Unveiling the Antiviral Properties of Panduratin A through SARS-CoV-2 Infection Modeling in Cardiomyocytes

Figures

• 

  Figure 1. Characterization of cultured cardiac myocytes. (a) Expression of cardiac markers in iPSC-CMs. Differentiated myocytes (>day20) were stained for expression of cardiac trophoninT (green), α-actinin (red) and MLC2V (red), respectively. (b) Expression of SARS-CoV-2 host receptors in iPSC-CMs (i–iii) and AC16 myocytes (iv–vi). Differentiated myocytes (>Day20) and AC16 cells were stained for expression of SARS-CoV-2 host cell markers ACE2 (red) and neurophilin-1 (green). Nuclei were counterstained with Hoechst 33342 (blue). Cells without primary antibodies were used as controls. Scale bar 50 µm. (c) Mean fluorescence intensity of cardiac markers (cTNT, α-actinin) and SARS-CoV-2 host receptors (ACE2, neuropilin-1) in iPSC-CM and AC16 cells. Statistical significance was analyzed using independent Student’s t-tests. p value < 0.01 was considered significant (**).

• 

  Figure 2. SARS-CoV-2 infectivity model of iPSC-CMs and AC16 cells. (a) iPSC-CMs (MOI: A = 12.5; B = 3.1; C = 0.78; D = 0.2; E = 0.05; F = 0.01; G = 0.003; H = 0.001; I = 1.9 × 10−4; J = 4.8 × 10−5; K = 1.2 × 10−5; L = Mock). Scale bar 50 µm. (b) Viral output of infected iPSC-CMs at different MOIs (left panel) and infectivity of SAR-CoV-2 on iPSCM (right panel). (c) AC16 cells (A = 12.5; B = 3.1; C = 0.78; D = 0.2; E = 0.05; F = 0.01; G = 0.003; H = 0.001; I = 1.9 × 10−4; J = 4.8 × 10−5; K = 1.2 × 10−5; L = Mock). Scale bar 50 µm. Data in figure (b) are represented as mean ± SEM values (n = 3 technical replicates), and values on the chart legend of figure (b) (left panel) are MOIs. Cells were inoculated with the SARS-CoV-2 virus, and infected cells were determined via the presence of NP (red). Microscopy images (10×) were captured 3 days post infection. A viral plaque assay was used to measure the viral output.

• 

  Figure 3. Modeling drug sensitivity and toxicity screening in cultured myocytes. (a) CC50 values of doxorubicin, remdesivir, and panA. Data are represented as mean ± SEM values (n = 3 independent experiments). (b) Representative figures illustrating the cytotoxic effects of doxorubicin, remdesivir, and panA at various concentrations (0–100 µM) on human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) and AC16 cells using an MTT cell viability assay. The percentages of cell viability after 48 h of treatment relative to the non-treated control (set as 100%). Each data point (black dot) indicates as mean ± SD values (n = 3 technical replicates). The red line represents the 50% cytotoxic concentration (CC50).

• 

  Figure 4. In vitro inhibition of SARS-CoV-2 in iPSC-CMs. (a) Myocytes were infected with SARS-CoV-2 in treatments using different concentrations of panA, remdesivir, or a dimethyl sulfoxide (DMSO) control. At 48 h post infection, cells were fixed and subjected to immunofluorescent staining by employing anti-NP (red) rabbit sera. The nuclei were stained with Hoechst dye (blue). (b) The left and middle panels show an evaluation of the anti-SARS-CoV-2 activity of panA. The graph shows the dose–response relationship, with the blue line indicating a 50% inhibition concentration (IC50) of 0.69 μM, and the red line representing the cytotoxic concentration that affects 50% of the cells (CC50) after 48 h of treatment, which was determined to be 10.09 μM. Each data point (black dot) refers to the mean ± SD (n = 3 technical replicates). (b) Right panel, illustration of viral output observed in SARS-CoV-2-infected iPS-CMs following treatment with panA at various concentrations (right symbols, µM). Error bars in the graphs represent SEM values (n = 3 technical replicates).