Regulation of human cardiomyocyte excitation-contraction coupling by human cardiac fibroblasts

Physiologically, cardiomyocytes develop features that enable them to meet the contractile demands of the healthy, adult heart. Among the structural and functional changes during development, there is an engagement of the sarcoplasmic reticulum as the main regulator of cytoplasmic Ca2+ cycling. In human cardiac disease and in ageing, there is progressive disengagement of the sarcoplasmic reticulum, reducing the efficiency of excitation-contraction coupling. In this project, we used human induced pluripotent stem cell- derived cardiomyocytes (hiPSC-CMs) to investigate the role of human cardiac fibroblasts in regulating cardiomyocyte Ca2+ cycling. In chapter 3, we use hiPSC-CMs with a genetically encoded Ca2+ indicator to perform optical recording of changes in hiPSC-CM intracellular Ca2+ in various co-culture setups with human cardiac fibroblasts. Co-culture setups that only allowed paracrine interactions between the two cell types led to prolongation of the hiPSC-CM Ca2+ transients. There was an abbreviation in Ca2+ transient duration when the two cell types were in direct physical contact, indicating an increase in Ca2+ cycling efficiency. In chapter 4, we investigated the role of the extracellular matrix in regulating hiPSC- CM Ca2+ cycling. As matrix proteins are known to form interactions with cardiomyocytes via integrin ligand-receptor interactions, we utilised synthetic peptides with the integrin-binding tripeptide motif, Arginine-Glycine-Aspartic Acid, to show that fibril-forming integrin ligands abbreviated hiPSC-CM Ca2+ transients by recruiting the sarcoplasmic reticulum to Ca2+ cycling. In chapter 5, we focus on the role of extracellular vesicles, which have emerged over the last decade as a major secretory vehicle for non-soluble paracrine interactions. A major limitation in the investigation of extracellular vesicles is that isolation techniques, and thus sample purity, varies considerably between studies. In chapter 5, we validated an ultrafiltration- and chromatography-based technique for the isolation of extracellular vesicles from cardiac fibroblast-conditioned culture media and showed that cardiac fibroblast extracellular vesicles significantly abbreviate the hiPSC-CM Ca2+ transient time to peak, indicating an increase in the efficiency of Ca2+-induced Ca2+-release. The findings of this project indicate that cardiac fibroblasts have differential effects on hiPSC-CM Ca2+ cycling depending on the modality of interaction. The findings also indicate that fibroblast-mediated modulation of hiPSC-CM Ca2+ cycling can be mediated by fibroblast-regulated turnover of the extracellular matrix. This project demonstrates the importance of the extracellular interactions in utilising hiPSC-CMs and understanding the modulators of cardiomyocyte structure and function.Open Acces

A unique bivalent binding and inhibition mechanism by the yatapoxvirus interleukin 18 binding protein.

Interleukin 18 (IL18) is a cytokine that plays an important role in inflammation as well as host defense against microbes. Mammals encode a soluble inhibitor of IL18 termed IL18 binding protein (IL18BP) that modulates IL18 activity through a negative feedback mechanism. Many poxviruses encode homologous IL18BPs, which contribute to virulence. Previous structural and functional studies on IL18 and IL18BPs revealed an essential binding hot spot involving a lysine on IL18 and two aromatic residues on IL18BPs. The aromatic residues are conserved among the very diverse mammalian and poxviruses IL18BPs with the notable exception of yatapoxvirus IL18BPs, which lack a critical phenylalanine residue. To understand the mechanism by which yatapoxvirus IL18BPs neutralize IL18, we solved the crystal structure of the Yaba-Like Disease Virus (YLDV) IL18BP and IL18 complex at 1.75 Å resolution. YLDV-IL18BP forms a disulfide bonded homo-dimer engaging IL18 in a 2∶2 stoichiometry, in contrast to the 1∶1 complex of ectromelia virus (ECTV) IL18BP and IL18. Disruption of the dimer interface resulted in a functional monomer, however with a 3-fold decrease in binding affinity. The overall architecture of the YLDV-IL18BP:IL18 complex is similar to that observed in the ECTV-IL18BP:IL18 complex, despite lacking the critical lysine-phenylalanine interaction. Through structural and mutagenesis studies, contact residues that are unique to the YLDV-IL18BP:IL18 binding interface were identified, including Q67, P116 of YLDV-IL18BP and Y1, S105 and D110 of IL18. Overall, our studies show that YLDV-IL18BP is unique among the diverse family of mammalian and poxvirus IL-18BPs in that it uses a bivalent binding mode and a unique set of interacting residues for binding IL18. However, despite this extensive divergence, YLDV-IL18BP binds to the same surface of IL18 used by other IL18BPs, suggesting that all IL18BPs use a conserved inhibitory mechanism by blocking a putative receptor-binding site on IL18

Kinetic analyses of the binding of IL18 with YLDV-IL18BP or ECTV-IL18BP.

