Abstract 123: Erythropoietin Enhances Abundance of Cardiomyogenic Precursors in vitro and in vivo

Recent data suggest that erythropoietin (Epo) can improve cardiac function following ischemia reperfusion injury. We found EpoR to be prominently expressed in embryonic rather than adult mouse heart. In the latter EpoR expression was confined to interstitial cells. We isolated the non-myocyte fraction of adult mouse hearts by enzymatic digestion and straining (pore size: 30 µm) and found a 4-fold higher EpoR expression compared to unselected cardiac cells (n=3). Flow cytometry (FC) revealed that 24±3% of this heterogeneous cell pool expressed EpoR (n=3) and 11±2% co-expressed αMHC. Half of the αMHC+ cells stained positive for Ki67, consistent with FC showing that 49±7% αMHC+/EpoR+ cells were in S and G2 phase (n=3; vs. 13±3% in αMHC-/EpoR-; n=3). Interestingly, co-culture with neonatal rat cardiomyocytes yielded EGFP+ cardiomyocyte-like cells. Since αMHC+ proliferated and differentiated in vitro we termed them cardiomyogenic precursors (CMPs). Epo treatment of the non-myocyte pool enhanced Akt phosphorylation (n=6/group) and increased CMP abundance in vitro (2-fold; n=3). Cell cycle arrest abrogated the aforementioned effect, suggesting that Epo influenced CMP proliferation. Finally, we tested the potential of Epo to protect against ischemia by enhancing CMP numbers in a model of myocardial infarction (MI). Following MI mice were treated twice with 2,000 U/kg Epo (i.p.). Ten weeks post MI echocardiography revealed blunted myocardial deterioration in mice receiving Epo (ΔEF=-11.18%, n=5) compared to control (ΔEF=-20.82%, n=6). FC revealed an enhanced αMHC+/cTnT+ cell pool in the Epo treated group (20.3±1.9% vs. 10.6±2.3% in control, n=8/6) 4 weeks post MI. In conclusion, we found that EpoR is expressed in a putative cardiomyogenic precursor cell pool in the adult heart. CMPs appear to proliferate in vitro and in vivo; with further enhanced proliferation under Epo administration. This may contribute to Epo mediated protection after MI and highlights the therapeutic effect of Epo upon ischemic cardiac injury. Whether these cells remain from embryogenesis or just constitute an immature myocyte population in the adult heart deriving from cardiomyocyte dedifferentiation needs to be further investigated.

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CTRP5-Overexpression Attenuated Ischemia-Reperfusion Associated Heart Injuries and Improved Infarction Induced Heart Failure

Frontiers in Pharmacology ◽

10.3389/fphar.2020.603322 ◽

2020 ◽

Vol 11 ◽

Author(s):

Meng Peng ◽

Yuan Liu ◽

Xiang-qin Zhang ◽

Ya-wei Xu ◽

Yin-tao Zhao ◽

...

Keyword(s):

Heart Failure ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Mouse Heart ◽

Related Protein ◽

Rat Cardiomyocytes ◽

Knock Out ◽

Cardiac Functions

Aims: C1q/tumor necrosis factor (TNF)-related protein 5 (CTRP5) belongs to the C1q/TNF-α related protein family and regulates glucose, lipid metabolism, and inflammation production. However, the roles of CTRP5 in ischemia/reperfusion (I/R) associated with cardiac injuries and heart failure (HF) needs to be elaborated. This study aimed to investigate the roles of CTRP5 in I/R associated cardiac injuries and heart failure.Materials and Methods: Adeno-associated virus serum type 9 （AAV9）vectors were established for CTRP5 overexpression in a mouse heart (AAV9-CTRP5 mouse). AAV9-CTRP5, AMPKα2 global knock out （AMPKα2−/−）and AAV9-CTRP5+ AMPKα2−/− mice were used to establish cardiac I/R or infarction associated HF models to investigate the roles and mechanisms of CTRP5 in vivo. Isolated neonatal rat cardiomyocytes (NRCMS) transfected with or without CTRP5 adenovirus were used to establish a hypoxia/reoxygenation (H/O) model to study the roles and mechanisms of CTRP5 in vitro.Key Findings: CTRP5 was up-regulated after MI but was quickly down-regulated. CTRP5 overexpression significantly decreased I/R induced IA/AAR and cardiomyocyte apoptosis, and attenuated infarction area, and improved cardiac functions. Mechanistically, CTRP5 overexpression markedly increased AMPKα2 and ACC phosphorylation and PGC1-α expression but inhibited mTORC1 phosphorylation. In in vitro experiments, CTRP5 overexpression could also enhance AMPKα2 and ACC phosphorylation and protect against H/O induced cardiomyocytes apoptosis. Finally, we showed that CTPR5 overexpression could not protect against I/R associated cardiac injuries and HF in AMPKα2−/− mice.Significance: CTRP5 overexpression protected against I/R induced mouse cardiac injuries and attenuated myocardial infarction induced cardiac dysfunction by activating the AMPKαsignaling pathway.

