Low THRB (thyroid hormone receptor beta) Promoter Methylation Levels in Peripheral Blood Leukocytes Induced By Systematic Inflammation Are Involved in Low Thyroid Hormone Function in Metabolic Syndrome.

[Figure: see text].

Triiodothyronine maintains cardiac transverse-tubule structure and function.

Subclinical hypothyroidism and low T3 syndrome are commonly associated with an increased risk of cardiovascular disease (CVD) and mortality. We examined effects of T3 on T-tubule (TT) structures, Ca2+ mobilization and contractility, and clustering of dyadic proteins. Thyroid hormone (TH) deficiency was induced in adult female rats by propyl-thiouracil (PTU; 0.025%) treatment for 8 weeks. Rats were then randomized to continued PTU or triiodo-L-thyronine (T3; 10 µg/kg/d) treatment for 2 weeks (PTU + T3). After in vivo echocardiographic and hemodynamic recordings, cardiomyocytes (CM) were isolated to record Ca2+ transients and contractility. TT organization was assessed by confocal microscopy, and STORM images were captured to measure ryanodine receptor (RyR2) cluster number and size, and L-type Ca2+ channel (LTCC, Cav1.2) co-localization. Expressed genes including two integral TT proteins, junctophilin-2 (Jph-2) and bridging integrator-1 (BIN1), were analyzed in left ventricular (LV) tissues and cultured CM using qPCR and RNA sequencing. The T3 dosage used normalized serum T3, and reversed adverse effects of TH deficiency on in vivo measures of cardiac function. Recordings of isolated CM indicated that T3 increased rates of Ca2+ release and re-uptake, resulting in increased velocities of sarcomere shortening and re-lengthening. TT periodicity was significantly decreased, with reduced transverse tubules but increased longitudinal tubules in TH-deficient CMs and LV tissue, and these structures were normalized by T3 treatment. Analysis of STORM data of PTU myocytes showed decreased RyR2 cluster numbers and RyR localizations within each cluster without significant changes in Cav1.2 localizations within RyR clusters. T3 treatment normalized RyR2 cluster size and number. qPCR and RNAseq analyses of LV and cultured CM showed that Jph2 expression was T3-responsive, and its increase with treatment may explain improved TT organization and RyR-LTCC coupling.

Ignoring a basic pathophysiological mechanism of heart failure progression will not make it go away.

A link between heart failure (HF) and low thyroid hormone (TH) function has been known for over a century. Nonetheless, there is a general belief that TH treatment of patients with HF may not be worth the risk. This is largely based on two clinical trials where heart patients were treated with excessive doses of TH analogs, not actual THs. Further complicating the matter is the fact that normalization of THs in noncardiac patients can often be challenging. This issue is not going away as noted by a steady increase in TH-HF citations in recent years. In this article, we discuss what we know and how we may move the field forward.

Cardioprotection by triiodothyronine following caloric restriction via long noncoding RNAs.

Severe caloric-restriction compromises thyroid hormone (TH) status, apparently to save energy and proteins for enduring stress stimulus. However, a persistent decrease in TH levels may compromise heart function. We hypothesized that supplementation of low dose active TH or targeting hypoxia-inducible factor-1-alpha, HIF-1α (a strong activator of deiodinase enzyme that degrades peripheral active THs) will prevent deterioration of cardiac performance. Adult mice were subjected to acute fasting based on institutional animal protocols with ad libitum access to water. The following groups were studied: Control mice with free access to food; severe caloric restriction fasting only group; Fasting with Triiodo-l-Thyronine (T3); Fasting with HIF-1α inhibitor (BAY). Cardiac hemodynamic and electrophysiological studies were performed and role of long noncoding RNAs were explored. Following severe caloric-restriction, we found that body weights, and heart weights to a partial extent, were decreased. Low-dose T3 treatment attenuated left ventricular hemodynamic impairment in indices of cardiac contractility and relaxation. In electrophysiology studies, fasting mice developed atrial tachyarrhythmias upon induction. This reverted to control levels following T3 treatment. There was a significant increase in atrioventricular conduction time and significant decrease in heart rate following fasting. Both these changes were attenuated following T3 treatment. Furthermore, BAY partially improved hemodynamics. Compared to the severe caloric-restriction group, both T3 and BAY reduced MALAT1 and GAS5 long noncoding RNA expression. These new findings indicate that T3 and BAY protect from cardiac decompensation secondary to acute severe caloric-restriction partly mediated by long noncoding RNAs.

