Mouse Model Of Cardiomyopathy Research Articles

In Experimental Dilated Cardiomyopathy Heart Failure and Survival Are Adversely Affected by a Lack of Sexual Interactions.

Nearly one in three people in the U.S. will develop heart failure (HF), characterized by fluid retention (edema) in the lungs and elsewhere. This leads to difficult breathing, deterioration of physical capacity, restriction of normal activities and death. There is little data about the safety and effects of sexual interactions in patients with HF. We tested whether a lack of sexual interactions affected pathophysiological outcomes in a pre-clinical mouse model of dilated cardiomyopathy that recapitulates the progressive stages of human HF. Male mice were randomly given access to, or deprived from, sexual interactions with female mice, which were confirmed by videography and generation of offspring. Cohousing with access to sexual interactions markedly prolonged survival, while cohousing without access to sexual activity did not. Sexual interactions improved systolic function, reduced HF-associated edema, altered transcription of heart contractile protein genes and decreased plasma testosterone levels. To determine whether testosterone levels contributed to survival, testosterone levels were experimentally reduced. Reduction of testosterone levels significantly prolonged survival. Taken together, in mice with dilated cardiomyopathy, sexual activity altered cardiac contractile gene transcription, improved systolic function, reduced edema and prolonged survival which may be in part due to lower testosterone levels.

Curcumin protects cardiomyopathy damage through inhibiting the production of reactive oxygen species in type 2 diabetic mice

Type 2 diabetes mellitus (DM)-induced cardiomyopathy is a multifactorial and complex disease involving oxidative stress, lipids, and fibrosis. It is based on metabolic disorders and microvascular disease and causes extensive focal necrosis of the heart muscle. Curcumin (CUR) is a natural polyphenol isolated from turmeric rhizomes and plays an important role in the antioxidant, anti-apoptotic and anti-inflammatory effects of diabetes. Therefore, we established a mouse model of diabetic cardiomyopathy (DCM) in type 2 diabetic db/db mice in our study. We divided the experiment into three groups: the control group, DM group and DM + CUR group.We performed cardiac dissection on mice treated in different conditions and conducted special pathological staining on isolated cardiac tissue. We were surprised to find that a high glucose environment can promote cardiomyocyte apoptosis by TUNEL assay. In addition, after detecting dihydroethiidine (DHE), hematoxylin-eosin (H&E) and Oil Red O staining, we unexpectedly found that CUR can inhibit the production of reactive oxygen species (ROS), reduce myocardial apoptosis, and myocardial lipid accumulation. CUR upregulated the expression of Bcl-2, and downstream the expression of Bax and Caspase-3 proteins by immunohistochemical determination and western blotting. Therefore, these results suggest that CUR has a certain protective effect on diabetic cardiomyopathy by inhibiting the production of ROS.

Central Role for Adipocyte Na,K-ATPase Oxidant Amplification Loop in the Pathogenesis of Experimental Uremic Cardiomyopathy.

Oxidative stress in adipocyte plays a central role in the pathogenesis of obesity as well as in the associated cardiovascular complications. The putative uremic toxin indoxyl sulfate induces oxidative stress and dramatically alters adipocyte phenotype in vitro. Mice that have undergone partial nephrectomy serve as an experimental model of uremic cardiomyopathy. This study examined the effects on adipocytes of administering a peptide that reduces oxidative stress to the mouse model. A lentivirus vector introduced the peptide NaKtide with an adiponectin promoter into the mouse model of experimental uremic cardiomyopathy, intraperitoneally. Then adipocyte-specific expression of the peptide was assessed for mice fed a standard diet compared with mice fed a western diet enriched in fat and fructose. Partial nephrectomy induced cardiomyopathy and anemia in the mice, introducing oxidant stress and an altered molecular phenotype of adipocytes that increased production of systemic inflammatory cytokines instead of accumulating lipids, within 4 weeks. Consumption of a western diet significantly worsened the adipocyte oxidant stress, but expression of NaKtide in adipocytes completely prevented the worsening. The peptide-carrying lentivirus achieved comparable expression in skeletal muscle, but did not ameliorate the disease phenotype. Adipocyte-specific expression of NaKtide, introduced with a lentiviral vector, significantly ameliorated adipocyte dysfunction and uremic cardiomyopathy in partially nephrectomized mice. These data suggest that the redox state of adipocytes controls the development of uremic cardiomyopathy in mice subjected to partial nephrectomy. If confirmed in humans, the oxidative state of adipocytes may be a therapeutic target in chronic renal failure.

