Mouse Myocytes Research Articles

Abstract Mo081: Genetic Deletion of Histone Lysine Demethylase KDM5A in Cardiomyocytes Attenuates LMNA-associated Dilated Cardiomyopathy

Background: Heart failure (HF) is a major cause of morbidity and mortality. Hereditary cardiomyopathies are the prototypic forms of HF. Notable among them is dilated cardiomyopathy (DCM) caused by mutations in the LMNA gene encoding Lamin A protein (LMNA-DCM). We have recently shown that KDM5A, a histone H3 lysine 4 demethylase, is activated in the hearts of human patients and mouse models of LMNA-DCM. Inhibition of the KDM5 family of proteins activates gene expression, including expression of genes involved in oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) in iPSC-derived cardiomyocytes. Thus, we hypothesized that activation of the KDM5A in the LMNA-DCM is pathogenic and contributes to cardiac dysfunction. Objective: The objective of this study is to determine the role of KDM5A in the pathogenesis of LMNA-DCM. Methods and Results: The Lmna and Kdm5a genes were concomitantly deleted in the cardiac myocytes (CMs) in mice using cre recombinase. CM-specific deletion of the Lmna gene ( Myh6-Cre:Lmna F/F ), representing a mouse model of LMNA-DCM, led to severe cardiac dysfunction, increased apoptosis, and premature death as published. The Myh6-Cre:Kdm5a F/F mice showed no discernible phenotype. Deletion of the Kdm5a gene in CMs in the LMNA-DCM mice (Myh6-Cre:Lmna F/F :Kdm5a F/F ) attenuated myocardial apoptosis, improved cardiac function, and prolonged survival. Specifically, left ventricular end-systolic and end-diastolic diameters (LVESD and LVEDD, respectively) were smaller and LV fractional shortening (LVFS) was greater in the Myh6-Cre: Lmna F/F :Kdm5a F/F mice as compared to the Myh6-Cre:Lmna F/F mice. Likewise, the median and maximum survival times were increased by ~ 40 to 50% in the Myh6-Cre:Lmna F/F :Kdm5a F/F . Analysis of the CM transcripts by RNA-sequencing showed dysregulation of 6609 genes in the Myh6-Cre: Lmna F/F CMs and 2791 genes were attenuated in the Myh6-Cre:Lmna F/F :Kdm5a F/F . Pathway analysis showed that genes involved in FAO and OXPHOS were the most suppressed in LMNA-DCM and were rescued upon Kdm5a deletion. Conclusions: Deletion of the Kdm5a gene in the LMNA-DCM mice imparts salubrious effects by prolonging survival and improving cardiac dysfunction, and upregulating genes involved in OXPHOS and FAO. Thus, activation of KDM5A is likely pathogenic and contributes to the phenotypic expression of LMNA-DCM.

Abstract Tu085: The β IV -spectrin/STAT3 Complex regulates the orientation of cardiac hypertrophic growth

Background: Cardiac hypertrophy is a major risk factor for adverse cardiovascular events including heart failure and arrhythmia. However, the precise orientation of cell and organ growth varies considerably depending on stress type and duration with important implications for cardiac function. Despite this, little is known regarding the mechanisms that regulate hypertrophic orientation. Here we evaluate the role of the cytoskeletal protein β IV -spectrin and signal transducer and activator of transcription (STAT3) in directing the orientation of pressure overload-induced hypertrophy. Methods: Transgenic mouse models with altered STAT3 signaling through modified interaction with its scaffolding partner β IV -spectrin, or phospho-regulation of STAT3 directly, were evaluated at baseline and transaortic constriction (TAC) for its role in promoting concentric versus eccentric morphologies and resulting impact on systolic function. Unbiased screening of gene expression from these structurally divergent states were evaluated for pathways responsible for directing myocyte length/width. These pathways were tested in vitro using primary mouse myocytes and in vivo to tune growth patterns for therapeutic intervention. Results: Loss of β IV -spectrin or direct STAT3 activation promoted a preferential increase in myocyte length over width, resulting in dilation of the left ventricular (LV) chamber (eccentric hypertrophy) and decreased systolic function. Conversely, preservation of β IV -spectrin favored an increase in myocyte width without LV dilation (concentric hypertrophy) and preserved systolic function in response to TAC. Differential expression of genes associated with microtubule dynamics were identified in concentric vs. eccentric hypertrophic states. In vitro assays revealed a relationship between β IV -spectrin/STAT3 signaling and microtubule stability which impacted spatial distribution of mRNA for the sarcomeric gene actc1 . Finally, intervention with pharmacologic STAT3 inhibition following chronic 6-week TAC successfully recovered concentric growth with improved systolic function. Conclusions: These data identify a novel and pivotal role for β IV -spectrin/STAT3 to modify microtubule dynamics and sarcomeric transcript distribution to direct myocyte geometry and therapeutically serve in the prevention or recovery from HF. These mechanisms further illustrate the unique separation of hypertrophic drivers from growth orientation as distinct pathways in cardiac remodeling.

