Maintaining proteostasis under mechanical stress.

Cell survival, tissue integrity and organismal health depend on the ability to maintain functional protein networks even under conditions that threaten protein integrity. Protection against such stress conditions involves the adaptation of folding and degradation machineries, which help to preserve the protein network by facilitating the refolding or disposal of damaged proteins. In multicellular organisms, cells are permanently exposed to stress resulting from mechanical forces. Yet, for long time mechanical stress was not recognized as a primary stressor that perturbs protein structure and threatens proteome integrity. The identification and characterization of protein folding and degradation systems, which handle force-unfolded proteins, marks a turning point in this regard. It has become apparent that mechanical stress protection operates during cell differentiation, adhesion and migration and is essential for maintaining tissues such as skeletal muscle, heart and kidney as well as the immune system. Here, we provide an overview of recent advances in our understanding of mechanical stress protection.

Proximity to injury, but neither number of nuclei nor ploidy define pathological adaptation and plasticity in cardiomyocytes.

The adult mammalian heart consists of mononuclear and binuclear cardiomyocytes (CMs) with various ploidies. However, it remains unclear whether a variation in ploidy or number of nuclei is associated with distinct functions and injury responses in CMs, including regeneration. Therefore, we investigated transcriptomes and cellular as well as nuclear features of mononucleated and binucleated CMs in adult mouse hearts with and without injury. To be able to identify the role of ploidy we analyzed control and failing human ventricular CMs because human CMs show a larger and disease-sensitive degree of polyploidization. Using transgenic Myh6-H2BmCh to identify mononucleated and binucleated mouse CMs, we found that cellular volume and RNA content were similar in both. On average nuclei of mononuclear CMs showed a 2-fold higher ploidy, as compared to binuclear CMs indicating that most mononuclear CMs are tetraploid. After myocardial infarction mononucleated and binucleated CMs in the border zone of the lesion responded with hypertrophy and corresponding changes in gene expression, as well as a low level of induction of cell cycle gene expression. Human CMs allowed us to study a wide range of polyploidy spanning from 2n to 16n. Notably, basal as well as pathological gene expression signatures and programs in failing CMs proved to be independent of ploidy. In summary, gene expression profiles were induced in proximity to injury, but independent of number of nuclei or ploidy levels in CMs.

Midbody Positioning and Distance Between Daughter Nuclei Enable Unequivocal Identification of Cardiomyocyte Cell Division in Mice.

RATIONALE: New strategies in the field of cardiac regeneration are directed at identifying proliferation-inducing substances to induce regrowth of myocardium. Current screening assays utilize neonatal cardiomyocytes and markers for cytokinesis, such as Aurora B-kinase. However, detection of cardiomyocyte division is complicated because of cell cycle variants, in particular, binucleation. OBJECTIVE: To analyze the process of cardiomyocyte binucleation to identify definitive discriminators for cell cycle variants and authentic cardiomyocyte division. METHODS AND RESULTS: Herein, we demonstrate by direct visualization of the contractile ring and midbody in Myh6 (myosin, heavy chain 6)-eGFP (enhanced green fluorescent protein)-anillin transgenic mice that cardiomyocyte binucleation starts by formation of a contractile ring. This is followed by irregular positioning of the midbody and movement of the 2 nuclei into close proximity to each other. In addition, the widespread used marker Aurora B-kinase was found to also label binucleating cardiomyocytes, complicating the interpretation of existing screening assays. Instead, atypical midbody positioning and the distance of daughter nuclei on karyokinesis are bona fide markers for cardiomyocyte binucleation enabling to unequivocally discern such events from cardiomyocyte division in vitro and in vivo. CONCLUSIONS: The 2 criteria provide a new method for identifying cardiomyocyte division and should be considered in future studies investigating cardiomyocyte turnover and regeneration after injury, in particular in the postnatal heart to prevent the assignment of false positive proliferation events.

The Transcription Factor ETV1 Induces Atrial Remodeling and Arrhythmia.

