Cardiac-specific Transgenic Mice Research Articles

Cardioprotective effects of nicorandil on mitochondrial apoptosis signaling in the failing heart of cardiac-specific Bcl-2-associated athanogene (BAG) 3 knockout mice

Abstract Background Bcl-2-associated athanogene (BAG) 3 is a known regulator of mitochondrial apoptotic cell death and autophagic protein degradation in the heart. Disruption of the BAG3 gene in a cardiac-specific manner can lead to cardiac failure in mice. However, therapeutic approaches for cardiac failure induced by BAG3 gene disruption remain poorly understood. Method and results We characterized mice with the ablated BAG3 gene using the Cre-recombinase-loxp site system in a cardiac-specific manner (BAG3f/f crossbred with cardiac specific Cre transgenic mice; BAG3 cKO) and examined the therapeutic effect of nicorandil, an antianginal drug, in BAG3 cKO mice. Cardiac BAG3 levels were markedly reduced in the cytoplasmic and mitochondrial fractions from the hearts of BAG3 cKO mice compared to BAG3f/f mice. BAG3cKO mice exhibited cardiac disease such as a reduction in fractional shortening detected by echocardiography, cardiac fibrosis at 6 months of age, and approximately 40% premature death by 6 months of age. Electron microscopy analysis confirmed cardiomyocyte loss and mitochondrial abnormalities in some areas of the heart section from BAG3cKO mice at 4.5 months of age. A marked decrease in the level of cytochrome C in the mitochondrial fraction and a slight increase in the cytoplasmic fraction, along with apoptotic cell death, were observed in hearts from BAG3 cKO mice at 4.5 months. Mitochondrial levels of anti-apoptotic factors, such as Bcl-2 and Bcl-xl, were reduced, while there was a marked increase in Bak1, an apoptotic inducible factor, in hearts from BAG3 cKO mice at 4.5 months of age. These results suggest that cardiac mitochondrial impairment and subsequent apoptotic cell death occurred in BAG3cKO mice. Nicorandil treatment (81 mg/L drinking water) initiated at 2 months of age prevented the reduction of fractional shortening, cardiac fibrosis, ultrastructural cardiac abnormalities, and premature death. Furthermore, nicorandil treatment prevented the decrease in mitochondrial cytochrome C and the increase in cytoplasmic cytochrome C in hearts from BAG3 cKO mice at 4.5 months of age. Nicorandil treatment also inhibited the elevation of mitochondrial Bak1 levels, reduction in Bcl-2 levels, and occurrence of apoptotic cell death in hearts from BAG3 cKO mice at 4.5 months of age. Conclusion Long-term treatment with nicorandil can inhibit cardiac disease induced by cardiac- specific BAG3 ablation through mitochondrial protection via the prevention of activation of mitochondrial apoptosis signaling.

Aberrant activation of p53-TRIB3 axis contributes to diabetic myocardial insulin resistance and sulforaphane protection

IntroductionInsulin resistance (IR) is associated with multiple pathological features. Although p53- or TRIB3-orchestrated IR is extensively studied in adipose tissue and liver, the role of p53-TRIB3 axis in myocardial IR remains unknown, and more importantly target-directed therapies of myocardial IR are missing. ObjectivesConsidering the beneficial effects of sulforaphane (SFN) on cardiovascular health, it is of particular interest to explore whether SFN protects against myocardial IR with a focus on the regulatory role of p53-TRIB3 axis. MethodsMouse models including cardiac specific p53-overexpressing transgenic (p53-cTg) mice and Trib3 knockout (Trib3-KO) mice, combined with primary cardiomyocytes treated with p53 activator (nutlin-3a) and inhibitor (pifithrin-α, PFT-α), or transfected with p53-shRNA and Trib3-shRNA, followed by multiple molecular biological methodologies, were used to investigate the role of p53-TRIB3 axis in SFN actions on myocardial IR. ResultsHere, we report that knockdown of p53 rescued cardiac insulin-stimulated AKT phosphorylation, while up-regulation of p53 by nutlin-3a or p53-cTg mice blunted insulin sensitivity in cardiomyocytes under diabetic conditions. Diabetic attenuation of AKT-mediated cardiac insulin signaling was markedly reversed by SFN in p53-Tgfl/fl mice, but not in p53-cTg mice. Importantly, we identified TRIB3 was elevated in p53-cTg diabetic mice, and confirmed the physical interaction between p53 and TRIB3. Trib3-KO diabetic mice displayed improved insulin sensitivity in the heart. More specifically, the AMPKα-triggered CHOP phosphorylation and degradation were essential for p53 on the transcriptional regulation of Trib3. ConclusionOverall, these results indicate that inhibiting the p53-TRIB3 pathway by SFN plays an unsuspected key role in the improvement of myocardial IR, which may be a promising strategy for attenuating diabetic cardiomyopathy (DCM) in diabetic patients.

Ubiquitin-specific protease 38 promotes inflammatory atrial fibrillation induced by pressure overload.

