Cardiomyocyte nuclear remodeling after mechanical unloading.

Cardiomyocytes increase DNA content in response to stress in humans. DNA content is reported to decrease in association with increased markers of proliferation in cardiomyocytes following left ventricular assist device (LVAD) unloading. However, cardiac recovery resulting in LVAD explant is rare. Thus, we sought to test the hypothesis that changes in DNA content with mechanical unloading occurs independent of cardiomyocyte proliferation by quantifying cardiomyocyte nuclear number, cell size, DNA content, and the frequency of cell-cycling markers using a novel imaging flow cytometry methodology comparing human subjects undergoing LVAD implantation or primary transplantation. We found that cardiomyocyte size was 15% smaller in unloaded versus loaded samples without differences in the percentage of mono-, bi-, or multinuclear cells. DNA content per nucleus was significantly decreased in unloaded hearts versus loaded controls. Cell-cycle markers, Ki67 and phospho-histon3 (H3P), were not increased in unloaded samples. In conclusion, unloading of failing hearts is associated with decreased DNA content of nuclei independent of nucleation state within the cell. As these changes were associated with a trend to decreased cell size but not increased cell-cycle markers, they may represent a regression of hypertrophic nuclear remodeling and not proliferation.NEW & NOTEWORTHY Our data suggest that increases in DNA content that occur with cardiomyocyte hypertrophy in heart failure may reverse with mechanical unloading.

Cell-Specific Pathways Supporting Persistent Fibrosis in Heart Failure.

BACKGROUND: Only limited data exist describing the histologic and noncardiomyocyte function of human myocardium in end-stage heart failure (HF). OBJECTIVES: The authors sought to determine changes in noncardiomyocyte cellular activity in patients with end-stage HF after left ventricular assist device (LVAD)-induced remodeling to identify mechanisms impeding recovery. METHODS: Myocardium was obtained from subjects undergoing LVAD placement and/or heart transplantation. Detailed histological analyses were performed, and, when feasible, mononuclear cells were isolated from fresh, dissociated myocardium for quantitative reverse transcription polymerase chain reaction studies. Echocardiographic and catheterization data were obtained during routine care. RESULTS: Sixty-six subjects were enrolled; 54 underwent 8.0 ± 1.2 months of LVAD unloading. Despite effective hemodynamic unloading and remodeling, there were no differences after LVAD use in capillary density (0.78 ± 0.1% vs. 0.9 ± 0.1% capillary area; n = 42 and 28, respectively; p = 0.40), cardiac fibrosis (25.7 ± 2.4% vs. 27.9 ± 2.4% fibrosis area; n = 44 and 31, respectively; p = 0.50), or macrophage density (80.7 ± 10.4 macrophages/mm2 vs. 108.6 ± 15 macrophages/mm2; n = 33 and 28, respectively; p = 0.1). Despite no change in fibrosis or myofibroblast density (p = 0.40), there was a 16.7-fold decrease (p < 0.01) in fibroblast-specific collagen expression. Furthermore, there was a shift away from pro-fibrotic/alternative pro-fibrotic macrophage signaling after LVAD use. CONCLUSIONS: Despite robust cardiac unloading, capillary density and fibrosis are unchanged compared with loaded hearts. Fibroblast-specific collagen expression was decreased and might be due to decreased stretch and/or altered macrophage polarization. Dysfunctional myocardium may persist, in part, from ongoing inflammation and poor extracellular matrix remodeling. Understanding these changes could lead to improved therapies for HF.

Cardiac macrophages adopt profibrotic/M2 phenotype in infarcted hearts: Role of urokinase plasminogen activator.

BACKGROUND: Macrophages (mac) that over-express urokinase plasminogen activator (uPA) adopt a profibrotic M2 phenotype in the heart in association with cardiac fibrosis. We tested the hypothesis that cardiac macs are M2 polarized in infarcted mouse and human hearts and that polarization is dependent on mac-derived uPA. METHODS: Studies were performed using uninjured (UI) or infarcted (MI) hearts of uPA overexpressing (SR-uPA), uPA null, or nontransgenic littermate (Ntg) mice. At 7days post-infarction, cardiac mac were isolated, RNA extracted and M2 markers Arg1, YM1, and Fizz1 measured with qrtPCR. Histologic analysis for cardiac fibrosis, mac and myofibroblasts was performed at the same time-point. Cardiac macs were also isolated from Ntg hearts and RNA collected after primary isolation or culture with vehicle, IL-4 or plasmin and M2 marker expression measured. Cardiac tissue and blood was collected from humans with ischemic heart disease. Expression of M2 marker CD206 and M1 marker TNFalpha was measured. RESULTS: Macs from WT mice had increased expression of Arg1 and Ym1 following MI (41.3±6.5 and 70.3±36, fold change vs UI, n=8, P<0.007). There was significant up-regulation of cardiac mac Arg1 and YM1 with MI in both WT and uPA null mice (n=4-9 per genotype and condition). Treatment with plasmin increased expression of Arg1 and YM1 in cultured cardiac macs. Histologic analysis revealed increased density of activated fibroblasts and M2 macs in SR-uPA hearts post-infarction with associated increases in fibrosis. Cardiac macs isolated from human hearts with ischemic heart disease expressed increased levels of the M2 marker CD206 in comparison to blood-derived macs (4.9±1.3). CONCLUSIONS: Cardiac macs in mouse and human hearts adopt a M2 phenotype in association with fibrosis. Plasmin can induce an M2 phenotype in cardiac macs. However, M2 activation can occur in the heart in vivo in the absence of uPA indicating that alternative pathways to activate plasmin are present in the heart. Excess uPA promotes increased fibroblast density potentially via potentiating fibroblast migration or proliferation. Altering macrophage phenotype in the heart is a potential target to modify cardiac fibrosis.

