Hygrothermal stress increases malignant arrhythmias susceptibility by remodeling connexin43 via AMPK

doi:10.21203/rs.3.rs-3249708/v1

Download PDF

Article

Hygrothermal stress increases malignant arrhythmias susceptibility by remodeling connexin43 via AMPK

https://doi.org/10.21203/rs.3.rs-3249708/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 29 Feb, 2024

Read the published version in Scientific Reports →

You are reading this latest preprint version

High mortality due to hygrothermal stress (high temperature and humidity, HHS) during heat waves is mostly linked to cardiovascular malfunction, the most serious of which are malignant arrhythmias. However, the mechanism associated with HHS leading to malignant arrhythmias remains unclear. Here, SD rats were exposed to 40°C and 85% humidity for constructing the HHS model, and the incidence of arrhythmic events, as well as the expression, phosphorylation, and distribution of Cx43 in the myocardium, were examined. The adenosine monophosphate-activated protein kinase (AMPK) activator, AICAR, was also administered to investigate the role played by AMPK in the process. Our results showed that HHS could induce malignant arrhythmias such as ventricular tachycardia (VT), ventricular fibrillation (VF), and severe atrioventricular block (AVB). Besides, HHS increased the distribution of "side-to-side" connections, decreased the phosphorylation of Cx43, and enhanced myocardial fibrosis. Furthermore, HHS also caused LKB1 and p-AMPK expression to be less abundant. While, pretreatment with AICAR could significantly ameliorate Cx43 remodeling and malignant arrhythmias, indicating that the HHS-induced arrhythmias is associated with the redistribution of gap junctions in cardiomyocytes, the dysfunction of intercellular conduction, and the organism's energy metabolism.

Health sciences/Cardiology

Health sciences/Diseases

Extremely high temperatures and humidity, as a severe stress element, will most likely have negative implications as global climates change. Acute hygrothermal stress (HHS) can result in heat stroke (HS), a deadly pyrogenic condition with a morbidity and fatality rate as high as 40% [1, 2]. Over the past several decades, the prevalence of HS has drastically grown; during the summer heat waves, the incidence ranges from 17.6 to 26.5 per 100,000 people, and up to 60% of patients with characteristic HS are hospitalized or discovered dead within one day of the reported beginning of illness [3, 4]. Furthermore, it is predicted that by 2030, the economic costs of heat stroke would surpass $2.4 billion[5]. The cardiovascular system is crucial to the systemic heat response and organ perfusion during sudden death brought on by HHS [6, 7]. The percentage of cardiovascular dysfunction in individuals with severe HS that had multiple organ failure can range from 43.4–65.2% [8]. Up to 90% of the overall increase in mortality during heat waves has been linked mostly to the cardiovascular system, with malignant arrhythmias serving as the primary reason [9, 10]. Arrhythmias do occur during HHS, but the underlying pathophysiologic and molecular processes are still poorly understood, and there aren't many effective and focused treatment options. It is vital to investigate the best course of action for its successful treatment to lower mortality.

Gap junctions, a unique membrane structure made of connexin (Cx), are the structural basis for electrical, metabolic, and mechanical coupling between neighboring cells. They are also an important channel for electrochemical signaling, maintenance of inter-cardiac communication, regulation, and assurance of normal cardiac rhythmicity by neighboring cardiomyocytes, which plays a crucial role in the development of cardiac arrhythmias [11–13].Cx43, the primary channel protein produced by cardiac working cells, is nearly exclusively present in the atrial and ventricular myocardium as well as the distal conduction system of all adult mammalian hearts. Its primary role is to influence the transmission of electrical impulses between cardiac myocytes. It [14, 15]. In the diseased condition, the decline in Cx43 expression and phosphorylation levels, as well as the aberrant distribution, can result in anisotropy of electrical signal conduction in cardiac myocytes, which can induce arrhythmias and even the occurrence of sudden cardiac death [16–18].

Arrhythmia development and improper energy metabolism in cardiomyocytes are intimately connected [19]. Adenosine monophosphate-activated protein kinase (AMPK), a serine-threonine kinase, controls energy metabolism as well as other cellular functions such as autophagy, apoptosis, oxidative stress, and inflammation [20–23]. Its activity has a significant role in a number of cardiovascular illnesses that cause arrhythmias by downregulating ion channels and gap junctions, changing arrhythmogenic circumstances, and causing electrophysiological dysfunction [24–26]. Meanwhile, as a key upstream kinase of AMPK, liver kinase B1 (LKB1) phosphorylates and activates AMPK to control cellular functions and energy metabolism [27, 28]. It has been shown in many studies that AMPK and Cx43 play a significant role in the emergence of arrhythmias, but it has not yet been determined whether HHS causes arrhythmias by controlling Cx43 expression via the LKB1/AMPK pathway[29–31].