Chronic Dantrolene Treatment Does Not Affect Hypertension, but Attenuates Sympathetic Stimulation Enhanced Atrial Fibrillation Inducibility in SHR.

BACKGROUND: Ryanodine receptor (RyR) dysfunction in skeletal muscle (RyR1) leads to malignant hyperthermia, and in cardiac muscle (RyR2) triggers cardiac arrhythmias. We hypothesized that RyR dysfunction in vascular smooth muscle could increase vascular resistance and hypertension, and may contribute to increased atrial fibrillation (AF) in hypertension. Thus, stabilizing RyR function with chronic dantrolene treatment may attenuate hypertension and AF inducibility in spontaneously hypertensive rats (SHR). METHODS: Male SHR (16 weeks old) were randomized into vehicle- (n = 10) and dantrolene-treated (10 mg/kg/day, n = 10) groups for 4 weeks. Wistar Kyoto (WKY, n = 11) rats served as controls. Blood pressures (BP) were monitored before and during the 4-week treatment. After 4-week treatment, direct BP, echocardiography, and hemodynamics were recorded. AF inducibility tests were performed in vivo at baseline and repeated under sympathetic stimulation (SS). RESULTS: Compared with WKY, SHR had significantly higher BP throughout the experimental period. Dantrolene treatment had no effect on BP levels in SHR (final systolic BP 212 ± 9 mm Hg in vehicle group vs. 208 ± 16 mm Hg in dantrolene group, P > 0.05). AF inducibility was very low and not significantly different between 5-month-old WKY and SHR at baseline. However, under SS, AF inducibility and duration were significantly increased in SHR (20% in WKY vs. 60% in SHR-vehicle, P<0.05). Dantrolene treatment significantly attenuated AF inducibility under SS in SHR (60% in vehicle vs. 20% in dantrolene, P < 0.05). CONCLUSIONS: Stabilizing RyR with chronic dantrolene treatment does not affect hypertension development in SHR. SHR has increased vulnerability to AF induction under SS, which can be attenuated with dantrolene treatment.

Chronic dantrolene treatment attenuates cardiac dysfunction and reduces atrial fibrillation inducibility in a rat myocardial infarction heart failure model.

BACKGROUND: Cardiac ryanodine receptor 2 (RyR2) dysfunction and elevated diastolic Ca2+ leak have been linked to arrhythmogenesis not only in inherited arrhythmia syndromes but also in acquired forms of heart disease including heart failure (HF) and atrial fibrillation (AF). Thus, stabilizing RyR2 may exert therapeutic effects in these conditions. OBJECTIVE: The purpose of this study was to investigate the effects of stabilizing RyR2 with chronic dantrolene treatment on HF development and AF inducibility in a myocardial infarction (MI)-induced HF model in rats. METHODS: MI was induced in adult Sprague-Dawley rats by ligation of the left anterior descending coronary artery. Two weeks after MI surgery, rats with large MI (≥40%) were randomly assigned to MI-vehicle (n = 14) or MI-dantrolene (10 mg/kg/d; n = 13) groups. Sham-surgery rats (n = 7) served as controls. RESULTS: Compared to the MI-vehicle group, 4-week dantrolene treatment significantly improved cardiac function, with increased left ventricular (LV) fractional shortening (19.48% ± 3.61% vs 15.43% ± 2.65%; P <.01), and decreased LV end-diastolic pressure (12.58 ± 8.52 mm Hg vs 21.91 ± 7.25 mm Hg; P <.01), left atrial diameter (4.97 ± 0.75 mm vs 6.09 ± 1.53 mm; P <.05), and fibrosis content (6.42% ± 0.78% vs 9.76% ± 2.25%; P <.001). Dantrolene significantly decreased AF inducibility (69% in MI-vehicle vs 23% in MI-dantrolene; P <.05). Dantrolene treatment was associated with reduced RyR2 phosphorylation and favorably altered gene expression involving ion channels, sympathetic signaling, oxidative stress, and inflammatory markers. CONCLUSION: Chronic dantrolene treatment attenuated LV dysfunction and reduced AF inducibility, which was associated with decreased RyR2 phosphorylation and normalization of many adverse changes in gene expression. Thus, stabilizing RyR2 with chronic dantrolene treatment is a promising novel strategy for decreasing AF in HF.

Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment.

Pre-clinical animal studies have shown that triiodothyronine (T3) replacement therapy improves cardiac contractile function after myocardial infarction (MI). We hypothesized that T3 treatment could prevent adverse post-infarction cardiomyocyte remodeling by maintaining transverse-tubule (TT) structures, thus improving calcium dynamics and contractility. METHODS: Myocardial infarction (MI) or sham surgeries were performed on female Sprague-Dawley rats (aged 12 wks), followed by treatment with T3 (5µg/kg/d) or vehicle in drinking water for 16 wks (n = 10-11/group). After in vivo echocardiographic and hemodynamic analyses, left ventricular myocytes were isolated by collagenase digestion and simultaneous calcium and contractile transients in single cardiomyocytes were recorded using IonOptix imaging. Live cardiomyocytes were stained with AlexaFluor-488 conjugated wheat germ agglutinin (WGA-488) or di-8-ANEPPS, and multiple z-stack images per cell were captured by confocal microscopy for analysis of TT organization. RTqPCR and immunoblot approaches determined expression of TT proteins. RESULTS: Echocardiography and in vivo hemodynamic measurements showed significant improvements in systolic and diastolic function in T3- vs vehicle-treated MI rats. Isolated cardiomyocyte analysis showed significant dysfunction in measurements of myocyte relengthening in MI hearts, and improvements with T3 treatment: max relengthening velocity (Vmax, um/s), 2.984 ± 1.410 vs 1.593 ± 0.325, p < 0.05 and time to Vmax (sec), 0.233 ± 0.037 vs 0.314 ± 0.019, p < 0.001; MI + T3 vs MI + Veh, respectively. Time to peak contraction was shortened by T3 treatment (0.161 ± 0.021 vs 0.197 ± 0.011 s., p < 0.01; MI + T3 vs MI + Veh, respectively). Analysis of TT periodicity of WGA- or ANEPPS-stained cardiomyocytes indicated significant TT disorganization in MI myocytes and improvement with T3 treatment (transverse-oriented tubules (TE%): 9.07 ± 0.39 sham, 6.94 ± 0.67 MI + Veh and 8.99 ± 0.38 MI + T3; sham vs MI + Veh, p < 0.001; MI + Veh vs MI + T3, p < 0.01). Quantitative RT-PCR showed that reduced expression of BIN1 (Bridging integrator-1), Jph2 (junctophilin-2), RyR2 (ryanodine receptor) and Cav1.2 (L-type calcium channel) in the failing myocardium were increased by T3 and immunoblot analysis further supporting a potential T3 effect on the TT-associated proteins, BIN1 and Jph2. In conclusion, low dose T3 treatment initiated immediately after myocardial infarction attenuated adverse TT remodeling, improved calcium dynamics and contractility, thus supporting the potential therapeutic utility of T3 treatment in heart failure.

Triiodothyronine Reduces Vascular Dysfunction Associated with Hypertension by Attenuating Protein Kinase G/Vasodilator-Stimulated Phosphoprotein Signaling.

