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(Kempers, M.J. et al., 2005. J Clin Endocrinol Metab)

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  1. Impaired cardiac function in baseline and following ischemia in adult offspring male rats with maternal hypothyroidism: The role of nitric oxide production pathways

  2. Hypothyroidism during pregnancy causes irreversible developmental effects on body systems of offspring. (Kempers, M.J. et al., 2005. J Clin Endocrinol Metab)

  3. Decrease of cardiac output • Decrease of heart rate (HR) Cardiovascular effects of thyroid hormone deficiency during pregnancy including: • Decrease of blood pressure • Decrease of intima-media thickness of arteries • Decrease of endothelial cells in aorta • Decrease of cardiac function • Decrease in tolerance to cardiac ischemia (Fouron, J.C., et al. 1982 Br Heart J) (Ghanbari, M., et al. 2015. J Endocrinol Invest) (Ghanbari, M., et al. Gen. 2016. Physiol. Biophys)

  4. Control MH Control MH Aorta Superior mesentric (Ghanbari, M., et al. Gen. 2016. Physiol. Biophys)

  5. However Molecular mechanisms of this state are still not fully understood

  6. Maturation of contractile proteins in heart Some studies have been shown in maternal hypothyroidism Postpone • Number of cytochrome c oxidase isoform in mitochondria of cardiomyocyte Decrease (Chizzonite RAZak R. et al. 1984. J Biol Chem) (Meehan J. et al. 1997. Biochem J)

  7. Nitric oxide (NO) is a gaseous messenger and free radical with many pivotal biological functions. (Sawyer, D.B., 2011. Am J Med Sci)

  8. Nitric oxide have direct impact on cardiac function in both physiological and pathological conditions. (Massion, P.B., et . 2003. Circ Res)

  9. Postganglionic parasympathetic fibers Norepinephrine Sympathetic varicosities (OrthoS) Epinephrine PDE3

  10. Antioxidant system regulates by nitric oxide via post-translational modifications pathways. Juan C. Begara-Moralrs. Frontiers in plant science. 2016

  11. Tyrosine nitration Post-translational modifications pathways including: • S-nitrosylation Mainly by Addition Nitrogen dioxide (NO2) Peroxynitrite (ONOO-) • Tyrosine nitration Tyrosine residues Mainly by Cysteine thiol • S-nitrosylation NO group Addition Change in function Proteins Juan C. Begara-Moralrs. Frontriers in plant science. 2016

  12. Several studies showed that NO bioavailability changes in aorta,heart and coronary arteriesof offspring adult rats with maternal hypothyroidism (MH). (Ghasemi, A, et al. 2013 . J Physiol Biochem) (Jeddi, S, et al. 2016. Nitric oxide) (Gaynullina, D.K, et al. 2018. Nitric oxide)

  13. Objective • Evaluation of the role of nitric oxide in decrease of baseline cardiac function in adult offspring rats with maternal hypothyroidism. • Evaluation of the role of nitric oxide in decrease of tolerance to ischemia–reperfusion injury in adult offspring rats with maternal hypothyroidism. • Evaluation of oxidant and antioxidant system status of the heart tissue in adult offspring rats with maternal hypothyroidism in baseline and following ischemia condition.

  14. Materials and methods Virgin female rats (200 ± 10 g, n = 50) were housed overnight with male rats (320 ± 20 g, n=50). Tap water Control Pregnant females were randomly distributed in two groups MH Tap water + 250 mg/L PTU Throughout pregnancy

  15. Materials and methods The weight of the offspring in both groups was measured weekly (A & D scale, EK-300i, Japan; sensitivity 0.01 g) from the first day of birth till 16 weeks-old.

  16. Materials and methods To assess the thyroid hormones status blood samples were obtained from both mothers (after delivery) and offspring (at birth and 4 months) in both group. Male Offspring At age of 4 month Tested

  17. Materials and methods 1.Control 2.MH 3.Control + Ischemia Adult offspring rats in both groups (MH and control) were divided: 4.MH + Ischemia 5.Control + Aminoguanidine (AG), 100 μmol/L , a selective inhibitor of iNOS 6.MH + AG (n=10-7 in each group) 7.Control + 7-nitroindazole , 100 μmol/L , a selective inhibitor of nNOS 8.MH + 7-nitroindazole

  18. Materials and methods 1.Control Without any intervention The heart of the was perfused with PBS solution containing NEM Groups 1 and 2 2.MH (n=10 in each group) Their hearts Stored at -80 °C until the time of performing biochemical tests

  19. Materials and methods 3.Control + Ischemia Perfusion Ischemia Reperfusion 4.MH + Ischemia Time (min) Their heart stored for biochemical tests 5.Control + AG Perfusion Perfusion + AG 6.MH + AG Time (min) 7.Control + 7-nitroindazole (7-NI) Perfusion Perfusion + 7-NI 8.MH + 7-nitroindazole Time (min)

  20. Left ventricular end diastolic pressure (LVEDP) Cardiac parameters in baseline and following ischemia: • Left ventricular developed pressure (LVDP) • Heart rate (HR) • Peak rates of positive and negative changes in left ventricular pressure (± dp/dt)

  21. Concentration of NOx in heart before and after ischemia By the Griess method • Concentration of 3 isoforms of NOS (eNOS, nNOS and iNOS) in heart before and after ischemia ELISA kits (ZellBio GmbH Germany) Biological parameters: 1.Control • Concentration of malondialdehyde (MDA) and total antioxidant capacity (TAC) in heart before and after ischemia kits (ZellBio GmbH Germany) 2.MH 3. Control + Ischemia • Activity of superoxide dismutase enzyme (SOD) and catalase enzyme (CAT) in heart before and after ischemia 4.MH + Ischemia kits (ZellBio GmbH Germany)

