requires an intact cardiac thyrotropin-releasing hormone (TRH) system to induce cardiac hypertrophy in mouse. Yjmcc (2018),
Animal models are an interesting tool to evaluate future consequences of early nutritional insults. We described in adult mice that: leptin induces cardiac cTRH, leading to cardiac damage regardless of blood pressure in hyperleptinemic Agouti. Conversely, cTRH in the ob/ob lacking leptin is similar to control and even do not have Left Ventricular Hypertrophy (LVH). Moreover, leptin restitution induces cTRH and later LVH. Also, in C57BL/6 a 3-week subanorexigenic leptin dose induced cTRH and sequent LVH. All this suggests that heart damage in obesity depends on the novel leptin-cTRH pathway. Plus, Leptin directly induces TRH in cell culture (Aisicovich 2019, AHA) To further investigate the impact of early dietary interventions in childhood, we studied rat litter size reduction, a model with a reported increase in body weight, fat, leptin, insulin and cholesterol levels (Stefanidis A, 2012) We hypothesize that leptin rise increases cTRH leading to cardiac damage, independently of hypertension. On postnatal day 4, Wistar rat litters of 11 pups at birth were adjusted to 4 male pups (overnourished ON) or 11 (control C). At weaning, we individualized rats with free access to water and standard diet up to 7 weeks old. The rise in body weight, food consumption, and fat in ON vs C (p<0.02 n=10 ANOVA) confirmed the model. There weren’t any blood pressure differences between groups, still LVH index increased (heart weight/tibia length) (p< 0.01 n=6) in ON vs C. As expected, in ON the higher leptin increased cTRH mRNA (p< 0.01 n=6) as protein [WB (p=0.05 n=3) IHQ (p<0.04 n=6)] vs C. According to cTRH actions, hypertrophic and fibrotic markers TGF-B and Collagens 1 and 3 mRNA increased (p<0.01, n=6) confirmed by IHQ and Sirius red staining (p< 0.01 n=6) Also, echocardiogram showed increases in LV end-diastolic (0.26±0.08 vs 0.61±0.07 ml p <0.01) and end-systolic (0.02±0.02 vs 0.11±0.01 ml p <0.01) volumes, and decreases in LV ejection fraction (94.5±2.2 vs 83.3±1.9 % p <0.01) and SF (54.4±2.6 vs 45.0±2.3 % p <0.02) This could be restored by cTRH inhibition as demonstrated in the cardiac infarct model (Schuman ML, 2021) Our results point out that early overnutrition induces evident cardiac damage already at 7 weeks, independently of blood pressure and mostly driven by the leptin-TRH pathway.
Cardiac TRH (cTRH) induces LVH and fibrosis. The adiponectin Leptin induces TRH in CNS. We hypothesized that in obesity, the increase of cTRH induced by hyperleptinemia is responsible for the LVH, mostly attributed to pressure load. We studied obese hyperleptinemic with mild hypertension Agouti mice, and found LVH with higher (p<0.05) cTRH, fibrotic (I/III col,TGFβ) and hypertrophic (BNP,βMHC) marker genes vs lean (BL/6J). We normalized pressure in Agouti mice with hydroclorothiazide (20 mg/kg/day) since weaning (n=9) vs non treated mice. Still both developed (p<0.05) LVH, higher cTRH, fibrotic and hypertrophic marker genes expression, suggesting that LVH would not be due to hypertension. Opposite to Agouti model, we used obese normotensive ob/ob mice, lacking Leptin expression, and without LVH despite their obesity. We treated them with Leptin (80 ug/kg/day) or saline since weaning for 15 days. Only the treated group developed LVH (p< 0.05, n=7) demonstrating that it is Leptin dependant. Also we found increase (p<0.05) in cTRH, col III, TGFβ and BNP suggesting that leptin-TRH interaction is required in obesity-induced LVH. Finally, we evaluated whether Leptin administration, (n=6, 10 ug/kg/day) could induce hypertrophy in lean C57 mice with and withouth native cTRH system, by previous siRNA injection (5ug). Diuretic was given to Leptin groups, to avoid its hypertensive effect. Leptin induced cTRH expression, not observed in the groups with siRNA-TRH (p<0.05).This probably induce fibrotic and hypertrophic markers, demonstrating that the novel interaction is functional also in mildy hyperleptinemia at normal weight status. To confirm TRH's Leptin induction we used cardiomyocytes cell line H9C2 (n=6) stimulated with Leptin (10 and 100 ng/ml). TRH expression and precursor content increased (p<0.05) post Leptin treatment with both concentrations. Moreover in primary cardiomyocytes culture from neonate rats, Leptin stimulus (80 ng/ml, 24 hs) increased (p<0.05) TRH content vs controls, confirming the direct TRH-Leptin induction in heart cells. We described a novel Leptin-cTRH pathway which explains Leptin-induced LVH, highly likely since early stages.
We have demonstrated TRH hyperactivity in the hypertrophied Left ventricle (LV) of SHR. Its specific inhibition attenuates hypertrophy development in spite of the significant higher pressure observed (Schuman M, Hypertension 2011). LV-TRH over-expression induces several features of the hypertrophied heart, including increases in the apoptotic index Bax/Bcl2, and the activated caspase 3 observed by immunohystochemistry (Schuman M, AJPH&C 2014). In addition, we have found that TRH expression was induced by D in primary cultures of cardiac cells. Based on these results we hypothesized that cTRH could participate in the D cardiotoxicity effects. Indeed, we used C57 adult males (n=10) with a single D or saline injection (200uL,10 mg/kg ip) which previously (24 h, under anesthesia) received an intracardiac injection of a specific TRH-siRNA to inhibit LV-TRH expression or scrambled Con-siRNA. Mice were sacrificed 2,4 and 7 days post D injection and body weight was measured. Genes expression was measured by real time PCR and protein by immunohystochemistry (ANOVA and Tukey test). Body weight showed a mild but not significant decrease in D treated animals. D significantly increased TRH gene expression and TRH protein content reaching the maximum at 7 days post injection (2d: 145%, 4d:190% and 7d: 250%) (p& 0.05), which were not observed in the groups with D+TRH-siRNA indicating the effectiveness of the specific TRH inhibition. Also at this time TRH inhibition attenuates (p&0.05) D-induced increase in the apoptotic index Bax/Bcl2 and the augmented activated caspase 3 content pointing out the participation of the cardiac TRH in the D-induced apoptosis. Similar results were observed with hypertrophic and fibrotic markers gene expression (BNP, BMHC and col III) which showed a significant increase (p& 0.05) only in the groups with D and the intact TRH system (D+Con-siRNA). Fibrosis results were confirmed by Sirius Red and Masson techniques. On the whole, we demonstrated for the first time that LV-TRH system is required for both, Doxorrubicin induction of apoptosis and consecutively hypertrophy and fibrosis in the mouse heart. Even more, we found that cTRH inhibition attenuates doxorrubicin induced damage suggesting a novel mechanisms in the cardiotoxicity injury.
Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.
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