Beta adrenergic (BAR) signaling is stimulated by the catecholamines norepinephrine (NE) and epinephrine (EPI) to control cardiac contractility. The beta-1-AR (B1AR) is the major cardiac subtype. B1AR signals canonically through the Gas subunit, leading to increases in cAMP and PKA; however, it can also signal via the Gai subunit, leading to increased NOS1, cGMP, and PKG levels. B1AR-NOS1 signaling leads to increased contractility and calcium cycling in cardiomyocytes. We found a novel complex within cardiomyocytes in which the B1AR, Cav1.2, and NOS1 proteins are held in proximity by the membrane scaffolding protein SAP97. In heart failure, there is a decrease in SAP97 expression and impaired B1AR signaling. Cardiac-specific deletion of SAP97 disrupts this complex. In the SAP97-cKO mice, there are minimal changes in B1AR-Gas; however, B1AR-Gai signaling is decreased, with a loss of B1AR-NOS1 signaling. Furthermore, we see a decrease in whole cell calcium currents and cardiac contractility. In mice lacking the NOS1, we see the same decrease in calcium currents. We hypothesize SAP97 is necessary to maintain B1AR modulation of Cav1.2 in cardiomyocytes. Using electrophysiology and biochemical techniques, we aim to elucidate the role of SAP97 in B1AR modulation of Cav1.2 and how this is impacted by the impairment of B1AR-Gai signaling. T32HL086350 RO1-HL147263. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Phosphorylation of myofilament proteins critically regulates beat-to-beat cardiac contraction and is typically altered in heart failure (HF). β-Adrenergic activation induces phosphorylation in numerous substrates at the myofilament. Nevertheless, how cardiac β-adrenoceptors (βARs) signal to the myofilament in healthy and diseased hearts remains poorly understood. The aim of this study was to uncover the spatiotemporal regulation of local βAR signaling at the myofilament and thus identify a potential therapeutic target for HF. Phosphoproteomic analysis of substrate phosphorylation induced by different βAR ligands in mouse hearts was performed. Genetically encoded biosensors were used to characterize cyclic adenosine and guanosine monophosphate signaling and the impacts on excitation-contraction coupling induced by β1AR ligands at both the cardiomyocyte and whole-heart levels. Myofilament signaling circuitry was identified, including protein kinase G1 (PKG1)-dependent phosphorylation of myosin light chain kinase, myosin phosphatase target subunit 1, and myosin light chain at the myofilaments. The increased phosphorylation of myosin light chain enhances cardiac contractility, with a minimal increase in calcium (Ca2+) cycling. This myofilament signaling paradigm is promoted by carvedilol-induced β1AR-nitric oxide synthetase 3 (NOS3)-dependent cyclic guanosine monophosphate signaling, drawing a parallel to the β1AR-cyclic adenosine monophosphate-protein kinase A pathway. In patients with HF and a mouse HF model of myocardial infarction, increasing expression and association of NOS3 with β1AR were observed. Stimulating β1AR-NOS3-PKG1 signaling increased cardiac contraction in the mouse HF model. This research has characterized myofilament β1AR-PKG1-dependent signaling circuitry to increase phosphorylation of myosin light chain and enhance cardiac contractility, with a minimal increase in Ca2+ cycling. The present findings raise the possibility of targeting this myofilament signaling circuitry for treatment of patients with HF.

