Upregulation of Yy1 Suppresses Dilated Cardiomyopathy caused by Ttn insufficiency

Truncating variants in TTN (TTNtv), coding for the largest structural protein in the sarcomere, contribute to the largest portion of familial and ambulatory dilated cardiomyopathy (DCM). TTN haploinsufficiency caused by TTNtv is suggested as the disease mechanism. However, it is unclear whether TTN insufficiency causes DCM. Moreover, it is unknown whether modulation of downstream pathways serves as a therapeutic strategy for DCM caused by TTN insufficiency. Here, we show that reduction of cardiac Ttn expression by adeno-associated virus mediated shRNA (Ttn shRNA) generated DCM in mouse, demonstrating impaired cardiac performance, enlarged left ventricle (LV) and reduced LV wall thickness. A screen of 10 dysregulated and selected genes identified that Yin Yang 1 (Yy1) significantly suppressed DCM caused by Ttn shRNA. Gene profiling by RNAseq showed Yy1 modulated cell growth related genes. Ttn insufficiency activated cardiomyocyte cell cycle reentry by upregulating of Ccnd1 and Ccnd2. Cardiomyocytes activated by Ttn insufficiency did not advance to S phase by EdU incorporation assay. Yy1 promoted cardiomyocyte cell cycle by further enhancing Ccnd1 and Ccnd2 and increasing DNA replication without undergoing cell division. Importantly, upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Our findings demonstrate that DCM caused by Ttn insufficiency can be treated by therapeutically promoting cardiac cell cycle.

Dilated cardiomyopathy (DCM) occurs as many as 1 in 250 people 1 . There are currently no approved therapeutic products indicated for DCM treatment. Typical treatments are those indicated for broader cardiovascular disease. As the disease progresses, patients have limited treatment options, such as surgical or other invasive interventions and heart transplant 2 . DCM will result in heart failure with reduced ejection fraction (EF), usually without prior ischemic. The walls of the left ventricle are thin and over-expanded, leading to improper contraction and insufficient blood being pumped by the dilated heart. DCM results from a variety of external factors, such as viral infection, alcohol abuse, exposure to cardiotoxic medications and pregnancy, as well as from genetic variants in a number of causal genes including TTN, LMNA, ACTC1, MHY7 and PLN [3][4][5][6][7][8][9] . Titin (coded by TTN) plays an important role in the contraction and relaxation of cardiac muscles by connecting Z-disc to the M-line in the sarcomere. TTN truncating variants (TTNtv) contribute up to 15% ambulatory DCM and 25% end-stage or familial DCM 3,8,9 . In DCM, TTNtv are significantly enriched most in A band as well as other regions including I-band, Z-disc or M-line with variable position-related odds ratios 3,10 . TTN haploinsufficiency caused by TTNtv is emerging as the potential disease mechanism. Rat models with TTNtv in Z-disc and A band did not result in change in titin protein levels and obvious cardiac performance under normal physiological conditions. It was not known whether Ttn insufficiency causes DCM in mouse.
Mammalian cardiomyocytes exit their cell cycle shortly after birth, preventing heart repair through cardiac regeneration 11 . Cell cycle reactivation is observed in a limited cardiomyocytes under physiological conditions 12,13 . As an emerging strategy for cardiac therapeutic regeneration, we and others showed that enhancing this process by removing cell cycle brakes or augmenting accelerators is beneficial for heart recovery from heart failure models caused by ischemia or pressure overload [14][15][16][17][18] . We previously observed that cardiomyocytes undergo an extra round of cell cycle in Mybpc3 deficient mice, suggesting cell cycle reactivation could compensate sarcomere insufficiency 19 . It was not known whether Ttn insufficiency could induce cardiac cell cycle reactivation. Moreover, it was unknown whether enhancing this process could be a therapeutic strategy for DCM caused by Ttn insufficiency. Here, we address these major gaps and identify therapeutic candidates that are vital for advancing potential hits into a therapeutic approach for DCM.

Upregulation of Yy1 modulates cardiac structural contractile and cell growth related gene expression.
