ß-Adrenergic Stimulation-Induced PVAT Dysfunction in Male Sex: A Role for 11ß-Hydroxysteroid Dehydrogenase-1.

Long-term ß-adrenoceptor (ß-AR) stimulation is a pathological mechanism associated with cardiovascular diseases resulting in endothelial and perivascular adipose tissue (PVAT) dysfunction. In this study, we aimed to identify whether ß-adrenergic signaling has a direct effect on PVAT. Thoracic aorta PVAT was obtained from male Wistar rats and cultured ex vivo with the ß-AR agonist isoproterenol (Iso; 1 µM) or vehicle for 24 hours. Conditioned culture medium (CCM) from Iso-treated PVAT induced a marked increase in aorta contractile response, induced oxidative stress, and reduced nitric oxide production in PVAT compared to vehicle. In addition, Iso-treated PVAT and PVAT-derived differentiated adipocytes exhibited higher corticosterone release and protein expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), an enzyme responsible for de novo synthesis of corticosterone. Macrophages exposed to Iso also exhibited increased corticosterone release in response to ß-AR stimulation. Incubation of Iso-treated PVAT and PVAT-derived differentiated adipocytes with ß3-AR antagonist restored aorta contractile function modulated by Iso-CCM and normalized 11ß-HSD1 protein expression. These results show that ß3-AR signaling leads to upregulation of 11ß-HSD1 in PVAT, thus increasing corticosterone release and contributing to impair the anticontractile function of this tissue.

Single-chain fluorescent integrators for mapping G-protein-coupled receptor agonists.

G protein-coupled receptors (GPCRs) transduce the effects of many neuromodulators including dopamine, serotonin, epinephrine, acetylcholine, and opioids. The localization of synthetic or endogenous GPCR agonists impacts their action on specific neuronal pathways. In this paper, we show a series of single-protein chain integrator sensors that are highly modular and could potentially be used to determine GPCR agonist localization across the brain. We previously engineered integrator sensors for the mu- and kappa-opioid receptor agonists called M- and K-Single-chain Protein-based Opioid Transmission Indicator Tool (SPOTIT), respectively. Here, we engineered red versions of the SPOTIT sensors for multiplexed imaging of GPCR agonists. We also modified SPOTIT to create an integrator sensor design platform called SPOTIT for all GPCRs (SPOTall). We used the SPOTall platform to engineer sensors for the beta 2-adrenergic receptor (B2AR), the dopamine receptor D1, and the cholinergic receptor muscarinic 2 agonists. Finally, we demonstrated the application of M-SPOTIT and B2AR-SPOTall in detecting exogenously administered morphine, isoproterenol, and epinephrine in the mouse brain via locally injected viruses. The SPOTIT and SPOTall sensor design platform has the potential for unbiased agonist detection of many synthetic and endogenous neuromodulators across the brain.

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

The redox-active defensive Selenoprotein T as a novel stress sensor protein playing a key role in the pathophysiology of heart failure.

Maladaptive cardiac hypertrophy contributes to the development of heart failure (HF). The oxidoreductase Selenoprotein T (SELENOT) emerged as a key regulator during rat cardiogenesis and acute cardiac protection. However, its action in chronic settings of cardiac dysfunction is not understood. Here, we investigated the role of SELENOT in the pathophysiology of HF: (i) by designing a small peptide (PSELT), recapitulating SELENOT activity via the redox site, and assessed its beneficial action in a preclinical model of HF [aged spontaneously hypertensive heart failure (SHHF) rats] and against isoproterenol (ISO)-induced hypertrophy in rat ventricular H9c2 and adult human AC16 cardiomyocytes; (ii) by evaluating the SELENOT intra-cardiomyocyte production and secretion under hypertrophied stimulation. Results showed that PSELT attenuated systemic inflammation, lipopolysaccharide (LPS)-induced macrophage M1 polarization, myocardial injury, and the severe ultrastructural alterations, while counteracting key mediators of cardiac fibrosis, aging, and DNA damage and restoring desmin downregulation and SELENOT upregulation in the failing hearts. In the hemodynamic assessment, PSELT improved the contractile impairment at baseline and following ischemia/reperfusion injury, and reduced infarct size in normal and failing hearts. At cellular level, PSELT counteracted ISO-mediated hypertrophy and ultrastructural alterations through its redox motif, while mitigating ISO-triggered SELENOT intracellular production and secretion, a phenomenon that presumably reflects the extent of cell damage. Altogether, these results indicate that SELENOT could represent a novel sensor of hypertrophied cardiomyocytes and a potential PSELT-based new therapeutic approach in myocardial hypertrophy and HF.

