Downregulation of cardiac PIASy inhibits Cx43 SUMOylation and ameliorates ventricular arrhythmias in a rat model of myocardial ischemia/reperfusion injury.

BACKGROUND: Dysfunction of the gap junction channel protein connexin 43 (Cx43) contributes to myocardial ischemia/reperfusion (I/R)-induced ventricular arrhythmias. Cx43 can be regulated by small ubiquitin-like modifier (SUMO) modification. Protein inhibitor of activated STAT Y (PIASy) is an E3 SUMO ligase for its target proteins. However, whether Cx43 is a target protein of PIASy and whether Cx43 SUMOylation plays a role in I/R-induced arrhythmias are largely unknown. METHODS: Male Sprague-Dawley rats were infected with PIASy short hairpin ribonucleic acid (shRNA) using recombinant adeno-associated virus subtype 9 (rAAV9). Two weeks later, the rats were subjected to 45 min of left coronary artery occlusion followed by 2 h reperfusion. Electrocardiogram was recorded to assess arrhythmias. Rat ventricular tissues were collected for molecular biological measurements. RESULTS: Following 45 min of ischemia, QRS duration and QTc intervals statistically significantly increased, but these values decreased after transfecting PIASy shRNA. PIASy downregulation ameliorated ventricular arrhythmias induced by myocardial I/R, as evidenced by the decreased incidence of ventricular tachycardia and ventricular fibrillation, and reduced arrythmia score. In addition, myocardial I/R statistically significantly induced PIASy expression and Cx43 SUMOylation, accompanied by reduced Cx43 phosphorylation and plakophilin 2 (PKP2) expression. Moreover, PIASy downregulation remarkably reduced Cx43 SUMOylation, accompanied by increased Cx43 phosphorylation and PKP2 expression after I/R. CONCLUSION: PIASy downregulation inhibited Cx43 SUMOylation and increased PKP2 expression, thereby improving ventricular arrhythmias in ischemic/reperfused rats heart.

Flavopiridol Mitigates the Progression of Monocrotaline-Induced Pulmonary Hypertension in Rats by Targeting Cyclin-Dependent Kinase 9.

PURPOSE: To investigate the role of cyclin-dependent kinase 9 (CDK9) and the therapeutic potential of a CDK9 inhibitor (flavopiridol) in monocrotaline (MCT)-induced pulmonary hypertension (PH). METHODS: For the in vivo experiments, rats with PH were established by a single intraperitoneal injection of MCT (60 mg/kg). After 2 weeks of MCT injection, rats were then treated with flavopiridol (5 mg/kg, i.p., twice a week) or vehicle for 2 weeks. For the in vitro experiments, human pulmonary artery smooth muscle cells (HPASMCs) were treated with flavopiridol (0.025-1 µM) or vehicle under hypoxic conditions. Hemodynamic recording, right ventricle histology, lung histology, and pulmonary arterial tissue isolation were performed. The expression levels of CDK9, RNA polymerase II, c-Myc, Mcl-1, and survivin were determined by qRT-PCR and western blotting, and the proliferation and apoptosis of rat pulmonary arterial tissues and/or HPASMCs were also assayed. RESULTS: Compared to the control group, CDK9 was upregulated in pulmonary arterial tissues from MCT-induced PH rats and hypoxic cultured HPASMCs. Upregulation of CDK9 was associated with enhanced phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNA pol II) at serine-2 (Ser-2), promoting the expression of prosurvival and antiapoptotic proteins (c-Myc, Mcl-1, and survivin). Furthermore, treatment with flavopiridol (5 mg/kg) significantly alleviated pulmonary artery remodeling and partially reversed the progression of MCT-induced PH. Consistently, flavopiridol (0.5 µM) treatment decreased the proliferation and induced the apoptosis of cultured HPASMCs under hypoxic conditions. As a result of CDK9 inhibition and subsequent inhibition of RNA pol II CTD phosphorylation at Ser-2, flavopiridol decreased c-Myc, Mcl-1, and survivin expression in isolated pulmonary small arteries, leading to cell growth inhibition and apoptosis. CONCLUSION: Flavopiridol mitigates the progression of MCT-induced PH in rats by targeting CDK9.

Cardiac-targeted PIASy gene silencing mediates deSUMOylation of caveolin-3 and prevents ischemia/reperfusion-induced Nav1.5 downregulation and ventricular arrhythmias.