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Overexpression of TIMP3 Protects Against Cardiac Ischemia/Reperfusion Injury by Inhibiting Myocardial Apoptosis Through ROS/Mapks Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000485401 ◽

2017 ◽

Vol 44 (3) ◽

pp. 1011-1023 ◽

Cited By ~ 16

Author(s):

Hui Liu ◽

Xibo Jing ◽

Aiqiao Dong ◽

Baobao Bai ◽

Haiyan Wang

Keyword(s):

Myocardial Infarct ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Tunel Staining ◽

Ros Production ◽

Rat Cardiomyocytes ◽

Doxorubicin Treatment ◽

Myocardial Apoptosis

Background/Aims: Myocardial ischemia/reperfusion (I/R) injury remains a great challenge in clinical therapy. Tissue inhibitor of metalloproteinases 3 (TIMP3) plays a crucial role in heart physiological and pathophysiological processes. However, the effects of TIMP3 on I/R injury remain unknown. Methods: C57BL/6 mice were infected with TIMP3 adenovirus by local delivery in myocardium followed by I/R operation or doxorubicin treatment. Neonatal rat cardiomyocytes were pretreated with TIMP3 adenovirus prior to anoxia/reoxygenation (A/R) treatment in vitro. Histology, echocardiography, in vivo phenotypical analysis, flow cytometry and western blotting were used to investigate the altered cardiac function and underlying mechanisms. Results: The results showed that upregulation of TIMP3 in myocardium markedly inhibited myocardial infarct areas and the cardiac dysfunction induced by I/R or by doxorubicin treatment. TUNEL staining revealed that TIMP3 overexpression attenuated I/R-induced myocardial apoptosis, accompanied by decreased Bax/Bcl-2 ratio, Cleaved Caspase-3 and Cleaved Caspase-9 expression. In vitro, A/R-induced cardiomyocyte apoptosis was abrogated by pharmacological inhibition of reactive oxygen species (ROS) production or MAPKs signaling. Attenuation of ROS production reversed A/R-induced MAPKs activation, whereas MAPKs inhibitors showed on effect on ROS production. Furthermore, in vivo or in vitro overexpression of TIMP3 significantly inhibited I/R- or A/R-induced ROS production and MAPKs activation. Conclusion: Our findings demonstrate that TIMP3 upregulation protects against cardiac I/R injury through inhibiting myocardial apoptosis. The mechanism may be related to inhibition of ROS-initiated MAPKs pathway. This study suggests that TIMP3 may be a potential therapeutic target for the treatment of I/R injury.

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Resveratrol Inhibits Ischemia-Induced Myocardial Senescence Signals and NLRP3 Inflammasome Activation

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/2647807 ◽

2020 ◽

Vol 2020 ◽

pp. 1-20

Author(s):

Hong Feng ◽

Shan-qi Mou ◽

Wen-jing Li ◽

Nan Zhang ◽

Zi-ying Zhou ◽

...

Keyword(s):

