Gene reprogramming in exercise-induced cardiac hypertrophy in swine: A transcriptional genomics approach

Highlights

• Exercise training in swine produced 16% cardiac left ventricle hypertrophy.

• Microarray analysis identified 339 upregulated and 408 downregulated genes.

• In promoter regions of these genes, 41 TF binding sites were overrepresented.

• DNA-binding assays with nuclear extracts identified 23 TFs with altered activity.

• Integrated analysis found YY1, PAX6 and GR as modulators of physiologic hypertrophy.

Abstract

Cardiac hypertrophy of the left ventricle (LV) in response to dynamic exercise-training (EX) is a beneficial adaptation to increased workload, and is thought to result from genetic reprogramming. We aimed to determine which transcription factors (TFs) are involved in this genetic reprogramming of the LV in swine induced by exercise-training. Swine underwent 3–6 weeks of dynamic EX, resulting in a 16% increase of LV weight/body weight ratio compared to sedentary animals (P = 0.03). Hemodynamic analysis showed an increased stroke volume index (stroke volume/body weight + 35%; P = 0.02). Microarray-analysis of LV tissue identified 339 upregulated and 408 downregulated genes (false discovery rate < 0.05). Of the human homologues of the differentially expressed genes, promoter regions were searched for TF consensus binding sites (TFBSs). For upregulated and downregulated genes, 17 and 24 TFBSs were overrepresented by > 1.5-fold (P < 0.01), respectively. In DNA-binding assays, using LV nuclear protein extracts and protein/DNA array, signal intensity changes > 2-fold were observed for 23 TF-specific DNA probes. Matching results in TFBS and protein/DNA array analyses were obtained for transcription factors YY1 (Yin Yang 1), PAX6 (paired box 6) and GR (glucocorticoid receptor). Notably, PAX6 and GR show lower signals in TFBS and protein/DNA array analyses upon exercise-training, whereas we previously showed higher signals for these factors in the remodeled LV of swine post-myocardial infarction (MI). In conclusion, we have identified transcription factors that may drive the genetic reprogramming underlying exercise-training induced LV hypertrophy in swine. PAX6 and GR are among the transcription factors that are oppositely regulated in LV hypertrophy after exercise-training and MI. These proteins may be at the base of the differences between pathological and physiological hypertrophy.

Introduction

Cardiac hypertrophy and remodeling is the response of the heart to an increased workload, stress or injury, in an attempt to maintain or increase cardiac output [1], [2]. Pathological hypertrophy as it occurs in hypertension, valvular diseases or after myocardial infarction (MI), is an independent risk factor for the later development of heart failure [3], [4]. In contrast, physiological hypertrophy such as that occurring during normal growth, pregnancy, or after prolonged exercise-training, is not associated with an increased risk for cardiovascular disease [5], [6]. Exercise-induced hypertrophy is not associated with increased fibrosis and apoptosis, and decreased capillary density, as is seen in various forms of pathological hypertrophy [7]. Physiological hypertrophy and pathological hypertrophy therefore have different cellular and molecular phenotypes which are thought to result from different genetic reprogramming [7], [8], [9].

The majority of studies into the molecular mechanisms underlying physiological hypertrophy have employed rodents subjected to treadmill running, voluntary wheel running or swim training. Various models of physiological hypertrophy in rodents have identified an important role for growth factors such as insulin-like growth factor (IGF-1), and phosphatidylinositol-3-kinase/Akt signaling [10]. Gene expression profiling has shown that expression of cell survival and fatty acid oxidation genes is altered after physiological hypertrophy, whereas genes involved in apoptosis and inflammation were mainly affected in pathological hypertrophy [8], [11], [12], [13]. However, there are significant differences in cardiac physiology between rodents and large laboratory animals [14], [15], such as swine, which in many aspects mimics human cardiac physiology and hemodynamics more closely than rodents [14]. Thus, similar to humans, prolonged exercise-training in swine produces cardiac hypertrophy [16], [17], increases coronary blood flow capacity [18], increases stroke volume [17], and decreases heart rate [16]. However, compared to murine models, studies in large animals into the molecular mechanisms of physiological hypertrophy, and the differences with pathological hypertrophy are scarce.

We hypothesized that LV hypertrophy results from genetic reprogramming through a limited number of transcription factors. We have previously reported on the changes in gene expression profiles, and implicated transcription factors, that underlie hypertrophy of the left ventricle (LV) during recovery from a MI [19]. Here, we studied the molecular pathways that underlie physiological hypertrophy and the TFs that drive these changes in LV hypertrophy caused by 3–6 weeks of dynamic exercise-training in swine. Using the changes in gene expression profiles, we explored the differentially expressed genes for common transcription factor binding sites (TFBSs), and combined these data with protein/DNA array analysis of protein extracts prepared from LV nuclei.