Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis. In this article, Mayerl and colleagues demonstrate that the thyroid hormone transporters MCT8 and OATP1C1 are unique gate-keepers in activated muscle stem cells. The expression of both transporters increases upon activation of muscle stem cells, while loss of MCT8 and OATP1C1 expression results in impaired muscle stem cell differentiation

Connexin45 is expressed in vascular smooth muscle but its function remains elusive.

Connexins (Cx) form gap junctions and allow the coordination of cellular behaviour. In vessels, expression of Cx40, Cx37, and Cx43 is well established and specifically Cx40 serves important functions in endothelial cells. In contrast, expression and physiological functions of Cx45 is unclear although its expression has been suggested in vascular smooth muscle (VSM). Therefore, we studied expression and function of Cx45 in vessels using different mice models allowing to identify and delete Cx45. Smooth muscle cell (SMC)-specific deletion was achieved by the Cre/loxP system using Cre-recombinase driven by a Nestin promoter. Deletion of Cx45 leads concomitantly to the expression of enhanced green fluorescence protein (EGFP) in these mice. Conduction of vasomotor responses was studied in cremasteric arterioles using intravital microscopy and arterial pressure was measured telemetrically. Cx45 is transcriptionally expressed in VSM as detected by EGFP expression in SMC-specific Cx45-deficient mice (Cx45fl/fl:Nestin-Cre) but not in endothelial cells (Cx45fl/fl:TIE2-Cre). Moreover, EGFP was located at VSM cell borders in arterioles of transgenic mice carrying an EGFP-tagged Cx45. Expectedly, arteriolar conduction of dilations evoked by the endothelium-dependent agonist acetylcholine were not different between Cx45fl/fl:Nestin-Cre mice and controls carrying homozygously a floxed Cx45 gene (Cx45fl/fl). Surprisingly, the amplitude of locally initiated endothelium-independent constrictions (K(+)) and dilations (adenosine) declined similarly with distance in both genotypes indicating an intact VSM conduction pathway also in mice being deficient for Cx45 in VSM. Arterial pressure was not different between freely moving Cx45fl/fl and Cx45fl/fl:Nestin-Cre mice during day or night. We conclude that Cx45 is physiologically expressed in VSM, but not in EC in murine arterioles. However, Cx45 is dispensable for the conduction of vasomotor responses along these arterioles. Possibly, other Cx functionally replace the lack of Cx45 in VSM. The reported role of Cx45 in renin secretion does not seem to alter arterial pressure in freely moving mice

Conduction of KCl-induced constrictions in mice deficient for Cx45 in vascular smooth muscle.

<p>Time courses of local and upstream constrictions evoked by depolarizing K⁺ stimulation. Brief local application of K⁺ (3 mol/L) induced a rapid, transient constriction of the arterioles at the local site (A). The constriction conducted to remote, upstream sites (B: 0.3, C: 0.66, D: 0.9 mm) with a decreasing amplitude that was similar in Cx45fl/fl and Cx45fl/fl:Nestin-Cre mice. In both genotypes amplitudes decreased with increasing distance from the application site. Six or 8 arterioles were studied in 4 animals of each group.</p

Arterial blood pressure in mice deficient for Cx45 in vascular smooth muscle.

<p>Arterial pressure measured by telemetry was not different between genotypes in conscious freely moving mice. A: Systolic, mean, and diastolic pressures as well as heart rate averaged for 24 h from measurements at day 5 to 7 after implantation of the telemetric device in Cx45fl/fl (white) and Cx45fl/fl:Nestin-Cre mice (grey) were not different from each other. B: Values obtained 12 to 14 days after implantation are separately depicted for day (plain) and night (hatched) indicating circadian rhythm which was pronounced in Cx45fl/fl:Nestin-Cre. Also at this time point pressures and heart rate were not different between genotypes. n = 8 animals each genotype, * <i>P</i><0.05 vs. day values.</p

Connexin45 expression in larger vessels.

<p>Expression of EGFP in smooth muscle cells of the gracilis artey (A, B) and the femoral artery (E, F) in Cx45fl/fl:Nestin-Cre mice but not in Cx45fl/fl:TIE2-Cre (C, gracilis artery marked with arrows) and Cx45fl/fl controls (D: gracilis artery, G, H: femoral artery). The vein (lumen marked by *) accompanying the gracilis artery was not stained in Cx45fl/fl:Nestin-Cre (A) or Cx45fl/fl:TIE2-Cre (C) compared to control (D). Nuclei are stained in blue (TO-PRO-3). Images are representative for at least n = 3 each genotype. Calibration bars are 100 (A, C, D, E, G) or 50 µm (B, F, H).</p

Conduction of vasomotor signals along the smooth muscle cell layer.

<p>Maximal amplitude of the constriction evoked by brief microapplication of depolarizing K⁺ solution (A) or application of adenosine (B, 10 mmol/L) are depicted as a function of distance from the stimulation site. A: The maximal amplitude of the K⁺-induced constriction decreased with distance and differences between genotypes were not observed (n = 6 or 8 arterioles in 4 animals each genotype). B: Likewise, the maximal amplitude of the adenosine-induced dilation decreased significantly with distance in Cx45fl/fl mice (n = 10 in 3 animals) and a similar trend was observed in less experiments in Cx45fl/fl:Nestin-Cre (n = 6 in 3 animals). Differences between genotypes were not observed. *: <i>P</i><0.05 vs. local site, #: <i>P</i><0.05 vs. 0.6 mm.</p

Conduction of endothelium-dependent dilations.

<p>Local and conducted dilations evoked by the endothelium-dependent agonist acetylcholine (ACh). Brief, local application of ACh (1 mmol/L) induced a transient local dilation (A) that was conducted to remote upstream sites (B: 0.6, C: 1.2 mm) without measurable delay in both genotypes. The maximal amplitude of the local dilation was achieved earlier and the dilation was shorter in Cx45fl/fl:Nestin-Cre animals reflecting most likely less vigourous stimulation. Nevertheless, the dilation conducted to remote sites up to the furthest distance studied also in these mice without attenuation of the amplitude. Six or 7 arterioles were studied in 4 or 5 animals of each genotype.</p

Thyroid Hormone Transporters MCT8 and OATP1C1 Control Skeletal Muscle Regeneration

Thyroid hormone (TH) transporters are required for the transmembrane passage of TH in target cells. In humans, inactivating mutations in the TH transporter MCT8 cause the Allan-Herndon-Dudley syndrome, characterized by severe neuromuscular symptoms and an abnormal TH serum profile, which is fully replicated in Mct8 knockout mice and Mct8/Oatp1c1 double-knockout (M/O DKO) mice. Analysis of tissue TH content and expression of TH-regulated genes indicate a thyrotoxic state in Mct8-deficient skeletal muscles. Both TH transporters are upregulated in activated satellite cells (SCs). In M/O DKO mice, we observed a strongly reduced number of differentiated SCs, suggesting an impaired stem cell function. Moreover, M/O DKO mice and mice lacking both transporters exclusively in SCs showed impaired skeletal muscle regeneration. Our data provide solid evidence for a unique gate-keeper function of MCT8 and OATP1C1 in SC activation, underscoring the importance of a finely tuned TH signaling during myogenesis.status: publishe