Dilated Cardiomyopathy Mice Research Articles

Human umbilical cord blood derived mesenchymal stem cells improve cardiac function in cTnTR141W transgenic mouse of dilated cardiomyopathy

Cell transplantation is a promising strategy in regenerative medicine. Beneficial effects of bone marrow mesenchymal stem cells (BM-MSCs) on heart disease have been widely reported. However, the MSCs in these studies have been mainly derived from autologous animals, and data on MSCs from human umbilical cord blood (UCB-MSCs) are still scarce. We investigated whether intramyocardial xenogeneic administration of UCB-MSCs is beneficial for preserving heart function in a cTnTR141W transgenic mouse of dilated cardiomyopathy (DCM). Cultured UCB-MSCs, which were identified by there morphology, differentiation and cell surface markers, were transplanted into cTnTR141W transgenic mice to examine apoptosis, fibrosis, vasculogenesis and the associated Akt pathway. Moreover, we measured the expression levels of VEGF and IGF-1, which are growth factors required for differentiation into cardiomyocytes, and are also involved in cardiac regeneration and improving heart function. One month after transplantation, MSCs significantly decreased chamber dilation and contractile dysfunction in the cTnTR141W mice. MSCs transplanted hearts showed a significant decrease in cardiac apoptosis and its regulation by the Akt pathway. Cardiac fibrosis and cytoplasmic vacuolisation were significantly attenuated in the MSCs group. Importantly, the levels of VEGF and IGF-1 were increased in the MSCs transplanted hearts. In vitro, the MSC-conditioned medium displayed anti-apoptotic activity in h9c2 cardiomyocytes subjected to hypoxia. These results further confirm the paracrine effects of MSCs. In conclusion, UCB-MSCs preserve cardiac function after intramyocardial transplantation in a DCM mouse, and this effect may be associated with reductions in cellular apoptosis, inflammation, hypertrophy and myocardial fibrosis; in addition to; up-regulation of Akt, VEGF and IGF-1; and enhanced angiogenesis.

Abstract 10260: Thrombin is a Novel Target of the Treatment of Dilated Cardiomyopathy

Introduction: A state of hypercoagulability has been observed in patients with dilated cardiomyopathy (DCM) compared to healthy subjects. In addition to being found in blood, thrombin is also expressed in the heart. Hypothesis: We hypothesize that thrombin in the heart tissue may contribute to the pathology of DCM and thrombin inhibition may be beneficial for the development of DCM. Methods: We evaluated the expression of thrombin by immunohistochemical analysis in the left ventricle of 5 patients with DCM undergoing Batista operation and that of 4 patients without heart disease serving as controls. The immunohistochemical staining was scored subjectively on a semi-quantitative scale of 0-4. We investigated the effects of the direct thrombin inhibitor, dabigatran, in the development of DCM in a mouse model carrying a deletion mutant of cardiac troponin T (DCM mouse) which causes human DCM, by using echocardiography and Kaplan-Meier method. We also estimated the apoptotic index using the terminal dUTP nick-end labeling (TUNEL) assay using the heart tissue from DCM mouse. Results: Immunohistochemical analysis showed strong thrombin expression in DCM patients compared to patients without heart disease [3.60 in DCM (n=5), 1.25 in control (n=4), p<0.001]. Dabigatran significantly improved impaired left ventricular function of DCM mouse by echocardiographic examination; fractional shortening was 15.6±5.7 % in DCM mouse and was 42.7±7.3 % in DCM with dabigatran (p=0.03, n=4). Dabigatran also significantly improved the poor outcome of DCM mouse (p=0.02, n=7) (Figure1). Although there were no significant differences, dabigatran tended to reduce the apoptotic index; the percentage of TUNEL-positive nuclei was 0.20±0.04 % in DCM mouse and was 0.08±0.05 % in DCM with dabigatran (p=0.11, n=4). Conclusion: Upregulation of tissue thrombin may be involved in the pathogenesis of DCM. The direct thrombin inhibitor, dabigatran, may be a possible treatment option against DCM.

