Coronary heart disease (CHD) is usually a leading cause of morbidity in people over 65 years of age; 40% of all deaths are due to this condition. a key feature of senescent cardiomyocytes and endothelial cells. Altered splicing of key cardiac or endothelial genes may contribute to the risk Rabbit Polyclonal to HTR2B of CHD in the human population. Introduction Cardiovascular disease is the major cause of mortality and morbidity in the developed world [1,2]. Age is one of the predominant risk factors for this group of disorders, and produces structural, molecular and functional changes in cardiac and vascular tissues [3C5]. The accumulation of senescent cells may be a contributor to cardiovascular pathology, since cells in the myocardium and vasculature are subject to cellular aging [6,7] and have known functions in cardiovascular dysfunction . Senescent cells are one of the major drivers of aging phenotypes; selective removal of such cells from transgenic mice results in rejuvenation and amelioration of multiple aging phenotypes [9,10]. Perfusion of aged myocardium with neonatal cells was able to restore more youthful patterns of gene expression to human cardiomyocyte progenitors or senescent rat myocytes, and these molecular changes were also associated with significant improvements in cardiovascular outcomes . Senescent cells display altered gene expression patterns compared with non-senescent cells . In particular, senescent cells and older organisms display reductions in cellular plasticity and adaptive capacity [13,14], which is usually in part determined by patterns of option mRNA processing. Over 95% of genes express more than one gene product (isoform) under different physiological conditions . Splice-site usage and patterns of alternative splicing are driven by a battery of splicing regulatory proteins termed as splicing factors [16,17]. Some studies have Z-DEVD-FMK reversible enzyme inhibition shown that genes encoding splicing factors are strongly dysregulated with aging in population studies and in senescent cells cultured isoform showed some evidence of a nominal association with prevalent and incident coronary heart disease (CHD) in participants from the InCHIANTI study of aging . This work provides evidence that dysregulation of option splicing may alter the transcriptomic output of aged cardiomyocytes or endothelial cells, and that these changes may be associated with the development of cardiovascular diseases. Methods Culture of early passage and senescent cardiomyocytes and endothelial cells Cultures of non-senescent and senescent endothelial cells and cardiomyocytes were used in the present study. Although the cultures were not from multiple isolates, each culture underwent senescence independently in three biological replicates to produce cultures that were not absolutely identical. Human aortic endothelial cells (HAoEC) and human cardiac myocytes (HCM) were seeded at a density of 6 103 cell/cm2 and were cultured in specific growth medium (C-22022 for HAoEC and C-22070 for HCM, PromoCell). Earlier passage proliferative cultures were at populace doubling (PD) = 24 for endothelial cells and PD = 28 for cardiomyocytes. For the production of senescent cultures, cells were counted and equal numbers of cells seeded at each passage in continuous culture until the growth of the culture slowed to less than 0.5 PD/week. This occurred at PD = 65 and PD = 75 for endothelial cells and cardiomyocytes respectively. Cells Z-DEVD-FMK reversible enzyme inhibition were maintained at 37C and 5% CO2 and used when not confluent to ensure that cessation of growth was not due to contact inhibition. Cell senescence Z-DEVD-FMK reversible enzyme inhibition was assessed in three biological replicates by the biochemical senescence marker senescence-associated -galactosidase (SA -Gal), tested in triplicate using a commercial kit (SigmaCAldrich, U.K.) according to manufacturers instructions, with a minimum of 300 cells assessed per replicate. Senescence was also quantified in molecular terms by assessing the expression of the cyclin-dependent kinase inhibitor 2A (expression was measured by qRTPCR relative to three empirically decided endogenous control genes (and list of splicing factor candidate genes were chosen on the basis that they were associated with human aging in populations and had been characterised in primary human fibroblasts and endothelial cells in our previous work [17,18]. The list of genes included the positive regulatory splicing factors and and the core spliceosomal factors and and endogenous control genes and normalised to their expression in RNA from early passage cells. Quantification of candidate.