Alpha-myosin Heavy Chain Promoter Research Articles

Abstract P3122: Latent Mouse Model Of HIV-1

Background: Antiretroviral therapy (ART) significantly improves the life span of people living with HIV/AIDS (PLWHA). However, HIV- associated cardiovascular diseases (CVDs) remain one of the leading causes of heart failure and comorbidity in PLWHA. Modern ART regimens reduce viral loads to undetectable levels in HIV+ patients while being less toxic. Nevertheless, HIV proteins are present in the plasma of PLWHA. The HIV protein, Nef, has been found circulating in the plasma of ART-treated patients within extracellular vesicles and in association with immune cells. We hypothesized that circulating HIV Nef proteins may cause an adverse effect on various organ functions. To further understand the effect of Nef on differing organ functions, we have generated a transgenic mouse model which expresses the Nef protein in the presence of Cre recombinase. Methods and Results: Nef transgenic mouse model was generated by cloning HIV-1 Nef ORF in CAG-Lox-CAT vector. Transgenic mice lines were crossed with the alpha myosin heavy chain promoter Cre mice to express Nef protein in the heart. Western blot data validated Nef protein expression in the adult mouse heart. Histological evaluation of the effect of Nef protein expression on heart morphology revealed cardiac hypertrophy. Furthermore, heart function analysis shows that Nef expressing mouse lines have depressed heart function. Conclusion: Our study suggests that Nef causes cardiac dysfunction that mimics cardiomyopathy secondary to latent HIV in PLWHA. Further, this mouse model could be useful in studying the effect of Nef protein in different organs or cells using cell-specific Cre recombinase. Moreover, Nef transgenic mice could be useful in generating new therapeutic tools to control comorbidity in PLWHA.

Targeted Genetic Reduction of Mutant Huntingtin Lessens Cardiac Pathology in the BACHD Mouse Model of Huntington's Disease.

Individuals affected by Huntington's disease (HD) present with progressive degeneration that results in a wide range of symptoms, including cardiovascular (CV) dysfunction. The huntingtin gene (HTT) and its product are ubiquitously expressed, hence, the cardiomyopathy could also be driven by defects caused by its mutated form (mHTT) in the cardiomyocytes themselves. In the present study, we sought to determine the contribution of the mHTT expressed in the cardiomyocytes to CV symptoms. We utilized the BACHD mouse model, which exhibits many of the HD core symptoms, including CV dysfunction. This model allows the targeted genetic reduction of mHTT expression in the cardiomyocytes while maintaining the expression of the mHTT in the rest of the body. The BACHD line was crossed with a line of mice in which the expression of Cre recombinase is driven by the cardiac-specific alpha myosin-heavy chain (Myh6) promoter. The offspring of this cross (BMYO mice) exhibited a dramatic reduction in mHTT in the heart but not in the striatum. The BMYO mice were evaluated at 6 months old, as at this age, the BACHD line displays a strong CV phenotype. Echocardiogram measurements found improvement in the ejection fraction in the BMYO line compared to the BACHD, while hypertrophy was observed in both mutant lines. Next, we examined the expression of genes known to be upregulated during pathological cardiac hypertrophy. As measured by qPCR, the BMYO hearts exhibited significantly less expression of collagen1a as well as Gata4, and brain natriuretic peptide compared to the BACHD. Fibrosis in the hearts assessed by Masson's trichrome stain and the protein levels of fibronectin were reduced in the BMYO hearts compared to BACHD. Finally, we examined the performance of the mice on CV-sensitive motor tasks. Both the overall activity levels and grip strength were improved in the BMYO mice. Therefore, we conclude that the reduction of mHtt expression in the heart benefits CV function in the BACHD model, and suggest that cardiomyopathy should be considered in the treatment strategies for HD.