To dissect the molecular mechanisms of Yy1 suppressing Ttn shRNA-induced DCM, we profiled control shRNA + EGFP (designated as control shRNA group, male, n = 4, dose = 0.2E + 13 vg/kg), Ttn shRNA + EGFP (designated as Ttn shRNA group, male, n = 4, dose = 0.2E + 13 vg/kg) and Ttn shRNA + Yy1 animals (designated as Yy1 treated group, male, n = 3, dose = 0.2E + 13 vg/kg) four weeks after virus transduction by RNAseq. To uncover signaling pathways regulated by Yy1, we found 134 genes significantly changed in Yy1 treated group compared to Ttn shRNA group (Fig. 3A). Among them, 80 genes were elevated, and 54 genes were reduced by Yy1. Hierarchical clustering of control shRNA, Ttn shRNA and Yy1 groups built significantly dysregulated genes into 4 regulated patterns. We analyzed gene list by Gene Set Enrichment Analysis (GSEA, Broad Institute). Gene ontology (GO) analysis identified top significantly enriched gene sets in category III including "positive regulation of growth" and "small molecule metabolic process" (Fig. 3B). Contractile fiber GO in category I included genes associated with sarcomere, such as Ankrd23, Fhl1 and Abra. The upregulation of four-and-a-half LIM domain protein 1 (Fhl1) and Ankyrin repeat domain-containing protein 23 (Ankrd23) by Ttn shRNA was www.nature.com/scientificreports www.nature.com/scientificreports/ suppressed by Yy1 (Fig. 3C). Importantly, Fhl1 and Ankrd23 interacted with Ttn protein, suggesting Yy1 modulates Ttn associated partners 31,32 . These results indicated Yy1 modulates Ttn partners and cardiac cell growth related gene expression which contributing to repair cardiac function.

Discussion
Ttn truncating variants are enriched in DCM patients suggesting a causal effect of TTN variants on DCM 3 . TTN Haploinsufficiency caused by TTNtv is emerging as the disease mechanism. To assess whether Ttn insufficiency causes DCM, we used shRNA to modulate Ttn expression. By reducing ~ 50% of Ttn expression, we generated a mouse model demonstrating a severe DCM phenotype, including ventricular wall thinning, dilated ventricular chambers and impaired cardiac function. To develop potential therapy for suppressing DCM caused by Ttn www.nature.com/scientificreports www.nature.com/scientificreports/ insufficiency, we screened 10 genes involved in different pathways. As a transcriptional regulator of TTN during direct cardiac reprogramming, we included Yy1 for the rescue experiments. Ttn expression was not induced by Yy1 in DCM hearts caused by Ttn shRNA, suggesting that Yy1 does not inhibit DCM through Ttn gene regulation in mouse hearts. Recently, Yy1 was shown to promote both Nkx2.5 expression, cardiac progenitor cell commitment and maintenance during early embryo development 34,35 . Cardiomyocyte-specific ablation of Yy1 mediated by Myh6-cre resulted in perinatal death of mutant mice, suggesting Yy1 plays an important role in early and late  www.nature.com/scientificreports www.nature.com/scientificreports/ cardiac lineage development 36 . Importantly, YY1 is shown to be upregulated in human idiopathic dilated cardiomyopathy (IDC) and heart failure 37,38 .
We detected over 35 fold increase of Ccnd1 and Ccnd2 in cardiomyocyte nuclei upon Ttn shRNA, indicating cardiomyocytes response to Ttn insufficiency by activating cell cycle reentry signals. We did not observed this reaction in another DCM model caused by Lmna insufficiency. Activated cardiomyocyte cell cycle induced by Ttn insufficiency does not advance to S phase. We found that upregulation of Yy1 promoted cardiac cell cycle reentry by further enhancing Ccnd1 and Ccnd2. Yy1 promoted cardiomyocyte cell cycle to S phase by a significant increase of EdU incorporation. However, we did not detect mitotic phase marker pH3 in cardiomyocytes, suggesting activated cardiomyocytes undergoing mitotic phase are limited. Importantly, Upregulation of Ccnd1 and Ccnd2 suppressed DCM caused by Ttn insufficiency. Taken together, Yy1 promotes cardiac cell cycle to facilitate to suppress Ttn shRNA-induced DCM.