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes.

We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

Effect and mechanism of gomisin D on the isoproterenol induced myocardial injury in H9C2 cells and mice.

We established myocardial injury models in vivo and in vitro to investigate the cardioprotective effect of gomisin D obtained from Schisandra chinensis. Gomisin D significantly inhibited isoproterenol-induced apoptosis and hypertrophy in H9C2 cells. Gomisin D decreased serum BNP, ANP, CK-MB, cTn-T levels and histopathological alterations, and inhibited myocardial hypertrophy in mice. In mechanisms research, gomisin D reversed ISO-induced accumulation of intracellular ROS and Ca2+. Gomisin D further improved mitochondrial energy metabolism disorders by regulating the TCA cycle. These results demonstrated that gomisin D had a significant effect on isoproterenol-induced myocardial injury by inhibiting oxidative stress, calcium overload and improving mitochondrial energy metabolism.

ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

25-hydroxycholesterol triggers antioxidant signaling in mouse atria.

Oxysterol, 25-hydroxycholesterol (25HC), is a potent regulator of immune reactions, its synthesis greatly increases by macrophages during inflammation. We hypothesize that 25HC can have cardioprotective effects by limiting consequences of excessive ß-adrenoceptor (ßAR) stimulation, particularly reactive oxygen species (ROS) production, in mouse atria. Isoproterenol, a ßAR agonist, increased extra- and intracellular levels of ROS. This enhancement of ROS production was suppressed by NADPH oxidase antagonists as well as 25HC. Inhibition of ß3ARs, Gi protein and protein kinase Cε prevented the effect of 25HC on isoproterenol-dependent ROS synthesis. Furthermore, 25HC suppressed isoproterenol-induced lipid peroxidation and mitochondrial ROS generation as well as ROS-dependent component of positive inotropic response to isoproterenol. Additionally, 25HC decreased mitochondrial ROS production and lipid peroxidation induced by antimycin A, a mitochondrial poison. Thus, 25HC exerts antioxidant properties alleviating mitochondrial dysfunction-induced and ßAR-dependent cardiac oxidative damage. In the latter case, 25HC can act via signaling mechanism engaging ß3ARs, Gi protein and protein kinase Cε.

RICH1 is a novel key suppressor of isoproterenol­ or angiotensin II­induced cardiomyocyte hypertrophy.

Cardiac hypertrophy is one of the key processes in the development of heart failure. Notably, small GTPases and GTPase­activating proteins (GAPs) serve essential roles in cardiac hypertrophy. RhoGAP interacting with CIP4 homologs protein 1 (RICH1) is a RhoGAP that can regulate Cdc42/Rac1 and F­actin dynamics. RICH1 is involved in cell proliferation and adhesion; however, to the best of our knowledge, its role in cardiac hypertrophy remains unknown. In the present study, the role of RICH1 in cardiomyocyte hypertrophy was assessed. Cell viability was analyzed using the Cell Counting Kit­8 assay and cells surface area (CSA) was determined by cell fluorescence staining. Reverse transcription­quantitative PCR and western blotting were used to assess the mRNA expression levels of hypertrophic marker genes, such as Nppa, Nppb and Myh7, and the protein expression levels of RICH1, respectively. RICH1 was shown to be downregulated in isoproterenol (ISO)­ or angiotensin II (Ang II)­treated H9c2 cells. Notably, overexpression of RICH1 attenuated the upregulation of hypertrophy­related markers, such as Nppa, Nppb and Myh7, and the enlargement of CSA induced by ISO and Ang II. By contrast, the knockdown of RICH1 exacerbated these effects. These findings suggested that RICH1 may be a novel suppressor of ISO­ or Ang II­induced cardiomyocyte hypertrophy. The results of the present study will be beneficial to further studies assessing the role of RICH1 and its downstream molecules in inhibiting cardiac hypertrophy.

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.

Detection of biomagnetic signals from induced pluripotent stem cell-derived cardiomyocytes using deep learning with simulation data.

