Isoproterenol Stimulation Research Articles

Effects of the restrictive cardiomyopathy-associated cardiac troponin I K178E mutation on cAMP signalling at the myofilament

Abstract Background Catecholaminergic stimulation of cardiac β-adrenergic receptors (β-ARs) and subsequent cAMP signalling control cardiac inotropy and lusitropy. cAMP signalling is known to be compartmentalised to optimise sympathetic control by producing heterogeneous cAMP signals and protein kinase A (PKA) activation at distinct subcellular sites. This signalling may be disrupted in diseases such as restrictive cardiomyopathy (RCM) characterised by diastolic dysfunction, which is associated with certain mutations including the K178E missense mutation in cardiac troponin I (TPNI). The impaired ventricular relaxation associated with this mutation is thought to arise from myofilament calcium hypersensitivity, which can be modulated by PKA-dependent TPNI phosphorylation. However, a detailed understanding of the pathological effects mediated by the K178E-TPNI mutation is lacking. Purpose This study aims to determine whether the K178E-TPNI mutation alters local cAMP/PKA signalling at TPNI, potentially affecting myofilament calcium sensitivity and cardiac myocyte relaxation. Methods Real-time imaging of cAMP was carried out using the Fluorescence Resonance Energy Transfer (FRET)-based sensor CUTie (cAMP Universal Tag for imaging experiments) [1] fused to wild type (WT)- or K178E-TPNI. The sensors were expressed by transfection in neonatal rat ventricular myocytes (NRVMs) and FRET-changes were recorded at the myofilament on application of the β-AR stimulant isoproterenol (ISO). We further assessed the involvement of cAMP hydrolysing phosphodiesterases (PDEs) in the regulation of cAMP levels selectively at TPNI. Results The peak FRET-change and FRET-change after 5mins on ISO application were significantly higher in NRVMs expressing the mutant compared to the WT sensor, as was the rate of cAMP accumulation. A strong trend showed that the rise in FRET following PDE4 inhibition was larger in NRVMs expressing the WT compared to the mutant sensor, suggesting the involvement of this isoform in degrading cAMP locally at TPNI and reduced PDE4 activity in the presence of the K178E mutation. Co-immunoprecipitation analysis in HEK293 cells demonstrated interaction between WT-TPNI and a PDE4D isoform, and a weaker interaction of this isoform with the K178E-TPNI mutant. Conclusions This study shows that the K178E-TPNI mutation attenuates the TPNI/PDE4D interaction and alters the magnitude and kinetics of the local cAMP response at the myofilament. This mechanistic insight may aid the development of therapeutics targeting the TPNI/PDE4D interaction to treat RCM-associated diastolic dysfunction.The TPNI-CUTie SensorFRET Response Trace

Ablation of three major phospho-sites in RyR2 preserves the global adrenergic response but creates an arrhythmogenic substrate.

Ryanodine receptor 2 (RyR2) is one of the first substrates undergoing phosphorylation upon catecholaminergic stimulation. Yet, the role of RyR2 phosphorylation in the adrenergic response remains debated. To date, three residues in RyR2 are known to undergo phosphorylation upon adrenergic stimulation. We generated a model of RyR2 phospho-ablation of all three canonical phospho-sites (RyR2-S2031A/S2808A/S2814A, triple phospho-mutant, TPM) to elucidate the role of phosphorylation at these residues in the adrenergic response. Cardiac structure and function, cellular Ca 2+ dynamics and electrophysiology, and RyR2 channel activity both under basal conditions and under isoproterenol (Iso) stimulation were systematically evaluated. We used echocardiography and electrocardiography in anesthetized mice, single-cell Ca 2+ imaging and whole-cell patch clamp in isolated adult cardiomyocytes, and biochemical assays. Iso stimulation produced normal chronotropic and inotropic responses in TPM mice as well as an increase in the global Ca 2+ transients in isolated cardiomyocytes. Functional studies revealed fewer Ca 2+ sparks in permeabilized TPM myocytes, and reduced RyR2-mediated Ca 2+ leak in intact myocytes under Iso stimulation, suggesting that the canonical sites may regulate RyR2-mediated Ca 2+ leak. TPM mice also displayed increased propensity for arrhythmia. TPM myocytes were prone to develop early afterdepolarizations (EADs), which were abolished by chelating intracellular Ca 2+ with EGTA, indicating that EADs require SR Ca 2+ release. EADs were also blocked by a low concentration of tetrodotoxin, further suggesting reactivation of the sodium current ( I Na ) as the underlying cause. Phosphorylation of the three canonical residues on RyR2 may not be essential for the global adrenergic responses. However, these sites play a vital role in maintaining electrical stability during catecholamine stimulation by fine-tuning RyR2-mediated Ca 2+ leak. These findings underscore the importance of RyR2 phosphorylation and a finite diastolic Ca 2+ leak in maintaining electrical stability during catecholamine stimulation.

