RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation

Figures

• 

  FIGURE 1. RRAD knockout did not affect the pluripotency nature of hESC and the ability of cardiac differentiation. (A) Schematic of human RRAD locus and editing site of designed gRNA-epiCRISPR/Cas9. (B) Sequencing data shows deletion of 55 bases in RRAD knockout hESC lines. (C) Immunostaining for the pluripotency markers SSEA4 and OCT4 of RRAD knockout hESC lines. Scalebar, 50 μm. (D) Schematic of cardiac differentiation using standard small molecule-based protocols. (E) Western blot shows RAD expression in WT and KO CMs at days 15. (F,G) Flow Cytometry for TNNT2 staining in representative WT and KO CMs at days 10 without purification. (H,I) Immunostaining for protein expression of MLC2v and MLC2a in WT and KO CMs at days 30. Scalebar, 50 μm. Data are expressed as means ± S.E.M. of three independent experiments. ns, not significant.

• 

  FIGURE 2. Phenotyping cardiac hypertrophy in RAD deficient CMs. (A) Images of α-actinin (green) and cTnT (red) immunostaining show an increased size in KO CMs at days 40. Scale bar, 50 μm. (B,C) Calibration of forward scatter (FSC;10000 cells/sample, n = 3) shows an increased size in KO CMs at days 40. (D) Images of α-actinin (green) and cTnT (red) immunostaining show disorganized arrangement of myofibrils in KO CMS. Scale bar, 50 μm. (E) Quantification of disorganized myofibrils in WT (n = 206) and KO CMs (n = 198). (F) Images of α-actinin/DAPI immunostaining. Scale bar, 50 μm. (G) Quantification of mono- and multi-nucleation in WT (n = 236) and KO (n = 262) CMs. Data are expressed as means ± S.E.M. of three independent experiments. **P < 0.01; ****P < 0.0001.

• 

  FIGURE 3. Transcriptome analysis in RAD deficient and WT CMs. (A) Heatmap of transcripts from WT and KO CMs (n = 2, respectively) show consistency within groups. Red: upregulated transcripts. Blue: downregulated transcripts. (B) Gene ontology (GO) term categories and distribution of differentially expressed genes. GO terms are divided into three categories: cellular component, molecular function and biological process. (C) Significantly enriched Kyoto Encyclopedia Genes and Genomes (KEGG) pathways in KO CMs.

• 

  FIGURE 4. RAD deficient CMs display abnormal regulation of intracellular calcium. (A) Representative line-scan images in WT and KO CMs. Red arrows indicate arrhythmia-like voltage waveforms observed in KO CMs but not in WT. (B) Peak amplitudes in WT and KO CMs (n = 30 cells per group). (C) Quantification of percentages for WT and KO CMs exhibit abnormal calcium regulation at days 20, 30, and 40 (n = 30 cells per group). (D) Representative Ca2 + transient from WT and KO CMs induced by caffeine exposure (n = 5 cells per group). (E–G) Peak amplitudes, duration and decay time after caffeine-induced Ca2 + transient in WT and KO CMs. (H) Representative line-scan images in WT and KO CMs after adrenergic stimuli by PE. Red arrows indicate arrhythmia-like voltage waveforms. (I) Quantification of WT and KO CMs exhibit arrhythmia-like voltage waveforms in response to PE. Data are expressed as means ± S.E.M. of three independent experiments. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

• 

  FIGURE 5. Increased hypertrophic signaling in RAD deficient CMs. (A) Heatmap shows expression changes of genes related to cardiac hypertrophic signaling pathways in WT and KO CMs at days 40. (B,C) Specific markers for cardiac hypertrophy NPPA, NPPB are significant upregulated in KO CMs. (D) Western blot shows expression of hypertrophic related proteins in WT and KO CMs, respectively. (E) Quantification of protein expression normalized by GAPDH in WT and KO CMs. Data are expressed as means ± S.E.M. of three independent experiments. ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

• 

  FIGURE 6. RAD deficient CMs show increased ICa–L and CaMKII activity. (A) Recording and identification of representative ICa–L from a series of step pulses between −50 mV to +60 mV from a holding potential of −80 mV. 200 nM nifedipine, a specific blocker of L-type voltage-gated Ca2 + channels, blocked the current. (B) Histogram analysis showing that the cell membance capacity (pF) was larger in KO CMs than that in WT CMs. (C) Typical ICa–L recording from WT CMs (black traces) and KO CMs (red traces). (D) I-V curves ICa–L showing that ICa–L density was significantly increased in KO CMs (n = 9, respectively). (E) Western blot shows expression of protein related to calcium regulation in WT and KO CMs, respectively. (F) Quantification of protein expression normalized by GAPDH in WT and KO CMs. (G) Western blot shows expression of calcineurin A, CaMKIIδ and phosphorylated CaMKII (Thr286) in WT and KO CMs, respectively. (H) Quantification of protein expression normalized by GAPDH in WT and KO CMs. Data are expressed as means ± S.E.M. of three independent experiments. ns, not significant; *P < 0.05; ***P < 0.001; ****P < 0.0001.

• 

  FIGURE 7. Calcium channel blocker prevents the development of cardiac hypertrophy in RAD deficient CMs. (A) Representative line-scan images of KO CMs treated with 100 nM verapamil for 10 days. (B) Quantification of percentages of KO CMs found to exhibit abnormal calcium regulation after treatment with verapamil (n = 30 cells per group). (C) Representative Ca2 + transient induced by caffeine of KO CMs after treated with verapamil (n = 5 cells per group). (D,E) Peak amplitudes and decay time of KO CMs after treated with verapamil. (F,G) Flow cytometry shows changes in cell size of WT, KO and verapamil treated KO CMs. (H) Heatmap shows expression changes of genes related to hypertrophy in WT, KO, and verapamil treated KO CMs. (I,J) Western blot shows expression of Cav1.2, SERCA2a, p-RyR2 (Ser2808, Ser2814), and p-CaMKII (Thr286) in WT, KO, and verapamil KO CMs. Quantification of protein expression normalized by GAPDH. Vera, verapamil. Data are expressed as means ± S.E.M. of three independent experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001.

• 

  FIGURE 8. A model for mechanisms of cardiac hypertrophy in RAD deficient CMs. RAD deficiency triggers hypertrophy-associated pathway via modulation of the cytoplasmic Ca2 +. NCX, sodium-calcium exchanger; LTCC, L-type calcium channel; CaM, Calmodulin; CaMKII, calmodulin-dependent kinase II; CnA, Calcineurin; SR, sarcoplasmic reticulum.