Objective: Recent studies have shown that intracellular b1- adrenoceptors (b1ARs) are associated with the (Sarco)endoplasmic reticulum calcium ATPase 2a (SERCA2a) on the sarcoplasmic reticulum (SR) membrane. However, the mechanism of how this plasma membrane- and SR-associated pool of b1ARs signals downregulation under chronic sympathetic stimulation is not well understood. We aim to study these subcellular β1AR signals in different cellular compartments in a heart failure (HF) mouse model with chronic infusion of isoproterenol (ISO). Results and Methods: We investigated the association of b1AR with L-type calcium channel (LTCC), ryanodine receptor 2 (RyR2), and SERCA2a by utilizing proximity ligation assay (PLA) and confocal imaging in HF induced by chronic ISO infusion (30 mg/kg/day, 2 weeks). Chronic ISO infusion resulted in a downregulation of b1AR, and decreased receptor association with LTCC and RyR2 at the tubular membrane. In comparison, the b1AR association with SERCA2a was increased; however, the local cAMP-PKA mediated phospholamban phosphorylation is downregulated. Using Förster Resonance Energy Transfer (FRET) with genetically encoded biosensors to probe subcellular β1AR-PKA signaling, we observed reduced local ISO-induced PKA signaling at the RyR2 but not the SERCA2a nanodomains. Meanwhile, the expression of phosphodiesterases (PDEs) was altered including the downregulation of PDE4D and PDE3A and the upregulation of PDE2A, PDE4A, and PDE4B, which alters the local PKA activities in individual subcellular nanodomains. Interestingly, the localized PKA signal induced by endogenous norepinephrine at the SERCA2a nanodomain was impaired. Inhibition of monoamine oxidase A rescued the norepinephrine-induced local PKA signaling at the SERCA2a nanodomain and myocyte sarcomere shortening. Conclusion: We have uncovered distinct remodeling of b1AR signaling at the RyR2 and SERCA2a nanodomains in mouse HF after a chronic infusion of ISO. Our study offers strategies to restore the subcellular nanodomain cardiac b1AR signaling and contractile function in HF associated with chronic adrenergic stress, pointing toward potential therapeutic targets. National Institutes of Health grants R01-HL147263 and HL162825, Veteran Affair Merit grants IK6BX005753, 01BX002900 and BX005100. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background Chronic sympathetic stimulation drives desensitization and downregulation of β1 adrenergic receptor (β 1 AR) in heart failure. We aim to explore the differential downregulation subcellular pools of β 1 AR signaling in the heart. Methods and Results We applied chronic infusion of isoproterenol to induced cardiomyopathy in male C57BL/6J mice. We applied confocal and proximity ligation assay to examine β 1 AR association with L‐type calcium channel, ryanodine receptor 2, and SERCA2a ((Sarco)endoplasmic reticulum calcium ATPase 2a) and Förster resonance energy transfer‐based biosensors to probe subcellular β 1 AR‐PKA (protein kinase A) signaling in ventricular myocytes. Chronic infusion of isoproterenol led to reduced β 1 AR protein levels, receptor association with L‐type calcium channel and ryanodine receptor 2 measured by proximity ligation (puncta/cell, 29.65 saline versus 14.17 isoproterenol, P <0.05), and receptor‐induced PKA signaling at the plasma membrane (Förster resonance energy transfer, 28.9% saline versus 1.9% isoproterenol, P <0.05) and ryanodine receptor 2 complex (Förster resonance energy transfer, 30.2% saline versus 10.6% isoproterenol, P <0.05). However, the β 1 AR association with SERCA2a was enhanced (puncta/cell, 51.4 saline versus 87.5 isoproterenol, P <0.05), and the receptor signal was minimally affected. The isoproterenol‐infused hearts displayed decreased PDE4D (phosphodiesterase 4D) and PDE3A and increased PDE2A, PDE4A, and PDE4B protein levels. We observed a reduced role of PDE4 and enhanced roles of PDE2 and PDE3 on the β 1 AR‐PKA activity at the ryanodine receptor 2 complexes and myocyte shortening. Despite the enhanced β 1 AR association with SERCA2a, the endogenous norepinephrine‐induced signaling was reduced at the SERCA2a complexes. Inhibiting monoamine oxidase A rescued the norepinephrine‐induced PKA signaling at the SERCA2a and myocyte shortening. Conclusions This study reveals distinct mechanisms for the downregulation of subcellular β 1 AR signaling in the heart under chronic adrenergic stimulation.

During the final preparation of Figure S2I and S9A, some panels were erroneously assembled by the au-thors. In Figure S2I, the images for "AAV9-Scramble"control actually represent "AAV9-LacZ"control. In Figure S9A, the images for the "Ad-FGF13 OE+Ad-IKB OE"actually represent "Ad-FGF13 NLS-OE."These errors do not affect the results or the conclusions of the paper. The authors apologize for any incon-venience caused to the readers.

Phosphodiesterases (PDEs) are a superfamily of enzymes that hydrolyze cyclic nucleotides, including cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Both cyclic nucleotides are critical secondary messengers in the neurohormonal regulation in the cardiovascular system. PDEs precisely control spatiotemporal subcellular distribution of cyclic nucleotides in a cell- and tissue-specific manner, playing critical roles in physiological responses to hormone stimulation in the heart and vessels. Dysregulation of PDEs has been linked to the development of several cardiovascular diseases, such as hypertension, aneurysm, atherosclerosis, arrhythmia, and heart failure. Targeting these enzymes has been proven effective in treating cardiovascular diseases and is an attractive and promising strategy for the development of new drugs. In this review, we discuss the current understanding of the complex regulation of PDE isoforms in cardiovascular function, highlighting the divergent and even opposing roles of PDE isoforms in different pathogenesis.

Review Unveiling Catecholamine Dynamics in Cardiac Health and Disease: Mechanisms, Implications, and Future Perspectives Wenjing Xiang, Xingyun Wang, Lei Li, Junhui Zeng, Haocheng Lu, and Ying Wang * Department of Pharmacology, School of Medicine, Southern University of Science and TechnologyShenzhen518055China * Correspondence: wangy6@sustech.edu.cn Received: 15 August 2023 Accepted: 21 September 2023 Published: 27 December 2023 Abstract: Catecholamines play a pivotal role in regulating both cardiac physiology and pathology, orchestrating the “Fight-or-flight” response through the activation of sympathetic nervous system (SNS) activation and subsequent stimulation of adrenergic receptor. However, chronic stress and various cardiac diseases can disrupt catecholamine balance, contributing to cardiac dysfunction. The synthesis, release, reuptake, and degradation of catecholamines intricately regulate their concentration. Notably, catecholamine dynamics is markedly altered in heart diseases, including heart failure, myocardial infarction, and arrhythmias. While β‍-adrenergic receptor blockers, which block catecholamines from binding to the adrenergic receptors, are widely used in clinical settings, the potential implication of directly manipulating catecholamine homeostasis for the treatment of cardiac diseases have not been extensively explored. This review provides an overview of catecholaminergic systems, and discusses their intricate synthesis, release, uptake, and metabolism within the heart. Additionally, the review highlights mechanisms underlying cardiac effects of catecholamine dysregulation, including contractile dysfunction, electrical remodeling, and cardiac remodeling. Moreover, the review emphasizes the importance of considering spatiotemporal and sexual heterogeneity in catecholamine dynamics for cardiac precision medicine. In terms of future perspectives, we believe that harnessing genetically encoded fluorescent biosensors to map the heterogenous for real-time imaging of catecholamine dynamics and conducting gender-specific dissection of catecholamine dynamics have significant potential to advance personalized management of cardiac diseases management.

