Giuseppina Nicolini

Early long-term L-T3 replacement rescues mitochondria and prevents ischemic cardiac remodelling in rats

3,5,3′‐Levo‐triiodothyronine (L‐T3) is essential for DNA transcription, mitochondrial biogenesis and respiration, but its circulating levels rapidly decrease after myocardial infarction (MI). The main aim of our study was to test whether an early and sustained normalization of L‐T3 serum levels after MI exerts myocardial protective effects through a mitochondrial preservation. Seventy‐two hours after MI induced by anterior interventricular artery ligation, rats were infused with synthetic L‐T3 (1.2 μg/kg/day) or saline over 4 weeks. Compared to saline, L‐T3 infusion restored FT3 serum levels at euthyroid state (3.0 ± 0.2 versus 4.2 ± 0.3 pg/ml), improved left ventricular (LV) ejection fraction (39.5 ± 2.5 versus 65.5 ± 6.9%), preserved LV end‐systolic wall thickening in the peri‐infarct zone (6.34 ± 3.1 versus 33.7 ± 6.21%) and reduced LV infarct‐scar size by approximately 50% (all P < 0.05). Moreover, L‐T3 significantly increased angiogenesis and cell survival and enhanced the expression of nuclear‐encoded transcription factors involved in these processes. Finally, L‐T3 significantly increased the expression of factors involved in mitochondrial DNA transcription and biogenesis, such as hypoxic inducible factor‐1α, mitochondrial transcription factor A and peroxisome proliferator activated receptor γ coactivator‐1α, in the LV peri‐infarct zone. To further explore mechanisms of L‐T3 protective effects, we exposed isolated neonatal cardiomyocytes to H2O2 and found that L‐T3 rescued mitochondrial biogenesis and function and protected against cell death via a mitoKATP dependent pathway. Early and sustained physiological restoration of circulating L‐T3 levels after MI halves infarct scar size and prevents the progression towards heart failure. This beneficial effect is likely due to enhanced capillary formation and mitochondrial protection.

Triiodothyronine Prevents Cardiac Ischemia/Reperfusion Mitochondrial Impairment and Cell Loss by Regulating miR30a/p53 Axis

Mitochondrial dysfunctions critically affect cardiomyocyte survival during ischemia/reperfusion (I/R) injury. In this scenario p53 activates multiple signaling pathways that impair cardiac mitochondria and promote cell death. p53 is a validated target of miR-30 whose levels fall under ischemic conditions. Although triiodothyronine (T3) rescues post-ischemic mitochondrial activity and cell viability, no data are available on its role in the modulation of p53 signaling in I/R. Here we test the hypothesis that early T3 supplementation in rats inhibits the post I/R activation of p53 pro-death cascade through the maintenance of miRNA 30a expression. In our model, T3 infusion improves the recovery of post-ischemic cardiac performance. At the molecular level, the beneficial effect of T3 is associated with restored levels of miR-30a expression in the area at risk (AAR) that correspond to p53 mRNA downregulation. The concomitant decrease in p53 protein content reduces Bax expression and limits mitochondrial membrane depolarization resulting in preserved mitochondrial function and decreased apoptosis and necrosis extent in the AAR. Also in primary cardiomyocyte culture of neonatal rats, T3 prevents both miR-30a downregulation and p53 raise induced by hypoxia. The regulatory effect of T3 is greatly suppressed by miR-30a knockdown. Overall these data suggest a new mechanism of T3-mediated cardioprotection that is targeted to mitochondria and acts, at least in part, through the regulation of miR-30a/p53 axis.

New Insights into Mechanisms of Cardioprotection Mediated by Thyroid Hormones

Heart failure represents the final common outcome in cardiovascular diseases. Despite significant therapeutic advances, morbidity and mortality of heart failure remain unacceptably high. Heart failure is preceded and sustained by a process of structural remodeling of the entire cardiac tissue architecture. Prevention or limitation of cardiac remodeling in the early stages of the process is a crucial step in order to ameliorate patient prognosis. Acquisition of novel pathophysiological mechanisms of cardiac remodeling is therefore required to develop more efficacious therapeutic strategies. Among all neuroendocrine systems, thyroid hormone seems to play a major homeostatic role in cardiovascular system. In these years, accumulating evidence shows that the “low triiodothyronine” syndrome is a strong prognostic, independent predictor of death in patients affected by both acute and chronic heart disease. In experimental models of cardiac hypertrophy or myocardial infarction, alterations in the thyroid hormone signaling, concerning cardiac mitochondrion, cardiac interstitium, and vasculature, have been suggested to be related to heart dysfunction. The aim of this brief paper is to highlight new developments in understanding the cardioprotective role of thyroid hormone in reverting regulatory networks involved in adverse cardiac remodeling. Furthermore, new recent advances on the role of specific miRNAs in thyroid hormone regulation at mitochondrion and interstitial level are also discussed.