Phosphoinositide 3-Kinase C2alpha controls cardiac contractility through regulation of beta2-adrenergic receptor recycling

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Doctoral fellowship Introduction Phosphoinositide 3-kinase C2α (PI3KC2α) is a ubiquitously expressed class II PI3K isoform, which has been previously shown to be involved in the control of vesicular trafficking. However, its role in the myocardium has not been investigated. The primary aim of this study is to elucidate the mechanism by which mechanism PI3KC2α controls cardiac pathophysiology. Methods Cardiomyocyte-specific PI3KC2α knockout (PI3KC2α KO) animals were generated by crossing mice expressing a tamoxifen-inducible Cre recombinase under the control of the αMHC promoter with PI3KC2αflox/flox mice. Zebrafish embryos were injected with a PI3KC2α (PI3KC2α morphants) or a control morpholino (control morphants) at 1-cell stage. HEK293 with stable overexpression of GFP-tagged β2-AR were generated and transfected with either scramble or PI3KC2α siRNAs. Results Our findings indicate that PI3KC2α KO mice display a reduced cardiac contractility compared to wildtype animals. Similarly, PI3KC2α morphants zebrafish exhibit lower heart rate and fractional shortening compared to controls, both at baseline and after isoproterenol (ISO) stimulation. A similar unresponsiveness to ISO was found in vivo in PI3KC2α KO mice where chronic treatment with the β-adrenergic receptor (β-AR) agonist failed to induce the classical β-AR-mediated remodeling, characterized by an increase of the left ventricular mass and of cardiomyocyte area. These findings suggest that PI3KC2α plays a critical role in the regulation of β-AR signaling. In agreement, cAMP levels failed to increase in response to ISO treatment in PI3KC2α morphants. Furthermore, silencing of PI3KC2α in HEK293-GFP-β2-AR cells resulted in an increased GFP-β2-AR internalization compared to control cells, both at baseline and after ISO stimulation. Interestingly, preliminary findings indicate that overexpression of constitutively active Rab11Q70L induced a redistribution of GFP-β2-AR at the plasma membrane in PI3KC2α-silenced cells. Conclusion Overall, we identify a key role for PI3KC2α in the control of cardiac contractility through the regulation of β2-AR trafficking to the plasma membrane through a Rab11-dependent mechanism.

Gene therapy with phosphodiesterases 2A and 4B ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.

Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.

Potential Role of Pig UCP3 in Modulating Adipocyte Browning via the Beta-Adrenergic Receptor Signaling Pathway.

Adipose tissue plays an important role in regulating body temperature and metabolism, with white adipocytes serving as storage units for energy. Recent research focused on the browning of white adipocytes (beige adipocytes), causing thermogenesis and lipolysis. The process of browning is linked to the activation of uncoupling protein (UCP) expression, which can be mediated by the β3 adrenergic receptor pathway. Transcriptional factors, such as peroxisome proliferator activated receptor γ (PPARγ) and PPARγ coactivator 1 alpha, play vital roles in cell fate determination for fat cells. Beige adipocytes have metabolic therapeutic potential to combat diseases such as obesity, diabetes mellitus, and dyslipidemia, owing to their significant impact on metabolic functions. However, the molecular mechanisms that cause the induction of browning are unclear. Therefore, research using animal models and primary culture is essential to provide an understanding of browning for further application in human metabolic studies. Pigs have physiological similarities to humans; hence, they are valuable models for research on adipose tissue. This study demonstrates the browning potential of pig white adipocytes through primary culture experiments. The results show that upregulation of UCP3 gene expression and fragmentation of lipid droplets into smaller particles occur due to isoproterenol stimulation, which activates beta-adrenergic receptor signaling. Furthermore, PPARγ and PGC-1α were found to activate the UCP3 promoter region, similar to that of UCP1. These findings suggest that pigs undergo metabolic changes that induce browning in white adipocytes, providing a promising approach for metabolic research with potential implications for human health. This study offers valuable insights into the mechanism of adipocyte browning using pig primary culture that can enhance our understanding of human metabolism, leading to cures for commonly occurring diseases.

Deficiency of heme oxygenase 1a causes detrimental effects on cardiac function.

