In vivo rescue of genetic dilated cardiomyopathy by systemic delivery of nexilin.

BACKGROUND: Dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. Multiple identified mutations in nexilin (NEXN) have been suggested to be linked with severe DCM. However, the exact association between multiple mutations of Nexn and DCM remains unclear. Moreover, it is critical for the development of precise and effective therapeutics in treatments of DCM. RESULTS: In our study, Nexn global knockout mice and mice carrying human equivalent G645del mutation are studied using functional gene rescue assays. AAV-mediated gene delivery is conducted through systemic intravenous injections at the neonatal stage. Heart tissues are analyzed by immunoblots, and functions are assessed by echocardiography. Here, we identify functional components of Nexilin and demonstrate that exogenous introduction could rescue the cardiac function and extend the lifespan of Nexn knockout mouse models. Similar therapeutic effects are also obtained in G645del mice, providing a promising intervention for future clinical therapeutics. CONCLUSIONS: In summary, we demonstrated that a single injection of AAV-Nexn was capable to restore the functions of cardiomyocytes and extended the lifespan of Nexn knockout and G645del mice. Our study represented a long-term gene replacement therapy for DCM that potentially covers all forms of loss-of-function mutations in NEXN.

Cardiac-specific deletion of heat shock protein 60 induces mitochondrial stress and disrupts heart development in mice.

Congenital heart diseases are the most common birth defects around the world. Emerging evidence suggests that mitochondrial homeostasis is required for normal heart development. In mitochondria, a series of molecular chaperones including heat shock protein 60 (HSP60) are engaged in assisting the import and folding of mitochondrial proteins. However, it remains largely obscure whether and how these mitochondrial chaperones regulate cardiac development. Here, we generated a cardiac-specific Hspd1 deletion mouse model by αMHC-Cre and investigated the role of HSP60 in cardiac development. We observed that deletion of HSP60 in embryonic cardiomyocytes resulted in abnormal heart development and embryonic lethality, characterized by reduced cardiac cell proliferation and thinner ventricular walls, highlighting an essential role of cardiac HSP60 in embryonic heart development and survival. Our results also demonstrated that HSP60 deficiency caused significant downregulation of mitochondrial ETC subunits and induced mitochondrial stress. Analysis of gene expression revealed that P21 that negatively regulates cell proliferation is significantly upregulated in HSP60 knockout hearts. Moreover, HSP60 deficiency induced activation of eIF2α-ATF4 pathway, further indicating the underlying mitochondrial stress in cardiomyocytes after HSP60 deletion. Taken together, our study demonstrated that regular function of mitochondrial chaperones is pivotal for maintaining normal mitochondrial homeostasis and embryonic heart development.

DELE1 is protective for mitochondrial cardiomyopathy.

Mitochondrial dysfunction in heart triggers an integrated stress response (ISR) through phosphorylation of eIF2α and subsequent ATF4 activation. DAP3 Binding Cell Death Enhancer 1 (DELE1) is a mitochondrial protein recently found to be critical for mediating mitochondrial stress-triggered ISR (MSR)-induced eIF2α-ATF4 pathway activation. However, the specific role of DELE1 in heart at baseline or in response to mitochondrial stress remains largely unknown. In this study, we report that DELE1 is dispensable for cardiac development and function under baseline conditions. Conversely, DELE1 is essential for mediating an adaptive response to mitochondrial dysfunction-triggered stress in the heart, playing a protective role in mitochondrial cardiomyopathy.

Mediator complex proximal Tail subunit MED30 is critical for Mediator core stability and cardiomyocyte transcriptional network.

Dysregulation of cardiac transcription programs has been identified in patients and families with heart failure, as well as those with morphological and functional forms of congenital heart defects. Mediator is a multi-subunit complex that plays a central role in transcription initiation by integrating regulatory signals from gene-specific transcriptional activators to RNA polymerase II (Pol II). Recently, Mediator subunit 30 (MED30), a metazoan specific Mediator subunit, has been associated with Langer-Giedion syndrome (LGS) Type II and Cornelia de Lange syndrome-4 (CDLS4), characterized by several abnormalities including congenital heart defects. A point mutation in MED30 has been identified in mouse and is associated with mitochondrial cardiomyopathy. Very recent structural analyses of Mediator revealed that MED30 localizes to the proximal Tail, anchoring Head and Tail modules, thus potentially influencing stability of the Mediator core. However, in vivo cellular and physiological roles of MED30 in maintaining Mediator core integrity remain to be tested. Here, we report that deletion of MED30 in embryonic or adult cardiomyocytes caused rapid development of cardiac defects and lethality. Importantly, cardiomyocyte specific ablation of MED30 destabilized Mediator core subunits, while the kinase module was preserved, demonstrating an essential role of MED30 in stability of the overall Mediator complex. RNAseq analyses of constitutive cardiomyocyte specific Med30 knockout (cKO) embryonic hearts and inducible cardiomyocyte specific Med30 knockout (icKO) adult cardiomyocytes further revealed critical transcription networks in cardiomyocytes controlled by Mediator. Taken together, our results demonstrated that MED30 is essential for Mediator stability and transcriptional networks in both developing and adult cardiomyocytes. Our results affirm the key role of proximal Tail modular subunits in maintaining core Mediator stability in vivo.