In this study, possible pathogenesis and intervention targets were identified for HHS-induced arrhythmias. Primarily, we established the HHS rat model in a unique temperature-controlled environment to observe the occurrence of arrhythmias. Then, in order to confirm the function of AMPK in the arrhythmia process brought on by HHS, we pretreated one group with AICAR, an AMPK agonist. Finally, to investigate the alteration of Cx43, AMPK, LKB1, and other associated components immunohistochemistry (IHC), western blotting (WB), and morphological and histological investigations were employed.

2.1 The progression of Tr and HR under HHS is slowed down by activating AMPK

After entering the HHS chamber, the Tr of rats climbed quickly from the Tcb to 40°C within 30 minutes, and the rate of increase of Tr reduced in the following 20 minutes before reaching (42.5±0.5)°C in about 80 minutes (Fig. 1A). In the meantime, the HR in rats was practically constant for 30 min from the start of the hygrothermal exposure, then climbed quickly within 30 min, peaking at (41.8 ± 0.15) °C, and then fell slightly until the modeling was complete (Fig. 1C). Yet, early treatment with an AMPK activator dramatically reduced the development of Tr and HR under HHS (Fig. 1B and D).

2.2 Activation of the AMPK by the AICAR injection attenuates arrhythmias in rats

Due to physiological changes such as electrolyte imbalances and dehydration, sinus arrhythmic events such as sinus tachycardia can happen in high temperature and high humidity environments. In the current study, we discovered an increased propensity for extreme HHS to cause fatal arrhythmias such as ventricular tachycardia (VT), ventricular flutter (VF), and high/third-degree atrioventricular block (AVB) (Fig. 2A.a). Additionally, we demonstrated that pre-injecting rats with AICAR dramatically reduced the occurrence of ventricular arrhythmias during HHS compared to untreated rats and that HHS also decreased the susceptibility of rats to AVB (Fig.2A.b and B).

2.3 Hormonal stress markers and inflammatory factors were both upregulated by HHS.

Following the end of the hygrothermal exposure, the serum expression levels of the hormone markers ACTH, CRH, CORT, and blood glucose were considerably higher in the HHS group than in the NC (Fig.3A and B). In addition, HHS had higher levels of IL-1β and TNF-α, which dramatically decreased after receiving AICAR (Fig. 3C).

2.4 HHS remodeled Cx43 by inhibiting activation of the LKB1/AMPK pathway

The expression and phosphorylation levels of the proteins Cx43 and AMPK during HHS were revealed by the western blot data. When compared to the NC group, the HHS group showed significant downregulation of p-Cx43, t-Cx43, p-AMPK, and LKB1 expression (Fig. 4A.a-f). Following pretreatment with AICAR, the expression of total Cx43 and the phosphorylation levels of Cx43 and AMPK were significantly higher in the A+HHS group (Fig. 4A.b-d). Nevertheless, AMPK activation does no effect on LKB1 expression (Fig. 4A.f), and there was no statistically significant difference in total AMPK across the three groups ( Fig. 4A.e). Meanwhile, immunohistochemical staining showed that the distribution of Cx43 in NC was centered on the intercalated discs (end-to-end junctions of cardiomyocytes) of cardiomyocytes, with a linear distribution perpendicular to the longitudinal axis of cardiomyocytes (Fig.4B.a). In contrast to NC, the Cx43-positive area in HHS myocardial tissues was greatly decreased (Fig. 4B.d), and the distribution of myocardial regional staining spots and plaques was random and primarily dispersed in the shape of side-to-side junctions (Fig. 4B.b). After receiving AICAR pretreatment in the A+HHS group, Cx43 expression and its "end-to-end" distribution ratio considerably increased when compared to HHS (Fig. 4B.c and d).

2.5 HHS leads to myocardial injury and fibrosis

H&E showed that the transverse stripe of cardiomyocytes in the NC group was clear, the nuclei of the cells were dense, the staining was homogeneous, and the pathological manifestations, such as the inflammatory cellular infiltration, were not seen (Fig.5a); whereas, in the HHS group, the myocardial tissue structure was blurred, cell gaps were enlarged, myofibrils were loosely arranged and disorganized, and some myofibrils were fractured and cleaved, with focal inflammatory cell infiltration and erythrocyte exudation (Fig.5A.b). Additionally, TEM analysis revealed that the NC group's myocardial myogenic fibers were neatly aligned, with regular Z lines and clear myofilaments; intercalated disks were clearly visible and mostly stepped; and mitochondria and nuclei had a normal structure (Fig. 5B.a-c). However, the ultrastructure of rat cardiomyocytes was significantly altered after HHS: the fiber gaps were widened, some fibers were even broken, and the Z line was blurred; intercalated discs were not clearly visible, and the connecting gaps were widened; mitochondria were swollen, cristae were broken, and vacuolated; and the nuclei were atrophied, and the nuclear membrane was irregular and jagged (Fig.5B.d-f). At the same time, Masson discovered that the HHS rats' cardiac tissue had a much higher level of fibrosis than the NC rats did ( Fig. 5C.d). Compared with the HHS group, the myocardial injury and fibrosis were significantly improved in the A+HHS group (Fig.5A.c, B.g-i, and C.c-d).

