Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Knock-Out

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6- Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Quantitative RT-PCR, Purification, Control, Western Blot, Immunofluorescence

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent mean ± SEM. *P < 0.05, 1-way ANOVA.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Patch Clamp, Comparison, Activation Assay

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1- EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n = 3) plotted adjacent to average fold change expression in PCs and VMs. Genes clustered into functional groups demonstrate that ETV1 regulates a PC transcriptome in neonatal cardiomyocytes.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Expressing, Transduction, RNA Sequencing, Comparison, Labeling, Functional Assay, Purification

Journal: Scientific reports

Article Title: ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes.

doi: 10.1038/s41598-018-28239-7

Figure Lengend Snippet: Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.

Article Snippet: P1 NRVM heart lysates were purified using Miltenyi Biotec rat neonatal cardiomyocyte isolation kit (Miltenyi Biotec, 130–105–420) according to the manufacture’s protocol.

Techniques: Activation Assay, Derivative Assay, Expressing, Transduction, Quantitative RT-PCR, Patch Clamp, Comparison

Journal: The Journal of Physiology

Article Title: Remuscularization of the failing heart

doi: 10.1113/JP273098

Figure Lengend Snippet: Schematic overview of strategies aiming at the remuscularization of the failing heart either by activation of endogenous mechanisms or by cardiomyocyte supplementation.

Article Snippet: Future strategies for enhanced therapeutic efficacy of cardiomyocyte grafts Despite some evidence for efficacy of tissue‐engineered and directly injected cardiomyocyte grafts in rat (Zimmermann et al . 2006 , Riegler et al . 2015 ; Qin et al . 2016 ), mouse (Didié et al . 2013 ), guinea pig (Weinberger et al . 2016 ) and macaque (Shiba et al . 2016 ) models, there is a clear need for an improvement of graft size.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Representative super-resolution imaging of TRAF2-icKO and TRAF2 floxed control hearts following immunohistochemical staining for polyubiquitin (red) and p62 (green) to identify protein aggregates, and COXIV (blue) to identify the inner mitochondrial membrane. B, C Line scan analysis was performed along the yellow lines shown in merged images in panel A to assess the localization of p62 and polyubiquitin relative to COXIV. TRAF2 floxed hearts demonstrate p62 and polyubiquitin outside the COXIV-positive region (arrows in panel B), whereas TRAF2-icKO hearts demonstrate p62 and ubiquitin inside the region delineated with COXIV (arrows in panel C), indicating localization within the mitochondria. D, E Representative images showing immunohistochemical staining for polyubiquitin (green), p62 (red), and COXIV (blue) in postnatal day 1 TRAF2 floxed neonatal mouse cardiac myocytes (NMCMs) treated with adenoviral Cre (Ad-Cre) or adenoviral LacZ (Ad-LacZ) control virus, 3 days after adenoviral treatment. Arrows in magnified inset (E) from Ad-Cre treated cells (marked by dotted line in D) point to increased localization of polyubiquitinated proteins and p62 within COXIV-delineated mitochondria in NMCMs with adenoviral Cre-mediated deletion of TRAF2.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Imaging, Control, Immunohistochemical staining, Staining, Membrane, Virus

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Representative images of immunohistochemical staining for mitochondrial marker COXIV (red) and GFP (green) in HEK293 cells transfected with GFP-tagged wild-type CRYAB (GFP-CRYAB-WT) or GFP-tagged CRYAB with the aggregation-prone R120G mutation (GFP-CRYAB-R120G). White arrows point to co-localization of COXIV with mutant GFP-CRYAB-R120G. B Representative images of induced pluripotent stem cell (iPSC)-derived cardiac myocytes from iPSC lines homozygous for knock-in of R120G mutation and isogenic controls expressing mKate-tagged α-actinin and stained with antibody against polyubiquitin. White arrows point to co-localization of α-actinin with polyubiquitin in aggregates. C Representative immunoblot demonstrating expression of CRYAB in mitochondria rich and cytosolic sub-cellular fractions from iPSC-derived cardiac myocytes homozygous for knock-in of R120G mutation (R120G) and isogenic controls (WT). GAPDH and COXIV are shown as markers for cytosol and mitochondrial fractions respectively, and Ponceau S staining is shown for protein loading control. D, E Quantitative evaluation of CRYAB abundance in mitochondrial (D) and cytosolic (E) fractions from experiments as shown in C, normalized as indicated and expressed as fold over control. P values shown are by t-test.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Immunohistochemical staining, Staining, Marker, Transfection, Mutagenesis, Derivative Assay, Knock-In, Expressing, Western Blot, Control

