Transaortic Constriction Research Articles

Abstract P413: Stretch-induced Caveolae-mediated Disruption Of Cyclic AMP Microdomains Leads To Arrhythmogenic Ca 2+ Mishandling In Mouse Atrial Myocytes

Background: Atrial fibrillation (AF) often occurs during hypertension and is associated with an increase in cardiomyocyte stretch. Mechanism of ectopic beats, that trigger AF, has been linked to Ca 2+ mishandling and leaky hyperphosphorylated ryanodine receptors (RyRs), while the underlying mechanisms remain elusive. Caveolae membrane structures are involved in cell mechanosensing processes and control the cAMP signaling pathway. We hypothesized that mechanical stretch disrupts caveolae, promoting cAMP production and sarcoplasmic reticulum Ca 2+ leak via augmentation of RyRs phosphorylation. Methods and Results: Cell size analysis and Ca 2+ dynamics measurements were performed by confocal imaging of isolated mouse atrial myocytes. Cell stretch was modeled by hypoosmotic swelling (from 310 mOsM to 220 mOsM to flatten caveolae structures) resulting in a ~30% increase in cell width (p<0.05) with no changes in cell length. Swelling resulted in a biphasic effect on Ca 2+ spark activity: a fast (<10 min of exposure) ~50% increase (p<0.001) followed by a slow decrease to the level observed in isotonic conditions (>30 min of exposure). Similarly, caveolae disruption via cholesterol depletion by 10 mM methyl-β-cyclodextrin (MβCD) led to 2-fold increase in Ca 2+ sparks frequency (p<0.001). Swelling- and MβCD-induced increases in atrial Ca 2+ spark activity were prevented via inhibition of cAMP production by adenylyl cyclases by 0.1mM SQ22536 or cAMP-dependent protein kinase A (PKA) by 1μM H-89. Then, we tested if this mechanism is present in atrial myocytes from pressure-overloaded (4-weeks transaortic constriction, TAC) mice. Atrial myocytes from TAC mice showed a 1.6 times higher Ca 2+ sparks frequency than wild-type myocytes (p<0.01), which was significantly reduced (p<0.01) to wild-type level after incubation with SQ22536. Conclusions: Our findings suggest that cell stretch increases spontaneous Ca 2+ spark activity through the disruption of caveolae and cAMP-mediated augmentation of PKA activity. This mechanism could be involved in the Ca 2+ mishandling and AF in pressure overloaded hearts.

Abstract P326: Nicotinamide Riboside Kinase-2 Deficiency Promotes Pressure Overload- Induced Dilated Cardiomyopathy And Heart Failure

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with a poor prognosis. Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin-binding protein, is predominantly expressed in skeletal muscle and upregulates in several heart failure models. Emerging pieces of evidence suggest that NRK-2 plays a key role in cardiac pathogenesis, however, its role in chronic pressure overload (PO)-induced cardiac remodeling is largely unknown. To investigate the potential role of NRK-2 in PO-induced DCM and delineate the underlying molecular mechanisms, NRK2 knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant LV chamber dilatation [LVIDd(mm); 5.32±0.36 vs. 4.75±0.39, P =0.001] and functional deterioration [LVEF(%); 20.53±8.81vs. 31.2±7.0, P =0.003] during the maladaptive cardiac remodeling phase (6 week). Consistently, NRK2 KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes ANP and BNP expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing AC16 cardiomyocytes displayed significantly attenuated fetal genes expression and, NRK-2 further suppressed the fetal gene expression when challenged with angiotensin II. Consistently, NRK-2 overexpression attenuated angiotensin II-induced cardiomyocyte death. Mechanistically, NRK-2 was identified as a critical regulator of JNK MAP kinase and NRK-2 overexpression markedly suppressed the angiotensin II-induced JNK activation. Overall, our results demonstrate that NRK-2 regulates dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.

B-011-05 PANORAMIC OPTICAL MAPPING AND MICRO COMPUTED TOMOGRAPHY FOR IN-DEPTH 3D CHARACTERIZATION OF THE ARRHYTHMOGENIC SUBSTRATE IN MURINE HEARTS

