Short-term Rapamycin Treatment Research Articles

A cohort study on IVF outcomes in infertile endometriosis patients: the effects of rapamycin treatment

Research questionCan rapamycin, used as an anti-senescence drug before IVF cycles, improve the IVF outcomes of women with endometriosis by reducing oxidative stress and senescence? DesignThis retrospective analysis involved 168 endometriosis patients undergoing two successive IVF cycles. The treatment group (n = 80) received 3 months of rapamycin before the second IVF cycle, while the non-treatment group (n = 88) had no adjuvant treatment. The first and second IVF cycles were compared between and within groups. Follicular fluid from both IVF cycles (treatment group n = 28, non-treatment group n = 32) were tested for senescence-associated and oxidative stress-related markers. ResultsComparing the follicular fluid from the second cycles, the rapamycin treatment group showed decreased markers of oxidative stress (8-hydroxydesoxyguanosine, malondialdehyde) and increased antioxidant markers (superoxidase dismutase, glutathione peroxidase) (all P < 0.001); the expression of senescence-related markers p16 and p21 was also significantly lower in the treatment group than in the non-treatment group (P < 0.001). Comparing the second IVF cycles, the treatment group needed fewer days of ovarian stimulation, with increased numbers of oocytes obtained and mature oocytes compared with the non-treatment group (all P < 0.001). Furthermore, rates of fertilization, implantation and clinical pregnancy were significantly higher in the treatment group (P = 0.008, 0.034 and 0.038 respectively), as was the live birth rate (P = 0.003). No structural abnormalities were seen in fetuses born to women treated with rapamycin. ConclusionsThis study suggested an association between short-term rapamycin treatment and improved pregnancy outcomes via IVF. This result needs to be further investigated in prospective randomized controlled clinical trials.

Effect of short-term rapamycin treatment in flies and mice

Effect of short-term rapamycin treatment in flies and mice

Short-term rapamycin treatment increases life span and attenuates aortic aneurysm in a murine model of Marfan-Syndrome

BackgroundMarfan syndrome (MFS) is a genetic disorder leading to medial aortic degeneration and life-limiting dissections. To date, there is no causal prevention or therapy. Rapamycin is a potent and selective inhibitor of the mechanistic target of rapamycin (mTOR) protein kinase, regulating cell growth and metabolism. The mgR/mgR mice represent an accepted MFS model for studying aortic pathologies to understand the underlying molecular pathomechanisms. This study investigated whether rapamycin inhibits the development of thoracic aortic aneurysms and dissections in mgR/mgR mice. MethodsIsolated primary aortic smooth muscle cells (mAoSMCs) from mgR/mgR mice were used for in vitro studies. Two mg kg/BW rapamycin was injected intraperitoneally daily for two weeks, beginning at 7–8 weeks of age. Mice were sacrificed 30 days post-treatment. Histopathological and immunofluorescence analyses were performed using adequate tissue specimens and techniques. Animal survival was evaluated accompanied by periodic echocardiographic examinations of the aorta. ResultsThe protein level of the phosphorylated ribosomal protein S6 (p-RPS6), a downstream target of mTOR, was significantly increased in the aortic tissue of mgR/mgR mice. In mAoSMCs isolated from these animals, expression of mTOR, p-RPS6, tumour necrosis factor α, matrix metalloproteinase-2 and -9 was significantly suppressed by rapamycin, demonstrating its anti-inflammatory capacity. Short-term rapamycin treatment of Marfan mice was associated with delayed aneurysm formation, medial aortic elastolysis and improved survival. ConclusionsShort-term rapamycin-mediated mTOR inhibition significantly reduces aortic aneurysm formation and thus increases survival in mgR/mgR mice. Our results may offer the first causal treatment option to prevent aortic complications in MFS patients.

Rapamycin treatment during development extends life span and health span of male mice and Daphnia magna.

Development is tightly connected to aging, but whether pharmacologically targeting development can extend life remains unknown. Here, we subjected genetically diverse UMHET3 mice to rapamycin for the first 45 days of life. The mice grew slower and remained smaller than controls for their entire lives. Their reproductive age was delayed without affecting offspring numbers. The treatment was sufficient to extend the median life span by 10%, with the strongest effect in males, and helped to preserve health as measured by frailty index scores, gait speed, and glucose and insulin tolerance tests. Mechanistically, the liver transcriptome and epigenome of treated mice were younger at the completion of treatment. Analogous to mice, rapamycin exposure during development robustly extended the life span of Daphnia magna and reduced its body size. Overall, the results demonstrate that short-term rapamycin treatment during development is a novel longevity intervention that acts by slowing down development and aging, suggesting that aging may be targeted already early in life.

