DNA-mediated immunization in a transgenic mouse model of the hepatitis B surface antigen chronic carrier state.

Transgenic mice expressing the sequences coding for the envelope proteins of the hepatitis B virus (HBV) in the liver have been used as a model of the HBV chronic carrier state. We evaluated the possibility of inducing a specific immune response to the viral envelope antigens and thus potentially controlling chronic HBV infection. Using HBV-specific DNA-mediated immunization in this transgenic model, we show that the immune response induced after a single intramuscular injection of DNA resulted in the complete clearance of circulating hepatitis B surface antigen and in the long-term control of transgene expression in hepatocytes. This response does not involve a detectable cytopathic effect in the liver. Adoptive transfer of fractionated primed spleen cells from DNA-immunized mice shows that T cells are responsible for the down-regulation of HBV mRNA in the liver of transgenic mice. To our knowledge, this is the first demonstration of a potential immunotherapeutic application of DNA-mediated immunization against an infectious disease and raises the possibility of designing more effective ways of treating HBV chronic carriers.     

Stimulation of endogenous neurogenesis by anti-EFRH immunization in a transgenic mouse model of Alzheimer's disease

Neurogenesis is a subject of intense interest and extensive research, but it stands at the center of a bitter debate over ethical and practical problems. Neurodegenerative diseases, such as Alzheimer's disease (AD), accompanied by a shifting balance between neurogenesis and neurodegeneration, are suitable for stimulation of neurogenesis for the benefit of diseased patients. We have previously shown that Abs against the EFRH sequence of beta-amyloid peptide (AbetaP) prevent aggregation and disaggregate AbetaP both in vitro and in vivo. EFRH, located in the soluble tail of the N-terminal region, acts as a regulatory site controlling both solubilization and disaggregation processes in the AbetaP molecule. Here we show that anti-EFRH immunotherapy of a platelet-derived amyloid precursor protein transgenic mouse model of AD stimulates endogenous neurogenesis, suggested by elevated numbers of BrdU-incorporated cells, most of which are colocalized with a marker of mature neurons, NeuN. These newly born neurons expressed the activity-dependent gene Zif268, indicating their functional integration and participation in response to synaptic input in the brain. These findings suggest that anti-amyloid immunotherapy may promote recovery from AD or other diseases related to AbetaP overproduction and neurotoxicity by restoring neuronal population, as well as cognitive functions in treated patients.     

Oral immunization of animals with transgenic cherry tomatillo expressing HBsAg

AIM: To investigate the expression of recombinant HBsAg(rHBsAg) in transgenic cherry tomatillo in order to explore the feasibility of producing HBV oral vaccine with cherry tomatillo by animal immune tests.METHODS: The recombinant plant expression vector containing HBsAg gene was constructed. Mediated with Agrobacterium tumefaciens, HBsAg gene was transferred into cotyledons of cherry tomatillo. Transformed cherry tomatillos were obtained through hygromycin delay-selection. Integrated DNA in transgenic cherry tomatillo was confirmed by hygromycin resistance selection, Gus detection, polymerase chain reaction (PCR) and dot blotting analysis. Antigenicity of rHBsAg was examined by ELISA and the immunogenicity of rHBsAg derived from transgenic cherry tomatillo tissues was confirmed by oral feed of transformed tissues to BALB/c mice primed with commercial HBV vaccines. Specific antibody titers in mice‘s serum were examined by ELISA every week.RESULTS: By far, 10 positive lines of transgenic cherry tomatillos containing HBsAg gene were obtained. Among different organs of the same transgenic cherry tomatillo,level of rHBsAg expressed in leaves was the highest with the yield up to 300ng/g fresh weight. And the rHBsAg expression level in fruits was about 10ng/g fresh weight.In animal immune tests, oral delivery with transgenic tissues to mice primed with commercial vaccine instead of naive mice resulted in significant immune response.CONCLUSION: The result of this animal immune test indicated the rHBsAg derived from transgenic cherry tomatillo possessed normal immunogenicity. This work demonstrated the feasibility to generate oral immunogenic rHBsAg in transgenic cherry tomatillo, and would provide some experimental approach for the production of low-cost oral vaccines using transgenic cherry tomatillo in large scale.     