Humans lacking heme oxygenase 1 (HMOX1) display growth retardation, haemolytic anaemia, and vulnerability to stress; however, cardiac function remains unclear. We aimed to explore the cardiac function of zebrafish lacking hmox1a at baseline and in response to stress. We generated zebrafish hmox1a mutants using CRISPR/Cas9 genome editing technology. Deletion of hmox1a increases cardiac output and further induces hypertrophy in adults. Adults lacking hmox1a develop myocardial interstitial fibrosis, restrain cardiomyocyte proliferation and downregulate renal haemoglobin and cardiac antioxidative genes. Larvae lacking hmox1a fail to respond to hypoxia, whereas adults are insensitive to isoproterenol stimulation in the heart, suggesting that hmox1a is necessary for cardiac response to stress. Haplodeficiency of hmox1a stimulates non-mitochondrial respiration and cardiac cell proliferation, increases cardiac output in larvae in response to hypoxia, and deteriorates cardiac function and structure in adults upon isoproterenol treatment. Intriguingly, haplodeficiency of hmox1a upregulates cardiac hmox1a and hmox1b in response to isoproterenol. Collectively, deletion of hmox1a results in cardiac remodelling and abrogates cardiac response to hypoxia and isoproterenol. Haplodeficiency of hmox1a aggravates cardiac response to the stress, which could be associated with the upregulation of hmox1a and hmox1b. Our data suggests that HMOX1 homeostasis is essential for maintaining cardiac function and promoting cardioprotective effects.

Artemisinin Attenuates Isoproterenol-induced Cardiac Hypertrophy via the ERK1/2 and p38 MAPK Signaling Pathways.

Artemisinin (ART) is mainly derived from Artemisia annua, a traditional Chinese medicinal plant, and has been found to affect cellular biochemical processes, such as proliferation, angiogenesis, and apoptosis, in addition to its antimalarial properties. However, its effect on cardiac hypertrophy and the underlying mechanisms remain unclear. This study aimed to investigate the effect of ART on cardiac hypertrophy and explore its possible mechanisms. A rat model was established by intraperitoneal injection of isoproterenol (ISO) for 3 days, and the degree of myocardial hypertrophy was compared among 5 groups: a control (CON) group, an ISO group, and groups treated with different doses of ART (7 mg/kg/d, 35 mg/kg/d, and 75 mg/kg/d). Echocardiography was used to evaluate cardiac function and structure. The cross-sectional area of cardiomyocytes was measured by hematoxylin and eosin (H&E) staining. The heart weight (HW), body weight (BW), and tail length were measured, and the HW/tail length ratio and the HW/BW ratio were calculated. H9c2 rat cardiomyocytes were cultured, and different amounts of ART were added 2 hours before ISO stimulation. Phalloidin staining was used to evaluate the degree of cell hypertrophy. The levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were quantified in rat plasma and cell supernatant using enzyme-linked immunosorbent assay (ELISA), while the expression levels of p- ERK1/2, p-JNK, and p-p38 MAPK were assessed in the myocardium and H9c2 cells via western blot analysis. Intragastric administration of ART at a dosage of 35 mg/kg/d or over mitigated the early-stage cardiac hypertrophy induced by ISO in rats led to a reduction in left ventricular posterior wall diastolic thickness, interventricular septal thickness at diastole, lowered ANP and BNP levels, as well as a decrease in HW/tail length and HW/BW ratio. In vitro studies demonstrated that ART at a concentration of 100 μM inhibited ISO-mediated hypertrophy of H9c2 cells. The ISO group showed a higher p-ERK/GAPDH ratio and p-p38 MAPK/GAPDH ratio than the control group both in vivo and in vitro. Although the p-JNK/GAPDH ratio was increased in the ISO group, there was no statistical difference. The p-ERK/GAPDH and p-p38/GAPDH ratios were significantly lower in the ART group than in the ISO group. The mechanism of ART against cardiac hypertrophy was related to inhibition of the ERK1/2 and p38 MAPK signaling pathways.

CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the β2-adrenergic receptor

In essence, the β2 adrenergic receptor (β2AR) plays an antiproliferative role by increasing the intracellular cyclic 3’,5’-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, β2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the β2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired β2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to β2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not β-arrestin2 or protein kinase A-mediated phosphorylation of β2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6′-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to β2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in β2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA.7HWufKwpH5ZfY_hx8fyY6aVideo

Rats with Postinfarction Heart Failure: Effects of Propranolol Therapy on Intracellular Calcium Regulation and Left Ventricular Function

