PICALM Regulating the Generation of Amyloid β-Peptide to Promote Anthracycline-Induced Cardiotoxicity

Abstract

Anthracyclines are chemotherapeutic drugs used to treat solid and hematologic malignancies. However, life-threatening cardiotoxicity, with cardiac dilation and heart failure, is a drawback. A combination of in vivo for single cell/nucleus RNA sequencing and in vitro approaches is used to elucidate the underlying mechanism. Genetic depletion and pharmacological blocking peptides on phosphatidylinositol binding clathrin assembly (PICALM) are used to evaluate the role of PICALM in doxorubicin-induced cardiotoxicity in vivo. Human heart tissue samples are used for verification. Patients with end-stage heart failure and chemotherapy-induced cardiotoxicity have thinner cell membranes compared to healthy controls do. Using the doxorubicin-induced cardiotoxicity mice model, it is possible to replicate the corresponding phenotype in patients. Cellular changes in doxorubicin-induced cardiotoxicity in mice, especially in cardiomyocytes, are identified using single cell/nucleus RNA sequencing. Picalm expression is upregulated only in cardiomyocytes with doxorubicin-induced cardiotoxicity. Amyloid β-peptide production is also increased after doxorubicin treatment, which leads to a greater increase in the membrane permeability of cardiomyocytes. Genetic depletion and pharmacological blocking peptides on Picalm reduce the generation of amyloid β-peptide. This alleviates the doxorubicin-induced cardiotoxicity in vitro and in vivo. In human heart tissue samples of patients with chemotherapy-induced cardiotoxicity, PICALM, and amyloid β-peptide are elevated as well.

Keywords: amyloid β‐peptide; cardiomyocytes; cardiotoxicity.

Figure 1

Histological performance of patients with chemotherapy‐induced cardiotoxicity. A) Natural history of the patients, from the diagnosis of malignancy to heart transplantation. B) Representative CMR image of patient No. 1, including LGE. C) Representative graphs of macro‐views of patients with chemotherapy‐induced cardiotoxicity. D) Representative graphs of hematoxylin and eosin (HE) staining of patients with chemotherapy‐induced cardiotoxicity. Scale bars, 2000 µm; scale bars for the magnified images, 50 µm. E–G) Representative and statistical graphs of patients with WGA/Masson/TUNEL‐stained chemotherapy‐induced cardiotoxicity. Scale bars, 2000 µm; scale bars for the magnified WGA/Masson/TUNEL‐stained images, 200/100/50 µm. H) Representative electron micrographs of cardiac muscles derived from the human heart. Scale bars, 1 µm; scale bars for the magnified images, 0.5 µm. NC, normal control; CICP, chemotherapy‐induced cardiotoxicity patient. n = 3/group. Statistical analyses were performed using an unpaired t‐test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 2

SnRNA‐seq and scRNA‐seq reveal the cellular landscape of DOX‐induced cardiotoxicity. A) Flowchart of our study including the experimental scheme of chronic cardiotoxicity induced by DOX, scRNA‐seq, snRNA‐seq, and analysis for data and experiment. B) Representative images and statistical graphs of cardiac echocardiography of mice injected with phosphate‐buffered saline (PBS) or DOX. EF: ejection fraction; FS: fractional shortening. C) The BW from weeks 6 to 10. D) Body and heart weight of DOX‐treated mice group on week 10. E,F) HE and changes in collagen volume fraction with and without DOX administration. Scan bars, 500 µm; scale bars for the magnified images, 50 µm. G) Representative and statistical graphs of WGA staining in DOX‐treated mice at the tissue level. Scan bars, 50 µm; scale bars for the magnified images, 20 µm. H) Representative and statistical graphs of TUNEL staining in DOX‐treated mice at the tissue level. Scan bars, 500 µm; scale bars for the magnified images, 50 µm. I) UMAP of 18956 nuclei, 24365 cells, and an integrated dataset combining snRNA‐seq and scRNA‐seq data after quality control and data filtering. J,K) Dot plots generated from the integrated dataset displaying characteristic marker genes of each identified cell population, and pie charts showing the proportion of cells within the snRNA‐seq, scRNA‐seq, and integrated datasets. NC, normal control (PBS‐treated mice); DOX, DOX‐treated mice. n = 5/group. Statistical analyses were performed using an unpaired t‐test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 3

