Transient pacing in pigs with complete heart block via myocardial injection of mRNA coding for the T-box transcription factor 18.

The adenovirus-mediated somatic transfer of the embryonic T-box transcription factor 18 (TBX18) gene can convert chamber cardiomyocytes into induced pacemaker cells. However, the translation of therapeutic TBX18-induced cardiac pacing faces safety challenges. Here we show that the myocardial expression of synthetic TBX18 mRNA in animals generates de novo pacing and limits innate and inflammatory immune responses. In rats, intramyocardially injected mRNA remained localized, whereas direct myocardial injection of an adenovirus carrying a reporter gene resulted in diffuse expression and in substantial spillover to the liver, spleen and lungs. Transient expression of TBX18 mRNA in rats led to de novo automaticity and pacemaker properties and, compared with the injection of adenovirus, to substantial reductions in the expression of inflammatory genes and in activated macrophage populations. In rodent and clinically relevant porcine models of complete heart block, intramyocardially injected TBX18 mRNA provided rate-adaptive cardiac pacing for one month that strongly correlated with the animal's sinus rhythm and physical activity. TBX18 mRNA may aid the development of biological pacemakers.

Tbx18 Orchestrates Cytostructural Transdifferentiation of Cardiomyocytes to Pacemaker Cells by Recruiting the Epithelial-Mesenchymal Transition Program.

Previously, we reported that heterologous expression of an embryonic transcription factor, Tbx18, reprograms ventricular cardiomyocytes into induced pacemaker cells (Tbx18-iPMs), though the key pathways are unknown. Here, we have used a tandem mass tag proteomic approach to characterize the impact of Tbx18 on neonatal rat ventricular myocytes. Tbx18 expression triggered vast proteome remodeling. Tbx18-iPMs exhibited increased expression of known pacemaker ion channels, including Hcn4 and Cx45 as well as upregulation of the mechanosensitive ion channels Piezo1, Trpp2 (PKD2), and TrpM7. Metabolic pathways were broadly downregulated, as were ion channels associated with ventricular excitation-contraction coupling. Tbx18-iPMs also exhibited extensive intracellular cytoskeletal and extracellular matrix remodeling, including 96 differentially expressed proteins associated with the epithelial-to-mesenchymal transition (EMT). RNAseq extended coverage of low abundance transcription factors, revealing upregulation of EMT-inducing Snai1, Snai2, Twist1, Twist2, and Zeb2. Finally, network diffusion mapping of >200 transcriptional regulators indicates EMT and heart development factors occupy adjacent network neighborhoods downstream of Tbx18 but upstream of metabolic control factors. In conclusion, transdifferentiation of cardiac myocytes into pacemaker cells entails massive electrogenic, metabolic, and cytostructural remodeling. Structural changes exhibit hallmarks of the EMT. The results aid ongoing efforts to maximize the yield and phenotypic stability of engineered biological pacemakers.

Engineered Cardiac Pacemaker Nodes Created by TBX18 Gene Transfer Overcome Source-Sink Mismatch.

Every heartbeat originates from a tiny tissue in the heart called the sinoatrial node (SAN). The SAN harbors only ≈10 000 cardiac pacemaker cells, initiating an electrical impulse that captures the entire heart, consisting of billions of cardiomyocytes for each cardiac contraction. How these rare cardiac pacemaker cells (the electrical source) can overcome the electrically hyperpolarizing and quiescent myocardium (the electrical sink) is incompletely understood. Due to the scarcity of native pacemaker cells, this concept of source-sink mismatch cannot be tested directly with live cardiac tissue constructs. By exploiting TBX18 induced pacemaker cells by somatic gene transfer, 3D cardiac pacemaker spheroids can be tissue-engineered. The TBX18 induced pacemakers (sphTBX18) pace autonomously and drive the contraction of neighboring myocardium in vitro. TBX18 spheroids demonstrate the need for reduced electrical coupling and physical separation from the neighboring ventricular myocytes, successfully recapitulating a key design principle of the native SAN. ß-Adrenergic stimulation as well as electrical uncoupling significantly increase sphTBX18s' ability to pace-and-drive the neighboring myocardium. This model represents the first platform to test design principles of the SAN for mechanistic understanding and to better engineer biological pacemakers for therapeutic translation.

Induced cardiac pacemaker cells survive metabolic stress owing to their low metabolic demand.