Vascular dysfunction associated with hypertension comprises hypercontractility and impaired vasodilation. We have previously demonstrated that triiodothyronine (T3), the active form of thyroid hormone, has vasodilatory effects acting through rapid onset mechanisms. In the present study, we examined whether T3 mitigates vascular dysfunction associated with hypertension. To test the direct effects of T3 in hypertensive vessels, aortas from female Dahl salt-sensitive (Dahl SS) rats fed a high-salt diet (8% NaCl, HS group) and their age-matched controls fed a standard low-salt diet (0.3% NaCl, LS group) for 16 weeks were isolated and used in ex vivo vascular reactivity studies. We confirmed that the HS group exhibited a higher systolic blood pressure in comparison with the control LS group and displayed aortic remodeling. Aortas from both groups were pretreated with T3 (0.1 µM) for 30 minutes at 37°C in a 5% CO2 incubator before functional vascular studies. T3 treatment significantly attenuated hypercontractility and improved impaired endothelium-dependent vasodilation in aortas from the HS group. These vascular improvements in response to T3 were accompanied by increased phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239, a vasodilatory factor of the cGMP-dependent protein kinase (PKG)/VASP signaling pathway in vascular smooth muscle cells. Moreover, increased production of reactive oxygen species in aortas from the HS group were significantly reduced by T3, suggesting a potential antioxidant effect of T3 in the vasculature. These results demonstrate that T3 can mitigate hypertension-related vascular dysfunction through the VASP signaling pathway and by reducing vascular ROS production. SIGNIFICANCE STATEMENT: This study demonstrates that triiodothyronine (T3) directly acts on vascular tone and has a beneficial effect in hypertension-induced vascular dysfunction. T3 augmented vasodilation and diminished vasoconstriction in blood vessels from hypertensive rats in association with activation of the protein kinase G/vasodilator-stimulated phosphoprotein signaling pathway that activates vascular relaxation and exerted an antioxidant effect. Collectively, these results show that T3 is a potential vasoprotective agent with rapid action on hypertension-related vascular dysfunction.

Western diet triggers Toll-like receptor 4 signaling-induced endothelial dysfunction in female Wistar rats.

Overconsumption of a diet rich in fat and carbohydrates, called the Western diet, is a major contributor to the global epidemic of cardiovascular disease. Despite previously documented cardiovascular protection exhibited in female rats, this safeguard may be lost under certain metabolic stressors. We hypothesized that female Wistar rats challenged by a Western diet composed of 21% fat and 50% carbohydrate (34.1% sucrose) for 17 wk would develop endothelial dysfunction via endothelial Toll-like receptor 4 (TLR4) signaling. Western diet-fed female rats exhibited dysregulation of metabolism, revealing increased body weight and abdominal fat, decreased expression of adiponectin in white adipose tissue, glucose intolerance, and impaired insulin sensitivity. Western diet exposure increased hepatic triglycerides and cholesterol alongside hepatic steatosis, categorizing nonalcoholic fatty liver disease. Moreover, a Western diet negatively affected vascular function, revealing hypertension, impaired endothelium-dependent vasorelaxation, aortic remodeling, and increased reactive oxygen species (ROS) production. Aortic protein expression of TLR4 and its downstream proteins were markedly increased in the Western diet-fed group in association with elevated serum levels of free fatty acids. In vitro experiments were conducted to test whether free fatty acids contribute to vascular ROS overproduction via the TLR4 signaling pathway. Cultured endothelial cells were stimulated with palmitate in the presence of TAK-242, a TLR4 signaling inhibitor. Palmitate-induced overgeneration of ROS in endothelial cells was abolished in the presence of TAK-242. Our data show that a Western diet induced endothelial dysfunction in female rats and suggest that endothelial TLR4 signaling may play a key role in abolishing female cardiovascular protection. NEW & NOTEWORTHY A Western diet induced elevated levels of free fatty acids, produced nonalcoholic fatty liver disease, and provoked endothelial dysfunction in female rats in association with Toll-like receptor 4 signaling-mediated vascular reactive oxygen species production. Limited consumption of a Western diet in premenopausal women may decrease their risk of cardiovascular complications.

Free triiodothyronine level correlates with statin responsiveness in acute myocardial infarction.