Cardiomyocyte‐Specific Transgenic MMP9 Overexpression Induces Cardiac Remodeling

Objective Activated matrix metalloproteinase-9 (MMP9), a collagenase, promotes cardiac remodeling leading to dysfunction. MMP9 is secreted by a wide variety of cells including cardiomyocytes. Although the role of MMP9 secreted from leukocytes and fibroblasts have been studied, very little is known about the role of cardiomyocyte-specific MMP9. Hypothesis Cardiomyocyte-specific MMP9 overexpression induces cardiac remodeling. Materials and Methods We have developed a novel cardiomyocytes-specific MMP9 transgenic (M9Tg) mouse strain. We used alpha-myosin heavy chain promoter and knocked in MMP9 gene using CRISPR-Cas9 method. After validation through genotyping, we performed gravimetric analyses and evaluated cardiac function by M-Mode echocardiography (short-axis), and hemodynamic changes by pressure-volume loop (open-chest, 4mm catheter, Transonic) studies. Results Eighteen-week old male M9Tg mice had a higher body weight and other organ's weight compared to the sibling WT. In four mice, the comparison of M9Tg/WT: body weight (g) = 26.8/24.8, heart weight (mg) = 119.0/107.0, Liver (mg) = 1309.5/1203.3, Kidney (mg) = 159.6/141.6, lung (mg) = 154.8/141.3). There was no significant change in the size of these organs relative to their body weight in M9Tg mice. The M-mode echocardiography analysis of three mice showed a decrease in cardiac output (WT= 15.8 ± 1.1, M9Tg =13.5 ± 0.8, P= 0.038) and a trend of decrease in fractional shortening (WT =27.4 ± 3.6, M9Tg = 24.9 ± 2.0, P = 0.231) and an increase in left ventricular mass (WT = 75.1 ± 4.1, M9Tg = 80.5 ± 3.8, P = 0.12). The pressure-volume loop study with two WT and three M9Tg mice showed an increase in dp/dt max (average, WT =5881.5, M9Tg = 7211.3 mmHg/s) and decrease in end systolic volume (WT = 22.1, M9Tg = 17.4 µL), suggesting a potential increase in cardiac contractility. Conclusion Cardiomyocyte-specific overexpression of MMP9 potentially increased left ventricular mass and cardiac contractility without altering fractional shortening. Further studies are ongoing to increase the sample size and examine the changes in the heart at the histological and molecular levels.

Susceptibility to Cardiac Arrhythmias and Sympathetic Nerve Growth in VEGF-B Overexpressing Myocardium

VEGF-B gene therapy is a promising proangiogenic treatment for ischemic heart disease, but, unexpectedly, we found that high doses of VEGF-B promote ventricular arrhythmias (VAs). VEGF-B knockout, alpha myosin heavy-chain promoter (αMHC)-VEGF-B transgenic mice, and pigs transduced intramyocardially with adenoviral (Ad)VEGF- B186 were studied. Immunostaining showed a 2-fold increase in the number of nerves per field (76 vs. 39 in controls, p< 0.001) and an abnormal nerve distribution in the hypertrophic hearts of 11- to 20-month-old αMHC-VEGF-B mice. AdVEGF-B186 gene transfer (GT) led to local sprouting of nerve endings in pig myocardium (141 vs. 78 nerves per field in controls, p< 0.05). During dobutamine stress, 60% of the αMHC-VEGF-B hypertrophic mice had arrhythmias as compared to 7% in controls, and 20% of the AdVEGF-B186-transduced pigs and 100% of the combination of AdVEGF-B186- and AdsVEGFR-1-transduced pigs displayed VAs and even ventricular fibrillation. AdVEGF-B186 GT significantly increased the risk of sudden cardiac death in pigs when compared to any other GT with different VEGFs (hazard ratio, 500.5; 95% confidence interval [CI] 46.4-5,396.7; p< 0.0001). In gene expression analysis, VEGF-B induced the upregulation of Nr4a2, ATF6, and MANF in cardiomyocytes, molecules previously linked to nerve growth and differentiation. Thus, high AdVEGF-B186 overexpression induced nerve growth in the adult heart via a VEGFR-1 signaling-independent mechanism, leading to an increased risk of VA and sudden cardiac death.