Septic cardiomyopathy is a life-threatening heart dysfunction caused by severe infection. Considering the complexity of the pathogenesis and high mortality, it was necessary to identify efficient biomarkers to guide the clinical practice. Based on the muti-microarray analysis, this study aimed to explore the pathogenesis of septic cardiomyopathy and the related immune landscape. The results showed that septic cardiomyopathy was organ dysfunction after extreme pro- and anti-inflammation. In this process, KLRG1, PRF1, BCL6, GAB2, MMP9, IL1R1, JAK3, IL6ST, and SERPINE1 were identified as the hub genes regulated the immune landscape of septic cardiomyopathy. Nine transcription factors regulated their expression: SRF, STAT1, SP1, RELA, PPARG, NFKB1, PPARA, SMAD3, and STAT3. Hub genes activated the Th17 cell differentiation pathway, JAK-STAT signaling pathway, and Cytokine-cytokine receptor interaction pathway. These were mainly involved in regulating inflammatory response, adaptive immune response, leukocyte-mediated immunity, cytokine-mediated immunity, immune effector process, myeloid cell differentiation, and T-helper cell differentiation. These nine hub genes can be seen as biomarkers for the early prediction of septic cardiomyopathy.

Improving the survival rate of cardiomyocytes is the key point to treat most of the heart diseases, and targeting autophagy is a potential advanced therapeutic approach. Monitoring autophagic activity in cardiomyocytes in situ will be useful for studying autophagy-related heart disease and screening autophagy-modulating drugs. Zebrafish, Danio rerio, has been proven as an animal model for studying heart diseases in situ. Taken the advantage of zebrafish, especially the imaging of intact animals, here we generated two stable transgenic zebrafish lines that specifically expressed EGFP-map1lc3b or mRFP-EGFP-map1lc3b in cardiomyocytes under the promoter of myosin light chain 7. We first used a few known autophagy-modulating drugs to confirm their usefulness. By quantifying the density of autophagosomes and autolysosomes, autophagy inducers and inhibitors showed their regulatory functions, which were consistent with previous studies. With the two lines, we then found a significant increase in the density of autophagosomes but not autolysosomes in zebrafish cardiomyocytes at the early developmental stages, indicating the involvement of autophagy in early heart development. To prove their applicability, we also tested five clinical statins by the two lines. And we found that statins did not change the density of autophagosomes but reduced the density of autolysosomes in cardiomyocytes, implying their regulation in autophagic flux. Our study provides novel animal models for monitoring autophagic activity in cardiomyocytes in situ, which could be used to study autophagy-related cardiomyopathy and drug screening.

We have recently identified a pool of intracellular β 1 adrenergic receptors (β 1 ARs) at the sarcoplasmic reticulum (SR) crucial for cardiac function. Here, we aim to characterize the integrative control of intracellular catecholamine for subcellular β 1 AR signaling and cardiac function. Using anchored Förster resonance energy transfer (FRET) biosensors and transgenic mice, we determined the regulation of compartmentalized β 1 AR-PKA signaling at the SR and plasma membrane (PM) microdomains by organic cation transporter 3 (OCT3) and monoamine oxidase A (MAO-A), two critical modulators of catecholamine uptake and homeostasis. Additionally, we examined local PKA substrate phosphorylation and excitation–contraction coupling in cardiomyocyte. Cardiac-specific deletion of MAO-A (MAO-A-CKO) elevates catecholamines and cAMP levels in the myocardium, baseline cardiac function, and adrenergic responses. Both MAO-A deletion and inhibitor (MAOi) selectively enhance the local β 1 AR-PKA activity at the SR but not PM, and augment phosphorylation of phospholamban, Ca 2+ cycling, and myocyte contractile response. Overexpression of MAO-A suppresses the SR-β 1 AR-PKA activity and PKA phosphorylation. However, deletion or inhibition of OCT3 by corticosterone prevents the effects induced by MAOi and MAO-A deletion in cardiomyocytes. Deletion or inhibition of OCT3 also negates the effects of MAOi and MAO-A deficiency in cardiac function and adrenergic responses in vivo. Our data show that MAO-A and OCT3 act in concert to fine-tune the intracellular SR-β 1 AR-PKA signaling and cardiac fight-or-flight response. We reveal a drug contraindication between anti-inflammatory corticosterone and anti-depressant MAOi in modulating adrenergic regulation in the heart, providing novel perspectives of these drugs with cardiac implications.