Spexin Diminishes Atrial Fibrillation Vulnerability by Acting on Galanin Receptor 2.

G protein-coupled receptors play a critical role in atrial fibrillation (AF). Spexin is a novel ligand of galanin receptors (GALRs). In this study, we investigated the regulation of spexin and GALRs on AF and the underlying mechanisms. Global spexin knockout (SPX-KO) and cardiomyocyte-specific GALRs knockout (GALR-cKO) mice underwent burst pacing electrical stimulation. Optical mapping was used to determine atrial conduction velocity and action potential duration. Atrial myocyte action potential duration and inward rectifying K+ current (IK1) were recorded using whole-cell patch clamps. Isolated cardiomyocytes were stained with Fluo-3/AM dye, and intracellular Ca2+ handling was examined by CCD camera. A mouse model of AF was established by Ang-II (angiotensin II) infusion. Spexin plasma levels in patients with AF were lower than those in subjects without AF, and knockout of spexin increased AF susceptibility in mice. In the atrium of SPX-KO mice, potassium inwardly rectifying channel subfamily J member 2 (KCNJ2) and sarcolipin (SLN) were upregulated; meanwhile, IK1 current was increased and Ca2+ handling was impaired in isolated atrial myocytes of SPX-KO mice. GALR2-cKO mice, but not GALR1-cKO and GALR3-cKO mice, had a higher incidence of AF, which was associated with higher IK1 current and intracellular Ca2+ overload. The phosphorylation level of CREB (cyclic AMP responsive element binding protein 1) was upregulated in atrial tissues of SPX-KO and GALR2-cKO mice. Chromatin immunoprecipitation confirmed the recruitment of p-CREB to the proximal promoter regions of KCNJ2 and SLN. Finally, spexin treatment suppressed CREB signaling, decreased IK1 current and decreased intracellular Ca2+ overload, which thus reduced the inducibility of AF in Ang-II-infused mice. Spexin reduces atrial fibrillation susceptibility by inhibiting CREB phosphorylation and thus downregulating KCNJ2 and SLN transcription by GALR2 receptor. The spexin/GALR2/CREB signaling pathway represents a novel therapeutic avenue in the development of agents against atrial fibrillation.

Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Calpain inhibition protects against atrial fibrillation by mitigating diabetes-associated atrial fibrosis and calcium handling dysfunction in type 2 diabetes mice

BackgroundDiabetes mellitus (DM) is a major risk factor for atrial structural remodeling and atrial fibrillation (AF). Calpain activity is hypothesized to promote atrial remodeling and AF. ObjectiveThe purpose of this study was to investigate the role of calpain in diabetes-associated AF, fibrosis, and calcium handling dysfunction. MethodsDM-associated AF was induced in wild-type (WT) mice and in mice overexpressing the calpain inhibitor calpastatin (CAST-OE) using high-fat diet feeding followed by low-dose streptozotocin injection (75 mg/kg). DM and AF outcomes were assessed by measuring blood glucose levels, fibrosis, and AF susceptibility during transesophageal atrial pacing. Intracellular Ca2+ transients, spontaneous Ca2+ release events, and intracellular T-tubule membranes were measured by in situ confocal microscopy. ResultsWT mice with DM had significant hyperglycemia, atrial fibrosis, and AF susceptibility with increased atrial myocyte calpain activity and Ca2+ handling dysfunction relative to control treated animals. CAST-OE mice with DM had a similar level of hyperglycemia as diabetic WT littermates but lacked significant atrial fibrosis and AF susceptibility. DM-induced atrial calpain activity and downregulation of the calpain substrate junctophilin-2 were prevented by CAST-OE. Atrial myocytes of diabetic CAST-OE mice exhibited improved T-tubule membrane organization, Ca2+ handling, and reduced spontaneous Ca2+ release events compared to littermate controls. ConclusionThis study confirmed that DM promotes calpain activation, atrial fibrosis, and AF in mice. CAST-OE effectively inhibits DM-induced calpain activation and reduces atrial remodeling and AF incidence through improved intracellular Ca2+ homeostasis. Our results support calpain inhibition as a potential therapy for preventing and treating AF in DM patients.