Activation of sarcolipin expression and altered calcium cycling in LMNA cardiomyopathy

Cardiomyopathy caused by A-type lamins gene (LMNA) mutations (LMNA cardiomyopathy) is associated with dysfunction of the heart, often leading to heart failure. LMNA cardiomyopathy is highly penetrant with bad prognosis with no specific therapy available. Searching for alternative ways to halt the progression of LMNA cardiomyopathy, we studied the role of calcium homeostasis in the evolution of this disease. We showed that sarcolipin, an inhibitor of the sarco/endoplasmic reticulum (SR) Ca2+ ATPase (SERCA) was abnormally elevated in the ventricular cardiomyocytes of mutated mice compared with wild type mice, leading to an alteration of calcium handling. This occurs early in the progression of the disease, when the left ventricular function was not altered. We further demonstrated that down regulation of sarcolipin using adeno-associated virus (AAV) 9-mediated RNA interference delays cardiac dysfunction in mouse model of LMNA cardiomyopathy. These results showed a novel role for sarcolipin on calcium homeostasis in heart and open perspectives for future therapeutic interventions to LMNA cardiomyopathy.

Empagliflozin prevents doxorubicin-induced myocardial dysfunction

BackgroundEmpagliflozin showed efficacy in controlling glycaemia, leading to reductions in HbA1c levels, weight loss and blood pressure, compared to standard treatment. Moreover, the EMPA-REG OUTCOME trial demonstrated a 14% reduction of major adverse cardiovascular events (MACE), a 38% reduction in cardiovascular (CV) death and a 35% reduction in the hospitalization rate for heart failure (HF). These beneficial effect on HF were apparently independent from glucose control. However, no mechanistic in vivo studies are available to explain these results, yet. We aimed to determine the effect of empagliflozin on left ventricular (LV) function in a mouse model of doxorubicin-induced cardiomyopathy (DOX-HF).MethodsMale C57Bl/6 mice were randomly assigned to the following groups: controls (CTRL, n = 7), doxorubicin (DOX, n = 14), DOX plus empagliflozin (DOX + EMPA, n = 14), or DOX plus furosemide (DOX + FURO group, n = 7). DOX was injected intraperitoneally. LV function was evaluated at baseline and after 6 weeks of treatment using high-resolution echocardiography with 2D speckle tracking (Vevo 2100). Histological assessment was obtained using Haematoxylin and Eosin and Masson’s Goldner staining.ResultsA significant decrease in both systolic and diastolic LV function was observed after 6 weeks of treatment with doxorubicin. EF dropped by 32% (p = 0.002), while the LS was reduced by 42% (p < 0.001) and the CS by 50% (p < 0.001). However, LV function was significantly better in the DOX + EMPA group, both in terms of EF (61.30 ± 11% vs. 49.24 ± 8%, p = 0.007), LS (− 17.52 ± 3% vs. − 13.93 ± 5%, p = 0.04) and CS (− 25.75 ± 6% vs. − 15.91 ± 6%, p < 0.001). Those results were not duplicated in the DOX + FURO group. Hearts from the DOX + EMPA group showed a 50% lower degree of myocardial fibrosis, compared to DOX mice (p = 0.03). No significant differences were found between the DOX + FURO and the DOX group (p = 0.103).ConclusionEmpagliflozin attenuates the cardiotoxic effects exerted by doxorubicin on LV function and remodelling in nondiabetic mice, independently of glycaemic control. These findings support the design of clinical studies to assess their relevance in a clinical setting.

