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Retinoid-related orphan receptor (RAR) gamma (RORγt)-expressing regulatory T cells (RORγt+ Tregs) play pivotal roles in preventing T cell hyperactivation and maintaining tissue homeostasis, in part by secreting the anti-inflammation cytokine interleukin-10 (IL-10). Here, we report that hypoxia-induced factor 1α (HIF1α) is the master transcription factor for Il10 in RORγt+ Tregs. This critical anti-inflammatory pathway is negatively regulated by an RNA binding protein DEAD box helicase 5 (DDX5). As a transcriptional corepressor, DDX5 restricts the expression of HIF1α and its downstream target gene Il10 in RORγt+ Tregs. T cell-specific Ddx5 knockout (DDX5ΔT) mice have augmented RORγt+ Treg suppressor activities and are better protected from intestinal inflammation. Genetic ablation or pharmacologic inhibition of HIF1α restores enteropathy susceptibility in DDX5ΔT mice. The DDX5-HIF1α-IL-10 pathway is conserved in mice and humans. These findings reveal potential therapeutic targets for intestinal inflammatory diseases.

FLNC, encoding filamin C, is one of the most mutated genes in dilated and hypertrophic cardiomyopathy. However, the precise role of filamin C in mammalian heart remains unclear. In this study, we demonstrated Flnc global (FlncgKO) and cardiomyocyte-specific knockout (FlnccKO) mice died in utero from severely ruptured ventricular myocardium, indicating filamin C is required to maintain the structural integrity of myocardium in the mammalian heart. Contrary to the common belief that filamin C acts as an integrin inactivator, we observed attenuated activation of β1 integrin specifically in the myocardium of FlncgKO mice. Although deleting β1 integrin from cardiomyocytes did not recapitulate the heart rupture phenotype in Flnc knockout mice, deleting both β1 integrin and filamin C from cardiomyocytes resulted in much more severe heart ruptures than deleting filamin C alone. Our results demonstrated that filamin C works in concert with β1 integrin to maintain the structural integrity of myocardium during mammalian heart development.

BackgroundLeft ventricular noncompaction cardiomyopathy (LVNC) was discovered half a century ago as a cardiomyopathy with excessive trabeculation and a thin ventricular wall. In the decades since, numerous studies have demonstrated that LVNC primarily has an effect on left ventricles (LVs) and is often associated with LV dilation and dysfunction. However, in part because of the lack of suitable mouse models that faithfully mirror the selective LV vulnerability in patients, mechanisms underlying the susceptibility of LVs to dilation and dysfunction in LVNC remain unknown. Genetic studies have revealed that deletions and mutations in PRDM16 (PR domain-containing 16) cause LVNC, but previous conditional Prdm16 knockout mouse models do not mirror the LVNC phenotype in patients, and the underlying molecular mechanisms by which PRDM16 deficiency causes LVNC are still unclear.MethodsPrdm16 cardiomyocyte-specific knockout (Prdm16cKO) mice were generated and analyzed for cardiac phenotypes. RNA sequencing and chromatin immunoprecipitation deep sequencing were performed to identify direct transcriptional targets of PRDM16 in cardiomyocytes. Single-cell RNA sequencing in combination with spatial transcriptomics was used to determine cardiomyocyte identity at the single-cell level.ResultsCardiomyocyte-specific ablation of Prdm16 in mice caused LV-specific dilation and dysfunction, as well as biventricular noncompaction, which fully recapitulated LVNC in patients. PRDM16 functioned mechanistically as a compact myocardium-enriched transcription factor that activated compact myocardial genes while repressing trabecular myocardial genes in LV compact myocardium. Consequently, Prdm16cKO LV compact myocardial cardiomyocytes shifted from their normal transcriptomic identity to a transcriptional signature resembling trabecular myocardial cardiomyocytes or neurons. Chamber-specific transcriptional regulation by PRDM16 was attributable in part to its cooperation with LV-enriched transcription factors Tbx5 and Hand1.ConclusionsThese results demonstrate that disruption of proper specification of compact cardiomyocytes may play a key role in the pathogenesis of LVNC. They also shed light on underlying mechanisms of the LV-restricted transcriptional program governing LV chamber growth and maturation, providing a tangible explanation for the susceptibility of LV in a subset of LVNC cardiomyopathies.

Mechanisms by which specific histone modifications regulate distinct gene networks remain little understood. We investigated how H3K79me2, a modification catalyzed by DOT1L and previously considered a general transcriptional activation mark, regulates gene expression during cardiogenesis. Embryonic cardiomyocyte ablation of Dot1l revealed that H3K79me2 does not act as a general transcriptional activator, but rather regulates highly specific transcriptional networks at two critical cardiogenic junctures: embryonic cardiogenesis, where it was particularly important for left ventricle-specific genes, and postnatal cardiomyocyte cell cycle withdrawal, with Dot1L mutants having more mononuclear cardiomyocytes and prolonged cardiomyocyte cell cycle activity. Mechanistic analyses revealed that H3K79me2 in two distinct domains, gene bodies and regulatory elements, synergized to promote expression of genes activated by DOT1L. Surprisingly, H3K79me2 in specific regulatory elements also contributed to silencing genes usually not expressed in cardiomyocytes. These results reveal mechanisms by which DOT1L successively regulates left ventricle specification and cardiomyocyte cell cycle withdrawal.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948-1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn's function in tubular system organization and normal cardiac function.