Abstract 11548: Nanofibrous Collagen Scaffolds Seeded With Human Pluripotent Stem Cell-derived Cardiomyocytes Induce Cardiac Remodeling in Dilated Cardiomyopathy

Introduction: Limited data are available on the effect of stem cells in non-ischemic dilated cardiomyopathy (DCM). Hypothesis: Since the diffuse nature of DCM calls for a broad distribution of cells, we tested the scaffold-based delivery of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) in a mouse model of DCM. Methods: (1) In vitro protocol: A clinical grade atelocollagen was electrospun and crosslinked under different conditions to optimize production of nanofibrous scaffolds. (2) In vivo protocol: The biocompatibility of acellular collagen scaffolds was first tested in C57BL6 (n=24) and αMHCMerCreMerSf/Sf (n=18) mice, a strain in which DCM is induced by a cardiac (αMHC)-specific and tamoxifen(T)-inducible invalidation of serum response factor (SRF). Scaffolds seeded with hiPSC-CM (obtained by a sequential activation/inhibition of the Wnt pathway) or kept acellular (controls) were then implanted in 8 and 6 αMHCMerCreMerSf/Sf mice, respectively. Results: As assessed by SEM and shear wave elastography, a 10% citric acid concentration and 150 min of baking at 150°C producing a scaffold with an average fiber thickness of 1400 nm, an average pore size of 2500 nm and an elasticity modulus of 10.1 kPa were associated with optimal hiPSC-CM differentiation and colonization of the scaffold. Seven and 14 days after implantation, the biocompatibility of cell-free scaffolds was confirmed by echocardiography and histology. Scaffolds seeded or not with hiPSC-CM were then grafted in DCM mice. After 14 days, heart function drastically decreased in hearts receiving cell-free scaffolds while it remained stable in the treated mice (p<0.001 for LVEDV, LVESV and EF). This pattern was associated with (1) a significantly (p<0.01) increased genomic and proteomic expression of SRF possibly reflecting an activation of resident stem cells (unaffected by T) and/or an increase in endothelial cells (also T-unaffected), in line with the greater vascularity of the scaffold, and (2) a reduced fibrosis consistent with the upregulation of several genes involved in extracellular matrix remodeling. Conclusion: These data suggest that a hiPSC-CM loaded electrospun collagen patch stabilizes function in DCM, most likely through paracrine signaling.

Omecamtiv Mecarbil, a Cardiac Myosin Activator, Increases Ca2+ Sensitivity in Myofilaments With a Dilated Cardiomyopathy Mutant Tropomyosin E54K.

Apart from transplant, there are no satisfactory therapies for the severe depression in contractility in familial dilated cardiomyopathy (DCM). Current heart failure treatments that act by increasing contractility involve signaling cascades that alter calcium homeostasis and induce arrhythmias. Omecamtiv mecarbil is a promising new inotropic agent developed for heart failure that may circumvent such limitations. Omecamtiv is a direct cardiac myosin activator that promotes and prolongs the strong myosin-actin binding conformation to increase the duration of systolic elastance. We tested the effect of omecamtiv on Ca(2+) sensitivity of myofilaments of a DCM mouse model containing a tropomyosin E54K mutation. We compared tension and ATPase activity of detergent-extracted myofilaments with and without treatment with 316 nM omecamtiv at varying pCa values. When transgenic myofilaments were treated with omecamtiv, the pCa50 for activation of tension increased from 5.70 ± 0.02 to 5.82 ± 0.02 and ATPase activity increased from 5.73 ± 0.06 to 6.07 ± 0.04. This significant leftward shift restored Ca(2+) sensitivity to levels no longer significantly different from controls. Proteomic studies lacked changes in sarcomeric protein phosphorylation. Our data demonstrate that omecamtiv can potentially augment cardiac contractility in DCM by increasing Ca(2+) sensitivity. The use of direct myosin activators addresses functional defects without incurring the adverse side effects of Ca(2+)-dependent treatments.

Wnt/β-Catenin Signaling Contributes to Skeletal Myopathy in Heart Failure via Direct Interaction With Forkhead Box O.

