Mitochondrial MICOS complex genes, implicated in hypoplastic left heart syndrome, maintain cardiac contractility and actomyosin integrity.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart disease (CHD) with a likely oligogenic etiology, but our understanding of the genetic complexities and pathogenic mechanisms leading to HLHS is limited. We performed whole genome sequencing (WGS) on 183 HLHS patient-parent trios to identify candidate genes, which were functionally tested in the Drosophila heart model. Bioinformatic analysis of WGS data from an index family of a HLHS proband born to consanguineous parents prioritized 9 candidate genes with rare, predicted damaging homozygous variants. Of them, cardiac-specific knockdown (KD) of mitochondrial MICOS complex subunit dCHCHD3/6 resulted in drastically compromised heart contractility, diminished levels of sarcomeric actin and myosin, reduced cardiac ATP levels, and mitochondrial fission-fusion defects. These defects were similar to those inflicted by cardiac KD of ATP synthase subunits of the electron transport chain (ETC), consistent with the MICOS complex's role in maintaining cristae morphology and ETC assembly. Five additional HLHS probands harbored rare, predicted damaging variants in CHCHD3 or CHCHD6. Hypothesizing an oligogenic basis for HLHS, we tested 60 additional prioritized candidate genes from these patients for genetic interactions with CHCHD3/6 in sensitized fly hearts. Moderate KD of CHCHD3/6 in combination with Cdk12 (activator of RNA polymerase II), RNF149 (goliath, E3 ubiquitin ligase), or SPTBN1 (ß-Spectrin, scaffolding protein) caused synergistic heart defects, suggesting the likely involvement of diverse pathways in HLHS. Further elucidation of novel candidate genes and genetic interactions of potentially disease-contributing pathways is expected to lead to a better understanding of HLHS and other CHDs.

Conserved transcription factors promote cell fate stability and restrict reprogramming potential in differentiated cells.

Defining the mechanisms safeguarding cell fate identity in differentiated cells is crucial to improve 1) - our understanding of how differentiation is maintained in healthy tissues or altered in a disease state, and 2) - our ability to use cell fate reprogramming for regenerative purposes. Here, using a genome-wide transcription factor screen followed by validation steps in a variety of reprogramming assays (cardiac, neural and iPSC in fibroblasts and endothelial cells), we identified a set of four transcription factors (ATF7IP, JUNB, SP7, and ZNF207 [AJSZ]) that robustly opposes cell fate reprogramming in both lineage and cell type independent manners. Mechanistically, our integrated multi-omics approach (ChIP, ATAC and RNA-seq) revealed that AJSZ oppose cell fate reprogramming by 1) - maintaining chromatin enriched for reprogramming TF motifs in a closed state and 2) - downregulating genes required for reprogramming. Finally, KD of AJSZ in combination with MGT overexpression, significantly reduced scar size and improved heart function by 50%, as compared to MGT alone post-myocardial infarction. Collectively, our study suggests that inhibition of barrier to reprogramming mechanisms represents a promising therapeutic avenue to improve adult organ function post-injury.

Foxm1 regulates cardiomyocyte proliferation in adult zebrafish after cardiac injury.

The regenerative capacity of the mammalian heart is poor, with one potential reason being that adult cardiomyocytes cannot proliferate at sufficient levels to replace lost tissue. During development and neonatal stages, cardiomyocytes can successfully divide under injury conditions; however, as these cells mature their ability to proliferate is lost. Therefore, understanding the regulatory programs that can induce post-mitotic cardiomyocytes into a proliferative state is essential to enhance cardiac regeneration. Here, we report that the forkhead transcription factor Foxm1 is required for cardiomyocyte proliferation after injury through transcriptional regulation of cell cycle genes. Transcriptomic analysis of injured zebrafish hearts revealed that foxm1 expression is increased in border zone cardiomyocytes. Decreased cardiomyocyte proliferation and expression of cell cycle genes in foxm1 mutant hearts was observed, suggesting it is required for cell cycle checkpoints. Subsequent analysis of a candidate Foxm1 target gene, cenpf, revealed that this microtubule and kinetochore binding protein is also required for cardiac regeneration. Moreover, cenpf mutants show increased cardiomyocyte binucleation. Thus, foxm1 and cenpf are required for cardiomyocytes to complete mitosis during zebrafish cardiac regeneration.

Zebrafish heart regenerates after chemoptogenetic cardiomyocyte depletion.

BACKGROUND: Zebrafish can regenerate adult cardiac tissue following injuries from ventricular apex amputation, cryoinjury, and cardiomyocyte genetic ablation. Here, we characterize cardiac regeneration from cardiomyocyte chemoptogenetic ablation caused by localized near-infrared excited photosensitizer-mediated reactive oxygen species (ROS) generation. RESULTS: Exposure of transgenic adult zebrafish, Tg(myl7:fapdl5-cerulean), to di-iodinated derivative of the cell- permeable Malachite Green ester fluorogen (MG-2I) and whole-body illumination with 660 nm light resulted in cytotoxic damage to about 30% of cardiac tissue. After chemoptogenetic cardiomyocyte ablation, heart function was compromised, and macrophage infiltration was detected, but epicardial and endocardial activation response was much muted when compared to ventricular amputation. The spared cardiomyocytes underwent proliferation and restored the heart structure and function in 45-60 days after ablation. CONCLUSIONS: This cardiomyocyte ablation system did not appear to activate the epicardium and endocardium as is noted in other cardiac injury models. This approach represents a useful model to study specifically cardiomyocyte injury, proliferation and regeneration in the absence of whole organ activation. Moreover, this system can be adapted to ablate distinct cell populations in any organ system to study their function in regeneration.