During prophase I of meiosis, chromosomes become organized as loop arrays around the proteinaceous chromosome axis. As homologous chromosomes physically pair and recombine, the chromosome axis is integrated into the tripartite synaptonemal complex (SC) as this structure's lateral elements (LEs). While the components of the mammalian chromosome axis/LE-including meiosis-specific cohesin complexes, the axial element proteins SYCP3 and SYCP2, and the HORMA domain proteins HORMAD1 and HORMAD2-are known, the molecular organization of these components within the axis is poorly understood. Here, using expansion microscopy coupled with 2-color stochastic optical reconstruction microscopy (STORM) imaging (ExSTORM), we address these issues in mouse spermatocytes at a resolution of 10 to 20 nm. Our data show that SYCP3 and the SYCP2 C terminus, which are known to form filaments in vitro, form a compact core around which cohesin complexes, HORMADs, and the N terminus of SYCP2 are arrayed. Overall, our study provides a detailed structural view of the meiotic chromosome axis, a key organizational and regulatory component of meiotic chromosomes.

RationaleMutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type-specific requirement for TBX20 in early myocardial development remains to be explored.ObjectiveHere, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development.Methods and resultsAblation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner.ConclusionsMyocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer.

RATIONALE:Myocardial infarction is a major cause of adult mortality worldwide. The origin(s) of cardiac fibroblasts that constitute the postinfarct scar remain controversial, in particular the potential contribution of bone marrow lineages to activated fibroblasts within the scar. OBJECTIVE:The aim of this study was to establish the origin(s) of infarct fibroblasts using lineage tracing and bone marrow transplants and a robust marker for cardiac fibroblasts, the Collagen1a1-green fluorescent protein reporter. METHODS AND RESULTS:Using genetic lineage tracing or bone marrow transplant, we found no evidence for collagen-producing fibroblasts derived from hematopoietic or bone marrow lineages in hearts subjected to permanent left anterior descending coronary artery ligation. In fact, fibroblasts within the infarcted area were largely of epicardial origin. Intriguingly, collagen-producing fibrocytes from hematopoietic lineages were observed attached to the epicardial surface of infarcted and sham-operated hearts in which a suture was placed around the left anterior descending coronary artery. CONCLUSIONS:In this controversial field, our study demonstrated that the vast majority of infarct fibroblasts were of epicardial origin and not derived from bone marrow lineages, endothelial-to-mesenchymal transition, or blood. We also noted the presence of collagen-producing fibrocytes on the epicardial surface that resulted at least in part from the surgical procedure.

Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals. NEW & NOTEWORTHY Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein and a central player of the cellular protein quality control system. BAG3 is prominently expressed in the heart and plays an essential role in cardiac protein homeostasis by interacting with chaperone heat shock proteins (HSPs) in large, functionally distinct multichaperone complexes. The BAG3 mutation of proline 209 to leucine (P209L), which resides in a critical region that mediates the direct interaction between BAG3 and small HSPs (sHSPs), is associated with cardiomyopathy in humans. However, the mechanism by which the BAG3 P209L missense mutation leads to cardiomyopathy remains unknown. To determine the molecular basis underlying the cardiomyopathy caused by the BAG3 P209L mutation, we generated a knockin (KI) mouse model in which the endogenous Bag3 gene was replaced with mutant Bag3 containing the P215L mutation, which is equivalent to the human P209L mutation. We performed physiological, histological, and biochemical analyses of Bag3 P209L KI mice to determine the functional, morphological, and molecular consequences of the P209L mutation. We found that Bag3 P209L KI mice exhibited normal cardiac function and morphology up to 16 mo of age. Western blot analysis further revealed that levels of sHSPs, stress-inducible HSPs, ubiquitinated proteins, and autophagy were unaffected in P209L mutant mouse hearts. In conclusion, the P209L mutation in Bag3 does not cause cardiomyopathy in mice up to 16 mo of age under baseline conditions. NEW & NOTEWORTHY Bcl-2-associated athanogene 3 (BAG3) P209L mutation is associated with human cardiomyopathy. A recent study reported that transgenic mice overexpressing human BAG3 P209L in cardiomyocytes have cardiac dysfunction. In contrast, our P209L mice that express mutant BAG3 at the same level as that of wild-type mice displayed no overt phenotype. Our results suggest that human cardiomyopathy may result from species-specific requirements for the conserved motif that is disrupted by P209L mutation or from genetic background-dependent effects.