Optical mapping of murine hearts is currently limited to the epicardium. Action potential duration (APD) heterogeneity is a key driver for initiation and persistence of reentry. When combined with micro CT, optical mapping can provide data on anatomical alterations and structural remodeling contributing to arrhythmogenesis. In this study, we developed a 3D visualization tool combining panoramic optical mapping (POM) with a convolutional neural network automatically segmenting the heart anatomy on micro CT for improved computational modeling and arrhythmia risk stratification in mice ex vivo. We performed high resolution POM with 4 simultaneous CMOS cameras on 17 Langendorff murine hearts: 7 control, 4 transaortic constriction (TAC), 5 transgenic (TG) SCN5A mutation at lidocaine binding site F1759A with spontaneous VF and 1 post LAD ligation. Activation and APD maps were projected onto 3D reconstructed cardiac silhouettes. We measured APD dispersion and the % of ventricular tissue exhibiting dispersion > 10 ms was analyzed. We then coregistered and overlaid the functional maps onto the reconstructed structural meshes obtained with the deep learning algorithm. The mean % of ventricular tissue with APD dispersion > 10 ms was significantly different (ANOVA, p<0.0001) across control, TAC, TG and LAD mice (0.6 ± 0.4 %, 4.8 ± 0.5 %, 12.9 ± 1.4 % and 13.9 %). Myocardial thickness on the TAC micro CT depicted LV hypertrophy, correlating with increased dispersion on POM. POM merged with micro CT allows us to visualize how anatomical markers correlate with arrhythmia mechanisms and thus offers improved understanding of disease progression.

B-PO04-006 CARDIAC SPECIFIC TSPO GENE SILENCING IMPROVES ELECTRICAL REMODELING AND POST-ISCHEMIC ARRHYTHMIAS IN HYPERTROPHIED HEARTS

Mice with cardiac specific conditional knockout of TSPO were found to be protected against mechanical dysfunction in a murine model of left ventricular hypertrophy (LVH) secondary to transaortic constriction (TAC). The role of TSPO in LVH related electrophysiological (EP) remodeling and arrhythmia remains unknown. We sought to determine the impact of cardiotropic AAV-mediated gene silencing of TSPO following onset of LVH as a therapeutic tool for reversing pre-established pathological remodeling and arrhythmia propensity. 8-wk old male mice underwent TAC and were monitored weekly by echocardiography. 3 wks following TAC, mice were randomized to receive AAV mediated gene transfer of short hairpin shRNA against TSPO (shTSPO, N=6, 5E10vg) or control (Ctrl, N =7). 6-wks following TAC, mice were sacrificed for detailed EP measurements using optical action potential mapping. Myocyte TSPO knockdown (35%, p=0.03) using shTSPO failed to reverse TAC-induced decrease in fractional shortening. shTSPO, however, prevented the increase in HW/BW ratio caused by TAC and significantly improved EP properties by blunting rate-dependent myocardial conduction slowing relative to Ctrl (A). Moreover, shTSPO suppressed post-ischemic episodes of VF in TAC hearts (0/4 shTSPO vs 2/5 Ctrl) likely by preventing APD overshoot during reperfusion (B). TAC-related VF in Ctrl but not shTSPO hearts was supported by focal activation and intermittent meandering rotors. Our findings reveal a potential role for myocardial TSPO gene silencing following onset of remodeling in improving EP properties and suppressing post-ischemic arrhythmias in LVH.

Constitutive protein kinase G activation exacerbates stress-induced cardiomyopathy.

Heart failure is associated with high morbidity and mortality, and new therapeutic targets are needed. Preclinical data suggest that pharmacological activation of protein kinase G (PKG) can reduce maladaptive ventricular remodelling and cardiac dysfunction in the stressed heart. However, clinical trial results have been mixed and the effects of long-term PKG activation in the heart are unknown. We characterized the cardiac phenotype of mice carrying a heterozygous knock-in mutation of PKG1 (Prkg1R177Q/+ ), which causes constitutive, cGMP-independent activation of the kinase. We examined isolated cardiac myocytes and intact mice, the latter after stress induced by surgical transaortic constriction or angiotensin II (Ang II) infusion. Cardiac myocytes from Prkg1R177Q/+ mice showed altered phosphorylation of sarcomeric proteins and reduced contractility in response to electrical stimulation, compared to cells from wild type mice. Under basal conditions, young PKG1R177Q/+ mice exhibited no obvious cardiac abnormalities, but aging animals developed mild increases in cardiac fibrosis. In response to angiotensin II infusion or fixed pressure overload induced by transaortic constriction, young PKGR177Q/+ mice exhibited excessive hypertrophic remodelling with increased fibrosis and myocyte apoptosis, leading to increased left ventricular dilation and dysfunction compared to wild type litter mates. Long-term PKG1 activation in mice may be harmful to the heart, especially in the presence of pressure overload and neurohumoral stress. This article is part of a themed issue on cGMP Signalling in Cell Growth and Survival. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.11/issuetoc.

