Multifaceted functions of Drp1 in hypoxia/ischemia-induced mitochondrial quality imbalance: from regulatory mechanism to targeted therapeutic strategy.

Hypoxic-ischemic injury is a common pathological dysfunction in clinical settings. Mitochondria are sensitive organelles that are readily damaged following ischemia and hypoxia. Dynamin-related protein 1 (Drp1) regulates mitochondrial quality and cellular functions via its oligomeric changes and multiple modifications, which plays a role in mediating the induction of multiple organ damage during hypoxic-ischemic injury. However, there is active controversy and gaps in knowledge regarding the modification, protein interaction, and functions of Drp1, which both hinder and promote development of Drp1 as a novel therapeutic target. Here, we summarize recent findings on the oligomeric changes, modification types, and protein interactions of Drp1 in various hypoxic-ischemic diseases, as well as the Drp1-mediated regulation of mitochondrial quality and cell functions following ischemia and hypoxia. Additionally, potential clinical translation prospects for targeting Drp1 are discussed. This review provides new ideas and targets for proactive interventions on multiple organ damage induced by various hypoxic-ischemic diseases.

Activated Drp1 regulates p62-mediated autophagic flux and aggravates inflammation in cerebral ischemia-reperfusion via the ROS-RIP1/RIP3-exosome axis.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) refers to a secondary brain injury that can occur when the blood supply to the ischemic brain tissue is restored. However, the mechanism underlying such injury remains elusive. METHODS: The 150 male C57 mice underwent middle cerebral artery occlusion (MCAO) for 1 h and reperfusion for 24 h, Among them, 50 MCAO mice were further treated with Mitochondrial division inhibitor 1 (Mdivi-1) and 50 MCAO mice were further treated with N-acetylcysteine (NAC). SH-SY5Y cells were cultured in a low-glucose culture medium for 4 h under hypoxic conditions and then transferred to normal conditions for 12 h. Then, cerebral blood flow, mitochondrial structure, mitochondrial DNA (mtDNA) copy number, intracellular and mitochondrial reactive oxygen species (ROS), autophagic flux, aggresome and exosome expression profiles, cardiac tissue structure, mitochondrial length and cristae density, mtDNA and ROS content, as well as the expression of Drp1-Ser616/Drp1, RIP1/RIP3, LC3 II/LC3 I, TNF-α, IL-1ß, etc., were detected under normal or Drp1 interference conditions. RESULTS: The mtDNA content, ROS levels, and Drp1-Ser616/Drp1 were elevated by 2.2, 1.7 and 2.7 times after CIRI (P < 0.05). However, the high cytoplasmic LC3 II/I ratio and increased aggregation of p62 could be reversed by 44% and 88% by Drp1 short hairpin RNA (shRNA) (P < 0.05). The low fluorescence intensity of autophagic flux and the increased phosphorylation of RIP3 induced by CIRI could be attenuated by ROS scavenger, NAC (P < 0.05). RIP1/RIP3 inhibitor Necrostatin-1 (Nec-1) restored 75% to a low LC3 II/LC3 I ratio and enhanced 2 times to a high RFP-LC3 after Drp1 activation (P < 0.05). In addition, although CIRI-induced ROS production caused no considerable accumulation of autophagosomes (P > 0.05), it increased the packaging and extracellular secretion of exosomes containing p62 by 4 - 5 times, which could be decreased by Mdivi-1, Drp1 shRNA, and Nec-1 (P < 0.05). Furthermore, TNF-α and IL-1ß increased in CIRI-derived exosomes could increase RIP3 phosphorylation in normal or oxygen-glucose deprivation/reoxygenation (OGD/R) conditions (P < 0.05). CONCLUSIONS: CIRI activated Drp1 and accelerated the p62-mediated formation of autophagosomes while inhibiting the transition of autophagosomes to autolysosomes via the RIP1/RIP3 pathway activation. Undegraded autophagosomes were secreted extracellularly in the form of exosomes, leading to inflammatory cascades that further damaged mitochondria, resulting in excessive ROS generation and the blockage of autophagosome degradation, triggering a vicious cycle.

Induction of Heat Shock Protein Genes is the Hallmark of Egg Heat Tolerance in Agasicles hygrophila (Coleoptera: Chrysomelidae).