Cardiac pacemaker cells of the sinoatrial node initiate each and every heartbeat. Compared with our understanding of the constituents of their electrical excitation, little is known about the metabolic underpinnings that drive the automaticity of pacemaker myocytes. This lack is largely owing to the scarcity of native cardiac pacemaker myocytes. Here, we take advantage of induced pacemaker myocytes generated by TBX18-mediated reprogramming (TBX18-iPMs) to investigate comparative differences in the metabolic program between pacemaker myocytes and working cardiomyocytes. TBX18-iPMs were more resistant to metabolic stresses, exhibiting higher cell viability upon oxidative stress. TBX18-induced pacemaker myocytes (iPMs) expensed a lower degree of oxidative phosphorylation and displayed a smaller capacity for glycolysis compared with control ventricular myocytes. Furthermore, the mitochondria were smaller in TBX18-iPMs than in the control. We reasoned that a shift in the balance between mitochondrial fusion and fission was responsible for the smaller mitochondria observed in TBX18-iPMs. We identified a mitochondrial inner membrane fusion protein, Opa1, as one of the key mediators of this process and demonstrated that the suppression of Opa1 expression increases the rate of synchronous automaticity in TBX18-iPMs. Taken together, our data demonstrate that TBX18-iPMs exhibit a low metabolic demand that matches their mitochondrial morphology and ability to withstand metabolic insult.

NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model.

Duchenne muscular dystrophy (DMD) is a neuromuscular disorder causing progressive muscle degeneration. Although cardiomyopathy is a leading mortality cause in DMD patients, the mechanisms underlying heart failure are not well understood. Previously, we showed that NF-κB exacerbates DMD skeletal muscle pathology by promoting inflammation and impairing new muscle growth. Here, we show that NF-κB is activated in murine dystrophic (mdx) hearts, and that cardiomyocyte ablation of NF-κB rescues cardiac function. This physiological improvement is associated with a signature of upregulated calcium genes, coinciding with global enrichment of permissive H3K27 acetylation chromatin marks and depletion of the transcriptional repressors CCCTC-binding factor, SIN3 transcription regulator family member A, and histone deacetylase 1. In this respect, in DMD hearts, NF-κB acts differently from its established role as a transcriptional activator, instead promoting global changes in the chromatin landscape to regulate calcium genes and cardiac function.

MyoD Regulates Skeletal Muscle Oxidative Metabolism Cooperatively with Alternative NF-κB.

MyoD is a key regulator of skeletal myogenesis that directs contractile protein synthesis, but whether this transcription factor also regulates skeletal muscle metabolism has not been explored. In a genome-wide ChIP-seq analysis of skeletal muscle cells, we unexpectedly observed that MyoD directly binds to numerous metabolic genes, including those associated with mitochondrial biogenesis, fatty acid oxidation, and the electron transport chain. Results in cultured cells and adult skeletal muscle confirmed that MyoD regulates oxidative metabolism through multiple transcriptional targets, including PGC-1ß, a master regulator of mitochondrial biogenesis. We find that PGC-1ß expression is cooperatively regulated by MyoD and the alternative NF-κB signaling pathway. Bioinformatics evidence suggests that this cooperativity between MyoD and NF-κB extends to other metabolic genes as well. Together, these data identify MyoD as a regulator of the metabolic capacity of mature skeletal muscle to ensure that sufficient energy is available to support muscle contraction.

An NF-κB--EphrinA5-Dependent Communication between NG2(+) Interstitial Cells and Myoblasts Promotes Muscle Growth in Neonates.

Skeletal muscle growth immediately following birth is critical for proper body posture and locomotion. However, compared with embryogenesis and adulthood, the processes regulating the maturation of neonatal muscles is considerably less clear. Studies in the 1960s predicted that neonatal muscle growth results from nuclear accretion of myoblasts preferentially at the tips of myofibers. Remarkably, little information has been added since then to resolve how myoblasts migrate to the ends of fibers. Here, we provide insight into this process by revealing a unique NF-κB-dependent communication between NG2(+) interstitial cells and myoblasts. NF-κB in NG2(+) cells promotes myoblast migration to the tips of myofibers through cell-cell contact. This occurs through expression of ephrinA5 from NG2(+) cells, which we further deduce is an NF-κB target gene. Together, these results suggest that NF-κB plays an important role in the development of newborn muscles to ensure proper myoblast migration for fiber growth.

A multicellular spheroid formation and extraction chip using removable cell trapping barriers.

This paper presents a multicellular spheroid chip capable of forming and extracting three-dimensional (3D) spheroids using removable cell trapping barriers. Compared to the conventional macro-scale spheroid formation methods, including spinning, hanging-drop, and liquid-overlay methods, the recent micro-scale spheroid chips have the advantage of forming smaller spheroids with better uniformity. The recent micro spheroid chips, however, have difficulties in extracting the spheroids due to fixed cell trapping barriers. The present spheroid chip, having two PDMS layers, uses removable cell trapping barriers, thereby making it easy to form and extract uniform and small-sized spheroids. We have designed, fabricated and characterized a 4 × 1 spheroid chip, where membrane cell trapping barriers are inflated at a pressure of 50 kPa for spheroid formation and are deflated at zero gauge pressure for simple and safe extraction of the spheroids formed. In this experimental study, the cell suspension of non-small lung cancer cells, H1650, is supplied to the fabricated spheroid chip in the pressure range 145-155 Pa. The fabricated spheroid chips collect the cancer cells in the cell trapping regions from the cell suspension at a concentration of 2 × 10(6) ml(-1), thus forming uniform 3D spheroids with a diameter of 197.2 ± 11.7 µm, after 24 h incubation at 5% CO(2) and 37°C environment. After the removal of the cell trapping barriers, the spheroids formed were extracted through the outlet ports at a cell inlet pressure of 5 kPa. The cells in the extracted spheroids showed a viability of 80.3 ± 7.7%. The present spheroid chip offers a simple and effective method of obtaining uniform and small-sized 3D spheroids for the next stage of cell-based biomedical research, such as gene expression analysis and spheroid inoculation in animal models.

