Inhibition of CTRP9, a novel and cardiac-abundantly expressed cell survival molecule, by TNFα-initiated oxidative signaling contributes to exacerbated cardiac injury in diabetic mice

Abstract

Recently identified as adiponectin (APN) paralogs, C1q/TNF-related proteins (CTRPs) share similar metabolic regulatory functions as APN. The current study determined cardiac expression of CTRPs, their potential cardioprotective function, and investigated whether and how diabetes may regulate cardiac CTRP expression. Several CTRPs are expressed in the heart at levels significantly greater than APN. Most notably, cardiac expression of CTRP9, the closest paralog of APN, exceeds APN by >100-fold. Cardiac CTRP9 expression was significantly reduced in high-fat diet-induced diabetic mice. In H9c2 cells, tumor necrosis factor-alpha (TNF-α) strongly inhibited CTRP9 expression (>60%), and significantly reduced peroxisome proliferator activated receptor-gamma (PPARγ), a known transcription factor promoting adiponectin expression. The inhibitory effect of TNF-α on PPARγ and CTRP9 was reversed by Tiron or rosiglitazone. CTRP9 knockdown significantly enhanced, whereas CTRP9 overexpression significantly attenuated simulated ischemia/reperfusion injury in H9c2 cells. In vivo CTRP9 administration to diabetic mice significantly attenuated NADPH oxidase expression and superoxide generation, reduced infarct size, and improved cardiac function. To the best of our knowledge, this is the first study providing evidence that downregulation of CTRP9, an abundantly expressed and novel cell survival molecule in the heart, by TNF-α-initiated oxidative PPARγ suppression contributes to exacerbated diabetic cardiac injury. Preservation of CTRP9 expression or augmentation of CTRP9-initiated signaling mechanisms may be the potential avenues for ameliorating ischemic diabetic cardiac injury.

Fig. 1

CTRP9 mRNA and protein are highly expressed in adult mouse heart. a The mRNA levels of APN and CTRPs in adult mouse heart. b The mRNA level of CTRP9 and APN in adipose tissue. c The mRNA level of CTRP9 and APN in cardiac tissue. d CTRP9 protein in adipose tissue, plasma and cardiac tissue determined by representative Western blots. e Detection of CTRP9 in culture medium of H9c2 cells (24 h) and adult cardiomyocytes (12 h). n = 5–8 per group

Fig. 2

CTRP9 expression is reduced in cardiac tissue from high-fat diet (HFD)-induced diabetic mice. a Fasting plasma glucose, b fasting plasma insulin and c HOMA Score before (0) and after 8 weeks normal (ND) or high-fat diet (HFD). d CTRP9 mRNA levels in cardiac tissue from HFD-fed groups. e CTRP9 protein levels of cardiac tissue from HFD-fed groups determined by representative Western blots. n = 5–8 heart per group. *p < 0.05, **p < 0.01 versus HFD 0 week group

Fig. 3

TNF-α significantly downregulates CTRP9 expression. a CTRP9 mRNA in H9c2 cells treated with TNF-α (10 ng/ml), high glucose (HG, 33 mM), and high lipid (HL, palmitate, 300 μM). The relative expression levels of CTRP9 transcript were normalized to the control group. b CTRP9 protein in H9c2 cells treated with TNF-α, HG, and HL. n = 8–10 per group. *p < 0.05, **p < 0.01 versus control group

Fig. 4

Antioxidant treatment reverses inhibitory effect of TNF-α on CTRP9 and PPARγ expression. a TNF-α inhibited CTRP9 mRNA expression via oxidative stress in H9c2 cells. b TNF-α inhibited CTRP9 protein via augmented oxidative stress. c TNF-α inhibited PPARγ expression via oxidative stress. n = 8–10 per group. *p < 0.05, **p < 0.01 versus control group

Fig. 5

TNF-α inhibits cardiac CTRP9 expression via decreased PPARγ. a PPARγ agonist rosiglitazone (RSG, 10 μM) reversed the effect of TNF-α upon CTRP9 mRNA levels in H9c2 cells. b RSG reversed the effect of TNF-α upon CTRP9 protein expression in H9c2 cells. n = 8–10 per group. *p < 0.05, **p < 0.01 versus control group. ^##p < 0.01 versus TNF-α group

Fig. 6

siRNA-mediated CTRP9 knockdown significantly increases simulated ischemia/reperfusion (SI/R) injury in H9c2 cells. a The effect of CTRP9-siRNA upon CTRP9 protein levels in H9c2 cells. b The effect of CTRP9 knockdown upon caspase-3 activity in SI/R H9c2 cells. c The effect of CTRP9 knockdown upon superoxide production in SI/R H9c2 cells. d The effect of CTRP9 overexpression upon caspase-3 activity in SI/R H9c2 cells. n = 8–10 per group. **p < 0.01 vs. sham group. ^##p < 0.01 versus SI/R scramble group (b) or versus SI/R group (c, d)

Fig. 7

CTRP9 reduces myocardial ischemia/reperfusion (MI/R) injury in high-fat diet (HFD)-induced diabetic mice. CTRP9 reduced cardiomyocyte apoptosis of HFD mice subjected to MI/R. Cardiomyocyte apoptosis determined by a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and b Caspase-3 activity. c CTRP9 reduced infarct size determined by Evans blue/2,3,5-triphenyl tetrazolium chloride double-staining technique in HFD mice subjected to MI/R. n = 5–8 hearts per group. **p < 0.01 vs. sham. ^##p < 0.01 versus vehicle group

Fig. 8

CTRP9 improves cardiac function in HFD-induced diabetic mice post myocardial ischemia/reperfusion (MI/R). a Left ventricular ejection fraction (LVEF), determined by echocardiography. b left ventricular end-diastolic pressure (LVEDP), determined by hemodynamic measurements. c Maximum rate of left ventricular pressure change (LV dP/dt_max), determined by hemodynamic measurements. n = 5–8 hearts per group. *p < 0.05, **p < 0.01 versus sham group, ^##p < 0.01 versus vehicle group

Fig. 9

CTRP9 reduces oxidative stress in ischemic/reperfused heart. a CTRP9 reduced gp91^phox determined by representative Western blots in HFD mice hearts subjected to MI/R. b CTRP9 reduced superoxide anion production of HFD mice subjected to MI/R. n = 5–8 hearts per group. **p < 0.01 versus sham. ^##p < 0.01 versus vehicle group