In gastrocnemius muscle from newborn rats the mRNA for the fast sarcoplasmic reticulum (SR) Ca(2+)-ATPase isoform (SERCA1) comprised over 90% of total SR Ca(2+)-ATPase mRNA content and increased ...5-fold between day 5 and 20 after birth, whereas in hypothyroid muscle the SERCA1 message level remained constant. Triiodothyronine (T3) treatment of 2-day-old euthyroid rats induced a precocious stimulation of SERCA1 mRNA levels, indicating that T3 is the determining factor in the stimulation of SERCA1 message levels and that this stimulation underlies the previously reported effect of the thyroid status on the neonatal development of SR Ca(2+)-ATPase activity. The low mRNA level for the slow SR Ca(2+)-ATPase isoform (SERCA2) was constant in both euthyroid and hypothyroid muscle development. Nevertheless, T3 treatment of hypothyroid neonates induced a transient stimulation of SERCA2 message levels, indicating that SERCA2 is responsive to higher levels of T3.
The activity of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) is reduced in the failing myocardium. Therefore, transfer of SERCA2a cDNA is considered as a therapeutical approach. The aim of this study ...was analysis of the long-term effect of SERCA2a overexpression in normal as well as pressure overload challenged myocardium of transgenic rats.
Independent transgenic rat lines were established expressing the rat SERCA2a cDNA specifically in the myocardium resulting in increased SERCA2a protein levels by 30-70%. Simultaneous measurements of isometric contraction and calcium transients were carried out in right ventricular papillary muscle preparations. Hemodynamic parameters were measured in hearts of unchallenged rats as well as 10 weeks after pressure overload induced by abdominal aortic banding.
Analysis of calcium handling and contractile parameters in isolated right ventricular papillary muscles revealed significant shortening of intracellular calcium transients and half maximal relaxation times (RT(50)). Assessing myocardial contractility in working heart preparations, both transgenic rat lines revealed elevated left ventricular pressure, improved systolic and diastolic parameters, attenuated negative force-frequency relation, and a dose-dependent beta-adrenergic effect. Aortic banding resulted in reduction of left ventricular pressure and worsening of contraction and relaxation parameters with no differences in mortality in both transgenic (+dP/dt 3084+/-96 vs. 3938+/-250 mmHg/s; RT(50) 47.0+/-1.2 vs. 36.7+/-1.4 ms) and wild-type rats (+dP/dt 2695+/-86 vs. 3297+/-122 mmHg/s; RT(50) 53.0+/-1.6 vs. 44.1+/-1.4). SERCA2a overexpressing hearts revealed improved hemodynamic parameters compared to wild-type controls. Acceleration of isovolumetric relaxation characterized by the index Tau was directly correlated to SERCA2a protein concentrations.
Overexpression of SERCA2a protein results in a positive inotropic effect under baseline conditions remaining preserved under pressure overload without affecting mortality. Therefore therapeutic transfer of SERCA2a may become a potential approach for gene therapy of congestive heart failure. Moreover, transgenic SERCA2a rats will be useful for studies of long-term SERCA2a overexpression in further cardiovascular disease models.
The effects of the thyroid status on the Ca++-transporting capabilities of rat slow skeletal muscle (m.soleus) were studied. The oxalate supported Ca++-uptake activity and Ca++-loading capacity of ...muscle homogenates from hyperthyroid rats showed an approximate 4.2 and 2.5 fold increase, respectively, as compared to values found in the hypothyroid group. Muscle homogenates of euthyroid rats gave intermediate values. The specific activity of oxalate supported Ca++ uptake, but not the Ca++-loading capacity, of membrane preparations enriched with respect to sarcoplasmic reticulum (SR) increased in proportion to the thyroid status. This was paralleled by a 3.5 fold increase in the amount of active Ca++ pumps in the SR preparations in the transition from hypothyroidism to hyperthyroidism as determined by measurement of Ca++-dependent 32P incorporation. These observations are not explained by differences in degree of purification of the examined SR preparations. Protein profiles of the membrane preparations obtained by gel electrophoresis indicated a thyroid-hormone dependent increase in Ca++-pump content relative to other SR proteins. The results suggest that thyroid hormone stimulates the proliferation of the SR and possibly also increases the Ca++-pump density in the SR membrane.
T3 action is locally modulated by the intracellular type 2 (D2) and type 3 (D3) iodothyronine-deiodinases which convert the prohormone T4 to active T3, and inactivate T4 and T3, respectively. The ...synchronized expression of D3 followed by D2 is necessary for proper muscle development and regeneration. D2 and D3 are selenoproteins containing selenium (Se) at their active site in the form of the rare amino acid selenocysteine (Sec) which is incorporated during translation by the recoding of an UGA stop codon. Consequently, dietary Se is essential for TH metabolism. During times of Se deficiency, selenocysteine lyase (Scly) decomposes Sec to Se allowing it to be recycled into new selenoproteins. We hypothesized that Scly is involved in preserving the production of the deiodinases and other selenoproteins when dietary Se levels are low. During the neonatal period when muscle is maturing, and deiodinase expression is high, we found that between P1 and P15 Scly expression increases 2-fold in soleus and 15-fold in the extensor digitalis longus (EDL) muscle. Further, we found that Scly mRNA expression was 2.5-fold higher in the adult slow oxidative soleus muscle versus the fast glycolytic EDL. Next, we fed male WT and Scly knockout (Scly
-/-
) mice a Se adequate or deficient diet and evaluated gene expression in the soleus and EDL via qPCR. In WT soleus, low Se diet decreased expression of many selenoproteins, likely in part due to nonsense-mediated decay of the non-translated UGA containing transcripts. In contrast, the expression of these selenoproteins was unchanged in the EDL. Scly
-/-
mice fed a Se adequate diet demonstrated a trend towards increased Dio2, SelenoN, SelenoM, SelenoP, Txnrd1, and Sephs2 expression in soleus when compared to WT mice on the same diet, but this was abolished under low Se conditions. In the soleus, diet had no effect on SelenoH in WT mice, while notably, SelenoH was significantly decreased in Scly
-/-
mice fed a low Se diet. In the EDL, expression of SelenoW and SelenoH was unchanged under low Se conditions in WT mice, but was decreased in Scly
-/-
. This suggests that the expression of a subset of genes relies upon the Se provided by Scly recycling when Se is limiting. Finally, to investigate the possible role of Scly in muscle differentiation, we induced the murine myoblast C2C12 cell line to differentiate via serum withdrawal in the presence of 100 nM total Se to control for the effects of decreased serum on Se levels. Scly mRNA expression reached peak induction by 48h after differentiation. Dio2, Gpx3, SelenoP, SelenoM, SelenoN, and the selenoprotein synthesis factor SBP2 exhibited similar expression profiles. Our results suggest a role for Scly in skeletal muscle development, differentiation, and adult muscle homeostasis. Further, expression of some muscle selenoproteins, including Dio2, appears to be sensitive to low dietary Se when Scly mediated Se recycling is impaired.