Insects are ecotothermic organisms. Their development, survival, reproduction as well as distribution and abundance are affected by temperature. Heat shock protein (HSP) gene expression is closely associated with temperature variation and influences the adaptation of organisms to adverse environments. The beetle Agasicles hygrophila has successfully been used for biological control of the invasive plant alligator weed (Alternanthera philoxeroides). As A. hygrophila populations are substantially inhibited by high temperatures in the summer, increasing global temperatures may limit the efficacy of this control agent. We previously established that A. hygrophila eggs have low tolerance to heat and this factored into the decreased numbers of A. hygrophila beetles at temperatures of 37.5°C and above. Here, we identified 26 HSP genes in A. hygrophila and examined the relationship between the transcript levels of these genes and heat tolerance. The temperature at which the expression of these 21 HSP genes peaked (Tpeak) was 37.5°C, which is in line with the limit of the high temperatures that A. hygrophila eggs tolerate. Therefore, we speculate that the Tpeak of HSP gene expression in eggs indicates the upper limit of temperatures that A. hygrophila eggs tolerate. This study identifies HSP genes as potential robust biomarkers and emphasizes that determining species' heat tolerance in their natural habitats remains an important consideration for biocontrol. HSP gene expression data provide information about a species' heat tolerance and may be used to predict its geographical distribution.

Behavioral effects of different attractants on adult male and female oriental fruit moths, Grapholita molesta.

BACKGROUND: Grapholita molesta (Busck) is a fruit pest worldwide. While sex pheromones-based technology for male attraction has made great progress in the monitoring or mass trapping of G. molesta, the attraction of males alone is not an effective reproductive control strategy. The integration of sex pheromones with female attractants, which have been reported to attract adult females and males, is therefore necessary. To determine a suitable dual-sex attractant for trapping G. molesta, combinations of four attractants [sex pheromones (ph), host-plant volatiles (pp), sugar-acetic acid-ethanol-water solutions (ss), and food lures (fl)] were screened using electroantennograms, wind tunnel experiments, and field trial tests. Various mediums and antioxidants were then added to the selected attractants combination (AC) and examined in the field. RESULTS: The responses of G. molesta to attractants differed significantly between the sexes. Increases in male activation behavior and trapping were motivated by ph, while pp and particularly ss increased adult antennae perception. In response to fl alone or together with ph, more male or female individuals were trapped. This indicates that ph, as a sex lure, and fl, as a host lure, may complete male and female attraction. Consequently, this combination is suggested. In the field trials, the additions of 10 µL of honey and 35% (184.3 µg) of 2,6-di-tert-butyl-4-methylphenol [BTH] (preservatives) to the AC (ph + fl) resulted in moth captures equal to that of a commercial sex pheromones lure. CONCLUSION: A dual-sex attractant composed of ph, fl, honey, and BTH is recommended for trapping G. molesta in the field. © 2020 Society of Chemical Industry.

Heat sensitivity of eggs attributes to the reduction in Agasicles hygrophila population.

Agasicles hygrophila has been introduced worldwide as a control agent for the invasive weed Alternanthera philoxeroides. However, global warming has potential impact on its controlling efficacy. The aim of this research was to explore the primary factors responsible for the greatly reduced A. hygrophila population in hot summers. To imitate the temperature conditions in summers, different developmental stages of A. hygrophila were treated with high temperatures from 32.5 °C to 45 °C for 1-5 h. Based on the survival rate, the heat tolerance of each developmental stage was ranked from lowest to highest as follows: egg, 1st, 2nd, 3rd instar larva, adult and pupa. Eggs showed the lowest heat tolerance with 37.5 °C as the critical temperature affecting larval hatching. Heat treatment of the A. hygrophila eggs at 37.5 °C for 1 h decreased the hatch rate to 24%. Our results indicated that when compared with the control at 25 °C, 1 h treatment at 37.5 °C prolonged the duration of the egg stage, shortened the duration of oviposition and total longevity, and changed the reproductive pattern of A. hygrophila. The net reproductive rate, intrinsic rate and finite rate were all significantly reduced. The results suggest that low heat tolerance of the eggs was the major factor responsible for the reduction of A. hygrophila populations, and the key temperature was 37.5 °C. Therefore, appropriate measures should be taken to protect eggs in order to maintain the efficacy of A. hygrophila in the biological control of A. philoxeroides in hot summers.

