Structural features of Yulink protein. a The multiple sequence alignment analysis was performed with Clustal Omega software (https://www.ebi.ac.uk/Tools/msa/clustalo/). The amino acid identity between human (NP_061878.3) and zebrafish (NP_958490.1) was 82%, and between human and mouse (NP_663349.2) was 98%. “Dark gray color” means that the residues in that column are identical in all sequences in the alignment. “Light gray color” means that the residues were conserved with strongly similar properties. *** refers to WD40 repeats. b Classification and similarity of homologs of Yulink proteins. The classification of these homologs of Yulink was shown with a phylogenetic tree. The percentages of identities in amino acid sequences for homologous proteins were compared with human Yulink. c WD40 repeats within YULINK predicted by four computer servers. The prediction for WD40 repeats were based on the CDD, UniProt, SMART, and InterPro computer servers. WD40 #1 to #4 were the common results based on prediction using the four computer servers. The WD40 A and B were identified through the annotation of the CDD server, whereas WD40 C was found with the UniProt server

Characterization of yulink-MO KD zebrafish morphants. a Expression of yulink in zebrafish using whole-mount in situ hybridization. Embryos were fixed overnight at 4 °C in 4% paraformaldehyde buffered with 1 × phosphate-buffered saline (PFA/PBS). After hybridization with Dig-labeled antisense or control sense RNA probes of yulink, embryos were incubated with anti-Dig antibody conjugated to AP and developed with NBT-BCIP reagents. The yulink was expressed in whole zebrafish embryo ubiquitously from 0.5 hpf (zygote stage) to 3 dpf (larval stage) with lateral overview. The black arrows point to heart regions at 24–30 hpf and 3 dpf with ventral overview. The black arrowheads point to yolk of embryos, hour post-fertilization (hpf); day post-fertilization (dpf). b After injection with yulink-MO to knockdown yulink expression, the yulink KD morphants presented with small eyes, a small head, abnormal blood circulation, and pericardial edema at 3 dpf. Increasing the yulink-MO dosage from 2.3 ng (n = 119) to 4.6 ng (n = 119 or 9.2 ng (n = 156) caused the proportion of severely affected embryos with pericardial edema to increase from 6 to 54% (purple). c Hemodynamic changes. Yulink KD morphants had a slower heart rate and reduced cardiac output, as compared to WT embryos at 2 dpf (** p < 0.01, Student’s t test). d Diagram of the EYFP-fusion construct with the yulink 5′-UTR region and partial coding region (amino acids 1–79). The position of the corresponding MO binding site is indicated by a bar. To demonstrate the specificity of the yulink-MO, embryos were injected with pYulink-EYFP plasmid (100 pg/embryo) alone, or together with yulink-MO or the mismatch control, yulink-5mmMO (4.6 ng/embryo). About 58% of embryos injected with pYulink-EYFP plasmid alone exhibited fluorescence at 1 dpf, while fluorescence was absent in embryos co-injected with yulink-MO. In contrast, co-injection with yulink-5mmMO did not affect EYFP expression

Yulink KD reduced Serca2 expression and induced irregular Ca²⁺ cycling in mouse HL-1 cardiomyocytes. a Representative line-scan images and spontaneous Ca²⁺ transient in Ctrl and Yulink KD mouse HL-1 cardiomyocytes. Red arrows indicate arrhythmia-like waveforms observed in Yulink KD HL-1 cardiomyocytes, but not in control. b Quantification of percentages for control and Yulink KD HL1 cardiomyocytes exhibiting irregular Ca²⁺ transients (n = 40). c The Ca²⁺ transient amplitude of HL-1 cardiomyocytes for Ctrl and Yulink KD confirms Yulink KD HL-1 cardiomyocytes exhibits a lower Ca²⁺ transient amplitude (n = 40). d The time constant for Ca²⁺ decay (Tau) of HL-1 cardiomyocytes for control and Yulink KD show that the time constant for Ca²⁺ decay is significantly larger in Yulink KD than in control HL-1 cardiomyocytes (n = 40, *p < 0.05, Student’s t test). e HL-1 cardiomyocytes were subjected to Yulink KD using Yulink-shRNA, and the effects on Yulink and Serca2 expression were assayed by qPCR and Western blot. Relative expression values were normalized to those of cells injected with control (Ctrl) vector (n = 3 for each group, **p < 0.01, Student’s t test)