The detection of spontaneous magnetic signals can be used for the non-invasive electrophysiological evaluation of induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We report that deep learning with a dataset that combines magnetic signals estimated using numerical simulation and actual noise data is effective in the detection of weak biomagnetic signals. To verify the feasibility of this method, we measured artificially generated magnetic signals that mimic cellular magnetic fields using a superconducting quantum interference device and attempted peak detection using a long short-term memory network. We correctly detected 80.0% of the peaks and the method achieved superior detection performance compared with conventional methods. Next, we attempted peak detection for magnetic signals measured from mouse iPS-CMs. The number of detected peaks was consistent with the spontaneous beats counted using microscopic observation and the average peak waveform achieved good similarity with the prediction. We also observed the synchronization of peak positions between simultaneously measured field potentials and magnetic signals. Furthermore, the magnetic measurements of cell samples treated with isoproterenol showed potential for the detection of chronotropic effects. These results suggest that the proposed method is effective and has potential application in the safety assessment of regenerative medicine and drug screening.

Arglabin: A mediator of inflammasome modulated and independent myocardial injury (PARA-AMI study).

BACKGROUND: Arglabin is a plant alkaloid (sesquiterpene lactone) that is used as an anticancer drug. It has potential anti-diabetic and anti-atherogenic effects. PURPOSE: Arglabin has drawn particular attention because of its therapeutic effects as an anti-inflammatory agent in multiple diseases. Since arglabin inhibits Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, concerns for cardiotoxic effects are valid. The present study was designed to investigate the protective effects of arglabin on the myocardium. STUDY DESIGN: This study was designed to evaluate the effect of arglabin on the myocardium in an experimental model of myocardial necrosis in rats. Different doses of arglabin (2.5, 5, and 10 µg/kg) were investigated as pre-treatment for 21 days in the isoproterenol (ISO) model of myocardial necrosis groups and per se groups. METHODS: On the 22nd day, hemodynamic, histopathological, electron microscopy, oxidative stress markers, inflammatory mediators, apoptotic markers, inflammasome mediators, and Western blot analysis were performed to evaluate the effects of arglabin. RESULTS: Arglabin pre-treatment showed improvement in hemodynamic parameters and histopathological findings at low doses in isoproterenol-induced myocardial necrosis model of rats. Arglabin administration altered myocardial structure and modulated myocardial function via activation of NFκB/MAPK pathway that led to myocardial injury with an increase in dose. CONCLUSION: Arglabin imparted partial cardio-protection via an inflammasome-dependent pathway and mediated injury through the inflammasome-independent pathway.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

The renal vasodilatation from ß-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels.

The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.

Mesenteric artery smooth muscle cells from hypertensive rats have increased microtubule acetylation.

The dynamic nature of the microtubule network is dependent in part by post-translational modifications (PTMs) - particularly through acetylation, which stabilizes the microtubule network. Whether PTMs of the microtubule network in vascular smooth muscle cells (VSMCs) contribute to the pathophysiology of hypertension is unknown. The aim of this study was to determine the acetylated state of the microtubule network in the mesenteric arteries of spontaneously hypertensive rats (SHR). Experiments were performed on male normotensive rats and SHR mesenteric arteries. Western blotting and mass spectrometry determined changes in tubulin acetylation. Wire myography was used to investigate the effect of tubacin on isoprenaline-mediated vasorelaxations. Isolated cells from normotensive rats were used for scanning ion conductance microscopy (SICM). Mass spectrometry and Western blotting showed that tubulin acetylation is increased in the mesenteric arteries of the SHR compared with normotensive rats. Tubacin enhanced the ß-adrenoceptor-mediated vasodilatation by isoprenaline when the endothelium was intact, but attenuated relaxations when the endothelium was denuded or nitric oxide production was inhibited. By pre-treating vessels with colchicine to disrupt the microtubule network, we were able to confirm that the effects of tubacin were microtubule-dependent. Using SICM, we examined the cell surface Young's modulus of VSMCs, but found no difference in control, tubacin-treated, or taxol-treated cells. Acetylation of tubulin at Lys40 is elevated in mesenteric arteries from the SHR. Furthermore, this study shows that tubacin has an endothelial-dependent bimodal effect on isoprenaline-mediated vasorelaxation.

Palmatine alleviates cardiac fibrosis by inhibiting fibroblast activation through the STAT3 pathway.