In the current work, we provided the first evidence of the function of Cx43 in controlling the development of arrhythmias, inflammation, and cardiac fibrosis brought on by HHS. The cardiac tissues of post-HHS arrhythmic rats showed substantially reduced Cx43 expression and phosphorylation as well as disorganized redistribution. Functionally, HHS caused cardiac fibrosis and inflammation, which made rats more vulnerable to dangerous arrhythmias. The HHS-induced index alterations were greatly mitigated by AMPK activation. From a mechanical standpoint, we showed that HHS prevented the LKB1/AMPK/Cx43 signaling pathway from being activated (Fig. 6).

Stress is defined as a sequence of physiological reactions initiated when the organism is aroused by numerous stressors in the internal and external surroundings, as well as several consequent changes in function and metabolism. The rhythm of the heart is impacted by stress, and in extreme circumstances, this can result in malignant arrhythmias or even abrupt cardiac death [32,33]. According to Benito B[34], high temperatures and humidity levels might result in oxidative stress, which can compromise heart function and trigger cardiomyocyte death. Acute stress was created using a model by Liu et al[35]. in goats, and it was shown that this stress resulted in a large rise in ventricular arrhythmias and altered ventricular electrophysiology. As a stressful disease that gravely jeopardizes life and health in the face of global warming, HS urgently requires the exploration of appropriate intervention targets and therapeutic strategies [36,37]. The effects of humidity were disregarded in earlier proposed animal models of HS [38–40], which established desert-like environments. However, research has shown that people feel hotter in high humidity than in low humidity[41]. For this reason, in this investigation, we consulted methodologies from the literature and took into account the impact of humidity before deciding on 40 °C and 85% humidity as the ambient parameters for HHS-induced arrhythmias. What is interesting is that, in addition to the previously described VT and VF, rats were also particularly prone to AVB under this stressful environment. We hypothesize that the cause of this might be that abnormal ATP release during the HHS state results in increased secretion of inflammatory substances like IL-1β and TNF-α, which facilitates the growth of myocardial fibrosis and, in turn, causes abnormal atrial impulse formation and conduction, which, in turn, triggers the emergence of AVB [42–44].

Gap junctions are implicated in the emergence of cardiac disorders such as arrhythmias, heart failure, and hypertrophy, according to mounting evidence [45, 46]. Gap junction protein Cx43, the major isoform expressed in ventricular myocytes, is crucial in defining cardiac excitation patterns and arrhythmia susceptibility [47]. In various types of stress models, the reduced expression and phosphorylation of Cx43 decreases myocardial excitability and conductivity, alters the conduction of electrical impulses, and increases vulnerability to ventricular arrhythmias [48–50]. In our research, we found that the HHS-induced reduction in t-Cx43 protein expression and its phosphorylated forms suggest that Cx43 may potentially be implicated in the occurrence of various arrhythmias, including AVB and VT/VF. Cx43, the primary channel protein expressed by myocardial working cells, is primarily found in the intramyocardial disc in the form of "end-to-end," ensuring the directionality of electrical impulse conduction. A small amount of it is also found in the connection between the cardiomyocytes' two sides, or the "side-to-side" connection, coordinating the transmission of electrical signals between adjacent parallel myocardium and preserving continuity and synchronicity. Slower conduction and higher electric heterogeneity, which eventually served as the foundation for the development and maintenance of VAs, were induced by the downregulation of Cx43 and aberrant distribution brought on by HHS, particularly the increase in side-to-side connections. Cxs, one of the less well-studied routes, can also lead to fibrosis [51,52]. Reduced Cx43 expression has been linked to increased collagen deposition in studies of non-cardiac tissues [53]. In the heart, Cx43 is strongly expressed in cardiomyocytes and fibrosis [54]. The amount of Cx43 expression has been linked to CF proliferation mechanistically [55]. We discovered that the dysregulation of cardiac gap junctions caused by Cx43 downregulation during the current HHS-induced arrhythmia correlated to increased fibrosis, which in turn further led to conduction anomalies and the genesis of conduction block.

We have further evidence that HHS changed the phosphorylation state of AMPK in the emergence of HHS-induced arrhythmias. We preinjected the rats with the AMPK agonist AICAR before the exposure in order to investigate the underlying mechanism of AMPK action in the development of HHS-induced arrhythmias. We discovered that Cx43 protein expression and phosphorylation were noticeably increased in myocardial tissue, and the "side-to-side" distribution was diminished compared to the HHS group. Additionally, HHS-induced inflammation, ultrastructural abnormalities, and fibrosis are all greatly reduced by activating AMPK. Most importantly, AMPK activation markedly decreased vulnerability to HHS-induced arrhythmias. These findings, which are consistent with research by Alesutan [56], suggest that AMPK regulates the phosphorylation of Cx43 and the development of arrhythmia via HHS. AMPK is a key regulator in the cardiac stress response that is triggered by increases in cytosolic Ca2+ concentration as well as the AMP/ATP concentration ratio and hence detects cellular Ca2+ overload and energy depletion [57]. HHS can trigger abnormal ATP release, whereas AMPK operates as an energy receptor in response to energy excess, and ATP competitively displaces AMP to decrease AMPK activity [58]. Several recent findings [59,60] have substantiated the relationship between the AMPK signaling system and Cx43 expression in CFs, implying a function for this route in the fibrotic response common to multiple cardiac diseases. The decrease in AMPK phosphorylation enhances the production of pro-inflammatory molecules such as IL-1β and TNF-α, which mediate the inflammatory response, induce Cx43 remodeling, and further promote the formation of cardiac fibrosis for arrhythmogenesis [61,62].

