Abstract
Thyroid hormone receptor α1 (TRα1) regulates body temperature and heart rate in humans and mice. In addition to its direct actions in target tissues, it also affects peripheral functions indirectly through the brain. While these central actions on peripheral tissues have been demonstrated for liver and brown fat, the consequences for cardiac functions are still enigmatic. Recently, a population of parvalbumin neurons has been discovered in the anterior hypothalamic area that depends on TRα1 for correct development and controls heart rate in a temperature-dependent manner. Here we test the hypothesis that not only developmental but also acute actions of TRα1 in hypothalamic parvalbumin neurons affect the central control of cardiovascular functions. We used an AAV-mediated stereotaxic approach to express a mutant TRα1R348C conditionally in hypothalamic parvalbumin cells, thus impairing TRα1 action specifically in these neurons. While this had no effect on metabolism or thermoregulation, using non-invasive radiotelemetry we observed a reduced heart rate both at 22°C and 30°C. Interestingly, heart rate was normalized when the animals were measured by ECG, which requires prior handling, suggesting that the impairment caused by the mutant TRα1 can be compensated in more stressful situations. Taken together, our data show that TRα1 signaling in hypothalamic parvalbumin neurons acutely affects the central control of heart rate, adding a novel mechanism to bradycardia in hypothyroidism. Furthermore, the data underline the importance of non-invasive recordings of in vivo functions in animal models with alterations in central thyroid hormone action.
Introduction
Thyroid hormone (TH) levels are known to influence heart rate and body temperature (1). An excess of TH, as seen in hyperthyroidism, causes numerous symptoms such as increased heart rate and heat production. Conversely, a constant deficit of TH, or hypothyroidism, has opposite effects (2). The action of TH is mediated by the binding of the active form of the hormone, 3,3′,5-triiodothyronine (T3), to its nuclear TH receptor (TR), which changes target gene expression in the respective tissues. There are two genes, THRA and THRB, coding for different TR isoforms, namely TH receptor α1 (TRα1) and TH receptor β (TRβ), which are differentially expressed throughout the body. TRβ is predominantly expressed in liver and kidney (2), while TRα1 is the main isoform in the brain and heart. To better understand the downstream effects of TRα1-mediated T3 signaling, a mouse model heterozygous for the TRα1R384C mutation has been generated (TRα1+/m mice). This mutation exchanges one amino acid in the ligand-binding domain, thereby reducing the binding affinity to T3 tenfold. This effectively decreased the downstream signaling of the receptor and mediates a hypothyroid state in TRα1-expressing tissue. The receptor can, however, be reactivated by high levels of T3 (3), allowing to distinguish between acute and developmental defects (4). The major phenotype of TRα1+/m mutants comprises of bradycardia, hypothermia, anxiety, and memory impairment (5, 6, 7, 8). While the anxiety-like behavior and cognition deficits are ameliorated after T3 treatment, other defects are not restored by reactivation of TRα1 signaling in the adult animal, e.g., bradycardia or hypothalamic neuronal development (9, 10, 11). The latter is particularly important, as T3 acts not only locally at the target tissues but also centrally in the brain, where it regulates the autonomic nervous system, which often acts synergistically with the peripheral actions of the hormone (12, 13). In particular, the PV neurons in the anterior hypothalamic area (AHA) require TRα1-mediated T3 signaling for development (9) and have been linked to the modulation of cardiovascular parameters, including blood pressure and heart rate (10, 14). These findings suggest that the central control of cardiac functions is impaired when TRα1 signaling is defective during development (10, 14). However, it is not yet known whether hypothalamic PV neurons also modulate their autonomic output to the heart in response to changes in T3 signaling acutely in the adult animal. To address this question, we used an adeno-associated virus (AAV)-based approach combined with the Cre/Lox system to inhibit TRα1 signaling specifically in the hypothalamic PV neurons. We then monitored the animals using telemetry transmitters and electrocardiogram (ECG) measurements, as well as infrared pictures. Our data show that impaired TRα1 signaling in hypothalamic PV neurons contributes to the bradycardia but has no influence on ECG interval lengths, body weight, food intake, or brown adipose tissue (BAT) thermogenesis. This suggests that hypothalamic PV neurons may be an important neuroanatomical substrate to facilitate changes in heart rate depending on TH signaling.
