Rig1 receptor plays a critical role in cardiac reprogramming via YY1 signaling.

We discovered that innate immunity plays an important role in the reprogramming of fibroblasts into cardiomyocytes. In this report, we define the role of a novel retinoic acid-inducible gene 1 Yin Yang 1 (Rig1:YY1) pathway. We found that fibroblast to cardiomyocyte reprogramming efficacy was enhanced by specific Rig1 activators. To understand the mechanism of action, we performed various transcriptomic, nucleosome occupancy, and epigenomic approaches. Analysis of the datasets indicated that Rig1 agonists had no effect on reprogramming-induced changes in nucleosome occupancy or loss of inhibitory epigenetic motifs. Instead, Rig1 agonists were found to modulate cardiac reprogramming by promoting the binding of YY1 specifically to cardiac genes. To conclude, these results show that the Rig1:YY1 pathway plays a critical role in fibroblast to cardiomyocyte reprogramming.

A novel Cbx1, PurB, and Sp3 complex mediates long-term silencing of tissue- and lineage-specific genes.

miRNA-based cellular fate reprogramming offers an opportunity to investigate the mechanisms of long-term gene silencing. To further understand how genes are silenced in a tissue-specific manner, we leveraged our miRNA-based method of reprogramming fibroblasts into cardiomyocytes. Through screening approaches, we identified three proteins that were downregulated during reprogramming of fibroblasts into cardiomyocytes: heterochromatin protein Cbx1, transcriptional activator protein PurB, and transcription factor Sp3. We show that knockdown of Cbx1, PurB, and Sp3 was sufficient to induce cardiomyocyte gene expression in fibroblasts. Similarly, gene editing to ablate Cbx1, PurB, and Sp3 expression induced fibroblasts to convert into cardiomyocytes in vivo. Furthermore, high-throughput DNA sequencing and coimmunoprecipitation experiments indicated that Cbx1, PurB, and Sp3 also bound together as a complex and were necessary to localize nucleosomes to cardiomyocyte genes on the chromosome. Finally, we found that the expression of these genes led to nucleosome modification via H3K27me3 (trimethylated histone-H3 lysine-27) deposition through an interaction with the polycomb repressive PRC2 complex. In summary, we conclude that Cbx1, PurB, and Sp3 control cell fate by actively repressing lineage-specific genes.

Insights from molecular signature of in vivo cardiac c-Kit(+) cells following cardiac injury and ß-catenin inhibition.

There is much interest over resident c-Kit(+) cells in tissue regeneration. Their role in cardiac regeneration has been controversial. In this study we aim to understand the in vivo behavior of cardiac c-Kit(+) cells at baseline and after myocardial infarction and in response to Sfrp2. This approach can accurately study the in vivo transcript expressions of these cells in temporal response to injury and overcomes the limitations of the in vitro approach. RNA-seq was performed with c-Kit(+) cells and cardiomyocytes from healthy non-injured mice as well as c-Kit(+) cells from 1 day post-MI and 12 days post-MI mice. When compared to in vivo c-Kit(+) cells isolated from a healthy non-injured mouse heart, cardiomyocytes were enriched in transcripts that express anion channels, cation channels, developmental/differentiation pathway components, as well as proteins that inhibit canonical Wnt/ß-catenin signaling. Myocardial infarction (MI) induced in vivo c-Kit(+) cells to transiently adopt the cardiomyocyte-specific signature: expression of a number of cardiomyocyte-specific transcripts was maximal 1 day post-MI and declined by 12 days post-MI. We next studied the effect of ß-catenin inhibition on in vivo c-Kit(+) cells by administering the Wnt inhibitor Sfrp2 into the infarct border zone. Sfrp2 both enhanced and sustained cardiomyocyte-specific gene expression in the in vivo c-Kit(+) cells: expression of cardiomyocyte-specific transcripts was higher and there was no decline in expression by 12 days post-MI. Further analysis of the biology of c-Kit(+) cells identified that culture induced a significant and irreversible change in their molecular signature raising questions about reliability of in vitro studies. Our findings provide evidence that MI induces in vivo c-Kit(+) cells to adopt transiently a cardiomyocyte-specific pattern of gene expression, and Sfrp2 further enhances and induces sustained gene expression. Our approach is important for understanding c-Kit(+) cells in cardiac regeneration and also has broad implications in the investigation of in vivo resident stem cells in other areas of tissue regeneration.

Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4.

In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca(2+)]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca(2+)]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca(2+)]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca(2+)]i through Ca(2+) influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca(2+) influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca(2+) influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca(2+) influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca(2+)]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn.

The aquaporin 1 C-terminal tail is required for migration and growth of pulmonary arterial myocytes.

Pulmonary arterial smooth muscle cell (PASMC) proliferation and migration are important contributors to the vascular remodeling that occurs during development of pulmonary hypertension. We previously demonstrated that aquaporin (AQP)1, a member of the water channel family of proteins, was expressed in PASMCs and was necessary for hypoxia-induced migration; however, the mechanism by which AQP1 controls this response is unclear. The C-terminal tail of AQP1 contains putative calcium (EF-hand) and protein binding sites. Thus, we wanted to explore whether the C-terminal tail or the EF-hand motif of AQP1 was required for migration and proliferation. Rat PASMCs were isolated from distal pulmonary arteries, and proliferation and migration were measured using BrdU incorporation and transwell filters, respectively. To deplete AQP1, PASMCs were transfected with AQP1 small interference RNA (siRNA) or nontargeting siRNA. Knockdown of AQP1 reduced basal proliferation and hypoxia-induced migration and proliferation in PASMCs. In subsequent experiments, wild-type AQP1, AQP1 lacking the entire cytoplasmic C-terminal tail, or AQP1 with a mutation in the EF-hand motif were expressed in PASMCs using adenoviral constructs. For all AQP1 constructs, infection increased AQP1 protein levels, water permeability, and the change in cell volume induced by hypotonic challenge. Infection with wild-type and EF-hand mutated AQP1, but not C-terminal-deleted AQP1, increased PASMC migration and proliferation. Our results suggest that AQP1 controls proliferation and migration in PASMCs and that the mechanism requires the C-terminal tail of the protein but is independent of water transport or the EF-hand motif.

Skeletal muscle differentiation induces wide-ranging nucleosome repositioning in muscle gene promoters.

In a previous report, we demonstrated that Cbx1, PurB and Sp3 inhibited cardiac muscle differentiation by increasing nucleosome density around cardiac muscle gene promoters. Since cardiac and skeletal muscle express many of the same proteins, we asked if Cbx1, PurB and Sp3 similarly regulated skeletal muscle differentiation. In a C2C12 model of skeletal muscle differentiation, Cbx1 and PurB knockdown increased myotube formation. In contrast, Sp3 knockdown inhibited myotube formation, suggesting that Sp3 played opposing roles in cardiac muscle and skeletal muscle differentiation. Consistent with this finding, Sp3 knockdown also inhibited various muscle-specific genes. The Cbx1, PurB and Sp3 proteins are believed to influence gene-expression in part by altering nucleosome position. Importantly, we developed a statistical approach to determine if changes in nucleosome positioning were significant and applied it to understanding the architecture of muscle-specific genes. Through this novel statistical approach, we found that during myogenic differentiation, skeletal muscle-specific genes undergo a set of unique nucleosome changes which differ significantly from those shown in commonly expressed muscle genes. While Sp3 binding was associated with nucleosome loss, there appeared no correlation with the aforementioned nucleosome changes. In summary, we have identified a novel role for Sp3 in skeletal muscle differentiation and through the application of quantifiable MNase-seq have discovered unique fingerprints of nucleosome changes for various classes of muscle genes during myogenic differentiation.

Modifying miRs for effective reprogramming of fibroblasts to cardiomyocytes.