<p>SPR analysis was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat-1002876-g009" target="_blank">Figure 9</a> with IL18 at 5 different concentrations. The binding curves were globally fitted with BiaEvaluation software to a 1∶1 binding model. The colored and black lines are the actual responses in RU and globally fitted curves, respectively.</p

YLDV-IL18BP:IL18 interface.

<p>A). Key residues of YLDV-IL18BP at the interface. YLDV-IL18BP binds nearly identical surface of IL18 as previously observed in ECTV-IL18BP inhibitory complex. IL18 is shown as surface representation and colored grey. YLDV-IL18BP is drawn as a ribbon diagram with β-sheets colored in yellow. Binding sites A, B and C on IL18 surface are colored red, orange and cyan respectively. YLDV-IL18BP residues involved in binding IL18 are shown as stick representations. Each insert details the interactions involved in the respective binding site between YLDV-IL18BP and IL18. B). Unique interactions at binding site A. Carbon atoms of YLDV-IL18BP and IL18 are colored in yellow and pink, respectively. The secondary structures of YLDV-IL18BP and IL18 are colored in cyan and green, respectively. Red dashed lines indicate H-bonds. C). Unique interactions at binding site C. YLDV-IL18BP P116 is involved in favorable hydrophobic interactions with IL18. Polar interactions at site C involve S105 and D110 residues of IL18. The coloring scheme is the same as in B.</p

X-Ray crystallographic data and refinement statistics.

*<p>WT, wild type; 3CM, triple-cysteine mutant.</p><p>Values in parentheses are for the highest resolution shell, 2.80 to 2.70 Å (WT), 1.78 to 1.75 Å (3CM).</p><p>R_sym = Σ |<i>I</i>_obs−<i>I</i>_avg|/Σ <i>I</i>_avg; <i>R</i>_work = Σ‖ <i>F</i>_obs |−|<i>F</i>_calc‖/Σ <i>F</i>_obs.</p><p><i>R</i>_free was calculated using 10% and 5% of data for the WT and 3CM complexes, respectively.</p

Biacore SPR analysis of IL18 mutants.

<p>Biotinylated YLDV-IL18BP and ECTV-IL18BP were captured on two different flow cells in a BIAcore streptavidin-coated CM5 chip, and their binding with IL18 was monitored simultaneously with a BIAcore 3000 sensor. The injection of IL18 started at ∼150 s and stopped at 900 s. The colored lines are the responses obtained with different IL18 mutants and normalized to a maximum of 100 RU (except for P57R, S105R for YLDV-IL18BP) for ease of comparison. YLDV-IL18BP and ECTV-IL18BP were expressed and secreted from mammalian cells and underwent <i>in vitro</i> biotinylation as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#s4" target="_blank">Materials and Methods</a>.</p

Kinetics and affinity constants of the binding of IL18 mutants with immobilized IL18BPs.

a<p>The kinetics and affinity constants were derived from 2 independent experiments similar to those shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat-1002876-g010" target="_blank">Figure 10</a>. <i>K</i> values are means ± standard deviations.</p>b<p>NF: binding data did not fit 1∶1 binding model.</p>c<p>NB: no binding.</p

Kinetics and affinity constants of the binding of YLDV-IL18BP mutants with immobilized IL18.

a<p>The kinetics and affinity constants were derived from 2 independent experiments similar to those shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat-1002876-g008" target="_blank">Figure 8</a>. <i>K</i> values are means ± standard deviations. All the mutants were derived from the monomeric form of IL18BP with the HVEC mutation.</p>b<p>NB: no binding.</p

Structure based sequence alignment of IL18 binding proteins.

<p>Structure based sequence alignment of various IL18BPs was created using the crystal structure of YLDV-IL18BP as the template. Lettering and numbering above alignment correspond to YLDV-IL18BP topology and numbering scheme. Colored stars above residues indicate the three binding sites on IL18 with which they interact, red: site A, orange: site B, cyan: site C. Solid black circles above residues indicate resides involved in homo-dimerization. The two intra-chain disulfide bonds are indicated with the green letters. The cysteine residue forming the unique inter-chain disulfide bond is indicated with a pink triangle. The sequence alignment was performed with the FATCAT server <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat.1002876-Ye1" target="_blank">[44]</a> and ClustalX <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat.1002876-Larkin1" target="_blank">[45]</a>, and the figure was created with ESPript <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002876#ppat.1002876-Gouet1" target="_blank">[46]</a>.</p