Furthermore, we showed that HHS influenced the protein expression of liver kinase B1 (LKB1), whereas LKB1 protein expression was not significantly altered after pretreatment with the AMPK pathway agonist. These findings suggest that in the process of HHS generating arrhythmias, liver kinase B1 (LKB1) works as an upstream master kinase to phosphorylate and activate AMPK, which impacts the apoptosis of cardiomyocytes and gap junctions [63]. Meanwhile, HHS considerably increased hypothalamus pituitary adrenal (HPA) hormone axis stress indexes. We hypothesize that this is because HHS stimulates the release of cortisol hormones by activating the HPA axis, which in turn activates the mitochondrial oxidative stress system, causing increased reactive oxygen species production, which leads to the dissociation of LKB1, reduces the interaction between LKB1 and AMPK, and decreases the level of phosphorylation of the T172 site, which inhibits AMPK activity [64]. AMPK activation may also protect cardiac gap junctions from remodeling in hyperglycemia, according to Li et al. [65].

There are several constraints to our study. Although rodents are commonly employed in cardiac arrhythmia research, they are not appropriate animal models for this subject due to differences in heart size, heart rate, and action potential structure when compared to humans. Furthermore, we found that the decreased expression and aberrant distribution of Cx43 in HHS-induced arrhythmic rats were connected with AMPK, although we did not study the particular mechanism by which AMPK regulates Cx43. Therefore, it is unclear whether AMPK controls Cx43 remodeling via the "ubiquitin-proteasome system" or by noncatalytic processes such as protein complex folding, protein interactions, or subcellular targeting [66,67]. Furthermore, both LKB1 and Cammk2 operate as upstream kinases for AMPK, however, the involvement of Ca2+ in HHS-induced arrhythmias was not investigated in this work. These crucial concerns need to be carefully handled in the future.

As a conclusion, our recent research has shown that the genesis of arrhythmias is significantly influenced by the lower expression of Cx43 in the cardiac tissues of HHS rats. For the development of the arrhythmias brought on by HHS as well as the underlying fibrotic and inflammatory responses in the myocardium, Cx43 expression, and distributional remodeling are necessary. To the best of our knowledge, the current work is the first to demonstrate that acute HHS not only causes ventricular arrhythmias but also increases susceptibility to other malignant arrhythmias like AVB, and in the meanwhile we demonstrate the pathophysiologic relationship of Cx43 in this context. Our research suggests that activating AMPK inhibits the emergence of arrhythmias brought on by HHS and may represent a unique strategy for preventing arrhythmias by concurrently inhibiting inflammatory and fibrotic responses.

4.1 Ethics statement

All experimental protocols and methods were performed in accordance with the relevant guidelines and regulations by General Hospital of Southern Theater Command (IAC11e approval No. 104110701). And the portion of the study involving live animals was conducted in compliance with ARRIVE guidelines.

4.2 Animals

We used 24 adult male Sprague-Dawley rats (280-350g, GUANGDONG MEDICAL LABORATORY ANIMAL CENTER, Guangzhou, China) that were 8 weeks old. All experimental animals were housed under standard experimental conditions with a controlled ambient temperature of (22±1)°C, relative humidity of (50±5)%, a light-dark cycle of 12 h, and adequate feed and water sources.

4.3 Experimental groups

Animals were divided into one of the following three groups at random: NC (normothermic controls) group: kept under standard experimental conditions throughout the entire experiment; HHS group: rats pretreated with 1ml PBS by intraperitoneal injection and received HHS; and AICAR (A) + HHS group (with AMPK agonist): rats were pretreated with AICAR, dissolved in 0.1% DMSO in PBS (phosphate buffered saline), and administered in a dose of 500 mg/kg by intraperitoneal injection 1 hour prior to the start of HHS (Fig. 7). Blood samples and heart tissue were taken at the conclusion of the experiment for biochemical analysis and histology.

