Serum response factor (SRF) regulates certain microRNAs that play a role in cardiac and skeletal muscle development. However, the role of SRF in the regulation of microRNA expression and microRNA biogenesis in cardiac hypertrophy has not been well established. In this report, we employed two distinct transgenic mouse models to study the impact of SRF on cardiac microRNA expression and microRNA biogenesis. Cardiac-specific overexpression of SRF (SRF-Tg) led to altered expression of a number of microRNAs. Interestingly, downregulation of miR-1, miR-133a and upregulation of miR-21 occurred by 7 days of age in these mice, long before the onset of cardiac hypertrophy, suggesting that SRF overexpression impacted the expression of microRNAs which contribute to cardiac hypertrophy. Reducing cardiac SRF level using the antisense-SRF transgenic approach (Anti-SRF-Tg) resulted in the expression of miR-1, miR-133a and miR-21 in the opposite direction. Furthermore, we observed that SRF regulates microRNA biogenesis, specifically the transcription of pri-microRNA, thereby affecting the mature microRNA level. The mir-21 promoter sequence is conserved among mouse, rat and human; one SRF binding site was found to be in the mir-21 proximal promoter region of all three species. The mir-21 gene is regulated by SRF and its cofactors, including myocardin and p49/Strap. Our study demonstrates that the downregulation of miR-1, miR-133a, and upregulation of miR-21 can be reversed by one single upstream regulator, SRF. These results may help to develop novel therapeutic interventions targeting microRNA biogenesis.

Purpose: To test the hypothesis that cardiac morphologic differences between Ames dwarf and wild-type littermates might correlate with the increased longevity observed in the Ames dwarf mice.Methods: Hearts removed from young adult (5-7 mo) and old (24-28 mo) Ames dwarf and wild-type littermates underwent histological and morphometric analysis. Measurements of cell size, nuclear size, and collagen content were made using computerized color deconvolution and particle analysis methodology.Results: In the young mice at six months of age, mean cardiomyocyte area was 46% less in Ames dwarf than in wild-type mice (p<0.0001). Cardiomyocyte size increased with age by about 52% in the wild-type mice and 44% in the Ames dwarf mice (p<0.001). There was no difference in nuclear size of the cardiomyocytes between the young adult wild-type and Ames dwarf mice. There was an age-associated increase in the cardiomyocyte nuclear size by approximately 50% in both the Ames and wild-type mice (p<0.001). The older Ames dwarf mice had slightly larger cardiomyocyte nuclei compared to wild-type (2%, p<0.05). The collagen content of the hearts in young adult Ames dwarf mice was estimated to be 57% less compared to wild-type littermates (p<0.05). Although collagen content of both Ames dwarf and wild-type mouse hearts increased with age, there was no significant difference at 24 months.Conclusions: In wild-type and Ames dwarf mice, nuclear size, cardiomyocyte size, and collagen content increased with advancing age. While cardiomyocyte size was much reduced in young and old Ames dwarf mice compared with wild-type, collagen content was reduced only in the young adult mice. Taken together, these findings suggest that Ames dwarf mice may receive some longevity benefit from the reduced cardiomyocyte cell size and a period of reduced collagen content in the heart during adulthood.

BackgroundThe p49/STRAP (or SRFBP1) protein was recently identified in our laboratory as a cofactor of serum response factor that contributes to the regulation of SRF target genes in the heart.ResultsIn the present study, we report that NDUFAB1, a nuclear encoded subunit of NADH dehydrogenase, represented the majority of the cDNA clones that interacted with p49/STRAP in multiple screenings using the yeast two-hybrid system. The p49/STRAP and NDUFAB1 proteins interacted and co-localized with each other in the cell. The p49/STRAP protein contains four classic nuclear localization sequence motifs, and it was observed to be present predominantly in the nucleus. Overexpression of p49/STRAP altered the intracellular level of NAD, and reduced the NAD/NADH ratio. Overexpression of p49/STRAP also induced the deacetylation of serum response factor.ConclusionThese data suggest that p49/STRAP plays a role in the regulation of intracellular processes such as cardiac cellular metabolism, gene expression, and possibly aging.