BACKGROUND: Abnormal myocardial Nav1.5 expression and function cause lethal ventricular arrhythmias during myocardial ischemia-reperfusion (I/R). Protein inhibitor of activated STAT Y (PIASy)-mediated caveolin-3 (Cav-3) SUMO modification affects Cav-3 binding to the voltage-gated sodium channel 1.5 (Nav1.5). PIASy activity is increased after myocardial I/R, but it is unclear whether this is attributable to plasma membrane Nav1.5 downregulation and ventricular arrhythmias. METHODS: Using recombinant adeno-associated virus subtype 9 (AAV9), rat cardiac PIASy was silenced using intraventricular injection of PIASy short hairpin RNA (shRNA). After two weeks, rat hearts were subjected to I/R and electrocardiography was performed to assess malignant arrhythmias. Tissues from peri-infarct areas of the left ventricle were collected for molecular biological measurements. RESULTS: PIASy was upregulated by I/R (P < 0.01), with increased SUMO2/3 modification of Cav-3 and reduced membrane Nav1.5 density (P < 0.01). AAV9-PIASy shRNA intraventricular injection into the rat heart downregulated PIASy after I/R, at both mRNA and protein levels (P < 0.05 vs. Scramble-shRNA + I/R group), decreased SUMO-modified Cav-3 levels, enhanced Cav-3 binding to Nav1.5, and prevented I/R-induced decrease of Nav1.5 and Cav-3 co-localization in the intercalated disc and lateral membrane. PIASy silencing in rat hearts reduced I/R-induced fatal arrhythmias, which was reflected by a modest decrease in the duration of ventricular fibrillation (VF; P < 0.05 vs. Scramble-shRNA + I/R group) and a significantly reduced arrhythmia score (P < 0.01 vs. Scramble-shRNA + I/R group). The anti-arrhythmic effects of PIASy silencing were also evidenced by decreased episodes of ventricular tachycardia (VT), sustained VT and VF, especially at the time 5-10 min after ischemia (P < 0.05 vs. Scramble-shRNA + IR group). Using in vitro human embryonic kidney 293 T (HEK293T) cells and isolated adult rat cardiomyocyte models exposed to hypoxia/reoxygenation (H/R), we confirmed that increased PIASy promoted Cav-3 modification by SUMO2/3 and Nav1.5/Cav-3 dissociation after H/R. Mutation of SUMO consensus lysine sites in Cav-3 (K38R or K144R) altered the membrane expression levels of Nav1.5 and Cav-3 before and after H/R in HEK293T cells. CONCLUSIONS: I/R-induced cardiac PIASy activation increased Cav-3 SUMOylation by SUMO2/3 and dysregulated Nav1.5-related ventricular arrhythmias. Cardiac-targeted PIASy silencing mediated Cav-3 deSUMOylation and partially prevented I/R-induced Nav1.5 downregulation in the plasma membrane of cardiomyocytes, and subsequent ventricular arrhythmias in rats. PIASy was identified as a potential therapeutic target for life-threatening arrhythmias in patients with ischemic heart diseases.

Sevoflurane Increases Hippocampal Theta Oscillations and Impairs Memory Via TASK-3 Channels.

Sevoflurane can induce memory impairment during clinical anesthesia; however, the underlying mechanisms are largely unknown. TASK-3 channels are one of the potential targets of sevoflurane. Accumulating evidence supports a negative role of intracranial theta rhythms (4-12 Hz) in memory formation. Here, we investigated whether TASK-3 channels contribute to sevoflurane-induced memory impairment by regulating hippocampal theta rhythms. In this study, the memory performance of mice was tested by contextual fear conditioning and inhibitory avoidance experiments. The hippocampal local field potentials (LFPs) were recorded from chronically implanted electrodes located in CA3 region. The results showed that sevoflurane concentration-dependently impaired the memory function of mice, as evidenced by the decreased time mice spent on freezing and reduced latencies for mice to enter the shock compartment. Our electrophysiological results revealed that sevoflurane also enhanced the spectral power of hippocampal LFPs (1-30 Hz), particularly in memory-related theta rhythms (4-12 Hz). These effects were mitigated by viral-mediated knockdown of TASK-3 channels in the hippocampal CA3 region. The knockdown of hippocampal TASK-3 channels significantly reduced the enhancing effect of sevoflurane on hippocampal theta rhythms and alleviated sevoflurane-induced memory impairment. Our data indicate that sevoflurane can increase hippocampal theta oscillations and impair memory function via TASK-3 channels.

Inhibition of RELM-ß prevents hypoxia-induced overproliferation of human pulmonary artery smooth muscle cells by reversing PLC-mediated KCNK3 decline.

