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1.  POPDC1S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking 
The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.
doi:10.1172/JCI79562
PMCID: PMC4701561  PMID: 26642364
2.  Modulation of Human Valve Interstitial Cell Phenotype and Function Using a Fibroblast Growth Factor 2 Formulation 
PLoS ONE  2015;10(6):e0127844.
Valve interstitial cells (VICs) are fibroblastic in nature however in culture it is widely accepted that they differentiate into a myofibroblastic phenotype. This study assessed a fibroblast culture media formulation for its ability to maintain the phenotype and function of VICs as in the intact healthy valve. Normal human VICs were cultured separately in standard DMEM and in fibroblast media consisting of FGF2 (10ng/ml), insulin (50ng/ml) and 2% FCS for at least a week. Cell morphology, aspect ratio, size, levels and distribution of protein expression, proliferation, cell cycle, contraction and migration were assessed. Some VICs and some valve endothelial cells expressed FGF2 in valve tissue and this expression was increased in calcified valves. VICs in DMEM exhibited large, spread cells whereas VICs in fibroblast media were smaller, elongated and spindly. Aspect ratio and size were both significantly higher in DMEM (p<0.01). The level of expression of α-SMA was significantly reduced in fibroblast media at day 2 after isolation (p<0.01) and the expression of α-SMA, SM22 and EDA-fibronectin was significantly reduced in fibroblast media at days 7 and 12 post-isolation (p<0.01). Expression of cytoskeletal proteins, bone marker proteins and extracellular matrix proteins was reduced in fibroblast media. Proliferation of VICs in fibroblast media was significantly reduced at weeks 1 (p<0.05) and 2 (p<0.01). Collagen gel contraction was significantly reduced in fibroblast media (p<0.05). VICs were found to have significantly fewer and smaller focal adhesions in fibroblast media (p<0.01) with significantly fewer supermature focal adhesions in fibroblast media (p<0.001). Ultrastructurally, VICs in fibroblast media resembled native VICs from intact valves. VICs in fibroblast media demonstrated a slower migratory ability after wounding at 72 hours (p<0.01). Treatment of human VICs with this fibroblast media formulation has the ability to maintain and to dedifferentiate the VICs back to a fibroblastic phenotype with phenotypic and functional characteristics ascribed to cells in the intact valve. This methodology is fundamental in the study of normal valve biology, pathology and in the field of tissue engineering.
doi:10.1371/journal.pone.0127844
PMCID: PMC4456368  PMID: 26042674
3.  Cardiac tissue slices: preparation, handling, and successful optical mapping 
Cardiac tissue slices are becoming increasingly popular as a model system for cardiac electrophysiology and pharmacology research and development. Here, we describe in detail the preparation, handling, and optical mapping of transmembrane potential and intracellular free calcium concentration transients (CaT) in ventricular tissue slices from guinea pigs and rabbits. Slices cut in the epicardium-tangential plane contained well-aligned in-slice myocardial cell strands (“fibers”) in subepicardial and midmyocardial sections. Cut with a high-precision slow-advancing microtome at a thickness of 350 to 400 μm, tissue slices preserved essential action potential (AP) properties of the precutting Langendorff-perfused heart. We identified the need for a postcutting recovery period of 36 min (guinea pig) and 63 min (rabbit) to reach 97.5% of final steady-state values for AP duration (APD) (identified by exponential fitting). There was no significant difference between the postcutting recovery dynamics in slices obtained using 2,3-butanedione 2-monoxime or blebistatin as electromechanical uncouplers during the cutting process. A rapid increase in APD, seen after cutting, was caused by exposure to ice-cold solution during the slicing procedure, not by tissue injury, differences in uncouplers, or pH-buffers (bicarbonate; HEPES). To characterize intrinsic patterns of CaT, AP, and conduction, a combination of multipoint and field stimulation should be used to avoid misinterpretation based on source-sink effects. In summary, we describe in detail the preparation, mapping, and data analysis approaches for reproducible cardiac tissue slice-based investigations into AP and CaT dynamics.
doi:10.1152/ajpheart.00556.2014
PMCID: PMC4551126  PMID: 25595366
heart; optical mapping; multiparametric; high spatial resolution; voltage- and calcium-sensitive dyes; live tissue slices
4.  Ablation of SGK1 Impairs Endothelial Cell Migration and Tube Formation Leading to Decreased Neo-Angiogenesis Following Myocardial Infarction  
PLoS ONE  2013;8(11):e80268.