BACKGROUND: Although thyroid hormone (TH) has important effects on lipid metabolism, the relationship between TH and statin responsiveness has never been investigated. We hypothesize that TH plays an important role in statin responsiveness in patients with acute myocardial infarction (AMI). METHODS: Consecutive 1091 hospitalized AMI patients in Fuwai hospital (Beijing, China) were enrolled into this current study. The study population was divided into three groups based on the intensity of statin treatment: low-intensity (n = 221), moderate-intensity (n = 712) and high-intensity (n = 158). Lipid levels were measured after statin therapy lasting for 10-14 days. The association between TH, lipid profile levels and achievement of low-density lipoprotein cholesterol (LDL-C) lowering goals was explored in patients with AMI on statin therapy. RESULTS: By general linear analysis, a significant linear trend between free triiodothyronine (FT3) and LDL-C level (linear coefficient r = -0.082, P = 0.001) and FT3 and total cholesterol (TC) level (r = -0.105, P = 0.031) was observed in the moderate-intensity statin group. A more apparent linear trend was detected in the high-intensity statin group (for LDL-C: r = -0.113, P = 0.005; for TC: r = -0.172, P = 0.029, respectively). However, no significant correlation was observed in the low-intensity statin group. Compared with the low-FT3 group (defined as FT3 < 1.79 pg/mL), the OR (95% CI) for attaining a LDL-C < 3.0mmol/L was found to be 2.217 (1.001-4.839) in the higher FT3 group (> 2.95 pg/mL). The OR (95% CI) for attaining the more intensive goal (LDL-C < 1.8mmol/L) was 2.836 (1.014-5.182). CONCLUSIONS: Our study reveals that variation in FT3 levels is related to the cholesterol-lowering responsiveness of statins in AMI patients. These findings suggest that low FT3 may be a factor responsible for lack of LDL-C goal attainment and patients' poor responsiveness to statin treatment.

Failing Hearts Are More Vulnerable to Sympathetic, but Not Vagal Stimulation-Induced, Atrial Fibrillation-Ameliorated with Dantrolene Treatment.

BACKGROUND: Both vagal (VS) and sympathetic (SS) stimulations can increase atrial fibrillation (AF) inducibility, with VS being known as more arrhythmogenic in normal hearts. Heart failure (HF) results in autonomic dysfunction (characterized by sympathetic activation and vagal withdrawal) and is associated with an increased AF incidence. This study investigated whether failing hearts, compared with normal control hearts, respond differently to autonomic stimulation-induced AF arrhythmogenesis and the effect of dantrolene on SS-enhanced AF in HF. METHODS AND RESULTS: A rat myocardial infarction (MI) HF model was used. In experiment 1, AF inducibility was compared in 9 MI-HF rats versus 10 sham-control animals at baseline, during VS, and during SS with isoproterenol infusion. In experiment 2, dantrolene treatment (n = 8) was compared with placebo-control (n = 9) on SS-induced AF inducibility in HF. Compared with the sham-control, baseline AF inducibility was higher in the MI-HF group. AF inducibility was augmented in both groups by autonomic stimulation. However, under VS the increased magnitude was less in the MI-HF group (49% ± 11% vs 80% ± 10%; P = .029), but under SS was significantly more (53% ± 8% vs 6% ± 7%; P < .001), compared with sham-control. Dantrolene significantly attenuated SS-enhanced AF in HF (69% ± 6% vs 29% ± 9%; P = .006). CONCLUSIONS: Failing hearts are less sensitive to VS, but more vulnerable to SS-induced AF compared with normal-control hearts. Dantrolene can significantly attenuate SS-enhanced AF in HF, indicating that cardiac ryanodine receptor dysfunction may play a critical role in SS-enhanced AF in HF, and stabilizing leaky ryanodine receptor with the use of dantrolene may be a new treatment option in this condition.

Comparison of Therapeutic Triiodothyronine Versus Metoprolol in the Treatment of Myocardial Infarction in Rats.