PICALM upregulation in CMs of DOX‐treated mice groups and patients with chemotherapy‐induced cardiotoxicity. A) UMAP plot for sub‐cluster analysis of CMs and cardiomyocyte cluster proportions in PBS‐ and DOX‐treated mice. B) Dot plots showing the markers for each cell subcluster. C) Heatmap displaying the top 20 upregulated and downregulated genes ranked by Log2 fold‐change between DOX and NC. D) A heat plot of the associations between upregulated genes and pathways determined by KEGG analysis. Squares indicate that the corresponding gene belongs to the corresponding pathway. E) Heat map showing dynamic changes in gene expression with pseudo‐time. The x‐coordinate from left to right represents the time from small to large, and the y‐coordinate represents the gene. Each point represents the expression of a specific gene at a specified pseudo‐time quantity (mean value). “Cluster” refers to the clustering of genes, clustering genes with similar or similar expression patterns into one cluster. F) Monocle dimension reduction diagram showing the expression of Picalm. G) Survival analysis of patients with breast cancer with high or low expression of PICALM in tumor samples according to TCGA database. H) Diagram showing the density distribution of different cell types over pseudo‐time. I) OMIHC representative and statistical graphs of PICALM expression in myocardial cells of DOX‐treated mice at different induction times at the tissue level. N > 25 CMs. Scan bars, 500 µm; scale bars for the magnified images, 20 µm. J) OMIHC representative and statistical graphs of PICALM expression in the myocardial cells of patients with chemotherapy‐induced cardiotoxicity. Scan bars, 2000 µm; scale bars for the magnified images, 200 µm. NC, normal control; DOX, DOX‐treated mice; CICP, chemotherapy‐induced cardiotoxicity patients. Mice: n = 5/group; human: n = 3/group. Student's t‐test was applied to analyze the differences between two groups. Multiple‐group comparisons were made by one‐way ANOVA followed by the Tukey test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 4

Amyloid β metabolic process pathway and related genes were the major issue in DOX‐induced cardiotoxicity mice and patients with chemotherapy‐induced cardiotoxicity. A) Bar plot for scGSVA analysis of CMs. B) Heat plot of the associations between genes and pathways. C) Violin plots showing the expression of Lpin1/Psap/Gnptab/App in each cardiomyocyte cluster. D,E) Representative and statistical graphs of APP expression at the myocardial cells in DOX‐induced cardiotoxicity group and patients. Scan bars, 50 µm; scale bars for the magnified images, 20 µm. F) Diagram showing the potential mechanisms of DOX induction in CMs. G,H) Representative and statistical graphs of Aβ40, PICALM expression at the CMs marked by TNNT2 in DOX‐induced cardiotoxicity mice, and a diagram showing the correlation of PICALM and Aβ40 in mice CMs. Scan bars, 1000 µm; scale bars for the magnified images, 200 µm. I,J) Representative and statistical graphs of Aβ40, PICALM expression at the CMs marked by TNNT2 in patients with chemotherapy‐induced cardiotoxicity, and a diagram showing the correlation of PICALM and Aβ40 in human CMs. Scan bars, 500 µm; scale bars for the magnified images, 50 µm. K) ELISA analysis of mouse serum Aβ40 level in DOX‐induced cardiotoxicity. L) ELISA analysis of human serum Aβ40 level in chemotherapy‐induced cardiotoxicity and plasma Aβ40 levels could distinguish CICP from non‐CICP (AUC = 0.94, P = 0.03). NC, normal control; DOX, DOX‐treated mice; CICP, chemotherapy‐induced cardiotoxicity patients. Mice: n = 5/group in RNA‐seq and n = 8/group in immunoimaging; human: n = 3/group. Student's t‐test was applied to analyze the differences between two groups. Multiple‐group comparisons were made by one‐way ANOVA followed by the Tukey test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 5

