A systems genetics approach identifies Trp53inp2 as a link between cardiomyocyte glucose utilization and hypertrophic response

Abstract

Cardiac failure has been widely associated with an increase in glucose utilization. The aim of our study was to identify factors that mechanistically bridge this link between hyperglycemia and heart failure. Here, we screened the Hybrid Mouse Diversity Panel (HMDP) for substrate-specific cardiomyocyte candidates based on heart transcriptional profile and circulating nutrients. Next, we utilized an in vitro model of rat cardiomyocytes to demonstrate that the gene expression changes were in direct response to substrate abundance. After overlaying candidates of interest with a separate HMDP study evaluating isoproterenol-induced heart failure, we chose to focus on the gene Trp53inp2 as a cardiomyocyte glucose utilization-specific factor. Trp53inp2 gene knockdown in rat cardiomyocytes reduced expression and protein abundance of key glycolytic enzymes. This resulted in reduction of both glucose uptake and glycogen content in cardiomyocytes stimulated with isoproterenol. Furthermore, this reduction effectively blunted the capacity of glucose and isoprotereonol to synergistically induce hypertrophic gene expression and cell size expansion. We conclude that Trp53inp2 serves as regulator of cardiomyocyte glycolytic activity and can consequently regulate hypertrophic response in the context of elevated glucose content.NEW & NOTEWORTHY Here, we apply a novel method for screening transcripts based on a substrate-specific expression pattern to identify Trp53inp2 as an induced cardiomyocyte glucose utilization factor. We further show that reducing expression of the gene could effectively blunt hypertrophic response in the context of elevated glucose content.

Keywords: Trp53inp2; glucose; hypertrophy; metabolic shift.

Fig. 1.

Framework for identification of glucose utilization-regulated transcripts. A: process of identification of Trp52inp2 as a transcript regulated by glucose utilization specifically. B: heat map for all 17,961 heart probes in 91 strains showing the correlation coefficient against circulating free fatty acids, glucose, and triglycerides. HMDP, Hybrid Mouse Diversity Panel.

Fig. 2.

Validation of candidate genes being regulated by substrate utilization. A–E: rat neonatal cardiomyocytes were treated for 16 h in basal media supplemented with the indicated amounts of glucose and probed for expression of Cdc42 (A), Tmem50 (B), Fkbp3 (C), Ppp2r4 (D), or Trp53inp2 (E). F–J: the same experimental design, except that media were supplemented with various amounts of palmitic acid conjugated to BSA; n = 6/group. All samples were normalized to B2M expression and expressed as means ± SE. ^aP < 0.05; ^abP < 0.001.

Fig. 3.

Trp53inp2 reduction suppresses expression of key glycolytic enzymes. Neonatal rat cardiomyocytes were transfected with either scrambled siRNA vector or siRNA targeting Trp52inp2. After 24 h, the cells were harvested and assessed for the mRNA expression of Trp53inp2 (A), hexokinase 2 (HK2; B), phosphofructokinase (PFK; C), and carnitine palmitoyltransferase (CPT1; D); n = 6/group. All samples were normalized to B2M expression and expressed as means ± SE. All gene expression quantifications of metabolic enzymes are accompanied by Western blots and corresponding quantification (E–G) of the same targets normalized to tubulin (n = 5). ^aP < 0.05; ^abP < 0.001 comparisons done between Iso and control groups at matched glucose contents. All samples are expressed as means ± SE.

Fig. 4.

Trp53inp2 gene is regulated by cardiac injury. A–C: Hybrid Mouse Diversity Panel (HMDP) assessing response to isoproterenol (Iso) infusion showed substantial variation among inbred strains (x-axis) in hypertrophic traits of left ventricle (LV) mass (A), fractional shortening (B), and Trp53inp2 gene expression (C). D: as a consequence of variation, Trp53inp2 showed striking correlations with relevant clinical traits (left). Quantitative PCR measurement of Trp53inp2 gene expression in model of carotid artery ligation comparing infarct vs. uninjured areas is shown; n = 4/group (right). All samples were normalized to B2M expression and expressed as means ± SE. ^aP < 0.05.

Fig. 5.

Trp53inp2 regulates isoproterenol (Iso)-induced glucose utilization. A: rat cardiomyocytes were treated with control (PBS) or 60 µM Iso in indicated concentrations of glucose and qPCR evaluated for Trp53inp2 expression. Samples were normalized to B2M. B and C: cardiomyocytes containing Trp53inp2 or control (cntrl) siRNA were evaluated for glucose uptake (B) and glycogen content (C) in both basal and Iso treatment (60 µM). Values were normalized to protein content; n = 10/group. All samples expressed as means ± SE. ^aP < 0.05; ^abP < 0.001. Veh, vehicle; KD, knockdown; AU, arbitrary units.

Fig. 6.

Reduction of Trp53inp2 blunts the synergistic hypertrophic action of glucose and isoproterenol (Iso). A–C: cells were transfected with scrambled control (open bars) or siRNA targeting Trp53inp2 (gray bars) and then treated with PBS or 60 μM Iso in either 5 or 25 mM glucose. Samples were then quantitative PCR-interrogated for expression of hypertrophic markers NppA (A), NppB (B), and phospholamban (Pln; C); n = 4. All samples were normalized to B2M. D–G: the same conditions as above, except brefeldin A was added to prevent secretion to immunoblot and quantify the same markers (n = 4). H and I: cells were subjected to the same conditions as above and then evaluated for changes in cell size, shown as quantification (H) and representative images (I); n = 15. All samples expressed as means ± SE. ^aP < 0.05; ^abP < 0.001.