BACKGROUND: Beta blockers are standard therapy for myocardial infarction (MI). Preclinical studies have shown efficacy and safety of thyroid hormone (TH) treatment of cardiovascular disorders. Since THs interact with the sympathoadrenergic system, this study aimed to compare triiodothyronine (T3) and metoprolol (Met) in the treatment of rats with MI on pathophysiology and TH-adrenergic signaling. METHODS: Female Sprague-Dawley rats aged 12 weeks underwent left anterior descending coronary artery ligation (MI) or sham surgeries. T3 (5 µg/kg/day) or Met (100 mg/kg/day) was given in drinking water immediately after surgery for eight weeks. At the terminal of the experiments, the rats were subjected to morphological, functional, and molecular examination. RESULTS: T3 and Met significantly enhanced left ventricular contractility (left ventricular fractional shortening 21.37 ± 2.58% and 21.14 ± 3.71%, respectively) compared to untreated MI (17.88 ± 1.23%), and decreased the incidence of inducible atrial tachyarrhythmia by 87.5% and 62.5%, respectively. Although both treatments showed efficacy, T3 but not Met showed statistically significant improvements compared to MI in arrhythmia duration, left atrial diameter (T3 vs. MI 4.33 ± 0.63 vs. 5.65 ± 1.32 mm; p < 0.05), fibrosis (6.1 ± 0.6%, 6.6 ± 0.6% vs. 8.2 ± 0.7%, T3, Met vs. MI, respectively), and aortic vasorelaxation responsiveness to acetylcholine (pD2 6.97 ± 0.22, 6.83 ± 0.21 vs. 6.66 ± 0.22, T3, Met vs. MI, respectively). Quantitative polymerase chain reaction showed that T3 and Met attenuated expression of genes associated with inflammation and oxidative stress and restored expression of ion channels and contractile proteins. CONCLUSION: These results support comparable efficacy of T3 and Met treatments, suggesting that T3 may provide a therapeutic alternative to standard ß-receptor blockade, especially for patients intolerant to treatment with ß-blockers after MI.

Long-term prognostic value of combined free triiodothyronine and late gadolinium enhancement in nonischemic dilated cardiomyopathy.

BACKGROUND: Thyroid dysfunction and myocardial fibrosis are both associated with cardiovascular events in patients with dilated cardiomyopathy (DCM). HYPOTHESIS: The combination of thyroid hormone (TH) and myocardial fibrosis (detected by late gadolinium enhancement [LGE]) is an independent and incremental predictor of adverse events in DCM. METHODS: We consecutively enrolled 220 idiopathic DCM patients with thyroid function and LGE assessment at Fuwai Hospital (China) from January 2010 to October 2011 and followed up through December 2015. Patients were divided into 4 groups according to the presence or absence of LGE and FT3 value (median level of 2.79 pg/mL): LGE-positive + FT3 < 2.79 pg/mL, LGE-positive + FT3 ≥ 2.79 pg/mL, LGE-negative + FT3 < 2.79 pg/mL, and LGE-negative + FT3 ≥ 2.79 pg/mL. RESULTS: During a median follow-up of 61 months, 56 patients (25.5%) died, with 27/56 (48.2%), 8/45 (17.8%), 12/54 (22.2%), and 9/65 (13.8%) among 4 groups (P = 0.009), respectively. Multivariable Cox regression analysis identified LGE-positive and FT3 < 2.79 pg/mL as a significant independent predictor of all-cause mortality (hazard ratio: 2.893, 95% confidence interval: 1.323-6.326, P = 0.008). Combining the predictive value of FT3 and LGE status significantly improved risk reclassification for all-cause mortality, as indicated by the net reclassification improvement (0.28; P = 0.005) and integrated discrimination improvement (0.058; P = 0.001). CONCLUSIONS: The findings suggest that the combination of FT3 and LGE yielded a more accurate predictive value for long-term prognosis in patients with DCM, which may improve patient selection for intensive interventions.

Ellagic Acid Reduces High Glucose-Induced Vascular Oxidative Stress Through ERK1/2/NOX4 Signaling Pathway.

