CaM kinase II regulates cardiac hemoglobin expression through histone phosphorylation upon sympathetic activation.

Sympathetic activation of ß-adrenoreceptors (ß-AR) represents a hallmark in the development of heart failure (HF). However, little is known about the underlying mechanisms of gene regulation. In human ventricular myocardium from patients with end-stage HF, we found high levels of phosphorylated histone 3 at serine-28 (H3S28p). H3S28p was increased by inhibition of the catecholamine-sensitive protein phosphatase 1 and decreased by ß-blocker pretreatment. By a series of in vitro and in vivo experiments, we show that the ß-AR downstream protein kinase CaM kinase II (CaMKII) directly binds and phosphorylates H3S28. Whereas, in CaMKII-deficient myocytes, acute catecholaminergic stimulation resulted in some degree of H3S28p, sustained catecholaminergic stimulation almost entirely failed to induce H3S28p. Genome-wide analysis of CaMKII-mediated H3S28p in response to chronic ß-AR stress by chromatin immunoprecipitation followed by massive genomic sequencing led to the identification of CaMKII-dependent H3S28p target genes. Forty percent of differentially H3S28p-enriched genomic regions were associated with differential, mostly increased expression of the nearest genes, pointing to CaMKII-dependent H3S28p as an activating histone mark. Remarkably, the adult hemoglobin genes showed an H3S28p enrichment close to their transcriptional start or end sites, which was associated with increased messenger RNA and protein expression. In summary, we demonstrate that chronic ß-AR activation leads to CaMKII-mediated H3S28p in cardiomyocytes. Thus, H3S28p-dependent changes may play an unexpected role for cardiac hemoglobin regulation in the context of sympathetic activation. These data also imply that CaMKII may be a yet unrecognized stress-responsive regulator of hematopoesis.

Effect of preculture and loading on expression of matrix molecules, matrix metalloproteinases, and cytokines by expanded osteoarthritic chondrocytes.

OBJECTIVE: One of the pathologic changes that occurs during osteoarthritis (OA) is the degeneration of the pericellular matrix (PCM). Since the PCM is likely to be involved in mechanotransduction, this study was undertaken to investigate the effects of PCM-like matrix accumulation in zonal OA chondrocytes and their influence on chondrocyte response to compression. METHODS: Superficial and middle/deep zone chondrocytes from macroscopically normal cartilage of OA knees were expanded and encapsulated in alginate gels. The effects of compression (short-term or long-term) and preculture on chondrocyte expression of various matrix molecules, cytokines, and matrix metalloproteinases (MMPs) were assessed. Additionally, nonexpanded chondrocytes were encapsulated in alginate and cultured in the presence or absence of transforming growth factor ß1 (TGFß1) and dexamethasone and analyzed following short-term compression experiments. RESULTS: Expanded OA chondrocytes (superficial and middle/deep zone) that were precultured for 2 weeks under free-swelling conditions prior to dynamic compression responded more sensitively to loading and had increased matrix accumulation, increased interleukin-1ß (IL-1ß) and IL-4 levels, and decreased levels of MMP-2 (in the middle/deep zone) compared to the nonloaded controls. Compression also decreased MMP-3 and MMP-13 levels even without preculture. Nonexpanded chondrocytes did not respond to compression, but differences in gene expression were found depending on the zone of harvest, time in culture, and medium composition. CONCLUSION: Our findings demonstrate that with predeposited PCM-like matrix, compressive stimulation can enhance matrix protein accumulation in expanded OA chondrocytes. Investigations into how PCM or other matrix components differentially affect this balance under mechanical loading may provide invaluable insight into OA pathogenesis and the use of expanded cells in tissue engineering and regenerative medicine-based applications.

Peripheral node addressin, a ligand for L-selectin is found in tumor cells and in high endothelial venules in endometrial cancer.

BACKGROUND: High endothelial venules (HEVs) are vessels specialized in the transport of lymphocytes shown to be implicated in various forms of cancer. They express peripheral node addressin (specifically recognized by the MECA-79 antibody). MECA-79 is also implicated in pregnancy through its expression by epithelial cells of the endometrium. However, the expression of MECA-79 by endothelial or epithelial (cancer) cells has never been studied in endometrial cancer. MATERIAL AND METHODS: In this retrospective study, we investigated the immunohistochemical expression of MECA-79 in 40 endometrioid adenocarcinoma hysterectomy specimens and compared it with its expression in 30 non-cancer hysterectomies. RESULTS: HEVs were found in 22% of tumor specimens and in none of the non-cancer hysterectomies (p = 0.005) and were positively associated with higher grade tumors (p = 0.04). MECA-70 was expressed in tumor cells of 70% of carcinomas and in epithelial cells of 46.6% of normal endometria (p = 0.04). It was inversely associated with parametrial invasion (p = 0.03) and larger tumors (statistical trend of p = 0.07). MECA-79 expression was not associated with overall or progression-free survival. CONCLUSION: MECA-79 is found in HEVs and tumor cells in endometrial endometrioid adenocarcinoma.