Patient-specific genomics and cross-species functional analysis implicate LRP2 in hypoplastic left heart syndrome.

Congenital heart diseases (CHDs), including hypoplastic left heart syndrome (HLHS), are genetically complex and poorly understood. Here, a multidisciplinary platform was established to functionally evaluate novel CHD gene candidates, based on whole-genome and iPSC RNA sequencing of a HLHS family-trio. Filtering for rare variants and altered expression in proband iPSCs prioritized 10 candidates. siRNA/RNAi-mediated knockdown in healthy human iPSC-derived cardiomyocytes (hiPSC-CM) and in developing Drosophila and zebrafish hearts revealed that LDL receptor-related protein LRP2 is required for cardiomyocyte proliferation and differentiation. Consistent with hypoplastic heart defects, compared to patents the proband's iPSC-CMs exhibited reduced proliferation. Interestingly, rare, predicted-damaging LRP2 variants were enriched in a HLHS cohort; however, understanding their contribution to HLHS requires further investigation. Collectively, we have established a multi-species high-throughput platform to rapidly evaluate candidate genes and their interactions during heart development, which are crucial first steps toward deciphering oligogenic underpinnings of CHDs, including hypoplastic left hearts.

Model system identification of novel congenital heart disease gene candidates: focus on RPL13.

Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.

Enhancing regeneration after acute kidney injury by promoting cellular dedifferentiation in zebrafish.

Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified 4-(phenylthio)butanoic acid (PTBA), a histone deacetylase inhibitor (HDI), as an enhancer of renal recovery, and showed that PTBA treatment increased RTEC proliferation and reduced renal fibrosis. Here, we investigated the regenerative mechanisms of PTBA in zebrafish models of larval renal injury and adult cardiac injury. With respect to renal injury, we showed that delivery of PTBA using an esterified prodrug (UPHD25) increases the reactivation of the renal progenitor gene Pax2a, enhances dedifferentiation of RTECs, reduces Kidney injury molecule-1 (Kim-1) expression, and lowers the number of infiltrating macrophages. Further, we found that the effects of PTBA on RTEC proliferation depend upon retinoic acid signaling and demonstrate that the therapeutic properties of PTBA are not restricted to the kidney but also increase cardiomyocyte proliferation and decrease fibrosis following cardiac injury in adult zebrafish. These studies provide key mechanistic insights into how PTBA enhances tissue repair in models of acute injury and lay the groundwork for translating this novel HDI into the clinic.This article has an associated First Person interview with the joint first authors of the paper.

Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration.

Zebrafish regenerate cardiac tissue through proliferation of pre-existing cardiomyocytes and neovascularization. Secreted growth factors such as FGFs, IGF, PDGFs and Neuregulin play essential roles in stimulating cardiomyocyte proliferation. These factors activate the Ras/MAPK pathway, which is tightly controlled by the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase. Here, we show that suppressing Dusp6 function enhances cardiac regeneration. Inactivation of Dusp6 by small molecules or by gene inactivation increased cardiomyocyte proliferation, coronary angiogenesis, and reduced fibrosis after ventricular resection. Inhibition of Erbb or PDGF receptor signaling suppressed cardiac regeneration in wild-type zebrafish, but had a milder effect on regeneration in dusp6 mutants. Moreover, in rat primary cardiomyocytes, NRG1-stimulated proliferation can be enhanced upon chemical inhibition of Dusp6 with BCI. Our results suggest that Dusp6 attenuates Ras/MAPK signaling during regeneration and that suppressing Dusp6 can enhance cardiac repair.

A genetically targetable near-infrared photosensitizer.

Upon illumination, photosensitizer molecules produce reactive oxygen species that can be used for functional manipulation of living cells, including protein inactivation, targeted-damage introduction and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. We describe a genetically encoded fluorogen-activating protein (FAP) that binds a heavy atom-substituted fluorogenic dye, forming an 'on-demand' activated photosensitizer that produces singlet oxygen and fluorescence when activated with near-infrared light. This targeted and activated photosensitizer (TAPs) approach enables protein inactivation, targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs provides a new spectral range for photosensitizer proteins that could be useful for imaging, manipulation and cellular ablation deep within living organisms.

Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration.

AIMS: After injury, the adult zebrafish can regenerate the heart. This requires the activation of the endocardium and epicardium as well as the proliferation of pre-existing cardiomyocytes to replace the lost tissue. However, the molecular mechanisms involved in this process are not completely resolved. In this work, we aim to identify the proteins involved in zebrafish heart regeneration and to explore their function. METHODS AND RESULTS: Using a proteomic approach, we identified Hyaluronan-mediated motility receptor (Hmmr), a hyaluronic acid (HA) receptor, to be expressed following ventricular resection in zebrafish. Moreover, enzymes that produce HA, hyaluronic acid synthases (has), were also expressed following injury, suggesting that this pathway may serve important functions in the regenerating heart. Indeed, suppression of HA production, as well as depletion of Hmmr, blocked cardiac regeneration. Mechanistically, HA and Hmmr are required for epicardial cell epithelial-mesenchymal transition (EMT) and their subsequent migration into the regenerating ventricle. Furthermore, chemical inhibition of Focal Adhesion Kinase (FAK) or inhibition of Src kinases, downstream effectors of Hmmr, also prevented epicardial cell migration, implicating a HA/Hmmr/FAK/Src pathway in this process. In a rat model of myocardial infarction, both HA and HMMR were up-regulated and localized in the infarct area within the first few days following damage, suggesting that this pathway may also play an important role in cardiac repair in mammals. CONCLUSION: HA and Hmmr are required for activated epicardial cell EMT and migration involving the FAK/Src pathway for proper heart regeneration.