AIMS: Although resistin-like molecule ß (RELM-ß) is involved in the pathological processes of various lung diseases, such as pulmonary inflammation, asthma and fibrosis, its potential roles in hypoxic pulmonary arterial hypertension (PAH) remain largely unknown. The study aims to investigate whether RELM-ß contributes to hypoxia-induced excessive proliferation of human pulmonary artery smooth muscle cells (PASMCs) and to explore the potential mechanisms of this process. MAIN METHODS: Human PASMCs were exposed to normoxia or hypoxia (1% O2) for 24 h. siRNA targeting RELM-ß was transfected into cells. Protein levels of KCNK3, RELM-ß, pSTAT3 and STAT3 were determined by immunoblotting. The translocation of NFATc2 and expression of KCNK3 were visualized by immunofluorescence. 5-ethynyl-2'-deoxyuridine assays and cell counting kit-8 assays were performed to assess the proliferation of PASMCs. KEY FINDINGS: (1) Chronic hypoxia significantly decreased KCNK3 protein levels while upregulating RELM-ß protein levels in human PASMCs, which was accompanied by excessive proliferation of cells. (2) RELM-ß could promote human PASMCs proliferation and activate the STAT3/NFAT axis by downregulating KCNK3 protein under normoxia. (3) Inhibition of RELM-ß expression effectively prevented KCNK3-mediated cell proliferation under hypoxia. (4) Phospholipase C (PLC) inhibitor U-73122 could not only prevent the hypoxia/RELM-ß-induced decrease in KCNK3 protein, but also inhibit the enhanced cell viability caused by hypoxia/RELM-ß. (5) Both hypoxia and RELM-ß could downregulate membrane KCNK3 protein levels by enhancing endocytosis. SIGNIFICANCE: RELM-ß activation is responsible for hypoxia-induced excessive proliferation of human PASMCs. Interfering with RELM-ß may alleviate the progression of hypoxic PAH by upregulating PLC-dependent KCNK3 expression.

Propofol Provides Cardiac Protection by Suppressing the Proteasome Degradation of Caveolin-3 in Ischemic/Reperfused Rat Hearts.

The mechanisms underlying propofol's cardioprotective role remain elusive. Caveolin-3 (Cav-3) has been shown to mediate both opioids- and volatile anesthetics-induced cardioprotection against ischemia/reperfusion (I/R) injury. We hypothesize that the cardioprotective role of propofol is mediated through Cav-3 and its regulation of PI3K/Akt/GSK3ß signal pathway. Rats or H9c2 cardiomyocytes were exposed to propofol before I/R or simulated ischemia/reperfusion (SI/R). Propofol pretreatment significantly decreased left ventricle infarct size in vivo (P < 0.05) and terminal deoxynucleotidyl transferase nick-end labeling-positive cells both in vivo and in vitro (P < 0.05), along with an increased Cav-3 protein expression and binding of Cav-3 to p85-subunit of PI3K. No significant change in Cav-3 mRNA expression in left ventricle tissues was found in either I/R or propofol-treated groups. Methyl-ß-cyclodextrin or Cav-3 siRNA was used to knockdown Cav-3 expression in vitro, which virtually abolished propofol-induced cardiac protection and PI3K/Akt/GSK3ß pathway activation. In contrast, MG132, a proteasome inhibitor, could significantly restore SI/R-induced Cav-3 decrease. It is concluded that Cav-3 mediates propofol-induced cardioprotection against I/R injury and the relevant PI3K/Akt/GSK3ß activation. The downregulation of Cav-3 under SI/R may be caused by proteasome degradation, and this process can be prevented by propofol.

Telmisartan reduced cerebral edema by inhibiting NLRP3 inflammasome in mice with cold brain injury.

The aim of this study was to investigate the possible beneficial role of telmisartan in cerebral edema after traumatic brain injury (TBI) and the potential mechanisms related to the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) pyrin domain-containing 3 (NLRP3) inflammasome activation. TBI model was established by cold-induced brain injury. Male C57BL/6 mice were randomly assigned into 3, 6, 12, 24, 48 and 72 h survival groups to investigate cerebral edema development with time and received 0, 5, 10, 20 and 40 mg/kg telmisartan by oral gavage, 1 h prior to TBI to determine the efficient anti-edemic dose. The therapeutic window was identified by post-treating 30 min, 1 h, 2 h and 4 h after TBI. Blood-brain barrier (BBB) integrity, the neurological function and histological injury were assessed, at the same time, the mRNA and protein expression levels of NLRP3 inflammasome, IL-1ß and IL-18 concentrations in peri-contused brain tissue were measured 24 h post TBI. The results showed that the traumatic cerebral edema occurred from 6 h, reached the peak at 24 h and recovered to the baseline 72 h after TBI. A single oral dose of 5, 10 and 20 mg/kg telmisartan could reduce cerebral edema. Post-treatment up to 2 h effectively limited the edema development. Furthermore, prophylactic administration of telmisartan markedly inhibited BBB impairment, NLRP3, apoptotic speck-containing protein (ASC) and Caspase-1 activation, as well as IL-1ß and IL-18 maturation, subsequently improved the neurological outcomes. In conclusion, telmisartan can reduce traumatic cerebral edema by inhibiting the NLRP3 inflammasome-regulated IL-1ß and IL-18 accumulation.

Pre- and Delayed Treatments With Ranolazine Ameliorate Ventricular Arrhythmias and Nav1.5 Downregulation in Ischemic/Reperfused Rat Hearts.

