A Molecular Analysis of Cardiac Hypertrophy

Abstract

Abstract :Cardiac hypertrophy has been identified as the most important independent risk factor for cardiovascular-related morbidity and mortality and is therefore regarded as a pathological condition. Despite this, beneficial physiological forms also appear to exist, such as in response to exercise, leading to maintained or improved cardiac function. The aim of this thesis was to examine two distinct rodent models, an endurance run-trained rat, and the DOCA-salt hypertensive rat, representing physiological and pathological hypertrophy, respectively, in order to develop a better understanding of the molecular changes associated with each condition. The thesis also examined the effect of dietary supplementation of L-arginine to the pathological model, a treatment that has been shown to ameliorate/prevent many of the cardiovascular impairments. Studies examined selected candidate genes (qRT-PCR), including conventional biomarkers of hypertrophy and exploratory analysis of adenosine-related genes (given adenosine’s established regulatory and protective role in the heart, yet minimally studied in cardiac hypertrophy), and explored global transcriptomic shifts via microarrays. The hypothesis of this work was that cardiac hypertrophy lies on a continuum, with similarities existing at the cardiac transcriptional level between early (adaptive) stages of pathological hypertrophy (DOCA-salt rat) and later stages of physiological hypertrophy (endurance run-trained rat). Examination of ten biomarkers of hypertrophy (ANF, BNP, -MHC, -MHC, cardiac -actin, skeletal -actin, SERCA2, PPAR, Coll I and III) revealed that the pathological model displayed alterations in the expression of many of these molecules in line with the literature. These changes were not observed in the physiological model. This therefore reinforces the value of conventional biomarkers in delineating pathological vs. physiological hypertrophies, and reveals fundamental differences in genesis of these two forms of hypertrophy. The adenosine system (receptors and purine handling molecules) was altered in the pathological hypertrophy model as evidenced by the modulation of genes corresponding to A3AR, Ada, and Adk, with a potential shift from purine salvage towards degradation of adenosine to inosine. Furthermore, this study represents the first report of altered regulation of the nucleoside transporter ENT3 in a pathological condition. None of these changes were seen in the physiological model with only modulation of the A2aAR evident. Examination of the transcriptional response to physiological hypertrophy revealed that short (6 week) and long (12 week) training programmes resulted in different profiles, likely reflecting progression of the hypertrophy process. The short programme stimulated genes associated with the mitochondria, oxidoreductase, receptor binding and coenzymemetabolismand repressed the expression of transcripts associated with phosphorylation, catalytic activity, defence/immunity and energy pathways. Thus, initial changes observed are primarily of a metabolic and signalling nature. In contrast, the longer programme resulted in shifts in protein handling and synthesis, and genes involved in structural molecule activity, nucleotide binding and cellular homeostasis. These patterns support a progression with time from initial metabolic adaptations to longer term shifts in protein phenotype and structural adaptations, consistent with longer term changes in heart structure. Similarly, the pathologicalmodel displayed different time-dependent gene expression profiles. Overall, the pattern of changewith early (2week) treatment is suggestive of changes in intracellular signalling and increasing transcriptional capacity with the later changes (at 4 weeks) indicative of structural adaptations (intra- and extracellularly) togetherwith an inflammation response. Genes coding for calciumhandling, ion channels, and gap junctions were altered throughout themodel andmay contribute to electrical conduction defects and cardiac dysfunction. The adrenergic signalling pathway was modulated as associated signalling molecules were down-regulated. The study revealed many expected and novel changes, of which further study should focus on: calcium regulation, metabolic regulation, gap junctions, and (as might be exii pected) signalling via the adrenergic pathway, insulin-like growth factor, PI3K, and Jak/STAT. L-Arginine modulated biomarker expression in pathological hypertrophy, with stimulation of PPAR and SERCA2 with little or no effect on the adenosine-related genes. L-Arginine affected the overall transcriptional response to DOCA-salt treatment, stimulating genes involved in cell growth andmaintenance, nerve transmission, heparin and glycosaminoglycan binding, peptide binding and protein targeting, as well as the repression of genes related to apoptosis (favouring a pro-apoptotic state), intracellular organisation and biogenesis, and enzyme inhibitor activity. The beneficial effects of L-arginine in the setting of pathological hypertrophy may be due to modulation of metabolism, improving calcium handling and overall enhancing cellular functioning. This work demonstrates that cardiac hypertrophy is clearly different at the transcriptional level depending upon the aetiology. This repudiated the hypothesis of the thesis that cardiac hypertrophy lies on a continuum with similarities existing at the cardiac transcriptional level between early (adaptive) stages of pathological hypertrophy and later stages of physiological hypertrophy. Whilst some of the data was in accordance with current knowledge of these states, novel changes were also discovered, contributing to our understanding of the molecular aspects of cardiac hypertrophy.