Methods
Animals
Male parvalbumin-Cre +/tg mice on a C57BL/6NCr background (Strain no. 008069, The Jackson Laboratory) were obtained from Gemeinsame Tierhaltung, University of Luebeck, Germany. The animals were at least 4 months old and single-housed at 22°C, followed by 30°C, under a 12 h light:12 h darkness cycle in a climate-controlled chamber (Flohr instruments, Netherlands). Animals had access to standard chow (#1314; Altromin, Germany) and water ad libitum. All experiments were conducted in accordance with the EU, in compliance with the ARRIVE guidelines, and approved by the MEKUN Schleswig-Holstein (Germany, 79/9-20).
Stereotaxic injection of an adeno associated virus
Parvalbumin-Cre +/tg mice were injected bilaterally with 200–500 nL of an AAV expressing a dominant negative TRα1 (floxed, CMV-TRα1R384C-IRES-Lyn/mCherry, 2.92 × 1012 GC/mL) or green fluorescent protein (GFP) (floxed, SYN1-NLS-EGFP, 2.69 × 1012 GC/mL) (VectorBuilder GmbH, Germany) into the AHA. The coordinates were measured from the bregma: anterior-posterior 0.75 mm, medial-lateral ±0.43 mm, and dorsal-ventral −5.25 mm. Transfection efficiency was 85 ± 18% for controls and 82 ± 17% for dominant negative TRα1 (P = 0.84, 8 PV-TRα1R384C mice were excluded from the analysis due to less than 40% transfection efficiency).
Implantation of telemetry transmitters
The mice were surgically equipped under isoflurane anesthesia with either a G2 or G2-HR E-mitter (Starr Life Sciences, USA), and were placed in their home cage on an ER-4000 receiver plate with a sampling interval of 30 s.
Immunofluorescence staining
Brains were fixed with 4% PFA, and coronal free-floating sections of 20 μm thickness were cut and blocked in 5% donkey serum in 0.3% Triton-X100. They were incubated with the anti-parvalbumin and anti-mCherry primary antibodies (anti-parvalbumin, 1:2,000, PV 27; Swant, Switzerland, and anti-mCherry, 1:1,000, AB0040; OriGene, Germany) or anti-GFP and anti-parvalbumin primary antibodies (anti-mCherry, 1:1,000; ab290; Abcam, UK and anti-parvalbumin, 1:2,000, PVG-213; Swant, Switzerland) in 5% NDS in 0.3% Triton-X100 overnight at 4°C, and with a secondary Alexa Fluor 488-labeled antibody (1:800, A-21206; Invitrogen, Thermo Fisher Scientific, Germany) and Alexa Fluor 594-labeled antibody (1:800, A-11058; Invitrogen, Thermo Fisher Scientific) in 0.3% Triton-X100 and mounted on glass slides with ProLong™ Diamond Antifade Mountant (Invitrogen, Thermo Fisher Scientific).
Infrared photography
Infrared pictures of the BAT, tail, and inner ear of freely moving animals were taken at the beginning of the light phase with FLIR T335 and T450 infrared cameras (emissivity 0.95). To reduce variation between measurements, the fur between the shoulder blades was brushed back with Vaseline to reveal the BAT (15).
Electrocardiogram (ECG)
ECG measurements were performed on the ECGenie tower (Mouse Specifics, Inc., USA) in awake animals. During recording, the mouse was placed on the tower for 10 min. The recordings were then analyzed with the ECGenie software.
Statistical analysis
Statistical analyses were performed using Microsoft Excel and GraphPad Prism 8 (USA). For comparisons of two groups, an unpaired Student’s t test was used. Two-way analysis of variance (ANOVA) with Sidak post hoc testing was performed for comparisons of more than two groups. Longitudinal locomotor activity, body temperature, and heart rate were analyzed at 6 h intervals using CircaCompare (16). Rhythmicity was determined based on a significance threshold of P < 0.05. For direct comparison of mesor and amplitude between groups, CircaCompare fits were utilized regardless of rhythmicity thresholds. Phase comparisons were conducted only when the parameter exhibited rhythmicity in both conditions (P < 0.05).