Response to Short-Term Cold Storage for Eggs of Agasicles hygrophila (Coleoptera: Chrysomelidae), a Biological Control Agent of Alligator Weed Alternanthera philoxeroides (Caryophyllales: Amaranthaceae).

The alligator weed flea beetle, Agasicles hygrophila Selman & Vogt (Coleoptera: Chrysomelidae) has been used very successfully for the biological control of the widely-distributed invasive weed Alternanthera philoxeroides (Mart.) Griseb (Caryophyllales: Amaranthaceae). In order to extend the 'shelf life' of natural enemies released in biological control programs, cold storage has proven to be a valuable commercial procedure. To determine a suitable low temperature for storage of A. hygrophila, we conducted short-term cold storage treatments of eggs (4°C for 0.5, 1, 2, 5 d, and 7.5, 10, 15°C for 5 d and a control of 25°C; all eggs were returned to 25°C after the treatments). We evaluated the effects of these treatments on the subsequent fitness of the populations based on a demographic analysis using group-reared age-stage two-sex life tables. For 5 d storage, temperatures below 10°C had lethal effects, which were also observed at 4°C for 2 d storage. Storage at 4°C for 0.5 d did not affect the fitness of A. hygrophila, but it did not prolong the developmental time. Storage at 10°C for 5 d significantly decreased rates of population increase compared with 25°C. A. hygrophila stored at 15°C for 5 d had similar age-(stage) specific survival rates, rates of population increase, increased longevity and reproductive capability to the controls at 25°C. It is concluded that there were no significant fitness costs after 5 d storage at 15°C, which is therefore potentially a suitable storage temperature for A. hygrophila eggs.

Melatonin alleviates hypoxia-induced cardiac apoptosis through PI3K/Akt pathway.

Hypoxia-induced apoptosis is an inevitable problem in cyanotic congenital heart disease. In the present study, we investigated effects of melatonin on hypoxic cardiomyocytes in vitro and in vivo, and explored its underlying mechanism. H9C2 cells were subjected to hypoxia for 48 hours. Mice were subjected to hypoxia treatment (10% O2) for 4 weeks. Cell viability was detected by the cell counting kit-8 assay. Cellular apoptosis was assessed by Annexin V/7 AAD assay. Western blotting was employed to determine the expression of Bcl-2, Bax, cleaved caspase 3, phosphorylation of PI3K, and AKT. Melatonin increased cell viability and alleviated apoptosis in hypoxic H9C2 cells and cardiomyocytes of hypoxia-treated mice. Melatonin pretreatment increased Bcl-2 and decreased cleaved caspase 3 and Bax levels. Moreover, melatonin activated the PI3K/Akt pathway. The protective effects of melatonin were abolished by a PI3K/Akt-inhibitor, LY294002. Our results demonstrated that melatonin confers cardioprotection by inhibiting apoptosis through the activation of PI3K/Akt signaling pathway in hypoxic cardiomyocytes.

Risk Factors for Acute Kidney Injury after Cardiovascular Surgery: Evidence from 2,157 Cases and 49,777 Controls - A Meta-Analysis.

PURPOSE: Cardiovascular surgery-associated acute kidney injury (AKI-CS) contributes to mortality and morbidity. However, risk factors accelerating its development are unclear. We identified risk factors for AKI-CS in patients with cardiopulmonary bypass in the hospital surgical intensive care unit to predict and minimize renal complication in future cardiac surgery. METHODS: We analyzed data from 14 case-control studies published prior to June 2014 and indexed in Science Citation Index, PubMed, and other databases to determine the major risk factors for AKI-CS. RESULTS: Analyzed risk factors were divided into three groups: preoperative, intraoperative and postoperative. Preoperative factors included: age (OR, 4.87; 95% CI, 3.50-6.24), NYHA class III/IV (OR, 2.53; 95% CI, 1.32-4.86), hypertension (OR, 1.68; 95% CI, 1.44-1.97), preoperative creatinine (OR, 0.66; 95% CI, 0.18-1.14), peripheral vascular disease (OR, 1.31 95% CI, 1.09-1.57), respiratory system disease (OR, 1.29; 95% CI, 1.10-1.50), diabetes mellitus (OR, 1.52; 95% CI, 1.07-2.16), and cerebrovascular disease (OR, 2.13; 95% CI, 1.11-4.09). Intraoperative factors were: cardiopulmonary bypass time (OR, 33.78; 95% CI, 23.15-44.41), aortic clamping time (OR, 13.24; 95% CI, 7.78-18.69), use of intra-aortic balloon pump (OR, 4.44; 95% CI, 2.37-8.30), and type of surgery (OR, 1.01; 95% CI, 0.43-2.39). Postoperative factors were: infection (OR, 3.58; 95% CI, 1.43-8.97), redo operation (OR, 2.57; 95% CI, 1.75-3.78), emergency surgery (OR, 4.76; 95% CI, 3.05-7.43), and low cardiac output (OR, 2.30; 95% CI, 1.05-5.04). CONCLUSIONS: Our results support that preoperative, intraoperative, and postoperative factors are associated with AKI-CS. Ejection fraction, BMI, acute myocardial infarction, type of surgery, and congestive heart failure were not absolutely associated with AKI.

