Premi Haynes

Altered ventricular torsion and transmural patterns of myocyte relaxation precede heart failure in aging F344 rats.

The purpose of this study was to identify and explain changes in ventricular and cellular function that contribute to aging-associated cardiovascular disease in aging F344 rats. Three groups of female F344 rats, aged 6, 18, and 22 mo, were studied. Echocardiographic measurements in isoflurane-anesthetized animals showed an increase in peak left ventricular torsion between the 6- and the 18-mo-old groups that was partially reversed in the 22-mo-old animals (P < 0.05). Epicardial, midmyocardial, and endocardial myocytes were subsequently isolated from the left ventricles of each group of rats. Unloaded sarcomere shortening and Ca(2+) transients were then measured in these cells (n = >75 cells for each of the nine age-region groups). The decay time of the Ca(2+) transient and the time required for 50% length relaxation both increased with age but not uniformly across the three regions (P < 0.02). Further analysis revealed a significant shift in the transmural distribution of these properties between 18 and 22 mo of age, with the largest changes occurring in epicardial myocytes. Computational modeling suggested that these changes were due in part to slower Ca(2+) dissociation from troponin in aging epicardial myocytes. Subsequent biochemical assays revealed a >50% reduction in troponin I phosphoprotein content in 22-mo-old epicardium relative to the other regions. These data suggest that between 18 and 22 mo of age (before the onset of heart failure), F344 rats display epicardial-specific myofilament-level modifications that 1) break from the progression observed between 6 and 18 mo and 2) coincide with aberrant patterns of cardiac torsion.

Transmural heterogeneity of cellular level power output is reduced in human heart failure.

Heart failure is associated with pump dysfunction and remodeling but it is not yet known if the condition affects different transmural regions of the heart in the same way. We tested the hypotheses that the left ventricles of non-failing human hearts exhibit transmural heterogeneity of cellular level contractile properties, and that heart failure produces transmural region-specific changes in contractile function. Permeabilized samples were prepared from the sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricular free wall of non-failing (n=6) and failing (n=10) human hearts. Power, an in vitro index of systolic function, was higher in non-failing mid-myocardial samples (0.59±0.06μWmg(-1)) than in samples from the sub-epicardium (p=0.021) and the sub-endocardium (p=0.015). Non-failing mid-myocardial samples also produced more isometric force (14.3±1.33kNm(-2)) than samples from the sub-epicardium (p=0.008) and the sub-endocardium (p=0.026). Heart failure reduced power (p=0.009) and force (p=0.042) but affected the mid-myocardium more than the other transmural regions. Fibrosis increased with heart failure (p=0.021) and mid-myocardial tissue from failing hearts contained more collagen than matched sub-epicardial (p<0.001) and sub-endocardial (p=0.043) samples. Power output was correlated with the relative content of actin and troponin I, and was also statistically linked to the relative content and phosphorylation of desmin and myosin light chain-1. Non-failing human hearts exhibit transmural heterogeneity of contractile properties. In failing organs, region-specific fibrosis produces the greatest contractile deficits in the mid-myocardium. Targeting fibrosis and sarcomeric proteins in the mid-myocardium may be particularly effective therapies for heart failure.

Numerical Evaluation of Myofiber Orientation and Transmural Contractile Strength on Left Ventricular Function

The left ventricle (LV) of the heart is composed of a complex organization of cardiac muscle fibers, which contract to generate force and pump blood into the body. It has been shown that both the orientation and contractile strength of these myofibers vary across the ventricular wall. The hypothesis of the current study is that the transmural distributions of myofiber orientation and contractile strength interdependently impact LV pump function. In order to quantify these interactions a finite element (FE) model of the LV was generated, which incorporated transmural variations. The influences of myofiber orientation and contractile strength on the Starling relationship and the end-systolic (ES) apex twist of the LV were assessed. The results suggest that reductions in contractile strength within a specific transmural layer amplified the effects of altered myofiber orientation in the same layer, causing greater changes in stroke volume (SV). Furthermore, when the epicardial myofibers contracted the strongest, the twist of the LV apex was greatest, regardless of myofiber orientation. These results demonstrate the important role of transmural distribution of myocardial contractile strength and its interplay with myofiber orientation. The coupling between these two physiologic parameters could play a critical role in the progression of heart failure.

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.

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

BackgroundFacioscapulohumeral 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.MethodsIsogenic 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.ResultsHuman 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.ConclusionsWe 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.

