Effects Of Levothyroxine Therapy On Left And Right Ventricular Function In Neonates With Congenital Hypothyroidism And The Mechanism Of It

Thyroid hormones are essential to proper development and differentiation of all cells of the human body. Congenital hypothyroidism (CH) is a common endocrine disease of pediatrics, which results in severe mental retardation and growth failure as well as other functional impairment because of the deficiency of thyroid hormone during human perinatal development. Recent research has demonstrated that cardiac function is impaired in children with CH, which can be fully or partly reversed by levothyroxine (L-T₄) substitution therapy. However, only few, contradictory data are available on cardiac function in neonates with CH.Cardiac muscle contraction and relaxation are regulated by the intracellular free calcium (Ca²⁺) concentration handling in excitation-contraction coupling. Sarcoplasmic reticulum Ca²⁺-ATPase (SERCA2a) and phospholamban (PLB) are the most important proteins located in SR in regulating calcium concentration in cardiac myocytes. Thyroid hormone is known to regulate myocyte contractility in part by changes in the expression of Ca²⁺ transport proteins. It was shown that SERCA2 mRNA and protein levels were both lower in adult hypothyroid animals, while PLB expression was higher in hypothyroid rodents. However, very fewstudies have been performed to evaluate the alteration of myocardial sarcoplasmic reticulum Ca²⁺-ATPase activity and Ca²⁺ transport proteins gene expression in neonatal hypothyroid rats. The aim of this study was to investigate the effects of L-T₄ substitution therapy on left and right ventricular function in neonates with congenital hypothyroidism as well as alteration of myocardial sarcoplasmic reticulum Ca²⁺-ATPase activity and Ca²⁺ thansport proteins gene expression in neonatal hypothyroid rats.Part One Effects of levothyroxine therapy on left and right ventricular function in neonates with congenital hypothyroidismObjectives1. To investigate left ventricular systolic and diastolic function as well as right ventricular function in neonates with congenital hypothyroidism.2. To investigate the effects of L-T4 substitution therapy on left and right ventricular function in neonates with congenital hypothyroidism.MethodsFifty full-term neonates with CH aged 17～28 days before and after 1 month of levothyroxine substitution therapy as well as 35 normal neonates aged 17～28 days underwent conventional M-mode echocardiography, pulsed-wave Doppler echocardiography (PWD), quantitative tissue velocity imaging (QTVI) and tissue tracking imaging (TTI). Serum hormone levels of TSH, TT₃, TT₄, FT₃ and FT₄ were measured with a standard chemiluminescent immunoassay. The following parameters were measured:1. M-mode echocardiography: left ventricular fractional shortening (LVFS), left ventricular ejection fraction (LVEF), left atrial diameter (LA), aortic diameter (Ao), and the LA/Ao ratio was calculated.2. Pulsed-wave Doppler echocardiography: peak early diastolic mitral and tricuspid inflow velocity (E), peak late diastolic mitral and tricuspid inflow velocity (A), and the E/A ratio was calculated.3. Quantitative tissue velocity imaging: peak systolic transmitral and transtricuspid annular velocity (Sa), peak early transmitral and transtricuspid annular velocity (Ea), peak late transmitral and transtricuspid annular velocity (Aa), and the Ea/Aa ratio was calculated.4. Tissue tracking imaging: peak systolic mitral annular displacement (Dm), peak systolic tricuspid annular displacement (Dt).Results1. Serum hormone levelsCompared with normal neonates, serum TSH levels in CH neonates were significantly higher, and TT₃, TT₄, FT₃ and FT₄ levels were significantly lower (P< 0.001, respectively). After 1 month of L-T₄ substitution therapy in the CH group, TT₃, TL₄, FT₃ and FT₄ levels all showed a significant increase and TSH levels a significant decrease (P< 0.001, respectively).2. Conventional Doppler echocardiography variablesM-mode echocardiography showed that LVEF and LVFS were significantly lower in CH neonates (P<0.01, respectively), whereas LA, AO, and LA/AO did not show statistically significant differences (P>0.05, respectively). PWD showed that CH neonates had significantly lower E and E/A of the mitral and tricuspid valve (P <0.001, respectively), while A did not differ between the two groups (P>0.05, respectively). After 1 month of L-T₄ therapy in the CH group, LVEF and LVFS significantly increased (P<0.01, respectively), while LA, AO and LA/AO did not change (P>0.05, respectively). In addition, L-T₄-treated neonates showed an improvement of both LV and RV diastolic function with a significant increase of E (P<0.001, respectively) and a subsequent increase of E/A (P<0.001, respectively),whereas A did not change with treatment (P>0.05, respectively).3. Quantitative tissue velocity imaging and tissue tracking imaging variablesQTVI showed that CH neonates had significantly lower Sa, Ea and Ea/Aa of both left and right ventricles (P<0.001, respectively). However, Aa of left and right ventricle did not differ significantly (P>0.05, respectively). After 1 month of substitution therapy, CH neonates showed a significant increase of Sa, Ea and a subsequent increase of Ea/Aa (P<0.00\, respectively), while Aa of mitral and tricuspid annular velocity remained unchanged. TTI showed that Dm and Dt in CH neonates were significantly lower than that in the control group (P<0.001, respectively), which significantly increased after 1 month of L-T4 substitution therapy (P<0.001, respectively).Conclusions1. Neonates with CH are associated with right ventricular subclinical systolic and diastolic dysfunction in addition to left ventricular dysfunction.2. Early L-T₄ substitution therapy is able to reverse the impairment of cardiac function fully.Part two Alteration of myocardial sarcoplasmic reticulum Ca²⁺-ATPase activity and Ca²⁺ transport proteins gene expression in neonatal hypothyroidratsObjectives1. To investigate the alteration of myocardial sarcoplasmic reticulum Ca²⁺-ATPase activity in neonatal hypothyroid rats.2. To investigate the alteration of myocardial sarcoplasmic reticulum Ca²⁺ transport proteins including SERCA2a and PLB mRNA expression in neonatalhypothyroid rats.3. To investigate the effect of L-T₄ substitution therapy on sarcoplasmicreticulum Ca²⁺-ATPase activity and Ca²⁺ transport proteins mRNA expression.Methods1. Animal treatmentHypothyroid group: Hypothyroidism was induced by the administration of propylthiouracil (PTU, 50mg/d) solution to the pregnant SD rats by gavages beginning on embryonic day 15 and continuing throughout the lactational period.L-T₄ treated group: A subgroup of neonatal hypothyroid rats were injected intraperitoneally with levothroxine (L-T₄, 2μg /100g) BW daily, starting from the day of birth.Control group: Other pregnant SD rats received normal saline instead of PTU.Rats of three groups were harvested at postnatal day 1, 3, 5 and 7 respectively (n=10). After measurement of serum thyroid hormone levels, the hearts were removed and the ventricles were weighed (HW).2. Measurement of sarcoplasmic reticulum Ca²⁺ -ATPase activitySR was prepared by differential centrifugation method. The concentration of SR protein was measured by Coomassie brilliant blue staining, and finally the. activity of SR Ca²⁺-ATPase was determined by the inorganic phosphorus method.3. Measurement of SERCA2a and PLB mRNA expressionIn reverse transcriptase-polymerase chain reaction (RT-PCR), the total RNA was subjected to reverse transcription. After isolated from ventricles tissue by using Trizol, the resulting DNA was amplified with sequence-specific primers and Taq DNA polymerase. Optical density of the bands was analyzed, β-actin was used as the reference transcript.Results1. Establishment of animal modelNeonatal hypothyroid rats had smaller body size and lighter body weight (BW) (P<0.001, respectively) except for day 1 (P>0.05). Furthermore, hypothyroidism was confirmed with significant lower serum FT₃ and FT₄ levels as well as higher serum TSH levels (P＜0.001, respectively). Neonatal L-T₄ treated rats showed an increase of serum thyroid hormone levels and a decrease of serum TSH levels (P＜0.001, respectively). However, it did not return to normal range till postnatal day 7 (P＜0.05).2. Heart weight(HW) and the ratio of HW to BW(HW/BW)Compared with the control group, neonatal hypothyroid rats showed a significant decrease in heart weight (HW) (P<0.05, respectively), while HW/BW did not show any statistically significant differences (P>0.05, respectively). Neonatal L-T₄ treated rats significantly increased HW and BW compared with the hypothyroid group (P＜0.001, respectively), and HW/BW was also increased compared with either the control or hypothyroid groups at postnatal day 7 (P＜0.05).3. Sarcoplasmic reticulum Ca²⁺-ATPase activityThe activity of sarcoplasmic reticulum Ca²⁺-ATPase in hypothyroid group was significantly lower than in the control group (P＜0.001, respectively), and L-T₄ treated group showed a significant increase compared with hypothyroid group (P＜0.05, respectively), however it did not reach the normal level till postnatal day 7 (P＜0.05).4. Expression of SERCA2a and PLB mRNANeonatal hypothyroid rats had significant lower SERCA2a mRNA (P＜0.05, respectively) and higher PLB mRNA levels (P＜0.001, respectively) and subsequent lower SERCA2a/PLB at each postnatal day (P＜0.001, respectively). Compared with hypothyroid group, the expression of SERCA2a mRNA significantlyincreased (P<0.05, respectively) and that of PLB mRNA significantly decreased (P<0.05, respectively) in L-T₄ treated group, which still did not reach the normal level till postnatal day 7 (P<0.05, respectively) except for SERCA2a mRNA of postnatal day 5 and 7 (P>0.05).Conclusions1. The deficiency of thyroid hormone results in decreased expression of SERCA2a mRNA as well as increased PLB mRNA, which contribute to SR Ca²⁺-ATPase activity reduction in neonatal rats. Those may be one of the most important mechanism of myocardial systolic and diastolic dysfunction.2. Early L-T₄ substitution therapy is able to reverse those dysfunctions fully or partly, which consequently improve cardiac function in neonatal hypothyroid rats in short postnatal time.