There are changes in the skeletal muscle of patients with chronic heart failure (CHF), such as volume reduction and fiber type shift toward fatigable type IIb fiber. Forkhead box O (FoxO) signaling plays a critical role in the development of skeletal myopathy in CHF, and functional interaction between FoxO and the Wnt signal mediator β-catenin was previously demonstrated. We have recently reported that serum of CHF model mice activates Wnt signaling more potently than serum of control mice and that complement C1q mediates this activation. We, therefore, hypothesized that C1q-induced activation of Wnt signaling plays a critical role in skeletal myopathy via the interaction with FoxO. Fiber type shift toward fatigable fiber was observed in the skeletal muscle of dilated cardiomyopathy model mice, which was associated with activation of both Wnt and FoxO signaling. Wnt3a protein activated FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt3a-induced fiber type shift was inhibited by suppression of FoxO1 activity, whereas Wnt3a-independent fiber type shift was observed by overexpression of constitutively active FoxO1. Serum of dilated cardiomyopathy mice activated both Wnt and FoxO signaling and induced fiber type shift toward fatigable fiber in C2C12 cells. Wnt inhibitor and C1-inhibitor attenuated FoxO activation and fiber type shift both in C2C12 cells and in the skeletal muscle of dilated cardiomyopathy mice. C1q-induced activation of Wnt signaling contributes to fiber type shift toward fatigable fiber in CHF. Wnt signaling may be a novel therapeutic target to prevent skeletal myopathy in CHF.

Overexpression of protein phosphatase 2A in a murine model of chronic myocardial infarction leads to increased adverse remodeling but restores the regulation of β-catenin by glycogen synthase kinase 3β

Background/ObjectivesIncreased activity of cardiac protein phosphatases is an important feature in human heart failure. Several different protein phosphatases (PP) are involved in the regulation of excitation–contraction-coupling of the myocardium. Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase consisting of a dimeric core enzyme and tissue-specific subunits. In this study we used transgenic mice overexpressing PP2A to further investigate the role of PP2A in cardiac remodeling after myocardial infarction. Methods and resultsAdult male CD-1 mice overexpressing the catalytic subunit α of PP2A (αMHC-PP2A; TG) underwent chronic LAD-ligation or sham surgery, respectively; wildtype littermates (WT) were used as controls. Cardiac function was determined by echocardiography before and 28days after LAD-ligation. 28days after MI, the animals were sacrificed and cardiac remodeling was analyzed in histological sections and by Western blots. PP2A overexpression leads to dilated cardiomyopathy in mice, and increased cardiomyocyte hypertrophy and fibrosis of the remote myocardium can be seen after myocardial infarction. However, we found an improved survival of TG in the subacute phase after MI in comparison to WT. On the molecular level, TG shows reduced expression of SERCA and CaMKII alpha both under basal condition as well 28days after MI. Additionally, the regulation of the Akt/GSK3β/β-catenin pathway is severely disturbed in TG at baseline where a significant activation of Akt is found that coincides with the typical phosphorylation of GSK3β. However, this does not lead to the accumulation of β-catenin — on the contrary: phosphorylation-induced degradation of β-catenin is significantly enhanced. ConclusionTransgenic overexpression of myocardial PP2A causes adverse remodeling which coincides with a disruption of the classical Akt/GSK3/β-catenin pathway under baseline conditions that is restored to normal values in chronic myocardial infarction. Even so overall survival of TG after myocardial infarction was not constrained and survival after day 2 post MI was improved.

Mitochondrial Fission and Fusion Factors Reciprocally Orchestrate Mitophagic Culling in Mouse Hearts and Cultured Fibroblasts

How mitochondrial dynamism (fission and fusion) affects mitochondrial quality control is unclear. We uncovered distinct effects on mitophagy of inhibiting Drp1-mediated mitochondrial fission versus mitofusin-mediated mitochondrial fusion. Conditional cardiomyocyte-specific Drp1 ablation evoked mitochondrial enlargement, lethal dilated cardiomyopathy, and cardiomyocyte necrosis. Conditionally ablating cardiomyocyte mitofusins (Mfn) caused mitochondrial fragmentation with eccentric remodeling and no cardiomyocyte dropout. Parallel studies in cultured murine embryonic fibroblasts (MEFs) and in vivo mouse hearts revealed that Mfn1/Mfn2 deletion provoked accumulation of defective mitochondria exhibiting an unfolded protein response, without appropriately increasing mitophagy. Conversely, interrupting mitochondrial fission by Drp1 ablation increased mitophagy and caused a generalized loss of mitochondria. Mitochondrial permeability transition pore (MPTP) opening in Drp1 null mitochondria was associated with mitophagy in MEFs and contributed to cardiomyocyte necrosis and dilated cardiomyopathy in mice. Drp1, MPTP, and cardiomyocyte mitophagy are functionally integrated. Mitochondrial fission and fusion have opposing roles during in vivo cardiac mitochondrial quality control.