Autophagy in cardiac myxoma: An important puzzle piece in understanding its inflammatory environment.

BACKGROUND: Cardiac myxomas are rare, predominantly sporadic tumors that can cause heart failure and systematic inflammatory symptoms, and increase the risk of emboli. Their pathophysiology remains poorly understood, but intra-tumoral inflammation and senescence seem to be implicated in it. One of the principal cellular mechanisms implicated in tumor progression is autophagy, largely unknown in myxomas. Thus, our study aimed to investigate the presence of autophagic markers in myxomas and to correlate it with their immune microenvironment. METHODS: Twenty-five cardiac myxomas were studied for the autophagic markers LC3B and p62/sequestosome 1 and were compared with markers of the immune microenvironment. RESULTS: Most myxomas showed expression of both autophagic markers. We found a positive correlation between LC3B and PD-L1, as well as CD163, and a negative correlation between LC3B and CD8, CD20, CD138, and CD117 infiltration. CONCLUSION: Our data not only confirm the presence of autophagic markers within cardiac myxomas but also suggest a possible association with their immune microenvironment.

Senescence, immune microenvironment, and vascularization in cardiac myxomas.

AIMS: Cardiac myxomas are rare tumors of incompletely elucidated pathogenesis. The aim of this study is to explore the possible presence of a senescence phenotype in cardiac myxomas, associated with an inflammatory and vasculogenic tumor microenvironment. METHODS AND RESULTS: This is a retrospective study of 29 cardiac myxomas with immunohistochemical detection of various inflammatory, vascular, and senescence markers. We show that all myxomas contain tumor cells in senescence overexpressing p16, and a fraction of senescent endothelial cells. Macrophages are the principal inflammatory cell population, followed by cytotoxic T cells, with fewer plasma cells, mastocytes, and B lymphocytes. These populations are found in different intratumoral localizations. Larger tumor volume is associated with a lower percentage of myxoid matrix, higher cellularity, higher macrophage, and lower number of mast cells as well as higher PD-L1 expression by inflammatory cells. Higher vascular density is associated with higher percentage of B cells, a lower number of macrophages and higher number of mastocytes, and lower PD-L1 expression by inflammatory cells. Tumors with higher vascular density and higher cellularity show higher amounts of p16 senescent endothelial cells. CONCLUSIONS: Myxoma tumor cells are in senescence and reside inside a tumor microenvironment with a distinct inflammatory profile rich in macrophages and cytotoxic T cells, and a rich vasculature, probably attributed to a senescence-associated secretory phenotype.

A proteolytic fragment of histone deacetylase 4 protects the heart from failure by regulating the hexosamine biosynthetic pathway.

The stress-responsive epigenetic repressor histone deacetylase 4 (HDAC4) regulates cardiac gene expression. Here we show that the levels of an N-terminal proteolytically derived fragment of HDAC4, termed HDAC4-NT, are lower in failing mouse hearts than in healthy control hearts. Virus-mediated transfer of the portion of the Hdac4 gene encoding HDAC4-NT into the mouse myocardium protected the heart from remodeling and failure; this was associated with decreased expression of Nr4a1, which encodes a nuclear orphan receptor, and decreased NR4A1-dependent activation of the hexosamine biosynthetic pathway (HBP). Conversely, exercise enhanced HDAC4-NT levels, and mice with a cardiomyocyte-specific deletion of Hdac4 show reduced exercise capacity, which was characterized by cardiac fatigue and increased expression of Nr4a1. Mechanistically, we found that NR4A1 negatively regulated contractile function in a manner that depended on the HBP and the calcium sensor STIM1. Our work describes a new regulatory axis in which epigenetic regulation of a metabolic pathway affects calcium handling. Activation of this axis during intermittent physiological stress promotes cardiac function, whereas its impairment in sustained pathological cardiac stress leads to heart failure.