Atrial structural and electrical remodelling is a major reason for the initiation and perpetuation of atrial fibrillation (AF). Ubiquitin-specific protease 38 (USP38) is a deubiquitinating enzyme, but its function in the heart remains unknown. The aim of this study was to investigate the effect of USP38 in pressure overload-induced AF. Cardiac-specific knockout USP38 and cardiac-specific transgenic USP38 mice and their corresponding control mice were used in this study. After 4 weeks with or without aortic banding (AB) surgery, atrial echocardiography, atrial histology, electrophysiological study, and molecular analysis were assessed. Ubiquitin-specific protease 38 knockout mice showed a remarkable improvement in vulnerability to AF, atrial weight and diameter, atrial fibrosis, and calcium-handling protein expression after AB surgery. Conversely, USP38 overexpression further increased susceptibility to AF by exacerbating atrial structural and electrical remodelling. Mechanistically, USP38 interacted with and deubiquitinated nuclear factor-kappa B (NF-κB), and USP38 overexpression increased the level of p-NF-κB in vivo and in vitro, accompanied by the upregulation of NOD-like receptor protein 3 (NLRP3) and inflammatory cytokines, suggesting that USP38 contributes to adverse effects by driving NF-κB/NLRP3-mediated inflammatory responses. Overall, our study indicates that USP38 promotes pressure overload-induced AF through targeting NF-κB/NLRP3-mediated inflammatory responses.

Abstract 17334: Association of BIN1 Deletion With Electrical and Structural Remodeling in a Transgenic BIN1 KO Mouse Model of Heart Failure-Novel Option for Effective Therapy Using VK-II-86

Introduction: Cardiac deletion of Bridging Integrator 1 (BIN1) membrane protein produces an age-related cardiomyopathy in mice. Our hypothesis was to determine whether electrical accompanies this structural remodeling and examine the utility of VK-II-86, a structural analog of carvedilol with minimum β-blocking activity, as a potential antiarrhythmic therapeutic strategy. Methods: A cardiac-specific transgenic BIN1 KO mouse was employed. Structural and electrical cardiac changes were assessed using echocardiography and EKG (Visual Sonics-Vevo Heart Image). Murine ventricular cardiomyocytes were studied using patch-clamp techniques to examine changes in ion channel function and to study the effects of VK-II-86 on the action potential (AP). Results: Loss of BIN1 (KO) significantly reduced Left Ventricular Ejection Fraction (LVEF) and Cardiac Output (CO) when compared to WT mice, with a relative reduction of nearly 20% and 35% in EF and CO, respectively. Heterozygotic mice (HET) also exhibited significant reduction in EF and CO compared to WT mice. Loss of BIN1 (KO and HET) significantly prolonged the QT interval on surface ECG by approximately 15%. This finding correlates with the increase in AP duration (APD) demonstrated in ventricular cardiomyocytes using current clamp. BIN1 deficient hearts displayed significant reduction in expression of I to , I K1 and peak I Na (80%, 40% and 20% respectively) as well as augmented expression of late I Na (50%). This electrical remodeling is likely responsible for the increased expression of early (EAD) and late (DAD) afterdepolarization observed in APs. In BIN1 deficient ventricular cardiomyocytes, the addition of 1 μM VK-II-86 prevented the development of APD prolongation and suppressed EAD and DAD development. Conclusions: Our study adds to the understanding the role of BIN1. We demonstrate the first-time electrical remodeling in the form of QT and APD prolongation associated with BIN1 deficiency, due to reduced I to and I K1 and increase in late I Na , an increase in EAD and DAD events. Additionally, VK-II-86 is shown to normalize APD in isolated cardiomyocytes and suppress afterdepolarization events, making it a potentially novel and effective therapeutic strategy in HF associated with BIN1 deficiency.

Increased cardiac PFK-2 protects against high-fat diet-induced cardiomyopathy and mediates beneficial systemic metabolic effects

A healthy heart adapts to changes in nutrient availability and energy demands. In metabolic diseases like type 2 diabetes (T2D), increased reliance on fatty acids for energy production contributes to mitochondrial dysfunction and cardiomyopathy. A principal regulator of cardiac metabolism is 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2), which is a central driver of glycolysis. We hypothesized that increasing PFK-2 activity could mitigate cardiac dysfunction induced by high-fat diet (HFD). Wild type (WT) and cardiac-specific transgenic mice expressing PFK-2 (GlycoHi) were fed a low fat or HFD for 16weeks to induce metabolic dysfunction. Metabolic phenotypes were determined by measuring mitochondrial bioenergetics and performing targeted quantitative proteomic and metabolomic analysis. Increasing cardiac PFK-2 had beneficial effects on cardiac and mitochondrial function. Unexpectedly, GlycoHi mice also exhibited sex-dependent systemic protection from HFD, including increased glucose homeostasis. These findings support improving glycolysis via PFK-2 activity can mitigate mitochondrial and functional changes that occur with metabolic syndrome.