Increasing biased G‐protein coupled receptor (GPCR) drugs are now under clinical investigation. More understanding of the biased β‐adrenergic receptor (β‐AR) signaling is urgently required for a better cardiovascular drug. Here we found that two nitric oxide synthase (NOS) isotypes, NOS1, and NOS3 coupled with β1ARs and transduce distinct β1AR‐cGMP signaling at sarcoplasmic reticulum and myofilament microdomain, respectively. Intriguingly, activation of NOS1 enhanced contractility and Ca2+ cycling, whereas NOS3 promoted contraction without affecting intracellular Ca2+. Quantitative proteomic revealed that NOS3 activation increased phosphorylation of myosin proteins including myosin binding protein C (MyBP‐C), myosin light chain kinase (MYLK) and myosin phosphatase target subunit 1 (MYPT1), to enhance contractility by Ca2+sensitization. Moreover, in heart failure, excessive adrenergic stimulation dissociated β1AR from NOS1 and impaired the NOS1‐cGMP signaling. Accordingly, stimulating NOS3 but NOS1 enhances systolic cardiac contraction in failing mice without rousing intracellular Ca2+. We propose NOS facilitate biased β1AR‐cGMP signaling via Ca2+‐dependent or independent mechanisms and define NOS3‐cGMP as specific therapeutic strategy targeting myofilament for heart failure.

RATIONALE:β1ARs (β1-adrenoceptors) exist at intracellular membranes and OCT3 (organic cation transporter 3) mediates norepinephrine entry into cardiomyocytes. However, the functional role of intracellular β1AR in cardiac contractility remains to be elucidated.OBJECTIVE:Test localization and function of intracellular β1AR on cardiac contractility.METHODS AND RESULTS:Membrane fractionation, super-resolution imaging, proximity ligation, coimmunoprecipitation, and single-molecule pull-down demonstrated a pool of β1ARs in mouse hearts that were associated with sarco/endoplasmic reticulum Ca2+-ATPase at the sarcoplasmic reticulum (SR). Local PKA (protein kinase A) activation was measured using a PKA biosensor targeted at either the plasma membrane (PM) or SR. Compared with wild-type, myocytes lacking OCT3 (OCT3-KO [OCT3 knockout]) responded identically to the membrane-permeant βAR agonist isoproterenol in PKA activation at both PM and SR. The same was true at the PM for membrane-impermeant norepinephrine, but the SR response to norepinephrine was suppressed in OCT3-KO myocytes. This differential effect was recapitulated in phosphorylation of the SR-pump regulator phospholamban. Similarly, OCT3-KO selectively suppressed calcium transients and contraction responses to norepinephrine but not isoproterenol. Furthermore, sotalol, a membrane-impermeant βAR-blocker, suppressed isoproterenol-induced PKA activation at the PM but permitted PKA activation at the SR, phospholamban phosphorylation, and contractility. Moreover, pretreatment with sotalol in OCT3-KO myocytes prevented norepinephrine-induced PKA activation at both PM and the SR and contractility.CONCLUSIONS:Functional β1ARs exists at the SR and is critical for PKA-mediated phosphorylation of phospholamban and cardiac contractility upon catecholamine stimulation. Activation of these intracellular β1ARs requires catecholamine transport via OCT3.

Aims beta-blockers are widely used in therapy for heart failure and hypertension. beta-blockers are also known to evoke additional diversified pharmacological and physiological effects in patients. We aim to characterize the underlying molecular signalling and effects on cardiac inotropy induced by beta-blockers in animal hearts. Methods and results Wild-type mice fed high-fat diet (HFD) were treated with carvedilol, metoprolol, or vehicle and echocardiogram analysis was performed. Heart tissues were used for biochemical and histological analyses. Cardiomyocytes were isolated from normal and HFD mice and rats for analysis of adrenergic signalling, calcium handling, contraction, and western blot. Biosensors were used to measure beta-blocker-induced cyclic guanosine monophosphate (cGMP) signal and protein kinase A activity in myocytes. Acute stimulation of myocytes with carvedilol promotes beta(1) adrenergic receptor (beta(1)AR)- and protein kinase G (PKG)-dependent inotropic cardiac contractility with minimal increases in calcium amplitude. Carvedilol acts as a biased ligand to promote beta(1)AR coupling to a G(i)-PI3K-Akt-nitric oxide synthase 3 (NOS3) cascade and induces robust beta(1)AR-cGMP-PKG signal. Deletion of NOS3 selectively blocks carvedilol, but not isoproterenol-induced beta(1)AR-dependent cGMP signal and inotropic contractility. Moreover, therapy with carvedilol restores inotropic contractility and sensitizes cardiac adrenergic reserves in diabetic mice with minimal impact in calcium signal, as well as reduced cell apoptosis and hypertrophy in diabetic hearts. Conclusion These observations present a novel beta(1)AR-NOS3 signalling pathway to promote cardiac inotropy in the heart, indicating that this signalling paradigm may be targeted in therapy of heart diseases with reduced ejection fraction.