Short-term oral rapamycin prevents age-related learning impairment in mice.

Effective treatments to prevent or delay age-related learning impairment are not generally available. In a preliminary preclinical study, mice 20 months of age were fed a diet containing 14 ppm rapamycin, an inhibitor of mTOR, for three months and then tested in a spatial navigation task. Mice fed the nonmedicated control diet showed learning impairment while mice fed the rapamycin diet were not learning impaired. This observation provides support for additional preclinical studies and suggests that short-term rapamycin treatment could be a possible strategy for preventing or delaying age-related cognitive impairment in people.

Small molecule H89 renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors.

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved Ser/Thr kinase that comprises two complexes, termed mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 phosphorylates S6K1 at Thr 389, whereas mTORC2 phosphorylates AKT at Ser 473 to promote cell growth. As the mTOR name implies it is the target of natural product called rapamycin, a clinically approved drug used to treat human disease. Short-term rapamycin treatment inhibits the kinase activity of mTORC1 but not mTORC2. However, the ATP-competitive catalytic mTOR inhibitor Torin1 was identified to inhibit the kinase activity of both mTORC1 and mTORC2. Here, we report that H89 (N-(2-(4-bromocinnamylamino) ethyl)-5-isoquinolinesulfonamide), a well-characterized ATP-mimetic kinase inhibitor, renders the phosphorylation of S6K1 and AKT resistant to mTOR inhibitors across multiple cell lines. Moreover, H89 prevented the dephosphorylation of AKT and S6K1 under nutrient depleted conditions. PKA and other known H89-targeted kinases do not alter the phosphorylation status of S6K1 and AKT. Pharmacological inhibition of some phosphatases also enhanced S6K1 and AKT phosphorylation. These findings suggest a new target for H89 by which it sustains the phosphorylation status of S6K1 and AKT, resulting in mTOR signaling.

A randomized control trial to establish the feasibility and safety of rapamycin treatment in an older human cohort: Immunological, physical performance, and cognitive effects

Inhibition of the mechanistic target of rapamycin (mTOR) pathway by rapamycin (RAPA), an FDA-approved immunosuppressive drug used as a clinical therapy to prevent solid organ allograft rejection, enhances longevity in mice. Importantly, RAPA was efficacious even when initiated in relatively old animals, suggesting that mTOR inhibition could potentially slow the progression of aging-associated pathologies in older humans (Harrison et al., 2009; Miller et al., 2011). However, the safety and tolerability of RAPA in older human subjects have not yet been demonstrated. Towards this end, we undertook a placebo-controlled pilot study in 25 generally healthy older adults (aged 70–95 years); subjects were randomized to receive either 1 mg RAPA or placebo daily. Although three subjects withdrew, 11 RAPA and 14 controls completed at least 8 weeks of treatment and were included in the analysis. We monitored for changes that would indicate detrimental effects of RAPA treatment on metabolism, including both standard clinical laboratory assays (CBC, CMP, HbA1c) and oral glucose tolerance tests (OGTTs). We also monitored parameters typically associated with aging that could potentially be modified by RAPA; these included cognitive function which was assessed by three different tools: Executive Interview-25 (EXIT25); Saint Louis University Mental Status Exam (SLUMS); and Texas Assessment of Processing Speed (TAPS). In addition, physical performance was measured by handgrip strength and 40-foot timed walks. Lastly, changes in general parameters of healthy immune aging, including serum pro-inflammatory cytokine levels and blood cell subsets, were assessed. Five subjects reported potential adverse side effects; in the RAPA group, these were limited to facial rash (1 subject), stomatitis (1 subject) and gastrointestinal issues (2 subjects) whereas placebo treated subjects only reported stomatitis (1 subject). Although no other adverse events were reported, statistically significant decrements in several erythrocyte parameters including hemoglobin (HgB) and hematocrit (Hct) as well as in red blood cell count (RBC), red blood cell distribution width (RDW), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) were observed in the RAPA-treatment group. None of these changes manifested clinically significant effects during the short duration of this study. Similarly, no changes were noted in any other clinical laboratory, cognitive, physical performance, or self-perceived health status measure over the study period. Immune parameters were largely unchanged as well, possibly due to the advanced ages of the cohort (70–93 years; mean age 80.5). RAPA-associated increases in a myeloid cell subset and in TREGS were detected, but changes in most other PBMC cell subsets were not statistically significant. Importantly, the OGTTs revealed no RAPA-induced change in blood glucose concentration, insulin secretion, and insulin sensitivity. Thus, based on the results of our pilot study, it appears that short-term RAPA treatment can be used safely in older persons who are otherwise healthy; a trial with a larger sample size and longer treatment duration is warranted.

