The similarities between the changes in cardiac gene expression in pathological ventricular hypertrophy and hypothyroidism suggest a role of impaired cardiac thyroid hormone (TH) action in the ...development of contractile dysfunction during chronic cardiac pressure overload. Here we studied the possible involvement of altered cardiac TH metabolism using a rat model of right-ventricular (RV) hypertrophy induced by pressure-overload. Pathological RV hypertrophy was indicated by decreased mRNA levels of sarcoplasmic reticulum(SR) Ca2-ATPase type 2a (SERCA2a) and myosin heavy chain a (MHCalpha), and increased levels of MHCbeta mRNA. Enzyme activity of type HI deiodinase (D3), which converts T4 and T3 to the inactive compounds rT3 and 3,3'-T2, respectively, was identified in ventricular tissue. This activity was stimulated up to five fold in hypertrophic RV, but remained unaltered in the non-hypertrophic left ventricle (LV). A low level of type Ideiodinase activity was also detected, which decreased significantly in both RV and LV. Stimulation of RV D3 activity was significantly higher in those animals in which hypertrophy progressed to heart failure, compared to animals that developed compensatory hypertrophy. The induction of a cardiac TR-degrading deiodinase maybe expected to result in reduced cellular levels of T3 and thereby contribute to a local hypothyroid state in the hypertrophic and, particularly, in the failing ventricle.
Relaxation of skeletal muscle requires the re-uptake of Ca2+, which is mediated by the sarcoplasmic reticulum Ca2+-ATPase (SERCA). Thyroid hormone (T3) stimulates the expression of the SERCA1 ...isoform, which is essential for fast skeletal muscle fiber phenotype. We have cloned and studied the first 962 base pairs of the 5′-flanking region of the rat SERCA1 gene. This sequence was tested for T3-regulated expression in transient transfection experiments using COS7 cells and for binding of thyroid hormone receptor (TR) α in mobility shift assays. A construct of the 5′-flanking region and a reporter gene was unresponsive to T3 in the absence of co-transfected thyroid hormone receptor. In the presence of TRα, a T3 induction ratio of almost 4.0 was found, and this induction ratio was doubled with co-transfection of an RXR expression plasmid. Analysis of progressive 5′-deletion fragments of the sequence indicated multiple regions involved in T3 responsiveness. Three regions, R1, R2, and R3, were identified that bound TR complexes in mobility shift assays and conferred T3 responsiveness to a heterologous promoter. The most potent of these thyroid hormone response elements, R3, increased the 2-fold background T3 stimulation of the thymidine kinase promoter to nearly 6-fold. Detailed analysis of this element showed that four TR-binding half-sites, comprising two independent thyroid hormone response elements, interact cooperatively to give the maximal T3 response. T3 regulation of SERCA1 expression is mediated by a complex thyroid hormone response element that may serve to provide a greater range of response in interaction with nuclear receptor partners or cell-specific transcription factors.
We have previously demonstrated an interaction between the major determinants of skeletal muscle phenotype by showing that continuous contractile activity represses the thyroid hormone (3,3', ...5-tri-iodothyronine; T3)-dependent transcriptional activity of fast-type sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase (SERCA1), a characteristic of the fast phenotype. Both the free cytosolic Ca2+ concentration (Ca2+i) and the myogenic determination factors MyoD and myogenin have been implicated as mediators of the effect of contractile activity on skeletal muscle phenotype. Using L6 cells we have shown that an increase in the steady-state Ca2+i above the resting level of 120 nM indeed can mimic the effect of contractile activity on T3-dependent SERCA1 expression. We now show that the repressing effect of increased Ca2+i on T3-dependent SERCA1 expression in L6 cells is exerted at a pre-translational level and is accompanied by increased myogenin mRNA expression. Myogenin overexpression in these cells revealed that increased expression of myogenin alone strongly decreases the T3-dependent stimulation of SERCA1 promoter activity. These results suggest a pathway for the regulation of skeletal muscle phenotype in which Ca2+i mediates the effect of contractile activity by regulating the expression of myogenin, which in turn interferes with transcriptional regulation by T3.
The iodothyronine deiodinases initiate or terminate thyroid hormone action and therefore are critical for the biological effects mediated by thyroid hormone. Over the years, research has focused on ...their role in preserving serum levels of the biologically active molecule T
3
during iodine deficiency. More recently, a fascinating new role of these enzymes has been unveiled. The activating deiodinase (D2) and the inactivating deiodinase (D3) can locally increase or decrease thyroid hormone signaling in a tissue- and temporal-specific fashion, independent of changes in thyroid hormone serum concentrations. This mechanism is particularly relevant because deiodinase expression can be modulated by a wide variety of endogenous signaling molecules such as sonic hedgehog, nuclear factor-κB, growth factors, bile acids, hypoxia-inducible factor-1α, as well as a growing number of xenobiotic substances. In light of these findings, it seems clear that deiodinases play a much broader role than once thought, with great ramifications for the control of thyroid hormone signaling during vertebrate development and metamorphosis, as well as injury response, tissue repair, hypothalamic function, and energy homeostasis in adults.