Cardiolipin Remodeling Defects Impair Mitochondrial Architecture and Function in a Murine Model of Barth Syndrome Cardiomyopathy.

BACKGROUND: Cardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in Tafazzin (TAZ), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking. METHODS: We performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated Taz cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group). RESULTS: Taz cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity. CONCLUSIONS: Our mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.

Factors associated with alveolar bone depth mesial to the mandibular third molars after orthodontic protraction.

INTRODUCTION: The objective of this research was to study the factors associated with the alveolar bone depth mesial to the mandibular third molars (M8) after the mandibular second (M7) and third molars were protracted into the space of the mandibular first molars (M6), which were newly extracted for orthodontic treatment or extracted more than 1 year before treatment. METHODS: This retrospective study included 57 adult patients (mean age 23.40 ± 4.40 years) in whom M6 were newly extracted for orthodontic treatment or extracted more than 1 year before treatment. The alveolar bone depth mesial to M8 was measured on posttreatment panoramic radiographs. The vertical, horizontal, and angular changes of M8 were measured on both pre- and posttreatment panoramic radiographs. Linear correlation and regression analyses were conducted to explore the factors associated with the alveolar bone depth mesial to M8. RESULTS: The alveolar bone conditions of M6 (R= -0.391, P <0.001) and the vertical movement directions of M8 (R= -0.433, P <0.001) were significant factors associated with the alveolar bone depth mesial to M8 after orthodontic protraction. CONCLUSIONS: Without considering the pretreatment periodontal status of M8, patients with M6 extracted exceeding 1 year before treatment and with M8 extruded after orthodontic protraction may exhibit deeper alveolar bone depth mesial to M8.

Homozygous G650del nexilin variant causes cardiomyopathy in mice.

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.

Deletion of IP3R1 by Pdgfrb-Cre in mice results in intestinal pseudo-obstruction and lethality.

BACKGROUND: Inositol 1,4,5-trisphosphate receptors (IP3Rs) are a family of intracellular Ca2+ release channels located on the membrane of endoplasmic reticulum, which have been shown to play critical roles in various cellular and physiological functions. However, their function in regulating gastrointestinal (GI) tract motility in vivo remains unknown. Here, we investigated the physiological function of IP3R1 in the GI tract using genetically engineered mouse models. METHODS: Pdgfrb-Cre mice were bred with homozygous Itpr1 floxed (Itpr1f/f) mice to generate conditional IP3R1 knockout (pcR1KO) mice. Cell lineage tracing was used to determine where Pdgfrb-Cre-mediated gene deletion occurred in the GI tract. Isometric tension recording was used to measure the effects of IP3R1 deletion on muscle contraction. RESULTS: In the mouse GI tract, Itpr1 gene deletion by Pdgfrb-Cre occurred in smooth muscle cells, enteric neurons, and interstitial cells of Cajal. pcR1KO mice developed impaired GI motility, with prolonged whole-gut transit time and abdominal distention. pcR1KO mice also exhibited lethality as early as 8 weeks of age and 50% of pcR1KO mice were dead by 40 weeks after birth. The frequency of spontaneous contractions in colonic circular muscles was dramatically decreased and the amplitude of spontaneous contractions was increased in pcR1KO mice. Deletion of IP3R1 in the GI tract also reduced the contractile response to the muscarinic agonist, carbachol, as well as to electrical field stimulation. However, KCl-induced contraction and expression of smooth muscle-specific contractile genes were not significantly altered in pcR1KO mice. CONCLUSIONS: Here, we provided a novel mouse model for impaired GI motility and demonstrated that IP3R1 plays a critical role in regulating physiological function of GI tract in vivo.

Genome-wide identification and functional analysis of long noncoding RNAs involved in the response to graphene oxide.

Long noncoding RNAs (lncRNAs), which are defined as noncoding RNAs having at least 200 nucleotides, can potentially regulate various biological processes. However, the roles of lncRNAs in regulating cellular response to engineered nanomaterials (ENMs) are still unclear. Using Hiseq 2000 sequencing technique, we performed a genome-wide screen to identify lncRNAs involved in the control of toxicity of graphene oxide (GO) using in vivo Caenorhabditis elegans assay system. HiSeq 2000 sequencing, followed by quantitative analysis, identified only 34 dysregulated lncRNAs in GO exposed nematodes. Bioinformatics analysis implies the biological processes and signaling pathways mediated by candidate lncRNAs involved in the control of GO toxicity. A lncRNAs-miRNAs network possibly involved in the control of GO toxicity was further raised. Moreover, we identified the shared lncRNAs based on the molecular regulation basis for chemical surface modifications and/or genetic mutations in reducing GO toxicity. We further provide direct evidence that these shared lncRNAs, linc-37 and linc-14, were involved in the control of chemical surface modifications and genetic mutations in reducing GO toxicity. linc-37 binding to transcriptional factor FOXO/DAF-16 might be important for the control of GO toxicity. Our whole-genome identification and functional analysis of lncRNAs highlights the important roles of lncRNAs based molecular mechanisms for cellular responses to ENMs in organisms.