Golgi‐resident β1‐adrenergic Receptor Signaling to Cardiac Hypertrophy in Cardiac Myocytes in Vitro and in Failing Hearts in Vivo

Pathological cardiac hypertrophy contributes to a higher risk of developing cardiac dysfunction, such as heart failure, one of the major causes of morbidity or mortality in the western world. Chronic stimulation of Gs-coupled β1-adrenergic receptor (β1-AR) signaling by catecholamines during long-term pathological stress induces cardiac hypertrophy and ultimately heart failure. However, the mechanism of action remains incompletely understood. GPCRs at different intracellular compartments are rapidly emerging as novel mediators of distinct local signaling outputs. We have demonstrated activation of Golgi-β1-ARs generates a local pool of cAMP which, in turn, stimulates Golgi-localized phosphatidylinositol 4-phosphate (PI4P) hydrolysis via EPAC dependent PLCε activation, leading to cardiac hypertrophy in a cellular model. This novel prohypertrophic, phosphoinositide hydrolysis pathway is not accessed by plasma membrane β1-AR stimulation. Remarkably, specific inhibition of activation of these preexisting Golgi-resident β1-ARs prevents hypertrophy induced by a physiological neurotransmitter norepinephrine (NE) in cardiac myocytes in vitro. These data defined significant and unique roles of Golgi-β1-ARs in regulating downstream hypertrophic signaling in a biologically relevant cellular model. To determine if Golgi-β1-ARs play an important role in a whole animal model of pathological cardiac hypertrophy, we developed a Golgi targeted construct, eNos-mApple-Nb80 to deliver the β1-AR inhibitor, Nb80, specifically to the Golgi apparatus without interrupting surface β1-ARs. To deliver our target genes into animal heart tissue, I adopted recombinant adeno-associated virus 9 (rAAV9) gene delivery system. We confirmed selective localization to the Golgi apparatus of cardiac myocytes and equal expression of eNos-mApple-Nb80 and eNos-mApple (negative control without Nb80) in mouse hearts. Additionally, activation of the prohypertrophic Golgi PI4P hydrolysis pathway by dobutamine was inhibited in cardiac myocytes isolated from animals expressing eNos-mApple-Nb80. To evaluate the effect of inhibition of Golgi-localized β1-ARs in a mouse model of pressure overload-induced cardiac hypertrophy, we will perform transaortic constriction (TAC) on eNos-mApple-Nb80 expressing mice as well as eNos-mApple and GFP controls. After 6-8 weeks animals will be assessed by echocardiography to measure ejection fraction, followed by sacrifice of the mice and evaluation of heart size, heart weight to tibia length, myocyte area, and interstitial fibrosis. Heart tissues will also be analyzed by western blotting and mRNA analysis for activation of key signal transduction pathways and expression of hypertrophic genes. This will help us elucidate the role of Golgi β1-ARs in cardiac hypertrophy, informing new strategies for the development of β-blocker therapies for heart failure.

Pressure Overload-Mediated Sustained PKR2 (Prokineticin-2 Receptor) Signaling in Cardiomyocytes Contributes to Cardiac Hypertrophy and Endotheliopathies.

[Figure: see text].

Interleukin-17 upregulation participates in the pathogenesis of heart failure in mice via NF-κB-dependent suppression of SERCA2a and Cav1.2 expression.

Interleukin-17 (IL-17), also called IL-17A, is an important regulator of cardiac diseases, but its role in calcium-related cardiac dysfunction remains to be explored. Thus, we investigated the influence of IL-17 on calcium handling process and its contribution to the development of heart failure. Mice were subjected to transaortic constriction (TAC) to induce heart failure. In these mice, the levels of IL-17 in the plasma and cardiac tissue were significantly increased compared with the sham group. In 77 heart failure patients, the plasma level of IL-17 was significantly higher than 49 non-failing subjects, and was negatively correlated with cardiac ejection fraction and fractional shortening. In IL-17 knockout mice, the shortening of isolated ventricular myocytes was increased compared with that in wild-type mice, which was accompanied by significantly increased amplitude of calcium transient and the upregulation of SERCA2a and Cav1.2. In cultured neonatal cardiac myocytes, treatment of with IL-17 (0.1, 1 ng/mL) concentration-dependently suppressed the amplitude of calcium transient and reduced the expression of SERCA2a and Cav1.2. Furthermore, IL-17 treatment increased the expression of the NF-κB subunits p50 and p65, whereas knockdown of p50 reversed the inhibitory effects of IL-17 on SERCA2a and Cav1.2 expression. In mice with TAC-induced mouse heart, IL-17 knockout restored the expression of SERCA2a and Cav1.2, increased the amplitude of calcium transient and cell shortening, and in turn improved cardiac function. In addition, IL-17 knockout attenuated cardiac hypertrophy with inhibition of calcium-related signaling pathway. In conclusion, upregulation of IL-17 impairs cardiac function through NF-κB-mediated disturbance of calcium handling and cardiac remodeling. Inhibition of IL-17 represents a potential therapeutic strategy for the treatment of heart failure.

Exchange proteins directly activated by cAMP mediate cardiac repolarization and arrhythmogenesis during chronic heart failure.