4.4 Basic physical parameters measure

Rat rectal temperature (Tr) was measured using an animal-specific electronic thermometer (OMRON Co., Ltd., Dalian), and Tr represented core body temperature. The anal temperature was recorded twice daily for seven days on twelve randomly chosen rats. The basic core temperature (Tcb) was calculated using the mean anal temperature. Additionally, baseline weight was assessed and recorded before each group of rats undergoing HHS, and again following the conclusion of modeling. Meanwhile, rats in each group were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg) 1 day before the experiment, fixed in the supine position, and then the limbs were dehairing and connected to the veterinary electrocardiogram machine (HB-A3, Zhuhai Hongbang Medical Technology Co., Ltd.) to record the basic electrocardiographic parameters, such as the base heart rate (BHR) and the standard limb-lead electrocardiographic waveforms. (Table 1).

Table 1 Basic physical parameters

Parameters	NC (n=8)	HHS (n=8)	A+HHS (n=8)	P-value
Weight, g	305±16.65	312.4±14.26	308±19.86	0.690
Tcb, ℃	36.9±0.15	37.0±0.21	36.9±0.19	0.564
BHR, beats/min	346.4±39.81	350.3±38.12	346.48.9±32.17	0.978

4.5 Hygrothermal exposure

According to the studied literature[38–40], the experimental rats were maintained normally at the hospital laboratory animal facility for a week to acclimate to the environment and fasted for a day before the stimulation. Prior to the hygrothermal stimulation, the artificial thermal climate simulation chamber (920 incubator, China Ningbo David medical equipment co., LTD.) was preheated to 40 °C with 85 % humidity, and then, except for the NC group, which was placed in a room temperature environment throughout the whole process, the rats in the HHS and the A+HHS group were put into the chamber in batches for the stimulation to prepare the HHS model. Tr was monitored transrectally throughout the modeling period (probe implanted into the anus at about 3-5 cm) and recorded every 10 minutes until Tr reached the (42.5±0.5)°C modeling success threshold, as well as the duration of any arrhythmic events that occurred during HHS.

4.6 ECG acquisition and analysis

Rat limbs were linked to the ECG machine during HHS, and the self-contained ECG module examined the ECG parameters. Arrhythmic events and changes in HR were recorded (feed speed, 25 mm/s, voltage, 10 mV).

4.7 ELISA

After cardiac monitoring, blood from the heart was drawn into sterile tubes and centrifuged at 2,000 rpm for 15 minutes at room temperature in order to separate the supernatant, which was then stored in the refrigerator at -80 °C to measure the level of serum stress hormone indicators CRH, ACTH, CORT and inflammatory factors TNF-α, IL-1β. On the day of the experiment, the ELISA kits (EK11288, EK12421, EK11067, EK1940, EK17664, SAB, America) were rewarmed at room temperature, followed the instructions, and measured the absorbance value (OD) of each well by enzyme meter (Thermo, Multiskan Go S / N: 1510-00633, Thermo Fisher technology company). The concentration and OD value of the standard was used as a reference to compute the concentration of the sample by drawing the standard curve with CurveExpert software.

4.8 Western blotting

The BCA protein assay kit (Beyotime, No. P0010, Shanghai, China) was used to measure the total proteins in the cardiac tissues after they had been extracted using cell lysis buffer (Beyotime, P0013, Shanghai, China). Then, PVDF membranes were blocked using Rapid Block (Beyotime, P0235, Shanghai) with anti-ser368-phosphorylated Cx43 (1:1000), anti-total Cx43 (1:1000), anti-thr172-phosphorylated AMPK (1:1000), anti-total AMPK (1:1000), and anti-lkb1 (1:1000) and were incubated overnight at 4 °C (all antibodies were purchased from CST, America). After that, it was treated with goat anti-rabbit antibody that had been HRP-labeled (1:3000, ab205718, Abcam, America). The results were finally visualized using ECL luminous solution (Merck Millipore, WBKLS0100, America). To view the images, chemiluminescence imaging equipment (Guangyi Biotechnology Co., Ltd., Guangzhou, China) was employed.

4.9 Morphological and Histological analysis

The atria and ventricles were embedded in paraffin and sliced to a thickness of 4 µm after being preserved with 4% paraformaldehyde. Sections were stained with HE and Masson maintenance staining for histopathological analysis, and immunohistochemistry (IHC) staining was done to observe Cx43 distribution. A BX-51 fluorescent digital imaging microscope (Olympus, Japan) was used to capture images and evaluate all of these histopathological alterations.

4.10 Transmission electron microscopy (TEM)

Left ventricular heart tissues (1 mm * 1 mm * 1 mm) were removed immediately after modeling was complete, placed in 2.5% glutaraldehyde fixative at 4 °C, fixed with osmium tetroxide solution for 2 hours at room temperature, dehydrated, replaced, baked at 60 °C for 48 hours, and cryo-microtomized at 40–60 nm on a microtomography machine (EM UC7, Leica). TEM (HT7700, Hitachi, Japan) was used to evaluate the stained sections after they had been double-stained with saturated uranyl acetate and lead citrate.