BACKGROUND/AIMS: Elevated production of reactive oxygen species (ROS) is linked to endothelial dysfunction and is one of the key contributors to the pathogenesis of diabetic vascular complications. Emerging evidence has indicated that ellagic acid (EA), a polyphenol found in fruits and nuts, possesses numerous biological activities including radical scavenging. However, whether EA exerts a vasculo-protective effect via antioxidant mechanisms in blood vessels exposed to diabetic conditions remains unknown. Accordingly, the goal of this current study was to determine whether EA decreases vascular ROS production and thus ameliorates endothelial dysfunction in the diabetic milieu. METHODS: Intact rat aortas and human aortic endothelial cells (HAEC) were stimulated with 30mM high glucose (HG) with and without EA co-treatment. Endothelium-dependent vasodilation was measured using a wire myograph. Gene and protein expression of non-phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 4 (NOX4) were detected using RT-PCR and western blotting, respectively. Oxidative stress was determined by measuring ROS levels using dihydroethidium (DHE) staining. RESULTS: Intact aortas exposed to HG condition displayed exacerbated ROS production and impairment of endothelium-dependent vasodilation, characterizing endothelial dysfunction. These effects were markedly reduced with EA treatment. HG enhanced ROS production in HAEC, paralleled by increased ERK1/2 activation and NOX4 expression. EA treatment blunted the increase of ROS generation, ERK1/2 activation and decreased NOX4. CONCLUSIONS: EA significantly decreases endothelial ROS levels and ameliorates the impairment of vascular relaxation induced by HG. Our results suggest that EA exerts a vasculo-protective effect under diabetic conditions via an antioxidant effect that involves inhibition of ERK1/2 and downregulation of NOX4.

Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelial cells accelerates coronary artery disease in a mouse model of familial hypercholesterolemia.

OBJECTIVE: Overexpression of tissue-nonspecific alkaline phosphatase (TNAP) in endothelium leads to arterial calcification in mice. The purpose of this study was to examine the effect of elevated endothelial TNAP on coronary atherosclerosis. In addition, we aimed to examine endogenous TNAP activity in human myocardium. APPROACH AND RESULTS: A vascular pattern of TNAP activity was observed in human non-failing, ischemic, and idiopathic dilated hearts (5 per group); no differences were noted between groups in this study. Endothelial overexpression of TNAP was achieved in mice harboring a homozygous recessive mutation in the low density lipoprotein receptor (whc allele) utilizing a Tie2-cre recombinase (WHC-eTNAP mice). WHC-eTNAP developed significant coronary artery calcification at baseline compared WHC controls (4312 vs 0µm2 alizarin red area, p<0.001). Eight weeks after induction of atherosclerosis, lipid deposition in the coronary arteries of WHC-eTNAP was increased compared to WHC controls (121633 vs 9330µm2 oil red O area, p<0.05). Coronary lesions in WHC-eTNAP mice exhibited intimal thickening, calcifications, foam cells, and necrotic cores. This was accompanied by the reduction in body weight and left ventricular ejection fraction (19.5 vs. 23.6g, p<0.01; 35% vs. 47%, p<0.05). In a placebo-controlled experiment under atherogenic conditions, pharmacological inhibition of TNAP in WHC-eTNAP mice by a specific inhibitor SBI-425 (30mg*kg-1*d-1, for 5 weeks) reduced coronary calcium (78838 vs.144622µm2) and lipids (30754 vs. 77317µm2); improved body weight (22.4 vs.18.8g) and ejection fraction (59 vs. 47%). The effects of SBI-425 were significant in the direct comparisons with placebo but disappeared after TNAP-negative placebo-treated group was included in the models as healthy controls. CONCLUSIONS: Endogenous TNAP activity is present in human cardiac tissues. TNAP overexpression in vascular endothelium in mice leads to an unusual course of coronary atherosclerosis, in which calcification precedes lipid deposition. The prevalence and significance of this mechanism in human atherosclerosis requires further investigations.

Modified Low-Dose Triiodo-L-thyronine Therapy Safely Improves Function Following Myocardial Ischemia-Reperfusion Injury.

