The SARS-CoV-2 Spike protein induces long-term transcriptional perturbations of mitochondrial metabolic genes, causes cardiac fibrosis, and reduces myocardial contractile in obese mice

Fig 1. LDL-c augments Spp viral entry which can be blocked by a SR-B1 inhibitor BLT-1. (A) Spp virus shows a higher maximal binding affinity to LDL-c than HDL-c. The biotinylated LDL-c and HDL-c were incubated with various quantities of Spp virus. The lipoprotein cholesterol bound virus was determined by mCherry RNA copies. (B-E) LDL-c significantly increases cellular uptake of Spp virus in HDMVEC (B), MØ (C), LDLr−/− MØ (D), and H9C2 at 2 hpi (E) (p<0.001 or 0.005). The addition of BLT-1 significantly blocks the LDL-c augmented Spp viral uptake in HDMVEC (B), MØ (C), LDLr−/− MØ (D), and H9C2 (E) at 2 hpi ( p<0.02 or 0.001).

Fig 2. Cardiac Spp viral accumulation in obese mice. (A) Outline of the animal treatment protocol. The C57BL/6J mice (2 months old) were fed normal chow or HFD. The viral administration was given at 3 months later (5 months old). The animals were remained on the same types of diets until being sacrificed at 2 hpi, 24 hpi, 3 wpi, 6 wpi, and 25 wpi. (B) Quantification of serum levels of total cholesterol, LDL/VLDL, and HDL in HFD mice and the NCF group. # indicates p < 0.01 for intergroup comparison. (C) The relative protein levels of SR-B1 in hearts and adipose tissues in HFD mice and the NCF group by Western blot. Blots from two animals per group were shown. # indicates p < 0.01 in HFD vs NCF group for SR-B1 with the 75Kd MW band (**). (D) Systemic dissemination of Spp virus in HFD mice. Viral load was determined by ratios of viral mCherry levels to housekeeping gene RPS18 levels in each tissue using a real time RT-PCR (Log 10 scale in Y-axis). & indicates statistical significance in HFD group as compared with normal mice group 2 hpi or 24 hpi. Dashed line: the average Spp level in the lungs at 24 hpi. *, indicates statistical significance at 24 hpi as compared with 2 hpi in adipose…

Fig 3. Transient administration of Spp virus induces long-term changes in cardiac transcriptome in obese mice. (A) Volcano plot showing the cardiac DEs in HFD mice 3 wpi as compared with control HFD mice. FDR < 0.05 (horizontal dashed line) is used for cut-off threshold. Right vertical dashed line: fold changes (FC) > 0.5 (increase); left vertical dashed line: FC < - 0.5 (decrease). Blue dots represent downregulated DEs (n=140) and red dots represent upregulated DEs (n=69). (B) Ranked heatmap showing the individual downregulated (left two panels) and upregulated DEs (right panel), which is ranked by FDR. (C) Scatter plot showing significant downregulations of gene families in ATP synthases and NDUFs (FDR < 0.05). Dashed lines in both panels represent FC = ± 0.5 levels. Blue dots represent downregulated DEs in ATP synthase (n = 10 out of 32) or NDUF (n = 9 out of 35) family.

Fig 4. Cardiac DE profiles in mitochondria respiratory chain complex in response to viral administration in obese mice. Normalized TPMs for individual DEs in ATP synthase (A), NDUF (B), and COX (C) families were plotted crossing six sample groups, e.g., NCF mice control or 24 hpi or 3 wpi, HFD mice control or 24 hpi or 3 wpi. The TPMs were extracted from RNA-seq datasets which was analyzed using a Rstudio package. As compared with HFD mouse controls, these genes typically showed significant decreases in HFD mice 3 wpi but not in other groups. &, FDR < 0.05 as compared with HFD mouse control group; #, FDR < 0.05 as compared with NCF control mouse group.

Fig 5. Upregulated cardiac DE profiles in response to viral administration in obese mice. Normalized TPMs for individual DEs in categories of stress pathway / kinase activity (A), glucose metabolism (B), and cardiovascular functions (C) were plotted crossing six sample groups, e.g., NCF mice control or 24 hpi or 3 wpi, HFD mice control or 24 hpi or 3 wpi. The TPMs were extracted from RNA-seq datasets which was analyzed using a Rstudio package. As compared with HFD mouse controls, these genes typically showed significant increases in HFD mice 3 wpi but not in other groups. &, FDR < 0.05 as compared with HFD mouse control group; #, FDR < 0.05 as compared with NCF control mouse group.

Fig 6. Long-term changes in cardiac DE transcripts in obese mice in response to viral administration. Real time RT-PCR assay was performed to evaluate the relative mRNA levels of representative downregulated (A) and upregulated (B) DE transcripts in the following groups, NCF or HFD mice post Spp administration for 6 or 24 wpi, age-matched NCF or HFD control mouse groups (8.5 or 13 months old). (C) Relative cardiac protein levels of some representative DEs such as ATP5C1, NDUFS3, NDUFA2, OGDH, PYGB, and STAT5A at 6 wpi using Western blot assay. #, p < 0.05 as compared with the age-matched control groups with matching ages using two sample t test.

Fig 7. Development of cardiac fibrosis and cardiac functional changes in obese mice in response to viral administration. (A) Masson’s trichrome staining on heart cross sections at 6 wpi. The development of focal fibrosis is evident in the HFD_SPP group (black boxed area). (B) a high magnification of the boxed area in the HFD_SPP group in (A). (C) a high magnification of the boxed area in (B). Blue arrow heads indicate the focal fibrotic areas. (D) Quantitative analysis of the percentage of fibrotic areas in each group. Mann-Whitney test was used to compare the differences among two groups. Whiskers are presented as mean ± S.D. with the diamond boxed showing interquartile ranges of the data. (E) Comparison of echo parameters such as EF (%), FS (%), LV Vol s (μL), and LVID s (mm) between the HFD mice 24 wpi and the age-matched HFD controls (13 months old). #, p < 0.05. (F) A proposed mechanistic model for cardiomyopathy in PACS under obese conditions which are likely resulted from multifactorial contributions: spike protein impairs lipid metabolisms and increases lipotoxicity-associated ferroptosis; lipids promote viral administration and facilitate cardiac viral accumulation; and viral…