In This Issue

In This Issue

Evidence that Sympathetic Activity is Increased Selectively to the Heart in a Mouse Model of Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM), the most common genetic heart disease, is characterized by cardiac hypertrophy, fibrosis, and increased risk of arrhythmias and sudden death, even in the absence of heart failure. We have studied young (&lt;20 wks) transgenic mice with cardiac‐specific expression of mutated alpha‐tropomyosin (Glu180Gly), an established model of HCM, to investigate autonomic mechanisms. In early experiments, abnormal heart rate and blood pressure responses to β‐adrenergic receptor blockade and ganglionic blockade indicated high cardiac accompanied by low vasomotor sympathetic tone in HCM vs. wild‐type (WT) mice. The aim of this study was to further characterize regional sympathetic activity in HCM vs. WT littermates by measuring expression/activity of tyrosine hydroxylase (TH, rate‐limiting enzyme for norepinephrine (NE) synthesis) and the norepinephrine transporter (NET) in sympathetic ganglia (stellate, superior cervical, celiac/mesenteric), and expression of β1 receptors in left ventricle (LV). Results (below) are expressed relative to WT (n=4 male mice each group). TH protein expression (Western blot) (standardized to neuronal content with PGP9.5) was significantly higher in stellate (cardiac) ganglia in HCM mice (HCM 162±12 vs. WT 100±7; p&lt;0.05), but not significantly different in superior cervical (HCM 82±4 vs. WT 100±14; p&gt;0.05) or celiac‐mesenteric (HCM 95±7 vs. WT 100±4; p&gt;0.05) ganglia. TH/PGP9.5 protein expression was also higher in the LV of HCM (135±15) vs. WT (100±1) mice (p&lt;0.05). LV β1 receptor protein expression was significantly lower in HCM (60±1) vs. WT (100±4) mice (p&lt;0.05), consistent with increased local NE levels. NET protein (immunofluorescence) was lower in stellate ganglia from HCM mice vs. WT (n=3 mice per group, 3 sections/mouse, p&lt;0.05). NET activity was assessed by measuring the rate of uptake of a fluorescent NET substrate (Molecular Devices) into individual sympathetic neurons isolated from stellate and superior cervical ganglia. NET activity measured in stellate neurons was significantly lower in HCM (4.5±0.3 a.u.*min−1, n=115 neurons, from 4 mice) vs. WT (5.3±0.4 a.u.*min−1, n=98 neurons, from 4 mice). NET activity in superior cervical ganglia did not differ between genotypes. In conclusion, increased TH expression (enhanced ‐NE synthesis) and decreased NET expression/activity (reduced NE reuptake) in stellate ganglia are consistent with increased cardiac sympathetic activity in HCM, whereas the lack of differences between HCM and WT in other sympathetic ganglia are consistent with normal sympathetic activity to non‐cardiac target organs in HCM.Support or Funding InformationNIH F32HL140880

Probenecid treatment improves outcomes in a novel mouse model of peripartum cardiomyopathy.

Probenecid has been used for decades in the treatment of gout but recently has also been found to improve outcomes in patients with heart failure via stimulation of the transient receptor potential vanilloid 2 (TRPV2) channel in cardiomyocytes. This study tested the use of probenecid on a novel mouse model of peripartum cardiomyopathy (PPCM) as a potential treatment option. A human mutation of the human heat shock protein 20 (Hsp20-S10F) in mice has been recently shown to result in cardiomyopathy, when exposed to pregnancies. Treatment with either probenecid or control sucrose water was initiated after the first pregnancy in both wild type and Hsp20-S10F mice. Serial echocardiography was performed during subsequent pregnancies and hearts were collected after the third pregnancies for staining and molecular analysis. Hsp20-S10F mice treated with probenecid had decreased mortality, hypertrophy, TRPV2 expression and molecular parameters of heart failure. Probenecid treatment also decreased apoptosis as evidenced by an increase in the level of Bcl-2/Bax. Probenecid improved survival in a novel mouse model of PPCM and may be an appropriate therapy for humans with PPCM as it has a proven safety and tolerability in patients with heart failure.

CIRCULATING FIBROCYTE LEVEL AS A MARKER OF MYOCARDIAL FIBROSIS FOLLOWING ACUTE ST ELEVATION MYOCARDIAL INFARCTION

Fibrocytes are bone marrow-derived progenitor cells that have been linked to myocardial fibrosis in mouse models of acute myocardial infarction (MI) and ischemic cardiomyopathy. Our hypothesis is that among survivors of ST elevation MI, the number and phenotype of circulating fibrocytes correlate

A role for connexin-43 in Duchenne muscular dystrophy cardiomyopathy.