Patients with heart failure may live longer if they receive chronic treatment with beta-adrenergic blocking medications. Unresolved are the mechanisms underlying the beneficial effects and if they may be applied to ischemic heart failure. Rats (n = 28) underwent echocardiographic-Doppler exams one and six weeks following a simulated operation or myocardial infarction (MI). After the first echocardiography, rats were randomized to either no therapy or 500 mg/l of propranolol in their drinking water. The noninfected left ventricular (LV) papillary muscles were used to record isometric contractions and intracellular Ca transients simultaneously. Untreated MI rats had a restrictive LV diastolic filling pattern, decreased systolic function (13% ± 2%), and significant LV dilatation (10.6 ± 0.4 mm vs. 8.9 ± 0.3 mm, MI vs. control). The LV chamber diameters of the propranolol-treated MI rats were 10.6 ± 0.5 mm, and systolic performance (13% ± 2%). Higher LV end-diastolic pressures (27 ± 2 mmHg vs. 20 ± 3 mmHg) and a more constrained LV diastolic filling pattern (increased ratio of early to late filling velocities and faster E wave deceleration rate) were seen in the propranolol-treated animals. Papillary muscle contractility in untreated MI rats was lower (1.6 ± 0.3 g mm²). Furthermore, the inotropic response to isoproterenol was attenuated, and Ca transients were extended. During isoproterenol stimulation, beta-adrenergic blocking administration had no effect on force development (1.6 ± 0.3 g mm²) or the length of Ca transients. Rats with postinfarction heart failure receiving chronic propranolol treatment did not have improvements in systolic function or LV remodeling. Propranolol exacerbated LV diastolic pressures and filling patterns. Additionally, there was no discernible improvement in intracellular contractility following treatment, Calcium control, or beta-adrenergic sensitivity in the myocardium that is not infarcted).

Estradiol-mediated small GTP-binding protein GDP dissociation stimulator induction contributes to sex differences in resilience to ferroptosis in takotsubo syndrome

BackgroundDeclining beneficial cardiovascular actions of estradiol (E2) have been associated with disproportionate susceptibility to takotsubo syndrome (TTS) in postmenopausal women. However, the underlying mechanisms between E2 and this marked disproportion remain unclear. SmgGDS (small GTP-binding protein GDP dissociation stimulator), as a key modulator of cardiovascular disease, plays protective roles in reducing oxidative stress and exerts pleiotropic effects of statins. Whether SmgGDS levels are influenced by E2 status and the effect of SmgGDS on sex differences in TTS are poorly understood. MethodsClinical data were reviewed from TTS inpatients. Echocardiography, immunofluorescence, and immunohistochemistry were performed together with expression analysis to uncover phenotypic and mechanism changes in sex differences in TTS-like wild-type (WT) and SmgGDS± mice. HL-1 cardiomyocytes were used to further examine and validate molecular mechanisms. ResultsIn 14 TTS inpatients, TTS had a higher incidence in postmenopausal women as compared to premenopausal women and men. In murine TTS, female WT mice exhibited higher cardiac SmgGDS levels than male WT mice. Ovariectomy reduced SmgGDS expression in female WT mice similar to that observed in male mice, whereas E2 replacement in these ovariectomized (OVX) female mice reversed this effect. The physiological importance of this sex-specific E2-mediated SmgGDS response is underscored by the disparity in cardiac adaptation to isoproterenol (ISO) stimulation between both sexes of WT mice. E2-mediated SmgGDS induction conferred female protection against TTS-like acute cardiac injury involving ferritinophagy-mediated ferroptosis. No such cardioprotection was observed in male WT mice and OVX female. A causal role for SmgGDS in this sex-specific cardioprotective adaptation was indicated, inasmuch as SmgGDS deficiency abolished E2-modulated cardioprotection against ferritinophagy and aggravates TTS progression in both sexes. Consistently, knockdown of SmgGDS in HL-1 cardiomyocytes exacerbated ferroptosis in a ferritinophagy-dependent manner and abrogated the protective role of E2 against ferritinophagy. Mechanistically, our findings revealed that SmgGDS regulated E2-dependent cardioprotective effects via AMPK/mTOR signaling pathway. SmgGDS deficiency abolished E2-conferred protection against ferritinophagy through activating AMPK/mTOR pathway, while treatment with recombinant SmgGDS in HL-1 cells significantly mitigated this pathway-associated ferritinophagy activity. ConclusionsThese results demonstrate that SmgGDS is a central mediator of E2-conferred female cardioprotection against ferritinophagy-mediated ferroptosis in TTS.

Abstract 15579: Stem Cell Modeling of a Candidate Genetic Variant in a Family With Complex Inherited Cardiomyopathy