Reprogramming scar fibroblasts into cardiomyocytes has been proposed to reverse the damage associated with myocardial infarction. However, the limited improvement in cardiac function calls for enhanced strategies. We reported enhanced efficacy of our miR reprogramming cocktail miR combo (miR-1, miR-133a, miR-208a, and miR-499) via RNA-sensing receptor stimulation. We hypothesized that we could combine RNA-sensing receptor activation with fibroblast reprogramming by chemically modifying miR combo. To test the hypothesis, miR combo was modified to enhance interaction with the RNA-sensing receptor Rig1 via the addition of a 5'-triphosphate (5'ppp) group. Importantly, when compared with unmodified miR combo, 5'ppp-modified miR combo markedly improved reprogramming efficacy in vitro. Enhanced reprogramming efficacy correlated with a type-I interferon immune response with strong and selective secretion of interferon ß (IFNß). Antibody blocking studies and media replacement experiments indicated that 5'ppp-miR combo utilized IFNß to enhance fibroblast reprogramming efficacy. In conclusion, miRs can acquire powerful additional roles through chemical modification that potentially increases their clinical applications.

Conservation of miR combo based direct cardiac reprogramming.

There is considerable interest in regenerating the injured heart by reprogramming resident fibroblasts into new functional cardiomyocytes. Cardiac reprogramming has been achieved via transcription factors or miRNAs. Transcription factor combinations appear to be species-specific as evidenced by the fact that combinations of transcription factors which are effective for the reprogramming of mouse fibroblasts are ineffective in pigs and humans. Whether miRNA based cardiac reprogramming suffers from the same limitation is unknown. We have previously demonstrated that mouse cardiac fibroblasts can be directly converted into cardiomyocytes both in vitro and in vivo via a combination of four microRNAs (miR-1, miR-133a, miR-208a and miR-499) termed "miR combo." To assess species-specificity, miR combo was transfected into cardiac fibroblasts isolated from the left ventricle of dogs, pigs and humans. QPCR analysis indicated that miR combo effectively reprogrammed fibroblasts from all of the tested mammalian species. Significant upregulation of cardiac developmental, sarcomere, and cardiac ion channel genes was observed. Through Actn2+ staining, we also found that miR combo transfection induced dog, pig and human cardiac fibroblasts to develop into cardiomyocyte-like cells. In conclusion, we have demonstrated that in contrast to transcription factor based approaches, miR combo effectively reprograms mammalian cardiac fibroblasts into cardiomyocyte-like cells.

Rho kinase and Na+ /H+ exchanger mediate endothelin-1-induced pulmonary arterial smooth muscle cell proliferation and migration.

Excessive production of endothelin-1 (ET-1) has been observed in almost all forms of pulmonary hypertension. ET-1, a highly potent vasoconstrictor, can also potentiate pulmonary arterial smooth muscle cell (PASMC) growth and migration, both of which contribute to the vascular remodeling that occurs during the development of pulmonary hypertension. Increasing evidence indicates that alkalinization of intracellular pH (pHi ), typically due to activation of Na+ /H+ exchange (NHE), is associated with enhanced PASMC proliferation and migration. We recently demonstrated that application of exogenous ET-1 increased NHE activity in murine PASMCs via a mechanism requiring Rho kinase (ROCK). However, whether ROCK and/or increased NHE activity mediate ET-1-induced migration and proliferation in PASMCs remains unknown. In this study, we used fluorescent microscopy in transiently cultured PASMCs from distal pulmonary arteries of the rat and the pH-sensitive dye, BCECF-AM, to measure changes in resting pHi and NHE activity induced by exposure to exogenous ET-1 (10-8  mol/L) for 24 h. Cell migration and proliferation in response to ET-1 were also measured using Transwell assays and BrdU incorporation, respectively. We found that application of exogenous ET-1 had no effect on NHE1 expression, but increased pHi , NHE activity, migration, and proliferation in rat PASMCs. Pharmacologic inhibition of NHE or ROCK prevented the ET-1-induced changes in cell function (proliferation and migration). Our results indicate that ET-1 modulates PASMC migration and proliferation via changes in pHi homeostasis through a pathway involving ROCK.