Serum and glucocorticoid inducible kinase 1 (SGK1) plays a pivotal role in early angiogenesis during embryonic development. In this study, we sought to define the SGK1 downstream signalling pathways in the adult heart and to elucidate their role in cardiac neo-angiogenesis and wound healing after myocardial ischemia. To this end, we employed a viable SGK1 knockout mouse model generated in a 129/SvJ background. Ablation of SGK1 in these mice caused a significant decrease in phosphorylation of SGK1 target protein NDRG1, which correlated with alterations in NF-κB signalling and expression of its downstream target protein, VEGF-A. Disruption of these signalling pathways was accompanied by smaller heart and body size. Moreover, the lack of SGK1 led to defective endothelial cell (ECs) migration and tube formation in vitro, and increased scarring with decreased angiogenesis in vivo after myocardial infarct. This study underscores the importance of SGK1 signalling in cardiac neo-angiogenesis and wound healing after an ischemic insult in vivo.
doi:10.1371/journal.pone.0080268
PMCID: PMC3827188  PMID: 24265802
5.  Cardiac fibrosis in mice expressing an inducible myocardial-specific Cre driver 
Disease Models & Mechanisms  2013;6(6):1470-1476.
SUMMARY
Tamoxifen-inducible Cre-mediated manipulation of animal genomes has achieved wide acceptance over the last decade, with numerous important studies heavily relying on this technique. Recently, a number of groups have reported transient complications of using this protocol in the heart. In the present study we observed a previously unreported focal fibrosis and depressed left-ventricular function in tamoxifen-treated αMHC-MerCreMer-positive animals in a Tβ4shRNAflox × αMHC-MerCreMer cross at 6–7 weeks following standard tamoxifen treatment, regardless of the presence of the floxed transgene. The phenotype was reproduced by treating mice from the original αMHC-MerCreMer strain with tamoxifen. In the acute phase after tamoxifen treatment, cell infiltration into the myocardium was accompanied by increased expression of pro-inflammatory cytokines (IL-1β, IL-6, TNFα, IFNγ, Ccl2) and markers of hypertrophy (ANF, BNP, Col3a1). These observations highlight the requirement for including tamoxifen-treated MerCreMer littermate controls to avert misinterpretation of conditional mutant phenotypes. A survey of the field as well as the protocols presented here suggests that controlling the parameters of tamoxifen delivery is important in avoiding the chronic MerCreMer-mediated cardiac phenotype reported here.
doi:10.1242/dmm.010470
PMCID: PMC3820269  PMID: 23929940
6.  Characterization of Porcine Aortic Valvular Interstitial Cell ‘Calcified’ Nodules 
PLoS ONE  2012;7(10):e48154.
Valve interstitial cells populate aortic valve cusps and have been implicated in aortic valve calcification. Here we investigate a common in vitro model for aortic valve calcification by characterizing nodule formation in porcine aortic valve interstitial cells (PAVICs) cultured in osteogenic (OST) medium supplemented with transforming growth factor beta 1 (TGF-β1). Using a combination of materials science and biological techniques, we investigate the relevance of PAVICs nodules in modeling the mineralised material produced in calcified aortic valve disease. PAVICs were grown in OST medium supplemented with TGF-β1 (OST+TGF-β1) or basal (CTL) medium for up to 21 days. Murine calvarial osteoblasts (MOBs) were grown in OST medium for 28 days as a known mineralizing model for comparison. PAVICs grown in OST+TGF-β1 produced nodular structures staining positive for calcium content; however, micro-Raman spectroscopy allowed live, noninvasive imaging that showed an absence of mineralized material, which was readily identified in nodules formed by MOBs and has been identified in human valves. Gene expression analysis, immunostaining, and transmission electron microscopy imaging revealed that PAVICs grown in OST+TGF-β1 medium produced abundant extracellular matrix via the upregulation of the gene for Type I Collagen. PAVICs, nevertheless, did not appear to further transdifferentiate to osteoblasts. Our results demonstrate that ‘calcified’ nodules formed from PAVICs grown in OST+TGF-β1 medium do not mineralize after 21 days in culture, but rather they express a myofibroblast-like phenotype and produce a collagen-rich extracellular matrix. This study clarifies further the role of PAVICs as a model of calcification of the human aortic valve.
doi:10.1371/journal.pone.0048154
PMCID: PMC3482191  PMID: 23110195
7.  Genetic or Pharmaceutical Blockade of Phosphoinositide 3-Kinase P110δ Prevents Chronic Rejection of Heart Allografts 
PLoS ONE  2012;7(3):e32892.