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: Representative images from induced pluripotent stem cell (iPSC)-derived cardiac myocytes (cultured in aggregates for 30 days) from iPSC lines homozygous for knock-in of R120G mutation and isogenic controls expressing mKate-tagged α-actinin and stained with antibody against DESMIN. Images demonstrate localization of DESMIN and α-actinin aggregates in R120G knock-in iPSC-derived cardiac myocytes (bottom panel), as compared with localization of these proteins in a sarcomeric pattern in control iPSC-derived cardiac myocytes (top panel). DAPI stained nuclei are blue.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Derivative Assay, Cell Culture, Knock-In, Mutagenesis, Expressing, Staining, Control

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Experimental approach to deplete TRAF2 in cardiac myocytes. One Traf2 floxed allele and the cardiac myocyte-specific MerCreMer tamoxifen-inducible Cre allele were introduced into CRYAB-R120G transgenic line to generate CRYAB-R120G mice with cardiac myocyte-specific inducible deletion of one Traf2 allele (termed R120G TRAF2-icHET). These mice were compared to R120G TRAF2 fl/+ mice (without the MerCreMer transgene) and to TRAF2 fl/+ and TRAF2-icHET control mice without CRYAB-R120G. Mice were treated with tamoxifen chow for one week starting at 8 weeks of age. Echocardiography was done at 20 weeks of age, and survival was monitored to 60 weeks. B Representative M-mode echocardiograms for 20-week-old mice in indicated groups. White arrows indicate left ventricular internal diastolic diameter, red arrows indicate left ventricular internal systolic diameter. C Assessment of left ventricular systolic function by fractional shortening in 20-week-old mice. Fractional shortening was reduced in R120G TRAF2-icHET mice versus R120G TRAF2 fl/+ controls. P values shown are by one-way ANOVA followed by Tukey’s test for multiple comparison testing between groups. D Kaplan-Meier survival analysis of R120G TRAF2-icHET mice, which exhibited earlier mortality versus CRYAB-R120G TRAF2 fl/+ mice. P value shown is by Mantel-Cox log-rank testing. E Representative hematoxylin and eosin, and Masson’s trichrome-stained images of hearts from 20-week-old mice from indicated groups modeled as in A. F Representative transmission electron microscopy of heart tissue from 20-week-old mice from indicated groups. CRYAB-R120G TRAF2-icHET hearts exhibit disrupted mitochondrial morphology with cristal rarefaction (white arrows) and mitochondrial protein aggregates (black arrowheads).

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Transgenic Assay, Control, Comparison, Staining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Left ventricular internal diastolic diameter by M-mode echocardiography in 20-week-old CRYAB-R120G mice with cardiac myocyte-specific inducible deletion of one Traf2 allele (termed R120G TRAF2-icHET; see for experimental schematic). These mice were compared to R120G TRAF2 fl/+ mice without the MerCreMer transgene, and to TRAF2 fl/+ and TRAF2-icHET control mice without CRYAB-R120G. Statistical comparisons were performed by one-way ANOVA followed by Tukey’s test for multiple comparison testing between groups. There were no statistically significant differences between groups in panel A. B Kaplan-Meier survival analysis of TRAF2-icHET mice and TRAF2 fl/+ controls. No statistically significant differences were detected by Mantel-Cox log-rank testing.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Control, Comparison