Cardiac fibrosis, the hallmark of cardiovascular disease, is characterized by excessive deposition of extracellular matrix in the heart. Emerging evidence indicates that cardiac fibroblasts (CFs) play pivotal roles in driving cardiac fibrosis. However, due to incomplete insights into CFs, there are limited effective approaches to prevent or reverse cardiac fibrosis currently. Palmatine, a protoberberine alkaloid extracted from traditional Chinese botanical remedies, possesses diverse biological effects. This study investigated the potential therapeutic value and mechanism of palmatine against cardiac fibrosis. Adult male C57BL/6 mice were treated with vehicle, isoproterenol (ISO), or ISO plus palmatine for one week. After echocardiography assessment, mice hearts were collected for histopathology, real-time polymerase chain reaction, and Western blot analyses. Primary rat CFs were utilized in vitro. Compared to control, ISO-treated mice exhibited cardiac hypertrophy and structural abnormalities; however, treatment with palmatine ameliorated these effects of ISO. Moreover, palmatine treatment mitigated ISO-induced cardiac fibrosis. Network pharmacology and molecular docking analysis showed that palmatine strongly binds the regulators of cardiac fibrosis including signal transducer and activator of transcription 3 (STAT3) and mammalian target of rapamycin. Furthermore, palmatine reduced the elevated fibrotic factor expressions and overactivated STAT3 induced by ISO, Transformed growth factor ß1 (TGF-ß1), or interleukin-6 both in vivo and in vitro. Additionally, blocking STAT3 suppressed the TGF-ß1-induced CF activation. Collectively, these data demonstrated that palmatine attenuated cardiac fibrosis partly by inhibiting fibroblast activation through the STAT3 pathway. This provides an experimental basis for the clinical treatment of cardiac fibrosis with palmatine.

Phosphorylation of RyR2 simultaneously expands the dyad and rearranges the tetramers.

We have previously demonstrated that type II ryanodine receptors (RyR2) tetramers can be rapidly rearranged in response to a phosphorylation cocktail. The cocktail modified downstream targets indiscriminately, making it impossible to determine whether phosphorylation of RyR2 was an essential element of the response. Here, we used the ß-agonist isoproterenol and mice homozygous for one of the following clinically relevant mutations: S2030A, S2808A, S2814A, or S2814D. We measured the length of the dyad using transmission electron microscopy (TEM) and directly visualized RyR2 distribution using dual-tilt electron tomography. We found that the S2814D mutation, by itself, significantly expanded the dyad and reorganized the tetramers, suggesting a direct link between the phosphorylation state of the tetramer and its microarchitecture. S2808A and S2814A mutant mice, as well as wild types, had significant expansions of their dyads in response to isoproterenol, while S2030A mutants did not. In agreement with functional data from these mutants, S2030 and S2808 were necessary for a complete ß-adrenergic response, unlike S2814 mutants. Additionally, all mutants had unique effects on the organization of their tetramer arrays. Lastly, the correlation of structural with functional changes suggests that tetramer-tetramer contacts play an important functional role. We thus conclude that both the size of the dyad and the arrangement of the tetramers are linked to the state of the channel tetramer and can be dynamically altered by a ß-adrenergic receptor agonist.

Surface entrenched ß-sitosterol niosomes for enhanced cardioprotective activity against isoproterenol induced cardiotoxicity in rats.

Cardiotoxicity (CT) is a severe condition that negatively impacts heart function. ß-sitosterol (BS) is a group of phytosterols and known for various pharmacological benefits, such as managing diabetes, cardiac protection, and neuroprotection. This study aims to develop niosomes (NS) containing BS, utilizing cholesterol as the lipid and Tween 80 as the stabilizer. The research focuses on designing and evaluating both conventional BS-NS and hyaluronic acid (HA) modified NS (BS-HA-NS) to enhance the specificity and efficacy of BS within cardiac tissue. The resulting niosomal formulation was spherical, with a size of about 158.51 ± 0.57 nm, an entrapment efficiency of 93.56 ± 1.48 %, and a drug loading of 8.07 ± 1.62 %. To evaluate cytotoxicity on H9c2 heart cells, the MTT assay was used. The cellular uptake of BS-NS and BS-HA-NS was confirmed by confocal microscopy on H9c2 cardiac cells. Administering BS-NS and BS-HA-NS intravenously at a dose of 10 mg/kg showed the ability to significantly decrease the levels of cardiac troponin-I (cTn-I), creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and lipid peroxidation (MDA). Tissue histopathology indicated a substantial potential for repairing cardiac tissue after treatment with BS-NS and BS-HA-NS and strong cardioprotection against ISO induced myocardial tissue damages. Thus, enhancing BS's therapeutic effectiveness through niosome surface modification holds promise for mitigating cardiac damage resulting from CT.