Decreased expression of the cardiac slow-twitch sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2), a major determinant of Ca2+ homeostasis, contributes to the abnormal intracellular Ca2+ ...handling in the failing heart. We investigated the contractility dependence of the effects of norepinephrine (NE) and thyroid hormone (T3) on SERCA2 expression in cultured neonatal heart cells under serum-free conditions. NE and T3 are associated with pathological and physiological forms of hypertrophy, respectively, whereas both hormones increase contractility. In contracting cultures, T3 increased SERCA2 protein and mRNA levels by 35 and 110%, respectively. The same stimulatory effects of T3 on SERCA2 expression were found in contraction-arrested cells. In contracting cultures, NE induced a decrease of SERCA2 protein and mRNA levels by 40 and 60%, respectively. In contrast, SERCA2 protein and mRNA levels were not decreased by NE in contraction-arrested cells, indicating that contractility is a prerequisite for the negative influence of NE on SERCA2 expression. Electrical stimulation at a fixed frequency in the presence and absence of NE demonstrated that the NE-induced increase in contraction frequency is unlikely to account for the decreased SERCA2 expression induced by NE. The results suggest that the effect of contractility on SERCA2 expression depends on the signal transduction pathways that are activated by NE and T3.
We studied the effect of thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) on the expression of sarcoplasmic reticulum (SR) fast- and slow-type Ca(2+)-ATPase isoforms, SERCA1 and SERCA2a, ...respectively, and total SR Ca(2+)-ATPase activity in rat skeletal muscle. Cross sections and homogenates of soleus and extensor digitorum longus muscles from hypo-, eu-, and hyperthyroid rats were examined, and expression of Ca(2+)-ATPase isoforms in individual fibers was compared with expression of fast (MHC II) and slow (MHC I) myosin heavy chain isoforms. In both muscles, T3 induced a coordinated and full conversion to a fast-twitch phenotype in one-half of the fibers that were slow twitch in the absence of T3. The conversion was partial in the other one-half of the fibers, giving rise to a mixed phenotype. The stimulation by T3 of total SERCA expression in all fibers was reflected by increased SR Ca(2+)-ATPase activity. The time course of the T3-induced changes of SERCA isoform expression was examined 1-14 days after the start of daily T3 treatment of euthyroid rats. SERCA1 expression was stimulated by T3 at a pretranslational level in all fibers. SERCA2a mRNA expression was transiently stimulated and disappeared in a subset of fibers. In these fibers SR Ca(2+)-ATPase activity was high because of high SERCA1 protein levels. These data suggest that the ultimate downregulation of SERCA2a expression, which is always associated with high SR Ca(2+)-ATPase activities, occurs at a pretranslational level.
Several studies suggest that the cardioprotective effect of sevoflurane depends on protein kinase C (PKC) activation, mitochondrial K(+)(ATP) channel (mitoK(+)(ATP)) opening, and reactive oxygen ...species (ROS). However, evidence for their involvement was obtained in separate experimental models. Here, we studied the relative roles of PKC, mitoK(+)(ATP), and ROS in sevoflurane-induced cardioprotection in one model. Rat trabeculae were subjected to simulated ischemia by applying metabolic inhibition (MI) through buffer containing NaCN, followed by 60-min reperfusion. Recovery of active force (F(a)) was assessed as percentage of pre-MI force. In time controls, F(a) amounted 60% +/- 5% at the end of the experiment. The recovery of F(a) after MI was reduced to 28% +/- 5% (P = 0.045 versus time control), whereas sevoflurane reversed the detrimental effect of MI (F(a) recovery, 67% +/- 8%; P = 0.01 versus MI). The PKC inhibitor chelerythrine, the mitoK(+)(ATP) inhibitor 5-hydroxy decanoic, and the ROS scavenger N-(2-mercaptopropionyl)-glycine all completely abolished the protective effect of sevoflurane (recovery of F(a), 31% +/- 8%, 33% +/- 8%, and 24% +/- 9% for chelerythrine, 5-hydroxy decanoic, and N-(2-mercaptopropionyl)-glycine, respectively). In conclusion, PKC activation, mitoK(+)(ATP) channel opening, and ROS production are all essential for sevoflurane-induced cardioprotection. These signaling events are arranged in series within a common signaling pathway, rather than in parallel cascades. Our findings implicate that the perioperative use of sevoflurane preserves cardiac function by preventing ischemia-reperfusion injury.
Protein kinase C, mitochondrial K(+)(ATP) channels and reactive oxygen species act within one downstream signaling pathway in mediating the cardioprotective effect of sevoflurane.
A spectrophotometric method is described for the determination of sarcoplasmic reticulum (SR) Ca2(+)-ATPase activity (EC 3.1.6.38) in unfractionated muscle homogenates. Conditions were established ...that give maximal SR Ca2(+)-ATPase activity, while eliminating Ca2(+)-dependent myofibrillar ATPase activity and reducing Ca2(+)-independent or background ATPase activity. High Ca2+ (20 mM) could be used to selectively inhibit the SR Ca2+ ATPase. Identification of the Ca2(+)-dependent ATPase activity in muscle homogenates as being SR Ca2+ ATPase was based on a comparison of several parameters using homogenate material and purified SR. The following parameters were compared and found to be the same in homogenate and SR: activation and inactivation between 0 and 20 mM Ca2+, temperature dependence, sensitivity toward Triton X-100, and the maximal level of inhibition of ATPase activity achieved by an antibody specific for SR Ca2+ ATPase. The method is illustrated with the analysis of homogenates prepared from freeze-dried muscle fibers and thin sections of muscles typically used in microscope analyses as well as an analysis of freshly prepared homogenates from various types of muscle, which shows a good correlation over a wide range between SR specific Ca2(+)-uptake and -ATPase activities. In addition, a simple, easily constructed cuvette is described which allows the analysis of less than 5 micrograms of tissue (wet weight) in a volume of 25 microliters.
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