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Representative immunoblot depicting expression of TRAF2, polyubiquitinated proteins and p62 in cytosolic and mitochondrial fractions from 20-week-old CRYAB-R120G mice with cardiac myocyte-specific inducible deletion of one Traf2 allele at 8 weeks of age (termed R120G TRAF2-icHET), and similarly treated R120G TRAF2 fl/+ mice, TRAF2-icHET and TRAF2 fl/+ as control as modeled in . GADPH was used to demonstrate separation of the cytosolic fraction, and LONP1, VDAC and COXIV were used as markers of the mitochondrial fraction. B Quantitation of TRAF2 levels in the cytosolic fractions from TRAF2-icHET and TRAF2 fl/+ mice treated as in A. P value shown is by Mann-Whitney test. C Quantitation of polyubiquitinated proteins in the mitochondrial fractions from R120G TRAF2-icHET and R120G TRAF2 fl/+ mice treated as in A. P value shown is by Mann-Whitney test.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Western Blot, Expressing, Control, Quantitation Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A Experimental approach to overexpress TRAF2 in cardiac myocytes. Wild-type (WT) and transgenic CRYAB-R120G mice were injected with AAV9 expressing TRAF2 (AAV9-TRAF2) or control AAV9 expressing GFP (AAV9-GFP). Cardiac troponin T promoter was employed in both viruses to specifically target cardiac myocytes. AAVs were injected via tail-vein at a dose of 7.0 X 10 11 viral genomes/mouse at 8 weeks of age. Echocardiography was performed at 20 weeks, and survival was monitored to 100 weeks. B Representative M-mode echocardiograms for 20-week-old mice in indicated groups modeled as in A. White arrows indicate left ventricular internal diastolic diameter; red arrows indicate left ventricular internal systolic diameter. C Assessment of systolic function by fractional shortening in 20-week-old mice. Left ventricular fractional shortening is improved in CRYAB-R120G mice treated with AAV9-TRAF2 versus AAV9-GFP. P values are by one-way ANOVA followed by Tukey’s test for multiple comparison testing between groups. D Survival of WT and CRYAB-R120G mice injected with AAV9-GFP and AAV9-TRAF2 viruses. CRYAB-R120G AAV9-TRAF2 mice exhibited improved survival versus CRYAB-R120G AAV9-GFP mice. P value shown is by Mantel-Cox log-rank testing. E Representative hematoxylin and eosin and Masson’s trichrome-stained images of hearts from 20-week-old mice from indicated groups modeled as in A. F Transmission electron microscopy of heart tissue from 20-week-old mice from indicated groups. AAV9-TRAF2 treatment improved mitochondrial morphology in CRYAB-R120G mice relative to AAV9-GFP treatment in these animals. Increased prevalence of mitochondria sequestered within double membranes, suggesting mitochondria undergoing mitophagy, was also seen in CRYAB-R120G AAV9-TRAF2 hearts (see inset). G, H High-resolution respirometry was performed to measure oxygen consumption [volume-specific oxygen flux (JO2)] in isolated, permeabilized cardiac fiber bundles from mice modeled as in A in the presence of succinate (to assess complex I+II in g) and FCCP (to assess maximal respiration in h). P values are by one-way ANOVA followed by Tukey’s post-hoc test for multiple comparison testing.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Transgenic Assay, Injection, Expressing, Control, Comparison, Staining, Transmission Assay, Electron Microscopy, Isolation

Journal: bioRxiv

Article Title: Mitophagy Facilitates Cytosolic Proteostasis to Preserve Cardiac Function

doi: 10.1101/2024.11.24.624947

Figure Lengend Snippet: A-E Representative immunoblots (A) of cytosolic and mitochondrial fractions from 20-week-old wild-type (WT) AAV9-GFP, WT AAV9-TRAF2, CRYAB-R120G AAV9-GFP, and CRYAB-R120G AAV9-TRAF2 hearts. Mice were injected with AAV virus at 8 weeks, as previously described in . Quantitation of cytosolic TRAF2, mitochondrial TRAF2, mitochondrial polyubiquitin, and mitochondrial p62 levels are shown in B, C, D, and E respectively, with normalization to total protein level as assessed by Ponceau S staining. Values shown as fold change over control. P values are by t-test for B, D and E; and by Mann-Whitney test for panel C. F Representative images depicting immunohistochemical analysis to assess DESMIN localization in cardiomyocytes. CRYAB-R120G AAV9-GFP hearts exhibited disruption of normal striated DESMIN pattern seen in WT AAV-GFP and WT AAV9-TRAF2 mice, with localization of DESMIN to aggregates. DESMIN localization (pseudo colored in white) was partially rescued with reduced aggregates in CRYAB-R120G AAV9-TRAF2 mice. G Quantitative assessment of protein aggregates seen by histology in mice treated as in A at 20 weeks of age. P values are by one-way ANOVA followed by Tukey’s post-hoc test.

Article Snippet: On the 2nd day, the culture medium was changed to the Rat Cardiomyocyte Culture Medium (Cell applications INC, CAT#R313-500) for at 3-5 days prior to staining.

Techniques: Western Blot, Injection, Virus, Quantitation Assay, Staining, Control, MANN-WHITNEY, Immunohistochemical staining, Disruption

Journal: Cell reports methods

Article Title: A size-exclusion-based approach for purifying extracellular vesicles from human plasma

doi: 10.1016/j.crmeth.2021.100055

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Skeletal Muscle Cell Growth Medium , Cell Applications , Cat# 151K-500.