Most sudden cardiac death in chronic heart failure (CHF) is caused by malignant ventricular arrhythmia (VA); however, the molecular mechanism remains unclear. This study aims to explore the effect of exchange proteins directly activated by cAMP (Epac) on VA in CHF and the potential molecular mechanism. Transaortic constriction was performed to prepare CHF guinea pigs. Epac activation model was obtained with 8-pCPT administration. Programmed electrical stimulation (PES) was performed to detect effective refractory period (ERP) or induce VA. Isolated adult cardiomyocytes were treated with 8-pCPT and (or) the Epac inhibitor. Cellular electrophysiology was examined by whole-cell patch clamp. With Epac activation, corrected QT duration was lengthened by 12.6%. The 8-pCPT increased action potential duration (APD) (APD50: 236.9 ± 18.07 ms vs. 328.8 ± 11.27 ms, p < 0.05; APD90: 264.6 ± 18.22 ms vs. 388.6 ± 6.47 ms, p < 0.05) and decreased rapid delayed rectifier potassium (IKr) current (tail current density: 1.1 ± 0.08 pA/pF vs. 0.7 ± 0.03 pA/pF, p < 0.05). PES induced more malignant arrhythmias in the 8-pCPT group than in the control group (3/4 vs. 0/8, p < 0.05). The selective Epac1 inhibitor CE3F4 rescued the drop in IKr after 8-pCPT stimulation (tail current density: 0.5 ± 0.02 pA/pF vs. 0.6 ± 0.03 pA/pF, p < 0.05). In conclusion, Epac1 regulates IKr, APD, and ERP in guinea pigs, which could contribute to the proarrhythmic effect of Epac1 in CHF.

Cardiac contractility modulation attenuates structural and electrical remodeling in a chronic heart failure rabbit model.

BackgroundCardiac contractility modulation (CCM) is non-excitatory electrical stimulation for improving cardiac function. This study aimed to evaluate the effects of CCM on structural and electrical remodeling in a rabbit model of chronic heart failure (CHF).MethodsThirty rabbits were randomly divided into the sham, CHF, and CCM groups. The CHF model was induced 12 weeks after trans-aortic constriction by pressure unloading and CCM was delivered to the myocardium for 4 weeks. Corrected QT intervals, the ventricular effective refractory period, and inducibility of ventricular tachycardia were measured by an electrophysiological examination. Connective tissue growth factor, galectin-3, Kv4.3, KCNQ1, KCNH2, and connexin 43 protein levels were measured by western blotting.ResultsThe CHF group had a significantly prolonged corrected QT interval and ventricular effective refractory period, and increased inducibility of ventricular tachycardia. Prominent myocardial fibrosis and increased hydroxyproline content were observed in the CHF group, but these were suppressed in the CCM group. Kv4.3, KCNQ1, KCNH2, and connexin 43 protein levels were significantly lower in the CHF group, but treatment with CCM partially restored their levels.ConclusionsCCM attenuates myocardial structural and electrical remodeling during CHF. These findings provide evidence for clinical use of CCM in treating CHF.

Exploring biomarkers and therapeutic targets for pressure overload induced heart failure based on microarray data.

Heart failure (HF) is an end stage heart condition with poor prognosis which brings about tremendous social medical cost. Along decades, mechanism and treatments of HF have been under restless research. In the present study, we first established pressure overload induced HF model using transaortic arch constriction (TAC) method in mice. The global expression profiles of long noncoding RNA (lncRNA), microRNA (miRNA) and messenger RNA (mRNA) were obtained by microarray probes, which were further confirmed by quantitative PCR (qPCR). Bioinformatics analysis was performed using multiple methods including volcano plotting, heatmapping and hierarchical clustering, Gene Ontology (GO) and pathway enrichment analysis, and competing endogenous RNA (ceRNA) regulatory network construction. Totally, 1,139 differentially expressed mRNAs (DEmRNAs), 3,830 lncRNAs (DElncRNAs) and 13 miRNAs (DEmiRNAs) were identified in HF group compared to control group, which could distinctly differentiate HF from normal control and were potential candidate biomarkers for HF. GO and pathway enrichment analysis revealed that multiple significant biological processes and pathways were involved in HF pathogenesis, such as extracellular matrix structural constituent, proteinaceous extracellular matrix, positive regulation of apoptotic process and integrin signaling pathway. Nine DElncRNAs, 3 DEmiRNAs and 25 DEmRNAs were filtrated out to construct a ceRNA network, which visually displayed their regulatory roles with therapeutic target potential. The present study identified differentially expressed RNAs that might be involved in the pathogenesis and progression of HF. The outcomes shed lights into the underlying mechanisms for HF and provided candidate biomarkers and intervention targets for further research.

CaMKIIδC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy.

CaMKII (Ca2+-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca2+ handling and CaMKII activation in hypertrophy and HF. To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca2+ content, Ca2+ transient amplitude, and [Ca2+] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca2+] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca2+-CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Extreme Acetylation of the Cardiac Mitochondrial Proteome Does Not Promote Heart Failure.