Results
To determine the contribution of TH signaling in AHA PV neurons, we used stereotaxic injection of an AAV into the AHA of PV-Cre mice. The transgene is activated upon recombination by Cre recombinase selectively in Cre+ cells but remains unexpressed in all other cell types, yielding a mouse model with cell type- and region-specific expression of a mutant TRα1 (termed PV-TRα1R384C mice), which inhibits cellular TR signaling due to its dominant negative properties. We equipped the mice with implantable telemetry transmitters to measure heart rate, body temperature, and locomotion in a continuous, disturbance-free manner (Fig. 1A). Immunofluorescence staining was used to confirm the AAV expression by targeting mCherry or GFP in hypothalamic slices (Fig. 1B), and only animals with detectable expression of the virus in the AHA were included in the analysis. Counterstaining against parvalbumin confirmed the specificity of the viral vector and the parvalbumin-Cre line. We then analyzed the animals at 22°C as well as 30°C (thermoneutrality), as this condition is more representative of the human situation since it alleviates the cold stress arising from rodent housing at room temperature (17) and switches cardiac control to predominantly parasympathetic (14, 17). Our data showed that the basic metabolic parameters body weight, food intake, and water intake were comparable between the groups at both temperatures (Fig. 1C, D, E; absolute weights 22°C: 27.8 ± 4.0 g (controls) and 30.1 ± 1.8 g (PV-TRα1R384C mice); 30°C: 28.9 ± 3.5 g (controls) and 31.2 ± 2.8 g (PV-TRα1R384C mice)).
The influence of hypothalamic parvalbumin TRα1 on food and water intake and body weight. (A) Experimental overview. (B) Immunofluorescence staining of controls and animals with a dominant negative TRα1. (C) Body weight change compared to starting body weight (controls: n = 11; PV-TRα1R384C: n = 7). (D) Food intake (controls: n = 11; PV-TRα1R384C: n = 7). (E) Water intake (controls: n = 9; PV-TRα1R384C: n = 7). Values are mean ± SD, two-way ANOVA with Sidak post hoc test.
Citation: European Thyroid Journal 14, 3; 10.1530/ETJ-25-0055
Given that hypothalamic T3 signaling has been linked to thermoregulation (13, 18), we tested if BAT activity was altered using infrared pictures to measure the BAT surface temperature (15). The BAT temperature did not differ between the groups, neither at 22°C nor at thermoneutrality (Fig. 2B and C), suggesting that the thermogenesis via BAT was not changed in PV-TRα1R384C mice. Tail temperature did also not differ between groups at either temperature (Fig. 2D and E). Core body temperature as measured with the implanted transmitter followed a diurnal pattern in both groups, with no significant differences in mesor (rhythm adjusted mean), phase (time of the highest level), or amplitude (rhythm strength) at 22°C (Fig. 2F, Supplementary Table 1 (see section on Supplementary materials given at the end of the article)). At 30°C, however, the PV-TRα1R384C mice lost the diurnal rhythm with lower mesor and amplitude compared to control mice (Fig. 2G, Supplementary Table 1). The movement activity was also rhythmic in all conditions, with a lower mesor at 22°C in the mutants, which normalized at 30°C (Fig. 2H and I, Supplementary Table 1). In conclusion, TH signaling in PV cells of the AHA seems to have no contribution to average body temperature, but dampens its diurnal rhythmicity at 30°C.
Influence of a dominant negative hypothalamic TRα1 on thermogenesis. (A) Representative infrared pictures of control (n = 12 at 22°C, n = 11 at 30°C) and PV-TRα1R384C animals (n = 7). (B) BAT surface temperature as measured by infrared pictures, normalized to the inner ear temperature (C). Values are mean ± SD (D) Tail base temperature as measured by infrared pictures, corrected by inner ear temperature (E). Values are mean ± SD (F) Core body temperature of controls (n = 10 at 22°C, n = 9 at 30°C) and PV-TRα1R384C animals (n = 6) at 22°C or (G) 30°C, measured by radiotelemetry. Shaded areas indicate lights off. Values are mean ± SEM. (H) Locomotion of controls (n = 11 at 22°C, n = 10 at 30°C) and PV-TRα1R384C animals (n = 7) at 22°C or (I) 30°C, measured by radiotelemetry. Shaded areas indicate lights off. Values are mean ± SEM.