Techniques: Purification, Recombinant, Sequencing, Modification, Mass Spectrometry, Saline, Isolation, Software, Pore Size

Journal: The Journal of Biological Chemistry

Article Title: Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

doi: 10.1016/j.jbc.2024.107254

Figure Lengend Snippet: Nesprin-2 redistributes from the NE to the sarcomere during myogenesis. A , schematic showing nesprin-2 giant gene and protein structure. The alternative novel stop codon is shown upstream of the KASH domain of nesprin-2 giant, which was generated by the retention of part of the intronic sequence (123 bp) immediately downstream of the penultimate exon 115, colored in pink ). The putative partial nesprin-2 KASH-less isoform was shown with the position of nesprin-2 N3 (Nes-2N3) antibody indicated, which was specifically generated against the SR52 close to the C-terminus of nesprin-2. B , immunofluorescence showed nesprin-2 localised to the NE at E12.5 and was present in the sarcomere in E16.5 and during development as observed (inserts, yellow arrowed ), colocalised with alpha-actinin ( red ) for the sarcomere and emerin ( red ) for the NE. C , Nesprin-2 ( green ) is present at the Z-disc and I band in the sarcomere in adult mouse cardiomyocytes, colocalised with α-actinin ( red ) and SERCA-2 ( red ). Myomesin ( red ) as a M-band marker, nuclei shown in blue with Dapi, Colocalisation quantification data is shown with Pearson’s coefficient for α-actinin (0.61), myomesin (0.10) and SERCA-2 (0.55), respectively. D , Western blot demonstrated dramatic changes in the production of nesprin-2 isoforms during mouse heart embryonic development, previously published nesprin isoforms are indicated ( , ). E , qPCR verified this partial nesprin-2 KASH-less isoform that contains the SR52-53, which is exclusively present in heart tissue.

Article Snippet: Neonatal rat cardiomyocyte (NRC) isolation was performed using a Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp) following the manufacturer's instructions.

Techniques: Generated, Sequencing, Immunofluorescence, Marker, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

doi: 10.1016/j.jbc.2024.107254

Figure Lengend Snippet: The binding between nesprin-2/telethonin is tightly regulated by telethonin phosphorylation. A , HA-tagged WT and S157/161A mutated telethonin protein with or without PKD pre-treatment were incubated with GST-nesprin-2 SR51-53 beads for a GST pull-down assay. The nesprin binding for wild-type (WT) or mutant recombinant telethonin proteins was quantified by densitometry with respect to the input material and expressed as a ratio of the value obtained for unphosphorylated WT telethonin. Five independent experiments were performed shown as mean ± SD, ∗∗∗∗ p < 0.0001. F (7, 32) =209.5 using one-way ANOVA analysis; Bonferroni post-hoc tests showed ∗∗∗∗ p < 0.0001 for WT- versus WT+, S157- versus S157+, S161- versus S161+ respectively. B , NRCs were infected with adenovirus carrying HA-tagged WT telethonin or S157/161A double mutants. Protein lysates were harvested 48 h after adenoviral infection and then subjected to immunoprecipitation using a telethonin antibody. Western blot showed nesprin-2 was significantly pulled down in neonatal cardiomyocytes with over-expressed unphosphorylatable S157/161A telethonin. HA antibody used on the same blot showed an equal amount of HA-tagged telethonin pulled down by telethonin antibody. The intensity of about ∼200 kDa nesprin pull-down bands was measured by densitometry with respect to the input material and relative binding to nesprin expressed as a ratio of the value obtained between mutant and WT telethonin. IP was repeated for five times. Student’s t test (unpaired, two-tailed) showed a significantly higher binding strength between nesprin and S157/161A double mutated telethonin than the WT. ∗∗∗∗ p < 0.0001.

Article Snippet: Neonatal rat cardiomyocyte (NRC) isolation was performed using a Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp) following the manufacturer's instructions.

Techniques: Binding Assay, Incubation, Pull Down Assay, Mutagenesis, Recombinant, Infection, Immunoprecipitation, Western Blot, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

doi: 10.1016/j.jbc.2024.107254

Figure Lengend Snippet: A potential role of nesprin-2 in regulating hypertrophic response. A , Western blot showed a ∼200 kDa nesprin-2 isoform was significantly up-regulated in the nuclear fraction of the H9C2 cells treated with phenylephrine, 25 kDa nesprin in the cytoplasmic fraction could be potential isoforms due to extensively alternative initiation and splicing or degradation products, further verification needed. B and C , Western blot and immunofluorescence showed nesprin-2 isoforms, especially those containing SR 52-53 redistributes from the sarcomere to the NE in hypertrophic cardiomyocytes in a transverse aortic constriction (TAC) mouse model ( arrowed ), and FHL-2 subcellular localization was affected shown in ( C , arrow head ).

Article Snippet: Neonatal rat cardiomyocyte (NRC) isolation was performed using a Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp) following the manufacturer's instructions.

Techniques: Western Blot, Immunofluorescence