Circumstantial evidence links the development of heart failure to posttranslational modifications of mitochondrial proteins, including lysine acetylation (Kac). Nonetheless, direct evidence that Kac compromises mitochondrial performance remains sparse. This study sought to explore the premise that mitochondrial Kac contributes to heart failure by disrupting oxidative metabolism. A DKO (dual knockout) mouse line with deficiencies in CrAT (carnitine acetyltransferase) and Sirt3 (sirtuin 3)-enzymes that oppose Kac by buffering the acetyl group pool and catalyzing lysine deacetylation, respectively-was developed to model extreme mitochondrial Kac in cardiac muscle, as confirmed by quantitative acetyl-proteomics. The resulting impact on mitochondrial bioenergetics was evaluated using a respiratory diagnostics platform that permits comprehensive assessment of mitochondrial function and energy transduction. Susceptibility of DKO mice to heart failure was investigated using transaortic constriction as a model of cardiac pressure overload. The mitochondrial acetyl-lysine landscape of DKO hearts was elevated well beyond that observed in response to pressure overload or Sirt3 deficiency alone. Relative changes in the abundance of specific acetylated lysine peptides measured in DKO versus Sirt3 KO hearts were strongly correlated. A proteomics comparison across multiple settings of hyperacetylation revealed ≈86% overlap between the populations of Kac peptides affected by the DKO manipulation as compared with experimental heart failure. Despite the severity of cardiac Kac in DKO mice relative to other conditions, deep phenotyping of mitochondrial function revealed a surprisingly normal bioenergetics profile. Thus, of the >120 mitochondrial energy fluxes evaluated, including substrate-specific dehydrogenase activities, respiratory responses, redox charge, mitochondrial membrane potential, and electron leak, we found minimal evidence of oxidative insufficiencies. Similarly, DKO hearts were not more vulnerable to dysfunction caused by transaortic constriction-induced pressure overload. The findings challenge the premise that hyperacetylation per se threatens metabolic resilience in the myocardium by causing broad-ranging disruption to mitochondrial oxidative machinery.

Pyrroloquinoline quinone can prevent chronic heart failure by regulating mitochondrial function.

Myocardial mitochondrial dysfunction is the leading cause of chronic heart failure (CHF). Increased reactive oxygen species (ROS) levels, disruption of mitochondrial biogenesis and mitochondrial Ca2+([Ca2+]m) homeostasis and reduction of the mitochondrial membrane potential (ΔΨm) cause myocardial mitochondrial dysfunction. Therefore, treating CHF by targeting mitochondrial function is a focus of current research. For the first time, this study investigated the effects of the strong antioxidant pyrroloquinoline quinone (PQQ) on mitochondrial function in a cardiac pressure overload model, and the mechanism by which PQQ regulates [Ca2+]m homeostasis was explored in depth. After transaortic constriction (TAC), normal saline and PQQ (0.4, 2 and 10 mg/kg) were administered intragastrically to Sprague Dawley (SD) rats for 12 weeks. In vitro, neonatal rat left ventricle myocytes (NRVMs) were pretreated with 200 nm angiotensin II (Ang II) with or without PQQ (1, 10 and 100 μM). Rat heart remodelling was verified by assessment of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) levels (qRT-PCR), cell surface area (wheat germ agglutinin (WGA) staining in vivo and α-actin in vitro) and echocardiography. Myocardial mitochondrial morphology was assessed by transmission electron microscopy. Western blotting was used to assess mitochondrial biogenesis [peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and transcription factor A, mitochondrial (TFAM)]. The ΔΨm was determined by tetraethyl benzimidazolyl carbocyanine iodide (JC-1) staining and flow cytometry, and ROS levels were measured by dichloro-dihydro-fluorescein diacetate (DCFH-DA) and MitoSOX Red staining. [Ca2+]m was measured by isolating rat mitochondria, and mitochondrial Ca2+ channel proteins [the mitochondrial Na+/Ca2+ exchanger (NCLX) and mitochondrial Ca2+ uniporter (MCU)] were detected by Western blot. In vivo and in vitro, PQQ pretreatment improved pressure overload-induced cardiac remodelling and cell hypertrophy, thus preventing the occurrence of CHF. PQQ also prevented mitochondrial morphology damage and reduced the PGC-1α and TFAM downregulation caused by TAC or Ang II. In addition, in NRVMs treated with Ang II + PQQ, PQQ regulated ROS levels and increased the ΔΨm. PQQ also regulated [Ca2+]m homeostasis and prohibited [Ca2+]m overloading by increasing NCLX expression. These results show that PQQ can prevent [Ca2+]m overload by increasing NCLX expression and thereby reducing ROS production and protecting the ΔΨm. At the same time, PQQ can increase PGC-1α and TFAM expression to regulate mitochondrial biogenesis. These factors can prevent mitochondrial dysfunction, thereby reducing cardiac damage caused by pressure overload and preventing the occurrence of CHF.