The cardiomyopathy of Duchenne muscular dystrophy (DMD) is an important cause of morbidity and mortality in affected males with this dreaded muscle disease. Previous studies have implicated changes in expression and subcellular localization of connexin-43 (Cx43), the major ventricular gap junction protein, in DMD cardiomyopathy. In this issue of the JCI, Himelman et al. explore how hypophosphorylation of Cx43 at a triplet of serine residues (S325/S328/S330) in the regulatory C-terminus contributes to multiple features of the cardiomyopathy phenotype. Using a mouse model of DMD cardiomyopathy in which phosphomimetic glutamic acids are substituted for serines at these residues in Cx43, Himelman et al. observed reduced gap junction remodeling and lateralization of Cx43 immunosignals, protection against isoproterenol-induced arrhythmias, and improved Ca2+ homeostasis. This study contributes to the understanding of pathologic Cx43 remodeling and encourages further research into developing strategic interventions to mitigate cardiac dysfunction and arrhythmias in DMD patients.

Mavacamten Decreases Maximal Force and Ca2+-Sensitivity of Contraction in Myocardial Strips From a Mouse Model for Hypertrophic Cardiomyopathy

Mavacamten Decreases Maximal Force and Ca2+-Sensitivity of Contraction in Myocardial Strips From a Mouse Model for Hypertrophic Cardiomyopathy

Progression and regression of left ventricular hypertrophy and myocardial fibrosis in a mouse model of hypertension and concomitant cardiomyopathy

BackgroundMyocardial fibrosis is observed in multiple cardiac conditions including hypertension and aortic stenosis. Excessive fibrosis is associated with adverse clinical outcomes, but longitudinal human data regarding changes in left ventricular remodelling and fibrosis over time are sparse because of the slow progression, thereby making longitudinal studies challenging. The purpose of this study was to establish and characterize a mouse model to study the development and regression of left ventricular hypertrophy and myocardial fibrosis in response to increased blood pressure and to understand how these processes reverse remodel following normalisation of blood pressure.MethodsWe performed a longitudinal study with serial cardiovascular magnetic resonance (CMR) imaging every 2 weeks in mice (n = 31) subjected to angiotensin II-induced hypertension for 6 weeks and investigated reverse remodelling following normalisation of afterload beyond 6 weeks (n = 9). Left ventricular (LV) volumes, mass, and function as well as myocardial fibrosis were measured using cine CMR and the extracellular volume fraction (ECV) s.ResultsIncreased blood pressure (65 ± 12 vs 85 ± 9 mmHg; p < 0.001) resulted in higher indices of LV hypertrophy (0.09 [0.08, 0.10] vs 0.12 [0.11, 0.14] g; p < 0.001) and myocardial fibrosis (ECV: 0.24 ± 0.03 vs 0.30 ± 0.02; p < 0.001) whilst LV ejection fraction fell (LVEF, 59.3 [57.6, 59.9] vs 46.9 [38.5, 49.6] %; p < 0.001). We found a strong correlation between ECV and histological myocardial fibrosis (r = 0.89, p < 0.001).Following cessation of angiotensin II and normalisation of blood pressure (69 ± 5 vs baseline 65 ± 12 mmHg; p = 0.42), LV mass (0.11 [0.10, 0.12] vs 0.09 [0.08, 0.11] g), ECV (0.30 ± 0.02 vs 0.27 ± 0.02) and LVEF (51.1 [42.9, 52.8] vs 59.3 [57.6, 59.9] %) improved but remained impaired compared to baseline (p < 0.05 for all). There was a strong inverse correlation between LVEF and %ECV during both systemic hypertension (r = − 0.88, p < 0.001) and the increases in ECV observed in the first two weeks of increased blood pressure predicted the reduction in LVEF after 6 weeks (r = − 0.77, p < 0.001).ConclusionsWe have established and characterized angiotensin II infusion and repeated CMR imaging as a model of LV hypertrophy and reverse remodelling in response to systemic hypertension. Changes in myocardial fibrosis and alterations in cardiac function are only partially reversible following relief of hypertension.

Cardioprotective effects of α-cardiac actin on oxidative stress in a dilated cardiomyopathy mouse model.

