Pitx2 maintains mitochondrial function during regeneration to prevent myocardial fat deposition

Abstract

Loss of the paired-like homeodomain transcription factor 2 (Pitx2) in cardiomyocytes predisposes mice to atrial fibrillation and compromises neonatal regenerative capacity. In addition, Pitx2 gain-of-function protects mature cardiomyocytes from ischemic injury and promotes heart repair. Here, we characterized the long-term myocardial phenotype following myocardial infarction (MI) in Pitx2 conditional-knockout (Pitx2 CKO) mice. We found adipose-like tissue in Pitx2 CKO hearts 60 days after MI induced surgically at postnatal day 2 but not at day 8. Molecular and cellular analyses showed the onset of adipogenic signaling in mutant hearts after MI. Lineage tracing experiments showed a non-cardiomyocyte origin of the de novo adipose-like tissue. Interestingly, we found that Pitx2 promotes mitochondrial function through its gene regulatory network, and that the knockdown of a key mitochondrial Pitx2 target gene, Cox7c, also leads to the accumulation of myocardial fat tissue. Single-nuclei RNA-seq revealed that Pitx2-deficient hearts were oxidatively stressed. Our findings reveal a role for Pitx2 in maintaining proper cardiac cellular composition during heart regeneration via the maintenance of proper mitochondrial structure and function.

Keywords: Adipogenesis; Cardiac regeneration; Mitochondria; Mouse; Myocardial infarction.

Fig. 1.

Adipose-like tissue in injured hearts with loss of Pitx2 in cardiomyocytes. (A-E) Trichrome staining showing scarring and an area of adipose-like tissue in the left ventricle of control (A,A′,B,B′) and Pitx2 CKO (C,C′,D,D′) hearts at 60 days after LAD occlusion performed at P2. Red, muscle; blue, collagen; white, adipose-like tissue. This adipose-like area is quantified in E. (F) Penetrance of adipose phenotype in Pitx2 CKO and control hearts. (G,G′,H,H′) Trichrome staining showing scarring in control (G,G′) and Pitx2 CKO (H,H′) hearts at 60 days after LAD occlusion performed at P8. (I,J) Quantification of the adipose-like (I) and scar areas (J) in hearts in which the LAD occlusion was performed at P8. *P<0.05, ***P<0.001; non-parametric (Mann–Whitney) test. NS, not significant.

Fig. 2.

Lipid accumulation and adipose marker expression in injured Pitx2 CKO hearts. (A-C) Oil Red O staining showing lipid droplets in Pitx2 CKO (B-B″) and control (A,A′) hearts at 60 days after LAD occlusion performed at P2. Black arrows in B″ indicate lipids. The positive-staining area is quantified in C. *P<0.05; non-parametric (Mann–Whitney) test. (D-E′) Immunostaining of cTnT (green) and C/EBPα (red; white arrows in E′) antibodies in control (D) and Pitx2 CKO (E,E′) hearts.

Fig. 3.

Perivascular localization of adipose-like tissue in regenerative Pitx2 CKO myocardium. (A,B) Representative images of normal coronary arteries in a regenerating control heart at 60 DPMI. (C,D) Representative images of adipose-like tissue located in close proximity to coronary vessels in regenerating Pitx2 CKO hearts. Arrow, coronary artery. (E) Phenotypic penetrance of the coexistence of adipose-like tissue and coronary arteries in Pitx2 CKO hearts compared with expected penetrance. *P<0.05; two-sided χ² test.

Fig. 4.

Loss of Pitx2 in ESCs promotes cell commitment to adipose lineage. (A) Flow chart showing the procedure carried out to assess the differentiation of control and Pitx2^nu/nu ESCs into cardiomyocytes. (B) qPCR was carried out to detect adipose marker expression in differentiated Pitx2^nu/nu and control cells. *P<0.05; non-parametric (Mann–Whitney) test. (C) Flow cytometry showing differentiated control and Pitx2^nu/nu ESCs stained with antibody against the adipose marker C/EBPα. (D) Quantification of the adipose fraction from C. Data are mean±s.d. (E) RNA-seq datasets from injured control and Pitx2 CKO myocardium were used to generate a heat map showing the expression of adipogenesis-relevant genes.

Fig. 5.

Compromised mitochondrial function contributes to adipose-like phenotype in injured neonatal mouse heart. (A) qPCR of Pparg in control and Pitx2 null P19 cell lines with or without 8 h of H₂O₂ treatment. (B) Seahorse assay shows oxygen consumption rate (OCR) of control and P19-Pitx2 null cell lines with readout normalized to the cell number in each assay. The x-axis indicates measurements 1-11. The dashed vertical lines indicate injections into media of the specific stressors oligomycin, FCCP and antimycin A/rotenone (A/R). (C) Western blot shows expression of ETC components in control and Pitx2 null P19 cell lines. (D,E) Trichrome staining of Cox7c^lacZ/+ and control mouse hearts that were subjected to LAD occlusion at P2 and analyzed at 60 days after occlusion. (F) Quantification of the amount of scarring seen in D,E. (G) Echocardiography shows the ejection fraction of control and Cox7c^lacZ/+ mice at 60 days after sham or LAD occlusion surgery. (H-J) Immunostaining for cTnT (green), C/EBPα (red) and DAPI (blue) in control (H) and Cox7c^lacZ/+ (I) hearts at 60 days after LAD occlusion. Asterisks indicate scar area. Arrows indicate C/EBPα positive cells. The percentage of C/EBPα⁺ scar cells is quantified in J. *P<0.05; non-parametric (Mann–Whitney) test. NS, not significant.

Fig. 6.

snRNA-seq on control and Pitx2 CKO cardiac tissue. (A) Diagram of snRNA-seq protocol. Samples were collected at 3 weeks after LAD occlusion on P2. Nuclei were isolated via density gradient centrifugation and nuclear snRNA-seq was performed. (B) Left, tSNE plot of 7849 cells showing the contribution of control (black) and Pitx2 CKO (red) samples. Right, the same tSNE as on the left but labeled by cell/cluster identity. (C) Average differential expression heat map for the top 1619 genes, with genes as rows and clusters as columns. Colors for each column match those in panel B, right. (D) Dot plot showing the average expression for each indicated gene (column) across all clusters (rows). FB, fibroblast; CM, cardiomyocyte; EC, endothelial cell; LEC, lymphatic endothelial cell; EndC, endocardial cell; EpiC, epicardial cell; SMC, (vascular) smooth muscle cell; Mφ, macrophage; PeC, pericyte.

Fig. 7.

Subclustering of cardiomyocytes shows differential subpopulations and gene expression between control and Pitx2 CKO groups. (A) Top, tSNE of 4136 subclustered cardiomyocytes (CM-1, CM-2 and CM-3 from Fig. 6) showing clusters c1-c4 in different colors. Bottom, χ² cluster composition analysis of CMs in Pitx2 CKO hearts compared with controls. Blue, cluster that significantly decreases in Pitx2 CKO hearts. Red, cluster that significantly increases in Pitx2 CKO hearts. Gray, no significant change in cluster composition between control and Pitx2 CKO conditions. (B) Differential expression of genes for clusters c1-c4 shown on a heat map, with the top candidate genes as rows and different cell clusters as columns. Expression of each gene was averaged across all cells in the indicated cluster. Low expression is indicated by cyan and high expression is shown in pink. (C) Gene ontology analysis of c3-enriched genes. (D) Expression plots for each gene projected across the tSNE. Pink indicates gene expression and white represents no expression. c3 is highlighted in green.