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq.

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (p-value < 1E-5) and enrichment score ≥ 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA Pol-II, including TATA-box, transcription initiator motif, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns were enriched with the Homeobox family of transcription factors and exhibited TA-rich motif sequences, suggesting potential motif-specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521 enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers, 3390 (12%) exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may dictate its function in myogenic differentiation and potentially other cellular and biological processes.

Natriuretic Peptide Receptor B Protects Against Atrial Fibrillation by Controlling Atrial cAMP Via Phosphodiesterase 2.

β-AR (β-adrenergic receptor) stimulation regulates atrial electrophysiology and Ca2+ homeostasis via cAMP-dependent mechanisms; however, enhanced β-AR signaling can promote atrial fibrillation (AF). CNP (C-type natriuretic peptide) can also regulate atrial electrophysiology through the activation of NPR-B (natriuretic peptide receptor B) and cGMP-dependent signaling. Nevertheless, the role of NPR-B in regulating atrial electrophysiology, Ca2+ homeostasis, and atrial arrhythmogenesis is incompletely understood. Studies were performed using atrial samples from human patients with AF or sinus rhythm and in wild-type and NPR-B-deficient (NPR-B+/-) mice. Studies were conducted in anesthetized mice by intracardiac electrophysiology, in isolated mouse atrial preparations using high-resolution optical mapping, in isolated mouse and human atrial myocytes using patch-clamping and Ca2+ imaging, and in mouse and human atrial tissues using molecular biology. Atrial NPR-B protein levels were reduced in patients with AF, and NPR-B+/- mice were more susceptible to AF. Atrial cGMP levels and PDE2 (phosphodiesterase 2) activity were reduced in NPR-B+/- mice leading to larger increases in atrial cAMP in the presence of the β-AR agonist isoproterenol. NPR-B+/- mice displayed larger increases in action potential duration and L-type Ca2+ current in the presence of isoproterenol. This resulted in the occurrence of spontaneous sarcoplasmic reticulum Ca2+ release events and delayed afterdepolarizations in NPR-B+/- atrial myocytes. Phosphorylation of the RyR2 (ryanodine receptor) and phospholamban was increased in NPR-B+/- atria in the presence of isoproterenol compared with the wildtypes. C-type natriuretic peptide inhibited isoproterenol-stimulated L-type Ca2+ current through PDE2 in mouse and human atrial myocytes. NPR-B protects against AF by preventing enhanced atrial responses to β-adrenergic receptor agonists.

Circulating myomiRNAs as biomarkers in patients with Cushing’s syndrome

PurposeImpairment of skeletal muscle mass and strength affects 40–70% of patients with active Cushing’s syndrome (CS). Glucocorticoid excess sustains muscle atrophy and weakness, while muscle-specific microRNAs (myomiRs) level changes were associated with muscle organization and function perturbation. The aim of the current study is to explore changes in circulating myomiRs in CS patients compared to healthy controls and their involvement in IGFI/PI3K/Akt/mTOR pathway regulation in skeletal muscle.MethodsC2C12, mouse myocytes, were exposed to hydrocortisone (HC), and atrophy-related gene expression was investigated by RT-qPCR, WB and IF to assess HC-mediated atrophic signalling. miRNAs were evaluated in HC-treated C2C12 by PCR Arrays. MyomiRs significantly overexpressed in C2C12 were investigated in 37 CS patients and 24 healthy controls serum by RT-qPCR. The anti-anabolic role of circulating miRNAs significantly upregulated in CS patients was explored in C2C12 by investigating the IGFI/PI3K/Akt/mTOR pathway regulation.ResultsHC induced higher expression of atrophy-related genes, miR-133a-3p, miR-122-5p and miR-200b-3p in C2C12 compared to untreated cells. Conversely, the anabolic IGFI/PI3K/Akt/mTOR signalling was reduced and this effect was mediated by miR-133a-3p. In CS patients miR-133a-3p and miR-200b-3p revealed higher circulating levels (p < 0.0001, respectively) compared to controls. ROC curves for miR-133a-3p (AUC 0.823, p < 0.0001) and miR-200b-3p (AUC 0.850, p < 0.0001) demonstrated that both myomiRs represent potential biomarkers to discriminate between CS and healthy subjects. Pearson’s correlation analysis revealed that circulating levels of miR-133a-3p are directly correlated with 24 h urinary-free cortisol level (r = 0.468, p = 0.004) in CS patients.ConclusionsHC induces atrophic signals by miR-133a-3p overexpression in mouse myocytes and humans. Circulating miR-133a-3p is promising biomarkers of hypercortisolism.

Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in isolated mouse ventricular myocytes.

The Bruton tyrosine kinase (BTK) inhibitor Ibrutinib is associated with a higher incidence of cardiotoxic side effects including heart failure (HF). Ibrutinib is capable of inhibiting PI3K/Akt signaling in neonatal rat ventricular cardiomyocytes when stimulated with insulin-like growth factor 1 (IGF-1). We therefore hypothesized that Ibrutinib might disrupt IGF-1-mediated activation of intracellular Ca handling in adult mouse cardiomyocytes by inhibiting PI3K/Akt signaling. Isolated ventricular myocytes (C57BL6/J) were exposed to IGF-1 at 10 nmol/L in the presence or absence of Ibrutinib (1 µmol/L) or Acalabrutinib (10 µmol/L; cell culture for 24 ± 2 h). Intracellular Ca handling was measured by epifluorescence (Fura-2 AM) and confocal microscopy (Fluo-4 AM). Ruptured-patch whole-cell voltage-clamp was used to measure ICa. Levels of key cardiac Ca handling proteins were investigated by immunoblots. IGF-1 significantly increased Ca transient amplitudes by ∼83% as compared to vehicle treated control cells. This was associated with unaffected diastolic Ca, enhanced SR Ca loading and increased ICa. Co-treatment with Ibrutinib attenuated both the IGF-1-mediated increase in SR Ca content and in ICa. IGF-1 treated cardiomyocytes had significantly increased levels of pS473Akt/Akt and SERCA2a expression as compared to cells concomitantly treated with IGF-1 and Ibrutinib. SR Ca release (as assessed by Ca spark frequency) was unaffected by either treatment. In order to test for potential off-target effects, second generation BTK inhibitor Acalabrutinib with greater BTK selectivity and lower cardiovascular toxicity was tested for IGF1-mediated activation of intracellular Ca handling. Acalabrutinib induced similar effects on Ca handling in IGF-1 treated cultured myocytes as Ibrutinib in regard to decreased Ca transient amplitude and slowed Ca transient decay, hence implying a functional class effect of BTK inhibitors in cardiac myocytes. Inhibition of BTK by Ibrutinib impairs IGF-1-dependent activation of intracellular Ca handling in adult ventricular mouse myocytes in the face of disrupted Akt signaling and absent SERCA2a upregulation.

Activation of Exchange Proteins directly Activated by cAMP (EPAC) lengthens action potential duration by inhibiting potassium currents in atrial cardiomyocytes