4.11 Statistical methods

To compare the three groups, a one-way ANOVA was performed using GraphPad Prism, version 8.0 (GraphPad Software Inc., San Diego, CA, USA). Image J, version 1.52v, from the National Institutes of Health in Rasband, USA, was used to analyze the outcomes of the WB, IHC, and Masson tests. All quantitative data were presented as mean standard deviation (x ± SD). Statistical significance was defined as a value of p < 0.05.

Author Contributions

Each author on the list has contributed significantly, directly, and intellectually to the work. The HHS model was developed by Jianing Chi, Pengfei Li, and Ningxia Wu, who also took part in sample gathering. The ECG data were examined and evaluated by Jiaman Hu, Hua Cai, Cailong Lin and Yingying Lai. Han Yang, Jianyu Huang, and Min Li carried out the histological analyses. Jianning Chi is the primary author of this manuscript. The final text has been reviewed and approved by all authors.

Competing interests

All authors declare no competing interests.

Data Availability Statement

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Funding

This research was funded by the General Logistics Department of the Chinese People's Liberation Army Project (No. 23BJZ07), the Guangzhou Science and Technology Program Project (No. 2023A03J0170), and the Army Medical Science and Technology Young Top Talents Cultivation Program Project (No. 20QNPY082).

Bouchama, A. & Knochel, J. P. Heat Stroke. New England Journal of Medicine 346, 1978–1988 (2002).
Jones, T. S. Morbidity and mortality associated with the July 1980 heat wave in St Louis and Kansas City, Mo. JAMA: The Journal of the American Medical Association 247, 3327–3331 (1982).
Argaud, L. Short- and Long-term Outcomes of Heatstroke Following the 2003 Heat Wave in Lyon, France. Archives of Internal Medicine 167, 2177 (2007).
Misset, B. et al. Mortality of patients with heatstroke admitted to intensive care units during the 2003 heat wave in France: A national multiple-center risk-factor study*. Critical Care Medicine 34, 1087–1092 (2006).
Meehl, G. A. & Tebaldi, C. More Intense, More Frequent, and Longer Lasting Heat Waves in the 21st Century. Science 305, 994–997 (2004).
Marchand, M. & Gin, K. The Cardiovascular System in Heat Stroke. CJC Open 4, 158–163 (2022).
Liu, J. et al. Heat exposure and cardiovascular health outcomes: a systematic review and meta-analysis. The Lancet Planetary Health 6, e484–e495 (2022).
Leon, L. R. & Bouchama, A. Heat Stroke. Comprehensive Physiology 5, 611–647 (2015).
Alahmad, B. et al. Associations Between Extreme Temperatures and Cardiovascular Cause-Specific Mortality: Results From 27 Countries. Circulation 147, 35–46 (2023).
Zipes, D. P. & Wellens, H. J. J. Sudden Cardiac Death. Circulation 98, 2334–2351 (1998).
Donahue, H. J., Qu, R. W. & Genetos, D. C. Joint diseases: from connexins to gap junctions. Nature reviews. Rheumatology 14, 42–51 (2017).
Lo, C. W. Role of Gap Junctions in Cardiac Conduction and Development. Circulation Research 87, 346–348 (2000).
Severs, N. Gap junction alterations in human cardiac disease. Cardiovascular Research 62, 368–377 (2004).
Reaume, A. et al. Cardiac malformation in neonatal mice lacking connexin43. Science 267, 1831–1834 (1995).
Gao, J. et al. Anti-arrhythmic effect of acupuncture pretreatment in the rats subjected to simulative global ischemia and reperfusion—involvement of intracellular Ca2+ and connexin 43. BMC complementary and alternative medicine 15, (2015).
Lillo, M. A. et al. Remodeled connexin 43 hemichannels alter cardiac excitability and promote arrhythmias. The Journal of general physiology 155, (2023).
Palatinus, J. A. et al. GJA1-20k Rescues Cx43 Localization and Arrhythmias in Arrhythmogenic Cardiomyopathy. Circulation Research 132, 744–746 (2023).
Sovari, A. A. et al. Mitochondria Oxidative Stress, Connexin43 Remodeling, and Sudden Arrhythmic Death. Circulation: Arrhythmia and Electrophysiology 6, 623–631 (2013).
Wong, Aaron K. F., Howie, J., Petrie, John R. & Lang, Chim C. AMP-activated protein kinase pathway: a potential therapeutic target in cardiometabolic disease. Clinical Science 116, 607–620 (2009).
Carling, D. The AMP-activated protein kinase cascade--a unifying system for energy control. Trends in Biochemical Sciences 29, 18–24 (2004).
Chenicheri Kizhakkeveettil Keerthana et al. The role of AMPK in cancer metabolism and its impact on the immunomodulation of the tumor microenvironment. Frontiers in immunology 14, (2023).
Sung, M. M. et al. AMPK deficiency in cardiac muscle results in dilated cardiomyopathy in the absence of changes in energy metabolism. Cardiovascular research 107, 235–245 (2015).
Harada, M., Nattel, S. N. & Nattel, S. AMP-Activated Protein Kinase. Circulation: Arrhythmia and Electrophysiology 5, 860–867 (2012).
Brown, S. M., Larsen, N. K., Thankam, F. G. & Agrawal, D. K. Regulatory role of cardiomyocyte metabolism via AMPK activation in modulating atrial structural, contractile, and electrical properties following atrial fibrillation. Canadian journal of physiology and pharmacology 99, 36–41 (2021).
Andersen, M. N. & Rasmussen, H. B. AMPK. Communicative & Integrative Biology 5, 480–484 (2012).
Zhang, Y. et al. Enhancing Fatty Acids Oxidation via L-Carnitine Attenuates Obesity-Related Atrial Fibrillation and Structural Remodeling by Activating AMPK Signaling and Alleviating Cardiac Lipotoxicity. Frontiers in pharmacology 12, (2021).
Kim, G. et al. LKB1 deletion causes early changes in atrial channel expression and electrophysiology prior to atrial fibrillation. Cardiovascular research 108, 197–208 (2015).
Kwan Long Mung, Eccleshall, W., Santio, N. M., Rivero-Müller, A. & Koskinen, P. J. PIM kinases inhibit AMPK activation and promote tumorigenicity by phosphorylating LKB1. Cell communication and signaling : CCS 19, (2021).
Shen, Z. et al. Theaflavin 3,3′‐digallate reverses the downregulation of connexin 43 and autophagy induced by high glucose via AMPK activation in cardiomyocytes. Journal of cellular physiology 234, 17999–18016 (2019).
