**4. Discussion**

In the current study, we characterized hairless SHR (SHRM) males and females adapted to cold and compared them with sex- and age-matched wild type SHR. While WKY rats were used as a reference normotensive strain to hypertensive rats, there were no significant differences in the biometric, registered blood samples and cardiac tissue parameters between the hairless SHR<sup>M</sup> and wild type SHR. However, compared to WKY rats, SHR exhibited significantly lower triglycerides and cholesterol but higher circulating T3, as well as higher heart and left ventricular weight, likewise previously reported [17].

Of interest, the enzyme histochemistry revealed an increase of alkaline phosphatase (AP) activity in the arterial portion of the heart capillary network in males and females of either SHR strain but more pronounced in hairless SHRM males when compared to WKY rats. Vascular AP is involved in purinergic metabolism and the production of adenosine, which is a potent vasodilator and anti-aggregator. However, enhanced purinergic signaling may be implicated in the adverse modulation of myocardial Cx43 channel functions in a pathological setting [9]. In contrast to AP, the activity of dipeptidyl peptidase-4 (DPP4), confined to the venous part of heart capillaries, was significantly reduced in hairless SHRM, regardless of the sex, but not in wild type SHR when compared to WKY rat hearts. Vascular DPP4 is a transmembrane serine protease that degrades vasoactive peptides and hormones and is also involved in collagen metabolism [30] and proinflammatory processes [31], while the inflammation deteriorates the Cx43 channel function [32]. Lower DPP4 activity attenuated capillary rarefaction and myocardial inflammation and contributed to a favorable metabolism in an ischemic heart [33]. Taken together, it appears that hairless SHRM may benefit comparing to wild type SHR.

Besides the apparent hypertrophy, the left ventricular tissue of males and females of both SHR strains was focally infiltrated with polymorphonuclears and exhibited perivascular and interstitial fibrosis. Such structural remodeling underlies the arrhythmogenic substrate that increased the propensity of the heart toward VF [13,22,34]. In this context, it should be emphasized that, compared to wild type SHR, the assessed extracellular matrix proteins collagen-1 and hydroxyproline were significantly lower in hairless SHRM males. In parallel, the markers of profibrotic pathways TGF-β1 and SMAD2/3 were lower as well. Unlike SHR males, the females of both SHR strains did not exhibit significant alterations of hydroxyproline, TGF-β1 and SMAD2/3 when compared to WKY rats. Our findings imply that ECM remodeling is less pronounced in hairless SHRM males, as well as in females of both SHR strains, when compared to sex-matched WKY rat hearts. Altogether, it appears that the hearts of hairless SHR<sup>M</sup> males and females might be less prone to developing malignant arrhythmias.

In addition to the arrhythmogenic substrate, one of the key factors impacting cardiac arrhythmias occurrence is disorders of Cx43, as previously reviewed [8,9,11]. Structural remodeling is linked with the abnormal and proarrhythmic topology of Cx43, resulting from the redistribution of Cx43 from the intercalated disc to the lateral sides of the hypertrophied cardiomyocytes, as shown previously [17], and in the current study as well. Indeed, compared to the WKY rat heart, "the lateralization" was significantly increased in SHR males but to lesser extent in hairless SHRM, as well as in females of either SHR strain. Apart from this, the levels of the total Cx43 protein and its variant phosphorylated at serine368 (that reduces Cx43 channel conduction and permeability [35]) were significantly decreased in SHR vs. WKY rat hearts (as was also previously demonstrated [17]) but not in hairless SHRM males and females. Moreover, the expression of the Cx43 variant phosphorylated at serine279, that also hampers Cx43 channel communication [26], exhibited a tendency to decrease in hairless SHR<sup>M</sup> males and females when compared to WKY rat hearts, while no changes were observed in wild type SHR. Finally, a myocardial abundance of β-catenin that impacted the Cx43 channel function [25] was increased in hairless SHR<sup>M</sup> males and females in contrast to reduced levels in wild type SHR when compared to WKY rat hearts. Both β-catenin and plakoglobin have been indispensable for maintaining mechanoelectrical coupling in the heart to prevent cardiac arrhythmias [36]. Altogether, the myocardial Cx43-related findings indicate that hairless SHR<sup>M</sup> might be less prone to developing malignant arrhythmias than wild type SHR, namely in males. It is important, because we have previously shown [37] that the threshold to induce ventricular fibrillation was significantly lower in wild type SHR males comparing to normotensive Wistar rats, while females, regardless the strain, are less prone to malignant arrhythmias, in part due to a higher level of Cx43 expression most likely induced by estrogen [38].

Considering the fact that the phosphorylation of Cx43 is required for Cx43 channel function, we were interested to find whether the myocardial expression of most relevant protein kinases interacting with Cx43 [26,27] differ between SHR rat strains. Indeed, the expression of PKCε was increased in hairless SHR<sup>M</sup> males and MAPK42/44 in SHR<sup>M</sup> females vs. sex-matched wild type SHR, while the expression of PKA was lower in hairless SHRM males vs. wild type SHR. These differences are challenging for further research, since the overexpression of protein kinases may increase Cx43 phosphorylation and vice versa, thereby impacting the channel function [39].