Nuclear Translocation ◽

Sirtuin 1 ◽

Neonatal Rat ◽

Tunel Staining ◽

Mouse Heart ◽

Inflammasome Activation ◽

Coronary Artery Ligation ◽

Rat Cardiomyocytes

Aims. The aim of this study was to investigate whether resveratrol (RSV) could ameliorate ischemia- and hypoxia-associated cardiomyocyte apoptosis and injury via inhibiting senescence signaling and inflammasome activation. Materials and Methods. Mice were treated with RSV by gastric tube (320 mg/kg/day) or vehicle one week before left coronary artery ligation or sham surgery until the end of the experiments. After pressure–volume loop analysis, mouse hearts were harvested for histopathological (including PSR, TTC, TUNEL staining, immunohistochemistry, and immunofluorescence) and molecular analysis by western blotting and RT-PCR. In addition, neonatal rat cardiomyocytes (NRCMs), cardiac fibroblasts (CFs), and macrophages were isolated for in vitro experiments. Key Findings. RSV treatment decreased mortality and improved cardiac hemodynamics. RSV inhibited the expression of senescence markers (p53, p16, and p19), inflammasome markers (NLRP3 and Cas1 p20), and nuclear translocation of NF-κB, hence alleviating infarction area, fibrosis, and cell apoptosis. RSV also inhibited expression of interleukin- (IL-) 1β, IL-6, tumor necrosis factor-α, and IL-18 in vivo. In in vitro experiment, RSV prevented hypoxia-induced NRCM senescence and apoptosis. After inhibition of sirtuin 1 (Sirt1) by EX27, RSV failed to inhibit p53 acetylation and expression. Moreover, RSV could inhibit expression of NLRP3 and caspase 1 p20 in NRCMs, CFs, and macrophages, respectively, in in vitro experiments. Significance. Our findings revealed that RSV protected against ischemia-induced mouse heart injury in vivo and hypoxia-induced NRCM injury in vitro via regulating Sirt1/p53-mediated cell senescence and inhibiting NLRP3-mediated inflammasome activation.

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Abstract 124: Function of the Cytoskeletal Proteins Alpha-catenins in Myocardial Growth Control

Circulation Research ◽

10.1161/res.113.suppl_1.a124 ◽

2013 ◽

Vol 113 (suppl_1) ◽

Author(s):

Jifen Li ◽

Sarah Carrante ◽

Roslyn Yi ◽

Frans van Roy ◽

Glenn L. Radice

Keyword(s):

Heart Development ◽

Growth Control ◽

Cytoskeletal Proteins ◽

Neonatal Rat ◽

Nuclear Accumulation ◽

Mouse Heart ◽

Cardiomyocyte Proliferation ◽

Rat Cardiomyocytes ◽

Neonatal Cardiomyocytes

Introduction: Mammalian heart possesses regenerative potential immediately after birth and lost by one week of age. The mechanisms that govern neonatal cardiomyocyte proliferation and regenerative capacity are poorly understood. Recent reports indicate that Yap-Tead transcriptional complex is necessary and sufficient for cardiomyocyte proliferation. During postnatal development, N-cadherin/catenin adhesion complex becomes concentrated at termini of cardiomyocytes facilitating maturation of a specialized intercellular junction structure, the intercalated disc (ICD). This process coincides with the time cardiomyocytes exit cell cycle soon after birth. Hypothesis: We hypothesize that coincident with maturation of ICD α-catenins sequester transcriptional coactivator Yap in cytosol thus preventing activation of genes critical for cardiomyocyte proliferation. Methods: We deleted αE-catenin / αT-catenin genes (α-cat DKO) in perinatal mouse heart and knockdown (KD) α-catenins in neonatal rat cardiomyocytes to study functional impact of α-catenins ablation on ICD maturation. Results: We previously demonstrated that adult α-cat DKO mice exhibited decrease in scar size and improved function post myocardial infarction. In present study, we investigated function of α-catenins during postnatal heart development. We found increase in the number of Yap-positive nuclei (58.7% in DKO vs. 35.8 % in WT, n=13, p<0.001) and PCNA (53.9% in DKO vs. 47.8%, n=8, p<0.05) at postnatal day 1 and day 7 of α-cat DKO heart, respectively. Loss of α-catenins resulted in reduction in N-cadherin at ICD at day 14. We observed an increase number of mononucleated myocytes and decrease number of binucleated myocytes in α-cat DKO compared to controls. Using siRNA KD, we were able to replicate α-cat DKO proliferative phenotype in vitro. The number of BrdU-positive cells was decreased in α-cat KD after interfering with Yap expression (2.91% in α-cat KD vs. 2.02% in α-cat/Yap KD, n>2500 cells, p<0.05), suggesting α-catenins regulate cell proliferation through Yap in neonatal cardiomyocytes. Conclusion: Our results suggest that maturation of ICD regulates α-catenin-Yap interactions in cytosol, thus preventing Yap nuclear accumulation and cardiomyocyte proliferation.

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Targeted disruption of PDE3B, but not PDE3A, protects murine heart from ischemia/reperfusion injury

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1416230112 ◽

2015 ◽

Vol 112 (17) ◽

pp. E2253-E2262 ◽

Cited By ~ 43

Author(s):

Youn Wook Chung ◽

Claudia Lagranha ◽

Yong Chen ◽

Junhui Sun ◽

Guang Tong ◽

...