Sacubitril/Valsartan Improves Left Ventricular Function in Chronic Pressure Overload Independent of Intact Cyclic Guanosine Monophosphate-dependent Protein Kinase I Alpha Signaling

BackgroundCombined angiotensin receptor/neprilysin inhibition with sacubitril/valsartan (Sac/Val) has emerged as a therapy for heart failure. The presumed mechanism of benefit is through prevention of natriuretic peptide degradation, leading to increased cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) signaling. However, the specific requirement of PKG for Sac/Val effects remains untested. Methods and ResultsWe examined Sac/Val treatment in mice with mutation of the cGMP-dependent protein kinase I (PKGI)α leucine zipper domain, which is required for cGMP-PKGIα antiremodeling actions in vivo. Wild-type (WT) or PKG leucine zipper mutant (LZM) mice were exposed to 56-day left ventricular (LV) pressure overload by moderate (26G) transaortic constriction (TAC). At day 14 after TAC, mice were randomized to vehicle or Sac/Val by oral gavage. TAC induced the same degree of LV pressure overload in WT and LZM mice, which was not affected by Sac/Val. Although LZM mice, but not WT, developed LV dilation after TAC, Sac/Val improved cardiac hypertrophy and LV fractional shortening to the same degree in both the WT and LZM TAC mice. ConclusionThese findings indicate the beneficial effects of Sac/Val on LV structure and function in moderate pressure overload. The unexpected finding that PKGIα mutation does not abolish the Sac/Val effects on cardiac hypertrophy and on LV function suggests that signaling other than natriuretic peptide– cGMP–PKG mediates the therapeutic benefits of neprilysin inhibition in heart failure.

Protein arginine methyltransferase 6 mediates cardiac hypertrophy by differential regulation of histone H3 arginine methylation

Heart failure remains a major cause of hospitalization and death worldwide. Heart failure can be caused by abnormalities in the epigenome resulting from dysregulation of histone-modifying enzymes. While chromatin enzymes catalyzing lysine acetylation and methylation of histones have been the topic of many investigations, the role of arginine methyltransferases has been overlooked. In an effort to understand regulatory mechanisms implicated in cardiac hypertrophy and heart failure, we assessed the expression of protein arginine methyltransferases (PRMTs) in the left ventricle of failing human hearts and control hearts. Our results show a significant up-regulation of protein arginine methyltransferase 6 (PRMT6) in failing human hearts compared to control hearts, which also occurs in the early phase of cardiac hypertrophy in mouse hearts subjected to pressure overload hypertrophy induced by trans-aortic constriction (TAC), and in neonatal rat ventricular myocytes (NRVM) stimulated with the hypertrophic agonist phenylephrine (PE). These changes are associated with a significant increase in arginine 2 asymmetric methylation of histone H3 (H3R2Me2a) and reduced lysine 4 tri-methylation of H3 (H3K4Me3) observed both in NRVM and in vivo. Importantly, forced expression of PRMT6 in NRVM enhances the expression of the hypertrophic marker, atrial natriuretic peptide (ANP). Conversely, specific silencing of PRMT6 reduces ANP protein expression and cell size, indicating that PRMT6 is critical for the PE-mediated hypertrophic response. Silencing of PRMT6 reduces H3R2Me2a, a mark normally associated with transcriptional repression. Furthermore, evaluation of cardiac contractility and global ion channel activity in live NRVM shows a striking reduction of spontaneous beating rates and prolongation of extra-cellular field potentials in cells expressing low-level PRMT6. Altogether, our results indicate that PRMT6 is a critical regulator of cardiac hypertrophy, implicating H3R2Me2a as an important histone modification. This study identifies PRMT6 as a new epigenetic regulator and suggests a new point of control in chromatin to inhibit pathological cardiac remodeling.