Enhanced late sodium current (late INa) and intracellular Nav1.5 redistribution contribute to ischemia/reperfusion (I/R)-induced arrhythmias. Ranolazine can reduce lethal arrhythmias by inhibiting late INa. However, little is known regarding its role in regulating the distribution of Nav1.5 during I/R. Therefore, we investigated the roles of ranolazine in post-I/R Nav1.5 expression and distribution in myocardium. Male Sprague Dawley rats were randomly assigned to 4 groups: sham, I/R, Ran Pre, and Ran Delay. Electrocardiogram and arterial pressure were recorded during the procedure. Nav1.5 mRNA and protein levels in peri-infarct cardiac tissue were determined by real-time polymerase chain reaction, Western blotting, and immunofluorescence. To further confirm the regulation of ranolazine on Nav1.5, GS967, another late INa inhibitor was used. Both pre- and delayed ranolazine treatments significantly reduced the incidence of severe ventricular arrhythmias, along with shortened corrected QT interval by 29.55% and QRS duration by 18.38% during I/R. The protein level of Nav1.5 decreased by 31.63% after I/R. Ranolazine and GS967 remained Nav1.5 protein expression and Nav1.5 redistribution on intercalated discs and lateral membranes, without affecting Nav1.5 mRNA level. In conclusion, upregulating Nav1.5 expression and redistribution on the intercalated discs and lateral membranes of cardiomyocytes may underlie the antiarrhythmic effects of ranolazine in I/R rats.

Two novel Brugada syndrome-associated mutations increase KV4.3 membrane expression and function.

The human cardiac fast transient outward K+ channel is composed of the KV4.3 α subunit encoded by KCND3 and the K+ channel­interacting protein 2 (KChIP2) ß subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in KV4.3 are associated with Brugada syndrome and they increase the KV4.3/KChIP2­encoded fast transient outward K+ current (Ito,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of Ito,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch­clamp recording was performed in HEK­293 cells expressing KV4.3­wild­type (WT) and KV4.3 mutants with KChIP2. The two individual mutant­encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed KV4.3/KChIP2­encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2­encoded channel inactivation. Western blotting showed that total KV4.3 protein was significantly augmented in HEK­293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the KV4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3­WT. Reverse transcription­polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain­of­function of KV4.3/KChIP2­encoded channels by increasing membrane protein expression and slowing channel inactivation.

Deletion of FoxO1 leads to shortening of QRS by increasing Na(+) channel activity through enhanced expression of both cardiac NaV1.5 and ß3 subunit.

Our in vitro studies revealed that a transcription factor, Forkhead box protein O1 (FoxO1), negatively regulates the expression of NaV1.5, a main α subunit of the cardiac Na(+) channel, by altering the promoter activity of SCN5a in HL-1 cardiomyocytes. The in vivo role of FoxO1 in the regulation of cardiac NaV1.5 expression remains unknown. The present study aimed to define the role of FoxO1 in the regulation of NaV1.5 expression and cardiac Na(+) channel activity in mouse ventricular cardiomyocytes and assess the cardiac electrophysiological phenotype of mice with cardiac FoxO1 deletion. Tamoxifen-induced and cardiac-specific FoxO1 deletion was confirmed by polymerase chain reaction (PCR). Cardiac FoxO1 deletion failed to result in either cardiac functional changes or hypertrophy as assessed by echocardiography and individual ventricular cell capacitances, respectively. Western blotting showed that FoxO1 was significantly decreased while NaV1.5 protein level was significantly increased in mouse hearts with FoxO1 deletion. Reverse transcription-PCR (RT-PCR) revealed that FoxO1 deletion led to an increase in NaV1.5 and Na(+) channel subunit ß3 mRNA, but not ß1, 2, and 4, or connexin 43. Whole patch-clamp recordings demonstrated that cardiac Na(+) currents were significantly augmented by FoxO1 deletion without affecting the steady-state activation and inactivation, leading to accelerated depolarization of action potentials in mouse ventricular cardiomyocytes. Electrocardiogram recordings showed that the QRS complex was significantly shortened and the P wave amplitude was significantly increased in conscious and unrestrained mice with cardiac FoxO1 deletion. NaV1.5 expression was decreased in the peri-infarct (border-zone) of mice with myocardial infarction and FoxO1 accumulated in the cardiomyocyte nuclei of chronic ischemic human hearts. Our findings indicate that FoxO1 plays an important role in the regulation of NaV1.5 and ß3 subunit expressions as well as Na(+) channel activity in the heart and that FoxO1 is involved in the modulation of NaV1.5 expression in ischemic heart disease.

Reactive oxygen species suppress cardiac NaV1.5 expression through Foxo1.