Keyword(s):

Connexin 43 ◽

Mitochondrial Permeability Transition ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Permeability Transition Pore ◽

Permeability Transition ◽

Mouse Heart ◽

Targeted Disruption

Although inhibition of cyclic nucleotide phosphodiesterase type 3 (PDE3) has been reported to protect rodent heart against ischemia/reperfusion (I/R) injury, neither the specific PDE3 isoform involved nor the underlying mechanisms have been identified. Targeted disruption of PDE3 subfamily B (PDE3B), but not of PDE3 subfamily A (PDE3A), protected mouse heart from I/R injury in vivo and in vitro, with reduced infarct size and improved cardiac function. The cardioprotective effect in PDE3B−/− heart was reversed by blocking cAMP-dependent PKA and by paxilline, an inhibitor of mitochondrial calcium-activated K channels, the opening of which is potentiated by cAMP/PKA signaling. Compared with WT mitochondria, PDE3B−/− mitochondria were enriched in antiapoptotic Bcl-2, produced less reactive oxygen species, and more frequently contacted transverse tubules where PDE3B was localized with caveolin-3. Moreover, a PDE3B−/− mitochondrial fraction containing connexin-43 and caveolin-3 was more resistant to Ca2+-induced opening of the mitochondrial permeability transition pore. Proteomics analyses indicated that PDE3B−/− heart mitochondria fractions were enriched in buoyant ischemia-induced caveolin-3–enriched fractions (ICEFs) containing cardioprotective proteins. Accumulation of proteins into ICEFs was PKA dependent and was achieved by ischemic preconditioning or treatment of WT heart with the PDE3 inhibitor cilostamide. Taken together, these findings indicate that PDE3B deletion confers cardioprotective effects because of cAMP/PKA-induced preconditioning, which is associated with the accumulation of proteins with cardioprotective function in ICEFs. To our knowledge, our study is the first to define a role for PDE3B in cardioprotection against I/R injury and suggests PDE3B as a target for cardiovascular therapies.

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Effects of the Delta Opioid Receptor Agonist DADLE in a Novel Hypoxia-Reoxygenation Model on Human and Rat-Engineered Heart Tissue: A Pilot Study

Biomolecules ◽

10.3390/biom10091309 ◽

2020 ◽

Vol 10 (9) ◽

pp. 1309

Author(s):

Sandra Funcke ◽

Tessa R. Werner ◽

Marc Hein ◽

Bärbel M. Ulmer ◽

Arne Hansen ◽

...

Keyword(s):

Clinical Trials ◽

Ischemia Reperfusion ◽

Heart Tissue ◽

Contractile Force ◽

Neonatal Rat ◽

Rat Cardiomyocytes ◽

Engineered Heart Tissue ◽

Hypoxia Reoxygenation

Intermittent hypoxia and various pharmacological compounds protect the heart from ischemia reperfusion injury in experimental approaches, but the translation into clinical trials has largely failed. One reason may lie in species differences and the lack of suitable human in vitro models to test for ischemia/reperfusion. We aimed to develop a novel hypoxia-reoxygenation model based on three-dimensional, spontaneously beating and work performing engineered heart tissue (EHT) from rat and human cardiomyocytes. Contractile force, the most important cardiac performance parameter, served as an integrated outcome measure. EHTs from neonatal rat cardiomyocytes were subjected to 90 min of hypoxia which led to cardiomyocyte apoptosis as revealed by caspase 3-staining, increased troponin I release (time control vs. 24 h after hypoxia: cTnI 2.7 vs. 6.3 ng/mL, ** p = 0.002) and decreased contractile force (64 ± 6% of baseline) in the long-term follow-up. The detrimental effects were attenuated by preceding the long-term hypoxia with three cycles of 10 min hypoxia (i.e., hypoxic preconditioning). Similarly, [d-Ala2, d-Leu5]-enkephalin (DADLE) reduced the effect of hypoxia on force (recovery to 78 ± 5% of baseline with DADLE preconditioning vs. 57 ± 5% without, p = 0.012), apoptosis and cardiomyocyte stress. Human EHTs presented a comparable hypoxia-induced reduction in force (55 ± 5% of baseline), but DADLE failed to precondition them, likely due to the absence of δ-opioid receptors. In summary, this hypoxia-reoxygenation in vitro model displays cellular damage and the decline of contractile function after hypoxia allows the investigation of preconditioning strategies and will therefore help us to understand the discrepancy between successful conditioning in vitro experiments and its failure in clinical trials.

