Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a ‘function follows form’ model of differentiation

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes

Doxorubicin-induced chronic dilated cardiomyopathy—the apoptosis hypothesis revisited

The chemotherapeutic agent doxorubicin (DOX) has significantly increased survival rates of pediatric and adult cancer patients. However, 10% of pediatric cancer survivors will 10–20 years later develop severe dilated cardiomyopathy (DCM), whereby the exact molecular mechanisms of disease progression after this long latency time remain puzzling. We here revisit the hypothesis that elevated apoptosis signaling or its increased likelihood after DOX exposure can lead to an impairment of cardiac function and cause a cardiac dilation. Based on recent literature evidence, we first argue why a dilated phenotype can occur when little apoptosis is detected. We then review findings suggesting that mature cardiomyocytes are protected against DOX-induced apoptosis downstream, but not upstream of mitochondrial outer membrane permeabilisation (MOMP). This lack of MOMP induction is proposed to alter the metabolic phenotype, induce hypertrophic remodeling, and lead to functional cardiac impairment even in the absence of cardiomyocyte apoptosis. We discuss findings that DOX exposure can lead to increased sensitivity to further cardiomyocyte apoptosis, which may cause a gradual loss in cardiomyocytes over time and a compensatory hypertrophic remodeling after treatment, potentially explaining the long lag time in disease onset. We finally note similarities between DOX-exposed cardiomyocytes and apoptosis-primed cancer cells and propose computational system biology as a tool to predict patient individual DOX doses. In conclusion, combining recent findings in rodent hearts and cardiomyocytes exposed to DOX with insights from apoptosis signal transduction allowed us to obtain a molecularly deeper insight in this delayed and still enigmatic pathology of DC

The RBL-2H3 cell line: its provenance and suitability as a model for the mast cell

The cell line, RBL-2H3, has been widely used as a mast cell model though much of the data is contradictory. The aim of this study is to assess the RBL-2H3 cell line as an in vitro model for degranulation studies. RBL-2H3 cells were stimulated with either dinitrophenylated-IgE, calcium ionophore A23187, compound 48/80, mast cell degranulating peptide or lipopolysaccharide and mediator (histamine, β-hexosaminidase, interleukin-13 and TNF-α) release was analysed. Toll-like receptors (TLR) mRNA expression in RBL-2H3 cells and rat peritoneal mast cells (RPMC) were compared by RT-PCR. TLR4 and CD14 surface proteins in RBL-2H3 were analysed by FACS. Mediator release was dependant on media composition and degranulation was stimulated by IgE and A23187. Degranulation was inhibited by quercetin, but not by cromoglycate or ketotifen. Transcripts encoding TLR3-5 and 6 were detected in RBL-2H3 cells whereas TLR1-6 and TLR8 were clearly seen in RPMC. While proteins were detected for TLR4 in RBL-2H3 cells, CD14 was largely absent. While RBL-2H3 cells may be useful as a model for mast cell IgE-mediated degranulation, other aspects may not be representative and they may share similarities with basophils rather than with other histamine-releasing cell types

Signal transduction analysis of the NLRP3-inflammasome pathway after cellular damage and its paracrine regulation

Activation of the NLRP3-inflammasome pathway and production of the inflammatory cytokine IL-1B after cellular damage caused by infarct or infection is a key process in several diseases such as acute myocardial infarction and inflammatory bowel disease. However, while the molecular triggers of the NLRP3-pathway after cellular damage are well known, the mechanisms that sustain or confine its activity are currently under investigation. We present here an Ordinary Differential Equation-based model that investigates the mechanisms of inflammasome activation and regulation in monocytes to predict IL-1β activation kinetics upon a two-step activation by Damage-Associate-Molecular-Particles (DAMP) and extracellular ATP. Assuming both activation signals to be concomitantly present or present with a delay of 12 h, the model predicted a transient IL-1β activation at different concentration levels dependent on signal synchronisation. Introducing a positive feedback loop mediated by active IL-1β resulted in a sustained IL-1β activation, hence arguing for a paracrine signalling between inflammatory cells to guarantee a temporally stable inflammatory response. We then investigate mechanisms that control termination of inflammation using two recently identified molecular intervention points in the inflammasome pathway. We found that a more upstream regulation, by attenuating production of the IL-1β-proform, was more potent in attenuating active IL-1β production than direct inhibition of the NLRP3-inflammasome. Interestingly, ablating this upstream negative feedback led to a high variability of IL-1β production in monocytes from different subjects, consistent with a recent pre-clinical study. We finally discuss the relevance and implications of our findings in disease models of acute myocardial infarction and spontaneous colitis

Quantitative proteomics and systems analysis of cultured H9C2 cardiomyoblasts during differentiation over time supports a "function follows form' model of differentiation

The rat cardiomyoblast cell line H9C2 has emerged as a valuable tool for studying cardiac development, mechanisms of disease and toxicology. We present here a rigorous proteomic analysis that monitored the changes in protein expression during differentiation of H9C2 cells into cardiomyocyte-like cells over time. Quantitative mass spectrometry followed by gene ontology (GO) enrichment analysis revealed that early changes in H9C2 differentiation are related to protein pathways of cardiac muscle morphogenesis and sphingolipid synthesis. These changes in the proteome were followed later in the differentiation time-course by alterations in the expression of proteins involved in cation transport and beta-oxidation. Studying the temporal profile of the H9C2 proteome during differentiation in further detail revealed eight clusters of co-regulated proteins that can be associated with early, late, continuous and transient up- and downregulation. Subsequent reactome pathway analysis based on these eight clusters further corroborated and detailed the results of the GO analysis. Specifically, this analysis confirmed that proteins related to pathways in muscle contraction are upregulated early and transiently, and proteins relevant to extracellular matrix organization are downregulated early. In contrast, upregulation of proteins related to cardiac metabolism occurs at later time points. Finally, independent validation of the proteomics results by immunoblotting confirmed hereto unknown regulators of cardiac structure and ionic metabolism. Our results are consistent with a 'function follows form' model of differentiation, whereby early and transient alterations of structural proteins enable subsequent changes that are relevant to the characteristic physiology of cardiomyocytes.status: publishe