Molecular Basis of Takotsubo Syndrome

IntroductionTakotsubo syndrome (TTS), also known as the “Broken Heart Syndrome” or “Apical Ballooning Syndrome is defined by its characteristic anomaly: when the heart contracts during systole, the apex of the heart dilates as the base of the heart contracts. Severe TTS can lead to cardiogenic shock and death in 3–4% of patients. There is no standard medical therapy for TTS because the mechanism underlying the development of the syndrome is unknown. Our goal is to determine the molecular mechanisms of TTS as a first step towards better treatment plans and outcomes.MethodsOur model of TTS is a Kv1.5 null mouse, with compromised coronary metabolic dilation, subjected to transaortic constriction (TAC). Two weeks after TAC, when Kv1.5 null mouse showed profound apical ballooning during ventricular contraction (echocardiography), myocardial blood flow (MBF) was measured by contrast echocardiography in the base and apex of the left ventricle of mice under control conditions and during acute administration of norepinephrine to increase cardiac work. Hearts were collected and gene expression (RNA deep sequencing) in both the apex and the base were performed and followed by bioinformatic analysis. Wild type (WT) and unstressed Kv1.5 null mice were used as controls. To increase the scientific rigor, we purposefully analyzed RNA expression from different animals with Real‐Time‐PCR, to confirm the sequencing data.ResultsA total of 3875 genes were identified by differentially expressed between TTS vs unstressed Kv1.5 null hearts. Gene Set Enrichment Analysis shows many families of genes downregulated (metabolism) and upregulated (inflammation, hypoxia, apoptosis) in the apical (ballooning) area of the heart in TTS (compared to the base of the heart). RT‐qPCR revealed significant upregulation of the genes for Postn, Lox, and C920009B18Rik (p&lt;0.01) and down‐regulation of the genes Ucp3, Acaa2, Pfkb1, Mir133a‐2 (p&lt;0.05). These significant changes in expression were also shown in the apex of TTS hearts as compared to the apex of control Kv1.5 null hearts (p&lt;0.01). We also found that in the mice with TTS, MBF was lower in the apex than in the base.ConclusionOur results suggest that the apex of the heart in TTS is receiving insufficient perfusion compared to the base, which may be one of the root problems in TTS. There is significant upregulation of structural genes in the apex of TTS hearts versus their bases and the apexes of normal hearts, whereas there is a significant downregulation of genes playing roles in metabolism. This confirms the importance of these specific genes in the development of the anomaly shown in TTS.

Cardiomyocyte‐generated Monocyte Chemoattractant Protein 1 Contributes to Cardiac Inflammation During Adverse Remodeling in Pressure Overload

Attempts to normalize cardiac function in response to stress often lead to cardiovascular diseases associated with adverse remodeling. Cardiac inflammation is a hallmark feature of adverse remodeling. Although it is established that ischemic injury induces cardiac inflammation through release of substances from dying cells, mechanisms for inflammation in the context of nonischemic injury have been elusive. Our lab has previously shown that the cardiomyocyte is the initial site of transcription of pro‐inflammatory cytokines and chemokines such as MCP‐1, IL‐6, Il‐1β and Il‐18, in response to pressure overload and that this occurs in the absence of cell death. In addition, the NLRP3 inflammasome is activated in cardiomyocytes resulting in activation of caspase‐1 and generation of active forms of Il‐1β and IL‐18. Pharmacological blockade of NFκ‐β, MCP‐1 signaling, or NLRP3 inflammasome activation resulted in decreases in cardiac macrophage accumulation, fibrosis, and dysfunction. These results suggest that cardiomyocyte‐generated inflammatory mediators play a key role in adverse cardiac remodeling in response to pressure overload. However, the extent to which cardiomyocytes contribute to immune cell recruitment and remodeling through MCP‐1 and inflammasome generated Il‐1β and IL‐18 remains elusive.To address this question, we made cardiomyocyte‐specific MCP‐1 knockout (MCP1) mice and are currently breeding cardiac specific NLRP‐3 knockout mice. MCP1 and wildtype control (Ctl) mice were subjected to transaortic constriction (TAC) or sham surgery. Blood was collected from the mice at various timepoints and analyzed for serum amylyloid A (SAA) as a marker of systemic inflammation. At the conclusion of 3 or14 days post‐surgery, whole hearts were collected for analysis of gene expression and NLRP3 priming using qPCR. Caspase‐1 activity was used to measure NLRP3 inflammasome activation.Pro‐inflammatory cytokines were upregulated in TAC mice versus sham controls. Inflammasome priming and activation, as measured by IL‐1β and IL‐18 gene expression and caspase‐1 activity respectively, were unaffected by cardiomyocyte‐specific MCP‐1 deletion at 3 days post‐surgery. However, both IL‐1β and IL‐18 gene expression were reduced at 14 days post‐surgery in MCP1 TAC mice compared to Ctl TAC. This coincided with a reduction of CD68+ macrophage accumulation in the myocardium suggesting that infiltrating macrophages, recruited through cardiomyocyte‐generated MCP‐1, contribute to inflammatory gene expression at this time. We are currently assessing inflammasome activation in cardiomyocytes and macrophages following TAC in Ctl and MCP1 mice. Parallel experiments using cardiomyocyte NLRP‐3 KO mice will reveal the importance of inflammasome products made in the myocyte compartment. In other studies, we are assessing the contribution of cardiomyocyte‐generated cytokines and chemokines to systemic inflammation by measuring serum amyloid A. MCP1 TAC animals displayed a reduction in SAA compared to Ctl TAC animals at 14 days post‐surgery suggesting that cardiomyocyte‐derived MCP‐1 can contribute to persistent inflammation in pressure overloaded murine hearts.Support or Funding InformationR01HL145459‐01 (JHB, SM)T32HL007444‐37 (RSR, JC)