IntroductionAtrial fibrillation (AF) is the most frequent sustained cardiac arrhythmia worldwide. Several mechanisms have been described in AF pathogenesis and sustainability such as dysregulation of calcium cycling and action potential (AP) duration. Effectors involved in these dysregulations are potential relevant therapeutic targets. Among them, the Exchange Proteins directly Activated by cAMP (EPAC) has been shown to increase arrhythmogenic activity in ventricular cardiomyocytes by lengthening of AP duration. However, the electrophysiological impact of EPAC at the atrial level remains to be established. ObjectiveTo evaluate the propensity of EPAC activation to induce atrial arrhythmogenic activity and by which mechanisms. MethodMyocytes from human right atrial appendages and wild type (WT) and EPAC1-/- mice atria were dissociated by enzymatic digestion. AP and potassium currents were recorded using the whole cell configuration of the patch clamp technique. EPAC proteins were pharmacologically activated by acute superfusion of 8-CPTAM (10μM), and the selective role of EPAC1 was evaluated by its selective inhibition by superfusion of AM-001 (20μM). ResultsIn mouse atrial myocytes, activation of EPAC by 8-CPTAM significantly increases AP duration at 90% of repolarization (APD90). Interestingly, this effect is higher in WT myocytes than in EPAC1-/-. Moreover, AM-001, which fails to correct AP lengthening in EPAC1-/- myocytes, reverses partially but significantly the effect of 8-CPTAM in WT myocytes. In human atrial myocytes, 8-CPTAM treatment lengthens APD90 by 32.3%. Additionally, in both WT mouse and human atrial myocytes, the treatment by 8-CPTAM induces an inhibition of both peak and sustained components of the K+ currents. Finally, effect of 8-CPTAM on K+ currents seems to be weaker in EPAC1-/- myocytes than in WT. ConclusionOur results show that EPAC activation lengthens AP duration in mouse and human atrial myocytes. Furthermore, inhibition of K+ currents participates in this AP duration modulation. Thus, these data suggest that EPAC proteins might be involved in atrial arrhythmogenic ectopic activity. Nevertheless, the role of each isoform of EPAC in these modulations and EPAC-induced regulation of other ionic currents remain to be specified.

A novel gene therapy for familial dilated cardiomyopathy based upon disruption of PP2A -- mAKAP-beta signalosomes

Familial Dilated Cardiomyopathy (DCM) is a common inherited cause of heart failure characterized by progressive left ventricular dilatation and systolic dysfunction. Despite the discovery of novel genetic mutations causing DCM, the downstream mechanisms resulting in heart failure are still unclear, and there remains a great unmet clinical need. Contributing to the phenotype of ventricular dilatation in DCM is the lengthening of cardiac myocytes, which exhibit increased overall length to width ratio. We have shown that increasing the phosphorylation of the transcription factor serine response factor (SRF) at serine residue 103 by displacement of protein phosphatase 2A (PP2A) from the perinuclear scaffold protein muscle A-kinase anchoring protein β (mAKAPβ) can inhibit myocyte lengthening and improve cardiac function following myocardial infarction. Notably, SRF S103 phosphorylation is decreased in both human heart failure and in the hearts of transgenic mice expressing the human DCM α-tropomyosin Glu54Lys (Tm-E54K) mutant allele. We now show that, accordingly, targeting of mAKAPβ-PP2A signalosomes constitutes a new therapeutic strategy for DCM. A self-complementary serotype 9 adeno-associated virus (AAV9sc) conferring cardiac-specific expression of a mAKAPβ fragment comprising the PP2A binding domain (PBD) was used to target the signalosomes in mice. AAV9sc.PBD administration into neonatal Tm-E54K mice provided long term preservation of cardiac function and prevented ventricular dilatation. At 16 weeks of age, ejection fraction was significantly improved 14%[MK1], and wall thickness was increased for AAV9sc.PBD TM-E54K mice when compared with control AAV9sc.GFP treated TM-E54K mice. Morphometry of acutely dissociated adult mouse myocytes showed that AAV9sc.PBD treatment restored myocyte length to width ratio to that of non-transgenic animals, while western blots showed increased SRF S103 phosphorylation. The results in mice have been corroborated by PBD expression in engineered heart tissues (EHT) generated from an induced pluripotent stem cell (iPSC) line for a DCM phospholamban R14del patient. PBD expression increased SRF S103 phosphorylation and improved tissue contractility. Taken together, this study suggests that increasing SRF S103 phosphorylation by disruption of mAKAPβ-PP2A signalosomes can constitute a novel therapeutic strategy for DCM. Funded by Department of NIH Grant R01HL146111 (Dr. Kapiloff), R01 HL139679 and Leducq Foundation CurePlan (Dr. Karakikes), and the NHLBI Gene Therapy Resource Program. This is the full abstract presented at the American Physiology Summit 2023 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.

A Compartmentalized Mathematical Model of the β1- and β2-Adrenergic Signaling Systems in Ventricular Myocytes from Mouse in Heart Failure.

