Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Infection of iPSC-CMs by SARS-CoV-2 in vitro results in increased cellular cytotoxicity. (A) Immunofluorescence staining shows infection of iPSC-derived cardiomyocytes. Nuclei (blue), α-Actinin (grey) and Nucleocapsid (red). Scale bar: 50 μm.(B) Quantification of infected cells at 24 h post-infection. (C) Infection of iPSC-derived cardiomyocytes is confirmed by expression of SARS-CoV-2 Spike RNA. (D) Western blot analysis confirms Spike Protein expression, quantified in (E). Full western blots in Suppl. Fig. 3A. (F) Cytotoxicity upon SARS-CoV-2 infection determined by activity of LDH released into the medium. Protein expression is normalized to β-actin, gene expression is normalized to GAPDH. n = 3 iPSC-CM differentiation rounds for all but (B), where n = 1 iPSC-CM differentiation round, statistical significance determined by Wilcoxon test (B), Kruskal-Wallis test (C, D), students t -test (F). p < 0.0001 = ****. Data are displayed as mean ± SD. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, In Vitro, Immunofluorescence, Staining, Derivative Assay, Expressing, Western Blot, Activity Assay, Gene Expression

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD. PDH phosphorylation is unaffected following SARS-CoV2 infection. (A) Cytotoxicity after Cyclosporine A (CsA) treatment [0.5 μM, 2 h] as determined by activity of LDH released into the medium. (B) Quantification of pyruvate dehydrogenase A1 (PDHA1) protein expression and its phosphorylation on serine 293. Values normalized to α-tubulin. (C) Representative Western Blot. (D) Quantification of CI = Complex I, CII = Complex II, CIII = Complex III, CIV = Complex IV and CV = Complex V normalized to α-tubulin. Full western blots in Suppl. Fig. 3B. (E) Protein expression of the respiratory chain following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3B. (F) Apoptosis rate determined by TUNEL staining 24 h post-infection with an MOI of 0.1. (G) Quantification of procaspase 3 and cleaved caspase 3 protein expression. Values normalized to α-tubulin. Corresponding western blots in Suppl. Fig. 3C. n = 3 iPSC-CM differentiation rounds for all but (A, B), where n = 1 iPSC-CM differentiation round. Statistical significance determined by two-way ANOVA (A, B), Mann-Whitney test (F) or t-test (D, G). p ≤ 0.05 = *, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Phospho-proteomics, Infection, Activity Assay, Expressing, Western Blot, TUNEL Assay, Staining, MANN-WHITNEY

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: SARS-CoV-2-infected cardiomyocytes exhibit upregulated necroptosis, but no evidence of mitochondrial permeability transition

doi: 10.1016/j.jmccpl.2025.100833

Figure Lengend Snippet: Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD. Increased necroptosis following SARS-CoV-2 infection. (A) Cytotoxicity after Necrostatin-1 (Nec-1) [2.5 μM] or MCC950 [0.05 μM] treatment as determined by activity of LDH released into the medium. (B) Spike- (C) RIP3 and (D) NLR family pyrin domain containing 3 (NLRP3) inflammasome gene expression following SARS-CoV-2 infection. Gene expression normalized to GAPDH. (E) Quantification of RIP3 and RIP3-S227 protein expression after Nec-1 treatment. (F) Representative WB for RIP3 and RIP3-S227 following SARS-CoV-2 infection. Full western blots in Suppl. Fig. 3D. (G) Quantification of MLKL and MLKL-S358 protein expression with a representative WB (H). Full western blots in Suppl. Fig. 3E. n = 3 iPSC-CM differentiation rounds, statistical significance determined by two-way ANOVA (A) or Kruskal-Wallis test (B–E) or Mann-Whitney (G). p < 0.01 = **, p < 0.0001 = ****. Data are displayed as mean ± SD.

Article Snippet: The following antibodies were used in a 1:500 dilution: α-Actinin (Sigma, #A7811) and SARS-CoV-2 Nucleocapsid (SinoBiological, #40143-T62).

Techniques: Infection, Activity Assay, Gene Expression, Expressing, Western Blot, MANN-WHITNEY