Citation: European Thyroid Journal 14, 3; 10.1530/ETJ-25-0055
Finally, we assessed the contribution of TH signaling in the hypothalamic PV neurons to central cardiovascular control, using ECG recordings in the awake mouse as well as telemetry heart rate data in a touch-free paradigm. The heart rate of both groups did not show a pronounced diurnal profile at 22°C, but was lower in the PV-TRα1R384C animals (Fig. 3A, Supplementary Table 1). At 30°C, however, we detected robust diurnal rhythms in both groups (Fig. 3B, Supplementary Table 1), again with a lower heart rate in the PV-TRα1R384C animals. The heart rate frequency distribution was not significantly different between the two groups, although it seemed that extreme heart rates occurred more frequently in the mutants (Fig. 3C and D). Interestingly, despite the different basic heart rate, there were no differences visible in the ECG, neither in the individual tracings nor in the analyzed parameters (Fig. 3E, F, G, H, I, J, K, L), suggesting that the bradycardia likely originated by slower atrial pacemaking rather than downstream effects. As the ECG requires the animals to be placed on the recording towers, the average heart rate is higher as compared to the data recorded by telemetry due to the more stressful condition involving handling (Fig. 3G). Taken together, the data demonstrate that PV cell-type specific expression of a dominant negative TRα1 in the AHA was sufficient to induce bradycardia in mice, but did not alter ECG intervals or thermogenesis.
Effect of a dominant negative TRα1 in PV cells of the hypothalamus on the cardiovascular system. (A) Heart rate of controls (n = 5) and PV-TRα1R384C animals at 22°C or (B) 30°C (n = 5 at 22°C, n = 4 at 30°C), measured by radiotelemetry. Shaded areas indicate lights off. Values are mean ± SEM. (C and D) Heart rate frequency distribution of control and PV-TRα1R384C animals. (E) Representative ECG tracings of control and (F) PV-TRα1R384C animals. (G, H, I, J, K, L) ECG parameters of control (n = 6) and PV-TRα1R384C animals (n = 7). Values are mean ± SD, two-way ANOVA with Sidak post hoc test.
Citation: European Thyroid Journal 14, 3; 10.1530/ETJ-25-0055
Discussion
To elucidate the role of impaired TH signaling in adult hypothalamic PV cells, which control, e.g. cardiovascular functions, we introduced a conditional dominant negative TRα1 into PV-Cre mice via stereotaxic AAV injection. This approach will antagonize all TR signaling in the AHA PV neurons despite the fact that both TR isoforms are likely expressed in the cells (19). While the expression of the mutant TRα1 in these neurons did not significantly affect general metabolism, including food and water consumption, body weight, and thermogenesis, it caused bradycardia at both room temperature and thermoneutrality. These data suggest that TH action in hypothalamic PV cells can indirectly influence heart rate, possibly in synergy with the well-known peripheral actions of the hormone in the heart.
Central action of thyroid hormone on heart rate
In our previous study, we found that in wild-type animals, a direct intracerebroventricular (i.c.v.) injection of T3 did not alter heart rate (11). This suggests that acute transient changes in central TH signaling are insufficient to affect cardiac functions, in contrast to what is observed for liver (20) and BAT (13). It is, however, not surprising that this observation differs from our current findings, as the treatment was only a single short-term elevation of T3 in the entire brain, while the AAV-based model facilitates a long-term and cell-specific hypothyroidism. Thus, a more comparable model of long-term impaired central TH signaling is TH transporter double knock-out (KO) mice, which lack both MCT8 and OATP1C1 (21). These transporters are essential for the delivery of TH across the blood–brain barrier, so animals with a combined deficiency exhibit central hypothyroidism, while the periphery is hyperthyroid. In these MCT8-OATP1C1 double knockout animals, the average heart rate was unaffected, but the heart rate frequency distribution broadened so that bradycardic and tachycardic bursts were more frequent (22). Interestingly, a similar phenomenon was observed in our AAV model at 30°C, where higher and lower heart rates occurred more frequently than in the controls. Despite the difference in average heart rate, which was only observed in our cell-specific receptor-mediated hypothyroidism, this suggests that central hypothyroidism generally impairs autonomic control, which then causes a broader corridor of allowed heart rates, possibly through the hypothalamic PV neurons.