Previous studies have confirmed that 50 µmol/l pinacidil postconditioning (PPC) activates the nuclear factor-E2 related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, which protects the myocardium from ischemia-reperfusion (IR) injury; however, whether this is associated with reactive oxygen species (ROS) generation remains unclear. In the present study, a Langendorff rat model of isolated myocardial IR was established to investigate the mechanism of PPC at different concentrations, as well as the association between the rat myocardial Nrf2-ARE signaling pathway and ROS. A total of 48 rats were randomly divided into the following six groups (n=8 per group): i) Normal; ii) IR iii) 10 µmol/l PPC (P10); iv) 30 µmol/l PPC (P30); v) 50 µmol/l PPC (P50); and vi) N-(2-mercaptopropionyl)-glycine (MPG; a ROS scavenger) + 50 µmol/l pinacidil (P50 + MPG). At the end of reperfusion (T3), compared with the IR group, the P10, P30 and P50 groups exhibited improved cardiac function, such as left ventricular development pressure, heart rate, left ventricular end-diastolic pressure, +dp/dtmax, myocardial cell ultrastructure and mitochondrial Flameng score. Furthermore, the P10 and P50 groups demonstrated the weakest and most marked improvements, respectively. Additionally, in the P10, P30 and P50 groups, the residual ROS content at the end of reperfusion was highly negatively correlated with relative expression levels of Nrf2 gene and protein. Higher pinacidil concentration was associated with higher ROS generation at 5 min post-reperfusion (T2), although this was significantly lower compared with the IR group, as well as with increased expression levels of antioxidant proteins and phase II detoxification enzymes downstream of the Nrf2 and Nrf2-ARE pathways. This result was associated with a stronger ability to scavenge ROS during reperfusion, leading to lower levels of ROS at the end of reperfusion (T3) and less myocardial damage. The optimal myocardial protective effect was achieved by 50 mmol/l pinacidil. However, cardiac function of the P50 + MPG group was significantly decreased, ultrastructure of cardiomyocytes was significantly impaired and the relative expression levels of genes and proteins in the Nrf2-ARE pathway were decreased. The aforementioned results confirmed that different PPC concentrations promoted early generation of ROS and activated the Nrf2-ARE signaling pathway following reperfusion, regulated expression levels of downstream antioxidant proteins and alleviated myocardial IR injury in rats. Treatment with 50 mmol/l pinacidil resulted in the best myocardial protection.