Mouse models of heart failure are extensively used to research human cardiovascular diseases. In particular, one of the most common is the mouse model of heart failure resulting from transverse aortic constriction (TAC). Despite this, there are no comprehensive compartmentalized mathematical models that describe the complex behavior of the action potential, [Ca2+]i transients, and their regulation by β1- and β2-adrenergic signaling systems in failing mouse myocytes. In this paper, we develop a novel compartmentalized mathematical model of failing mouse ventricular myocytes after TAC procedure. The model describes well the cell geometry, action potentials, [Ca2+]i transients, and β1- and β2-adrenergic signaling in the failing cells. Simulation results obtained with the failing cell model are compared to those from the normal ventricular myocytes. Exploration of the model reveals the sarcoplasmic reticulum Ca2+ load mechanisms in failing ventricular myocytes. We also show a larger susceptibility of the failing myocytes to early and delayed afterdepolarizations and to a proarrhythmic behavior of Ca2+ dynamics upon stimulation with isoproterenol. The mechanisms of the proarrhythmic behavior suppression is investigated and sensitivity analysis is performed. The developed model can explain the existing experimental data on failing mouse ventricular myocytes and make experimentally testable predictions of a failing myocyte's behavior.

Bioengineered peptibodies as blockers of ion channels

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

Membrane Estrogen Receptor β Is Sufficient to Mitigate Cardiac Cell Pathology.

Estrogen acting through estrogen receptor β (ERβ) has been shown to oppose the stimulation of cardiac myocytes and cardiac fibroblasts that results in cardiac hypertrophy and fibrosis. Previous work has implicated signal transduction from ERβ as being important to the function of estrogen in this regard. Here we address whether membrane ERβ is sufficient to oppose key mechanisms by which angiotensin II (AngII) stimulates cardiac cell pathology. To do this we first defined essential structural elements within ERβ that are necessary for membrane or nuclear localization in cells. We previously determined that cysteine 418 is the site of palmitoylation of ERβ that is required and sufficient for cell membrane localization in mice and is the same site in humans. Here we determined in Chinese hamster ovarian (CHO) cells, and mouse and rat myocytes and cardiac fibroblasts, the effect on multiple aspects of signal transduction by expressing wild-type (WT ) or a C418A-mutant ERβ. To test the importance of the nuclear receptor, we determined a 4-amino acid deletion in the E domain of ERβ that strongly blocked nuclear localization. Using these tools, we expressed WT and mutant ERβ constructs into cardiomyocytes and cardiac fibroblasts from ERβ-deleted mice. We determined the ability of estrogen to mitigate cell pathology stimulated by AngII and whether the membrane ERβ is necessary and sufficient.

Abstract P1061: Crosstalk Between Acetylation And Methylglyoxal Modifications On Myofilament Proteins Affects Sarcomere Function

Diabetes doubles the risk of developing heart failure, independent of coronary artery disease and hypertension. The mechanisms connecting these two diseases are poorly understood, although we have previously shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis byproduct that can irreversibly modify lysine (K) and arginine (R) residues in a process called glycation. We found MGO glycation of the myofilament decreased calcium sensitivity and maximum calcium activated force in diabetes. Here we hypothesized MGO may further impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to impact cardiac function. To assess this hypothesis, non-failing human left ventricular (LV) myocardial tissue was exposed to MGO (100 μM) or vehicle and then the treated with acetic anhydride in acetonitrile (300 μM, Ac 2 O-ACN) overnight. Ac 2 O-ACN increased myofilament protein acetylation in both groups, but this effect was greatly reduced in samples pre-treated with MGO, suggesting these two PTMs do compete for a subset of lysine residues. To discover the specific sites of this acetylation-glycation crosstalk, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes, which revealed sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Importantly, these glycation-acetylation crosstalk sites were found in human samples and the data suggest the balance of these may change in disease states. To explore the functional consequence(s) of this crosstalk, we incubated human LV isolated skinned myocytes with Ac 2 O-ACN (300 μM, overnight), which enhanced function by significantly increasing calcium sensitivity. However, pre-treatment with MGO (100 μM) completely blocked this functional effect of acetylation. Together, these data demonstrate that glycation and acetylation compete for certain myofilament lysine residues, and that glycation can block the beneficial functional effects of acetylation. These results provide insight into the molecular mechanisms behind diabetic cardiomyopathy and the utility of targeting acetylation clinically.