Abstract 303: Fast Skeletal Myosin Binding Protein-c Expression in Heart Failure

Background: Heart failure is a devastating disease affecting nearly 5 million Americans. We recently identified the upregulation of fast skeletal myosin binding protein-C (fsMyBP-C) in models of heart failure. fsMyBP-C is a homologue of cardiac MyBP-C, which is normally expressed in the heart. This family of proteins regulate contractility by modulating thick and thin filament interactions. However, the functional role(s) of fsMyBP-C expression in the heart is unknown. Methods: To understand the role of fsMyBP-C expression in the heart, we generated cardiac specific transgenic mice expressing fsMyBP-C under the control of the alpha myosin heavy chain promoter. Mice underwent serial echocardiography to monitor cardiac function. Western blotting and qRT-PCR were performed to determine transgene expression levels. Additionally, fsMyBP-C localization in cardiomyocytes was established by immunofluorescence. Furthermore, to understand the temporal expression profiles of the MyBP-C homologues, we performed qRT-PCR on 9 muscle groups at different developmental stages in healthy mice. Results: qRT-PCR demonstrated an upregulation of the slow MyBP-C transcript in the developing heart, while fsMyBP-C transcript levels increased steadily following birth in WT mice. At 3 months of age, transgenic mice displayed a significant increase in ventricular dimensions compared to NTG littermates by echocardiography (LVID d : 3.72 ± 0.07 vs. 4.17 ± 0.09 mm, p&lt;0.01; LVID s : 2.44 ± 0.09 vs. 2.81 ± 0.09, p&lt;0.05), with no changes in wall thickness. This was accompanied by a 25% increase in stroke volume (p&lt;0.01) and a 30% increase in cardiac output (p&lt;0.05). fsMyBP-C correctly localized to the C-zone of the sarcomere. Furthermore, protein levels showed mild expression of fsMyBP-C, in line with the small upregulation seen in diseased hearts, while qRT-PCR confirmed overexpression of fsMyBP-C in the ventricles. Conclusion: Mice expressing fsMyBP-C in the heart display a mild hypertrophic phenotype at 3 months, with preserved function, possibly indicating compensated hypertrophy. Ongoing studies investigate cardiac function at later stages and cardiac specific conditional knockout of fsMyBP-C to determine the long-term effect of fsMyBP-C regulation in the heart.

Cardiac-Specific Deletion of Pyruvate Dehydrogenase Impairs Glucose Oxidation Rates and Induces Diastolic Dysfunction.

Obesity and type 2 diabetes (T2D) increase the risk for cardiomyopathy, which is the presence of ventricular dysfunction in the absence of underlying coronary artery disease and/or hypertension. As myocardial energy metabolism is altered during obesity/T2D (increased fatty acid oxidation and decreased glucose oxidation), we hypothesized that restricting myocardial glucose oxidation in lean mice devoid of the perturbed metabolic milieu observed in obesity/T2D would produce a cardiomyopathy phenotype, characterized via diastolic dysfunction. We tested our hypothesis via producing mice with a cardiac-specific gene knockout for pyruvate dehydrogenase (PDH, gene name Pdha1), the rate-limiting enzyme for glucose oxidation. Cardiac-specific Pdha1 deficient (Pdha1Cardiac−/−) mice were generated via crossing a tamoxifen-inducible Cre expressing mouse under the control of the alpha-myosin heavy chain (αMHC-MerCreMer) promoter with a floxed Pdha1 mouse. Energy metabolism and cardiac function were assessed via isolated working heart perfusions and ultrasound echocardiography, respectively. Tamoxifen administration produced an ~85% reduction in PDH protein expression in Pdha1Cardiac−/− mice versus their control littermates, which resulted in a marked reduction in myocardial glucose oxidation and a corresponding increase in palmitate oxidation. This myocardial metabolism profile did not impair systolic function in Pdha1Cardiac−/− mice, which had comparable left ventricular ejection fractions and fractional shortenings as their αMHC-MerCreMer control littermates, but did produce diastolic dysfunction as seen via the reduced mitral E/A ratio. Therefore, it does appear that forced restriction of glucose oxidation in the hearts of Pdha1Cardiac−/− mice is sufficient to produce a cardiomyopathy-like phenotype, independent of the perturbed metabolic milieu observed in obesity and/or T2D.