Background: We have shown that thyroid hormones (THs) are cardioprotective and can be potentially used as safe therapeutic agents for diabetic cardiomyopathy and permanent infarction. However, no reliable, clinically translatable protocol exists for TH treatment of myocardial ischemia-reperfusion (IR) injury. We hypothesized that modified low-dose triiodo-L-thyronine (T3) therapy would confer safe therapeutic benefits against IR injury. Methods: Adult female rats underwent left coronary artery ligation for 60 min or sham surgeries. At 2 months following surgery and T3 treatment (described below), the rats were subjected to functional, morphological, and molecular examination. Results: Following surgery, the rats were treated with T3 (8 µg/kg/day) or vehicle in drinking water ad libitum following IR for 2 months. Oral T3 significantly improved left ventricular (LV) contractility, relaxation, and relaxation time constant, and decreased beta-myosin heavy chain gene expression. As it takes rats ~6 h post-surgery to begin drinking water, we then investigated whether modified T3 dosing initiated immediately upon reperfusion confers additional improvement. We injected an intraperitoneal bolus of T3 (12 µg/kg) upon reperfusion, along with low-dose oral T3 (4.5 µg/kg/day) in drinking water for 2 months. Continuous T3 therapy (bolus + low-dose oral) enhanced LV contractility compared with oral T3 alone. Relaxation parameters were also improved compared to vehicle. Importantly, these were accomplished without significant increases in hypertrophy, serum free T3 levels, or blood pressure. Conclusions: This is the first study to provide a safe cardiac therapeutic window and optimized, clinically translatable treatment-monitoring protocol for myocardial IR using commercially available and inexpensive T3. Low-dose oral T3 therapy supplemented with bolus treatment initiated upon reperfusion is safer and more efficacious.

Triiodothyronine Potentiates Vasorelaxation via PKG/VASP Signaling in Vascular Smooth Muscle Cells.

BACKGROUND/AIMS: Vascular relaxation caused by Triiodothyronine (T3) involves direct activation of endothelial cells (EC) and vascular smooth muscle cells (VSMC). Activation of protein kinase G (PKG) has risen as a novel contributor to the vasorelaxation mechanism triggered by numerous stimuli. We hypothesize that T3-induced vasorelaxation involves PKG/vasodilator-stimulated phosphoprotein (VASP) signaling pathway in VSMC. METHODS: Human aortic endothelial cells (HAEC) and VSMC were treated with T3 for short (2 to 60 minutes) and long term (24 hours). Nitric oxide (NO) production was measured using DAF-FM. Expression of protein targets was determined using western blot. For functional studies, rat aortas were isolated and treated with T3 for 20 minutes and mounted in a wire myograph. Relaxation was measured by a concentration-dependent response to acetylcholine (ACh) and sodium nitroprusside (SNP). RESULTS: Aortas stimulated with T3 exhibited augmented sensitivity to ACh and SNP-induced relaxation, endothelium-dependent and endothelium-independent responses, respectively. T3 directly increased vasorelaxation, which was abolished in the presence of a PKG inhibitor. T3 markedly induced phosphorylation of Akt, eNOS and consequently increased NO production in EC. Likewise, T3 induced phosphorylation of VASP at serine 239 via the PKG pathway in VSMC. CONCLUSION: Our findings have uncovered a PKG/VASP signaling pathway in VSMC as a key molecular mechanism underlying T3-induced vascular relaxation.

Free Triiodothyronine Level Correlates with Myocardial Injury and Prognosis in Idiopathic Dilated Cardiomyopathy: Evidence from Cardiac MRI and SPECT/PET Imaging.

Thyroid dysfunction is associated with poor prognosis in heart failure, but theories of mechanisms are mainly based on animal experiments, not on human level. We aimed to explore the relation between thyroid function and myocardial injuries in idiopathic dilated cardiomyopathy (IDCM) using cardiac magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT) and positron emission tomography (PET). Myocardial fibrosis was detected by late gadolinium enhancement (LGE) MRI, and myocardial perfusion/metabolism was evaluated by 99mTc-MIBI SPECT /18F-FDG PET imaging. Across the quartiles of FT3, decreased percentage of segments with LGE and perfusion/metabolism abnormalities were found. As for FT4 and TSH levels, no significant distribution trend of myocardial injuries could be detected. In logistic analysis, FT3 was independently associated with the presence of LGE (OR: 0.140, 95% CI: 0.035-0.567), perfusion abnormalities (OR: 0.172, 95% CI: 0.040-0.738) and metabolism abnormalities (OR: 0.281, 95% CI: 0.081-0.971). After a median follow-up of 46 months, LGE-positive and FT3 < 2.77 pg/mL was identified as the strongest predictor of cardiac events (HR: 8.623, 95% CI: 3.626-16.438). Low FT3 level is associated with myocardial fibrosis and perfusion/metabolism abnormalities in patients with IDCM. The combination of FT3 level and LGE provides useful information for assessing the prognosis of IDCM.