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Mechanical stimulation of organogenic cardiomyocyte growth in vitro

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.5.c1284 ◽

1996 ◽

Vol 270 (5) ◽

pp. C1284-C1292 ◽

Cited By ~ 38

Author(s):

H. H. Vandenburgh ◽

R. Solerssi ◽

J. Shansky ◽

J. W. Adams ◽

S. A. Henderson

Keyword(s):

Mechanical Load ◽

Mechanical Stretch ◽

Neonatal Rat ◽

Cardiomyocyte Hypertrophy ◽

Heart Cells ◽

Mechanical Stimuli ◽

Rat Cardiomyocytes ◽

Morphological Alterations

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.

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Myricetin Alleviates Pathological Cardiac Hypertrophy via TRAF6/TAK1/MAPK and Nrf2 Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/6304058 ◽

2019 ◽

Vol 2019 ◽

pp. 1-14 ◽

Cited By ~ 4

Author(s):

Hai-han Liao ◽

Nan Zhang ◽

Yan-yan Meng ◽

Hong Feng ◽

Jing-jing Yang ◽

...

Keyword(s):

Cardiac Hypertrophy ◽

Pressure Overload ◽

Mapk Signaling ◽

Neonatal Rat ◽

Mouse Heart ◽

Pathological Cardiac Hypertrophy ◽

Pathological Hypertrophy ◽

Nrf2 Signaling Pathway

Myricetin (Myr) is a common plant-derived polyphenol and is well recognized for its multiple activities including antioxidant, anti-inflammation, anticancer, and antidiabetes. Our previous studies indicated that Myr protected mouse heart from lipopolysaccharide and streptozocin-induced injuries. However, it remained to be unclear whether Myr could prevent mouse heart from pressure overload-induced pathological hypertrophy. Wild type (WT) and cardiac Nrf2 knockdown (Nrf2-KD) mice were subjected to aortic banding (AB) surgery and then administered with Myr (200 mg/kg/d) for 6 weeks. Myr significantly alleviated AB-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction in both WT and Nrf2-KD mice. Myr also inhibited phenylephrine- (PE-) induced neonatal rat cardiomyocyte (NRCM) hypertrophy and hypertrophic markers’ expression in vitro. Mechanically, Myr markedly increased Nrf2 activity, decreased NF-κB activity, and inhibited TAK1/p38/JNK1/2 MAPK signaling in WT mouse hearts. We further demonstrated that Myr could inhibit TAK1/p38/JNK1/2 signaling via inhibiting Traf6 ubiquitination and its interaction with TAK1 after Nrf2 knockdown in NRCM. These results strongly suggested that Myr could attenuate pressure overload-induced pathological hypertrophy in vivo and PE-induced NRCM hypertrophy via enhancing Nrf2 activity and inhibiting TAK1/P38/JNK1/2 phosphorylation by regulating Traf6 ubiquitination. Thus, Myr might be a potential strategy for therapy or adjuvant therapy for malignant cardiac hypertrophy.

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Abstract 15925: Newt Exosomes are Bioactive on Mammalian Heart, Enhancing Proliferation of Rat Cardiomyocytes and Improving Recovery After Myocardial Infarction

Circulation ◽

10.1161/circ.132.suppl_3.15925 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Eleni Tseliou ◽

Liu Weixin ◽

Jackelyn Valle ◽

Baiming Sun ◽

Maria Mirotsou ◽

...

Keyword(s):