Abstract 13219: ER-Selective Autophagy Alleviates ER Stress-Mediated Myocardial Injury

Introduction: Increasing lines of evidence suggest endoplasmic reticulum-selective autophagy (ER-phagy) as a cell-protective pathway to mediate degradation of damaged ER. However, the role of ER-phagy in cardiomyocytes and its molecular mechanisms remain unsolved. Hypothesis: Considering that various cardiac loads cause ER stress which contributes to the development of heart failure, we hypothesized that ER-phagy would be a compensatory system for protecting cardiomyocytes against ER stress. Methods and Results: To evaluate ER-phagy in cardiomyocytes, we generated lentivirus (LV) and cardiac-specific transgenic mice (Tg-mice) harboring ss-RFP-GFP-KDEL sequence, an ER-phagy reporter possessing an N-terminal ER signal sequence followed by tandem fluorescence sequences and ER retention signal. Fluorescent microscopic analyses and immunoblotting demonstrated that amino acid deprivation of LV-ss-RFP-GFP-KDEL-tranduced H9c2 (tfH9c2) cells markedly elevated the amount of RFP fragments, suggesting that ER-phagy is increased in tfH9c2 cells. 48 hours of food starvation markedly induced ER-phagy in Tg-mice hearts, as well. Treatment with Doxorubicin (Dox), a cardiotoxic compound inducing ER stress, activated ER-phagy in tfH9c2 cells as evidenced by the increase in RFP fragments detected by immunoblotting (Control vs. Dox: 1.0±0.2a.u. vs. 31.6±6.3a.u.; p<0.05). Consistently, treatment with Dox (20 mg/kg) significantly induced ER-phagy in myocardium of Tg-mice compared with those of saline-treated ones (Saline vs. Dox: 1.0±0.05a.u. vs. 2.5±0.2a.u.; p<0.05). qRT-PCR analyses revealed that treatment with Dox enhanced the expression of CCPG1, an ER-phagy receptor, in tfH9c2 cells (Control vs. Dox: 1.0±0.05a.u. vs. 2.1±0.2a.u.; p<0.05). Furthermore, LV-CCPG1 transduction-mediated silencing CCPG1 of tfH9c2 cells resulted in the reduction of ER-phagy activity, accumulation of pro-apoptotic proteins including cleaved caspase 3 and deterioration of cell survival (Cell viability: Control vs. Knockdown: 79.0±3.3% vs. 62.4±2.7%; p<0.05) in response to Dox treatment. Conclusions: These results suggest that ER-phagy plays a protective role in cardiomyocytes against Dox toxicity, possibly through CCPG1-mediated signaling.

Inhibiting miR-22 Alleviates Cardiac Dysfunction by Regulating Sirt1 in Septic Cardiomyopathy.

High morbidity and mortality are the most typical characteristics of septic cardiomyopathy. We aimed to reveal the role of miR-22 in septic cardiomyopathy and to explore the underlying mechanisms. miR-22 cardiac-specific knockout (miR-22cKO) mice and miR-22 cardiac-specific transgenic (miR-22cOE) mice were subjected to a cecal ligation and puncture (CLP) operation, while a sham operation was used in the control group. The echocardiogram results suggested that miR-22cKO CLP mice cardiac dysfunction was alleviated. The serum LDH and CK-MB were reduced in the miR-22cKO CLP mice. As expected, there was reduced apoptosis, increased autophagy and alleviated mitochondrial dysfunction in the miR-22cKO CLP mice, while it had contrary role in the miR-22cOE group. Inhibiting miR-22 promoted autophagy by increasing the LC3II/GAPDH ratio and decreasing the p62 level. Additionally, culturing primary cardiomyocytes with lipopolysaccharide (LPS) simulated sepsis-induced cardiomyopathy in vitro. Inhibiting miR-22 promoted autophagic flux confirmed by an increased LC3II/GAPDH ratio and reduced p62 protein level under bafilomycin A1 conditions. Knocking out miR-22 may exert a cardioprotective effect on sepsis by increasing autophagy and decreasing apoptosis via sirt1. Our results revealed that targeting miR-22 may become a new strategy for septic cardiomyopathy treatment.

Amino terminus of cardiac myosin binding protein-C regulates cardiac contractility