Mechanisms of bone marrow-derived cell therapy in ischemic cardiomyopathy with left ventricular assist device bridge to transplant.

BACKGROUND: Clinical trials report improvements in function and perfusion with direct injection of bone marrow cells into the hearts of patients with ischemic cardiomyopathy. Preclinical data suggest these cells improve vascular density, which would be expected to decrease fibrosis and inflammation. OBJECTIVES: The goal of this study was to test the hypothesis that bone marrow stem cells (CD34+) will improve histological measurements of vascularity, fibrosis, and inflammation in human subjects undergoing left ventricular assist device (LVAD) placement as a bridge to cardiac transplantation. METHODS: Subjects with ischemic cardiomyopathy who were scheduled for placement of an LVAD as a bridge to transplantation underwent bone marrow aspiration the day before surgery; the bone marrow was processed into cell fractions (bone marrow mononuclear cells, CD34+, and CD34-). At LVAD implantation, all fractions and a saline control were injected epicardially into predetermined areas and each injection site marked. At the time of transplantation, injected areas were collected. Data were analyzed by paired Student t test comparing the effect of cell fractions injected within each subject. RESULTS: Six subjects completed the study. There were no statistically significant differences in complications with the procedure versus control subjects. Histological analysis indicated that myocardium injected with CD34+ cells had decreased density of endothelial cells compared to saline-injected myocardium. There were no significant differences in fibrosis or inflammation between groups; however, density of activated fibroblasts was decreased in both CD34+ and CD34- injected areas. CONCLUSIONS: Tissue analysis does not support the hypothesis that bone marrow-derived CD34+ cells promote increased vascular tissue in humans with ischemic cardiomyopathy via direct injection.

Urokinase plasminogen activator induces pro-fibrotic/m2 phenotype in murine cardiac macrophages.

OBJECTIVE: Inflammation and fibrosis are intertwined in multiple disease processes. We have previously found that over-expression of urokinase plasminogen activator in macrophages induces spontaneous macrophage accumulation and fibrosis specific to the heart in mice. Understanding the relationship between inflammation and fibrosis in the heart is critical to developing therapies for diverse myocardial diseases. Therefore, we sought to determine if uPA induces changes in macrophage function that promote cardiac collagen accumulation. METHODS AND RESULTS: We analyzed the effect of the uPA transgene on expression of pro-inflammatory (M1) and pro-fibrotic (M2) genes and proteins in hearts and isolated macrophages of uPA overexpressing mice. We found that although there was elevation of the pro-inflammatory cytokine IL-6 in hearts of transgenic mice, IL-6 is not a major effector of uPA induced cardiac fibrosis. However, uPA expressing bone marrow-derived macrophages are polarized to express M2 genes in response to IL-4 stimulation, and these M2 genes are upregulated in uPA expressing macrophages following migration to the heart. In addition, while uPA expressing macrophages express a transcriptional profile that is seen in tumor-associated macrophages, these macrophages promote collagen expression in cardiac but not embryonic fibroblasts. CONCLUSIONS: Urokinase plasminogen activator induces an M2/profibrotic phenotype in macrophages that is fully expressed after migration of macrophages into the heart. Understanding the mechanisms by which uPA modulates macrophage function may reveal insights into diverse pathologic processes.

Differentiation and fiber type-specific activity of a muscle creatine kinase intronic enhancer.

