Stabilised beta-catenin in postnatal ventricular myocardium leads to dilated cardiomyopathy and premature death.

Beta-catenin is a component of the intercalated disc in cardiomyocytes, but can also be involved in signalling and activation of gene transcription. We wanted to determine how long-term changes in beta-catenin expression levels would affect mature cardiomyocytes. Conditional transgenic mice that either lacked beta-catenin or that expressed a non-degradable form of beta-catenin in the adult ventricle were created. While mice lacking beta-catenin in the ventricle do not have an overt phenotype, mice expressing a non-degradable form develop dilated cardiomyopathy and do not survive beyond 5 months. A detailed analysis could reveal that this phenotype is correlated with a distinct localisation of beta-catenin in adult cardiomyocytes, which cannot be detected in the nucleus, no matter how much protein is present. Our report is the first study that addresses long-term effects of either the absence of beta-catenin or its stabilisation on ventricular cardiomyocytes and it suggests that beta-catenin's role in the nucleus may be of little significance in the healthy adult heart.

Activation of a HIF1alpha-PPARgamma axis underlies the integration of glycolytic and lipid anabolic pathways in pathologic cardiac hypertrophy.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

Essential role of developmentally activated hypoxia-inducible factor 1alpha for cardiac morphogenesis and function.

Development of the mammalian heart is governed by precisely orchestrated interactions between signaling pathways integrating environmental cues and a core cardiac transcriptional network that directs differentiation, growth and morphogenesis. Here we report that in mice, at about embryonic day (E)8.5 to E10.0, cardiac development proceeds in an environment that is hypoxic and characterized by high levels of hypoxia-inducible factor (HIF)1alpha protein. Mice lacking HIF1alpha in ventricular cardiomyocytes exhibit aborted development at looping morphogenesis and embryonic lethality between E11.0 to E12.0. Intriguingly, HIF1alpha-deficient hearts display reduced expression of the core cardiac transcription factors Mef2C and Tbx5 and of titin, a giant protein that serves as a template for the assembly and organization of the sarcomere. Chromatin immunoprecipitation experiments revealed that Mef2C, Tbx5, and titin are direct target genes of HIF1alpha in vivo. Thus, hypoxia signaling controls cardiac development through HIF1alpha-mediated transcriptional regulation of key components of myofibrillogenesis and the cardiac transcription factor network, thereby providing a mechanistic basis of how heart development, morphogenesis, and function is coupled to low oxygen tension during early embryogenesis.

Re-expression of proteins involved in cytokinesis during cardiac hypertrophy.

Cardiomyocytes stop dividing after birth and postnatal heart growth is only achieved by increase in cell volume. In some species, cardiomyocytes undergo an additional incomplete mitosis in the first postnatal week, where karyokinesis takes place in the absence of cytokinesis, leading to binucleation. Proteins that regulate the formation of the actomyosin ring are known to be important for cytokinesis. Here we demonstrate for the first time that small GTPases like RhoA along with their downstream effectors like ROCK I, ROCK II and Citron Kinase show a developmental stage specific expression in heart, with high levels being expressed in cardiomyocytes only at stages when cytokinesis still occurs (i.e. embryonic and perinatal). This suggests that downregulation of many regulatory and cytoskeletal components involved in the formation of the actomyosin ring may be responsible for the uncoupling of cytokinesis from karyokinesis in rodent cardiomyocytes after birth. Interestingly, when the myocardium tries to adapt to the increased workload during pathological hypertrophy a re-expression of proteins involved in DNA synthesis and cytokinesis can be detected. Nevertheless, the adult cardiomyocytes do not appear to divide despite this upregulation of the cytokinetic machinery. The inability to undergo complete division could be due to the presence of stable, highly ordered and functional sarcomeres in the adult myocardium or could be because of the inefficiency of degradation pathways, which facilitate the division of differentiated embryonic cardiomyocytes by disintegrating myofibrils.

Probing the role of septins in cardiomyocytes.

Heart growth in the embryo is achieved by division of differentiated cardiomyocytes. Around birth, cardiomyocytes stop dividing and heart growth occurs only by volume increase of the individual cells. Cardiomyocytes seem to lose their capacity for cytokinesis at this developmental stage. Septins are GTP-binding proteins that have been shown to be involved in cytokinesis from yeast to vertebrates. We wanted to determine whether septin expression patterns can be correlated to the cessation of cytokinesis during heart development. We found significant levels of expression only for SEPT2, SEPT6, SEPT7 and SEPT9 in heart, in a developmentally regulated fashion, with high levels in the embryonic heart, downregulation around birth and no detectable expression in the adult. In dividing embryonic cardiomyocytes, all septins localize to the cleavage furrow. We used drugs to probe for the functional interactions of SEPT2 in dividing embryonic cardiomyocytes. Differences in the effects on subcellular septin localization in cardiomyocytes were observed, depending whether a Rho kinase (ROCK) inhibitor was used or whether actin and myosin were targeted directly. Our data show a tight correlation of high levels of septin expression and the ability to undergo cytokinesis in cardiomyocytes. In addition, we were able to dissect the different contributions of ROCK signaling and the actomyosin cytoskeleton to septin localization to the contractile ring using cardiomyocytes as an experimental system.

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes.

The contractile tissue of the heart is composed of individual cardiomyocytes. During mammalian embryonic development, heart growth is achieved by cell division while at the same time the heart is already exerting its essential pumping activity. There is still some debate whether the proliferative activity is carried out by a less differentiated, stem cell-like type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of these completely different dynamic tasks, contraction and cell division. Our analysis of triple-stained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes are indeed able to proliferate. However, to go through cell division, a disassembly of the contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps with Z-disk and thin (actin)-filament-associated proteins getting disassembled before disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen. Another interesting observation was that the cell-cell contacts remain seemingly intact during division, probably reflecting the requirement of intact integration sites of the individual cells in the contractile tissue. Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex disassembly-reassembly process might also provide a mechanistic explanation, why cardiomyocytes cede to divide postnatally.

Subcellular targeting of metabolic enzymes to titin in heart muscle may be mediated by DRAL/FHL-2.

During sarcomere contraction skeletal and cardiac muscle cells consume large amounts of energy. To satisfy this demand, metabolic enzymes are associated with distinct regions of the sarcomeres in the I-band and in the M-band, where they help to maintain high local concentrations of ATP. To date, the mechanism by which metabolic enzymes are coupled to the sarcomere has not been elucidated. Here, we show that the four and a half LIM-only protein DRAL/FHL-2 mediates targeting of the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase by interaction with the elastic filament protein titin in cardiomyocytes. Using yeast two-hybrid assays, colocalisation experiments, co-immunoprecipitation and protein pull-down assays, we show that DRAL/FHL-2 is bound to two distinct sites on titin. One binding site is situated in the N2B region, a cardiac-specific insertion in the I-band part of titin, and the other is located in the is2 region of M-band titin. We also show that DRAL/FHL-2 binds to the metabolic enzymes creatine kinase, adenylate kinase and phosphofructokinase and might target these enzymes to the N2B and is2 regions in titin. We propose that DRAL/FHL-2 acts as a specific adaptor protein to couple metabolic enzymes to sites of high energy consumption in the cardiac sarcomere.