Fig 1: Colocalization and protein interactions of ALPK3 and myomesins. Confocal micrographs of hiPSC-CMs expressing eGFP-TTN (green) and ALPK3-FLAG (probed with anti-FLAG antibody; red). A, ALPK3-FLAG introduced by transient plasmid transfection into hiPSC-CMs expressing eGFP-TTN. Scale bar=25 µm. B, Magnified regions of A display ALPK3-FLAG (red) localizing to sarcomere M-bands between Z-discs (green, eGFP-TTN). Scale bar=5 µm. C, ALPK3-FLAG (red) localizes to nuclear envelope. Scale bar=5 µm. D, Depth-encoded (Z-stack) maximum intensity projection of nucleus (C) showing ALPK3 localization at nuclear envelope. E, MYOM1, stained with anti-myomesin 1 antibody (red) localizes to M-bands between Z-discs (green, eGFP-TTN). Scale bar=5 µm. F, MYOM1 (red) is also found at the nucleus (N) and nuclear envelope (NE). Scale bar=5 µm. G, MYOM2, stained with anti-myomesin 2 antibody (red), localizes to the nuclear envelope. Scale bar=5 µm. H, hiPSC-CMs transfected with plasmid expressing MYOM2-HA (red) shows MYOM2 at M-bands between Z-discs (green, eGFP-TTN). Scale bar=5 µm. I, Immunoprecipitation (IP) studies of ALPK3-FLAG, FOXI3-FLAG (negative control), and myomesins (MYOM1-HA, MYOM2-HA) in transfected HEK293T cells. IP of ALPK3-FLAG results in pulldown of MYOM2-HA. ALPK3 indicates a-kinase 3; eGFP, endogenous GFP; hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; MYOM1, myomesin-1; MYOM2, myomesin-2; and TTN, titin.
Fig 2: The results of RT-PCR and the graphs (CT curves) to verify the expression of characteristic genes at the mRNA level; (A), (E) ACLY; (B), (F) ADH1B; (C), (G) MYOM1; (D), (H) PTGFR; **p < 0.01.
Fig 3: Confocal micrographs of WT and ALPK3-deficient hiPSC-CMs and murine cardiomyocytes stained with MYOM1 antibody (red) demonstrate myomesin-1 accumulation in mutant cells. Top, MYOM1 accumulates outside the M-band and around the nucleus (N) in ALPK3ins/del hiPSC-CMs versus WT. Scale bar=5 µm. Bottom, Myom1 accumulates outside the M-band in Alpk3–/– mouse cardiomyocytes (left ventricular cardiomyocytes isolated from mice age 7–9 weeks). Scale bar=5 µm (see also Figure S7 for confocal micrographs of entire cardiomyocytes). ALPK3 indicates a-kinase 3; hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; MYOM1, myomesin-1; and WT, wild type.
Fig 4: ALPK3 variants dysregulate myomesin and thick filament proteins in hiPSC-CMs, mice, and human tissues. A and B, Volcano plots of protein expression in ALPK3ins/del hiPSC-CMs (n=7) versus WT (n=8) and ALPK3dIC/dIC hiPSC-CMs (n=4) versus WT (n=5587 total proteins). Dashed lines indicate cutoffs for differential expression: |log2-fold change|=0.67 and unadjusted t-test P value <0.01. Sarcomere M-band and thick filament proteins are highlighted in red (see also Supplemental Data S3). C, Overlap of proteins with altered expression in ALPK3ins/del and ALPK3dIC/dIC hiPSC-CMs. D, Fold-change of M-band and thick filament proteins in ALPK3sIC/sIC (n=4), ALPK3dIC/dIC (n=4), ALPK3del/del (n=4), and ALPK3ins/del (n=7) hiPSC-CMs relative to WT (n=12). E, Serial echocardiographic measurements (x axis, age in weeks) of WT and Alpk3 mutant mice. No measurements are reported in Alpk3–/– mice after 14 weeks because of early lethality. F, Transverse cardiac sections showing ventricular enlargement of Alpk3–/– heart at 14 weeks (Masson trichrome stain, scale bar=1 mm). G, Volcano plot of protein expression in LV tissues harvested at postnatal day 8 from Alpk3–/– vs WT mice (n=5678 total proteins; see also Supplemental Data S3). Proteins are identified by their human homologues. Dashed lines and labels are as in A and B (WT, n=4; Alpk3+/–, n=3, Alpk3–/–, n=3). H, Upper, Western blotting analysis of LV tissues from human subjects with compound heterozygous variants in ALPK3 (PT1-PT2) compared with adult healthy control tissues (C1-C5) probed with (blot 1) myomesin-1 (MYOM1, blot 1), tubulin (TUBB), and myomesin-2 (MYOM2, blot 2) antibodies (n=3 technical replicates per sample; see also Table S2). Lower, Western blotting quantification of MYOM1 and MYOM2 expression in human ALPK3-deficient subjects and ALPK3-normal subjects (normalized to TUBB levels). Data are mean±SEM. ALPK3 indicates a-kinase 3; dIC, dual interlobe cleft variant; hiPSC-CM, human induced pluripotent stem cell–derived cardiomyocyte; IVSd, interventricular septal thickness at end-diastole; LV, left ventricle; LVIDd, LV internal dimension at end diastole; LVPWd, LV posterior wall thickness at end-diastole; sIC, single interlobe cleft variant; and WT, wild type.
Supplier Page from Abcam for Anti-MYOM1 antibody [EPR17322]