Na(V)1.5 is a cardiac voltage-gated Na(+) channel αsubunit and is encoded by the SCN5a gene. The activity of this channel determines cardiac depolarization and electrical conduction. Channel defects, including mutations and decrease of channel protein levels, have been linked to the development of cardiac arrhythmias. The molecular mechanisms underlying the regulation of Na(V)1.5 expression are largely unknown. Forkhead box O (Foxo) proteins are transcriptional factors that bind the consensus DNA sequences in their target gene promoters and regulate the expression of these genes. Comparative analysis revealed conserved DNA sequences, 5'-CAAAACA-3' (insulin responsive element, IRE), in rat, mouse and human SCN5a promoters with the latter two containing two overlapping Foxo protein binding IREs, 5'-CAAAACAAAACA-3'. This finding led us to hypothesize that Foxo1 regulates Na(V)1.5 expression by directly binding the SCN5a promoter and affecting its transcriptional activity. In the present study, we determined whether Foxo1 regulates Na(V)1.5 expression at the transcriptional level and also defined the role of Foxo1 in hydrogen peroxide (H(2)O(2))-mediated Na(V)1.5 suppression in HL-1 cardiomyocytes using chromatin immunoprecipitation (ChIP), constitutively nuclear Foxo1 expression, and RNAi Foxo1 knockdown as well as whole cell voltage-clamp recordings. ChIP with anti-Foxo1 antibody and follow-up semi-quantitative PCR with primers flanking Foxo1 binding sites in the proximal SCN5a promoter region clearly demonstrated enrichment of DNA, confirming Foxo1 recruitment to this consensus sequence. Foxo1 mutant (T24A/S319A-GFP, Foxo1-AA-GFP) was retained in nuclei, leading to a decrease of Na(V)1.5 expression and Na(+) current, while silencing of Foxo1 expression by RNAi resulted in the augmentation of Na(V)1.5 expression. H(2)O(2) significantly reduced Na(V)1.5 expression by promoting Foxo1 nuclear localization and this reduction was prevented by RNAi silencing Foxo1 expression. These studies indicate that Foxo1 negatively regulates Na(V)1.5 expression in cardiomyocytes and reactive oxygen species suppress Na(V)1.5 expression through Foxo1.

ERKs/p53 signal transduction pathway is involved in doxorubicin-induced apoptosis in H9c2 cells and cardiomyocytes.

The cardiotoxic effects of doxorubicin, a potent chemotherapeutic agent, have been linked to DNA damage, oxidative mitochondrial damage, and nuclear translocation of p53, but the exact molecular mechanisms causing p53 transactivation and doxorubicin-induced cardiomyopathy are not clear. The present study was carried out to determine whether extracellular signal-regulated kinases (ERKs), which are known to be activated by DNA damaging agents, are responsible for doxorubicin-induced p53 activation and oxidative mitochondrial damage in H9c2 cells. Cell death was measured by terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling, annexin V-fluorescein isothiocyanate, activation of caspase-9 and -3, and cleavage of poly(ADP-ribose) polymerase (PARP). We found that doxorubicin produced cell death in H9c2 cells in a time-dependent manner, beginning at 6 h, and these changes are associated decreased expression of Bcl-2, increases in Bax and p53 upregulated modulator of apoptosis-alpha expression, and collapse of mitochondria membrane potential. The changes in cell death and Bcl-2 family proteins, however, were preceded by earlier activation and nuclear translocation of ERKs, followed by increased phosphorylation at Ser15 and nuclear translocation of the phosphorylated p53. The functional importance of ERK1/2 and p53 in doxorubicin-induced toxicity was further demonstrated by the specific ERK inhibitor U-0126 and p53 inhibitor pifithrin (PFT)-alpha, which abrogated the changes in Bcl-2 family proteins and cell death produced by doxorubicin. U-0126 blocked the phosphorylation and nuclear translocation of both ERK1/2 and p53, whereas PFT-alpha blocked only the changes in p53. Doxorubicin and ERK inhibitors produced similar changes in ERK1/2-p53, PARP, and caspase-3 in neonatal rat cultured cardiomyocytes. Thus we conclude that ERK1/2 are functionally linked to p53 and that the ERK1/2-p53 cascade is the upstream signaling pathway responsible for doxorubicin-induced cardiac cell apoptosis. ERKs and p53 may be considered as novel therapeutic targets for the treatment of doxorubicin-induced cardiotoxicity.

Pro-apoptotic effects of anti-beta1-adrenergic receptor antibodies in cultured rat cardiomyocytes: actions on endoplasmic reticulum and the prosurvival PI3K-Akt pathway.