Oral immunization with hepatitis B surface antigen expressed in transgenic plants

Oral immunogenicity of recombinant hepatitis B surface antigen (HBsAg) derived from yeast (purified product) or in transgenic potatoes (uncooked unprocessed sample) was compared. An oral adjuvant, cholera toxin, was used to increase immune responses. Transgenic plant material containing HBsAg was the superior means of both inducing a primary immune response and priming the mice to respond to a subsequent parenteral injection of HBsAg. Electron microscopy of transgenic plant samples revealed evidence that the HBsAg accumulated intracellularly; we conclude that natural bioencapsulation of the antigen may provide protection from degradation in the digestive tract until plant cell degradation occurs near an immune effector site in the gut. The correlate of protection from hepatitis B virus infection is serum antibody titers induced by vaccination; the protective level in humans is 10 milliunits/ml or greater. Mice fed HBsAg-transgenic potatoes produced HBsAg-specific serum antibodies that exceeded the protective level and, on parenteral boosting, generated a strong long-lasting secondary antibody response. We have also shown the effectiveness of oral delivery by using a parenteral prime-oral boost immunization schedule. The demonstrated success of oral immunization for hepatitis B virus with an "edible vaccine" provides a strategy for contributing a means to achieve global immunization for hepatitis B prevention and eradication.     

Hyperdynamic circulatory syndrome in a mouse model transgenic for SerpinB3

Introduction and objectivesSerpinB3 is a cysteine protease inhibitor involved in several biological activities. It is progressively expressed in chronic liver disease, but not in normal liver. The role in vascular reactivity of this serpin, belonging to the same family of Angiotensin II, is still unknown. Our aim was to evaluate the in vivo and in vitro effects of SerpinB3 on systemic and splanchnic hemodynamics.Material and methodsDifferent hemodynamic parameters were evaluated by ultrasonography in two colonies of mice (transgenic for human SerpinB3 and C57BL/6J controls) at baseline and after chronic carbon tetrachloride (CCl₄) treatment. In vitro SerpinB3 effect on mesenteric microvessels of 5 Wistar-Kyoto rats was analyzed measuring its direct action on: (a) preconstricted arteries, (b) dose–response curves to phenylephrine, before and after inhibition of angiotensin II type 1 receptors with irbesartan. Hearts of SerpinB3 transgenic mice and of the corresponding controls were also analyzed by morphometric assessment.ResultsIn SerpinB3 transgenic mice, cardiac output (51.6 ± 21.5 vs 30.1 ± 10.8 ml/min, p = 0.003), hepatic artery pulsatility index (0.85 ± 0.13 vs 0.65 ± 0.11, p < 0.001) and portal vein blood flow (5.3 ± 3.2 vs 3.1 ± 1.8 ml/min, p = 0.03) were significantly increased, compared to controls. In vitro, recombinant SerpinB3 had no direct hemodynamic effect on mesenteric arteries, but it increased their sensitivity to phenylephrine-mediated vasoconstriction (p < 0.01). This effect was suppressed by inhibiting angiotensin II type-1 receptors.ConclusionsIn transgenic mice, SerpinB3 is associated with a hyperdynamic circulatory syndrome-like pattern, possibly mediated by angiotensin receptors.     

Prophylactic DNA vaccine for hepatitis C virus (HCV) infection: HCV-specific cytotoxic T lymphocyte induction and protection from HCV-recombinant vaccinia infection in an HLA-A2.1 transgenic mouse model

DNA vaccines express antigens intracellularly and effectively induce cellular immune responses. Because only chimpanzees can be used to model human hepatitis C virus (HCV) infections, we developed a small-animal model using HLA-A2.1-transgenic mice to test induction of HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs) and protection against recombinant vaccinia expressing HCV-core. A plasmid encoding the HCV-core antigen induced CD8(+) CTLs specific for three conserved endogenously expressed core peptides presented by human HLA-A2.1. When challenged, DNA-immunized mice showed a substantial (5-12 log(10)) reduction in vaccinia virus titer compared with mock-immunized controls. This protection, lasting at least 14 mo, was shown to be mediated by CD8(+) cells. Thus, a DNA vaccine expressing HCV-core is a potential candidate for a prophylactic vaccine for HLA-A2.1(+) humans.     