The complete mitochondrial genome of the flea beetle Agasicles hygrophila.

To provide molecular markers for population genetic analysis of the flea beetle Agasicles hygrophila, we determined its mitochondrial genome (mitogenome) for the first time. The mitogenome of A. hygrophila was 15 917 bp in length with an AT content of 75.15%. It had the typical set of 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and an AT-rich control region. Compared with the ancestral mitogenome of insects, no gene rearrangement occurred in A. hygrophila. Incomplete stop codons were present in PCGs of A. hygrophila. All tRNA genes except for trnS(AGN) could form the typical clover-leaf secondary structures. The phylogenetic analysis indicated that A. hygrophila was close to other species belonging to the same family of Chrysomelidae.

The application of on-pump beating-heart surgery for partial atrioventricular septal defect: a report of 87 cases.

BACKGROUND: Partial atrioventricular septal defect (P-AVSD) is a common congenital heart disease. Because of the presence of left and right atrioventricular valve deformities and the shift in the atrioventricular node and cardiac conduction bundle, the surgical repair of P-AVSD is difficult. This study was performed to compare the effects on the coronary sinus septum in the left versus the right atrium during surgical treatment for P-AVSD and report our experiences regarding the application of on-pump beating heart surgery under mild hypothermia for patients with P-AVSD. MATERIALS AND METHODS: The effects of on-pump beating heart surgery were analyzed retrospectively in 87 P-AVSD patients. Of the 87 total patients, 84 with anterior mitral leaflet cleft underwent valvuloplasty and 3 underwent mitral valve replacement. Seventy-seven patients underwent tricuspid valve annuloplasty, 2 underwent tricuspid valve replacement, and 1 underwent left superior vena cava ligation, and 3 patients with atrial fibrillation were treated with radiofrequency ablation. Patients with an ostium primum atrial septal defect underwent autologous pericardial modified Kirklin repair. Of these, 46 patients had their coronary sinus septum separated into the left atrium and 41 had their coronary sinus retained in the right atrium. Fingertip oxygen saturation was compared between patients in whom the coronary sinus was separated to the left atrium and those in whom the coronary sinus was retained in the right atrium. RESULTS: There was 1 postoperative early death (1.15%) due to respiratory failure, and 1 patient had a III degree atrioventricular block (1.15%) and underwent implantation of a permanent pacemaker. The fingertip oxygen saturation levels of the left atrium group were 96.81 ± 3.17 preoperatively, 95.37 ± 4.62 at 7 days postoperatively, and 94.53 ± 4.95 at 3 months postoperatively. Those of the right atrium group were 98.53 ± 2.84 preoperatively, 97.19 ± 3.57 at 7 days postoperatively, and 96.89 ± 4.19 at 3 months postoperatively. During the follow-up period, which ranged from 3 months to 7 years, the cardiac function was adequately restored. CONCLUSIONS: On-pump beating heart surgery under mild hypothermia is a safe and feasible method. The retention of the coronary sinus in the right atrium might maintain oxygen saturation.

Sequential loss of genetic variation in flea beetle Agasicles hygrophila (Coleoptera: Chrysomelidae) following introduction into China.

