation was measured as a function of added calcium. As has been 114 reported previously, the speed of regulated thin filament translocation increased 115 sigmoidally with increasing Ca 2+ concentration (43), ( Figure 1B). Data were fit with the Hill 116 equation to obtain the pCa50 (i.e., the concentration of calcium necessary for half-117 maximal activation). Consistent with previous studies using mouse cardiac, rabbit cardiac, 118 and rabbit skeletal muscle fibers (31, 33, 44), DK210 shows a right-shifted curve (pCa50 119 = 5.7 ± 0.1) compared to the WT (pCa50 = 6.1 ± 0.1; p < 0.0001), meaning more calcium 120 is needed for the same level of activation. This suggests that the mutant could show 121 impaired force production during a calcium transient. 122 7 123 Molecular mechanism of DK210-induced changes in thin filament regulat...

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Section: Resultssupporting

confidence: 72%

Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Clippinger¹,

Cloonan²,

Greenberg³

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Furthermore, the causative role of many gene mutations remains controversial in HCM, one example being ACTN2, which encodes a-actinin 2. The aactinin 2 protein is located at the Z-disk of the sarcomere in homodimers fulfilling three main functions: sarcomere formation, anchoring/crosslinking of actin thin filaments, and interaction with titin (Gautel & Djinovic-Carugo, 2016;Chopra et al, 2018). Several ACTN2 variants have been associated with HCM, but only a few could be validated as disease-causing with altered function (Theis et al, 2006;Chiu et al, 2010;Girolami et al, 2014;Haywood et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

et al. 2019

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Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease accompanied by structural and contractile alterations. We identified a rare c.740C>T (p.T247M) mutation in ACTN2, encoding α‐actinin 2 in a HCM patient, who presented with left ventricular hypertrophy, outflow tract obstruction, and atrial fibrillation. We generated patient‐derived human‐induced pluripotent stem cells (hiPSCs) and show that hiPSC‐derived cardiomyocytes and engineered heart tissues recapitulated several hallmarks of HCM, such as hypertrophy, myofibrillar disarray, hypercontractility, impaired relaxation, and higher myofilament Ca2+ sensitivity, and also prolonged action potential duration and enhanced L‐type Ca2+ current. The L‐type Ca2+ channel blocker diltiazem reduced force amplitude, relaxation, and action potential duration to a greater extent in HCM than in isogenic control. We translated our findings to patient care and showed that diltiazem application ameliorated the prolonged QTc interval in HCM‐affected son and sister of the index patient. These data provide evidence for this ACTN2 mutation to be disease‐causing in cardiomyocytes, guiding clinical therapy in this HCM family. This study may serve as a proof‐of‐principle for the use of hiPSC for personalized treatment of cardiomyopathies.

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“…In the cardiac sarcomere, alpha actinin-2 (referred henceforth as actinin) is the major structural component of the Z-disk, where it is essential for sarcomere assembly and function 8 . Actinin regulates sarcomere assembly by providing a scaffold for protein-protein interactions (PPIs) such as with titin (TTN) and actin through CaM and AB domains, respectively 8–10 . Secondary to these interactions, the thin and thick filament sarcomere systems are organized to promote twitch contraction.…”

Section: Mainmentioning

confidence: 99%

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

Thakar

et al. 2020

Preprint

Self Cite

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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

show abstract

Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions

Cited by 141 publications

References 56 publications

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Disrupted Mechanobiology Links the Molecular and Cellular Phenotypes in Familial Dilated Cardiomyopathy

Disease modeling of a mutation in α‐actinin 2 guides clinical therapy in hypertrophic cardiomyopathy

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

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