Oral immunization with a live recombinant attenuated Salmonella typhimurium protects mice against Toxoplasma gondii

The natural site of infection for T. gondii is the mucosal surface of the intestine, so the protective immunity obtained after natural infection with T. gondii points to the importance of developing a vaccine that stimulates mucosal defences. In this study, an aroA- and aroD- attenuated strain of Salmonella typhimurium (BRD509) has been used to deliver the recombinant eukaryotic plasmid pSAG(1-2)/CTA2/B expressing a multi-antigenic gene encoding SAG1 and SAG2 of T. gondii linked to A2/B subunits of cholera toxin as a candidate oral T. gondii vaccine. Immunoblot analysis showed compound gene expression in HeLa cells in vitro and intragastric immunization of mice with the recombinant salmonella resulted in the induction of humoral and Th1 type cellular immune responses and afforded protection against RH strain T. gondii challenge. Anti-T. gondii IgG values increased markedly in the BRD509/pSAG(1-2)-CTA2/B immunized group; these values were significantly higher than those in the negative controls (P = 0.008). With CTA2/B genetic adjuvant, the T. gondii-specific response was predominantly Th1, indicating that the CTA(2)/B genetic adjuvant was able to overcome the strong Th2-bias of the antigen (IgG2a > IgG1). Antigen-specific T cell proliferative responses and CTL activity were significantly enhanced when cholera toxin CTA2/B genetic adjuvant was used (P = 0.009; P = 0.006). Culture supernatants from antigen-stimulated splenocytes from mice in these groups were also examined by ELISA for Th1- and Th2-type cytokines; mean IFN-gamma levels produced after oral immunization with BRD509/pSAG(1-2)-CTA2/B were about nine-fold higher than after immunization with BRD509/pSAG(1-2) (P = 0.007). On the other hand, the levels of IL-4 were low for all groups and no increase was seen in the presence of CTA2/B genetic adjuvant. When the immunized mice were intraperitoneally challenged with 10(3) tachyzoites of the highly virulent RH strain, the survival time of the mice immunized with BRD509/pSAG(1-2)-CTA2/B was markedly longer than other groups (P = 0.003) and a 40% survival rate was achieved. This is the first report that demonstrates that an oral attenuated salmonella DNA vaccine can induce protective immunity against the acute phase of T. gondii infection.     

Amino acid-modified calcitonin immunization induces tumor epitope-specific immunity in a transgenic mouse model for medullary thyroid carcinoma

Up to now, no relevant tumor antigen has been identified in medullary thyroid carcinoma (MTC). The aim of the present study was to prove the concept of an immunization with an amino acid-modified calcitonin (CT) for the treatment of MTC in a transgenic mouse model. Amino acid-modified (human) CT has been chosen for vaccination because of its higher binding affinity to the murine H2-Kb-MHC molecule. Mice were immunized over 6 months with monthly injections of amino acid-modified CT-pulsed dendritic cells. For enumeration of tumor epitope-specific CD8+ cytotoxic T cells, tetramer analyses were performed. CT peptide-treated mice revealed a mean 0.73 +/- 0.45 and 0.91 +/- 0.59% positive cells, depending on the two tetramers tested, whereas no increase was seen in control protein-immunized mice (0.08-0.12% tetramer-positive cells). Importantly, the subset of CT-specific CD8+ T cells also showed a high expression of interferon-gamma. In line with these results, CT-immunized mice also showed an intratumor infiltration with CD8+ T lymphocytes. Importantly, we also found a diminished tumor outgrowth of -57% and a decrease of the serum CT levels (2.0 +/- 0.1 pg/ml) compared with control protein-immunized Ret/Cal mice (3.0 +/- 0.4 pg/ml). In summary, we show that amino acid-modified CT is recognized from the immune system leading to a specific antitumor immune response and a diminished tumor outgrowth in transgenic MTC mice. The results are of potential importance because they might be applicable to patients with metastatic spread of a MTC.     

