Impaired Reorganization of Centrosome Structure Underlies Human Infantile Dilated Cardiomyopathy

Abstract

Background: During cardiomyocyte maturation, the centrosome, which functions as a microtubule organizing center in cardiomyocytes, undergoes dramatic structural reorganization where its components reorganize from being localized at the centriole to the nuclear envelope. This developmentally programmed process, referred to as centrosome reduction, has been previously associated with cell cycle exit. However, understanding of how this process influences cardiomyocyte cell biology, and whether its disruption results in human cardiac disease, remains unknown. We studied this phenomenon in an infant with a rare case of infantile dilated cardiomyopathy (iDCM) who presented with left ventricular ejection fraction of 18% and disrupted sarcomere and mitochondria structure.

Methods: We performed an analysis beginning with an infant who presented with a rare case of iDCM. We derived induced pluripotent stem cells from the patient to model iDCM in vitro. We performed whole exome sequencing on the patient and his parents for causal gene analysis. CRISPR/Cas9-mediated gene knockout and correction in vitro were used to confirm whole exome sequencing results. Zebrafish and Drosophila models were used for in vivo validation of the causal gene. Matrigel mattress technology and single-cell RNA sequencing were used to characterize iDCM cardiomyocytes further.

Results: Whole exome sequencing and CRISPR/Cas9 gene knockout/correction identified RTTN, the gene encoding the centrosomal protein RTTN (rotatin), as the causal gene underlying the patient's condition, representing the first time a centrosome defect has been implicated in a nonsyndromic dilated cardiomyopathy. Genetic knockdowns in zebrafish and Drosophila confirmed an evolutionarily conserved requirement of RTTN for cardiac structure and function. Single-cell RNA sequencing of iDCM cardiomyocytes showed impaired maturation of iDCM cardiomyocytes, which underlie the observed cardiomyocyte structural and functional deficits. We also observed persistent localization of the centrosome at the centriole, contrasting with expected programmed perinuclear reorganization, which led to subsequent global microtubule network defects. In addition, we identified a small molecule that restored centrosome reorganization and improved the structure and contractility of iDCM cardiomyocytes.

Conclusions: This study is the first to demonstrate a case of human disease caused by a defect in centrosome reduction. We also uncovered a novel role for RTTN in perinatal cardiac development and identified a potential therapeutic strategy for centrosome-related iDCM. Future study aimed at identifying variants in centrosome components may uncover additional contributors to human cardiac disease.

Keywords: cardiomyopathy, dilated; centrosome; induced pluripotent stem cells; myocytes, cardiac.

Figure 1. CMs derived from the iDCM patient’s iPSCs recapitulate cardiac defects.

a, TEM images of hearts. Left, TEM of normal heart showing well-organized myofilaments, distinct Z-lines (Z; arrowheads) and mitochondria (Mito) with distinct cristae. Right, EM of the iDCM patient’s heart showing severely disorganized myofibrils with indistinct Z-lines and dysmorphic mitochondria (Mito) without appreciable cristae. Scale bar = 1 μm. b, iPSC-CM from a healthy control exhibiting organized myofilaments with distinct Z-lines (Z; arrowheads) (top), whereas the iPSC-CM derived from the iDCM patient (bottom) exhibit disorganized myofibrils without distinct Z-lines (Z). Scale bar = 0.5 μm. c, Representative immunofluorescence images of α-actinin staining of control (top) and iDCM (bottom) CMs. Scale bar = 10 μm. d, Compared with control-CMs derived from 2 independent healthy control iPSC lines (Control-1, −2), iDCM-CMs from 2 independent iPSC lines (iDCM-1, −2) exhibited significantly reduced cell shortening (~11% vs ~8%). p value by one-way ANOVA and post-hoc Tukey *p < 0.01, **p < 0.05. n = 28 Control 1-CMs, 39 Control 2-CMs, 59 iDCM 1-CMs, 48 iDCM 2-CMs. Center line = median; whiskers = 1.5IQR. e, TEM images of iPSC-CMs focused on mitochondria. Left, mitochondria of control iPSC-CMs are easily distinguishable and have distinct cristae, whereas mitochondria (arrowheads) from iDCM-CMs appear larger and swollen, without clear cristae. Scale bar = 0.5 μm. f, Fluorescence-activated cell sorting analysis demonstrating that iDCM-CMs have quantitatively lower TMRM uptake and hence lower mitochondrial membrane potentials than control iPSC-CMs. TEM images of CMs were acquired at 45 days of differentiation. Other studies were done at 35 days of differentiation.