Lee, C.-C., Chen, W.-T., Chen, S. & Lee, T.-M. Dapagliflozin attenuates arrhythmic vulnerabilities by regulating connexin43 expression via the AMPK pathway in post-infarcted rat hearts. Biochemical Pharmacology 192, 114674 (2021).
Qiu, J., Zhou, S. & Liu, Q. Phosphorylated AMP-activated protein kinase slows down the atrial fibrillation progression by activating Connexin43. International Journal of Cardiology 208, 56–57 (2016).
Steptoe, A. & Kivimäki, M. Stress and Cardiovascular Disease. Nature Reviews Cardiology 9, 360–370 (2012).
Michalsen, A. Acute stress and ventricular arrhythmias. European Heart Journal 22, 712 (2001).
Benito, B. et al. Cardiac Arrhythmogenic Remodeling in a Rat Model of Long-Term Intensive Exercise Training. Circulation 123, 13–22 (2011).
Liu, J., Wang, Y., Shan, Z. & Guo, H. Influence of acute stress on cardiac electrophysiological stability in male goats. Acta Cardiologica 67, 325–330 (2012).
Bouchama, A. et al. Classic and exertional heatstroke. Nature Reviews Disease Primers 8, 1–23 (2022).
Epstein, Y. & Yanovich, R. Heatstroke. New England Journal of Medicine 380, 2449–2459 (2019).
Yuan, F. et al. Z-DNA binding protein 1 promotes heatstroke-induced cell death. Science 376, 609–615 (2022).
Zhou, R. ou, Liu, J. W., Zhang, D. & Zhang, Q. Heatstroke model for desert dry-heat environment and observed organ damage. The American Journal of Emergency Medicine 32, 573–579 (2014).
Miyamoto, K. et al. A novel mouse model of heatstroke accounting for ambient temperature and relative humidity. Journal of intensive care 9, (2021).
RAMANATHAN, N. L. & BELDING, H. S. Physiological Evaluation of the WBGT Index for Occupational Heat Stress. American Industrial Hygiene Association Journal 34, 375–383 (1973).
Stoffels, M. et al. ATP-Induced IL-1Î2 Specific Secretion: True Under Stringent Conditions. Frontiers in Immunology 6, (2015).
Zhao, M. et al. Targeting fibrosis: mechanisms and clinical trials. Signal Transduction and Targeted Therapy 7, (2022).
Nguyen, M.-N., Kiriazis, H., Gao, X. & Du, X.-J. Cardiac Fibrosis and Arrhythmogenesis. Comprehensive Physiology 7, 1009–1049 (2017).
Chu, M. et al. Increased Cardiac Arrhythmogenesis Associated With Gap Junction Remodeling With Upregulation of RNA-Binding Protein FXR1. Circulation 137, 605–618 (2018).
Luc Leybaert et al. Connexin hemichannels as candidate targets for cardioprotective and anti-arrhythmic treatments. The Journal of clinical investigation 133, (2023).
Maarten De Smet et al. Cx43 hemichannel microdomain signaling at the intercalated disc enhances cardiac excitability. The Journal of clinical investigation 131, (2021).
Beardslee, M. A. et al. Dephosphorylation and Intracellular Redistribution of Ventricular Connexin43 During Electrical Uncoupling Induced by Ischemia. Circulation Research 87, 656–662 (2000).
Rohr, S. Role of gap junctions in the propagation of the cardiac action potential. Cardiovascular Research 62, 309–322 (2004).
Betsuyaku, T. et al. Spontaneous and inducible ventricular arrhythmias after myocardial infarction in mice. Cardiovascular Pathology 13, 156–164 (2004).
Fontes, M. S. C., van Veen, T. A. B., de Bakker, J. M. T. & van Rijen, H. V. M. Functional consequences of abnormal Cx43 expression in the heart. Biochimica et Biophysica Acta (BBA) - Biomembranes 1818, 2020–2029 (2012).
Fu, X. et al. Specialized fibroblast differentiated states underlie scar formation in the infarcted mouse heart. Journal of Clinical Investigation 128, 2127–2143 (2018).
Nattel, S., Burstein, B. & Dobrev, D. Atrial remodeling and atrial fbrillation: mechanisms and implications. Circulation: Arrhythmia and Electrophysiology 1, 62–73 (2008).
Mollenhauer, M. et al. Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling. Circulation research 121, 56–70 (2017).
Jansen, J. A. et al. Reduced Cx43 Expression Triggers Increased Fibrosis Due to Enhanced Fibroblast Activity. Circulation: Arrhythmia and Electrophysiology 5, 380–390 (2012).
Ioana Alesutan et al. AMP-Activated Protein Kinase α1 Regulates Cardiac Gap Junction Protein Connexin 43 and Electrical Remodeling Following Pressure Overload. Cellular Physiology and Biochemistry 35, 406–418 (2015).
Zaha, V. G. & Young, L. H. AMP-Activated Protein Kinase Regulation and Biological Actions in the Heart. Circulation Research 111, 800–814 (2012).
Yan, Y. et al. Structure of an AMPK complex in an inactive, ATP-bound state. Science 373, 413–419 (2021).
Evangelos-Panagiotis Daskalopoulos, Cécile Dufeys, Bertrand, L., Christophe Beauloye & Horman, S. AMPK in cardiac fibrosis and repair: Actions beyond metabolic regulation. Journal of Molecular and Cellular Cardiology 91, 188–200 (2016).
Cécile Dufeys et al. AMPKα1 deletion in myofibroblasts exacerbates post-myocardial infarction fibrosis by a connexin 43 mechanism. Basic research in cardiology 116, (2021).
Xiang, H.-C. et al. AMPK activation attenuates inflammatory pain through inhibiting NF-κB activation and IL-1β expression. Journal of neuroinflammation 16, (2019).
Lucero, C. et al. TNF-α Plus IL-1β Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway. International journal of molecular sciences 23, 10097–10097 (2022).
Ozcan, C., Dixit, G. & Li, Z. Activation of AMP-Activated Protein Kinases Prevents Atrial Fibrillation. Journal of cardiovascular translational research 14, 492–502 (2020).
Bagath, M. et al. The impact of heat stress on the immune system in dairy cattle: A review. Research in Veterinary Science 126, 94–102 (2019).
Li, X. et al. Apelin Ameliorates High Glucose-Induced Downregulation of Connexin 43 via AMPK-Dependent Pathway in Neonatal Rat Cardiomyocytes. Aging and disease 9, 66 (2018).
Leithe, E. & Rivedal, E. Ubiquitination of Gap Junction Proteins. Journal of Membrane Biology 217, 43–51 (2007).
Baskin, K. K. & Taegtmeyer, H. AMP-Activated Protein Kinase Regulates E3 Ligases in Rodent Heart. Circulation Research 109, 1153–1161 (2011).