RATIONALE: Structural and electrophysiological remodeling of the atria are recognized consequences of sustained atrial arrhythmias, such as atrial fibrillation. The identification of underlying key molecules and signaling pathways has been challenging because of the changing cell type composition during structural remodeling of the atria. OBJECTIVE: Thus, the aims of our study were (1) to search for transcription factors and downstream target genes, which are involved in atrial structural remodeling, (2) to characterize the significance of the transcription factor ETV1 (E twenty-six variant 1) in atrial remodeling and arrhythmia, and (3) to identify ETV1-dependent gene regulatory networks in atrial cardiac myocytes. METHODS AND RESULTS: The transcription factor ETV1 was significantly upregulated in atrial tissue from patients with permanent atrial fibrillation. Mice with cardiac myocyte-specific overexpression of ETV1 under control of the myosin heavy chain promoter developed atrial dilatation, fibrosis, thrombosis, and arrhythmia. Cardiac myocyte-specific ablation of ETV1 in mice did not alter cardiac structure and function at baseline. Treatment with Ang II (angiotensin II) for 2 weeks elicited atrial remodeling and fibrosis in control, but not in ETV1-deficient mice. To identify ETV1-regulated genes, cardiac myocytes were isolated and purified from mouse atrial tissue. Active cis-regulatory elements in mouse atrial cardiac myocytes were identified by chromatin accessibility (assay for transposase-accessible chromatin sequencing) and the active chromatin modification H3K27ac (chromatin immunoprecipitation sequencing). One hundred seventy-eight genes regulated by Ang II in an ETV1-dependent manner were associated with active cis-regulatory elements containing ETV1-binding sites. Various genes involved in Ca2+ handling or gap junction formation ( Ryr2, Jph2, Gja5), potassium channels ( Kcnh2, Kcnk3), and genes implicated in atrial fibrillation ( Tbx5) were part of this ETV1-driven gene regulatory network. The atrial ETV1-dependent transcriptome in mice showed a significant overlap with the human atrial proteome of patients with permanent atrial fibrillation. CONCLUSIONS: This study identifies ETV1 as an important component in the pathophysiology of atrial remodeling associated with atrial arrhythmias.

Role of Mononuclear Cardiomyocytes in Cardiac Turnover and Regeneration.

PURPOSE OF REVIEW: The typical remodeling process after cardiac injury is scarring and compensatory hypertrophy. The limited regeneration potential of the adult heart is thought to be due to the post-mitotic status of postnatal cardiomyocytes, which are mostly binucleated and/or polyploid. Nevertheless, there is evidence for cardiomyocyte turnover in the adult heart. The purpose of this review is to describe the recent findings regarding the proliferative potential of mononuclear cardiomyocytes and to evaluate their function in cardiac turnover and disease. RECENT FINDINGS: There is overwhelming evidence from carbon-dating in humans and multi-isotope imaging mass spectrometry in mice that there is a very low but detectable level of turnover of cardiomyocytes in the heart. The source of this renewal is not clear, but recent evidence points to a population of mononuclear, diploid cardiomyocytes that are still capable of authentic cell division. Controversy arises when their role in cardiac repair is considered, as some studies claim that they contribute to repair by cell division while other studies do not find evidence for hyperplasia but hypertrophy. Stimulation of the mononuclear cardiomyocyte population has been proposed as a therapeutic strategy in cardiac disease. The studies reviewed here agree on the existence of a low annual cardiomyocyte turnover rate which can be attributed to the proliferation of mononuclear cardiomyocytes. Potential roles of mononucleated cardiomyocytes in cardiac repair after injury are discussed.

PDK4 Inhibits Cardiac Pyruvate Oxidation in Late Pregnancy.

RATIONALE: Pregnancy profoundly alters maternal physiology. The heart hypertrophies during pregnancy, but its metabolic adaptations, are not well understood. OBJECTIVE: To determine the mechanisms underlying cardiac substrate use during pregnancy. METHODS AND RESULTS: We use here 13C glucose, 13C lactate, and 13C fatty acid tracing analyses to show that hearts in late pregnant mice increase fatty acid uptake and oxidation into the tricarboxylic acid cycle, while reducing glucose and lactate oxidation. Mitochondrial quantity, morphology, and function do not seem altered. Insulin signaling seems intact, and the abundance and localization of the major fatty acid and glucose transporters, CD36 (cluster of differentiation 36) and GLUT4 (glucose transporter type 4), are also unchanged. Rather, we find that the pregnancy hormone progesterone induces PDK4 (pyruvate dehydrogenase kinase 4) in cardiomyocytes and that elevated PDK4 levels in late pregnancy lead to inhibition of PDH (pyruvate dehydrogenase) and pyruvate flux into the tricarboxylic acid cycle. Blocking PDK4 reverses the metabolic changes seen in hearts in late pregnancy. CONCLUSIONS: Taken together, these data indicate that the hormonal environment of late pregnancy promotes metabolic remodeling in the heart at the level of PDH, rather than at the level of insulin signaling.