Proangiogenic Effect of TSH in Human Microvascular Endothelial Cells through Its Membrane Receptor

CONTEXT TSH, which acts via specific cell membrane TSH receptors (TSHR), is regarded as a thyroid-specific growth factor. Recently, the presence of TSHR has been reported in extrathyroid tissues, but the role of TSH in nonthyroid tissue is unknown. OBJECTIVE Our objective was to study the direct effect of TSH on angiogenesis in a human dermal microvascular endothelial cell line (HMEC-1). Parallel experiments were conducted with human primary cardiac microvascular endothelial cells (HMVEC-C). DESIGN TSHR in HMEC-1 was measured by immunofluorescence, Western blot, and RT-PCR and its functional activity by variation of intracellular cAMP concentrations. The expression of some angiogenic genes and angiogenic signaling pathways was also evaluated after TSH treatment. Assays of cell proliferation and capillary network formation on collagen or Matrigel were performed in HMEC-1 cells and HMVEC-C. RESULTS We showed the presence of TSHR in HMEC-1 cells. Increased intracellular cAMP concentrations after TSH treatment indicated the TSHR to be functional. TSH enhanced proliferation and stimulated capillary network formation in HMEC-1, whereas antibodies against vascular endothelial growth factor (VEGF) and TSHR abolished this effect. TSH increased AAMP, VEGF, and eNOS expression. TSH induced phosphorylation of protein kinase S6K1, whereas TSHR blocking antibodies inhibited the phosphorylation of the protein kinase S6K1. A similar effect of TSH on capillary network formation was observed in HMVEC-C. CONCLUSION Our findings provide strong evidence for a direct effect of TSH on angiogenesis through its receptor, via cAMP-mammalian target of rapamycin signaling and indicate that this effect is VEGF dependent.

Mitochondria as Key Targets of Cardioprotection in Cardiac Ischemic Disease: Role of Thyroid Hormone Triiodothyronine

Ischemic heart disease is the major cause of mortality and morbidity worldwide. Early reperfusion after acute myocardial ischemia has reduced short-term mortality, but it is also responsible for additional myocardial damage, which in the long run favors adverse cardiac remodeling and heart failure evolution. A growing body of experimental and clinical evidence show that the mitochondrion is an essential end effector of ischemia/reperfusion injury and a major trigger of cell death in the acute ischemic phase (up to 48–72 h after the insult), the subacute phase (from 72 h to 7–10 days) and chronic stage (from 10–14 days to one month after the insult). As such, in recent years scientific efforts have focused on mitochondria as a target for cardioprotective strategies in ischemic heart disease and cardiomyopathy. The present review discusses recent advances in this field, with special emphasis on the emerging role of the biologically active thyroid hormone triiodothyronine (T3).

Cardioprotection and thyroid hormones

The evolution of cardiac disease after an acute ischemic event depends on a complex and dynamic network of mechanisms alternating from ischemic damage due to acute coronary occlusion to reperfusion injury due to the adverse effects of coronary revascularization till post-ischemic remodeling. Cardioprotection is a new purpose of the therapeutic interventions in cardiology with the goal to reduce infarct size and thus prevent the progression toward heart failure after an acute ischemic event. In a complex biological system such as the human one, an effective cardioprotective strategy should diachronically target the network of cross-talking pathways underlying the disease progression. Thyroid system is strictly interconnected with heart homeostasis, and recent studies highlighted its role in cardioprotection, in particular through the preservation of mitochondrial function and morphology, the antifibrotic and proangiogenetic effect and also to the potential induction of cell regeneration and growth. The objective of this review was to highlight the cardioprotective role of triiodothyronine in the complexity of post-ischemic disease evolution.

LH, Progesterone, and TSH can Stimulate Aldosterone In Vitro: A Study on Normal Adrenal Cortex and Aldosterone Producing Adenoma

Endocrine factors different from ACTH or angiotensin II can stimulate aldosterone secretion and have a role in the pathophysiology of hyperaldosteronism. Aldosterone may increase in luteotropic/progestogenic and in hypothyroid states; LH and, occasionally, TSH receptors have been detected in normal adrenal cortex and aldosterone-producing adenoma. The aim of the study was to compare adrenal contents of LH and TSH receptors between normal cortex and aldosterone-producing adenoma and to evaluate the ability of LH, its product progesterone, and TSH to stimulate aldosterone secretion in vitro from primary adrenocortical cells. Surgical aldosterone-producing adenoma fragments from 19 patients and adrenal cortex fragments from 10 kidney donors were used for Western blotting and cell cultures. LH (n=26), TSH (n=19) and progesterone (n=8) receptor proteins were investigated; LH receptor-mRNA was also tested in 8 samples. Aldosterone responses in vitro to LH, progesterone, and TSH stimulation were assayed. LH and TSH receptors were more expressed in adenoma than normal cortex (p<0.01, p<0.05, respectively); progesterone receptor was observed in 6/8 samples. Aldosterone increased after in vitro stimulation with LH (5/12 adenoma, 1/7 normal cells), progesterone (4/5 adenoma, 5/6 normal cells), and TSH (3/5 adenoma and 3/5 normal cells). LH and TSH receptors were more expressed in aldosterone producing adenoma than normal adrenal cortex. LH, progesterone, and TSH can stimulate aldosterone in vitro. Similar mechanisms could participate in vivo in the aldosterone increase in lutheotropic, progestogenic, or hypothyroid states and may exist in both normal adrenal cortex and adenoma in responsive individuals.