表达幽门螺杆菌尿素酶B亚单位的减毒鼠伤寒杆菌口服疫苗的制备

目的制备抗幽门螺杆菌(Hp)尿素酶B亚单位(UreB)减毒鼠伤寒杆菌活菌疫苗,观察其免疫效果.方法构建表达UreB的原核表达载体PTc01-UreB并转化减毒鼠伤寒杆菌SL3261,得到重组菌SL3261/PTc01-UreB.应用抗Hp菌体蛋白兔血清行Western-blot检测UreB在SL3261中的表达.将SL3261/PTc01-UreB口服免疫Balb/c小鼠,12周后应用ELISA检测肠液和血清中的特异性抗体反应.SL3261/PTc01-UreB在Luria-Bertani培养液中连续传代60代以确定其稳定性.结果成功构建PTc01-UreB原核表达载体.Western-blot显示,其转化减毒鼠伤寒杆菌SL3261后能表达相对分子质量约61×103的蛋白,与HpUreB亚单位相符,具有抗原性.口服免疫小鼠后,在肠液和血清中可分别检测到针对UreB的特异性IgA和IgG抗体.体外连续培养60代未见PTc01-UreB质粒丢失及对宿主细胞毒性.结论表达HpUreB的减毒鼠伤寒杆菌SL3261/PTc01-UreB可用作抗Hp感染口服疫苗.     

Histopathology of alpha 1-antitrypsin liver disease in a transgenic mouse model.

Transgenic mice were constructed using human alpha 1-antitrypsin M and Z genomic clones. Livers of the M lineage mice showed slight cellular pleomorphism and immunohistochemically demonstrable finely granular alpha 1-antitrypsin material in hepatocytes. Z lineage mice with five gene copies per haploid mouse genome (Z#1) demonstrated fine granular alpha 1-antitrypsin material and a few large globules. In contrast, Z lineage mice with 12 gene copies per haploid mouse genome (Z#2) demonstrated hepatocytes filled with homogeneous, eosinophilic globules that were strongly reactive with diastase and periodic acid-Schiff and antibody to alpha 1-antitrypsin. Scattered microscopic polymorphonuclear leukocyte accumulations were seen that contained extracellular alpha 1-antitrypsin material, but there was neither histological nor serological evidence of mouse infectious hepatitis. In young animals, small clusters of hepatocytes lacking alpha 1-antitrypsin material were seen. These cells were the dominant population in older animals and formed nodular arrangements. Fibrosis was not demonstrable in neonatal and young animals or in any of the controls, but perisinusoidal fibrosis was seen in older Z#2 mice. Groups of hepatocytes without alpha 1-antitrypsin material showed dysplastic changes. We conclude that the transgenic mouse is a reliable and useful model in which to study the effects of alpha 1-antitrypsin in the liver because it demonstrates changes similar to those in the human disease.     

Oral immunization with live, avirulent fla+ strains of Salmonella protects mice against subsequent oral challenge with Salmonella typhimurium

Some strains of Salmonella, when fed to mice, establish a nonlethal, limited infection in the Peyer's patches of the small intestine. When such mice are later challenged orally with a normally lethal dose of virulent Salmonella typhimurium, a protective effect of the prior vaccination is seen. In an effort to characterize the determinant(s) of the avirulent strains responsible for this protective effect, we examined the cell envelope protein profiles of five such protective strains and of eight strains of Salmonella that were both nonpersistent and nonprotective when fed to mice. The protective strains produced high levels of flagellin. We made otherwise isogenic fla+ and fla- derivatives of two such strains and showed that although the fla- derivatives colonized mice as well as did the fla+ strains, the fla- derivatives given orally showed a much reduced ability to protect mice from S. typhimurium challenge. Both fla+ and fla- strains induced cellular immunity in vaccinated mice.     

Prostate cancer in a transgenic mouse.

Progress toward understanding the biology of prostate cancer has been slow due to the few animal research models available to study the spectrum of this uniquely human disease. To develop an animal model for prostate cancer, several lines of transgenic mice were generated by using the prostate-specific rat probasin promoter to derive expression of the simian virus 40 large tumor antigen-coding region. Mice expressing high levels of the transgene display progressive forms of prostatic disease that histologically resemble human prostate cancer, ranging from mild intraepithelial hyperplasia to large multinodular malignant neoplasia. Prostate tumors have been detected specifically in the prostate as early as 10 weeks of age. Immunohistochemical analysis of tumor tissue has demonstrated that dorsolateral prostate-specific secretory proteins were confined to well-differentiated ductal epithelial cells adjacent to, or within, the poorly differentiated tumor mass. Prostate tumors in the mice also display elevated levels of nuclear p53 and a decreased heterogeneous pattern of androgen-receptor expression, as observed in advanced human prostate cancer. The establishment of breeding lines of transgenic mice that reproducibly develop prostate cancer provides an animal model system to study the molecular basis of transformation of normal prostatic cells and the factors influencing the progression to metastatic prostate cancer.     