Gerald W. Dorn II: Thinker, Teacher, Tinkerer.

Cardiologist and molecular and cell biologist Gerald Dorn, MD, has built his research career crafting interesting questions and teaching himself techniques needed to find answers. Using approaches including genetic and physiological manipulation in animal models, development of mini-mouse technology to elucidate cardiac mysteries, and human genetics/genomics, Dorn is helping to unravel cellular and molecular mechanisms underlying cardiomyopathy and heart failure.1,2 Gerald W. Dorn II Dorn, 57, is founding director of the Center for Pharmacogenomics at the Washington University in St. Louis School of Medicine, where he joined the faculty in 2008. As a teenager Dorn sped through high school, starting college two years early. He earned his medical degree and trained in internal medicine, pharmacology, and clinical and interventional cardiology at the Medical University of South Carolina (MUSC) in Charleston, where two key mentors sparked in him a passion for patient care and research. After a couple of years at the University of Texas Health Science Center at San Antonio, Dorn moved to the University of Cincinnati, where an American Heart Association Established Investigator Award kick-started his research program. From 1990 to 2008 Dorn rose through the ranks at Cincinnati, capitalizing on the institution’s strength in mouse cardiac transgenesis. Dorn’s current interests include molecular mechanisms of cell death,3 microRNA regulation of cardiac genes,4 and mitochondrial dynamism5—work that has expanded to Parkinson’s disease. Building on an upbringing that valued lifelong growth, and drawing from his own broad experience, Dorn now leverages the holism of systems biology to fill in what he calls his field’s “infinitely large shades of gray.” My father was a career naval aviator. Whenever he moved, we moved. So I’m not really from anywhere. We spent a lot of time on both coasts. As a child I lived, in addition to …

Диференціювання індукованих плюрипотентних стовбурових клітин в кардіоміоцити в суспензійній культурі з постійним перемішуванням та в спеціальних планшетах з мікролунками

In order to enhance the differentiation of induced pluripotent cells into cardiomyocytes, we compared two methods of embryoid bodies formation: differentiation in rotating suspension culture and formation of embryoid bodies from a predetermined number of pluripotent stem cells in microwells of AggreWell plates. We used transgenic murine induced pluripotent stem cell line AT25. Cell line expressed IRES-flanked enhanced green fluorescent protein (eGFP) under the control of cardiac alpha myosin heavy chain promoter (αMHC). We applied flow cytometry and fluorescence microscopyin order to test the efficiency of differentiation processes. Thus, differentiation of pluripotent stem cells in AggreWell plates without adding differentiation factors was more effective than differentiation in rotating suspension culture. However, we obtained the most amounts of cardiomyocytes on the 11-th day in rotating suspension culture with ascorbic acid, after we applied dorsomorfin, DMSO, ascorbic acid, G-CSF with the above-mentioned methods. The amount of GFP + cells was 2,71 ± 0,07%.

