Hygrothermal stress increases malignant arrhythmias susceptibility by inhibiting the LKB1-AMPK-Cx43 pathway

Abstract

High mortality due to hygrothermal stress during heat waves is mostly linked to cardiovascular malfunction, the most serious of which are malignant arrhythmias. However, the mechanism associated with hygrothermal stress leading to malignant arrhythmias remains unclear. The energy metabolism regulated by liver kinase B1 (LKB1) and adenosine monophosphate-activated protein kinase (AMPK) and the electrical signaling based on gap junction protein, connexin43 (Cx43), plays important roles in the development of cardiac arrhythmias. In order to investigate whether hygrothermal stress induces arrhythmias via the LKB1-AMPK-Cx43 pathway, Sprague-Dawley rats were exposed to high temperature and humidity for constructing the hygrothermal stress model. A final choice of 40 °C and 85% humidity was made by pre-exploration based on different gradient environmental conditions with reference to arrhythmia event-inducing stability and risk of sudden death. Then, the incidence of arrhythmic events, as well as the expression, phosphorylation at Ser368, and distribution of Cx43 in the myocardium, were examined. Meanwhile, the adenosine monophosphate-activated protein kinase activator, Acadesine, was also administered to investigate the role played by AMPK in the process. Our results showed that hygrothermal stress induced malignant arrhythmias such as ventricular tachycardia, ventricular fibrillation, and severe atrioventricular block. Besides, hygrothermal stress decreased the phosphorylation of Cx43 at Ser368, induced proarrhythmic redistribution of Cx43 from polar to lateral sides of the cardiomyocytes, and also caused LKB1 and phosphorylated-AMPK expression to be less abundant. While, pretreatment with Acadesine significantly actived the LKB1-AMPK-Cx43 pathway and thus ameliorated malignant arrhythmias, indicating that the hygrothermal stress-induced arrhythmias is associated with the redistribution of gap junctions in cardiomyocytes and the organism's energy metabolism.

Figure 1

Tr and HR trends under HHS are impacted by AMPK activation. (A) Tr trends through time for each group of rats. (B) The time for Tr to reach (42.5 ± 0.5)°C was significantly longer in AICAR pretreated rats than in the HHS group (n = 8). ****P < 0.0001. (C) HR trends over time during hygrothermal exposure. (D) HR at the end of modeling was significantly lower in the A + HHS group than in the HHS (n = 8). ****P < 0.0001.

Figure 2

HHS causes malignant arrhythmias, whereas AICAR lowers the occurrence and severity of arrhythmias. (A) Typical ECG recordings for HHS or A + HHS. (a) The main types of malignant arrhythmias seen in the HHS group were ventricular tachycardia, ventricular fibrillation, and high degree of AV block; (b) After AICAR pretreatment, the A + HHS group did not exhibit malignant arrhythmias, only showing sinus rhythm abnormalities. (B) Number of rats with arrhythmias in each group (n = 8).

Figure 3

Hygrothermal exposure causes acute stress in rats and raises levels of inflammation. (A) Elisa test was used to quantify the stress hormones. (a–c) CRH, ACTH and CORT were significantly elevated in serum of rats after HHS. (B) HHS significantly increased blood glucose levels in rats. (C) Elisa test for serum inflammatory factors. (a, b) Expression of IL-1β and TNF-α were significantly increased by HHS exposure, but levels decreased markedly after AICAR pretreatment.****P < 0.0001.

Figure 4

HHS affects Cx43-pS368 expression and distribution via the LKB1/AMPK pathway. (A) Results of western blot. (a) Western Blot demonstration of p-Cx43, t-Cx43, p-AMPK, t-AMPK, and LKB1 in cardiac tissues from NC, HHS, and A + HHS. (b–f) Quantification of the p-Cx43, t-Cx43, p-AMPK, t-AMPK, and LKB1 level (n = 4). (B) IHC staining of Cx43-pS368. (a) Normal Cx43-pS368 expression in the NC group was high and basically distributed in the transverse junctions, i.e., "end-to-end"; (b) Cx43-pS368 expression was reduced and changed to "side-to-side" distribution in the HHS group; (c) Improvement in the expression and distribution of Cx43-pS368 in the A + HHS group after pretreatment with AICAR; (d) Quantification of the positive area in myocardial tissues from three groups (n = 7). Scale bar = 50 μm. ns P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5

Activation of the AMPK by AICAR injection inhibits HHS-induced myocardial injury and fibrosis. (A) Representative HE staining results of rat myocardial tissues in all groups. (a) Normal expression of myocardial structure of NC group; (b) Alterations in Myocardial Structure in the HHS Group; (c) Improvements in myocardial structure in the A + HHS group. (B) Ultrastructural deformation of rat cardiomyocytes in each group was shown by TEM imaging. (a–c) Normal ultrastructure of the NC group; (d–f) Ultrastructural alterations in the HHS group; (g–i) Improvements of ultrastructure of A + HHS group. (C) Typical Masson staining for fibrosis that stained in blue. (a) Normal Masson staining manifestation in NC group; (b) Significant fibrotic changes in HHS group; (c) Improvement of fibrosis in A + HHS group; (d) Quantification of the fibrotic area of the Masson-stained sections (n = 6). (D) Western Blot Analysis of ST2, a Marker of Myocardial Fibrosis. (a) Western Blot demonstration of ST2; (b) Quantification of the ST2 level (n = 4). ****P < 0.0001.

Figure 6

The possible process through which HHS modifies Cx43 and causes arrhythmia is depicted in a cartoon. By triggering the HPA axis, HHS raises blood glucose levels and suppresses the activity of LKB1. This, in turn, hampers the expression of AMPK, or HHS directly inhibits AMPK expression. Ultimately, these mechanisms contribute to the development of arrhythmias by further influencing the remodeling of Cx43 and initiating fibrosis after inflammatory responses.

Figure 7

The molding process diagram. Rats categorized as NC, HHS, and A + HHS groups received pretreatment with either 1 ml of PBS or AICAR. Subsequently, the HHS and A + HHS groups were subjected to stress exposure within a simulation chamber, while real-time monitoring of arrhythmia was conducted using an electrocardiogram machine. At the conclusion of the modeling, blood was collected from the heart, and left ventricular myocardial tissues were isolated for subsequent experimental analysis.