Introduction: Inherited cardiomyopathies, such as arrhythmogenic right (or left) ventricular cardiomyopathy (ARVC; ALVC) and dilated cardiomyopathy (DCM), can lead to sudden cardiac death. Mechanisms of arrhythmogenesis in these cardiomyopathies are not fully understood, with ~ 20% of patients carrying a pathogenic variant in a validated cardiomyopathy-associated gene. Aims: Here, we investigate a family with highly variable expression of arrhythmogenic cardiomyopathy. The two affected siblings have differential ventricle involved and arrhythmia burden: with one fulfilling criteria for ARVC, and the other for ALVC. Their father has a non-ischemic cardiomyopathy with ventricular arrhythmias and a family history of sudden death. The maternal side is unaffected. The sibling with ALVC harbors a paternally inherited rare missense variant of uncertain significance (VUS) in plakoglobin ( JUP) (c.356C>T; p.Pro119Leu), an ARVC-associated gene that does not segregate in the sibling with ARVC. Hypothesis: Study of patient-specific inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CM) can define an elusive genetic etiology as well molecular pathways associated with cardiomyopathy in the affected family members. Methods: We reprogrammed iPSC lines from 3 affected family members and 2 unaffected maternal controls and differentiated them into cardiomyocytes. Results: Differentiation to iPSC-CMs resulted in transcriptionally upregulated markers of cardiac stress and contractile function in affected family members ( NPPA, NPPB, MYH7 ). iPSC-CM harboring JUP c.356C>T did not differ in plakoglobin RNA or protein expression. Hypertrophy was observed in the iPSC-CMs from affected family members after isoproterenol stimulation. Whole exome sequencing results revealed rare candidate variants (MAF<5x10 -5 , REVEL>0.7) that segregated in affected family members. Conclusions: iPSC-CMs from affected family members recapitulate cardiomyopathy phenotypes. Affected family members harbor novel candidate variants not previously associated with cardiomyopathies. Future work will utilize base editing to evaluate for compound contributions of several novel VUS on cardiomyopathy phenotypes in iPSC-CM from family members.

Abstract 17623: Estrogen-Mediated Small GTP-Binding Protein GDP Dissociation Stimulator Induction Contributes to Sex Differences in Resilience to Ferroptosis in Takotsubo Syndrome

Introduction: Declining estrogen (E2) has been associated with disproportionate susceptibility to takotsubo syndrome (TTS) in postmenopausal women. Small GTP-binding protein GDP dissociation stimulator (SmgGDS), as a key modulator of cardiovascular disease, plays protective roles in reducing oxidative stress and exerts pleiotropic effects of statins. Whether SmgGDS levels are influenced by E2 status and the effect of SmgGDS on sex differences in TTS are poorly understood. Methods: Clinical data were reviewed from TTS inpatients. Echocardiography, immunofluorescence, and immunohistochemistry were performed together with expression analysis to uncover phenotypic and mechanism changes in sex differences in TTS-like wild-type ( WT ) and SmgGDS +/- mice. HL-1 cardiomyocytes were used to further examine molecular mechanisms. Results: In 14 TTS inpatients, TTS had a higher incidence in postmenopausal women as compared to premenopausal women and men. In murine TTS, female WT mice exhibited higher cardiac SmgGDS levels than male WT mice. Ovariectomy reduced SmgGDS expression in female WT mice, whereas E2 replacement reversed this effect. The physiological importance of this sex-specific E2-mediated SmgGDS response is underscored by the disparity in cardiac adaptation to isoproterenol (ISO) stimulation between both sexes of WT mice. E2-mediated SmgGDS induction conferred female cardioprotection against ferritinophagy-mediated ferroptosis. No such cardioprotection was observed in male WT mice and ovariectomized (OVX) female mice. A causal role for SmgGDS in this sex-specific cardioprotective adaptation was indicated, inasmuch as SmgGDS deficiency abolished E2-modulated cardioprotection against ferritinophagy and aggravated TTS progression in both sexes. Consistently, knockdown of SmgGDS in HL-1 cardiomyocytes exacerbated ferritinophagy and abrogated the protective role of E2 against ferritinophagy. Mechanistically, our findings revealed that SmgGDS deficiency abolished E2-conferred protection against ferritinophagy through activating AMPK/mTOR pathway. Conclusions: These results demonstrate SmgGDS is a central mediator of E2-conferred female cardioprotection against ferritinophagy-mediated ferroptosis in TTS.

Inhibition of the mitochondrial respiratory chain reduces catecholamine‑stimulated lipolysis via increasing lactate production in 3T3‑L1 adipocytes.

Adipose tissue serves a significant role in the regulation of energy metabolism in the body. The re‑esterification of the fatty acids generated during lipolysis is critical for efficient lipolysis. However, the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis is not yet fully understood. The present study aimed to assess the effect of the intracellular energy state on lipolytic activity and fatty acid re‑esterification during lipolysis. 3T3‑L1 adipocytes were incubated with mitochondrial respiratory chain inhibitors, oligomycin A or rotenone, during isoproterenol stimulation; and glycerol, glucose and lactate concentrations in the medium were measured. Western blot analysis was performed to examine the phosphorylation levels of cAMP‑dependent protein kinase A (PKA). The results showed that inhibition of mitochondrial ATP synthesis decreased catecholamine‑stimulated lipolysis without affecting PKA signaling. The inhibition of mitochondrial respiration increased glucose uptake and lactate production, indicating that a large amount of glucose taken up into the cell was preferentially used for ATP production rather than for re‑esterification. In conclusion, the results of the present study suggested that the energy state during lipolysis may influence lipolytic activity by suppressing fatty acid re‑esterification.