Heart regeneration and the cardiomyocyte cell cycle.

Cardiovascular disease and in particular, heart failure are still main causes of death; therefore, novel therapeutic approaches are urgently needed. Loss of contractile substrate in the heart and limited regenerative capacity of cardiomyocytes are mainly responsible for the poor cardiovascular outcome. This is related to the postmitotic state of differentiated cardiomyocytes, which is partly due to their polyploid nature caused by cell cycle variants. As such, the cardiomyocyte cell cycle is a key player, and its manipulation could be a promising strategy for enhancing the plasticity of the heart by inducing cardiomyocyte proliferation. This review focuses on the cardiac cell cycle and its variants during postnatal growth, the different regenerative responses of the heart in dependance of the developmental stage and on manipulations of the cell cycle. Because a therapeutic goal is to induce authentic cell division in cardiomyocytes, recent experimental approaches following this strategy are also discussed.

Visualization of endothelial cell cycle dynamics in mouse using the Flt-1/eGFP-anillin system.

Endothelial cell proliferation is a key process during vascular growth but its kinetics could only be assessed in vitro or ex vivo so far. To enable the monitoring and quantification of cell cycle kinetics in vivo, we have generated transgenic mice expressing an eGFP-anillin construct under control of the endothelial-specific Flt-1 promoter. This construct labels the nuclei of endothelial cells in late G1, S and G2 phase and changes its localization during the different stages of M phase, thereby enabling the monitoring of EC proliferation and cytokinesis. In Flt-1/eGFP-anillin mice, we found eGFP+ signals specifically in Ki67+/PECAM+ endothelial cells during vascular development. Quantification using this cell cycle reporter in embryos revealed a decline in endothelial cell proliferation between E9.5 to E12.5. By time-lapse microscopy, we determined the length of different cell cycle phases in embryonic endothelial cells in vivo and found a M phase duration of about 80 min with 2/3 covering karyokinesis and 1/3 cytokinesis. Thus, we have generated a versatile transgenic system for the accurate assessment of endothelial cell cycle dynamics in vitro and in vivo.

Deciphering the Epigenetic Code of Cardiac Myocyte Transcription.

RATIONALE: Epigenetic mechanisms are crucial for cell identity and transcriptional control. The heart consists of different cell types, including cardiac myocytes, endothelial cells, fibroblasts, and others. Therefore, cell type-specific analysis is needed to gain mechanistic insight into the regulation of gene expression in cardiac myocytes. Although cytosolic mRNA represents steady-state levels, nuclear mRNA more closely reflects transcriptional activity. To unravel epigenetic mechanisms of transcriptional control, cell type-specific analysis of nuclear mRNA and epigenetic modifications is crucial. OBJECTIVE: The aim was to purify cardiac myocyte nuclei from hearts of different species by magnetic- or fluorescent-assisted sorting and to determine the nuclear and cellular RNA expression profiles and epigenetic marks in a cardiac myocyte-specific manner. METHODS AND RESULTS: Frozen cardiac tissue samples were used to isolate cardiac myocyte nuclei. High sorting purity was confirmed for cardiac myocyte nuclei isolated from mice, rats, and humans. Deep sequencing of nuclear RNA revealed a major fraction of nascent, unspliced RNA in contrast to results obtained from purified cardiac myocytes. Cardiac myocyte nuclear and cellular RNA expression profiles showed differences, especially for metabolic genes. Genome-wide maps of the transcriptional elongation mark H3K36me3 were generated by chromatin-immunoprecipitation. Transcriptome and epigenetic data confirmed the high degree of cardiac myocyte-specificity of our protocol. An integrative analysis of nuclear mRNA and histone mark occurrence indicated a major impact of the chromatin state on transcriptional activity in cardiac myocytes. CONCLUSIONS: This study establishes cardiac myocyte-specific sorting of nuclei as a universal method to investigate epigenetic and transcriptional processes in cardiac myocytes of different origins. These data sets provide novel insight into cardiac myocyte transcription.