Adrenergic stimulation is important for the cold acclimation [28,29], and adrenoceptors are directly involved in Cx43 mediated inter-myocyte communication [40]. An increase of β2-AR and β3-AR in normotensive rats exposed to cold acclimation at 8 ◦C for 5 weeks was reported [7]. Of interest, we revealed a distinct expression of β-adrenoceptors in the SHR vs. WKY strains. There was a decrease of cardiac dominant β1-AR protein in males, as well as females, and a decrease of β2-AR protein in males of both SHR strains when compared to WKY rats. It suggests the possible downregulation of these receptors and, hence, the attenuation of their undesirable impact on Cx43 in both SHR strains. There was no difference in β3-AR expression among WKY, wild type SHR and hairless SHRM males, unlike hairless SHR<sup>M</sup> females, that exhibited an increased expression of β3-AR comparing to WKY or wild type SHR. Increased β3-AR may be beneficial due its anti-fibrotic and anti-hypertrophic signaling [29].

Taken into consideration another hormonal factor impacting thermogenesis [3], such as thyroid hormones, we were interested in registering potential differences in the responses of hairless SHR<sup>M</sup> vs. wild type SHR to alter the thyroid status. The latter is known to affect the propensity of the heart to arrhythmias, in part due to modulation of the myocardial expression of Cx43 [41].

We demonstrated that the expression of Cx43 and its variant phosphorylated at serine368, as well as PKCε, were increased in hairless SHRM; similar to wild type SHR males and females in response to their hypothyroid status. It might contribute to a lower susceptibility of the hypothyroid SHR heart to malignant arrhythmias, as it was reported in hypothyroid normotensive rats [42]. On the other hand, an undesirable response to the hypothyroid status was an increase of the myocardial matrix proteins, collagen and hydroxyproline in males of both SHR strains and, to a lesser extent, in SHR<sup>M</sup> females. Of interest, the hyperthyroid status did not affect the markers of the extracellular matrix in either SHR strain but resulted in the suppression of Cx43 and its serine368 variant in hairless SHRM males, as well as in wild type SHR males. It may predict a higher susceptibility to arrhythmias due to hyperthyroidism, as reported in normotensive rats and humans [41,42].

## **5. Conclusions**

The results of this study indicate differences in the levels of the cardiac pro-arrhythmia factors between the hearts of hairless (in particular, in males) vs. wild type SHR while not in the response to an induced hyperthyroid or hypothyroid status. The main findings, i.e., upregulation of myocardial Cx43, along with the suppression of fibrotic factors, suggests that hairless SHR<sup>M</sup> are more likely to benefit from adaption to the cold most due to suppression of a malignant arrhythmia risk. Increased thermogenesis induced by the cold might be a possible cause for observed cardiac differences in the SHRM and wild type SHR. The findings are a challenge to explore the mechanisms by which adaptation to cold triggers a cardiac response.

#### *Limitations*

We did not assess either the function of the hairless SHR<sup>M</sup> heart or its susceptibility to malignant arrhythmias. This should be explored in the next study to provide the evidence that the adaptation of hypertensive rats to the cold may attenuate their higher susceptibility to lethal cardiac arrhythmias. It might be challenging for clinical trials and the innovative management of hypertensive patients.

**Author Contributions:** Conceptualization, N.T. and S.P.; methodology, K.A., B.S.B., M.S., H.R. and S.P.; validation, N.T., K.A., B.S.B. and S.P.; investigation, K.A., B.S.B., M.S. and H.R.; resources, N.T.; data curation, K.A., B.S.B., H.R., S.P. and N.T.; writing—original draft preparation, K.A. and N.T.; writing—review and editing, N.T., K.A., B.S.B., M.S., H.R. and S.P.; visualization, N.T., K.A. and S.P.; supervision, N.T. and S.P.; project administration, S.P. and N.T. and funding acquisition, N.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by VEGA grants 2/0002/20 and 2/0158/19; Slovak Research and Development Agency under the Contract no. APVV-21-0410, APVV-18-0548 and APVV-19-0317 and EU Structural Fund ITMS 26230120009 from the Ministry of Education.

**Institutional Review Board Statement:** The maintenance and handling of animals were performed in accordance with the "Guide for the Care and Use of Laboratory Animals" published by US National Institutes of Health (NIH publication No 85-23, revised 1996) and approved by the Ethical Committee of the Institute of Physiology v.v.i., (approval code—49/17/45.0, approved date—16 February 2017) Academy of Sciences of the Czech Republic, Prague.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors thank Ing. Michal Pravenec and his team for providing the hairless SHR, as well as for the excellent technical assistance to Karla Bohunova from the Institute of Physiology, v.v.i., AS CR, Prague.

**Conflicts of Interest:** There are no conflict of interest.