The flea beetle (Agasicles hygrophila) was imported to Florida, USA and then introduced from Florida into China in 1987 as a biological control agent for the invasive plant alligator weed (Alternanthera philoxeroides). The initial beetle population was subsequently used for sequential introductions in other areas of China, but little is known about the genetic consequences of the introductions. In this study, the genetic diversity and population structure of five beetle populations, the source Florida population, three intentionally introduced China populations and one accidentally dispersed China population, were examined using amplified fragment length polymorphisms. The results showed a clear pattern of decreasing genetic diversity with the sequential introductions. The diversity was highest in the Florida population followed by the first introduction to Chongqing and then in Kunming and Fuzhou. The lowest diversity was found in the accidentally dispersed Guangzhou population that was first recorded in 1996. Both loci parameters and Nei's genetic diversity showed a high variation among these populations. Genetic differentiation among populations was further verified by the GST statistic (0.136-0.432). Beetles in Kunming had the highest gene flow with those in Guangzhou, and therefore lowest differentiation and closest genetic distance. These data show that sequential introduction influenced the genetic diversity of populations in China. Genetic diversity should be considered in planning introduction and long-term maintenance of populations.

Research on the practical parameters of sex pheromone traps for the oriental fruit moth.

BACKGROUND: The oriental fruit moth (OFM) is a worldwide fruit-boring insect pest. In China, OFM monitoring traps use a sex pheromone lure, but their overall design is varied. As such, there is a critical need to develop a standardised OFM trap design. In this field study, ten different trap shapes in varying combinations of colours and sizes (such as trap length and surface area) were examined. RESULTS: The results showed that there was no significant difference in the trapping efficiency between eight colours. The ship-shaped trap could kill more OFM in a short period, whereas the automatic watering basin trap could be more effective in the long run. The optimal trapping diameter of the basin trap was 25 cm. The trapping efficiency of triangle traps with diameters of less than 10 cm was better than that of triangle traps with diameters of over 30 cm. The trapping number of pasteboard traps obviously declined when the surface area increased, and the pasteboard trap with a single board possessed excellent trapping efficiency. CONCLUSION: The results provide useful information for the design of standardised sex pheromone traps for monitoring as well as trapping of OFM in the field.

Increased expression of connective tissue growth factor and transforming growth factor-beta-1 in atrial myocardium of patients with chronic atrial fibrillation.

OBJECTIVES: This study aimed to investigate the roles of connective tissue growth factor (CTGF) and transforming growth factor-ß1 (TGF-ß1) in atrial fibrosis in patients with chronic atrial fibrillation. METHODS: Up to 40 cases involving simple mitral valve replacement surgery were divided into 2 groups: the chronic atrial fibrillation (cAF) group (n = 28) and the sinus rhythm group (n = 12). Echocardiography was used to measure the cardiac cavity size and analyze the cardiac function. Right atrial specimens were obtained during the operation. The collagen volume fraction in the atrial specimens was examined. The mRNA and protein levels of TGF-ß1, Smad3 and CTGF were also investigated. RESULTS: Compared with the sinus rhythm group, the cAF group had higher collagen content in the right atrial tissues. The mRNA and protein levels of TGF-ß1, Smad3 and CTGF were also significantly elevated in the cAF group (p < 0.05). The mRNA and protein levels of TGF-ß1 and CTGF in the cAF group correlated positively with the collagen volume fraction. The positive correlation between the expression of TGF-ß1 and CTGF was also demonstrated. CONCLUSIONS: CTGF is upregulated via the TGF-ß1/Smad pathway in the atrial myocardium of cAF patients. Furthermore, the TGF-ß1/Smad pathway may play an important role in the structural remodeling during atrial fibrosis.

Pivotal role of activating transcription factor 6α in myocardial adaptation to chronic hypoxia.