  22. Left ventricular end diastolic pressure (LVEDP) Cardiac parameters in baseline and following AG administration: • Left ventricular developed pressure (LVDP) • Heart rate (HR) • Peak rates of positive and negative changes in left ventricular pressure (± dp/dt)

  23. Left ventricular end diastolic pressure (LVEDP) Cardiac parameters in baseline and following 7-nitroindazole administration: • Left ventricular developed pressure (LVDP) • Heart rate (HR) • Peak rates of positive and negative changes in left ventricular pressure (± dp/dt)

  24. Statistical analysis Data were analyzed by Graph Pad Prism software (Version 6) and values were expressed as mean ± SEM. Student’s t-test was used to compare thyroid hormones, cardiac function in basal condition, oxidant and antioxidant components between MH and control groups in serum. Two-way ANOVA followed by Bonferroni test was used for comparing changes in weight gain, cardiac function parameters after ischemia between groups in different times. One-way ANOVA was used for biological parameters in left ventricular before and after ischemia in both group. A value of p <0.05 was considered statistically significant.

  25. Results

  26. Results (n=40 in each group) *P < 0.05, †P < 0.01, #P < 0.001

  27. Results

  28. Results (n=10 in each group) *P < 0.05

  29. Results (n=10 in each group) *P < 0.05

  30. Results (n~8 in each group) *P < 0.05, †P < 0.01, #P < 0.001

  31. Results

  32. Results

  33. Discussion In our study, decrease of NOx in serum was in line with strong decrease NO in plasma of newborns with congenital hypothyroidism. (Rodriguez-Arnao MD. et al., 2003, Eur Cytokine Netw)

  34. In baseline condition Cardiomyocyte eNOS = NOx nNOS iNOS = ICa,L RyR2 T tubule - Sarcoplasmic reticulum Ca+2 - Voltage-operated calcium channels (ICa,L) Ryanodine receptors (RyR2)

  35. In baseline condition Cardiomyocyte Sarcoplasmic reticulum PKA + S-nitrosylation + PLN + Directly NO SERCA nNOS S-nitrosylation Ca+2 Ca+2 nNOS Sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) Phospholamban (PLN)

  36. Discussion Some studies showed that during fetal life, nNOS is a target for THs, and both of mRNA expression and protein levels of this enzyme strongly increase in neocortex of offspring rats with MH. (Sinha RA. et al., 2008, Endocrinology)

  37. After ischemia TNFɑ mRNA instability Cardiomyocyte AC eNOS NOx nNOS Overproduction of NO cAMP - iNOS - PKA PDE3 NO ONOO- T tubule - ICa,L cGMP RyR2 PKG ONOO- Sarcoplasmic reticulum + Ca+2 TnI Relaxation Voltage-operated calcium channels (ICa,L) Ryanodine receptors (RyR2)

  38. Discussion • The high level of NO due to increased iNOS level in left ventricular of MH after ischemia give rise to more cytoxicity via formatting more ONOO- following an over production of O2- by iNOS. (Dhalla NS. et. al. Cardiovasc Res. 2000) • Reduction of endogenous antioxidant compounds capable to decrease the recovery of cardiac function after reperfusion (Dianat M. et. al. Iran Red Crescent Med J. 2014)

  39. Superoxide radicals (O2-) SOD undergo inactivation by peroxynitrite-mediated nitration. (Demicheli et al., free radbiomed, 2007) S-nitrosoglutathione (GSNO), formed by S-nitrosylation of the antioxidant GSH SOD activity is increased after GSNO treatment (Sehrawat et al., Front. Plant Sci, 2013)

  40. Catalase is a key enzyme that regulates H2O2 levels. • Catalase is one of the first antioxidant enzymes to be analyze to check how its activity can be modulated by NO donors. (Clark et al., Plant Microbe Interact. 2000) It is known that catalase can be nitrated and S-nitrosylated in vitro both Inhibit activity of catalase (Ortega-Galisteo, A. P, J. Exp. Bot2012, )

  41. In baseline condition Cardiomyocyte NOx + GSNO SOD S-nitrosylation GSH nNOS CAT - Directly NO Negative correlation S-nitrosylation MDA TAC

  42. Discussion The CAT activity result in left ventricular is comparable with decreased activity of this enzyme in other tissuse in offspring with MH including cerebrum, cerebellum and medulla oblongata. (Ahmed OM. et al., 2012, Int J Dev Neurosci)

  43. Conclusion • The negative inotropic effect which observed in MH group in basal condition may be due to the higher level of NOx which attributed to increase of nNOS isoform in left ventricular tissue. • The decrease of tolerance to ischemia which observed in MH group compared to control at least partly due to the increase of iNOS concentration and decrease of eNOS after ischemia. • The increase of NOx in left ventricular as well as decrease in TAC and CAT enzyme activity may be a part of the mechanism of the decrease of tolerance to ischemia in MH.

  44. Thanks for your attention,,,

  45. Some studies have shown the decrease of cardiac function and increase of injury caused by reperfusion may occur By induction and activation of some mediators such as iNOS enzyme; the excess NO production by iNOS acts as negative inotrope by myocardial cGMP production or reactive oxygen species. Wildhirt SM., et al . Cardiovasc Res, 1999