Involvement of activated SUMO-2 conjugation in cardiomyopathy

Sumoylation is a posttranslational modification that regulates a wide spectrum of cellular activities. Cardiomyopathy is the leading cause of heart failure. Whether sumoylation, particularly SUMO-2/3 conjugation, is involved in cardiomyopathy has not been investigated. We report here that SUMO-2/3 conjugation was elevated in the human failing hearts, and we investigated the impact of increased SUMO-2 conjugation on heart function by using the gain-of-function approach in mice, in which cardiac specific expression of constitutively active SUMO-2 was governed by alpha myosin heavy chain promoter (MHC-SUMO-2 transgenic, SUMO-2-Tg). Four of five independent SUMO-2-Tg mouse lines exhibited cardiomyopathy with various severities, ranging from acute heart failure leading to early death to the development of chronic cardiomyopathy with aging. We further revealed that SUMO-2 directly regulated apoptotic process by at least partially targeting calpain 2 and its natural inhibitor calpastatin. SUMO conjugation to calpain 2 promoted its enzymatic activity, and SUMO attachment to calpastatin mainly promoted its turnover and altered its subcellular distribution. Thus, enhanced SUMO-2 conjugation led to increased apoptosis and played a pathogenic role in the development of cardiomyopathy and heart failure.

The influence of dexamethasone administration on the protection against doxorubicin-induced cardiotoxicity in purified embryonic stem cell-derived cardiomyocytes

Embryonic stem cells (ESCs) have various uses in drug toxicity, as they can be easily differentiated in vitro. However, one of the major obstacles in the assessment of these differentiated cells is the presence of a heterogeneous cell population. To circumvent this problem, purified ESC-derived desired cells by means of the tissue-specific GFP and/or antibiotic resistance gene expression has been proposed. Therefore, this study aimed to assess the role of doxorubicin (DOX) in cardiotoxicity by using genetically engineered purified ESC-derived cardiomyocytes under the control alpha-myosin heavy chain promoter. The results revealed that ESCs are suitable for evaluation of DOX cardiotoxicity. This study showed that DOX cardiotoxicity was reduced as detected by beating cardiomyocytes and caspase activity only by pretreatment with dexamethasone (DEX), not during or post-DOX treatment. DEX influence appears to be mediated via glucocorticoid receptor and enhances cardiomyocyte-specific gene expression. Therefore, for the general assessment of cytotoxicity, non-genetically engineered ESC-derived cardiomyocytes are sufficient but for the molecular assessment of DOX-induced toxicity, genetically engineered purified ESC-derived cardiomyocytes are necessary.

Abstract 149: Effect of Pentraxin 3 on Pressure Overload--Induced Left Ventricular Hypertrophy

Backgrounds: Left ventricular (LV) adverse remodeling is enhanced by inflammatory cytokines. Pentraxin 3 (PTX3), which is one of the pentraxin superfamily same as C-reactive protein, is rapidly produced in response to inflammatory signals, and plasma PTX3 levels are increased in patients with heart failure. Previously we reported that PTX3 deficient mice demonstrated the suppression of hypertrophic cardiac remodeling after pressure overload. This study aimed to examine the cardiac specific influence of PTX3 on LV hypertrophy and dysfunction in response to pressure overload. Methods and Results: We created transgenic (TG) mice with cardiac-specific overexpression of PTX3 using alpha-myosin heavy chain promoter. We performed transverse aortic constriction (TAC) or sham operation on TG and wild type (WT) littermate mice. Expression of PTX3 was increased after TAC in WT mice, and immunohistochemistry proved that PTX3 was produced mainly from the interstitial tissue of the heart and infiltrating macrophages. Activation of extracellular signal-regulated kinase (ERK) and nuclear factor kappa-B was enhanced in TG mice compared to WT mice after TAC. Interleukin-6 production in heart tissue was augmented in TG mice compared to WT mice. Echocardiography and cardiac catheterization revealed that adverse remodeling with LV dysfunction was enhanced with increased interstitial fibrosis in TG mice compared to WT mice. Real time RT-PCR demonstrated that gene expressions related to cardiac hypertrophy and fibrosis were augmented in TG mice compared to WT mice. Next we isolated neonatal rat cardiomyocytes and fibroblasts. PTX3 expressions were increased after H 2 O 2 stimulation in cardiomyocytes and fibroblasts. Phosphorylation of ERK by recombinant PTX3 stimulation was more potent in cardiac fibroblasts than in cardiomyocytes. Conclusions: The local inflammatory mediator PTX3 directly modulates the hypertrophic response and LV dysfunction following increased afterload, and cardiac fibroblast might be important target cells of PTX3 stimulation.

