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.

Oncogenic KRAS suppresses store-operated Ca2+ entry and ICRAC through ERK pathway-dependent remodelling of STIM expression in colorectal cancer cell lines.

The KRAS GTPase plays a fundamental role in transducing signals from plasma membrane growth factor receptors to downstream signalling pathways controlling cell proliferation, survival and migration. Activating KRAS mutations are found in 20% of all cancers and in up to 40% of colorectal cancers, where they contribute to dysregulation of cell processes underlying oncogenic transformation. Multiple KRAS-regulated cell functions are also influenced by changes in intracellular Ca2+ levels that are concurrently modified by receptor signalling pathways. Suppression of intracellular Ca2+ release mechanisms can confer a survival advantage in cancer cells, and changes in Ca2+ entry across the plasma membrane modulate cell migration and proliferation. However, inconsistent remodelling of Ca2+ influx and its signalling role has been reported in studies of transformed cells. To isolate the interaction between altered Ca2+ handling and mutated KRAS in colorectal cancer, we have previously employed isogenic cell line pairs, differing by the presence of an oncogenic KRAS allele (encoding KRASG13D), and have shown that reduced Ca2+ release from the ER and mitochondrial Ca2+ uptake contributes to the survival advantage conferred by oncogenic KRAS. Here we show in the same cell lines, that Store-Operated Ca2+ Entry (SOCE) and its underlying current, ICRAC are under the influence of KRASG13D. Specifically, deletion of the oncogenic KRAS allele resulted in enhanced STIM1 expression and greater Ca2+ influx. Consistent with the role of KRAS in the activation of the ERK pathway, MEK inhibition in cells with KRASG13D resulted in increased STIM1 expression. Further, ectopic expression of STIM1 in HCT 116 cells (which express KRASG13D) rescued SOCE, demonstrating a fundamental role of STIM1 in suppression of Ca2+ entry downstream of KRASG13D. These results add to the understanding of how ERK controls cancer cell physiology and highlight STIM1 as an important biomarker in cancerogenesis.

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 DCM.