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation.

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.

The Treatment Benefit of Ghrelin on a Mouse Model of Inherited Dilated Cardiomyopathy Caused by Troponin Mutation

The therapeutic effect of ghrelin has been reported in humans as well as in animal models of chronic heart failure. However, little is known about the therapeutic efficacy of ghrelin for the treatment of inherited forms of dilated cardiomyopathy (DCM). We aim to examine whether ghrelin is beneficial for the treatment of inherited DCM with a deletion mutation ΔK210 in the cardiac troponin T (cTnT) gene using a knock-in mouse model. Ghrelin (150 μg/kg/day) was administered subcutaneously to the mouse model of inherited DCM. The therapeutic effects were examined on the basis of survival and myocardial remodeling. Ghrelin administration prolonged the life span of DCM mice compared to the saline-treated controls. Echocardiography data showed that ghrelin reduced left ventricular (LV) end-diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart-to-body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. Ghrelin has therapeutic benefits for the treatment of DCM with ΔK210 mutation in cTnT. Importantly, these cardiovascular benefits of ghrelin are likely linked to the suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

Stage-Dependent Benefits and Risks of Pimobendan in Genetic Dilated Cardiomyopathy Mice with Progressive Heart Failure

The Ca2+ sensitizer pimobendan is a unique inotropic agent that, compared with traditional inotropes, improves cardiac contractility with less oxygen consumption and potentially fewer adverse effects on myocardial remodelling and arrhythmia. However, clinical trials report contradictory effects of pimobendan in heart failure (HF) patients. We provide mechanistic experimental evidence of the efficacy of pimobendan using a novel mouse model of progressive HF.A knock-in mouse model of human genetic dilated cardiomyopathy, which shows a clear transition from compensatory to end-stage HF at a fixed time during growth, was used to evaluate the efficacy of pimobendan and explore the underlying molecular and cellular mechanisms.Pimobendan prevented myocardial remodelling in compensated HF and significantly extended the life span in both compensated and end-stage HF, but dose-dependently increased the sudden death in end-stage HF. In cardiomyocytes isolated from end-stage HF mice, pimobendan induced triggered activity probably due to early or delayed afterdepolarizations via markedly up-regulated electrogenic sodium/calcium exchanger 1 activation. The L-type Ca2+ channel blocker verapamil decreased the incidence of triggered activity, suggesting that this was from overly elevated cytoplasmic Ca2+ through increased Ca2+ entry by phosphodiesterase 3 inhibition under diminished sarcoplasmic reticulum Ca2+ reuptake and increased Ca2+ leakage from sarcoplasmic reticulum in end-stage HF.Pimobendan is beneficial irrespective of HF stage, but increases sudden cardiac death in end-stage HF with extensive remodelling ofCa2+ handling. Reduction of cytoplasmic Ca2+ elevated by phosphodiesterase 3 inhibition may decrease the risk of sudden cardiac death.

IL-22-producing Th22 cells play a protective role in CVB3-induced chronic myocarditis and dilated cardiomyopathy by inhibiting myocardial fibrosis.

BackgroundA new subset of T helper (Th) cells, named IL-22-producing Th22 cells, was identified recently. Th22 cells have been implicated in immunity and inflammation. However, the role of these cells in the progression from acute viral myocarditis (AVMC) to dilated cardiomyopathy (DCM) and myocardial fibrosis remains unknown.MethodsBALB/c mice were repeatedly i.p. infected with Coxsackie virus B3 (CVB3) to establish models of AVMC, chronic myocarditis and DCM. On week 2, 12 and 24 post initial injection, the percentage of splenic Th22 cells, the levels of plasma IL-22, cardiac IL-22 receptor (IL-22R) expression, and indicators of myocardial fibrosis were measured. Further, mice with AVMC and chronic myocarditis were treated with an anti-IL-22 neutralizing antibody (Ab). The collagen volume fraction (CVF), the percentage of splenic Th22 cells, plasma IL-22 levels, cardiac IL-22R expression and indicators of myocardial fibrosis were then monitored.ResultsCompared to control mice at the same time points, AVMC, chronic myocarditis and DCM mice have higher percentage of splenic Th22 cells, higher plasma IL-22 levels, increased cardiac IL-22R, as well as increased collagen typeI-A1 (COL1-A1), collagen type III-A1 (COL3-A1) and matrix metalloproteinase-9 (MMP9) expression. However, the expression of tissue inhibitor of metalloproteinase-1(TIMP-1) was decreased. Treatment of AVMC and chronic myocarditis mice with an anti-IL-22 Ab decreased the survival rate and exacerbated myocardial fibrosis. The percentage of splenic Th22 cells, plasma IL-22 levels and cardiac IL-22R expression also decreased in anti-IL-22 Ab treatment group as compared to IgG and PBS treated groups of AVMC and chronic myocarditis mice. Moreover, increased expression of COL1-A1, COL3-A1, MMP9 but decreased expression of TIMP-1 were observed in anti-IL-22 Ab mouse group.ConclusionsTh22 cells play an important role in the pathogenesis of CVB3-induced mouse chronic myocarditis and DCM. IL-22 is a myocardium-protective cytokine by inhibiting myocardial fibrosis. Therefore, Th 22 cells may be considered as potential therapeutic targets for DCM.