The expression of α-cardiac actin, a major constituent of the cytoskeleton of cardiomyocytes, is dramatically decreased in a mouse model of dilated cardiomyopathy triggered by inducible cardiac-specific serum response factor (Srf) gene disruption that could mimic some forms of human dilated cardiomyopathy. To investigate the consequences of the maintenance of α-cardiac actin expression in this model, we developed a new transgenic mouse based on Cre/LoxP strategy, allowing together the induction of SRF loss and a compensatory expression of α-cardiac actin. Here, we report that maintenance of α-cardiac actin within cardiomyocytes temporally preserved cytoarchitecture from adverse cardiac remodeling through a positive impact on both structural and transcriptional levels. These protective effects were accompanied in vivo by the decrease of ROS generation and protein carbonylation and the downregulation of NADPH oxidases NOX2 and NOX4. We also show that ectopic expression of α-cardiac actin protects HEK293 cells against oxidative stress induced by H2 O2 . Oxidative stress plays an important role in the development of cardiac remodeling and contributes also to the pathogenesis of heart failure. Taken together, these findings indicate that α-cardiac actin could be involved in the regulation of oxidative stress that is a leading cause of adverse remodeling during dilated cardiomyopathy development.

Unravelling the ultrastructural details of αT-catenin-deficient cell-cell contacts between heart muscle cells by the use of FIB-SEM.

The intercalated disc is an important structure in cardiomyocytes, as it is essential to maintain correct contraction and proper functioning of the heart. Adhesion and communication between cardiomyocytes are mediated by three main types of intercellular junctions, all residing in the intercalated disc: gap junctions, desmosomes and the areae compositae. Mutations in genes that encode junctional proteins, including αT-catenin (encoded by CTNNA3), have been linked to arrhythmogenic cardiomyopathy and sudden cardiac death. In mice, the loss of αT-catenin in cardiomyocytes leads to impaired heart function, fibrosis, changed expression of desmosomal proteins and increased risk for arrhythmias following ischemia-reperfusion. Currently, it is unclear how the intercalated disc and the intercellular junctions are organised in 3D in the hearts of this αT-catenin knockout (KO) mouse model. In order to scrutinise this, ventricular cardiac tissue of αT-catenin KO mice was used for volume electron microscopy (VEM), making use of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), allowing a careful 3D reconstruction of the intercalated disc, including gap junctions and desmosomes. Although αT-catenin KO and control mice display a comparable organisation of the sarcomere and the different intercalated disc regions, the folds of the plicae region of the intercalated disc are longer and more narrow in the KO heart, and the pale region between the sarcomere and the intercalated disc is larger. In addition, αT-catenin KO intercalated discs appear to have smaller gap junctions and desmosomes in the plicae region, while gap junctions are larger in the interplicae region of the intercalated disc. Although the reason for this remodelling of the ultrastructure after αT-catenin deletion remains unclear, the excellent resolution of the FIB-SEM technology allows us to reconstruct details that were not reported before. LAY DESCRIPTION: Cardiomyocytes are cells that make up the heart muscle. As the chief cell type of the heart, cardiomyocytes are primarily involved in the contractile function of the heart that enables the pumping of blood around the body. Cardiac muscle cells are connected to each other at their short end by numerous intercellular junctions forming together a structure called the intercalated disc. These intercellular junctions comprise specific protein complexes, which are crucial for both intercellular adhesion and correct contraction of the heart. Imaging by conventional electron microscopy (EM) revealed a heavily folded intercalated disc with apparently random organization of the intercellular junctions. However, this conclusion was based on analysis in two dimensions (2D). 3D information of these structures is needed to unravel their true organization and function. In the present study, we used a more contemporary technique, called volume EM, to image and reconstruct the intercalated discs in 3D. By this approach, EM images are made from a whole block of tissue what differs significantly from classical EM methods that uses only one very thin slice for imaging. Further, we analyzed in comparison to normal mice also a mouse model for cardiomyopathy in which a specific protein of the cardiac intercellular junctions, αT-catenin, is absent. Volume EM revealed that in the hearts of these mice with cardiomyopathy, the finger-like folds of the intercalated disc are longer and thinner compared to control hearts. Also the intercellular junctions on the folded parts of the intercalated disc are smaller and their connection to the striated cytoskeleton seems further away. In conclusion, our volume EM study has expanded our understanding of 3D structures at the intercalated discs and will pave the way for more detailed models of disturbed cell-cell contacts associated with heart failure.

