**1. Introduction**

The regulation of body temperature is fundamental to the maintenance of homeostasis, and adaptive thermogenesis, tightly regulated by the central nervous system, is essential for survival [1,2]. Catecholamines and thyroid hormones (TH) are the most important factors impacting thermogenesis. TH activate thermogenesis in brown adipose tissue, and white adipose tissue can undergo browning via adrenergic stimulation [3]. The adaptation to cold results in the deiodination of thyroxine (T4) and causes an increase of the triiodothyronine (T3) levels in blood. Higher T3 leads to an increase in expression of uncoupling protein 2, following an increase in heat production [4,5].

Cardiovascular system responses to thermoregulatory challenges and cold acclimation may induce cardio-protection [6,7]. However, there is still a gap in the knowledge on the underlying mechanisms by which a temperature challenge or adaptation to cold induces

**Citation:** Andelova, K.; Szeiffova Bacova, B.; Sykora, M.; Pavelka, S.; Rauchova, H.; Tribulova, N. Cardiac Cx43 Signaling Is Enhanced and TGF-β1/SMAD2/3 Suppressed in Response to Cold Acclimation and Modulated by Thyroid Status in Hairless SHRM. *Biomedicines* **2022**, *10*, 1707. https://doi.org/10.3390/ biomedicines10071707

Academic Editor: Julie Chan

Received: 6 June 2022 Accepted: 7 July 2022 Published: 14 July 2022

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cardiac responses. Moreover, molecular mechanisms linked with cardio-protection to maintain heart function in response to cold adaptation are poorly elucidated as well.

The heart is an electromechanical pump, and gap junction channels formed by connexin-43 (Cx43) ensure coupling between cardiac myocytes to enable the transmission of electrical and molecular signals, resulting in coordinated contractions [8,9], while the downregulation of Cx43 and/or channel dysfunction, as well as abnormal Cx43 topology, jeopardize synchronous cardiac electromechanical functions and render the heart prone to developing arrhythmias, including potentially lethal ventricular fibrillation (VF) [10,11]. Moreover, reduced inter-myocyte coupling and communication activate pro-fibrotic signaling, followed by excessive extracellular matrix (ECM) deposition [12], that profoundly contribute to arrhythmogenesis and heart mechanical failure [13].

Of interest, an increased myocardial expression of Cx43 was demonstrated in hibernators during cold acclimatization and hibernation [14]. The upregulation of Cx43 appears to be a mechanism by which the hibernators avoid lethal arrhythmia during hibernation and arousal [15], despite marked changes in body temperature, which, in non-hibernating subjects and humans, induce VF. The questions arise as to whether the upregulation of cardiac Cx43 in response to cold adaptation is a general phenomenon occurring in non-hibernating animals and whether there are sex-related differences.

Genetically, hairless strains of rodents represent rewarding models to study adaptive thermogenesis. It has been reported that hairless coisogenic spontaneously hypertensive rats (SHRM), harboring the mutant desmoglein-4 gene, exhibit increased thermogenesis due to a metabolic adaptation to cold [16]. The standard ambient temperature of 22 ◦C lies well below thermoneutrality for such rats due to the diminished insulating capacity. However, the response of the heart to cold acclimation was not investigated in this strain. It would be of a great interest, since hypertension increases the arrhythmia risk, and SHR has been shown as prone to VF due to the downregulation of myocardial Cx43, its abnormal topology and ECM alterations (fibrosis) [13]. Moreover, SHR differ in Cx43 and ECM responses to altered the thyroid status when compared to normotensive rats [17].

Taken together, we aimed to investigate whether there are specific arrhythmia-related differences between the hearts of hairless vs. wild type SHR and in the response to the induced hyperthyroid or hypothyroid status. In this context, our main objective was to explore the expression of myocardial Cx43 and its topology, as well as the key markers of fibrogenesis in hairless SHR<sup>M</sup> males and females. Additionally, the expression levels of the selected proteins that may modulate Cx43 channel's function were examined in the heart of both SHR strains.