The bitter taste receptor (TAS2R) agonist denatonium promotes a strong relaxation of rat corpus cavernosum

Bitter taste receptors (TAS2R) are found in numerous extra-oral tissues, including smooth muscle (SM) cells in both vascular and visceral tissues. Upon activation, TAS2R stimulate the relaxation of the SM. Nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) signaling pathway is involved in penile erection, and type 5 phosphodiesterase (PDE5) inhibitors, a cGMP-specific hydrolase are used as first-line treatments for erectile dysfunction (ED). Nevertheless, PDE5 inhibitors are ineffective in a considerable number of patients, prompting research into alternative pharmacological targets for ED. Since TAS2R agonists regulate SM contractility, this study investigates the role of TAS2Rs in rat corpus cavernosum (CC). We performed immunohistochemistry to detect TAS2R10, isometric force recordings for TAS2R agonists denatonium and chloroquine, the slow-release H2S donor GYY 4137, the NO donor SNAP, the β-adrenoceptor agonist isoproterenol and electrical field stimulation (EFS), as well as measurement of endogenous hydrogen sulfide (H2S) production. The immunofluorescence staining indicated that TAS2R10 was broadly expressed in the CC SM and to some extent in the nerve fibers. Denatonium, chloroquine, SNAP, and isoproterenol cause potent dose-dependent SM relaxations. H2S production was decreased by NO and H2S synthase inhibitors, while it was enhanced by denatonium. In addition, denatonium increased the relaxations induced by GYY 4137 and SNAP but failed to modify EFS- and isoproterenol-induced responses. These results suggest neuronal and SM TAS2R10 expression in the rat CC, where denatonium induces a strong SM relaxation per se and promotes the H2S- and NO-mediated inhibitory gaseous neurotransmission. Thus, TAS2R10 might represent a valuable therapeutic target in ED.

Abstract P3146: Cardiac Stress-induced Arrhythmias And Myocardial Infarction Via Specific Genetically Phosphodeleted Sites In Connexin 43 Channels

Connexin 43 (Cx43) gap junction remodeling is observed in various cardiac pathologies and is linked with arrhythmogenesis, myocardial infarction, and sudden death. Here, we aim to characterize the contribution of Cx43 phosphorylation toward mislocalization at the intercalated disc. For this, we used two genetically phospho-deleted Cx43 knock-in mouse lines with serine residues replaced by alanine in the corresponding sites for phosphorylation via mitogen-activated protein kinase (MAPK) (S255/262/279/282A; Cx43MK4) or Ca 2+/ calmodulin protein kinase II (CaMKII) (S325A/S328A/S330A; Cx43S3A). Ca 2+ signaling, cardiac electrical signals, and arrhythmias were assessed via optical mapping and telemetry in wild type, Cx43MK4, and Cx43S3A mice. Upon basal conditions or isoproterenol stimulation, wild-type animals did not display arrhythmogenic behaviors. However, after isoproterenol treatment, Cx43S3A mice showed increased QTc and deadly arrhythmias compared to wild-type animals. Isoproterenol challenge only increased the number of single PVCs in Cx43MK4 mice when compared to wild-type. Isoproterenol also evoked myocardial infarction in Cx43S3A hearts, which is negligible in Cx43MK4 and wild-type mice. Interestingly, GAP19, a Cx43 hemichannel blocker, reduced lethal arrhythmias and myocardial infarction in Cx43S3A mice. Isoproterenol challenge induced an increase in arrhythmogenic Ca 2+ events in both Cx43S3A and Cx43MK4 hearts compared to wild-type mice. However, Cx43S3A also displayed prolonged Ca 2+ decay transients that were reduced in the presence of Gap19. Our results suggest that the absence of Cx43 phosphorylation by CaMKII leads to lateralization and opening of Cx43 hemichannels, and consequently severe arrhythmias and myocardial infarction. On the other hand, the absence of MAPK phosphorylation promotes mainly a reduction of Cx43 levels at the intercalated disc region that is only associated with mild arrhythmias.