Background:To identify in vivo new cardiac binding sites of serum response factor (SRF) in genes and to study the response of these genes to mild over-expression of SRF, we employed a cardiac-specific, transgenic mouse model, with mild over-expression of SRF (Mild-O SRF Tg).Methodology:Microarray experiments were performed on hearts of Mild-O-SRF Tg at 6 months of age. We identified 207 genes that are important for cardiac function that were differentially expressed in vivo. Among them the promoter region of 192 genes had SRF binding motifs, the classic CArG or CArG-like (CArG-L) elements. Fifty-one of the 56 genes with classic SRF binding sites had not been previously reported. These SRF-modulated genes were grouped into 12 categories based on their function. It was observed that genes associated with cardiac energy metabolism shifted toward that of carbohydrate metabolism and away from that of fatty acid metabolism. The expression of genes that are involved in transcription and ion regulation were decreased, but expression of cytoskeletal genes was significantly increased. Using public databases of mouse models of hemodynamic stress (GEO database), we also found that similar altered expression of the SRF-modulated genes occurred in these hearts with cardiac ischemia or aortic constriction as well.Conclusion and significance:SRF-modulated genes are actively regulated under various physiological and pathological conditions. We have discovered that a large number of cardiac genes have classic SRF binding sites and were significantly modulated in the Mild-O-SRF Tg mouse hearts. Hence, the mild elevation of SRF protein in the heart that is observed during typical adult aging may have a major impact on many SRF-modulated genes, thereby affecting cardiac structure and performance. The results from our study could help to enhance our understanding of SRF regulation of cellular processes in the aged heart.

Treatment of lung cancer is evolving from the use of cytotoxic drugs to drugs that interrupt metabolic pathways specific to a malignancy. We report here in that the metabolic phenotype of lung cancer can be determined in lymph node aspirates harboring malignant tumor cells. Knowledge about metabolic activity of malignant tumor cells may aide to personalize therapy.

Lung cancer is the leading cause of cancer-related deaths in US. For the majority of patients (70%), the extent of the disease precludes complete surgical resection and treatment relies solely on radiation, chemotherapy or a combination of both. Unfortunately, these treatments improve survival only minimally and are accompanied by considerable adverse side effects. Despite immense research efforts over the last 20 years, the mean survival of lung cancer patients has only increased by 13 days. This relatively poor mean survival is attributed to inadequate therapy selection. Current therapy selection is mainly based on the histopathologic examination of needle biopsies obtained during bronchoscopy. There is growing concern that these classifications and stratification are insufficient to predict treatment response of individual tumors. To complement current histopathological-based tumor classification, we investigated the suitability of metabolomics to improve tumor classification. Therefore, metabolomic profiles were obtained from various lung cancer cell-lines. Data were analyzed for molecular features characteristic for cell-lines derived from adenocarcinoma (AdenoCa) or squamous cell carcinoma (SqCCa). Data analyses revealed 78 features that are capable of clearly separating AdenoCa, SqCCa, and lung fibroblasts from each other. Subsequently, the studies were extended to specimen from 30 lung cancer patients. Similar to the cell-line experiments, metabolomic profiles were obtained and analyzed for characteristic features of patients with AdenoCa, SqCCa, or non-malignant specimen. The analysis of the human specimen revealed 21 unique features that are potentially suitable biomarkers for tumor classification. Our data suggest that metabolomic profiling is a promising approach to classify and distinguish between individual tumor cell-lines and to distinguish patients with AdenoCa from patients with SqCCa. Citation Format: Daniel R. Sappington, Scott Helms, Ishwori B. Dhakal, Eric R. Siegel, Thaddeus Bartter, Gunnar Boysen. Molecular characterization of lung tumors based on metabolomic profiling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3236. doi:10.1158/1538-7445.AM2013-3236

Lung cancer is the leading cause of cancer-related deaths in the US and worldwide. The majority of lung cancer patients are diagnosed with an advanced stage, precluding curable surgical resection, and therapy is limited to thoracic radiation and chemotherapy. The efficacies have improved in recent years due to patient pre-selection based on the cytology of fine needle aspirates of lymph nodes, which harbor circulating tumor cells. We report herein the evaluation of lymph node aspirates for the presence of metabolite features that are suitable to distinguish patients with lung cancer from patients without malignancies and to separate patients with adenocarcinoma from patients with squamous cell carcinoma. The results provide evidence that patient groups can be separated based on their metabolite profiles. Multivariate statistical analyses (PLS and OPLS) produce a set of consensus features that can be further developed into clinical biomarkers. Two of the features were identified as kynurenine and oxidized glutathione, and their identities were confirmed in a second larger sample set. In addition, the accompanying metabolites glutamate, glutamine and tryptophan were included in the validation experiment to utilize relative metabolite ratios as indicators for corresponding enzyme activities. The validation results demonstrate significant differences in metabolites and metabolite ratios between lymph node aspirates harboring a circulating tumor cell and aspirates negative for malignancy. Subsequently, a second set of lymph node samples were analyzed for an extended panel of metabolites and of these 42 were suitable biomarker to be statistically significant to distinguish between at least one of the pair wise comparisons. Together the results demonstrate that metabolite profiles in lymph node aspirates are suitable to differentiate patients with adenocarcinoma from patients with squamous cell carcinoma. Citation Format: Daniel R. Sappington, Scott A. Helms, Eric Siegel, Susanne K. Jeffus, Teka Bartter, Thaddeus Bartter, Gunnar Boysen. Diagnosis of lung tumor types based on metabolomic profiles in lymph node aspirates. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 547. doi:10.1158/1538-7445.AM2015-547