HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy

Abstract

Human mutations that truncate the massive sarcomere protein titin [TTN-truncating variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By combining functional analyses with RNA sequencing, we explain why truncations in the A-band domain of TTN cause DCM, whereas truncations in the I band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS cell-derived cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodeling.

Copyright © 2015, American Association for the Advancement of Science.

Figures

Fig. 1. Engineered iPS-CM microtissues with TTN mutations have impaired intrinsic contractility and responses to stress

(A) Schematic of the cardiac sarcomere with TTN (orange), thick filaments (gold rods with white globular heads), and thin filaments (green, coiled ovals). TTN protein segments (z-disc, red; I-band, blue; A-band, green; M-band, gold) with location of human (“p” patient-derived; “c” CRISRP/CAS9-derived) mutations. (B) Images (bright field (left) and fluorescent (right); green, Phalloidin, Alexa Fluor® 488; blue, DAPI) of iPS-CMT suspended between two polydimethylsiloxane (PDMS) pillars from top-down (upper) and side (lower) views. Scale bar, 50 μm. (C) Representative force tracing of pWT (black) and pP22582fs+/− (red) CMT over three twitch cycles. (D) Mean force of pW976R+/−, pA22352fs+/−, and pP22582fs+/− iPS-CMTs compared to pWT iPS-CMTs (N=5). (E) Mean force produced by isogenic iPS-CMTs with heterozygous or homozygous I-band (cV6382fs) or A-band (cN22577fs) TTNtv (N > 4 CMTs). (F) Mean force produced by pWT and pP22582fs+/− CMTs in response to increased pillar stiffness (0.2 to 0.45 μN/μm; N>11 CMTs). Difference (Δ) in force generation between pP22528fs+/− and pWT measured at low and high stiffness. (G) Isoproterenol-induced force (N>5) and (H) spontaneous beating rate (N >5, Hz) in pP22582fs+/− versus pWT CMTs (N>5). Significance assessed by Student’s t test (D–H); data are means +/− SEM (D-H).

Fig. 2. Sarcomere abnormalities in TTNtv iPS-CMs

(A) pWT, pP22582fs+/− and cT33520fs−/− iPS-CMs stained with TTN specific antibody (9D10; green) and nuclei (DAPI; blue). (40X; Scale bar=20 μm). (B) pWT and pP22582fs+/− iPS-CMs were patterned on 25 μm × 25 μm grids, stained for z-discs (α-actinin A; green) and nuclei (DAPI; blue). (40X; Scale bar=20 μm). (C) pWT, pP22582fs+/−, and cT33520fs−/− CMTs stained for α-actinin A (green) and F-actin (red) (40X; Scale bar, 20 μm). (D) Sarcomere organization (ɛ) quantified by 2-dimensional Fourier Transform (FFT) analysis of pWT, pP22582fs+/− and cT33520fs−/− iPS-CMs (N>5 per genotype). (E) Sarcomere length (μm) measured by intensity profiles of α-actinin in pWT and pP22582fs+/− iPS-CMs (N>22). (F) Protein electropherograms of lysates from pP22582fs+/− and pWT iPS-CMs and human LV stained with Coomassie Blue (For additional blots, see fig S6). Sizes of TTN isoforms and fragments are: N2BA fetal, ~3700 kDa; N2BA adult, ~3300 kDa; N2B, ~3000kDa; TTNtv, ~2500 kDa; degraded TTN, ~1800–2200 kDa. Obscurin size is ~700 kDa. Western blots (WB) probed with amino-terminus TTN antibody (T12, fig. S6). TTNtv protein (~2500 kDa) was detected in pP22582fs+/− iPS-CMs. (G) Representative micrographs of H&E stained tissue from the left ventricles (LV) of control and P22582fs+/− patients (arrow highlights region of disorganized sarcomere; Scale bar=20 μm). (H) Sarcomere organization (ɛ) quantified by FFT analysis of LV tissue from control and P22582fs+/− patients (n>15 per genotype). Significance assessed by Student’s t test (D, E and H)); data are means +/− SEM (D, E and H).

Fig. 3. TTN regulates iPS-CM signalling and RNA expression

(A)Upstream transcriptional regulators were identified by Ingenuity pathway analysis of differentially regulated genes (normalized ratio >1.2 and <0.8 and p<0.01; table S1C) using RNAseq from cWT, cN22577fs+/−, and cN22577fs−/− iPS-CMs. Data is plotted as Z-score of enrichment (Z-scores cut-off ≥3.5 and≤3.5). (B–D) Comparison of cWT, cN22577fs+/−, and cN22577fs−/−iPS-CMs normalized expression (FPKM) of (B) β(MYH7) and α(MYH6) myosin heavy chain ratios; (C) atrial (NPPA) and brain natriuretic (NPPB) peptides; (D) TGF-β1 and VEGF-A in cWT, cN22577fs+/−, and cN22577fs−/− iPS-CMs. (E) Densitometry of western blots (N>4) of pWT and pP22582fs+/− lysates, normalized for protein loading, and probed with antibodies to phosphorylated JNK (p46, p54), ERK, p38, and AKT. (F) Representative western blots of pWT and pP22582fs+/− iPS-CMs lysates probed for (p)-JNK(T183/Y185), p-ERK(T202/Y204), p-p38(T180/Y182) and p-AKT(T308) and total JNK, ERK, p38 and AKT. (G) Densitometry of western blots (N>4) normalized to protein loading of TGF-β1-3 and VEGF. (H) Representative lanes from western blots from pWT and pP22582fs+/− iPS-CMs probed for TGF-β1-3, VEGF, and GAPDH. (I) Mean twitch force (μN) generated by pP22582fs+/− iPS-CMTs pre-treated with 50ng/ml VEGF (N>4 CMTs). Significance assessed by Bayesian p-values (B-D) or Student’s t test (E, G and I); data are means +/− SEM (E, G and I).