Abstract 11977: Tranilast, Transient Receptor Potential Vanilloid 2 Antagonist, Ameliorates End-Stage Heart Failure of Mice With Dilated Cardiomyopathy

Introduction: Abnormal intracellular Ca2+ handling seems to be involved in the pathogenesis of idiopathic dilated cardiomyopathy (DCM). We have found up-regulation of the expression of transient receptor potential vanilloid 2 (TRPV2), a calcium-permeable cation channel, in the sarcolemma of myocardium of animal and human DCM. Hypothesis: We hypothesized an orally active TRPV2 antagonist, tranilast, ameliorated heart failure symptoms of DCM mice. Methods: We used 4C30 mice (created by National Institute of Biomedical Innovation, Japan), which has abnormal myocardial calcium handling, as a model of DCM. Sixteen 4C30 mice of 25 weeks old with end-stage heart failure were given no drug (control) or 20 mg/kg/day of carvediol (group C) or 400 mg/kg/day of tranilast (group T) or both of them (group B) for 2 weeks. Results: Blood pressure and heart rate were similar among the 4 groups. Echocardiography demonstrated tranilast improved fractional shortening (in %). Control: 6.2±2.5; Group C: 14.2±5.6, NS; Group T: 16.6±2.3, p<0.05; Group B: 17.2±3.2, p<0.05. Tranilast also improved cardiac hypertrophy measured with heart-to-body weight ratio (HW/BW in mg/g). Control: 12.4±2.5; Group C: 11.5±3.4, NS; Group T: 8.6±0.9, p<0.05; Group B: 5.9±1.1, p<0.01. Sarcolemmal expression of TRPV2 measured with immunostaining in 4C30 mice increased twice as much as syngeneic C57BL/6J. Tranilast, not carvedilol, halved the expression of TRPV2, corresponding to reduction in [Ca2+]i of isolated cardiomyocytes. Consistent with those changes, CaMKII phosphorylation reduced in the 4C30 mice after treatment of tranilast. Conclusions: Tranilast ameliorated heart failure symptoms of 4C30 mice, possibly due to the inhibition of Ca2+ influx through TRPV2.

Survival benefit of ghrelin in the heart failure due to dilated cardiomyopathy.

Although ghrelin has been demonstrated to improve cardiac function in heart failure, its therapeutic efficacy on the life expectancy remains unknown. We aim to examine whether ghrelin can improve the life survival in heart failure using a mouse model of inherited dilated cardiomyopathy (DCM) caused by a deletion mutation ΔK210 in cardiac troponin T (cTnT). From 30 days of age, ghrelin (150 μg/kg) was administered subcutaneously to DCM mice once daily, control mice received saline only. The survival rates were compared between the two groups for 30 days. After 30-day treatment, functional and morphological measurements were conducted. Ghrelin-treated DCM mice had significantly prolonged life spans compared with saline-treated control DCM mice. Echocardiography showed that ghrelin reduced left ventricular (LV) end-diastolic dimensions and increased LV ejection fraction. Moreover, histoanatomical data revealed that ghrelin decreased the heart-to-body weight ratio, prevented cardiac remodeling and fibrosis, and markedly decreased the expression of brain natriuretic peptide. Telemetry recording and heart rate variability analysis showed that ghrelin suppressed the excessive cardiac sympathetic nerve activity (CSNA) and recovered the cardiac parasympathetic nerve activity. These results suggest that ghrelin has therapeutic benefits for survival as well as for the cardiac function and remodeling in heart failure probably through suppression of CSNA and recovery of cardiac parasympathetic nerve activity.

