TRANSMURAL HETEROGENEITY OF CELLULAR LEVEL CARDIAC CONTRACTILE PROPERTIES IN AGING AND HEART FAILURE

The left ventricle of the heart relaxes when it fills with blood and contracts to eject blood into circulation to meet the body’s metabolic demands. Dysfunction in either relaxation or contraction of the left ventricle can lead to heart failure. Transmural heterogeneity is thought to contribute to normal ventricular wall motion but it is not well understood how transmural modifications affect the failing left ventricle. The overall hypothesis of this dissertation is that normal left ventricles exhibit transmural heterogeneity in cellular level contractile properties and with aging and heart failure there are region-specific changes in cellular level contractile mechanisms. Age is the biggest risk factor associated with heart failure and therefore we investigated transmural changes in Ca2+ handling and contractile proteins in aging F344 rats before the onset of heart failure. We found that in 22-month old F344 rats there is a region-specific decrease in cardiac troponin I phosphorylation in the sub-epicardium that may contribute to slowed myocyte relaxation in the sub-epicardial cells of the same age. We then investigated the transmural patterns of contractile properties in myocardial tissue samples from patients with heart failure. Force and power output reduced most significantly in the samples from the mid-myocardial region when compared to sub-epicardium and sub-endocardium of the failing hearts. There was a region-specific increase in fibrosis is the mid-myocardium of the failing hearts. Myocardial power output was correlated with key sarcomeric proteins including cardiac troponin I, desmin and myosin light chain-1. The results in this dissertation reveal novel region-specific modifications in contractile properties in aging and heart failure. These transmural effects can potentially contribute to disruption in normal wall motion and lead to ventricular dysfunction

Sporadic DUX4 expression in FSHD myocytes is associated with incomplete repression by the PRC2 complex and gain of H3K9 acetylation on the contracted D4Z4 allele

Abstract Background Facioscapulohumeral muscular dystrophy 1 (FSHD1) has an autosomal dominant pattern of inheritance and primarily affects skeletal muscle. The genetic cause of FSHD1 is contraction of the D4Z4 macrosatellite array on chromosome 4 alleles associated with a permissive haplotype causing infrequent sporadic expression of the DUX4 gene. Epigenetically, the contracted D4Z4 array has decreased cytosine methylation and an open chromatin structure. Despite these genetic and epigenetic changes, the majority of FSHD myoblasts are able to repress DUX4 transcription. In this study we hypothesized that histone modifications distinguish DUX4 expressing and non-expressing cells from the same individuals. Results FSHD myocytes containing the permissive 4qA haplotype with a long terminal D4Z4 unit were sorted into DUX4 expressing and non-expressing groups. We found similar CpG hypomethylation between the groups of FSHD-affected cells suggesting that CpG hypomethylation is not sufficient to trigger DUX4 expression. A survey of histone modifications present at the D4Z4 region during cell lineage commitment revealed that this region is bivalent in FSHD iPS cells with both H3K4me3 activating and H3K27me3 repressive marks present, making D4Z4 poised for DUX4 activation in pluripotent cells. After lineage commitment, the D4Z4 region becomes univalent with H3K27me3 in FSHD and non-FSHD control myoblasts and a concomitant increase in H3K4me3 in a small fraction of cells. Chromatin immunoprecipitation (ChIP) for histone modifications, chromatin modifier proteins and chromatin structural proteins on sorted FSHD myocytes revealed that activating H3K9Ac modifications were ~ fourfold higher in DUX4 expressing FSHD myocytes, while the repressive H3K27me3 modification was ~ fourfold higher at the permissive allele in DUX4 non-expressing FSHD myocytes from the same cultures. Similarly, we identified EZH2, a member of the polycomb repressive complex involved in H3K27 methylation, to be present more frequently on the permissive allele in DUX4 non-expressing FSHD myocytes. Conclusions These results implicate PRC2 as the complex primarily responsible for DUX4 repression in the setting of FSHD and H3K9 acetylation along with reciprocal loss of H3K27me3 as key epigenetic events that result in DUX4 expression. Future studies focused on events that trigger H3K9Ac or augment PRC2 complex activity in a small fraction of nuclei may expose additional drug targets worthy of study

Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures

Abstract Background Facioscapulohumeral muscular dystrophy (FSHD) is most commonly inherited in an autosomal dominant pattern and caused by the abnormal expression of DUX4 in skeletal muscle. The DUX4 transcription factor has DNA binding domains similar to several paired class homeotic transcription factors, but only myogenic factors PAX3 and PAX7 rescue cell viability when co-expressed with DUX4 in mouse myoblasts. This observation suggests competition for DNA binding sites in satellite cells might limit muscle repair and may be one aspect of DUX4-associated myotoxicity. The competition hypothesis requires that DUX4 and PAX3/7 be expressed in the same cells at some point during development or in adult tissues. We modeled myogenesis using human isogenic iPS and ES cells and examined expression patterns of DUX4, PAX3, and PAX7 to determine if conditions that promote PAX3 and PAX7 expression in cell culture also promote DUX4 expression in the same cells. Methods Isogenic iPSCs were generated from human fibroblasts of two FSHD-affected individuals with somatic mosaicism. Clones containing the shortened FSHD-causing D4Z4 array or the long non-pathogenic array were isolated from the same individuals. We also examined myogenesis in commercially available hES cell lines derived from FSHD-affected and non-affected embryos. DUX4, PAX3, and PAX7 messenger RNAs (mRNAs) were quantified during a 40-day differentiation protocol, and antibodies were used to identify cell types in different stages of differentiation to determine if DUX4 and PAX3 or PAX7 are present in the same cells. Results Human iPS and ES cells differentiated into skeletal myocytes as evidenced by Titin positive multinucleated fibers appearing toward the end of a 40-day differentiation protocol. PAX3 and PAX7 were expressed at similar times during differentiation, and DUX4 positive nuclei were seen at terminal stages of differentiation in cells containing the short D4Z4 arrays. Nuclei that expressed both DUX4 and PAX3, or DUX4 and PAX7 were not observed after examining immunostained nuclei at five different time points during myogenic differentiation of pluripotent cells. Conclusions We conclude that DUX4, PAX3, and PAX7 have distinct expression patterns during myogenic differentiation of stem cells. Our findings are consistent with the hypothesis that muscle damage in FSHD is due to DUX4-mediated toxicity causing destruction of terminally differentiated myofibers. While these studies examine DUX4, PAX3, and PAX7 expression patterns during stem cell myogenesis, they should not be generalized to tissue repair in adult muscle tissue

Additional file 8: Figure S8. of Expression patterns of FSHD-causing DUX4 and myogenic transcription factors PAX3 and PAX7 are spatially distinct in differentiating human stem cell cultures

DUX4 and PAX7 are expressed in distinct cell types during myogenic differentiation of human ES cells with FSHD. A, B, C, D, E and F) Images of hESC-FSHD from D40 of the differentiation protocol stained with antibodies to both PAX7 and DUX4. Arrows indicate representative DUX4 positive nuclei counted. (DOCX 6109 kb

A Protocol for Collecting Human Cardiac Tissue for Research

This manuscript describes a protocol at the University of Kentucky that allows a translational research team to collect human myocardium that can be used for biological research. We have gained a great deal of practical experience since we started this protocol in 2008, and we hope that other groups might be able to learn from our endeavors. To date, we have procured ~4000 samples from ~230 patients. The tissue that we collect comes from organ donors and from patients who are receiving a heart transplant or a ventricular assist device because they have heart failure. We begin our manuscript by describing the importance of human samples in cardiac research. Subsequently, we describe the process for obtaining consent from patients, the cost of running the protocol, and some of the issues and practical difficulties that we have encountered. We conclude with some suggestions for other researchers who may be considering starting a similar protocol