An anti-beta(1)-adrenergic receptor antibody against the second extracellular receptor loop (beta(1)-EC(II)) has been shown to cause myocyte apoptosis and dilated cardiomyopathy in animals. We report in this review that the anti-beta(1)-EC(II) antibody increases intracellular Ca(++) transients and exerts a direct apoptotic effect in cultured neonatal rat cardiomyocytes. Both Fab and Fc fragments are required for the full expression of the apoptotic effects of the anti-beta(1)-EC(II) antibody. Our studies further suggest that the anti-beta(1)-EC(II)-antibody acts primarily on the cardiac beta(1)-adrenergic receptor and its post-receptor activation of Ca(++)/Calmodulin dependent protein kinase II (CaMKII) and p-38 mitogen-activated protein kinase (MAPK), leading to endoplasmic reticulum stress as evidenced by the increased expressions of GRP78 and CHOP, as well as the increased processing of the initiator procaspase-12. Also, observed with the apoptotic effect of anti-beta(1)-EC(II) antibody is reduced activity of the phosphatidylinositol (PI) 3-kinase/Akt/STAT3 signaling pathway. Our results suggest that agents that block the activation of p38-MAPK/endoplasmic reticulum stress or reverse the suppression of the prosurvival PI3K/Akt/STAT3 pathway may be explored as potential novel therapeutic modalities in the treatment of dilated cardiomyopathy.

Darbepoetin alfa exerts a cardioprotective effect in autoimmune cardiomyopathy via reduction of ER stress and activation of the PI3K/Akt and STAT3 pathways.

Dilated human cardiomyopathy is associated with suppression of the prosurvival phosphatidylinositol-3-kinase (PI3K)/Akt and STAT3 pathways. The present study was carried out to determine if restoration of the PI3K/Akt and STAT3 activity by darbepoetin alfa improved cardiac function or reduced cardiomyocyte apoptosis in rabbit autoimmune cardiomyopathy induced by a peptide corresponding to the second extracellular loop of the ss(1)-adrenergic receptor (ss(1)-EC(II)). We found that ss(1)-EC(II) immunization produced progressive LV dilation, systolic dysfunction and myocyte apoptosis as measured by TUNEL, single-stranded DNA antibody, and active caspase-3. These changes were associated with activation of p38 mitogen-activated protein kinase (MAPK), endoplasmic reticulum stress markers (GRP78 and CHOP), and increased cleavage of procaspase-12, as well as decreased phosphorylation of Akt and STAT3, and decreased Bcl2/Bax ratio. As expected, darbepoetin alfa treatment increased phosphorylation of Akt and STAT3. It also increased the myocardial expression of erythropoietin receptor which was reduced in the failing myocardium, and improved cardiac function in the ss(1)-EC(II)-immunized animals. The latter was associated with reductions of myocyte apoptosis and cleaved caspase-3, as well as reversal of increased phosphorylation of p38-MAPK, increased ER stress, and decline in Bcl2/Bax ratio. The anti-apoptotic effects of darbepoetin alfa via Akt and STAT activation were also demonstrated in cultured cardiomyocytes treated with the anti-ss(1)-EC(II) antibody. These effects of darbepoetin alfa in vitro were prevented by LY294002 and STAT3 peptide inhibitor. Thus, we conclude that darbepoetin alfa improves cardiac function and prevents progression of dilated cardiomyopathy probably by activating the PI3K/Akt and STAT3 pathways and reducing ER stress.

Adoptive passive transfer of rabbit beta1-adrenoceptor peptide immune cardiomyopathy into the Rag2-/- mouse: participation of the ER stress.

Auto-antibodies against the beta(1)-adrenoceptors are present in 30-40% of patients with dilated cardiomyopathy. Recently, a synthetic peptide corresponding to a sequence of the second extracellular loop of the human beta(1)-adrenoceptor (beta(1)-EC(II)) has been shown to produce endoplasmic reticulum (ER) stress, myocyte apoptosis and cardiomyopathy in immunized rabbits. To study the direct cardiac effects of anti-beta(1)-EC(II) antibody in intact animals and if they are mediated via beta(1)-adrenoceptor stimulation, we administered IgG purified from beta(1)-EC(II)-immunized rabbits to recombination activating gene 2 knock-out (Rag2(-/-)) mice every 2 weeks with and without metoprolol treatment. Serial echocardiography and cardiac catheterization showed that beta(1)-EC(II) IgG reduced cardiac systolic function after 3 months. This was associated with increase in heart weight, myocyte apoptosis, activation of caspase-3, -9 and -12, and increased ER stress as evidenced by upregulation of GRP78 and CHOP and cleavage of ATF6. The Rag2(-/-) mice also exhibited increased phosphorylation of CaMKII and p38 MAPK. Metoprolol administration, which attenuated the phosphorylation of CaMKII and p38 MAPK, reduced the ER stress, caspase activation and cell death. Finally, we employed the small-interfering RNA technology to reduce caspase-12 in cultured rat cardiomyocytes. This reduced not only the increase of cleaved caspase-12 but also of the number of myocyte apoptosis produced by beta(1)-EC(II) IgG. Thus, we conclude that ER stress plays an important role in cell death and cardiac dysfunction in beta(1)-EC(II) IgG cardiomyopathy, and the effects of beta(1)-EC(II) IgG are mediated via the beta(1)-adrenergic receptor.