Chronic rejection is the major cause of long-term heart allograft failure, characterized by tissue infiltration by recipient T cells with indirect allospecificity. Phosphoinositol-3-kinase p110δ is a key mediator of T cell receptor signaling, regulating both T cell activation and migration of primed T cells to non-lymphoid antigen-rich tissue. We investigated the effect of genetic or pharmacologic inactivation of PI3K p110δ on the development of chronic allograft rejection in a murine model in which HY-mismatched male hearts were transplanted into female recipients. We show that suppression of p110δ activity significantly attenuates the development of chronic rejection of heart grafts in the absence of any additional immunosuppressive treatment by impairing the localization of antigen-specific T cells to the grafts, while not inducing specific T cell tolerance. p110δ pharmacologic inactivation is effective when initiated after transplantation. Targeting p110δ activity might be a viable strategy for the treatment of heart chronic rejection in humans.
doi:10.1371/journal.pone.0032892
PMCID: PMC3316549  PMID: 22479345
8.  Increased cardiogenesis in P19-GFP teratocarcinoma cells expressing the propeptide IGF-1Ea 
Highlights
► In this study, we explored the function of IGF-1Ea propeptide in inducing cardiogenesis of stem cells. ► IGF-1Ea promoted cardiac mesodermal induction in uncommitted cells. ► Under differentiation condition, IGF-1Ea increased expression of cardiac differentiation markers. ► Furthermore, it promoted formation of finely organized sarcomeric structure. ► IGF-1Ea propeptide may be a good candidate to improve production of cardiomyocytes from pluripotent cells.
The mechanism implicated in differentiation of endogenous cardiac stem cells into cardiomyocytes to regenerate the heart tissue upon an insult remains elusive, limiting the therapeutical goals to exogenous cell injection and/or gene therapy. We have shown previously that cardiac specific overexpression of the insulin-like growth factor 1 propeptide IGF-1Ea induces beneficial myocardial repair after infarct. Although the mechanism is still under investigation, the possibility that this propeptide may be involved in promoting stem cell differentiation into the cardiac lineage has yet to be explored. To investigate whether IGF-1Ea promote cardiogenesis, we initially modified P19 embryonal carcinoma cells to express IGF-1Ea. Taking advantage of their cardiomyogenic nature, we analyzed whether overexpression of this propeptide affected cardiac differentiation program. The data herein presented showed for the first time that constitutively overexpressed IGF-1Ea increased cardiogenic differentiation program in both undifferentiated and DMSO-differentiated cells. In details, IGF-1Ea overexpression promoted localization of alpha-actinin in finely organized sarcomeric structure compared to control cells and upregulated the cardiac mesodermal marker NKX-2.5 and the ventricular structural protein MLC2v. Furthermore, activated IGF-1 signaling promoted cardiac mesodermal induction in undifferentiated cells independently of cell proliferation. This analysis suggests that IGF-1Ea may be a good candidate to improve both in vitro production of cardiomyocytes from pluripotent stem cells and in vivo activation of the differentiation program of cardiac progenitor cells.
doi:10.1016/j.bbrc.2011.11.028
PMCID: PMC3407877  PMID: 22100652
Pluripotent stem cells; Cardiac differentiation; IGF-1Ea
9.  Revealing New Mouse Epicardial Cell Markers through Transcriptomics 
PLoS ONE  2010;5(6):e11429.
Background
The epicardium has key functions during myocardial development, by contributing to the formation of coronary endothelial and smooth muscle cells, cardiac fibroblasts, and potentially cardiomyocytes. The epicardium plays a morphogenetic role by emitting signals to promote and maintain cardiomyocyte proliferation. In a regenerative context, the adult epicardium might comprise a progenitor cell population that can be induced to contribute to cardiac repair. Although some genes involved in epicardial function have been identified, a detailed molecular profile of epicardial gene expression has not been available.
Methodology
Using laser capture microscopy, we isolated the epicardial layer from the adult murine heart before or after cardiac infarction in wildtype mice and mice expressing a transgenic IGF-1 propeptide (mIGF-1) that enhances cardiac repair, and analyzed the transcription profile using DNA microarrays.