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) regulates cardiac contraction through modulation of actomyosin interactions mediated by the protein's amino terminal (N′)-region (C0-C2 domains, 358 amino acids). On the other hand, dephosphorylation of cMyBP-C during myocardial injury results in cleavage of the 271 amino acid C0-C1f region and subsequent contractile dysfunction. Yet, our current understanding of amino terminus region of cMyBP-C in the context of regulating thin and thick filament interactions is limited. A novel cardiac-specific transgenic mouse model expressing cMyBP-C, but lacking its C0-C1f region (cMyBP-C∆C0-C1f), displayed dilated cardiomyopathy, underscoring the importance of the N′-region in cMyBP-C. Further exploring the molecular basis for this cardiomyopathy, in vitro studies revealed increased interfilament lattice spacing and rate of tension redevelopment, as well as faster actin-filament sliding velocity within the C-zone of the transgenic sarcomere. Moreover, phosphorylation of the unablated phosphoregulatory sites was increased, likely contributing to normal sarcomere morphology and myoarchitecture. These results led us to hypothesize that restoration of the N′-region of cMyBP-C would return actomyosin interaction to its steady state. Accordingly, we administered recombinant C0-C2 (rC0-C2) to permeabilized cardiomyocytes from transgenic, cMyBP-C null, and human heart failure biopsies, and we found that normal regulation of actomyosin interaction and contractility was restored. Overall, these data provide a unique picture of selective perturbations of the cardiac sarcomere that either lead to injury or adaptation to injury in the myocardium.

Abstract 16602: Mitolamban is a Novel Cardiac-Enriched Mitochondrial Microprotein That Regulates Respiratory Complex III

Introduction: Emerging evidence suggests that many RNA molecules currently annotated as noncoding contain short open reading frames that code for functional small proteins called microproteins. Microproteins play critical roles in a diverse range of essential biological processes. To identify novel cardiac-expressed microproteins, we used a comparative genomics approach and identified mitolamban (Mtlbn) as a highly conserved 47-amino acid transmembrane protein that is abundantly expressed in the heart. Methods and Results: Subcellular localization studies showed that Mtlbn localized specifically to the inner mitochondrial membrane. Immunoprecipitations and mass spectrometry analysis indicated that Mtlbn interacted with subunits of complex III of the electron transport chain (ETC) and we observed the presence of Mtlbn in complex III respiratory supercomplexes (SCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). Cardiac-specific Mtlbn overexpressing transgenic (TG) mice and Mtlbn gene-deleted (knockout, KO) mice were generated to dissect the molecular function of this protein in the heart. Mtlbn TG mice developed cardiomyopathy and died prematurely with histological, biochemical and ultrastructural pathologic features. Metabolomic analysis indicated that hearts from TG mice had signs of increased oxidative stress and mitochondrial dysfunction. While Mtlbn KO mice had normal cardiac function and histological appearance, BN-PAGE analysis of purified mitochondria from KO hearts showed altered complex III composition. Functional assessment of purified heart mitochondria from Mtlbn KO mice revealed a reduction in complex III activity and metabolomic analysis of KO heart tissue indicated an altered metabolite profile consistent with deficiencies in complex III activity. Conclusions: Mtlbn is a novel heart-enriched microprotein that localizes to the inner mitochondrial membrane where it interacts with complex III of the ETC. Our data indicate that Mtlbn serves as a critical regulator of mitochondrial ETC activity through a direct role in affecting complex III function.

Novel genomic targets of valosin-containing protein in protecting pathological cardiac hypertrophy

Pressure overload-induced cardiac hypertrophy, such as that caused by hypertension, is a key risk factor for heart failure. However, the underlying molecular mechanisms remain largely unknown. We previously reported that the valosin-containing protein (VCP), an ATPase-associated protein newly identified in the heart, acts as a significant mediator of cardiac protection against pressure overload-induced pathological cardiac hypertrophy. Still, the underlying molecular basis for the protection is unclear. This study used a cardiac-specific VCP transgenic mouse model to understand the transcriptomic alterations induced by VCP under the cardiac stress caused by pressure overload. Using RNA sequencing and comprehensive bioinformatic analysis, we found that overexpression of the VCP in the heart was able to normalize the pressure overload-stimulated hypertrophic signals by activating G protein-coupled receptors, particularly, the olfactory receptor family, and inhibiting the transcription factor controlling cell proliferation and differentiation. Moreover, VCP overexpression restored pro-survival signaling through regulating alternative splicing alterations of mitochondrial genes. Together, our study revealed a novel molecular regulation mediated by VCP under pressure overload that may bring new insight into the mechanisms involved in protecting against hypertensive heart failure.

Abstract 437: A Novel Variant Cell Cycle Related Kinase Protect Heart Against Ischemia by Promoting Cardiomyocytes Survival