Effects of candesartan on electrical remodeling in the hearts of inherited dilated cardiomyopathy model mice.

Inherited dilated cardiomyopathy (DCM) is characterized by dilatation and dysfunction of the ventricles, and often results in sudden death or heart failure (HF). Although angiotensin receptor blockers (ARBs) have been used for the treatment of HF, little is known about the effects on postulated electrical remodeling that occurs in inherited DCM. The aim of this study was to examine the effects of candesartan, one of the ARBs, on cardiac function and electrical remodeling in the hearts of inherited DCM model mice (TNNT2 ΔK210). DCM mice were treated with candesartan in drinking water for 2 months from 1 month of age. Control, non-treated DCM mice showed an enlargement of the heart with prolongation of QRS and QT intervals, and died at t1/2 of 70 days. Candesartan dramatically extended the lifespan of DCM mice, suppressed cardiac dilatation, and improved the functional parameters of the myocardium. It also greatly suppressed prolongation of QRS and QT intervals and action potential duration (APD) in the left ventricular myocardium and occurrence of ventricular arrhythmia. Expression analysis revealed that down-regulation of Kv4.2 (Ito channel protein), KChIP2 (auxiliary subunit of Kv4.2), and Kv1.5 (IKur channel protein) in DCM was partially reversed by candesartan administration. Interestingly, non-treated DCM heart had both normal-sized myocytes with moderately decreased Ito and IKur and enlarged cells with greatly reduced K+ currents (Ito, IKur IK1 and Iss). Treatment with candesartan completely abrogated the emergence of the enlarged cells but did not reverse the Ito, and IKur in normal-sized cells in DCM hearts. Our results indicate that candesartan treatment suppresses structural remodeling to prevent severe electrical remodeling in inherited DCM.

In vivo effects of propyl gallate, a novel Ca2 + sensitizer, in a mouse model of dilated cardiomyopathy caused by cardiac troponin T mutation

AimsWe have previously demonstrated that propyl gallate has a Ca2+ sensitizing effect on the force generation in membrane-permeabilized (skinned) cardiac muscle fibers. However, in vivo beneficial effects of propyl gallate as a novel Ca2+ sensitizer remain uncertain. In the present study, we aim to explore in vivo effects of propyl gallate. Main methodsWe compared effects of propyl gallate on ex vivo intact cardiac muscle fibers and in vivo hearts in healthy mice with those of pimobendan, a clinically used Ca2+ sensitizer. The therapeutic effect of propyl gallate was investigated using a mouse model of dilated cardiomyopathy (DCM) with reduced myofilament Ca2+ sensitivity due to a deletion mutation ΔK210 in cardiac troponin T. Key findingsPropyl gallate, as well as pimobendan, showed a positive inotropic effect. Propyl gallate slightly increased the blood pressure without changing the heart rate in healthy mice, whereas pimobendan decreased the blood pressure probably through vasodilation via inhibition of phosphodiesterase and increased the heart rate. Propyl gallate prevented cardiac remodeling and systolic dysfunction and significantly improved the life-expectancy of knock-in mouse model of DCM with reduced myofilament Ca2+ sensitivity due to a mutation in cardiac troponin T. On the other hand, gallate, a similarly strong antioxidant polyphenol lacking Ca2+ sensitizing action, had no beneficial effects on the DCM mice. SignificanceThese results suggest that propyl gallate might be useful for the treatment of inherited DCM caused by a reduction in the myofilament Ca2+ sensitivity.

Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation.