Improved Biomarker and Imaging Analysis for Characterizing Progressive Cardiac Fibrosis in a Mouse Model of Chronic Chagasic Cardiomyopathy.

BackgroundChronic chagasic cardiomyopathy (CCC), caused by Trypanosoma cruzi infection, is an important public health problem attributable to progressive cardiomyopathy in patients, for which there is no cure. Chronic chagasic cardiomyopathy is characterized by myocarditis and cardiac fibrosis, which leads to life‐threatening arrhythmogenic and circulatory abnormalities. This study aimed to investigate cardiac fibrosis progression in a mouse model of chronic chagasic cardiomyopathy.Methods and ResultsCardiac cells infected with T cruzi produced significantly higher concentrations of transforming growth factor‐β (TGF‐β), connective tissue growth factor, endothelin‐1, and platelet‐derived growth factor‐D than noninfected controls. Female Balb/c mice infected with T cruzi were compared with naïve mice. TGF‐β genes and other TGF‐β superfamily genes, as well as connective tissue growth factor, endothelin‐1, and platelet‐derived growth factor, were upregulated in infected mouse hearts. Serum concentrations of TGF‐β, connective tissue growth factor, and platelet‐derived growth factor‐D were higher in infected mice and correlated with cardiac fibrosis. Strain analysis performed on magnetic resonance images at 111 and 140 days postinfection and echocardiography images at 212 days postinfection revealed significantly elevated left ventricular strain and cardiac fibrosis and concomitantly significantly decreased cardiac output in infected mice.ConclusionsTGF‐β, connective tissue growth factor and platelet‐derived growth factor‐D are potentially useful biomarkers of cardiac fibrosis, as they correlate with cardiac fibrosis. Strain analysis allows for use of noninvasive methods to measure fibrosis in the chronic stages of chagasic cardiomyopathy in a mouse model. These findings can be applied as noninvasive tools to study the effects of interventions and/or therapeutics on cardiac fibrosis development when using a mouse model of chronic chagasic cardiomyopathy.

Wisteria floribunda agglutinin staining for the quantitative assessment of cardiac fibrogenic activity in a mouse model of dilated cardiomyopathy

Cardiac fibrosis is a typical phenomenon in failing hearts for most cardiac diseases, including dilated cardiomyopathy (DCM), and its specific detection and quantification are crucial for the analysis of cardiac remodeling. Since cardiac fibrosis is characterized by extensive remodeling of the myocardial extracellular matrix (ECM), in which glycoproteins are the major components, we assumed that fibrosis-related alterations in the cardiac glycome and glycoproteome would be suitable targets for the detection of cardiac fibrosis. Here, we compared protein glycosylation between heart tissues of normal and DCM model mice by laser microdissection-assisted lectin microarray. Among 45 lectins, Wisteria floribunda agglutinin (WFA) was selected as the most suitable lectin for staining cardiac fibrotic tissues. Although the extent of WFA staining was highly correlated (r > 0.98) with that of picrosirius red staining, a common collagen staining method, WFA did not bind to collagen fibers. Further histochemical analysis with N-glycosidase revealed that WFA staining of fibrotic tissues was attributable to the binding of WFA to N-glycoproteins. Using a mass spectrometry-based approach, we identified WFA-binding N-glycoproteins expressed in DCM hearts, many of which were fibrogenesis-related ECM proteins, as expected. In addition, the identified glycoproteins carrying WFA-binding N-glycans were detected only in DCM hearts, suggesting their cooperative glycosylation alterations with disease progression. Among these WFA-binding ECM N-glycoproteins, co-localization of the collagen α6(VI) chain protein and WFA staining in cardiac tissue sections was confirmed with a double-staining analysis. Collectively, these results indicate that WFA staining is more suitable for the quantitative assessment of cardiac fibrogenic activity than current collagen staining methods. Furthermore, given that plasma WFA-binding glycoprotein levels were significantly correlated with the echocardiographic parameters for left ventricular remodeling, cardiac WFA-binding glycoproteins are candidate circulating glyco-biomarkers for the quantification and monitoring of cardiac fibrogenesis.