Si‐Picalm can alleviate DOX‐induced hiPSC‐CMs damage and Aβ peptide production. A) Cell viability in different groups. Cells were treated with the indicated concentration of DOX (0–10 µm). After incubation, cell viability was measured by CCK8 assay. B,C) Statistical analysis of vesicle area, PICALM and Aβ40 level of hiPSC‐CMs treated with 1 µM DOX for 0, 12, 24, and 36 h with confocal microscope (Zeiss) by using ImageJ plugins.^{[ 23 ]} D) The statistical graphs determined using an IncuCyte imaging system. H9c2 cells pre‐stained with 0.5 µM LysoTracker Red for 30 min were treated with 1 µm DOX for 24 h. E) The statistical graphs of LPIN1 expression determined by confocal. F) The WGA staining of DOX‐treated cells and statistical graphs of vesicles area. G) Representative images of ROS and calcium concentration of hiPSC‐CMs treated with DOX or Aβ40 and statistical analysis. Scan bars, 50 µm. H) Representative images of propidium iodide (PI)‐staining of hiPSC‐CMs treated with DOX or Aβ40 and statistical analysis of apoptosis and necrotic cell percentage. Scan bars, 200 µm. I) Statistical analysis of Aβ40 level of hiPSC‐CMs using ELISA in the medium. J) The western blot of the hiPSC‐CMs groups including SI‐NC, SI‐PICALM, DOX+SI‐NC, DOX+SI‐PICALM, and statistical graphs of PICALM, APP, and Aβ40 expression. K,L) Representative images and statistical analysis of WGA/LPIN1 staining in the hiPSC‐CMs groups. Scan bars, 200 µm. M) Representative images and statistical analysis of lysosome tracking in the hiPSC‐CMs groups. Scan bars, 50 µm. N) The cell viability in groups. O,P) Representative images and statistical analysis of TUNEL assay in the hiPSC‐CMs groups. Scan bars, 200 µm. DOX, DOX‐treated cells; SI‐NC, SI‐GFP; OE‐NC, overexpression vector. Student's t‐test was applied to analyze the differences between two groups. Multiple‐group comparisons were made by one‐way ANOVA followed by the Tukey test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 6

Genetic depletion of PICALM attenuated DOX‐induced cardiotoxicity in vivo. A) The treatment experiment design. B) Survival analysis of the three groups. C) Representative and statistical graphs of EF and FS evaluated by echocardiography of normal control, DOX induction on wild‐type mice and DOX induction of Picalm knockout mice. D,E) Representative and statistical graphs of HE and Masson staining of normal control, DOX induction on wild‐type mice and DOX induction of Picalm knockout mice. F) The IHC images of TUNEL of normal control, DOX induction on wild‐type mice and DOX induction of Picalm knockout mice. G) Representative and statistical graphs of WGA, Aβ40, PICALM expression at the CMs marked by TNNT2 in normal control, DOX induction on wild‐type mice and DOX induction of Picalm knockout mice. NC, normal control, n = 5; DOX, DOX‐treated mice, n = 5; DOX+KO, DOX‐treated Picalm knockout mice, n = 6. Student's t‐test was applied to analyze the differences between two groups. Multiple‐group comparisons were made by one‐way ANOVA followed by the Tukey test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.

Figure 7

DOX‐induced cardiotoxicity is suppressed by anti‐PICALM antibody BP. A) Treatment experiment design. B) Representative and statistical graphs of EF and FS evaluated by echocardiography in the control, DOX, and DOX + PICALM BP group. C,D) Representative and statistical graphs of HE and Masson staining of the control, DOX, and DOX + PICALM BP group. E) IHC images of TUNEL of the control, DOX, and DOX + PICALM BP group. F) Representative graphs of WGA, Aβ40, PICALM expression at the CMs marked by TNNT2 in the control, DOX, and DOX + PICALM BP group. G) Statistical graphs of TUNEL‐positive cells, and PICALM, Aβ40 expression at the CMs marked by TNNT2 in the control group, DOX, and DOX + PICALM BP group. DOX, DOX‐treated mice. Mice: n = 4/group. Student's t‐test was applied to analyze the differences between two groups. Multiple‐group comparisons were made by one‐way ANOVA followed by the Tukey test. ^* P < 0.05; ^** P < 0.01; ^*** P < 0.001. Data are presented as the mean ± SD.