Our recent study showed insufficiency of Mybpc3, another causal gene for sarcomeric cardiomyopathy, induces an extra round of cardiomyocyte cell cycle during neonatal stage 19 . It is now of great interest to know whether this reactivation of cardiac cell cycle is a common mechanism for sarcomere deficiency and enhancing of this process by Yy1 or other regulators is able to suppress cardiovascular disease related to sarcomere. Reactivation of cardiomyocyte cell cycle does not always lead to cell number increase. Cytokinesis is still rarely detected in adult hearts upon regenerative enhancement. Yy1 might promote hypertrophic growth coupled with cell cycle reentry as Ccnd2 is a mediator for Myc overexpression or exercise-induced hypertrophic growth in cardiomyocytes 39 . Recently, Yy1 is suggested to serve as a structural regulator between enhancer and promoter interactions and facilitates gene expression 40 . Apart from modulating cell growth, upregulation of Yy1 might indeed reinstate or promote enhancer -promoter interaction to restore gene regulatory network dysregulated by Ttn insufficiency.
Our selected candidates included many therapeutic targets for heart failure including Atp2a2, Bcl2, Sod2 and Sirt3 23,24,28,41 . Most of them failed to protect DCM induced by Ttn shRNA, suggesting root causes and disease mechanisms should be taken into account for DCM prevention and treatment strategies. It is of great interest to know whether Yy1 is able to suppress DCM caused by other genes including LMNA, MYH7 and PLN. To translate our research, one concern is whether upregulation of Yy1 could cause any cardiac defects. Previous study showed overexpression of Yy1 induced a relative marginal hypertrophy cardiomyopathy only in male mice. Here, we specified the Yy1 expression by cardiac specific promoter, virus dose and postnatal transduction. In contrast to a previous study, no detectable impairment of cardiac structure and performance was observed after ~ 5 months of virus transduction 37 . Taken together, our findings provide a strong supporting evidence for translational research.

Materials and Methods
Animal protocols. All mice were maintained and studied using protocols approved by the Institutional Animal Care and Use Committee (IACUC) of National University of Singapore. Animal work was undertaken in accordance with Singapore National Advisory Committee for Laboratory Animal Research guidelines. Relevant national and institutional guidelines and regulations must be consulted before commencement of any animal work. All studies were conducted in male C57BL/6JINV (Jax) mice. For virus injection, 50 µl viruses were injected into thoracic cavity of 10 days old pups via insulin syringe, avoiding the heart and lungs. For EdU injection, EdU (Sigma, 900584) was dissolved in saline and 5 mg/kg EdU was delivered to mice by intraperitoneal injection for two weeks after AAV transduction. For heart harvesting, mouse was anesthetized by 2% isoflurane and the heart was exposed by opening chest. After that, 15% KCl was injected into inferior vena cava to achieve asystole at diastole, then the heart was rapidly isolated and flushed with D-PBS through LV to wash out blood. Half of the apex was isolated and immersed in RNALater (Qiagen, 76104) at room temperature for RNA extraction, while the other half was snap frozen in liquid nitrogen for protein extraction. The rest part of heart was fixed in 4% paraformaldehyde for 24 hours and subsequently embedded by paraffin. echocardiogram (echo) and surface electrocardiogram (ecG). Cardiac dimension and function of mice three and/or four weeks after virus transduction were achieved by echocardiography (VisualSonics, Vevo 2100, 40 Mhz-550S probe). All mice were shaved to expose chest area one day before experiment. During echo, 1.5% isoflurane with oxygen were applied to each mouse, and cine of 300 frames of both B mode and M mode (left parasternal long and short axes) were recorded when heart rate was around 450-500 bpm. Measurements were processed by Vevo ® LAB (VisualSonics Inc.). LV tracings were averaged from at least 3 consecutive heart beats of M-mode. LVDD (LV diastolic dimensions), LVWT (LV posterior wall thickness), EF (ejection fraction) and FS (fractional shortening) were obtained from short axis images. cell culture and transfection. HEK293T cells were cultured at 37 °C with 5% CO2 and maintained in DMEM (Hyclone) supplemented with 10% FBS, 1 mM sodium pyruvate and 10 µg/ml gentamicin. Transfection of shRNA constructs and other plasmids was performed using PEI (Polysciences. Inc, 24765-2) according to manufacturer's instructions. cardiomyocyte isolation. We adapted protocol from Ackers-Johnson et al. In brief, the heart was perfused with warm EDTA, perfusion buffer and collagenase buffer through LV when aorta was clamped, then heart tissue became softened and swollen. After that, heart tissue was teared into pieces and dissociated by pipetting. Cell suspension was then passed through 100 µm strainer and processed to three rounds of gravity settling. Cardiomyocytes, in the pellet, were harvested for RNA extraction.