MiR-34b/c regulates doxorubicin-induced myocardial cell injury through ITCH

ABSTRACT Objective: To determine the underlying mechanism of miR-34b/c in regulating doxorubicin (Dox)-induced myocardial cell injury. Methods: The viability of mouse myocardial cells HL-1 was detected by MTT assay. The apoptosis of HL-1 cells was detected by TUNEL assay. mRNA expressions of ITCH, TNF-α and IL-6 were measured by qRT-PCR. Protein levels of ITCH, NF-κB, TNF-α and IL-6 were measured by western blot. Dual luciferase assay was performed to detect the regulation of miR-34b/c on ITCH. Mouse model of cardiomyopathy was induced by intraperitoneal injection of Dox. Results: Dox reduced HL-1 cell viability and activated NF-κB pathway in HL-1 cells. miR-34b/c expressions were gradually up-regulated and ITCH expression was gradually down-regulated in Dox-treated HL-1 cells. miR-34b/c expression had negative correlation with the mRNA expression of ITCH. Besides, ITCH was a target of miR-34b/c. miR-34b/c mimic reduced cell viability, suppressed ITCH expression, increased TNF-α and IL-6 level, and promoted NF-κB expression in nucleus and cytoplasm of HL-1 cells. Whereas silencing miR-34 protected HL-1 cells through regulating ITCH. Finally, we demonstrated miR-34 antagomir-protected myocardial cells in mouse model of cardiomyopathy. Conclusion: miR-34b/c decreased HL-1 cell viability and promoted the secretion of proinflammatory cytokines in Dox-induced myocardial cells through ITCH/NF-κB pathway.

P6274Role of the nicotinamide riboside kinase 2 in NAD metabolism in the heart in basal and pathological condition

Abstract Background NAD is a major coenzyme in energy metabolism and a substrate for SIRT1 and PARP1 enzymes involved in the response to energy and oxidative stress. We have shown the beneficial effects of nicotinamide riboside (NR), a new type of vitamin B3, on cardiac function and remodelling in a mouse model of dilated cardiomyopathy (DCM) triggered by deletion of the SRF transcription factor in the heart (Srf-HKO) (1). This functional improvement correlated with protection of NAD metabolism and a robust increase in cardiac expression of the Nicotinamide Riboside Kinase 2 (NMRK2) that phosphorylates NR to generate nicotinamide mononucleotide (NMN), an immediate precursor of NAD. Purpose We aim to understand the role of the NMRK2-mediated NAD biosynthetic pathway in the heart at baseline and in the DCM context. Methods We generated Nmrk2-KO mice that we bred with Srf-HKO to generate double KO mice (db-KO). We analysed cardiac function and remodelling by echocardiography and quantified myocardial NAD levels at baseline and following NR supplementation in food. Results Nmrk2KO mice developed a progressive eccentric remodelling of LV and decline in EF with aging. At 24-mo, we observed a reduction of myocardial NAD levels (−40% compared to wild type, p&lt;0.05) and of LVEF (61%, SD 6.3% in Nmrk2-KO vs 78%, SD 1.5% in WT, p&lt;0.05). To assess the contribution of cardiac Nmrk2 induction to NR response in DCM, we compared SrfH-KO and db-KO mice fed with control diet (CD) or NR supplemented diet for 40 days starting at young age (2-mo). NR reduced the extent of LV eccentric remodelling and drop in EF as well as the thinning of the LV posterior wall in both genotypes (2-way ANOVA, diet effect, p&lt;0.01). Myocardial NAD levels were more reduced in db-KO mice under CD diet (−22% compared to control mice, p&lt;0.05) than in Srf-HKO mice (−11%, non-significant), when we previously showed a 25% drop in myocardial NAD in aged SrfHKO mice (1). NR partially preserved cardiac NAD pool in db-KOmice (−10% compared to controls, non-significant). Parallel pathways for NMN synthesis were studied. Nampt gene expression was significantly repressed in db-KO mice fed with CD or NR diet compared to control mice (−50% in average, p&lt;0.01), when there was only a trend toward lower expression in SrfHKO mice (−40% in average, p&gt;0.05). Nmrk1 gene expression trended to increase in all groups compared to wild-type control mice. Conclusion We show that NMRK2 pathway plays a role in the maintenance of basal cardiac function and NAD levels when relying on the endogenous myocardial NR pool. In contrast, the beneficial effect of a therapeutic dose of NR is not affected by the lack of NMRK2 suggesting compensation by NMRK1 in the heart and/or that NR beneficial effects on cardiac function could be mediated through its action on systemic metabolism. Aging appears as an aggravating factor for the loss of myocardial NAD coenzyme in DCM. Acknowledgement/Funding Agence Nationale pour la Recherche, Fondation de France