Integrated miRNA-mRNA networks underlie attenuation of chronic ß-adrenergic stimulation-induced cardiac remodeling by minocycline.

Adverse cardiac remodeling contributes to heart failure development and progression, partly due to inappropriate sympathetic nervous system activation. Although ß-adrenergic receptor (ß-AR) blockade is a common heart failure therapy, not all patients respond, prompting exploration of alternative treatments. Minocycline, an FDA-approved antibiotic, has pleiotropic properties beyond antimicrobial action. Recent evidence suggests it may alter gene expression via changes in miRNA expression. Thus, we hypothesized that minocycline could prevent adverse cardiac remodeling induced by the ß-AR agonist isoproterenol, involving miRNA-mRNA transcriptome alterations. Male C57BL/6J mice received isoproterenol (30 mg/kg/day sc) or vehicle via osmotic minipump for 21 days, along with daily minocycline (50 mg/kg ip) or sterile saline. Isoproterenol induced cardiac hypertrophy without altering cardiac function, which minocycline prevented. Total mRNA sequencing revealed isoproterenol altering gene networks associated with inflammation and metabolism, with fibrosis activation predicted by integrated miRNA-mRNA sequencing, involving miR-21, miR-30a, miR-34a, miR-92a, and miR-150, among others. Conversely, the cardiac miRNA-mRNA transcriptome predicted fibrosis inhibition in minocycline-treated mice, involving antifibrotic shifts in Atf3 and Itgb6 gene expression associated with miR-194 upregulation. Picrosirius red staining confirmed isoproterenol-induced cardiac fibrosis, prevented by minocycline. These results demonstrate minocycline's therapeutic potential in attenuating adverse cardiac remodeling through miRNA-mRNA-dependent mechanisms, especially in reducing cardiac fibrosis. NEW & NOTEWORTHY We demonstrate that minocycline treatment prevents cardiac hypertrophy and fibrotic remodeling induced by chronic ß-adrenergic stimulation by inducing antifibrotic shifts in the cardiac miRNA-mRNA transcriptome.

Sex- and age-dependent susceptibility to ventricular arrhythmias in the rat heart ex vivo.

The incidence of life-threatening ventricular arrhythmias, the most common cause of sudden cardiac death (SCD), depends largely on the arrhythmic substrate that develops in the myocardium during the aging process. There is a large deficit of comparative studies on the development of this substrate in both sexes, with a particular paucity of studies in females. To identify the substrates of arrhythmia, fibrosis, cardiomyocyte hypertrophy, mitochondrial density, oxidative stress, antioxidant defense and intracellular Ca2+ signaling in isolated cardiomyocytes were measured in the hearts of 3- and 24-month-old female and male rats. Arrhythmia susceptibility was assessed in ex vivo perfused hearts after exposure to isoproterenol (ISO) and hydrogen peroxide (H2O2). The number of ventricular premature beats (PVBs), ventricular tachycardia (VT) and ventricular fibrillation (VF) episodes, as well as intrinsic heart rate, QRS and QT duration, were measured in ECG signals recorded from the surfaces of the beating hearts. After ISO administration, VT/VFs were formed only in the hearts of males, mainly older ones. In contrast, H2O2 led to VT/VF formation in the hearts of rats of both sexes but much more frequently in older males. We identified several components of the arrhythmia substrate that develop in the myocardium during the aging process, including high spontaneous ryanodine receptor activity in cardiomyocytes, fibrosis of varying severity in different layers of the myocardium (nonheterogenic fibrosis), and high levels of oxidative stress as measured by nitrated tyrosine levels. All of these elements appeared at a much greater intensity in male individuals during the aging process. On the other hand, in aging females, antioxidant defense at the level of H2O2 detoxification, measured as glutathione peroxidase expression, was weaker than that in males of the same age. We showed that sex has a significant effect on the development of an arrhythmic substrate during aging. This substrate determines the incidence of life-threatening ventricular arrhythmias in the presence of additional stimuli with proarrhythmic potential, such as catecholamine stimulation or oxidative stress, which are constant elements in the pathomechanism of most cardiovascular diseases.