Background and objects: Our previous studies identified a novel variant of cell cycle-related kinase in heart (cardiac CCRK or cCCRK) which is different from generic CCRK found in other tissues in terms of its amino acid sequence, substrates, protein-protein interaction and tissue distribution. cCCRK has been found dramatically decreased in the failed heart tissues, while overexpress of cCCRK in cardiac myocytes in vitro protected the cells against induced cell death. Therefore, we tested the hypothesis here that cCCRK acts as a potential candidate of cell survival promotor providing cardiaoprotection in vivo upon cardiac ischemic stress. Methods and results: A cardiac specific transgenic (TG) mouse was generated with an alpha- MHC premotor in which cCCRK was overexpressed by 3.5 folds compared to wild type mice (WT). Both cCCRK TG and the litter-mated WT mice were subjected to an 45 minutes myocardial ischemia followed by an 24 hours reperfusion (IR), and the infarct size was measured by 2,3,5-Triphenyltetrazolium chloride (TTC) staining. We found that despite the similar risk area, infarct size was significantly reduced by 50% in the TG mice verse WT mice (p<0.05). The apoptosis in the adjacent area of the heart tissues measured by TUNEL was reduced by 30% in TG mice vs WT (p<0.05). Mechanistically, the survival-promoting signaling pathway, including phosphorylated extracellular-signal-regulated kinase (p-ERK), phosphorylated Mitogen-activated protein kinase/ERK kinase (p-MEK), and P21, was activated in the heart tissues from TG vs WT mice (p<0.05 vs WT). Overexpression of cCCRK in cardiac myocytes in vitro reduced chelerythrine-induced apoptosis reflected by a reduction of caspase 3 activity, this protection was abolished by the Inhibition of MEK with the treatment of its inhibitor U1206 vs vehicle controls (p<0.05 vs vehicle). Conclusion: This study demonstrated that overexpression of a variant of CCRK in heart provides protection against ischemic stress by activating survival signaling.

Abstract 551: Distinct Transcriptional Roles for Wildtype vs. Truncated Nrf2 in the Heart of Transgenic Mice

Background: Nuclear erythroid-2 like factor-2 (Nrf2), a redox-sensitive transcriptional regulator of cytoprotective and antioxidant genes. Despite the basal transcription of antioxidants/redox genes, forced activation of Nrf2 may lead to “reductive stress” (RS). In this study, we hypothesized that controlled vs. sustained activation of Nrf2 differentially regulates the myocardial transcriptome, which elucidates the transition of a physiological adaptation to pathological process. Methods: Cardiac specific Nrf2 transgenic mice expressing full length Nrf2 (FL-TG) and truncated Nrf2 (TR-TG), and their non-transgenic littermates (NTG) in the C57/BL6J backround at the age of 6-8 months were used for this study. Next generation RNA sequencing was performed using the myocardial mRNA obtained from FL-TG, TR-TG and NTG mice (n=3/group). Validation of the NGS data was carried out by qPCR analysis using specific primers for the targeted genes(n=4 to 6/group). Results: Analysis of the NGS data uncovered eccentric genomic profiles in the myocardium of mice with a 310 differentially regulated genes (DEGs) in FL-TG ( vs. NTG) and 472 DEGs in TR-TG ( vs. NTG). A closer evaluation reveals 397 and 237 DEGs commonly shared between FL-TG and TR-TG, respectively. STRING analysis revealed genomic networks involve in oxidoreductase, antioxidant and glutathione transferase were differentially altered between TL-TG vs. TR-TG groups. For instance, within the glutathione synthesis pathway, Gclm transcript was upregulated 6.0 fold (FL-TG) and 8.0 fold (TR-TG) when compared to NTG. Indeed, a significant douwnregulation of stress response genes Hspa1b (5.0 & 2.0 fold) and Hsph1 (2.0 & 2.2 fold) was apparent in between the groups. An eccentric cytoskeletal disarrangement ( Efnb3, Map1a, Acta ), an impaired protein quality control ( Bin1,Myl4 ) and a reduced cardiac function ( Timp4) were significantly downregulated in both the groups. Conclusion: These findings conclude that transgenic expression of wild-type vs. mutant forms of Nrf2 in the heart uniquely regulate the myocardial transcriptome, which might result in independent pathological processes.

Genomic characterization reveals novel mechanisms underlying the valosin-containing protein-mediated cardiac protection against heart failure

Chronic hypertension is a key risk factor for heart failure. However, the underlying molecular mechanisms are not fully understood. Our previous studies found that the valosin-containing protein (VCP), an ATPase-associated protein, was significantly decreased in the hypertensive heart tissues. In this study, we tested the hypothesis that restoration of VCP protected the heart against pressure overload-induced heart failure. With a cardiac-specific transgenic (TG) mouse model, we showed that a moderate increase of VCP was able to attenuate chronic pressure overload-induced maladaptive cardiac hypertrophy and dysfunction. RNA sequencing and a comprehensive bioinformatic analysis further demonstrated that overexpression of VCP in the heart normalized the pressure overload-stimulated hypertrophic signals and repressed the stress-induced inflammatory response. In addition, VCP overexpression promoted cell survival by enhancing the mitochondria resistance to the oxidative stress via activating the Rictor-mediated-gene networks. VCP was also found to be involved in the regulation of the alternative splicing and differential isoform expression for some genes that are related to ATP production and protein synthesis by interacting with long no-coding RNAs and histone deacetylases, indicating a novel epigenetic regulation of VCP in integrating coding and noncoding genomic network in the stressed heart. In summary, our study demonstrated that the rescuing of a deficient VCP in the heart could prevent pressure overload-induced heart failure by rectifying cardiac hypertrophic and inflammatory signaling and enhancing the cardiac resistance to oxidative stress, which brought in novel insights into the understanding of the mechanism of VCP in protecting patients from hypertensive heart failure.