Early and short-term triiodothyronine supplementation prevents adverse post-ischemic cardiac remodeling: role of transforming growth factor-β1 and anti-fibrotic miRNA signaling.

Activation of transforming growth factor (TGF)-β1 signaling in the ischemia/reperfusion (I/R) injured myocardium leads to dysregulation of miR-29-30-133, favoring the profibrotic process that leads to adverse cardiac remodeling (CR). We have previously shown that timely correction of the postischemic low-T3 syndrome (Low-T3S) exerts antifibrotic effects, but the underlying molecular players are still unknown. Here we hypothesize that a prompt, short-term infusion of T3 in a rat model of post I/R Low-T3S could hamper the early activation of the TGFβ1-dependent profibrotic cascade to confer long-lasting cardioprotection against adverse CR. Twenty-four hours after I/R, rats that developed the Low-T3S were randomly assigned to receive a 48-h infusion of 6 µg/kg/d T3 (I/R-L+T3) or saline (I/R-L) and sacrificed at 3 or 14 d post-I/R. Three days post-I/R, Low-T3S correction favored functional cardiac recovery. This effect was paralleled by a drop in TGFβ1 and increased miR-133a, miR-30c and miR-29c in the infarcted myocardium. Consistently, connective transforming growth factor (CTGF) and matrix metalloproteinase-2 (MMP-2), validated targets of the above miRNAs, were significantly reduced. Fourteen days post-I/R, the I/R-L+T3 rats presented a significant reduction of scar size with a better preservation of cardiac performance and LV chamber geometry. At this time, TGFβ1 and miR-29c levels were in the normal range in both groups, whereas miR-30c-133a, MMP-2 and CTGF remained significantly altered in the I/R group. In conclusion, the antifibrotic effect exerted by T3 in the early phase of postischemic wound healing triggers a persistent cardioprotective response that hampers the progression of heart dysfunction and adverse CR.

TSH induces co-localization of TSH receptor and Na/K-ATPase in human erythrocytes.

Thyroid stimulating hormone (TSH) binds to a specific TSH receptor (TSHR) which activates adenylate cyclase and increases cAMP levels in thyroidal cells. Recent studies have reported the presence of TSH receptor in several extra‐thyroidal cell types, including erythrocytes. We have previously suggested that TSH is able to influence the erythrocyte Na/K‐ATPase ouabain binding properties through a receptor mediated mechanism. The direct interaction of TSH receptor with the Na/K‐pump and a functional role of TSHR in erythrocytes was not demonstrated. The interaction of TSH receptor with Na/K‐pump and a TSHR functional role are not yet demonstrated in erythrocytes. In this study, we examined the interaction between the two receptors after TSH treatment using immunofluorescence coupled to confocal microscopy and a co‐immunoprecipitation technique. The cAMP dependent signalling after TSH treatment was measured to verify TSHR functionality. We found that TSH receptor and Na/K‐ATPase are localized on the membranes of both erythrocytes and erythrocyte ghosts; TSH receptor responds to TSH treatment by increasing intracellular cAMP levels from two to tenfold. In ghost membranes TSH treatment enhances up to three fold co‐localization of TSHR with Na/K‐ATPase and co‐immunoprecipitation confirms their direct physical interaction. In conclusion our results are compatible with the existence, in erythrocytes, of a functional TSHR that interacts with Na/K‐ATPase after TSH treatment, thus suggesting a novel cell signalling pathway, potentially active in local circulatory control. Copyright

Low T3 State Is Correlated with Cardiac Mitochondrial Impairments after Ischemia Reperfusion Injury: Evidence from a Proteomic Approach

Mitochondria are major determinants of cell fate in ischemia/reperfusion injury (IR) and common effectors of cardio-protective strategies in cardiac ischemic disease. Thyroid hormone homeostasis critically affects mitochondrial function and energy production. Since a low T3 state (LT3S) is frequently observed in the post infarction setting, the study was aimed to investigate the relationship between 72 h post IR T3 levels and both the cardiac function and the mitochondrial proteome in a rat model of IR. The low T3 group exhibits the most compromised cardiac performance along with the worst mitochondrial activity. Accordingly, our results show a different remodeling of the mitochondrial proteome in the presence or absence of a LT3S, with alterations in groups of proteins that play a key role in energy metabolism, quality control and regulation of cell death pathways. Overall, our findings highlight a relationship between LT3S in the early post IR and poor cardiac and mitochondrial outcomes, and suggest a potential implication of thyroid hormone in the cardio-protection and tissue remodeling in ischemic disease.