No competing interests reported.

Download PDF

Journal Publication

published 29 Feb, 2024

Read the published version in Scientific Reports →

Editorial decision: Revision requested
30 Dec, 2023
Reviewers agreed at journal
18 Dec, 2023
Reviews received at journal
11 Oct, 2023
Reviewers agreed at journal
04 Oct, 2023
Reviewers invited by journal
07 Sep, 2023
Editor assigned by journal
07 Sep, 2023
Editor invited by journal
16 Aug, 2023
Submission checks completed at journal
16 Aug, 2023
First submitted to journal
09 Aug, 2023

You are reading this latest preprint version

Hygrothermal stress increases malignant arrhythmias susceptibility by remodeling connexin43 via AMPK

Status:

Journal Publication

Version 1

Abstract

Figures

1. Introduction

2. Results

2.1 The progression of Tr and HR under HHS is slowed down by activating AMPK

2.2 Activation of the AMPK by the AICAR injection attenuates arrhythmias in rats

2.3 Hormonal stress markers and inflammatory factors were both upregulated by HHS.

2.4 HHS remodeled Cx43 by inhibiting activation of the LKB1/AMPK pathway

2.5 HHS leads to myocardial injury and fibrosis

3. Discussion

Methods

4.1 Ethics statement

4.2 Animals

4.3 Experimental groups

4.4 Basic physical parameters measure

4.5 Hygrothermal exposure

4.6 ECG acquisition and analysis

4.7 ELISA

4.8 Western blotting

4.9 Morphological and Histological analysis

4.10 Transmission electron microscopy (TEM)

4.11 Statistical methods

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1