Over-expression of YULINK, PPARγ and SERCA2 rescued the phenotypes of mouse Yulink KD HL-1 cardiomyocytes. Control (Ctrl), YULINK, PPARγ or SERCA2 plasmids (carrying BFP as indicator of expression marker) were transfected or electroporated into Yulink KD cells, and then analyzed for Ca²⁺ cycling using fluorescent Ca²⁺ dye, Rhod-2 AM. a Over-expression of YULINK or PPARγ. Representative line-scan images and spontaneous Ca²⁺ transient in Yulink KD mouse HL-1 cardiomyocytes. Arrhythmia-like waveforms were observed in Yulink KD cardiomyocytes with Ctrl vector (red arrows). After over-expression of YULINK (YULINK-OE) or PPARγ (PPARγ-OE) in Yulink KD cells, normal waveforms were observed. The result of Ca²⁺ spark analysis was consistent with the increase of Ca²⁺ transient amplitudes (3.1 ± 0.5 for YULINK-OE or 3.2 ± 0.3 for PPARγ-OE, vs. 1.9 ± 0.35 for Ctrl). The cells with YULINK or PPARγ over-expression also exhibited a decrease of the percentages of irregular Ca²⁺ transients (8 ± 2% for YULINK-OE or 10 ± 2% for PPARγ-OE, vs. 27 ± 3% for Ctrl). The cells with YULINK or PPARγ over-expression also exhibited a reduction of the Ca²⁺ decay (Tau) rate (430 ± 42 ms for YULINK-OE or 450 ± 51 ms for PPARγ-OE, vs. 605 ± 47 ms for Ctrl). Ctrl (black bars), YULINK-OE (orange bars) and PPARγ-OE (blue bars) (n = 40, each). *p < 0.05, Student’s t test. b Over-expression of SERCA2. Representative line-scan images and spontaneous Ca²⁺ transients in Yulink KD mouse HL-1 cardiomyocytes. Arrhythmia-like waveforms were observed in Yulink KD cardiomyocytes with control vector (Ctrl, red arrows). After over-expression of SERCA2 (SERCA2-OE) in Yulink KD cells, normal waveforms were observed. The result of Ca²⁺ spark analysis was consistent with the increase of Ca²⁺ transient amplitudes (3.2 ± 0.45 for SERCA2-OE vs. 2.02 ± 0.3 for Ctrl). The cells with SERCA2-OE also exhibited a decrease of the percentages of irregular Ca²⁺ transients (11 ± 3% for SERCA2-OE vs. 32 ± 5% for Ctrl). The cells with SERCA2-OE also exhibited a reduction of the Ca²⁺ decay (Tau) rate (412 ± 50 ms for SERCA2-OE vs. 635 ± 78 ms for Ctrl). Ctrl vector (black bars), SERCA2-OE (gray bars) (n = 35, each). *p < 0.05, Student’s t test

Effect of Yulink KD on PPARγ activity in HL-1 cardiomyocytes. a The DNA-binding activity of nuclear PPARγ in HL-1 cardiomyocytes was significantly reduced by Yulink KD using Yulink-shRNA (left panel); values normalized to those in cells treated with Ctrl vector (relative PPARγ activity) are shown. (n = 3, ** p < 0.01, Student’s t test). b The nuclear PPARγ expression level (Red) was decreased in Yulink KD cardiomyocytes. In the immunofluorescence assay, PPARγ was stained red, and nuclei were stained with DAPI (blue). c The amounts of total, cytoplasmic, and nuclear PPARγ were determined by Western blot, and normalized to an internal control (GAPDH or Histone H1) (right panel). Total PPARγ protein was decreased by 30%, while nuclear PPARγ was decreased by 85% in Yulink KD cardiomyocytes. d KD of Yulink decreases the uptake of the PPARγ ligand 15d-PGJ2-biotin in cardiomyocytes. Cells were incubated in 1 μM 15d-PGJ2-biotin for 3 h, and then fixed and stained with streptavidin-Alexa Fluor647 (red). Signals were detected using flow cytometry and immunofluorescence. Black lines indicate cells incubated without ligand; red lines indicate cells incubated with ligand in flow cytometric analyses. In the immunofluorescence assay, 15d-PGJ2-biotin was stained red, and nuclei were stained with Hoechst 33342 (blue). The majority of cardiomyocytes transfected with control vector took up the 15d-PGJ2-biotin (upper panel), while only 2% of Yulink KD cardiomyocytes took up the ligand (bottom panel)