Nonapoptotic neurodegeneration in a transgenic mouse model of Huntington's disease

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder characterized by personality changes, motor impairment, and subcortical dementia. HD is one of a number of diseases caused by expression of an expanded polyglutamine repeat. We have developed several lines of mice that are transgenic for exon 1 of the HD gene containing an expanded CAG sequence. These mice exhibit a defined neurological phenotype along with neuronal changes that are pathognomonic for the disease. We have previously observed the appearance of neuronal intranuclear inclusions, but did not find evidence for neurodegeneration. In this study, we report that all lines of these mice develop a late onset neurodegeneration within the anterior cingulate cortex, dorsal striatum, and of the Purkinje neurons of the cerebellum. Dying neurons characteristically exhibit neuronal intranuclear inclusions, condensation of both the cytoplasm and nucleus, and ruffling of the plasma membrane while maintaining ultrastructural preservation of cellular organelles. These cells do not develop blebbing of the nucleus or cytoplasm, apoptotic bodies, or fragmentation of DNA. Neuronal death occurs over a period of weeks not hours. We also find degenerating cells of similar appearance within these same regions in brains of patients who had died with HD. We therefore suggest that the mechanism of neuronal cell death in both HD and a transgenic mouse model of HD is neither by apoptosis nor by necrosis.     

Peptide-induced T-cell tolerance to prevent autoimmune diabetes in a transgenic mouse model.

A synthetic peptide corresponding to an immunodominant epitope of lymphocytic choriomeningitis virus glycoprotein (LCMV GP) was used to prime or to tolerize CD8+ T cells in vivo, dependent on mode of immunization. Peptide-specific tolerance was then examined in transgenic mice expressing LCMV GP in the beta islet cells of the pancreas; these mice develop CD8+ T-cell-mediated diabetes within 8-14 days after LCMV infection. Specific peptide-induced tolerance prevented autoimmune destruction of beta islet cells and diabetes in this transgenic mouse model.     

HCV—specific cytokine induction in monocytes of patients with different oputcomes of hepatitis C

AIM：Cytokine release by macrophages critically determines the type of immune response to an antigen Therefore.we studied hepatitis C virus (HCV0-Specific induction of interleukins-1β,-10,-12(IL-1β,il-10,IL-12),and tumor necrosis factor-α(TNF-α) in monocytes.METHODS:Intracellular cytokine expression was studied by flow cytometry in 23 patients with chronic hepatitis C,14 anti-HCV seropositives without viremia and 11 controls after stimulation of peripheral blood mononuclear cells with recombinant core,NS3,NS4 NS5a and NS5b proteins .RESULTS:Patients with HCV viremia revealed greater spontaneous exprssion of IL-1β,TNF-α,and IL-10,Furthermore,greater than twofold higher IL-10 epression was induced by the HCV antigens in chronic hepatitis C than in the other two groups (P&lt;0.05) In contrast,neither IL-12 noir TNF-α was induced preferentially.CONCLUSION:In chonic hepatitis C antigen-specific cytokine induction in monocytes is apparently shifted towards predominant IL-10 induction-not counterbalanced by antiviral type 1 cytokines,This may contribute to persistent viral replication.     

结蛋白相关心肌病转基因小鼠闰盘重塑

目的　探讨结蛋白相关心肌病 (DRC)闰盘相关蛋白表达和分布的变化。方法　以 1个月DRC转基因小鼠为模型 ,采用Western印迹和免疫标记共聚焦显微镜观察闰盘相关蛋白在心室中的表达和分布。结果　与野生型结蛋白转基因小鼠和非转基因小鼠相比 ,突变型结蛋白小鼠 pan cadherin、α catenin、β catenin总蛋白升高 (P <0 0 5 ) ;α catenin和 β catenin以胞浆部分显著 (P <0 0 5 ) ;pan cadherin膜蛋白部分和胞浆部分升高 (P <0 0 5 ) ,而细胞骨架蛋白 (TIF)部分降低 ;Cx4 3总蛋白和TIF则明显降低 (P <0 0 5 ) ,存在着再分布。野生型结蛋白小鼠与非转基因小鼠比较 ,各蛋白均无显著变化。免疫标记证实这一结果。结论　DRC早期已发生闰盘重塑 ,闰盘蛋白表达和分布改变可能促进心律失常和心功能衰竭的发生。     