Principal Findings
Expression of epithelial genes such as basonuclin, dermokine, and glycoprotein M6A are highly enriched in the epicardial layer, which maintains expression of selected embryonic genes involved in epicardial development in mIGF-1 transgenic hearts. After myocardial infarct, a subset of differentially expressed genes are down-regulated in the epicardium representing an epicardium-specific signature that responds to injury.
Conclusion
This study presents the description of the murine epicardial transcriptome obtained from snap frozen tissues, providing essential information for further analysis of this important cardiac cell layer.
doi:10.1371/journal.pone.0011429
PMCID: PMC2893200  PMID: 20596535
10.  A naturally occurring calcineurin variant inhibits FoxO activity and enhances skeletal muscle regeneration 
The Journal of Cell Biology  2007;179(6):1205-1218.
The calcium-activated phosphatase calcineurin (Cn) transduces physiological signals through intracellular pathways to influence the expression of specific genes. Here, we characterize a naturally occurring splicing variant of the CnAβ catalytic subunit (CnAβ1) in which the autoinhibitory domain that controls enzyme activation is replaced with a unique C-terminal region. The CnAβ1 enzyme is constitutively active and dephosphorylates its NFAT target in a cyclosporine-resistant manner. CnAβ1 is highly expressed in proliferating myoblasts and regenerating skeletal muscle fibers. In myoblasts, CnAβ1 knockdown activates FoxO-regulated genes, reduces proliferation, and induces myoblast differentiation. Conversely, CnAβ1 overexpression inhibits FoxO and prevents myotube atrophy. Supplemental CnAβ1 transgene expression in skeletal muscle leads to enhanced regeneration, reduced scar formation, and accelerated resolution of inflammation. This unique mode of action distinguishes the CnAβ1 isoform as a candidate for interventional strategies in muscle wasting treatment.
doi:10.1083/jcb.200704179
PMCID: PMC2140042  PMID: 18086917
11.  Carbon monoxide is a major contributor to the regulation of vascular tone in aortas expressing high levels of haeme oxygenase-1 
British Journal of Pharmacology  1998;125(7):1437-1444.
The contribution of haeme oxygenase-derived carbon monoxide (CO) to the regulation of vascular tone in thoracic aorta was investigated following induction of the inducible isoform of haeme oxygenase (HO-1).Isometric smooth muscle contractions were recorded in isolated rat aortic ring preparations. Rings were incubated in the presence of the nitric oxide (NO) donor S-nitroso-N-acetyl penicillamine (SNAP, 500 μM) for 1 h, then repetitively washed and maintained for a further 4 h prior to producing a concentration-response curve to phenylephrine (PE, 1–3000 nM).Treatment with SNAP resulted in increased mRNA and protein expression of aortic HO-1 and was associated with a significant suppression of the contractile response to PE (P<0.05 vs control). Immunohistochemical staining procedures revealed marked HO-1 expression in the endothelial layer and, to a lesser extent, in smooth muscle cells.Induction of HO-1 in SNAP-treated rings was associated with a higher 14CO release compared to control, as measured by scintillation counting after incubation of aortas with [2-14C]-L-glycine, the precursor of haeme. Guanosine 3′,5′-monophosphate (cyclic GMP) content was also greatly enhanced in aortas expressing high levels of HO-1.Incubation of aortic rings with the NO synthase inhibitor, NG-monomethyl-L-arginine (100 μM), significantly (P<0.05) increased the contractile response to PE in controls but failed to restore PE-mediated contractility in SNAP-treated rings. In contrast, the selective inhibitor of haeme oxygenase, tin protoporphyrin IX (SnPP-IX, 10 μM), restored the pressor response to PE in SNAP-treated rings whilst markedly reducing CO and cyclic GMP production.We conclude that up-regulation of the HO-1/CO pathway significantly contributes to the suppression of aortic contractility to PE. This effect appears to be mediated by the elevation of cyclic GMP levels and can be reversed by inhibition of the haeme oxygenase pathway.
doi:10.1038/sj.bjp.0702212
PMCID: PMC1565726  PMID: 9884071
Haeme oxygenase-1; carbon monoxide; NO donors; aortic contractility; tin protoporphyrin IX; cyclic GMP

Results 1-11 (11)