Yulink regulated Serca2 expression through the PPARγ pathway. a Treatment with 50 μM rosiglitazone for 6 h rescued and enhanced Serca2 expression at mRNA and protein levels in Yulink KD cardiomyocytes by qPCR (left panel) and Western blot (right panel). Serca2 mRNA levels normalized to values in cells treated with Ctrl vector. (n = 3, **p < 0.01, Student’s t test). Protein levels normalized to internal control, GAPDH. b Treatment with 50 mM pioglitazone (PPARγ agonist) for 12 h increased Serca2 mRNA expression in Yulink KD cardiomyocytes, as shown by qPCR. Data for cardiomyocytes treated with Ctrl vector and Yulink KD cardiomyocytes are shown in blue and red, respectively. Before pioglitazone treatment, Serca2 expression was decreased in Yulink KD cardiomyocytes (n = 3, **p < 0.01, Student’s t test). Pioglitazone increased relative Serca2 expression in Yulink KD cardiomyocytes from 0.25 to 0.82 (n = 3, **p < 0.01, Student’s t test). In Ctrl vector-treated cardiomyocytes, the expressions of Serca2 before and after pioglitazone were 1 and 0.81, respectively (n = 3). c Gene expression and protein levels in PPARγ-shRNA KD cardiomyocytes and cells treated with Ctrl Vector were quantified by qRT-PCR and Western blot, respectively. The mRNA expressions of PPARγ and Serca2 were significantly lower in PPARγ-shRNA KD cardiomyocytes (0.36 and 0.51, respectively) compared to values of 1 in cells treated with Ctrl vector (n = 3, ** p < 0.01, Student’s t test). Decreased total cellular PPARγ and SERCA2 protein levels were also observed in PPARγ-shRNA KD cardiomyocytes; values normalized to GAPDH. d KD of Yulink resulted in obvious decreases in nuclear levels of PPARα and PPARβ/δ (left panel, n = 3, ** p < 0.01, Student’s t test), but treatment with the PPARα agonist GW7647 (middle panel) or PPAR β/δ agonist GW0742 (right panel) did not statistical increase Serca2 expression in Control vector-treated or Yulink KD cardiomyocytes (n = 3)

Assessment of arrhythmia and irregular Ca²⁺ regulation in YULINK KD iPSC-derived human cardiomyocytes. a Representative Ca²⁺ scans and spontaneous Ca²⁺ transient in control and YULINK KD iPSC-derived human cardiomyocytes. Red arrows indicate abnormal arrhythmia-like Ca²⁺ waveforms observed in YULINK KD cardiomyocytes. b Percentages of the irregular Ca²⁺ transients in control and YULINK KD iPSC-derived human cardiomyocytes (n = 40). c Bar graph comparison of Ca²⁺ transient amplitude of human iPSC-derived cardiomyocytes for control and YULINK KD confirmed that YULINK KD human cardiomyocytes exhibited a lower Ca²⁺ transient amplitude (n = 40). d The time constant for Ca²⁺ decay (Tau) of human iPSC-derived cardiomyocytes for control and YULINK KD showed that the time constant for Ca²⁺ decay was significantly larger in YULINK KD than in control iPSC-derived human cardiomyocytes (n = 40). e Quantification of spontaneous beating rate of control and YULINK KD human cardiomyocytes (n = 40). (*p < 0.05, Student’s t test). f Human cardiomyocytes were subjected to YULINK KD using YULINK-shRNA and the effects on YULINK and SERCA2 expression were assayed by Western blot

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.