HomeCirculation ResearchVol. 129, No. 10Monoamine Oxidases Desensitize Intracellular β1AR Signaling in Heart Failure Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyRedditDiggEmail Jump toFree AccessLetterPDF/EPUBMonoamine Oxidases Desensitize Intracellular β1AR Signaling in Heart Failure Ying Wang, Meimi Zhao, Qian Shi, Bing Xu, Chaoqun Zhu, Minghui Li, Vaseem Mir, Donald M. Bers and Yang K. Xiang Ying WangYing Wang https://orcid.org/0000-0002-8566-875X Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Meimi ZhaoMeimi Zhao Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Qian ShiQian Shi Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Bing XuBing Xu Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). VA Northern California Health Care System, Mather, CA (B.X., Y.K.X) , Chaoqun ZhuChaoqun Zhu Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Minghui LiMinghui Li Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Vaseem MirVaseem Mir Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). , Donald M. BersDonald M. Bers Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). and Yang K. XiangYang K. Xiang Correspondence to: Yang K. Xiang, PhD, Pharmacology, UC Davis, CA 95616. Email E-mail Address: [email protected] https://orcid.org/0000-0003-1786-9143 Department of Pharmacology, University of California, Davis, CA (Y.W., M.Z., Q.S., B.X., C.Z., M.L., V.M., D.M.B., Y.K.X.). VA Northern California Health Care System, Mather, CA (B.X., Y.K.X) Originally published17 Sep 2021https://doi.org/10.1161/CIRCRESAHA.121.319546Circulation Research. 2021;129:965–967is related toMeet the First AuthorsOther version(s) of this articleYou are viewing the most recent version of this article. Previous versions: September 17, 2021: Ahead of Print Meet the First Author, see p 893Desensitization of β1AR (β1 adrenergic receptor) and depressed cardiac contractility are hallmarks of heart failure (HF). Therefore, clinical drugs have been primarily aimed at rescuing β1ARs at the plasma membrane in therapy. This paradigm has been challenged by emerging evidence of functioning intracellular β1ARs at the sarcoplasmic reticulum (SR).1 The SR-β1AR regulates local PKA (protein kinase A) phosphorylation of PLB (phospholamban) and excitation-contraction coupling. Thus, enhancing β1AR signaling at the SR represents an appealing approach for effectively improving contractility in HF. We found that elevation of MAO-A (monoamine oxidase A) in HF prevents local β1AR-PKA-PLB signaling at the SR. Inhibition of MAO-A rescues local β1AR signaling, phosphorylation of PLB, and excitation-contraction-coupling in HF.We applied chronic intraperitoneal injection of β-agonist isoproterenol to induce HF. We examined SR-β1AR association with SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) using proximity ligation assay (Figure [A]). HF adult ventricular myocytes (AVMs) displayed more proximity ligation assay signals between β1AR and SERCA2a than non-HF cells, suggesting an increased β1AR association with SERCA2a in failing hearts.1 There was minimal proximity ligation assay signal between β1AR and ryanodine receptor 2 in non-HF and HF AVMs (Figure [A]). Failing hearts usually have low catecholamine contents associated with contractile dysfunction.2 Catecholamines are imported via OCT (organic cation transporter)1 and degraded by MAOs in hearts.1,2 Transcriptomic analysis of patients with dilated cardiomyopathy4 revealed significant downregulation of β1AR (ADRB1) and upregulation of MAO-A, but no change in MAO-B, OCT3, and COMT (catechol-O-methyltransferase, another catecholamine degradation enzyme; Figure [B]).Download figureDownload PowerPointFigure. Elevated monoamine oxidase A impairs intracellular β1AR signaling in failing myocytes.A, Proximity ligation assay assay of adult ventricular myocytes (AVMs) co-stained with anti-β1AR/SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) or anti-β1AR/RyR2 (ryanodine receptor 2) antibodies. Representative 3-dimensional images were randomly selected and quantified with Image J. B, Volcano plot of transcriptome mRNA from human heart failure (HF) patients with dilated cardiomyopathy relative to non-HF patients (GSE3586). Dot-plot shows the mRNA expression of MAO-A (monoamine oxidase A) from non-HF and HF patients. C, Schematic of intracellular sarcoplasmic reticulum (SR)-β1AR signaling in non-HF and HF AVMs. AVMs expressing PKA (protein kinase A) biosensors anchored on the plasma membrane and SR were pretreated with MAOi (MAO-A inhibitor; clorgyline, 5 µmol/L, 5 min) before stimulation with EPI (epinephrine, 1 µmol/L). Traces show time courses of the changes in fluorescence resonance energy transfer (FRET) YFP/CFP (yellow fluorescent protein, YFP emission intensity divide Cyan fluorescent protein, CFP emission intensity) ratio. Dot-plots show maximal increases in FRET ratio. D, Detection of PKA phosphorylation of PLB (phospholamban) at serine 16 (pS16) and LTCC (L-type Ca2+ channel) Cav1.2 at serine 1928 (pS1928) after stimulation with EPI (1 µmol/L) or dobutamine (1 µmol/L) in the presence of MAOi. E, HF AVMs were preloaded with Ca2+ indicator Fluo-4 (2 µmol/L), paced at 1 Hz, and pretreated with MAOi. Ca2+ transient (CaT) and sarcomere shortening (SS) were recorded in response to EPI, NE, or DOB. Dot-plots show maximal changes in SS, CaT amplitude (F/F0), and Ca2+ decay (Tau). Dot-plots show mean±SEM of the number of AVMs from mice (indicated). For D, P values were obtained in paired comparisons only after a significance found in a nonparametric Kruskal-Wallis test. All other data passed Shapiro-Wilk normality test. P values were obtained after 2-way ANOVA analysis followed by Tukey test (A, C, and E) or Student t test (B). AU indicates arbitrary unit; and DOB, dobutamine.We employed fluorescence resonance energy transfer-based AKAR3 (A kinase activity reporter 3)1 to assess the impacts of MAO-A on local PKA activity at the plasma membrane or SR (Figure [C]). In non-HF AVMs, epinephrine-induced robust increases in PKA activity at the plasma membrane and SR. MAOi (MAO-A inhibitor) clorgyline enhanced PKA activity only at the SR. In HF AVMs, epinephrine-induced negligible PKA activation, and MAOi selectively enhanced PKA activation at the SR. These observations imply that the upregulated MAO-A in HF limits local β1AR-PKA activation. Inhibition of MAO-A rescues intracellular β1AR-PKA signaling at the SR.Activation of SR-β1AR promotes PKA phosphorylation of PLB, a SERCA2a regulator, to enhance Ca2+ transients. MAOi selectively enhanced PKA phosphorylation of PLB in non-HF AVMs after stimulation with epinephrine but not dobutamine, a nonsubstrate for MAO (Figure [D]). In HF AVMs, MAOi enhanced epinephrine-induced increases in PKA phosphorylation of PLB at the SR but not L-type Ca2+ channel at the plasma membrane (Figure [D]).Norepinephrine, epinephrine, and dobutamine promoted little contractile response in HF AVMs. MAOi significantly rescued norepinephrine and epinephrine but not dobutamine-induced excitation-contraction (E-C) coupling (Figure [E]). Collectively, our results indicate inhibition of MAO-A restores SR-localized β1AR-PKA-PLB signaling and excitation-contraction-coupling in HF.The expression of MAO-A is increased by HF-associated pathological stresses including inflammation, aging, and reactive oxidative species. The expression of MAO-A is also regulated by hormones such as thyroid and estrogen, which affects SR calcium handling and cardiac contractility.4 Our data indicate that MAO-A inhibitors may hold promise in rescuing SR-β1AR signaling and enhancing cardiac contractility while reducing oxidative stress in HF.2Data AvailabilityThe methods, data, and materials are available upon request. C57BL/6J male mice (2–4-month-old) were randomly assigned for intraperitoneal injection of saline or ISO (30 mg/kg per day, 14 days) and blinded for data analysis. Animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and the protocols approved by the University of California Davis Institutional Animal Care and Use Committee (IACUC).Article InformationSources of FundingThis work was supported by NIH-HL147263 and Veteran affair BX005100 (Y.K. Xiang) and American Heart Association postdoctoral fellowships (Y. Wang and Q. Shi).DisclosuresNone.FootnotesFor Sources of Funding and Disclosures, see page 967.Correspondence to: Yang K. Xiang, PhD, Pharmacology, UC Davis, CA 95616. Email [email protected]eduReferences1. Wang Y, Shi Q, Li M, Zhao M, Reddy Gopireddy R, Teoh JP, Xu B, Zhu C, Ireton KE, Srinivasan S, et al.. Intracellular β1-Adrenergic receptors and organic cation transporter 3 mediate phospholamban phosphorylation to enhance cardiac contractility.Circ Res. 2021; 128:246–261. doi: 10.1161/CIRCRESAHA.120.317452LinkGoogle Scholar2. Kaludercic N, Takimoto E, Nagayama T, Feng N, Lai EW, Bedja D, Chen K, Gabrielson KL, Blakely RD, Shih JC, et al.. Monoamine oxidase A-mediated enhanced catabolism of norepinephrine contributes to adverse remodeling and pump failure in hearts with pressure overload.Circ Res. 2010; 106:193–202. doi: 10.1161/CIRCRESAHA.109.198366LinkGoogle Scholar3. Barth AS, Kuner R, Buness A, Ruschhaupt M, Merk S, Zwermann L, Kääb S, Kreuzer E, Steinbeck G, Mansmann U, et al.. Identification of a common gene expression signature in dilated cardiomyopathy across independent microarray studies.J Am Coll Cardiol. 2006; 48:1610–1617. doi: 10.1016/j.jacc.2006.07.026CrossrefMedlineGoogle Scholar4. Trivieri MG, Oudit GY, Sah R, Kerfant BG, Sun H, Gramolini AO, Pan Y, Wickenden AD, Croteau W, Morreale de Escobar G, et al.. Cardiac-specific elevations in thyroid hormone enhance contractility and prevent pressure overload-induced cardiac dysfunction.Proc Natl Acad Sci U S A. 2006; 103:6043–6048. doi: 10.1073/pnas.0601072103CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesMeet the First AuthorsCirculation Research. 2021;129:892-894 October 29, 2021Vol 129, Issue 10Article InformationMetrics Download: 437 © 2021 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.121.319546PMID: 34530626 Originally publishedSeptember 17, 2021 Keywordsphosphorylationcatecholamineepinephrineheart failuresarcoplasmic reticulumPDF download Advertisement SubjectsBasic Science ResearchCell Signaling/Signal TransductionContractile FunctionHeart Failure