Cardiac hypertrophy, an adaptive process that responds to increased wall stress, is characterized by the enlargement of cardiomyocytes and structural remodeling. It is stimulated by various growth signals, of which the mTORC1 pathway is a well-recognized source. Here, we show that loss of Flcn, a novel AMPK-mTOR interacting molecule, causes severe cardiac hypertrophy with deregulated energy homeostasis leading to dilated cardiomyopathy in mice. We found that mTORC1 activity was upregulated in Flcn-deficient hearts, and that rapamycin treatment significantly reduced heart mass and ameliorated cardiac dysfunction. Phospho-AMP-activated protein kinase (AMPK)-alpha (T172) was reduced in Flcn-deficient hearts and nonresponsive to various stimulations including metformin and AICAR (5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide). ATP levels were elevated and mitochondrial function was increased in Flcn-deficient hearts, suggesting that excess energy resulting from up-regulated mitochondrial metabolism under Flcn deficiency might attenuate AMPK activation. Expression of Ppargc1a, a central molecule for mitochondrial metabolism, was increased in Flcn-deficient hearts and indeed, inactivation of Ppargc1a in Flcn-deficient hearts significantly reduced heart mass and prolonged survival. Ppargc1a inactivation restored phospho-AMPK-alpha levels and suppressed mTORC1 activity in Flcn-deficient hearts, suggesting that up-regulated Ppargc1a confers increased mitochondrial metabolism and excess energy, leading to inactivation of AMPK and activation of mTORC1. Rapamycin treatment did not affect the heart size of Flcn/Ppargc1a doubly inactivated hearts, further supporting the idea that Ppargc1a is the critical element leading to deregulation of the AMPK-mTOR-axis and resulting in cardiac hypertrophy under Flcn deficiency. These data support an important role for Flcn in cardiac homeostasis in the murine model.

Phosphoinositide-Dependent Kinase 1 and mTORC2 Synergistically Maintain Postnatal Heart Growth and Heart Function in Mice

The protein kinase Akt plays a critical role in heart function and is activated by phosphorylation of threonine 308 (T308) and serine 473 (S473). While phosphoinositide-dependent kinase 1 (PDK1) is responsible for Akt T308 phosphorylation, the identities of the kinases for Akt S473 phosphorylation in the heart remain controversial. Here, we disrupted mTOR complex 2 (mTORC2) through deletion of Rictor in the heart and found normal heart growth and function. Rictor deletion caused significant reduction of Akt S473 phosphorylation but enhanced Akt T308 phosphorylation, suggesting that a high level of Akt T308 phosphorylation maintains Akt activity and heart function. Deletion of Pdk1 in the heart caused significantly enhanced Akt S473 phosphorylation that was suppressed by removal of Rictor, leading to worsened dilated cardiomyopathy (DCM) and accelerated heart failure in Pdk1-deficient mice. In addition, we found that increasing Akt S473 phosphorylation through deletion of Pten or chemical inhibition of PTEN reversed DCM and heart failure in Pdk1-deficient mice. Investigation of heart samples from human DCM patients revealed changes similar to those in the mouse models. These results demonstrated that PDK1 and mTORC2 synergistically promote postnatal heart growth and maintain heart function in postnatal mice.

Calponin1 inhibits dilated cardiomyopathy development in mice through the εPKC pathway

BackgroundCalponin1 (CNN1) is involved in the regulation of smooth muscle contraction in physiological situation and it also expresses abnormally in a variety of pathological situations. We found that the expression of CNN1 decreased significantly in the heart tissue of a cTnTR141W transgenic dilated cardiomyopathy (DCM) mouse model and an adriamycin (ADR)-induced DCM mouse model, suggesting that CNN1 is involved in the pathogenesis of DCM. However, the role of CNN1 on cardiac function, especially on pathogenesis of DCM, has not been clarified. In this study, we tested whether rescued expression of CNN1 could prevent the development of DCM and investigated its possible mechanisms. Methods and resultsThe DCM phenotypes were significantly improved with the transgenic expression of CNN1 in the cTnTR141W×CNN1 double transgenic (DTG) mice, which was demonstrated by the survival, cardiac geometry and function analyses, as well as microstructural and ultrastructural observations based on echocardiography and histology examination. The expression of CNN1 could also resist the cardiac geometry breakage and dysfunction in the ADR-induced DCM mice model. Meanwhile, the epsilon isoform of protein kinase C (εPKC) activator and inhibitor could reverse the activation of εPKC/ERK/mTOR pathway and DCM phenotypes in the cTnTR141W and cTnTR141W×CNN1 double transgenic (DTG) mice. ConclusionsεPKC/ERK/mTOR pathway activation induced by the rescued expression of CNN1 contributed to the improvement of cardiac dysfunction and pathological changes observed in the DTG mice. CNN1 could be a therapeutic target to prevent the development of DCM and heart failure (HF).