Figure 2.. Compound heterozygous mutations in RTTN lead to iDCM phenotype.

a, Whole-exome sequencing of the proband and his parents revealed that the patient was a compound heterozygote for a G1321D missense mutation from the father and an in-frame deletion (p.1921–1925) from the mother. b, RTTN knockout (KO) CMs displayed disorganized myofibrils with indistinct Z-lines (top), whereas the RTTN gene-corrected (GC) CMs (bottom) appear similar to the healthy control-CMs seen in Figure 1b. Scale bar = 0.5 μm. c, RTTN KO-CMs (top) displayed globular or punctate mitochondria, as visualized by MitoTracker (green), and disorganized myofilaments, as visualized by α-actinin staining (red). By contrast, RTTN GC-CMs (bottom) displayed networks of elongated mitochondria (green) and organized myofilaments, similar to healthy control-CMs. Scale bar = 20 μm. d, RTTN KO-CMs from two independent KO lines (KO-1, −2) displayed reduced cell shortening similar to the iDCM-CMs. By contrast, RTTN GC-CMs from three isogenic GC lines (GC-1, −2, −3) displayed restored shortening, comparable to healthy control-CMs. n = 28 Control 1-CMs, 39 Control 2-CMs, 59 iDCM 1-CMs, 48 iDCM 2-CMs, 43 KO 1-CMs, 29 KO 2-CMs, 32 GC 1-CMs, 35 GC 2-CMs. Center line = median; whiskers = 1.5IQR. e, FACS analysis of TMRM staining demonstrating reduced mitochondrial membrane potential in RTTN KO-CMs and restored mitochondrial membrane potential in RTTN GC-CMs.

Figure 3.. RTTN is an evolutionarily conserved causal gene for iDCM.

a, Left, representative images of embryos at 48 hpf. (i) uninjected embryo; (ii) embryo injected with translational blocking morpholino (MO); embryos injected with CRISPRi #1 (iii) and #2 (iv). Right, corresponding enlarged images of the heart. Arrowhead, outline of heart. Incidence of pericardial edema with impaired tail circulation in 48-hpf embryos. Results n ≥ 101 embryos from n ≥ 7 biological replicates using embryos from n ≥ 2 breeding pairs. Vehicle was 0.08% phenol red + PBS in ultrapure water. ***p <0.00017 by Fisher’s exact test with Bonferroni Correction; N.S. = not significant. b, Quantification of immunofluorescent imaging of actin and actinin in adult heart (segment A4) from control (w1118), ana3^+/− and ana3^−/− flies. Statistical results of normalized cardiac fiber density (N = 10). * P <0.05; ** P <0.01. c, OCT images for heart of control, ana3+/− and ana3−/− flies. Arrowheads indicate\ the end-diastolic diameter (EDD). Statistical analysis for EDD (μm) and percent fractional shortening (FS) obtained from the optimal computed tomography data. Each datapoint represents the average of measurements from three heartbeats randomly selected within a two-second time frame for each fly. Center line = median; whiskers = 1.5IQR for each genotype. *P <0.05.

Figure 4.. RTTN mutation leads to impaired maturation via disrupted reorganization of the centrosome and microtubule network formation.

a, Heat map showing expression of genes related to CM maturation in control and iDCM-CMs. b, Dot plot of fold enrichment (fe) of gene ontology (GO) pathways. up = upregulated in iDCMs; down = downregulated in iDCMs. c, Shannon entropy score showing relative maturation score of control and iDCM-CMs. d, Top, In the control CM with a normally organized sarcomere (α-actinin, red), PCNT (green) is redistributed to the perinuclear region (arrowheads). Bottom, In the RTTN mutant (iDCM) CM with a disorganized sarcomere (α-actinin, red), PCNT (green) remains localized to the centrosome. Quantification of PCNT distribution in D36 control and iDCM-CMs by a blinded observer with categories of centriolar, split, or perinuclear. PCNT was perinuclear in 21.65% of control-CMs, whereas it was perinuclear in only 7.26% of iDCM-CMs. Perinuclear vs centriolar p = 0.0021 by two-tailed Fisher’s exact test. n = 97 control-CMs, 124 iDCM-CMs. e, Left, Control CM displaying a prominent network of thick microtubule (MT) fibers (α-tubulin, green) emanating from the perinuclear region, with an organized sarcomere (cardiac TnT, red). Right, by contrast, iDCM CM displayed thinner, shorter and fainter MT fibers without a clear organizing center, and a disorganized sarcomere. Quantification of CMs with grossly normal and abnormal MT network by a blinded observer. The MT network was grossly normal in 86% of control-CMs, whereas only 48% of iDCM-CMs had normal MT networks. n= 28 Control-CMs, 23 iDCM-CMs. p=0.006 by two-tailed Fisher’s exact test. Scale bar = 50 μm.

Figure 5.. Pharmacological treatment to induce centrosomal reorganization restores CM structure and function in iDCM.

a, Schematic detailing C19 treatment at differentiation day 30 (d30) control-CMs and iDCM-CMs. b, Quantification of centriolar, split, and perinuclear MTOC localization in D32 CMs by a blinded observer. PCNT was perinuclear in 23.1% of control-CMs, 7.6% of iDCM-CMs, and 23.6% of iDCM+C19 CMs. p = 0.0082 by Fisher Exact test. c, Representative images of untreated iDCM-CMs (top) and iDCM-CMs treated with C19, which showed increased levels of sarcomere formation. Scale bar = 50 μm. d, Boxplots showing % cell shortening in control, D40 iDCM, and iDCM C19 treated CMs. n = 9 control, 32 iDCM, 36 iDCM + C19 from two differentiations. p value = 0.0006 by one-way ANOVA. Post hoc Tukey correction p value of iDCM vs iDCM + C19 = 0.001, control vs iDCM + C19 = 0.682 (NS). Center line = median; whiskers = 1.5IQR. e, Working model of CM maturation cascade showing RTTN-mediated centrosome reduction is upstream of other canonical maturation events.