Empagliflozin Ameliorates Ouabain-Induced Na+ and Ca2+ Dysregulations in Ventricular Myocytes in an Na+-Dependent Manner.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are a novel class of glucose-lowering agents that have improved clinical outcomes in patients with heart failure; however, their therapeutic mechanisms remain elusive. Although contradictory results have been reported, it has been proposed that improving Na+ homeostasis may be the underlying mechanism of action of SGLT2 inhibitors in heart failure treatment. This study explored whether empagliflozin ameliorates Na+ and Ca2+ handling disorders induced by ouabain in an Na+-dependent manner. Isolated ventricular myocytes of mice were incubated with ouabain to establish a cellular model of Na+ overload. Effects of empagliflozin on Na+ and Ca2+ handling were evaluated using an ionOptix system and a confocal microscope. Distinct cytosolic Na+ levels were established by incubating different ouabain concentrations (10, 50, and 100μmol/L). In the absence of ouabain, 1μmol/L empagliflozin had a negligible impact on Na+ and Ca2+ handling in ventricular myocytes. Ouabain (50μmol/L) significantly enhanced cytosolic Na+ levels and dysregulated Ca2+ handling, including an increased Ca2+ transient amplitude, elevated Ca2+ content in the sarcoplasmic reticulum, and enhanced spontaneous Ca2+ release normalized by treatment with 1μmol/L empagliflozin within 10min. All Na+ and Ca2+ handling abnormalities induced by ouabain were reversed by 1μmol/L empagliflozin. The efficacy of empagliflozin was more potent at higher cytosolic Na+ levels. Pretreatment with the Na+/H+ exchanger (NHE) inhibitor (1μmol/L cariporide) abolished the effects of empagliflozin. Empagliflozin ameliorates ouabain-induced Na+ and Ca2+ handling disorders in a cytosolic Na+-dependent manner, potentially by inhibiting the NHE.

Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice.

The Cre-LoxP technology, including the tamoxifen (TAM) inducible MerCreMer (MCM), is increasingly used to delineate gene function, understand the disease mechanisms, and test therapeutic interventions. We set to determine the effects of TAM-MCM on cardiac myocyte transcriptome.Expression of the MCM was induced specifically in cardiac myocytes upon injection of TAM to myosin heavy chain 6-MCM (Myh6-Mcm) mice for 5 consecutive days. Cardiac function, myocardial histology, and gene expression (RNA-sequencing) were analyzed 2 weeks after TAM injection. A total of 346 protein coding genes (168 up- and 178 down-regulated) were differentially expressed. Transcript levels of 85 genes, analyzed by a reverse transcription-polymerase chain reaction in independent samples, correlated with changes in the RNA-sequencing data. The differentially expressed genes were modestly enriched for genes involved in the interferon response and the tumor protein 53 (TP53) pathways. The changes in gene expression were relatively small and mostly transient and had no discernible effects on cardiac function, myocardial fibrosis, and apoptosis or induction of double-stranded DNA breaks.Thus, TAM-inducible activation of MCM alters cardiac myocytes gene expression, provoking modest and transient interferon and DNA damage responses without exerting other discernible phenotypic effects. Thus, the effects of TAM-MCM on gene expression should be considered in discerning the bona fide changes that result from the targeting of the gene of interest.

Mettl3 deficiency leads to the upregulation of Cav1.2 and increases arrhythmia susceptibility in mice.

Methyltransferase-like 3 (Mettl3) is a component of methyltransferase complex that mediates mA modification of RNAs, and participates in multiple biological processes. However, the role of Mettl3 in cardiac electrophysiology remains unknown. This study aims to explore the ventricular arrhythmia susceptibility of Mettl3 mice and the underlying mechanisms. Mice were anesthetized with 2% avertin (0.1 mL/ body weight) for echocardiography and programmed electrical pacing. Whole-cell patch clamp technique was used to examine the electrophysiological property of cardiomyocytes. The expression of Cav1.2 was determined by qRT-PCR and western blot analysis. The mA medication of mRNA was examined by MeRIP-Seq and MeRIP-qPCR. No differences are found in the morphology and function of the hearts between Mettl3 mice and wild-type (WT) controls. The QT and QTc intervals of Mettl3 mice are significantly longer. High-frequency electrical stimulation showed that heterozygous knockout of Mettl3 increases ventricular arrhythmia susceptibility. The whole-cell patch-clamp recordings showed that the APD is prolonged in Mettl3 ventricular myocytes and more EADs were observed. The density of is substantially increased in ventricular myocytes of Mettl3 mice. The pore-forming subunit of L-type calcium channel Cav1.2 is upregulated in Mettl3 mice, while the mRNA of its coding gene does not change. MeRIP-Seq and MeRIP-qPCR showed that the mA methylation of mRNA is decreased in cultured Mettl3-knockdown cardiomyocytes and Mettl3 hearts. Collectively, deficiency of Mettl3 increases ventricular arrhythmia susceptibility due to the upregulation of Cav1.2 by reducing mA modification onmRNA in mice. This study highlights the role of mA modification in the regulation of cardiac electrophysiology.