Involvement of Autophagy in Cardiac Remodeling in Transgenic Mice with Cardiac Specific Over-Expression of Human Programmed Cell Death 5

Programmed cell death 5 (PDCD5) is a cytosolic protein suppressing growth of multiple types of cancer cells through activating p53. We hypothesized that PDCD5 plays an essential role in cardiac remodeling and function. PDCD5 was significantly up-regulated in the hearts from mice subjected to angiotensin II treatment or transverse aortic constriction. Thus, we generated transgenic mice over-expressing human PDCD5 under the control of alpha myosin heavy chain promoter to examine the role of PDCD5 in cardiac remodeling. Transgenic founder died spontaneously displayed enlarged heart. The high PDCD5 over-expressing line (10-fold) showed reduced survival rate, increase in heart weight normalized to body weight. Real-Time RT-PCR analysis revealed fetal gene program was up-regulated. Echocardiography and histopathological examination showed characteristics of dilated cardiomyopathy and heart failure in transgenic mice. Western blot and immunohistochemistry analysis showed autophagy was dramatically increased in transgenic mice as compared to WT littermates control mice, while apoptosis remained unchanged. The enhanced autophagy in high over-expressing line was associated with significant increase in p53 activity and its downstream target damage-regulated autophagy modulator expression. The low over-expressing line (3.5-fold) appeared normal, but was more susceptible to angiotensin II-induced cardiac hypertrophy. This study is the first providing evidence that PDCD5 plays an important role in cardiac remodeling.

Abstract P066: Imaging Autophagy in Living Mice

Autophagy is a homeostatic response to cellular stress. It has been shown to be potently upregulated in the heart in response to a variety of interventions. However, to date, it has not been possible to monitor autophagy without sacrificing the animal. Here we report the use of the Caliper Life Sciences Spectrum In Vivo Imaging System (IVIS) to image autophagy in homozygous transgenic mice expressing mCherryLC3 under control of the alpha myosin heavy chain promoter. Autophagy was stimulated by the administration of rapamycin (2mg/kg), and autophagosomal flux was blocked by administration of chloroquine (10mg/kg) ip. Mice were imaged at baseline and four hours later, using a protocol of 3 acquisitions of 15 seconds each. Total flux was 3.19±0.72 before drug administration and 3.93+1.10 after 4 hr, p&lt;;0.01, n=14. These results show for the first time imaging of autophagy in hearts of live mice.

Targeted Sprouty1 overexpression in cardiac myocytes does not alter myocardial remodeling or function

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (alpha-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male alpha-MHC-Spry1 mice compared to control mice. Ventricular function of alpha-MHC-Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.

Intergenic Bidirectional Promoter of Cardiac Myosin Heavy Chain (MHC) Genes in Rats

The rat heart expresses two MHC isoforms: beta (b) and alpha (a); these genes are arranged in tandem on the chromosome. We have hypothesized that there is an intergenic bidirectional promoter between these two genes that both promotes transcription of the a isoform and natural antisense of the b isoform. This intergenic bidirectional promoter may hold the key to the coordinated and antithetical regulation of a and b MHC genes that has been long recognized in the field of cardiac muscle regulation. Using a method of in vivo injection of promoter‐reporter plasmids, we have tested promoter activities of intergenic sequence in antisense and sense direction and found that activity requires an intact NF1 site! In fact, NF1 mutation caused a dramatic decrease in activity of the full length alpha promoter, which is very active in rat heart. NF1 site, is not conserved in the sequence of larger mammals. This is especially noteworthy because there is a divergence of b MHC expression in large vs. small mammals: the human and “large” mammals express the b isoform primarily in ventricular muscle whereas the small rodents, eg. the rat, express the a isoform primarily. Yet, both humans and rats express the beta MHC in “slow” fibers of skeletal muscles. To further understand the regulation of isoforms genes, we also tested tissue‐specificity and species specificity of alpha MHC promoter in rat skeletal muscle and heart. Supported by ADA and NIHHBL