Aims: The N6-methyladenosine (m6A) modification plays an important role in various biological processes, but its role in atherosclerosis remains unknown. The aim of this study was to investigate the role and mechanism of m6A modification in endothelial cell inflammation and its influence on atherosclerosis development. Methods: We constructed a stable TNF-α-induced endothelial cell inflammation model and assessed the changes in the expression of m6A modification-related proteins to identify the major factors involved in this process. The m6A-modified mRNAs were identified by methylated RNA immunoprecipitation (RIP) sequencing and forkhead box O1 (FOXO1) was selected as a potential target. Through cytological experiments, we verified whether methyltransferase-like 14 (METTL14) regulates FOXO1 expression by regulating m6A-dependent mRNA and protein interaction. The effect of METTL14 on atherosclerosis development in vivo was verified using METTL14 knockout mice. Results: These findings confirmed that METTL14 plays major roles in TNF-α-induced endothelial cell inflammation. During endothelial inflammation, m6A modification of FOXO1, an important transcription factor, was remarkably increased. Moreover, METTL14 knockdown significantly decreased TNF-α-induced FOXO1 expression. RIP assay confirmed that METTL14 directly binds to FOXO1 mRNA, increases its m6A modification, and enhances its translation through subsequent YTH N6-methyladenosine RNA binding protein 1 recognition. Furthermore, METTL14 was shown to interact with FOXO1 and act directly on the promoter regions of VCAM-1 and ICAM-1 to promote their transcription, thus mediating endothelial cell inflammatory response. In vivo experiments showed that METTL14 gene knockout significantly reduced the development of atherosclerotic plaques. Conclusion: METTL14 promotes FOXO1 expression by enhancing its m6A modification and inducing endothelial cell inflammatory response as well as atherosclerotic plaque formation. Decreased expression of METTL14 can inhibit endothelial inflammation and atherosclerosis development. Therefore, METTL14 may serve as a potential target for the clinical treatment of atherosclerosis.

Rationaleβ1AR‐adrenergic receptor (β1AR) plays a central role in sympathetic regulation of cardiac excitation‐contraction (EC) coupling. Stimulation of β1AR by catecholamines enhances cardiomyocyte contraction and Ca2+transient via the cAMP‐protein kinase A (PKA) pathway. Organic cation transporter 3 (OCT3) plays a role in cleaning up the extracellular catecholamines and transporting a portion of catecholamine into myocardium. Recent progress found that β1AR is also present at intracellular membranes. However, how the intracellular β1AR was activated, and the role of OCT3 in regulating intracellular β1AR signaling and EC‐coupling remains unknown.ObjectiveTo characterize the presence and activation of intracellular β1AR‐adrenergic receptors and their impact on cardiac EC‐couplingMethods and ResultsWe used a genetic deletion of OCT3 to inhibit the entrance of catecholamine into the cells. We used isolated myocytes to measure β1AR‐induced contractility and Ca2+transients. We used PKA activity reporters to detect local activation via intracellular β1AR. We found that catecholamine‐induced inotropic effects were diminished in OCT3‐knock out hearts. OCT3 deficiency significantly attenuated norepinephrine (NE) induced PKA activities at the sarcoplasmic reticulum, myocyte contractility, and Ca2+transient. Additional data show β1AR associates with sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) at the sarcoplasmic reticulum (SR). Inhibition of OCT3 blocked the transportation of NE, and attenuated β1AR‐induced PKA activity at the SR and EC‐coupling.ConclusionOur finding demonstrates a previously unrecognized intracellular β1AR‐mediated local PKA activity at SR that is gated by OCT3. Upon catecholamine stimulation, this SR‐localized β1AR signal plays a critical role in promoting E‐C coupling and cardiac contractile function. Thus, internal β1AR signaling may presents a novel therapeutic target in cardiac diseases treatment.Support or Funding InformationNIH HL113413VABX009200