Atrial Natriuretic Peptide Affects Cardiac Remodeling, Function, Heart Failure, and Survival in a Mouse Model of Dilated Cardiomyopathy

Dilated cardiomyopathy is a frequent cause of heart failure and death. Atrial natriuretic peptide (ANP) is a biomarker of dilated cardiomyopathy, but there is controversy whether ANP modulates the development of heart failure. Therefore, we examined whether ANP affects heart failure, cardiac remodeling, function, and survival in a well-characterized, transgenic model of dilated cardiomyopathy. Mice with dilated cardiomyopathy with normal ANP levels survived longer than mice with partial ANP (P<0.01) or full ANP deficiency (P<0.001). In dilated cardiomyopathy mice, ANP protected against the development of heart failure as indicated by reduced lung water, alveolar congestion, pleural effusions, etc. ANP improved systolic function and reduced cardiomegaly. Pathological cardiac remodeling was diminished in mice with normal ANP as indicated by decreased ventricular interstitial and perivascular fibrosis. Mice with dilated cardiomyopathy and normal ANP levels had better systolic function (P<0.001) than mice with dilated cardiomyopathy and ANP deficiency. Dilated cardiomyopathy was associated with diminished cardiac transcripts for NP receptors A and B in mice with normal ANP and ANP deficiency, but transcripts for NP receptor C and C-type natriuretic peptide were selectively altered in mice with dilated cardiomyopathy and ANP deficiency. Taken together, these data indicate that ANP has potent effects in experimental dilated cardiomyopathy that reduce the development of heart failure, prevent pathological remodeling, preserve systolic function, and reduce mortality. Despite the apparent overlap in physiological function between the NPs, these data suggest that the role of ANP in dilated cardiomyopathy and heart failure is not compensated physiologically by other NPs.

Abstract 18794: More Than a Biomarker_ANP Modulates Heart Failure, Cardiac Function and Survival in Dilated Cardiomyopathy

Although atrial natriuretic peptide (ANP) is an established biomarker for heart failure, there is significant controversy whether ANP modifies or affects the progression of heart failure in patients with reduced contractile function and dilated cardiomyopathy (DCM). Therefore we examined the effects of ANP on survival, heart failure progression, fibrosis and cardiac function in a well-established model of DCM caused by the cardiac-targeted expression of a dominant negative CREB transgene. By comparison to DCM mice with normal ANP levels (DCM, ANP+/+) mice with partial ANP deficiency (DCM, ANP+/-) had reduced survival (p&lt;0.001) and, mice with full ANP deficiency (DCM, ANP-/-), had even greater reductions in survival than those with normal ANP or partial ANP deficiency (p&lt;0.001). Radiography showed that ANP-deficient DCM mice developed more severe heart failure with pleural effusions and pronounced cardiomegaly. These mice showed increased alveolar congestion (22.9±1.5% vs. 57.3±3.2%, p&lt;0.001) and increased heart weights (heart:body weight 0.54±0.04% vs. 0.95±0.05%). ANP-deficient DCM mice showed a more marked reduction in cardiac function as evidenced by decreased EF% (30.7±3.2 % vs. 16.0 ±1.5 %, p&lt;0.001) and enhanced cardiac dilation (LVIDd 4.51±0.11 mm vs. 5.85±0.10 mm, p&lt;0.001; LVIDs 3.9±0.1 mm vs. 5.0 mm±0.2, p&lt;0.001). ANP-deficient DCM mice had more severe cardiac interstitial fibrosis (4.54±0.3 % vs. 12.58±0.62%, p&lt;0.01) and perivascular fibrosis (ratio to lumen area 0.58±0.07 vs. 2.53±0.26, p&lt;0.01) as well as eccentric cardiomyocyte hypertrophy (cross sectional area 662±12 μm2 vs. 905±23 μm2, p&lt;0.001). Levels of cGMP were significantly reduced in ANP-deficient DCM mice (p&lt;0.001). We conclude that ANP profoundly modulates the progression of DCM in mice by reducing the severity of heart failure, improving contractility, reducing ventricular dilation, preventing cardiac fibrosis and extending survival. Thus ANP is not only a key biomarker, but it also modulates the progression of heart failure, cardiac function and remodeling in DCM.