Hypoxic states are generally associated with cardiovascular disease. Adaptation to chronic hypoxia is one well-defined means of improving cardiac tolerance to certain kinds of stresses. However, the details of the mechanisms underlying myocardial adaptation to chronic hypoxia are still poorly understood. Hypoxia stresses the endoplasmic reticulum and activates unfolded protein response. However, the behavior of individual signaling pathways can vary markedly over time. By examining myocardial samples from patients with cyanotic congenital cardiac defects, we detected endoplasmic reticulum stress and found that, out of all the components of the unfolded protein response, only activating transcription factor 6α limb was activated in cyanotic patients. The activation of activating transcription factor 6α and expression of glucose regulated protein 78 were notably induced in cardiac myocytes cultured for prolonged hypoxia (1% O(2) for 48 h). When the activation of activating transcription factor 6α under prolonged hypoxia was blocked by chemical inhibitor Brefeldin A, the rate of apoptosis among cardiac myocytes increased and levels of cleaved caspase 3 and cleaved poly ADP ribose polymerase also increased significantly. After the expression of activating transcription factor 6α was knocked down, the activity of cardiac myocytes under prolonged hypoxia decreased and the phosphorylation of c-Jun NH2-terminal kinases increased during the re-oxygenation process (after 72 h of hypoxia). Together, these results indicate that activating transcription factor 6α plays a pivotal role in myocardial adaptation to chronic hypoxia and that the activation of activating transcription factor 6α is one possible mechanism of myocardial preconditioning.

[Genetic diversity of introduced natural enemy Agasicles hygrophila (Coleoptera: Chrysomelidae)].

Flea beetle Agasicles hygrophila Selman & Vogt, the most effective natural enemy of alligator weed Alternanthera philoxeroideis, is originated from South America, and has been introduced into China for more than 20 years to control the weed. In this study, the technique of random amplification of polymorphic DNA (RAPD) was applied to assess the genetic diversity of four A. hygrophila geographic populations in Guangzhou, Chongqing, Kunming, and Fuzhou of China, and, taking the population in Florida of US as the reference, the relationships between the genetic differentiation and geographic differentiation among and within the populations were explored at molecular level. Thirteen primers that produced clear and reproducible products were selected from 111 random primers to amplify the 25 samples of the five A. hygrophila populations. The total polymorphic rate was 42.0%, and the polymorphic rate of Florida population was significantly higher than that of four China populations. The genetic differentiation among the four China populations was 25.5%. The cluster analysis by UPGMA indicated that there was a positive correlation between the genetic distance and geographic distance of different A. hygrophila geographic populations.

Increase of macrophage migration inhibitory factor (MIF) expression in cardiomyocytes during chronic hypoxia.

BACKGROUND: Macrophage migration inhibitory factor (MIF) might play an important role in the myocardium during chronic hypoxia because MIF protects the heart during myocardial ischemia by activating 5'-adenosine monophosphate activated protein kinase (AMPK). METHODS: We investigated 35 infants with cyanotic or acyanotic cardiac defects and H9c2 embryonic rat cardiomyocytes to examine the effect of chronic hypoxia on the expression of MIF in vivo and in vitro, respectively. RESULTS: We found out an increase of endogenous cardiac MIF expression positively correlated with degree of hypoxia. Also, AMPK activation was elevated while MIF expression was increased in cells exposed to long periods of hypoxia in vitro. There was no significant difference in the growth ratio of cells cultivated in long periods of hypoxia and normoxia. CONCLUSIONS: The expression of MIF is significantly increased in cardiomyocytes exposed to chronic hypoxia, and the activation of AMPK was increased accordingly.

[Effects of rapid atrial pacing on L-type calcium channel subunits and potassium channel Kv4.3 expressions in rabbits].

OBJECTIVE: To investigate rapid atrial pacing (RAP) induced atrial ultrastructural changes and mRNA and protein expression changes of L-type calcium channel subunits and potassium channel Kv4.3 in a rabbit model. METHODS: Thirty-six rabbits were electrically paced at a frequency of 600 beats/min for durations ranging from 0 - 48 h via bipolar endocardial leads through surgical techniques. Ultrastructural changes of the atrium were observed through a transmission electron microscope (TEM), L-type calcium channel subunits and potassium channel Kv4.3 expressions at mRNA and protein levels were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Atrial ultrastructure changes characterized by mitochondrial vacuolization, myofilament lysis, and glycogen accumulation were detected obvious at 3 h post pacing. Down-regulated mRNA expression of Ca(2+) channel beta1 and alpha1 subunits was observed 6 h post pacing, Kv4.3 mRNA down-regulation occurred 24 h post pacing, auxiliary subunit alpha2 was not affected by pacing. Protein expression of alpha1c subunit and potassium channel Kv4.3 paralleled their mRNA expression changes. CONCLUSION: RAP induced ultrastructural changes of the atrium and down-regulated mRNA and protein expressions of L-type calcium channel subunits and potassium channel Kv4.3 occurred thereafter in response to intracellular calcium overload induced by RAP.