Cardiomyocyte apoptosis in autoimmune cardiomyopathy: mediated via endoplasmic reticulum stress and exaggerated by norepinephrine.

Evidence suggests that the autoimmune cardiomyopathy produced by a peptide corresponding to the sequence of the second extracellular loop of the beta(1)-adrenergic receptor (beta(1)-EC(II)) is mediated via a biologically active anti-beta(1)-EC(II) antibody, but the mechanism linking the antibody to myocyte apoptosis and cardiac dysfunction has not been well elucidated. Since the beta(1)-EC(II) autoantibody is a partial beta(1)-agonist, we speculate that the cardiomyopathy is produced by the beta(1)-receptor-mediated stimulation of the CaMKII-p38 MAPK-ATF6 signaling pathway and endoplasmic reticulum (ER) stress, and that excess norepinephrine (NE) exaggerates the cardiomyopathy. Rabbits were randomized to receive beta(1)-EC(II) immunization, sham immunization, NE pellet, or beta(1)-EC(II) immunization plus NE pellet for 6 mo. Heart function was measured by echocardiography and catheterization. Myocyte apoptosis was determined by terminal deoxytransferase-mediated dUTP nick-end labeling and caspase-3 activity, whereas CaMKII, MAPK family (JNK, p38, ERK), and ER stress signals (ATF6, GRP78, CHOP, caspase-12) were measured by Western blot, immunohistochemistry, and kinase activity assay. beta(1)-EC(II) immunization produced progressive LV dilation, systolic dysfunction, and myocyte apoptosis. These changes were associated with activation of GRP78 and CHOP and increased cleavage of caspase-12, as well as increased CaMKII activity, increased phosphorylation of p38 MAPK, and nucleus translocation of cleaved ATF6. NE pellet produced additive effects. In addition, KN-93 and SB 203580 abolished the induction of ER stress and cell apoptosis produced by the beta(1)-EC(II) antibody in cultured neonatal cardiomyocytes. Thus ER stress occurs in autoimmune cardiomyopathy induced by beta(1)-EC(II) peptide, and this is enhanced by increased NE and caused by activation of the beta(1)-adrenergic receptor-coupled CaMKII, p38 MAPK, and ATF6 pathway.

Cardiac sympathetic neuroprotective effect of desipramine in tachycardia-induced cardiomyopathy.

Cardiac sympathetic transmitter stores are reduced in the failing heart. In this study, we proposed to investigate whether the reduction of cardiac sympathetic neurotransmitters was associated with increased interstitial norepinephrine (NE) and reactive oxygen species in congestive heart failure (CHF), using a microdialysis technique and salicylate to detect .OH generation. Rabbits with and without rapid ventricular pacing (340 beats/min) were randomized to receive desipramine (10 mg/day) or placebo for 8 wk. Rapid pacing produced left ventricular dilation and systolic dysfunction. The failing myocardium also showed reduced tissue contents of NE and tyrosine hydroxylase protein and activity. In contrast, myocardial interstitial NE was increased in CHF (0.89 +/- 0.11 ng/ml) compared with the sham-operated animals (0.26 +/- 0.03 ng/ml). In addition, cardiac oxidative stress was increased in CHF animals as measured by myocardial interstitial .OH radical, tissue oxidized glutathione, and oxidized mitochondrial DNA. Desipramine treatment produced significant NE uptake inhibition as evidence by an exaggerated pressor response and a greater increase of myocardial interstitial NE in response to intravenous NE infusion but no significant effects on cardiac function or hemodynamics in sham-operated or CHF animals. However, desipramine treatment attenuated the reductions of tissue NE and tyrosine hydroxylase protein and activity in CHF. Desipramine also prevented the reduction of tyrosine hydroxylase produced by NE in PC12 cells. Thus the reduction of cardiac sympathetic neurotransmitters is related to the increased interstitial NE and tissue oxidative stress in CHF. Also, normal neuronal uptake of NE is required for NE or its oxidized metabolites to exert their neurotoxic effects.

Norepinephrine-induced oxidative stress causes PC-12 cell apoptosis by both endoplasmic reticulum stress and mitochondrial intrinsic pathway: inhibition of phosphatidylinositol 3-kinase survival pathway.