Myocardial hypertrophy reduces transmural variation in mitochondrial function

There is growing evidence that some cellular properties of mammalian hearts are transmurally heterogeneous, varying systematically from the inner (sub-endocardium) to the outer (sub-epicardium) region of the left ventricular wall. For example, action potential duration (Lou et al., 2011), calcium sensitivity (Cazorla et al., 2005; Haynes et al., 2014), mitochondria with faster sedimentation rate (Whitty et al., 1976), and β myosin heavy chain isoform (Stelzer et al., 2008) are significantly greater in the sub-endocardium than in the sub-epicardium of the left ventricular wall. Transmural differences in the phosphorylation of myosin light chain-2 (Davis et al., 2001), the dynamics of Ca2+ handling and contraction (Campbell et al., 2013), and myocyte orientation (Streeter et al., 1969; Schmid et al., 2005) have also been shown. These heterogeneities may be important for ventricular function (Ingels, 1997; Sengupta et al., 2006). For example, ventricular torsion (the wringing motion of the heart) augments systolic ejection and has been linked to transmural heterogeneities in myocardial architecture, action potential duration, and contractile properties (Streeter et al., 1969; Evangelista et al., 2011; Campbell et al., 2013). Data from several labs now show that transmural variation in cellular-level properties can be disrupted in diseased human (Lou et al., 2011; Haynes et al., 2014) and animal (Humphrey et al., 1988; Cazorla et al., 2005) hearts. This raises a fundamental question. Does the loss of transmural variation cause the disease, or is it a consequence of remodeling? One way of answering this question is to determine how transmural heterogeneity changes during the development of cardiac disease. These data might ultimately help the field to develop better therapies for heart failure. The recent study by Kindo et al. (2012) in this journal investigated whether transmural variation in mitochondrial function precedes heart failure. The authors induced mild left ventricular hypertrophy in rats by banding the abdominal aorta for 6-weeks to induce pressure overload. The treated rats did not show clinical symptoms of heart failure (depressed ejection fraction and fractional shortening) but exhibited clear mitochondrial dysfunction when compared to the sham animals. One of the important findings was that in the sham animals, the sub-epicardial tissue had ~55% greater mitochondrial respiratory chain complex IV activity than the sub-endocardium. This transmural gradient was reduced in the rats that had been subjected to pressure overload. Specifically, complex IV activity was lower in the sub-epicardium of these animals, which suggests that the sub-epicardium was more affected by the remodeling. Dysfunction in complex IV activity of the electron transport chain can disrupt the proton gradient needed for ATP synthesis and may compromise energy dependent processes including cross-bridge cycling, and the pumping of Ca2+ ions (Carley et al., 2014). Although previous studies have shown that heart failure is associated with mitochondrial dysfunction (Rosca et al., 2011; Carley et al., 2014), the work of Kindo et al. (2012) is the first to show that transmural region-dependent mitochondrial dysfunction precedes overt ventricular failure. These new data are important and augment prior studies that have focused primarily on ischemic tissue. For example, a study by Humphrey et al. (1988) showed that after 25 min of global ischemia the myocytes from the sub-endocardium had lower ATP levels than myocytes from the sub-epicardium. Another study investigated a long term effect of ischemia in rat hearts by ligating a coronary artery and examining the animals after 12 weeks. The activities of complex I and complex IV were decreased in the sub-endocardial tissue (Andre et al., 2013). These two studies are particularly interesting because they suggest that ischemia may produce the biggest detriments in sub-endocardial issue. In contrast, Kindo et al. (2012) studied non-ischemic remodeling and showed that sub-epicardium was more affected. The sensitivity of the sub-epicardium to adaptations prior to heart failure is further supported by data that describe relaxation dynamics in myoctyes isolated from Fisher 344 rats of different ages. Cells from the sub-epicardium showed greater age-dependent changes in relaxation dynamics than cells from other regions (Campbell et al., 2013). One possibility is that ischemic and non-ischemic remodeling produces different transmural effects. However more data are clearly required to test this hypothesis. Myocytes in the sub-epicardium and sub-endocardium are aligned close to the base to apex axis while myocytes in the mid-myocardium (middle transmural region) are circumferentially arranged (Streeter et al., 1969; Greenbaum et al., 1981). A recent study by Haynes et al. (2014) showed that isometric force is higher in the mid-myocardium than in the sub-epicardium and sub-endocardium of non-failing humans hearts. Most of this transmural variation was lost in diseased human organs. Cazorla et al. (2005) performed similar experiments using rat tissue but did not show significant transmural effects in force production. Other studies in pigs (Stelzer et al., 2008; Van Der Velden et al., 2011) have only investigated the sub-epicardium and the sub-endocardium. Dissecting the ventricular wall into two, as opposed to three or more sections, may hide important transmural effects as myocytes arranged in orthogonal directions may undergo different stress patterns during the cardiac cycle. A definitive test of this hypothesis probably requires analyzing samples from multiple transmural regions from many locations along the base to apex axis. However, this would require large numbers of experiments and a design that could be hard to reproduce in different labs because of its complexity. The importance of the findings reported by Kindo et al. (2012) reinforce the significance of documenting the anatomical source of myocardial samples that are used in basic science experiments. Transmural variation is also likely to be important in clinical settings. For example, Wachtell et al. (2010) have shown that fractional shortening of the middle transmural region is a better predictor of clinical endpoints than the shortening of other regions, or than traditional global measures of ventricular function such as ejection fraction. Improved understanding of the transmural variation that can contribute to these effects may help scientists and clinicians to develop better therapies for patients with heart disease. The recent work by Kindo et al. is an important step in this process.

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

BackgroundFacioscapulohumeral 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.ResultsFSHD 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.ConclusionsThese 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.