1888-P: Activation of Myocardial Mitochondrial Akt1 Remotely Modulated Body Composition and Adiposity through LXR/RXR Signaling

Insulin resistance contributed to the development of type 2 diabetes (T2D) and is a major risk factor for diabetic cardiomyopathy (DCM). We have previously reported that insulin resistance led to impaired Akt1 translocation into myocardial mitochondria in DCM. To study the role of mitochondrial Akt1 in DCM, we have generated two cardiac-specific inducible Cre-lox transgenic mice models that expresses (1) a mitochondria-targeting dominant negative Akt (CAMDAKT) or (2) a mitochondria-targeting constitutively active Akt (CAMCAKT) upon Tamoxifen induction (T). T-CAMDAKT mice showed LV dysfunction with increased heart failure markers, while T-CAMCAKT mice restored LV dysfunction to normal in the T2D model. In a T2D model induced by high fat/high fructose diet with relative insulin deficiency, T-CAMCAKT mice showed lower body fat mass (16.6% vs. 26.5%, p<0.001) and higher body lean mass (77.1% vs. 68.1%, p<0.001). To study the mechanism underlying the changes of body composition, we performed transcriptomic profiling and analysis in gonadal white adipose tissues (WAT) by bulk RNA sequencing. When compared to the control T2D mice, 544 genes were upregulated and 1077 genes were downregulated in the WAT of T-CAMCAKT T2D mice. The result indicated enhanced lipid catabolism, triglyceride catabolism, HDL assembly, and downregulated critical nuclear transcription. Pathway analysis revealed the Liver X receptor/Retinoid X receptor signaling was activated in WAT of T-CAMCAKT mice as compared to the controls. This was accompanied by increased fatty acid oxidation and apolipoproteins expression. These findings suggest that impaired mitochondrial Akt signaling in DCM could remotely increase adiposity by reducing lipid catabolism in WAT through modulating LXR/RXR signaling. Disclosure Y. Chen: None. A. Ta: None. E. Tom: None. Y. Chen: None. P.H. Wang: Board Member; Self; Dianavi. Funding National Institutes of Health (R01HL096987)

Leukocyte immunoglobulin-like receptor B4 protects against cardiac hypertrophy via SHP-2-dependent inhibition of the NF-κB pathway.

Cardiac hypertrophy is a complex pathological process, and the molecular mechanisms underlying hypertrophic remodeling have not been clearly elucidated. Leukocyte immunoglobulin-like receptor B4 (lilrb4) is an inhibitory transmembrane protein that is necessary for the regulation of various cellular signaling pathways. To investigate whether lilrb4 plays a role in cardiac hypertrophy, we performed aortic banding in lilrb4 knockout mice, lilrb4 cardiac-specific transgenic mice, and their wild-type littermates. Cardiac hypertrophy was evaluated by echocardiographic, hemodynamic, pathological, and molecular analyses. We found that lilrb4 was expressed both in myocardial tissue and on cultured cardiomyocytes under basal conditions, but the expression was obviously decreased in mouse hearts following aortic banding and in cardiomyocytes treated with angiotensin II. Lilrb4 disruption aggravated cardiac hypertrophy, fibrosis, and dysfunction in response to pressure overload. Conversely, the cardiac overexpression of lilrb4 led to the opposite effects. Moreover, lilrb4 overexpression inhibited angiotensin II-induced cardiomyocyte hypertrophy in vitro. Mechanistically, we determined that the cardioprotective effect of lilrb4 was mediated through an interaction with SHP-2, the preservation of phosphorylated SHP-2, and the inhibition of the NF-κB pathway. In addition, SHP-2 knockdown in cardiomyocytes eliminated the inhibitory effects of lilrb4 on angiotensin II-induced hypertrophy and NF-κB activation. Our results suggest that lilrb4 protects against pathological cardiac hypertrophy via the SHP-2-dependent inhibition of the NF-κB pathway and may act as a potential therapeutic target for cardiac hypertrophy. KEY MESSAGES: Lilrb4 expression is decreased by hypertrophic stimuli. Lilrb4 protects against pathological cardiac hypertrophy. Lilrb4 interacts with SHP-2 and inhibits NF-κB pathway.

In vivo elongation of thin filaments results in heart failure.

A novel cardiac-specific transgenic mouse model was generated to identify the physiological consequences of elongated thin filaments during post-natal development in the heart. Remarkably, increasing the expression levels in vivo of just one sarcomeric protein, Lmod2, results in ~10% longer thin filaments (up to 26% longer in some individual sarcomeres) that produce up to 50% less contractile force. Increasing the levels of Lmod2 in vivo (Lmod2-TG) also allows us to probe the contribution of Lmod2 in the progression of cardiac myopathy because Lmod2-TG mice present with a unique cardiomyopathy involving enlarged atrial and ventricular lumens, increased heart mass, disorganized myofibrils and eventually, heart failure. Turning off of Lmod2 transgene expression at postnatal day 3 successfully prevents thin filament elongation, as well as gross morphological and functional disease progression. We show here that Lmod2 has an essential role in regulating cardiac contractile force and function.