Developmental action of thyroid hormone on parvalbumin neurons
Similar to our findings, TRα1+/m mice, which have a knock-in of the dominant negative TRα1 in the endogenous locus, thus expressing the mutant TR globally, also display bradycardia (14). This has, however, been attributed to direct actions of the mutant TRα1 in the heart (11). Nevertheless, their central control of cardiovascular functions is also impaired (14), but primarily as a consequence of a developmental impairment in heart and brain (10, 11), including a near total loss of hypothalamic PV neurons (23) caused by impaired TH signaling in the second half of pregnancy (9). Interestingly, in the double transporter KO mice, hypothalamic PV neurons are not affected, as the blood–brain barrier has not yet closed, which allows for a sufficient supply of TH to the developing hypothalamus in this critical period. Consequently, the comparison between the full TRα1+m mice and the AAV model is not meaningful, and further studies with more specific expression of TRα1 in adult Cre mice are required to dissect the individual actions in the different tissues independently of developmental alterations.
Possible mechanisms by which central TH signaling affects the heart
Systemic hyperthyroidism achieved by oral T3 treatment reduces parasympathetic (PSNS) activity in both wild-type and TRα1 mutant mice without affecting SNS (11), indicating that any central effects of TH are likely primarily mediated via PSNS and not SNS. Consequently, one could speculate that increased PSNS activity could be the underlying mechanism for the bradycardia in the PV-TRα1R384C animals seen in our experiments. However, we did not find any alterations in heart rate variability as an indicator of SNS to PSNS ratio. Likewise, none of the other ECG parameters were altered, suggesting that hypothalamic PV neurons might primarily act on the sinoatrial node, but do not affect other signal transmission aspects within the heart. Most interestingly, when recorded on the ECG tower, the difference in heart rate was reversed. This is likely caused by the fact that this setup poses an acutely stressful condition unlike long-term non-invasive telemetry, which is also reflected in the much higher average heart rate determined in the ECG towers in both genotypes. This suggests that centrally induced bradycardia might be overwritten by an anxiety-mediated increase in heart rate, possibly even on the level of the PV neurons, as a recent study found that mice exposed to a threat exhibit increased activity in these neurons (24). Consequently, the ECG recorded heart rate variability may also be affected by the more stressful situation. This suggests that further studies directly recording SNS and PSNS activity in our AAV model under non-stressed conditions would be required to dissect the underlying mechanism in greater detail.
Conclusion
In summary, our data show that impaired T3 signaling in PV neurons of the anterior hypothalamus leads to bradycardia and a less stringent control of allowed heart rates in mice, while there are no obvious changes in general metabolism and a minor defect in nocturnal body temperature upregulation. While these data provide a possible mechanism for how acute central TH signaling affects heart rate control, they also suggest that handling stress is a major confounding factor that can potentially mask any effects and underline the need for non-invasive recordings of body functions in mouse models with central alterations in TH signaling. Moreover, given that hypothalamic PV neurons also regulate blood pressure, it remains to be elucidated whether this parameter could also be affected in the presented animal model.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ETJ-25-0055.
Declaration of interest
J M is a consultant of Rare Thyroid Therapeutics International Ab, Sweden. The other authors declare no conflicts of interest.
Funding
This work was supported by the German Research Council (grants TRR296 LocoTact and Mi1242/9-1 to JM).
Author contribution statement
B K, R D, and S C S performed the experiments; B K, R D, L A, and H O analyzed the data; J M and R D designed the study; B K and J M drafted the manuscript; all authors read, corrected, and approved the final version.
Data availability
Data are available from the corresponding author upon reasonable request.
Acknowledgements
We thank Julia Resch for technical assistance and the GTH Lübeck for excellent animal caretaking.
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