Norepinephrine (NE) induces endoplasmic reticulum (ER) unfolded protein response and reduces maturation and translocation of NE transporter to cell membrane via enhanced formation of reactive oxygen species in PC-12 cells. In the present study, we investigated whether ER stress is also implicated in the proapoptotic effect of NE. We found that the apoptotic effect of NE was associated with increased processing of ER-resident pro-caspase-12, cleavage of caspase-9 and -3, and mitochondrial release of cytochrome c. ER stress was evidenced by upregulation of ER chaperone GRP78 and transcription factor CHOP and the translocation of XBP-1 from the ER to the nucleus by NE. NE also reduced phospho-Akt (Ser473), indicating suppression of the phosphatidylinositol 3-kinase (PI3-kinase)/Akt survival pathway. Similar results were produced by thapsigargin. NGF, which promotes the PI3-kinase/Akt activity, reduced the effects of NE and thapsigargin on apoptosis and activation of caspase-12 and -3. However, the effects of NE, but not of thapsigargin, were abolished by pretreatment with SOD and catalase. In contrast, the PI3-kinase inhibitors LY-294002 and wortmannin abolished the protective effects of both SOD/catalase and NGF on NE-induced apoptosis. The functional importance of caspase-12 activation was supported by the use of Z-ATAD-FMK, which reduced the NE-induced processing of caspase-12 and cell apoptosis, but the caspase-12, -9, and -3 inhibitors had no effects on the increase in cytosolic cytochrome c produced by NE. In contrast, the release of mitochondrial cytochrome c was abolished by SOD/catalase and NGF. These results indicate that NE induced cell apoptosis by both ER stress and a mitochondrial death pathway and that the effects of NE were mediated via oxidative stress and inhibition of the PI3-kinase/Akt survival pathway.

Norepinephrine induces endoplasmic reticulum stress and downregulation of norepinephrine transporter density in PC12 cells via oxidative stress.

Cardiac norepinephrine (NE) uptake is reduced in cardiomyopathy. This change is associated with a decrease of NE transporter (NET) receptor and can be reproduced in PC12 cells by extracellular NE. To study whether this effect of NE is mediated via impaired glycosylation and trafficking of NET in the endoplasmic reticulum (ER), we measured the distribution of glycosylated 80-kDa NET and unglycosylated 46-kDa NET in the membrane and cytosolic fractions of PC12 cells. We found that NE decreased glycosylated NET in both membrane and cytosolic fractions and increased cytosolic unglycosylated NET protein. Similar results were produced by tunicamycin and thapsigargin, two agents that induce ER stress by inhibiting N-glycosylation of membrane proteins and disrupting calcium homeostasis, respectively. Also, like the ER stressors, NE not only increased phosphorylation of both the alpha-subunit of eukaryotic initiation factor-2 and its upstream RNA-dependent protein kinase-like ER kinase over 12 h of treatment but also increased ER chaperone molecule glucose-regulated protein 78 and the nuclear transcription factor C/EBP homologous protein. Antioxidants superoxide dismutase and catalase prevented the downregulation of NET proteins and induction of ER stress signals produced by NE but not by tunicamycin or thapsigargin. The results indicate that the downregulation of membrane NET by NE is mediated by decreased N-glycosylation of NET proteins secondary to induction of ER stress pathways by NE-derived oxidative metabolites. Interventions involving the ER stress pathways may provide novel therapeutic strategies for the treatment of sympathetic dysfunction in heart failure.

Importance of antioxidant and antiapoptotic effects of beta-receptor blockers in heart failure therapy.

The present study was carried out to determine whether beneficial effects of carvedilol in congestive heart failure (CHF) are mediated via its beta-adrenergic blocking, antioxidant, and/or alpha-adrenergic blocking action. Rabbits with heart failure induced by rapid cardiac pacing were randomized to receive subcutaneous carvedilol, metoprolol, propranolol plus doxazosin, or placebo pellets for 8 wk and compared with sham-operated rabbits without pacing. We found rapid cardiac pacing produced clinical heart failure, left ventricular dilation, and decline of left ventricular fractional shortening. This was associated with an increase in left ventricular end-diastolic pressure, decrease in left ventricular first derivative of left ventricular pressure, and myocyte hypertrophy. Tissue oxidative stress measured by GSH/GSSG was increased in the heart with increased oxidation product of mitochondrial DNA, 8-oxo-7,8-dihydro-2'-deoxyguanosine, increase of Bax, decrease of Bcl-2, and increase of apoptotic myocytes as measured by anti-single-stranded DNA monoclonal antibody. Administration of carvedilol and metoprolol, which had no effect in sham animals, attenuated cardiac ventricular remodeling, cardiac hypertrophy, oxidative stress, and myocyte apoptosis in CHF. In contrast, propranolol plus doxazosin, which has less antioxidant effects, produced smaller effects on left ventricular function and myocyte apoptosis. In all animals, GSH/GSSG correlated significantly with changes of left ventricular end-diastolic dimension (r = -0.678, P < 0.0001), fractional shortening (r = 0.706, P < 0.0001), and apoptotic myocytes (r = -0.473, P = 0.0001). Thus our findings suggest antioxidant and antiapoptotic actions of carvedilol and metoprolol are important determinants of clinical beneficial effects of beta-receptors in the treatment of CHF.