RNAseq library preparation and next generation sequencing. Total RNA from left ventricular tissue of male mice (n = 3 per group) was achieved to establish RNAseq library. RNA samples were pre-treated with Truseq Stranded Total RNA Library Prep kit (Illumina, RS-122-2201) to remove abundant cytoplasmic rRNA. The remaining intact RNA was fragmented using a chemical mix, followed by first-and second-strand cDNA synthesis using random hexamer primers. End-repaired fragments were ligated with a unique illumina adapter. All individually indexed samples were subsequently pooled together and multiplexed for sequencing. Libraries were sequenced using the Illumina Hiseq. 2000 sequencing system and paired-end 101 bp reads were generated for analysis. RNAseq data was deposited to NCBI. For selecting potential therapeutic genetic candidates, 3101 genes (log fold change <−0.59 or >0.7, FDR <0.05) were identified from DCM group (Ttn shRNA) compared to control group (Ctrl shRNA). For dissecting molecular mechanism of Yy1, 134 genes (P < 0.005 and FDR <0.2) were selected from Yy1 treated group (Ttn shRNA + Yy1) compared to Ttn shRNA group (Ttn shRNA + EGFP). Differentially expressed genes were uploaded to Morpheus for Hierarchical clustering and color-coded heat-map. Gene ontology (GO) of each category were analyzed by Gene Set Enrichment Analysis (GSEA, Broad Institute).
Quantitative real-time PCR (qPCR). Transcription level were quantified by qPCR. cDNA was synthesized using Maxima First Strand kit (ThermoFisher, K1641) and qPCR was carried out by KAPA SYBR Fast qPCR Master Mix kit (KAPA Biosystems, KR0389). All qPCR primers are listed as follows: Histological and immunostaining analysis. Heart samples were fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin and sectioned at 5 µm intervals. Paraffin samples were further treated with xylene (to remove paraffin), re-hydrated, and permeabilized in 0.1%(v/v) Triton-X100 in PBS. Hematoxylin and eosin (HE) was applied to observe myocyte architecture and Masson trichrome (MT) to identify cardiac fibrosis. Fibrosis was quantified by Image J. The percentage of fibrosis was calculated as the blue-stained areas divided by total ventricular area. As for immunostaining, boiled citric acid was used for Ccnd1, Ccnd2, EdU and pH3. Primary antibodies: Ccnd1 (Abcam, ab16663), Ccnd2 (Cell Signaling Technology, 3741), pH3 (Cell Signaling Technology, 9718) and cTnI (Abcam, ab8295). Other labeling dyes: DAPI for nucleus (ThermoFisher, D1306) and EdU Imaging kit (ThermoFisher, C10229). All the positive signals from three completed cross sections were counted for each heart sample, and data were normalized to total nucleus number.
Statistical analyses. Statistical analysis was achieved by Prism Graphpad 7.0. P value between two groups was performed by two-tailed, unpaired T-test with Weltch correction, and one-way ANOVA with Tukey's multiple comparisons test for multiple groups. Quantitative data were shown as mean ± SD. ns, non-significant, P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.