Abstract 14286: Crosstalk Between Methylglyoxal and Acetylation Modifications on Actin, Myosin, and Myosin Light Chain Proteins Affects Sarcomere Functionality

Diabetes affects ten percent of Americans. Risk of developing heart failure in these patients is doubled, independent of coronary artery disease and hypertension. However, the mechanistic connection between these two diseases is poorly understood. We have shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis product that can irreversibly modify lysine and arginine residues, a process called glycation. Moreover, we found MGO glycation of the myofilament decreased both calcium sensitivity and maximum calcium activated force (Fmax). Here, we hypothesized that MGO may also negatively impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to be beneficial to cardiac function. Using non-failing human left ventricular (LV) myocardial tissue that had been exposed to acute treatment of MGO (500 μM) or vehicle and subsequently treated with a high dose of acetic anhydride in acetonitrile (300μM) overnight, we found that compared to the vehicle treated samples, MGO inhibited overall acetylation, suggesting that these two PTMs do compete for a subset of lysine residues in the heart. Next, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes to identify the specific overlapping residues that acetylation and glycation compete for. This analysis identified multiple sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Our preliminary data support a contractile consequence to this crosstalk. Incubation of mouse LV isolated skinned myocytes with acetic anhydride in acetonitrile (300μM, overnight) increased both calcium sensitivity and Fmax. However, exposure to MGO first (500μM) blocked these beneficial functions acetylation (300μM, overnight) in mouse myocytes. Together, we believe these data demonstrate that glycation and acetylation do compete for residues in the myofilament, and that glycation is capable of blocking the beneficial functional effects of protein acetylation. If these results are confirmed and extended in vivo, they could have important consequences in diabetes and in the utility of using HDAC inhibitors clinically.

Differences in molecular phenotype in mouse and human hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is characterized by phenotypic heterogeneity. We investigated the molecular basis of the cardiac phenotype in two mouse models at established disease stage (mouse-HCM), and human myectomy tissue (human-HCM). We analyzed the transcriptome in 2 mouse models with non-obstructive HCM (R403Q-MyHC, R92W-TnT)/littermate-control hearts at 24 weeks of age, and in myectomy tissue of patients with obstructive HCM/control hearts (GSE36961, GSE36946). Additionally, we examined myocyte redox, cardiac mitochondrial DNA copy number (mtDNA-CN), mt-respiration, mt-ROS generation/scavenging and mt-Ca2+ handling in mice. We identified distinct allele-specific gene expression in mouse-HCM, and marked differences between mouse-HCM and human-HCM. Only two genes (CASQ1, GPT1) were similarly dysregulated in both mutant mice and human-HCM. No signaling pathway or transcription factor was predicted to be similarly dysregulated (by Ingenuity Pathway Analysis) in both mutant mice and human-HCM. Losartan was a predicted therapy only in TnT-mutant mice. KEGG pathway analysis revealed enrichment for several metabolic pathways, but only pyruvate metabolism was enriched in both mutant mice and human-HCM. Both mutant mouse myocytes demonstrated evidence of an oxidized redox environment. Mitochondrial complex I RCR was lower in both mutant mice compared to controls. MyHC-mutant mice had similar mtDNA-CN and mt-Ca2+ handling, but TnT-mutant mice exhibited lower mtDNA-CN and impaired mt-Ca2+ handling, compared to littermate-controls. Molecular profiling reveals differences in gene expression, transcriptional regulation, intracellular signaling and mt-number/function in 2 mouse models at established disease stage. Further studies are needed to confirm differences in gene expression between mouse and human-HCM, and to examine whether cardiac phenotype, genotype and/or species differences underlie the divergence in molecular profiles.