Funding:
NIH-NIDDK
Acute myocardial infarction (MI) evokes production of circulating pro‐inflammatory cytokines, which can result in neuroinflammation. This study aimed to investigate signs of neuroinflammation in mice ...by measuring brain TNF‐alpha expression and microglia activation after MI.
Male 14 week old C57 Bl/6 mice were subjected to MI or sham surgery. Two weeks later, infarct was verified by ECHO‐cardiography, and mice were sacrificed. Brain was processed for western blot on TNF‐alpha, and TNF‐receptors.
Heart failure in MI mice was confirmed by increased lung dry weight (35.2±2.2 vs 30.1±0.7 mg), and reduced fractional shortening (19±3 vs 46±1%). TNF‐alpha precursor expression was significantly increased in MI versus sham (9.79±0.60 vs 6.99±0.64 AU) and was associated with higher TNF‐R1 (5.92±2.39 vs 1.88±0.36 AU) and lower TNF‐R2 expression (0.71±0.61 vs 3.01±0.95 AU). Brain tissue stained for microglia showed increased numbers of microglia in the dentate gyrus of the hippocampus (17.1±0.8 vs 14.7±0.8 per high power field) combined with a decreased ratio of processes/cell body surface area (0.071±0.004 vs 0.098±0.007), indicating a higher degree of activation.
MI in mice induces increased TNF‐alpha expression at higher TNF‐R1 and lower TNF‐R2 expression in the brain, indicating a persistent pro‐inflammatory state. Spatially altered number of microglia may suggest localization of this inflammation.
The distribution of phospholipids and fatty acyl composition of individual phospholipids in sarcoplasmic reticulum from fast skeletal muscle of hypothyroid and euthyroid (control) rats have been ...determined. Hypothyroidism resulted in a 24% decrease in the phosphatidylethanolamine (PE) content and a concomitant increase in the phosphatidylcholine (PC) content of the sarcoplasmic reticulum. The amounts of other phospholipids and cholesterol remained unaffected. Fatty acyl compositions of PE and PC were quantitatively different, but hypothyroidism affected these compositions similarly. Changes included an increase in the proportions of docosahexaenoic (22:6(n - 3)), arachidonic (20:4(n - 6)), icosatrienoic (20:3(n - 6)) and stearic (18:0) acids and a decrease in those of linoleic (18:2(n - 6)), palmitic (16:0) and oleic (18:1(n - 9)) acids. The effects of hypothyroidism on the phospholipid distribution could be reversed by treatment of hypothyroid animals with thyroid hormone for a period of 14 days (10 micrograms T3/100 g body weight per 2 days). The fatty acyl composition of the phospholipids was also restored to the euthyroid values by this treatment. Exceptions were 18:2 and 22:6 in PE, in which case reversal was significant but not complete, and 18:2, 20:4 and 22:6 in PC. The levels of these acids in PC were not reversed to the euthyroid values after the 14-day treatment, but rather the opposite occurred.
A fraction containing a protein-glycogen complex was isolated from rat skeletal muscle in order to study the effect of hypothyroidism on phosphorylase activation in this structural and functional ...unit of the glycogenolytic process. The total activities of phosphorylase and phosphorylase phosphatase in euthyroids and hypothyroids were the same in the fraction containing the protein-glycogen complex (P2 suspension). Hypothyroidism selectively lowered the maximal phosphorylase kinase activity in glycogen particles in the P2 suspension by 40%. Addition of Mg2+ (10 mM), ATP (2 mM), and Ca2+ (5 mM) rapidly stimulated phosphorylase b to a conversion resulting from phosphorylase kinase activation. Hypothyroidism reduced the rate of phosphorylase a formation by 50-70% in the P2 suspension. Glucose 6-phosphate (0.4-1.4 mM) inhibited the rate of phosphorylase a formation and this inhibition was similar for eu- and hypothyroids. There was a shift from 5.2 to 5.8 in the free Ca2+ concentration (pCaF) for half-maximal activation of phosphorylase in the P2 suspension of hypothyroids. A sixfold higher steady-state level of phosphorylase in euthyroids compared to hypothyroids was observed at a pCaF of 5.5. The Ca2+ sensitivity of the phosphorylase kinase, however, was not changed by hypothyroidism. These results provide further insight into the different time course of the phosphorylase activation in skeletal muscle during tetanic stimulation observed in euthyroidism and hypothyroidism (W. J. Leijendekker et al. (1985) Metabolism 34, 437-441).
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