Studies on the immunogenicity of hCEA in a transgenic mouse model

BACKGROUND AND AIMS: Immunization protocols in mice have shown that the tumor-associated antigen hCEA could be a target for active immunization; however, human CEA is foreign to mice. Success may depend in part on a simple anti-xenoresponse. Using hCEA-transfected syngeneic tumor cells in hCEA-transgenic mice should bypass this problem and allow testing for new vaccination strategies. MATERIALS AND METHODS: We established a hCEA transgenic model of the haplotype H2(d), which may differ from other haplotypes in cytokine production and in effectiveness of antigen presentation, and tested two vaccination protocols in wild-type and transgenic mice. RESULTS: Syngeneic wild-type mice built up an immune response with high antibody titers; only 65% of animals developed solid tumors after tumor challenge. In contrast, hCEA-transgenic mice developed no antibody response and accepted the tumor in more than 90% of cases, thus demonstrating the role of human CEA as a foreign antigen. Accordingly, active immunization using tumor lysate or lymphocytes loaded with hCEA resulted in a CTL response and tumor-rejection in up to 80% of wild-type mice. hCEA-transgenic mice could be induced with both immunization protocols to build up a CTL response, although the number of CTL were much lower and the cytotoxic response weaker than in wild-type mice. In vivo hCEA-transgenic mice rejected hCEA-positive tumors only after immunization with the tumor lysate in about 60% whereas there was no rejection of tumors after immunization with the human hCEA-loaded autologous lymphocytes. CONCLUSION: The findings clearly show the importance of transgenic models when testing the effects of immunization towards human tumor associated antigens such as hCEA because results differ in wild-type and transgenic mice.     

Therapeutic immunization protects dopaminergic neurons in a mouse model of Parkinson's disease

Degeneration of the nigrostriatal dopaminergic pathway, the hallmark of Parkinson's disease, can be recapitulated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. Herein, we demonstrate that adoptive transfer of copolymer-1 immune cells to MPTP recipient mice leads to T cell accumulation within the substantia nigra pars compacta, suppression of microglial activation, and increased local expression of astrocyte-associated glial cell line-derived neurotrophic factor. This immunization strategy resulted in significant protection of nigrostriatal neurons against MPTP-induced neurodegeneration that was abrogated by depletion of donor T cells. Such vaccine treatment strategies may provide benefit for Parkinson's disease.     

Investigation of a transgenic mouse model of familial dilated cardiomyopathy

We have investigated a transgenic mouse model of inherited dilated cardiomyopathy that stably expresses the ACTC E361G mutation at around 50% of total actin in the heart. F-actin isolated from ACTC E361G mouse hearts was incorporated into thin filaments with native human tropomyosin and troponin and compared with NTG mouse actin by in vitro motility assay. There was no significant difference in sliding speed, fraction of filaments motile or Ca²⁺-sensitivity (ratio EC₅₀ E361G/NTG = 0.95 ± 0.08). The Ca²⁺-sensitivity of force in skinned trabeculae from ACTC E361G mice was slightly higher than NTG (EC₅₀ E361G/NTG = 0.78 ± 0.04). The molecular phenotype was revealed when troponin was dephosphorylated; Ca²⁺-sensitivity of E361G-containing thin filaments was now lower than NTG (EC₅₀ E361G_dPTn/NTG_dPTn = 2.15 ± 0.09). We demonstrated that this was due to uncoupling of Ca²⁺-sensitivity from troponin I phosphorylation by comparing Ca²⁺-sensitivity of phosphorylated and dephosphorylated thin filaments. For NTG actin-containing thin filaments EC₅₀ native/dPTn = 3.0 ± 0.3 but for E361G-containing thin filaments EC₅₀ native/dPTn = 1.04 ± 0.07.We studied contractility in isolated myocytes and found no significant differences under basal conditions. We measured cardiac performance by cine-MRI, echocardiography and with a conductance catheter over a period of 4 to 18 months and found minimal systematic differences between NTG and ACTC E361G mice under basal conditions. However, the increase in septal thickening, ejection fraction, heart rate and cardiac output following dobutamine treatment was significantly less in ACTC E361G mice compared with NTG. We propose that the ACTC E361G mutation uncouples myofilament Ca²⁺-sensitivity from Troponin I phosphorylation and blunts the response to adrenergic stimulation, leading to a reduced cardiac reserve with consequent contractile dysfunction under stress, leading to dilated cardiomyopathy.