Hyperoxia but not AOX expression mitigates pathological cardiac remodeling in a mouse model of inflammatory cardiomyopathy

Constitutive expression of the chemokine Mcp1 in mouse cardiomyocytes creates a model of inflammatory cardiomyopathy, with death from heart failure at age 7–8 months. A critical pathogenic role has previously been proposed for induced oxidative stress, involving NADPH oxidase activation. To test this idea, we exposed the mice to elevated oxygen levels. Against expectation, this prevented, rather than accelerated, the ultrastructural and functional signs of heart failure. This result suggests that the immune signaling initiated by Mcp1 leads instead to the inhibition of cellular oxygen usage, for which mitochondrial respiration is an obvious target. To address this hypothesis, we combined the Mcp1 model with xenotopic expression of the alternative oxidase (AOX), which provides a sink for electrons blocked from passage to oxygen via respiratory complexes III and IV. Ubiquitous AOX expression provided only a minor delay to cardiac functional deterioration and did not prevent the induction of markers of cardiac and metabolic remodeling considered a hallmark of the model. Moreover, cardiomyocyte-specific AOX expression resulted in exacerbation of Mcp1-induced heart failure, and failed to rescue a second cardiomyopathy model directly involving loss of cIV. Our findings imply that mitochondrial involvement in the pathology of inflammatory cardiomyopathy is multifaceted and complex.

Abstract 615: The Cardiac Myosin Inhibitor, CK-3773274, Reduces Contractility in the R403q Mouse Model of Hypertrophic Cardiomyopathy

Cardiac sarcomere hypercontractility appears to underlie pathological hypertrophy and fibrosis in select genetic hypertrophic cardiomyopathies. The small molecule, CK-3773274, is a novel cardiac myosin inhibitor that decreases contractility in vitro and in healthy animals in vivo . The objective of this study was to evaluate the effect of CK-3773274 in the genetic R403Q mouse model of hypertrophic cardiomyopathy. At approximately 40 weeks of age, left ventricular wall dimensions were determined by echocardiography in male wild type (WT) and heterozygous R403Q mice. As an indicator of cardiac hypertrophy, R403Q mice had significantly greater septal and posterior wall thickness than WT mice (septal wall WT: 0.93 ± 0.03 mm vs. R403Q: 1.22 ± 0.08 mm; posterior wall WT: 0.84 ± 0.04 mm vs. R403Q: 1.09 ± 0.04 mm; mean ± SEM, p&lt; 0.05). R403Q mice were treated with single oral doses of CK-3773274 ranging from 0.25 to 1.5 mg/kg and fractional shortening (FS) and heart rate were assessed at select time points over a 24-hour period. One hour after dose administration, CK-3773274 significantly reduced FS in a dose-related fashion relative to pre-dose baseline values (FS % R403Q baseline: 55.5 ± 2%; 0.25 mg/kg: 43.9 ± 2%; 1 mg/kg: : 27.3 ± 2%; 1.5 mg/kg: 13.7 ± 1%; mean ±SEM, p&lt;0.05 vs. baseline at all doses) without any changes to heart rate. At all dose levels, fractional shortening returned to baseline values by 24 hours. The plasma concentration at 10% and 50% reduction of FS relative to baseline (IC 10 and IC 50 ) was 0.11 and 0.78 μM, respectively. In summary, single oral dose administration of CK-3773274 reduced fractional shortening in a dose and concentration-dependent manner in the genetic R403Q mouse model of hypertrophic cardiomyopathy. Cardiac myosin inhibition may be a viable approach to reduce underlying hypercontractility of the cardiac sarcomere in hypertrophic cardiomyopathies.