Identification of the heart as the critical site of adenosine mediated embryo protection

BackgroundOur understanding of the mechanisms that protect the developing embryo from intrauterine stress is limited. Recently, adenosine has been demonstrated to play a critical role in protecting the embryo against hypoxia via adenosine A1 receptors (A1ARs), which are expressed in the heart, nervous system, and other sites during development. However, the sites of A1AR action that mediate embryo protection are not known. To determine if the heart is a key site of adenosine-mediated embryo protection, A1ARs were selectively deleted in the embryonic heart using a Cre-LoxP system in which the alpha-myosin heavy chain promoter drives Cre-recombinase expression and excision of the A1AR gene from cardiomyocytes.ResultsWith increasing exposure of maternal hypoxia (10% O2) from 48-96 hours beginning at embryonic day (E) 8.5, embryo viability decreased in the cardiac-A1AR deleted embryos. 48 hours of hypoxia reduced embryonic viability by 49% in embryos exposed from E10.5-12.5 but no effect on viability was observed in younger embryos exposed to hypoxia from E8.5-10.5. After 72 hours of hypoxia, 57.8% of the cardiac-A1AR deleted embryos were either dead or re-absorbed compared to 13.7% of control littermates and after 96 hours 81.6% of cardiac-A1AR deleted embryos were dead or re-absorbed. After 72 hours of hypoxia, cardiac size was reduced significantly more in the cardiac-A1AR deleted hearts compared to controls. Gene expression analysis revealed clusters of genes that are regulated by both hypoxia and A1AR expression.ConclusionsThese data identify the embryonic heart as the critical site where adenosine acts to protect the embryo against hypoxia. As such these studies identify a previously unrecognized mechanism of embryo protection.

Cardiac Specific Overexpression of N-RAP in Transgenic Mice

The muscle specific protein NRAP plays a role in myofibril assembly and is upregulated in mouse models of dilated cardiomyopathy. We sought to determine if increased N-RAP expression would directly lead to a cardiomyopathy phenotype. Novel transgenic lines were developed using the tet-off system with transgenic N-RAP expression requiring the tetracycline transactivator (tTA). tTA was introduced by mating the N-RAP transgenic animals with well-characterized animals carrying the tTA transgene controlled by the cardiac specific alpha-myosin heavy chain promoter. Multiple founder lines were examined and lines showing the most significant increase in NRAP expression were used for further investigation. N-RAP expression in theses animals was up to 2.5 times greater than control littermates as determined by western blot analysis. Histological examination of hearts from ∼12 week old transgenic mice showed no structural defects compared to control littermates. Additionally, examination of these hearts by immunofluorescence microscopy revealed normal myofibrillar structure and localization of the transgenic protein to intercalated disks, as normally seen with the endogenous protein. Protein markers for cardiomyopathy were examined by qPCR and revealed no difference between non-transgenic and transgenic animals. Echocardiography and magnetic resonance imaging of the N-RAP transgenic animals revealed no significant structural or functional differences when compared to control littermates at 12 weeks of age. Based on these data, it does not appear that overexpression of N-RAP directly leads to an observable cardiac phenotype. The alternative hypothesis that upregulation of N-RAP in dilated cardiomyopathy is compensatory remains to explored.