Epigenetic changes induced by reactive oxygen species in hepatocellular carcinoma: methylation of the E-cadherin promoter.

BACKGROUND & AIMS: In addition to genetic alterations, epigenetic changes underlie tumor progression and metastasis. Promoter methylation can silence tumor suppressor genes, and reactive oxygen species (ROS) promote DNA damage, although the relationship between ROS and epigenetic changes in cancer cells is not clear. We sought to determine whether ROS promote hypermethylation of the promoter region of E-cadherin, a regulator of the epithelial-to-mesenchymal transition, in hepatocellular carcinoma (HCC) cells. METHODS: HCC cells were exposed to H(2)O(2) or stably transfected to express Snail, a transcription factor that down-regulates E-cadherin expression. E-cadherin and Snail expression levels were examined by real-time reverse-transcriptase polymerase chain reaction and immunoblot analyses. The methylation status of E-cadherin was examined by methyl-specific polymerase chain reaction, bisulfite sequencing, and chromatin immunoprecipitation. The interactions between Snail, histone deacetylase 1, and DNA methyltransferase 1 were assessed by immunoprecipitation/immunoblot and immunofluorescence analyses. ROS-induced stress, E-cadherin expression, Snail expression, and E-cadherin promoter methylation were confirmed in HCC tissues by immunoblot, immunohistochemistry, and methyl-specific polymerase chain reaction analyses. RESULTS: We demonstrated that ROS induce hypermethylation of the E-cadherin promoter by increasing Snail expression. Snail induced DNA methylation of the E-cadherin promoter by recruiting histone deacetylase 1 and DNA methyltransferase 1. In human HCC tissues, we observed a correlation among ROS induction, E-cadherin down-regulation, Snail up-regulation, and E-cadherin promoter methylation. CONCLUSIONS: These findings provide novel mechanistic insights into epigenetic modulations induced by ROS in the process of carcinogenesis. They are potentially relevant to understanding the activity of ROS in silencing various tumor suppressor genes and in subsequent tumor progression and metastasis.

A novel splice variant of occludin deleted in exon 9 and its role in cell apoptosis and invasion.

The tight junction protein occludin participates in cell adhesion and migration and has been shown to possess antitumorigenic properties; however, the exact mechanism underlying these effects is poorly understood. In liver cell lines, we identified an occludin splice variant deleted in exon 9 (Occ(DeltaE9)). Furthermore, comparison analysis of wild-type occludin (Occ(WT)) and Occ(DeltaE9) revealed that exon 9 played important roles in the induction of mitochondria-mediated apoptosis and the inhibition of invasion, along with the downregulation of matrix metalloproteinase expression. In addition, by using the calcium indicator X-rhod-1, and the inositol trisphosphate receptor inhibitor 2-aminoethoxydiphenyl borate, we found that Occ(WT) but not Occ(DeltaE9) increased calcium release from the endoplasmic reticulum. In conclusion, our results showed that occludin mediates apoptosis and invasion by elevating the cytoplasmic calcium concentration and that exon 9 of occludin is an important region that mediates these effects.

HBx modulates iron regulatory protein 1-mediated iron metabolism via reactive oxygen species.

Hepatitis B virus X protein (HBx) is involved in viral metabolism and progression of liver disease. Iron metabolism plays a significant role in liver disease. In this report, to elucidate the relationship between iron metabolism and HBx, we established the Huh7 cell lines in which HBx was stably expressed (Huh7-HBx). In Huh7-HBx, we observed that transferrin receptor 1 (TfR1) expression decreased and ferritin heavy chain (FtH) expression increased as well as reactive oxygen species (ROS) level increased. We also found that these modulations were caused by the downregulation of iron regulatory protein 1 (IRP1). Furthermore, the levels of total iron and labile iron pool (LIP) were altered in Huh7-HBx. In addition, antioxidant N-acetylcystein (NaC) increased IRP1 expression by depleting HBx-induced ROS. We also confirmed these alterations of TfR1 and FtH in the primary hepatocytes of HBx transgenic mice and in HepG2.2.15 cells that constitutively replicate the intact HBV genome. In conclusion, these results suggest that HBx modulates iron metabolism via ROS leading to pathological status in liver diseases.