Bag3 P209L myopathies and efficacy of blocking signaling pathways with the therapeutic peptide, MMI-0100

Background and Hypothesis: The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which play a critical role in cellular processes, including protein degradation and turnover. Over 30 Bag3 mutations have been identified, including a Proline209Leucine (P209L) missense mutation which causes a severe childhood cardiomyopathy. The mechanism by which Bag3 mutations causes cardiomyopathy is currently unknown, but the p38/MAPK signaling cascade has been shown to be altered in our animal model that coexpresses the human Bag3 P209L gene. We hypothesized the cell-permeant peptide, MMI-0100, which is known to inhibit MAPK-activated protein kinase 2 (MK2) activity in the p38/MAPK signaling cascade, would alleviate or reduce cardiac dysfunction.
 Experimental Design: Echocardiographic analysis of cardiac function of cardiac-specific Bag3 P209L transgenic (and wildtype littermate control) mice (20 – 22 months of age) was assessed by high-resolution ultrasound echocardiography (VisualSonics Vevo 2100, MS550D probe, cardiology package) to document the established disease-related cardiac dysfunction at baseline. Mice were then treated with 100mg/kg/day MMI-0100 nebulized daily for 30 days. Follow-up echocardiography was performed at 10, 20, and 30 days of MMI-0100 treatment. Echocardiographic analysis was performed to determine the systolic function (EF%, FS%), chamber dimensions, and wall thickness in systole and diastole using Vevo 2100 Workstation software package.
 Results: Blinded analysis of echocardiographic data identified that Bag3 P209L Tg+ mice had a baseline cardiac dysfunction compared to wildtype controls at 20 months of age (WT= 76% EF, 44% FS; Tg+= 66% EF, 36% FS). MMI-0100 treatment significantly attenuated this dysfunction by 20 days of MMI-0100 treatment (WT= 79% EF, 47% FS; Tg+= 80% EF, 48% FS), consistent with demonstrating for the first time MK2’s role in mediating p38 signaling in the pathogenesis of Bag3 P209L cardiomyopathy.
 Conclusion and Potential Impact: The MMI-0100 peptide has proven efficacious in several animal models of fibrosis driven by p38 signaling, as MK2 is a p38 downstream mediator. Future studies seek to translate the use of the MMI-0100 peptide in pediatric patients with Bag3 P209L cardiomyopathy through compassionate use FDA pathways.

Abstract 917: Actively Blocking Nuclear Localization of G Protein-Couple Receptor 5 Blunts Hypertrophy and Heart Failure After TAC

Introduction: The induction of heart failure (HF) after aortic banding (TAC) in mice encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy and contractile dysfunction. Previously, our lab has shown that the nuclear targeting of GRK5, which is required for maladaptive hypertrophy, is controlled by its N-terminal (NT) calmodulin-binding domain. Hypothesis: We hypothesize that expression of a peptide encoding the NT of GRK5 will act as a GRK5-calmodulin inhibitor preventing the nuclear accumulation of GRK5 and inhibit its pathological activities. Methods: Neonatal rat ventricular cardiomyocytes (NRCM) overexpressing GRK5-NT or control virus were subjected to α-adrenergic stress with 50μM phenylephrine (PE) or vehicle for 30 minutes. Subcellular fractionation and western blotting were used to detect nuclear GRK5. Novel cardiac-specific transgenic mice were generated expressing HA-tagged GRK5-NT and these mice along with their control mice were characterized 8 weeks after TAC or sham surgery. Echocardiography, fibrosis, and lung and heart weight to tibia length metrics were used to evaluate hypertrophy and heart failure response post-TAC. Other cohorts were subjected to TAC for 1 week followed by Western blotting to assess acute signaling and nuclear accumulation of GRK5. Results: In vitro, expression of the GRK5-NT peptide reduced PE-induced nuclear accumulation of GRK5. In both NRCM and tissue, the GRK5-NT peptide was found to bind to calmodulin, which disrupts the endogenous calmodulin-GRK5 interaction. GRK5-NT expression abrogates nuclear GRK5 accumulation following hypertrophic stress in vitro and in vivo. Following 8 weeks of TAC, cardiac function, hypertrophy, and pulmonary congestion were significantly attenuated in GRK5-NT mice compared to NLC mice. Conclusion: We show that the GRK5-NT peptide binds to calmodulin and prevents nuclear accumulation of GRK5 following hypertrophic stress. Our in vivo data show that cardiac-restricted expression of GRK5-NT prevents HF by partially restoring cardiac dysfunction, hypertrophy, and pulmonary congestion following 8 weeks of TAC. Further in vitro work will reveal the calmodulin-dependence of GRK5-NT and the peptide’s effects on G-protein signaling.