Myocardial Infarction ◽

Critical Role ◽

Dermal Fibroblasts ◽

Neonatal Rat ◽

Cardiomyocyte Proliferation ◽

Rat Cardiomyocytes ◽

Mesodermal Cell ◽

Wistar Kyoto

Introduction: Adult newts can regenerate amputated cardiac tissue (and whole limbs) without fibrosis, unlike adult mammals which lack such regenerative capacity. Exosomes are nanoparticles which mediate intercellular communication and play a critical role in therapeutic regeneration. Hypothesis: We isolated exosomes from a newt mesodermal cell line, and evaluated their bioactivity in rat models. Methods: A1 cells, derived from the amputated limb buds of Notopthalmus viridescense (Brockes JP, 1988), were expanded in culture. Exosomes were isolated by polyethylene glycol precipitation of A1-conditioned serum-free media (or media conditioned by human dermal fibroblasts [DF] as a control) followed by centrifugation. Bioactivity was tested in vitro on neonatal rat ventricular myocytes (NRVM), and in vivo on acute myocardial infarction in Wistar-Kyoto rats (250μg or 500μg of A1-exosomes or vehicle [placebo] injected intramyocardially). Functional and histological analyses were performed 3 weeks after therapy. Results: A1-conditioned media yielded ~2.8±1Billion particles/ml of 129±1.1 nm diameter. In vitro, A1-exosomes increased the proliferative capacity of NRVM compared to DF-exosomes (4.98±0.89% vs 0.77±0.33%, p=0.035). Priming of DFs with A1-exosomes increased SDF-1 secretion compared to DF-exosomes (755±117pg/ml vs.368±21pg/ml, p=0.03). In vivo, both A1-exosome doses increased cardiac function compared to placebo (EF= 46±1% in 250μg, 49±4% in 500μg vs 36±1% in placebo, p=0.045 by ANOVA). Scar size was markedly decreased (11±1% in 250μg, 9±2% in 500μg vs 18±2% in placebo, p=0.006 by ANOVA), and infarct wall thickness was increased after A1-exosome treatment (1.7±0.11mm in 250μg, 1.85±0.16mm in 500μg vs 1.17±0.11mm in Placebo, p=0.01 by ANOVA). Donor-specific antibodies were present at barely detectable levels in the serum of animals that had been injected with A1-exosomes. Conclusions: Newt exosomes stimulate rat cardiomyocyte proliferation and improve functional and structural outcomes in rats with myocardial infarction. Characterization of the RNA and protein content of newt exosomes, now in progress, may provide clues regarding conserved (or newt-unique) molecular mediators of therapeutic benefit.

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Abstract 1828: Dyxin/Lmcd1 Mediates Cardiac Hypertrophy Both In Vitro And In Vivo

Circulation ◽

10.1161/circ.118.suppl_18.s_393-a ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Derk Frank ◽

Robert Frauen ◽

Christiane Hanselmann ◽

Christian Kuhn ◽

Rainer Will ◽

...

Keyword(s):

Transgenic Mice ◽

Cardiac Hypertrophy ◽

Neonatal Rat ◽

Heart Weight ◽

Cardiomyocyte Hypertrophy ◽

The Novel ◽

Rat Cardiomyocytes ◽

A Genome

In order to identify new molecular mediators of cardiomyocyte hypertrophy, we performed a genome wide mRNA microarray screen of biomechanically stretched neonatal rat cardiomyocytes (NRCM). We found the novel sarcomeric LIM protein Dyxin/Lmcd1 being significantly upregulated (5.6x, p<0.001). Moreover, Dyxin was also significantly induced in several mouse models of myocardial hypertrophy including aortic banding, calcineurin overexpression and angiotensin stimulation, suggesting a potential role as a mediator of cardiac hypertrophy. To further test this hypothesis, we adenovirally overexpressed Dyxin in NRCM which potently induced cellular hypertrophy (150%, p<0.001) and the hypertrophic gene program (ANF, BNP). Consistent with an induction of calcineurin signalling, the calcineurin-responsive gene Rcan1– 4 (MCIP1.4) was found significantly upregulated (3.2x, p<0.001). Conversely, knockdown of Dyxin (−75% on protein level) via miRNA completely blunted the hypertrophic response to hypertrophic stimuli, including stretch and PE (both p<0.001). Furthermore, PE-mediated activation of calcineurin signaling (Upregulation of Rcan1– 4 by 7.3x, p<0.001) was completely blocked by knockdown of Dyxin. To confirm these results in vivo, we next generated transgenic mice with cardiac-restricted overexpression of Dyxin using the α -MHC promoter. Despite normal cardiac function as assessed by echocardiography, adult transgenic mice displayed significant cardiac hypertrophy in morphometrical analyses (3.9 vs. 3.5 mg/g LV/heart weight, n=8–11, p<0.05). This finding was supplemented by a robust induction of the hypertrophic gene program including ANF (3.7-fold, n=6, p=0.01) and α -skeletal actin (2.8-fold, n=6, p<0.05). Likewise, Rcan1– 4 was found upregulated (+112%, n=5, p<0.05), Taken together, we show that the novel sarcomeric z-disc protein Dyxin/Lmcd1 is significantly upregulated in several models of cardiac hypertrophy and potently induces cardiomyocyte hypertrophy both in vitro and in vivo. Mechanistically, Lmcd1/Dyxin appears to signal through the calcineurin pathway.

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