BackgroundChronic stimulation of β adrenergic receptor (βAR) has been implicated in cardiac remodeling including hypertrophy and fibrosis associated with heart failure. Previous studies indicate a role of β2AR on inhibiting collagen production in cultured cardiac fibroblasts, however, our recent studies show that global deletion of β2AR prevent cardiac fibrosis in diabetic cardiomyopathy. Therefore, this study aims to determine the specific role of cardiomyocyte β2AR in cardiac function and the development of cardiac fibrosis in vivo.Methods2‐month old male β2AR flox/flox (f/f) mice (n=10) and cardiomyocyte‐specific knockout of β2AR (CKO) mice (n=10) were fed with either normal chow (NC), or high fat diet (HFD) for 6 months to induce obesity and diabetes. Echocardiography was performed using a Vevo 2100 imaging system from VisualSonics. Biochemistry and histological analyses were performed on heart tissues. Real‐time PCR was performed for mRNA expression examination.ResultsWhile cardiac function was normal at 2‐month old CKO mice, a significant decrease in cardiac contractility was observed in CKO mice compared to f/f mice at 8‐month old age (EF%: CKO 48.43 ± 1.314 vs. f/f 52.69 ± 0.8270, p<0.05). Moreover, HFD feeding exacerbated cardiac dysfunction in CKO mice when compared to f/f mice (EF%: CKO 42.93 ± 0.69 vs. f/f 46.79 ± 0.76, p<0.001). CKO mice also exhibited diastolic dysfunction (IVRT: CKO 20.23 ± 0.30 vs. f/f 16.83 ± 0.23, p<0.0001), which was exacerbated after HFD feeding (IVRT, CKO 24.75 ± 0.50 vs. f/f 19.94 ± 0.45, p<0.0001). The observed cardiac dysfunction was associated with severe cardiac fibrosis in 8‐month old CKO mice, and HFD feeding leads to further exacerbation in cardiac fibrosis. Cardiac fibrosis was validated with elevated protein expression of connective tissue growth factor (CTGF), and significant upregulations of mRNA expression of CTGF, α smooth muscle actin (α‐SMA), and collagen 1 in CKO HFD mice.ConclusionsThis study elucidates that cardiomyocyte β2AR is essential to maintain normal cardiac function and plays a protective role against the development of cardiac fibrosis in aging and HFD feeding in vivo.Support or Funding InformationNIH HL113413, HL147264, VABX002900

RATIONALE:Cardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.OBJECTIVE:We aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.METHODS AND RESULTS:The integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.CONCLUSIONS:These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

RationaleHeart failure as one of the leading causes of morbidity worldwide is hallmarked by dysregulated myocardial calcium (Ca2+) and cardiomyocyte apoptosis. There exists an urgent unmet need of developing novel therapies for preventing and reversing HF progression by correcting Ca2+ dysregulation and inhibiting apoptosis. However, the mechanisms underlying Ca2+ regulation in physiological and pathological conditions remain incompletely elucidated.ObjectiveTo identify how a synaptic associated protein 97 (SAP97) controls cardiac intracellular Ca2+ in both physiological and HF development.Methods and ResultsWe used genetic deletion (cardiomyocyte specific SAP97 knock out mice, SAP97‐cKO) to determine the essential role of SAP97 in regulating the Ca2+ channel activity and how this contributes to the heart failure process. Proximity ligation assay (PLA) and confocal imaging technology were applied to identify a novel SAP97/protein Kinase A (PKA)/ L‐type calcium channel (LTCC) protein machinery complex. We use fluorescence resonance energy transfer (FRET) and living cell imaging to analyze the dynamic spatial changes of Ca2+ homeostasis under the sympathetic adrenergic control of SAP97/LTCC complex. Our study found that a SAP97 binds LTCC and modulates PKA‐dependent LTCC activation via scaffolding PKA and phosphodiesterase 4D8 (PDE4D8) in cardiomyocyte. Disruption of this complex impaired the negative feedback control of cAMP/PKA by PDE4D8, therefore augments PKA‐dependent LTCC activation and promotes Ca2+ dysregulation. Moreover, genetic deletion of SAP97 enhances cAMP/PKA activity and induces Ca2+ dysregulation, which promotes cardiomyocyte apoptosis and HF development in mice.ConclusionSAP97 mediated local control of cAMP/PKA gradients regulates LTCC and Ca2+ homeostasis in failing heart, therefore offer a novel SAP97/PKA/Ca2+ signaling axis as a therapeutic target for heart failure prevention and treatment.Support or Funding InformationThis study was supported by NIH grants HL113413 and HL147264 and VA Merit grant 01BX002900 to Y.K.X.