Changes in gene expression caused by genetic elimination of high molecular weight FGF2 are associated with prevention of stress‐induced cardiac systolic dysfunction

Fibroblast growth factor 2 (FGF2) is produced as high (&gt;20 kDa, Hi‐) and low molecular weight (18 kDa, Lo) isoforms in the heart. While administered Lo‐FGF2 has been established as a cardioprotective agent in multiple models of cardiac injury, there is limited information about the role of Hi‐FGF2 in the heart. To investigate the effect of endogenous Hi‐FGF2 we compared cardiac transcriptome (35240 targets, GeneChip™ Mouse Gene 2.0 ST Array (Affymetrix, 902119)) and systolic function between Hi‐FGF2 knock‐out mice, FGF2(Lo), expressing only Lo‐FGF2, and wild type mice, FGF2(WT), expressing both Hi‐FGF2 and Lo‐FGF2, under ‘normal’ (sham‐operated) and “stress” (pressure overload) conditions. Transaortic constriction surgery (TAC) was used to induce pressure overload. Echocardiography was done at baseline and at 4–8 weeks post‐surgery. Microarray analysis showed that under normal conditions, the absence of Hi‐FGF2 promoted changes in relative levels of 118 transcripts, including those associated with circadian rhythm regulation and heat shock protein (HSP70)‐associated apoptosis regulation. Comparative analysis of TAC surgery‐induced gene expression changes showed that 275 transcripts were differentially affected by the presence or absence of Hi‐FGF2 expression, most prominently the orphan nuclear receptor NR1D1 which is linked to regulation of circadian rhythm and metabolism. Under non‐stress conditions, systolic function was unaffected by endogenous Hi‐FGF2 expression. Pressure overload stress caused a decline in systolic function in the presence of endogenous Hi‐FGF2, at 4–8 weeks post‐TAC surgery, accompanied by increases in markers of myocardial stress/damage including B‐type natriuretic peptide (BNP) and the pro‐cell death protein BCL2/adenovirus E1B 19 kDa protein‐interacting protein‐3, Bnip3. In the absence of endogenous Hi‐FGF2, mice were protected from stress‐induced loss of systolic function and increases in BNP and Bnip3. TAC surgery induced Hi‐FGF2‐independent increases in: cardiac mass (heart weight/tibia length); cardiac fibrosis; transcripts linked to exracellular matrix remodeling. Increased cardiomyocyte size, however, was only observed in FGF2(WT) but not FGF2(Lo) hearts post‐TAC. It is suggested that elimination of endogenous Hi‐FGF2 elicits cardioprotection by increasing cardiac HSP70 (pre‐TAC surgery); and NR1D1, after TAC surgery.Support or Funding InformationFunding (EK, PAC, DJ) was provided by the Canadian Institutes for Health Research (FRN‐74733) and the Molson Women’s Heart Health Foundation (EK). MPC was supported by a CIHR Open Operating Grant (MOP136862). NK and RSN were the recipients of a Bank of Montreal studentship award via the St. Boniface Hospital Albrechtsen Research Centre and University of Manitoba funding to PAC.

Fibroblast growth factor-inducible 14 mediates macrophage infiltration in heart to promote pressure overload-induced cardiac dysfunction

AimsHeart failure (HF) is characterized by compromised cardiac structure and function. Previous work has identified a link between upregulation of pro-inflammatory cytokines and HF. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a pro-inflammatory cytokine, which binds to fibroblast growth factor inducible 14 (Fn14), a ubiquitously expressed cell-surface receptor. The objective of this study was to investigate the role of TWEAK/Fn14 pathway in promoting cardiac inflammation under non ischemic stress conditions. Main methodsWild type (WT) and Fn14 knock out (Fn14−/−) mice were subjected to pressure overload [transaortic constriction (TAC)] for 1 or 6 weeks. A subset of WT TAC animals were treated with the Fn14 antagonist L524-0366. Cardiac function was measured by echocardiography. Cardiac fibrosis and macrophage infiltration were quantified using immunohistochemistry and flow cytometry, respectively. Cardiac fibroblasts were isolated for quantifying TWEAK-induced chemokine release. Key findingsFn14−/− mice displayed improved cardiac function, reduced fibrosis and lower macrophage infiltration in heart compared to WT following TAC. L524-0366 mitigated maladaptive remodeling with TAC. TWEAK induced secretion of the pro-inflammatory chemokine, monocyte chemoattractant protein 1 from WT but not Fn14−/− fibroblasts in vitro, in part through activation of non-canonical NF-κB signaling. Finally, Fn14 expression was increased in mouse following TAC and in human failing hearts. SignificanceOur findings support an important role for the TWEAK/Fn14 promoting macrophage infiltration and fibrosis in heart under non-ischemic stress, with potential for therapeutic intervention to improve cardiac function in the setting of HF.