IL-6/gp130/STAT3 signaling contributed to the activation of the PERK arm of the unfolded protein response in response to chronic β-adrenergic stimulation

Prolonged activation of the PERK branch of the unfolded protein response (UPR) promotes cardiomyocytes apoptosis in response to chronic β-adrenergic stimulation. STAT3 plays a critical role in β-adrenergic functions in the heart. However, whether STAT3 contributed to β-adrenoceptor-mediated PERK activation and how β-adrenergic signaling activates STAT3 remains unclear. This study aimed to investigate whether STAT3-Y705 phosphorylation contributed to the PERK arm activation in cardiomyocytes and if IL-6/gp130 signaling was involved in the chronic β-AR-stimulation-induced STAT3 and PERK arm activation. We found that the PERK phosphorylation was positively associated with STAT3 activation. Wild-type STAT3 plasmids transfection activated the PERK/eIF2α/ATF4/CHOP pathway in cardiomyocytes while dominant negative Y705F STAT3 plasmids caused no obvious effect on PERK signaling. Stimulation with isoproterenol produced a significant increase in the level of IL-6 in the cardiomyocyte's supernatants, while IL-6 silence inhibited PERK phosphorylation but failed to attenuate STAT3 activation in response to isoproterenol stimulation. Gp130 silence attenuated isoproterenol-induced STAT3 activation and PERK phosphorylation. Inhibiting IL-6/gp130 pathway by bazedoxifene and inhibiting STAT3 by stattic both reversed isoproterenol-induced STAT3-Y705 phosphorylation, ROS production, PERK activation, IRE1α activation, and cardiomyocytes apoptosis in vitro. Bazedoxifene (5 mg/kg/day by oral gavage once a day) exhibited similar effect as carvedilol (10 mg/kg/day by oral gavage once a day) on attenuating chronic isoproterenol (30 mg/kg by abdominal injection once a day, 7 days) induced cardiac systolic dysfunction, cardiac hypertrophy and fibrosis in C57BL/6 mice. Meanwhile, bazedoxifene attenuates isoproterenol-induced STAT3-Y705 phosphorylation, PERK/eIF2α/ATF4/CHOP activation, IRE1α activation, and cardiomyocytes apoptosis to a similar extend as carvedilol in the cardiac tissue of mice. Our results showed that chronic β-adrenoceptor-mediated stimulation activated the STAT3 and PERK arm of the UPR at least partially via IL-6/gp130 pathway. Bazedoxifene has great potential to be used as an alternative to conventional β-blockers to attenuate β-adrenoceptor-mediated maladaptive UPR.

What triggers eNOS translocation to the mitochondria in the heart: Adrenergic or mechanical stress?

The translocation of eNOS from the cytosol to subsarcolemmal (SSM) and intermyofibrillar (IMF) mitochondrial subfractions has been recently reported as a key trigger of exercise training-induced cardioprotection. The cardiac response to exercise is determined by both intrinsic mechanical and extrinsic adrenergic-dependent signaling pathways. Whether eNOS translocation in response to a single bout of exercise is due to mechanical stretch of the cardiac muscle or adrenergic stress remains to be studied. The aim of this work was to evaluate the impact of cardiac adrenergic or mechanical stress on the translocation of eNOS from the cytosol to the mitochondria. Mechanical stress was obtained on isolated perfused hearts by adjusting the diastolic pressure at 5 (low-stretch) or 15 (high-stretch) mmHg. The β-adrenergic stress was induced in low or high-stretch isolated hearts perfused with 10 nM Isoproterenol for 5 min. The hearts were quick-frozen. The cytosolic, SSM, and IMF + Myofilaments subfractions were obtained by differential centrifugations. The level of eNOS was analyzed by Western blot. High-stretch tended to increase the level of eNOS in both IMF and SSM subfractions without reaching statistical significance. In contrast, Isoproterenol stimulation increased markedly the level of eNOS both in SSM and IMF in low-stretch hearts, with no additional impact of high-stretch. These results suggest that β-adrenergic receptors stimulation rather than mechanical stress of the cardiac muscle can trigger the translocation of eNOS. These results obtained ex-vivo were confirmed in vivo by treating the rats with a single injection of Isoproterenol (1 mg/kg, 30 min) that increased the level of eNOS in mitochondrial subfractions. In this preliminary work, we reported that myocardial acute adrenergic stress, rather than acute stretch, is associated with eNOS translocation from cytosol to mitochondrial subfractions. The role of the eNOS-NO pathway in the normal response of the heart to adrenergic stress remain however to be studied and will be the aim of further studies.

Decreased levels of s-nitrosylated Cx43 prevent arrhythmogenicity and myocardial infarction upon cardiac stress in duchenne muscular dystrophy.

