Heart and Skeletal Muscle Disease
Heart and skeletal muscle disease are leading causes of death and disability. The Huggins Lab investigates genetic and biological mechanisms of heart and cardiovascular disease using a two pronged approach. One prong uses molecular and cellular biology techniques complemented by the use of animal models to study genetic regulators of muscle development and their role in muscle disease. The other prong relies on informatics methods to identify genes associated with human diseases and traits. Both approaches complement each other as molecular and cellular biology studies help create hypotheses to be tested in human genetic databases and genes identified through human association studies become the focus of molecular studies designed to explore mechanisms underlying the human trait and disease. The convergence of the two approaches offers an exciting platform for discovering important biological mechanisms and genetic human variations that contribute to disease.
Analysis of Muscle Development and Disease
Causative factors of heart and skeletal muscle disease include environmental insults such as ischemia/infarction, toxins, drug exposure and atypical genetic changes. One of the characteristic features of the diseased heart muscle is a reversion of gene expression to a pattern which was present in the developing embryo called the “fetal gene program”. This event affirms an interesting role for developmental regulators and gene transcription in diseased muscle. We have explored the role of transcriptional regulators that regulate the switch between the fetal and adult gene expression programs. The laboratory is currently focusing on one particular regulator, called FOG-2, which has the ability to modulate several DNA-binding transcription factors, and when over-expressed in the heart produces fibrosis and heart failure.
Figure 1. Transgenic over-expression of FOG-2 in the heart produces fibrosis and activation of the fetal gene program. (a) Histological analysis of collagen content analyzed by Sirius Red staining: dark staining indicates fibrosis. Diffuse interstitial staining indicates increased collagen content of MHC-FOG-2 transgenic (Tg) compared with non-transgenic (NTg) mouse hearts. (b) Northern blots demonstrates increased atrial natriuretic factor (ANF) and collagen transcripts in Tg compared with NTg mice. For more information, see Rouf, et al, 2008.
Muscle cell differentiation occurs in a conducive environment through a carefully orchestrated series of transcription factors. Environmental insults and in particular excessive alcohol exposure damages muscle through complex genetic and cellular mechanisms. One approach we have taken to study muscle cell differentiation is the development of a high content cellular phenotyping system capable of analyzing the events of muscle cell differentiation on each cell (Figure 2).
Figure 2. Quantitation of muscle cell differentiation using the ImageExpress (Molecular Devices). Determining the full spectrum of muscle cell differentiation by conventional imaging and quantitation tools is limited by the large number of measurements that must be recorded. To address this issue we have customized existing computer-based cellular and nuclear analyses to comprehensively study muscle cell differentiation in a 96-well plate format. The image demonstrates computer identified and demarcated multi-nucleated muscle tubes formed in the mouse C2C12 cell line after seven days under differentiating conditions. The muscle tubes are stained for the troponin protein (green) and nuclei are counter-stained with DAPI (blue). Image provided by Michelle Arya.
Identifying Genes Associated with Human Disease
Within the past one to two decades the human genome sequence has been completed and a map of genetic markers suitable for studying disease associations has been established. Both candidate gene and genome-wide association studies are performed in the Huggins laboratory using data generated within the laboratory or obtained through public repositories. Identifying the genetic contributors to bicuspid aortic valve (BAV), a highly heritable congenital heart defect, is one focus of the laboratory. By comparing genotypes from DNA samples collected from subjects with BAV treated at Tufts Medical Center with control samples several loci were found to be associated with BAV through the use of gene network/pathway informatics tools (Figure 3). Genes and loci identified by candidate gene and genome-wide association approaches then become the subject of further study. AXIN1 is an intriguing gene to be associated with a congenital abnormality of the aortic valve because of the known role of the Wnt-b-catenin pathway and great vessel and valve development.
Figure 3. BAV Associated SNPs Spanning PDIA2 and AXIN1. Data from three approaches showing the location of SNPs associated with BAV. The four graphical data-panes indicate RR cM/Mb: relative recombination rate in centimorgans per megabase. STRING –log(p), CANDID –log(p), and fitSNPs –log(p): -log10 uncorrected p-values observed in each of the three indicated schemes. For more information, see Wooten et al, 2010.