Expression and Regulation of Shark NCX Gene in Transgenic Mouse Heart

Inward currents generated by the mammalian cardiac Na+-Ca2+-exchanger (NCX1.1) have arrhythmogenic potential, especially in the failing heart where expression of NCX is up-regulated. We hypothesize that arrhythmogenesis might be alleviated if NCX were subject to the same cAMP mediated regulation (suppression of Ca2+-influx, but enhancement of Ca2+-efflux on NCX) as found in the native shark ventricle. To test this hypothesis, we created heterozygous transgenic mice that express the shark NCX protein with a myc-tag, under the control of the alpha-myosin heavy chain promoter (α-MHC). The construct was evaluated by expression and functionality prior to production of transgenic lines. The expression of the transgene was confirmed by immunocytochemistry staining using DAPI and myc-FITC antibody on transfected HL-1 cells. Using dual laser confocal microscopy, the pattern of staining was consistent with NCX expression at the protein level. To determine the functionality of the transgene, HL-1 cells were co-transfected with the shark NCX transgene and GFP. GFP positive cells were incubated with Fluo-4 AM and imaged confocally. These cells showed NCX activity in response to withdrawal and readmission of [Na+]o consistent with electrophysiological data of native shark myocytes.Echocardiography and ECG studies on transgenic mice showed no remarkable cardiac phenotype, but analysis of initial voltage-clamp and western blot studies verify robust exchanger currents and expression levels. Our findings show that shark NCX is functional in the transgenic mouse producing no discernable cardiac pathology, but it remains to be determined as yet whether shark NCX can confer anti-arrhythmic properties to the mammalian heart.

DGAT1 Expression Increases Heart Triglyceride Content but Ameliorates Lipotoxicity

Intracellular lipid accumulation in the heart is associated with cardiomyopathy, yet the precise role of triglyceride (TG) remains unclear. With exercise, wild type hearts develop physiologic hypertrophy. This was associated with greater TG stores and a marked induction of the TG-synthesizing enzyme diacylglycerol (DAG) acyltransferase 1 (DGAT1). Transgenic overexpression of DGAT1 in the heart using the cardiomyocyte- specific alpha-myosin heavy chain (MHC) promoter led to approximately a doubling of DGAT activity and TG content and reductions of approximately 35% in cardiac ceramide, 26% in DAG, and 20% in free fatty acid levels. Cardiac function assessed by echocardiography and cardiac catheterization was unaffected. These mice were then crossed with animals expressing long-chain acyl-CoA synthetase via the MHC promoter (MHC-ACS), which develop lipotoxic cardiomyopathy. MHC-DGAT1XMHC-ACS double transgenic male mice had improved heart function; fractional shortening increased by 74%, and diastolic function improved compared with MHC-ACS mice. The improvement of heart function correlated with a reduction in cardiac DAG and ceramide and reduced cardiomyocyte apoptosis but increased fatty acid oxidation. In addition, the survival of the mice was improved. Our study indicates that TG is not likely to be a toxic lipid species directly, but rather it is a feature of physiologic hypertrophy and may serve a cytoprotective role in lipid overload states. Moreover, induction of DGAT1 could be beneficial in the setting of excess heart accumulation of toxic lipids.

Losartan reduces mortality in a model of heart failure

Calsequestrin (CSQ) is the main Ca2+ store in the cardiac sarcoplasmic reticulum (SR). Overexpression of CSQ selectively in the heart of transgenic mice (TG) under the control of the alpha myosin heavy chain promoter leads to hypertrophy, fibrosis, heart failure, cardiac arrhythmias and ultimately premature death compared to littermate controls (WT). We hypothesized that losartan, an inhibitor of angiotensin II receptors, may be able to abrogate these detrimental effects. Hence, TG and WT were treated for about 2 or 5 months perorally with losartan (5 mg/Kg body weight) or solvent alone. Under control conditions (solvent alone), none of the WT died within the observation period whereas all TG died within 9 months. In the treatment group the mortality of TG was significantly reduced: mean life span was raised from 116 d to 192.5 d (n=18). Likewise, the degree of fibrosis was reduced. Hemodynamic parameters (pressure; dp/dt) were improved. However, the relative heart weight remained unchanged. Likewise, the expression of CSQ in the heart was unchanged by losartan treatment. In conclusion, the renin angiotensin system is apparently involved in heart failure due to CSQ overexpression, because its blockade improves survival (supported by the DFG).