Abstract 877: Thioredoxin-1 Maintains Cardiac Function and Metabolic Gene Expression via mTOR Signaling

Thioredoxin-1 (Trx1) is a 12 kDa oxidoreductase that reduces proteins with disulfide bonds through thiol disulfide exchange reactions. Several lines of evidence show that Trx1 protects cardiomyocytes against stress, such as ischemia and pressure overload. However, due to the embryonic lethality of systemic Trx1 knockout mice, the role of endogenous Trx1 in cardiac function has not been tested with loss-of-function mice. In this study, we generated cardiac-specific Trx1 knockout (Trx1cKO) mice, using Trx1floxflox and αMHC-Cre transgenic mice, to elucidate the physiological role of Trx1 in the postnatal heart. The Trx1cKO mice were viable but started dying at 13 days of age. The median survival age was 27 days. Echocardiographic analyses indicated cardiac dysfunction in Trx1cKO mice, evidenced by reduced ejection fraction (WT: 0.78; Trx1cKO: 0.50*; p<0.05 vs. WT). RNA-sequencing and pathway analyses revealed that metabolic genes involved in energy production were most prominently downregulated in Trx1cKO mice. Mitochondrial morphological abnormality and increased oxidative stress were evident in Trx1cKO mice. Among Trx1 substrates possibly involved in metabolic gene expression, mechanistic target of rapamycin (mTOR) was oxidized and inhibited in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mTOR signaling, as evidenced by reduced phosphorylation of mTOR substrates, such as S6K and 4EBP1. Trx1 knockdown in cardiomyocytes inhibited mitochondrial protein expression, respiration and ATP production (relative ATP production in isolated mitochondria; control: 1; Trx1 knockdown: 0.69*; p<0.05 vs. control), suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F mutant is resistant against oxidation and inactivation induced by downregulation of Trx1. Trx1 knockdown suppressed reporter gene activity driven by the promoter sequences of endogenous metabolic genes, such as Ndufa1 and Ndufs1, which was rescued by mTOR-C1483F. These results suggest that Trx1 is essential for cardiac function and metabolism, possibly through reduction of mTOR at Cys1483.

Abstract 168: Remote Ischemic Preconditioning Rescues Cardiac Dysfunction in Atg5 Knockdown Mice

Objective: Remote ischemic pre-conditioning (RIPC) is highly cardioprotective in models of ischemia-reperfusion (IR) injury but has not been tested in genetically modified mice. This study was design to test whether RIPC rescues cardiomyopathy induced by knockdown of autophagy-related 5 protein (Atg5). Approach and Results: Atg5 knockdown was induced by feeding tamoxifen diet for two weeks in Atg5 flox/flox Cre +/- (KD) mice which were created by crossing Atg5 flox/flox and heterozygous cardiac-specific Cre recombinase transgenic (Cre +/- ) mice. Atg5 flox/flox Cre -/- mice served as controls (CONT). Cardiac function was evaluated by echocardiography 28 days after tamoxifen induction. Autophagy proteins were determined by Western blot analysis. RIPC was induced by four cycles of five minutes of hind limb ischemia with tourniquet followed by five minutes reperfusion. Tamoxifen feeding knocked down Atg5 to 32% (CONT 0.91 ± 0.09 vs KD 0.29 ± 0.01, p < 0.01, n = 7), leading to 96% decrease of autophagy activity indicated by LC3-II/LC3-I ratio (CONT 0.96 ± 0.07 vs 0.06 ± 0.03, p < 0.01, n = 7). This was associated with significant cardiac dysfunction (LV ejection fraction, CONT 59.78% ± 3.26 vs KD 47.13% ± 2.50, n = 8 and 10, p < 0.01) and increased heart weight to body weight ratio (CONT 0.50 ± 0.02 vs KD 0.57 ± 0.01, n = 8 and 10, p < 0.01). RIPC increased levels of Atg5 in Atg5 KD mice by 183% (Sham 1.00 ± 0.16 vs RIPC 1.83 ± 0.14, p < 0.01, n = 11 and 13), leading to 148% increase of autophagy activity (Sham 0.99 ± 0.13 vs RIPC 1.47 ± 0.18, p < 0.05, n = 6 and 7), and resulted in rescued cardiac function in terms of heart weight (CONT 0.50 ± 0.01 vs KD 0.53 ± 0.10, n = 11 and 13 ) and LV ejection fraction (CONT 55.64% ± 2.45 vs 52.48% ±1.61, n = 11 and 13). RIPC also suppressed mTOR phosphorylation by 62% compared with sham procedure in Atg5 KD mice (Sham 1.00 ± 0.18 vs RIPC 0.45 ± 0.08 p < 0.01 n = 11 and 13) which was increased by 650% in Atg5 KD compared with CONT mice (CONT 1.23 ± 0.14 vs KD 6.35 ± 0.09, p < 0.01, n = 7). Conclusion: We conclude that RIPC rescues Atg5 knockdown-induced cardiomyopathy. Our data adds to the emerging data suggesting benefits of RIPC beyond acute cardioprotection in IR injury.