Connexin43 (Cx43) plays a critical role in action potential propagation and cardiac contractility. In healthy cardiomyocytes, Cx43 is located at the intercalated disk, however, in cardiac diseases such as hypertrophic and dilated cardiomyopathy, it is remodelled to the lateral side of ventricular cardiomyocytes. In Duchenne muscular dystrophy (DMD) mice, lateralized cardiac Cx43 is found forming hemichannels at the plasma membrane that activate upon cardiac stress evoking triggered activity and arrhythmogenic behaviours. S-nitrosylated levels of Cx43 strongly correlate with changes in membrane excitability in cardiomyocytes from DMD mice. To unequivocally demonstrate a direct role of S-nitrosylated Cx43 in DMD cardiomyopathy, we developed a specific knockin mouse line, in which the Cx43 site for S-nitrosylation, cysteine 271, was specifically substituted for a serine (Cx43C271S). We then crossed the C271S mouse line with DMD mice to obtain DMD:Cx43C271S+/- mice with reduced levels of S-nitrosylated Cx43. Littermate mice (4-6-month-old) were used to assess isoproterenol-induced cardiac stress and, consequently, heart dysfunction in WT, DMD and DMD:Cx43C271S+/- mice. DMD mice displayed increased number of arrhythmogenic events, but without changes in RR and QRS complexes within 1-h post-isoproterenol treatment. However, elevated number of deadly arrhythmias, bradycardia, and increased QRS complex duration were detected in DMD mice for 24-h following isoproterenol stimulation. Strikingly, the number of arrhythmogenic events were significantly reduced in DMD:Cx43C271S+/- mice. β-adrenergic stimulation with isoproterenol induced cardiac hypertrophy, myocardial infarction, and 40% mortality in DMD mice, which was prevented in DMD:Cx43C271S+/- mice. Therefore, these findings strongly suggest that S-nitrosylation of Cx43 proteins at site cysteine 271 represents a fundamental nitric oxide-mediated mechanism involved in the induction of arrythmias and myocardial infarction in DMD mice after β-adrenergic stress.

Calcitriol Suppressed Isoproterenol-induced Proliferation of Cardiac Fibroblasts via Integrin β3/FAK/Akt Pathway.

Cardiac fibroblasts (CFs) proliferation and extracellular matrix deposition are important features of cardiac fibrosis. Various studies have indicated that vitamin D displays an anti-fibrotic property in chronic heart diseases. This study explored the role of vitamin D in the growth of CFs via an integrin signaling pathway. MTT and 5-ethynyl-2'-deoxyuridine assays were performed to determine cell viability. Western blotting was performed to detect the expression of proliferating cell nuclear antigen (PCNA) and integrin signaling pathway. The fibronectin was observed by ELISA. Immunohistochemical staining was employed to evaluate the expression of integrin β3. The PCNA expression in the CFs was enhanced after isoproterenol (ISO) stimulation accompanied by an elevated expression of integrin beta-3 (β3). The blockade of the integrin β3 with a specific integrin β3 antibody reduced the PCNA expression induced by the ISO. Decreasing the integrin β3 by siRNA reduced the ISO-triggered phosphorylation of FAK and Akt. Both the FAK inhibitor and Akt inhibitor suppressed the PCNA expression induced by the ISO in the CFs. Calcitriol (CAL), an active form of vitamin D, attenuated the ISO-induced CFs proliferation by downregulating the integrin β3 expression, and phosphorylation of FAK and Akt. Moreover, CAL reduced the increased levels of fibronectin and hydroxyproline in the CFs culture medium triggered by the ISO. The administration of calcitriol decreased the integrin β3 expression in the ISO-induced myocardial injury model. These findings revealed a novel role for CAL in suppressing the CFs growth by the downregulation of the integrin β3/FAK/Akt pathway.

Toll-like receptor-2 in cardiomyocytes and macrophages mediates isoproterenol-induced cardiac inflammation and remodeling.

Heart failure (HF) is the leading cause of morbidity and mortality worldwide. Activation of the innate immune system initiates an inflammatory response during cardiac remodeling induced by isoproterenol (ISO). Here, we investigated whether Toll-like receptor-2 (TLR2) mediates ISO-induced inflammation, hypertrophy, and fibrosis. TLR2 was found to be increased in the heart tissues of mouse with HF under ISO challenge. Further, cardiomyocytes and macrophages were identified as the main cellular sources of the increased TLR2 levels in the model under ISO stimulation. The effect of TLR2 deficiency on ISO-induced cardiac remodeling was determined using TLR2 knockout mice and bone marrow transplantation models. In vitro studies involving ISO-treated cultured cardiomyocytes and macrophages showed that TLR2 knockdown significantly decreased ISO-induced cell inflammation and remodeling via MAPKs/NF-κB signaling. Mechanistically, ISO significantly increased the TLR2-MyD88 interaction in the above cells in a TLR1-dependent manner. Finally, DAMPs, such as HSP70 and fibronectin 1 (FN1), were found to be released from the cells under ISO stimulation, which further activated TLR1/2-Myd88 signaling and subsequently activated pro-inflammatory cytokine expression and cardiac remodeling. In summary, our findings suggest that TLR2 may be a target for the alleviation of chronic adrenergic stimulation-associated HF. In addition, this paper points out the possibility of TLR2 as a new target for heart failure under ISO stimulation.