BACKGROUND: Hundreds of genes, including muscle creatine kinase (MCK), are differentially expressed in fast- and slow-twitch muscle fibers, but the fiber type-specific regulatory mechanisms are not well understood. RESULTS: Modulatory region 1 (MR1) is a 1-kb regulatory region within MCK intron 1 that is highly active in terminally differentiating skeletal myocytes in vitro. A MCK small intronic enhancer (MCK-SIE) containing a paired E-box/myocyte enhancer factor 2 (MEF2) regulatory motif resides within MR1. The SIE's transcriptional activity equals that of the extensively characterized 206-bp MCK 5'-enhancer, but the MCK-SIE is flanked by regions that can repress its activity via the individual and combined effects of about 15 different but highly conserved 9- to 24-bp sequences. ChIP and ChIP-Seq analyses indicate that the SIE and the MCK 5'-enhancer are occupied by MyoD, myogenin and MEF2. Many other E-boxes located within or immediately adjacent to intron 1 are not occupied by MyoD or myogenin. Transgenic analysis of a 6.5-kb MCK genomic fragment containing the 5'-enhancer and proximal promoter plus the 3.2-kb intron 1, with and without MR1, indicates that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers (types I and IIa, respectively), but is not required for expression in fast-twitch muscle fibers (types IIb and IId). CONCLUSIONS: In this study, we discovered that MR1 is critical for MCK expression in slow- and intermediate-twitch muscle fibers and that MR1's positive transcriptional activity depends on a paired E-box MEF2 site motif within a SIE. This is the first study to delineate the DNA controls for MCK expression in different skeletal muscle fiber types.

Embryonic and tissue-specific regulation of myostatin-1 and -2 gene expression in zebrafish.

Myostatin is a member of the TGF-beta superfamily and a potent negative regulator of muscle growth and development in mammals. Its expression is limited primarily to skeletal muscle in mammals, but occurs in many different fish tissues, although quantitative measurements of the embryonic and tissue-specific expression profiles are lacking. A recent phylogenetic analysis of all known myostatin genes identified a novel paralogue in zebrafish, zfMSTN-2, and prompted the reclassification of the entire subfamily to include MSTN-1 and -2 sister clades in the bony fishes. The differential expression profiles of both genes were therefore determined using custom RNA panels generated from pooled (100-150/sampling) embryos at different stages of development and from individual adult tissues. High levels of both transcripts were transiently present at the blastula stage, but were undetectable throughout gastrulation (7 hpf). Levels of zfMSTN-2 peaked during early somitogenesis (11 hpf), returned to basal levels during late somitogenesis and did not begin to rise again until hatching (72 hpf). By contrast, zfMSTN-1 mRNA levels peaked during late somitogenesis (15.5-19 hpf), returned to baseline at 21.5 hpf and eventually rose 25-fold by 72 hpf. In adults, both transcripts were present in a wide variety of tissues, including some not previously known to express myostatin. Expression of zfMSTN-1 was highest in brain, muscle, heart and testes and was 1-3 log orders above that in other tissues. It was also greater than zfMSTN-2 expression in most tissues, nevertheless, levels of both transcripts increased almost 600-fold in spleens of fish subjected to stocking stress. Myostatin expression was also detected in mouse spleens, suggesting that myostatin may influence immune cell development in mammals as well as fish. These studies indicate that zfMSTN-1 and -2 gene expression is differentially regulated in developing fish embryos and in adult tissues. The increased expression of both genes in spleens from stressed fish is further supportive of an immunomodulatory role and may explain increased disease susceptibility associated with stocking stress.

Oil red-O stains non-adipogenic cells: a precautionary note.

Bovine adipofibroblasts, 3T3-L1 cells, L-6 myogenic cells, and sheep satellite cells were allowed to proliferate for 48 h. Oil red-O (ORO) was dissolved in three different solvents isopropanol, propylene glycol and triethyl phosphate. At 48 h, the proliferative cultures were stained with the three stains. ORO stain prepared in both propylene glycol and triethyl phosphate resulted in bright red droplets appearing in all cultures, whereas ORO dissolved in isopropanol was not taken up by any of the cells. These data suggest that certain preparations of ORO may stain cells in non-adipogenic lineages as well as undifferentiated pre-adipocytes. Caution must be exercised when choosing solvents for ORO in differentiation studies using cells of the fat/adipose lineage.

Coulter counter use in the enumeration of muscle and fat stem cells.

Although the manual counting chamber (hemacytometer) is the gold standard for counting cells, this method is subject to great variability due to the 'human factor'. The automated cell counter (Coulter Counter) can enumerate cells in less time and with greater accuracy than the hemacytometer by removing many of the steps in which errors are made. While the Coulter Counter (and others of its type) has been used for many years in the cell culture field, there have been few studies to validate its use with specific cell types. We conducted several experiments in which we assessed the accuracy of the Coulter Counter over counts made with a hemacytometer as well as validated its use for the counting of satellite cells and preadipocytes.