A randomized controlled trial to establish effects of short-term rapamycin treatment in 24 middle-aged companion dogs.

Age is the single greatest risk factor for most causes of morbidity and mortality in humans and their companion animals. As opposed to other model organisms used to study aging, dogs share the human environment, are subject to similar risk factors, receive comparable medical care, and develop many of the same age-related diseases humans do. In this study, 24 middle-aged healthy dogs received either placebo or a non-immunosuppressive dose of rapamycin for 10weeks. All dogs received clinical and hematological exams before, during, and after the trial and echocardiography before and after the trial. Our results showed no clinical side effects in the rapamycin-treated group compared to dogs receiving the placebo. Echocardiography suggested improvement in both diastolic and systolic age-related measures of heart function (E/A ratio, fractional shortening, and ejection fraction) in the rapamycin-treated dogs. Hematological values remained within the normal range for all parameters studied; however, the mean corpuscular volume (MCV) was decreased in rapamycin-treated dogs. Based on these results, we will test rapamycin on a larger dog cohort for a longer period of time in order to validate its effects on cardiac function and to determine whether it can significantly improve healthspan and reduce mortality in companion dogs.

Allogeneic Hair Transplantation with Enhanced Survival by Anti-ICAM-1 Antibody with Short-Term Rapamycin Treatment in Nonhuman Primates

Allogeneic Hair Transplantation with Enhanced Survival by Anti-ICAM-1 Antibody with Short-Term Rapamycin Treatment in Nonhuman Primates

Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice.

The FDA approved drug rapamycin increases lifespan in rodents and delays age-related dysfunction in rodents and humans. Nevertheless, important questions remain regarding the optimal dose, duration, and mechanisms of action in the context of healthy aging. Here we show that 3 months of rapamycin treatment is sufficient to increase life expectancy by up to 60% and improve measures of healthspan in middle-aged mice. This transient treatment is also associated with a remodeling of the microbiome, including dramatically increased prevalence of segmented filamentous bacteria in the small intestine. We also define a dose in female mice that does not extend lifespan, but is associated with a striking shift in cancer prevalence toward aggressive hematopoietic cancers and away from non-hematopoietic malignancies. These data suggest that a short-term rapamycin treatment late in life has persistent effects that can robustly delay aging, influence cancer prevalence, and modulate the microbiome.

Short-term rapamycin treatment in mice has few effects on the transcriptome of white adipose tissue compared to dietary restriction

Rapamycin, a drug that has been shown to increase lifespan in mice, inhibits the target of rapamycin (TOR) pathway, a major pathway that regulates cell growth and energy status. It has been hypothesized that rapamycin and dietary restriction (DR) extend lifespan through similar mechanisms/pathways. Using microarray analysis, we compared the transcriptome of white adipose tissue from mice fed rapamycin or DR-diet for 6 months. Multidimensional scaling and heatmap analyses showed that rapamycin had essentially no effect on the transcriptome as compared to DR. For example, only six transcripts were significantly altered by rapamycin while mice fed DR showed a significant change in over 1000 transcripts. Using ingenuity pathway analysis, we found that stearate biosynthesis and circadian rhythm signaling were significantly changed by DR. Our findings showing that DR, but not rapamycin, has an effect on the transcriptome of the adipose tissue, suggesting that these two manipulations increase lifespan through different mechanisms/pathways.

Rapamycin decreases DNA damage accumulation and enhances cell growth of WRN‐deficient human fibroblasts

Werner syndrome (WS), caused by mutations at the WRN helicase gene, is a progeroid syndrome characterized by multiple features consistent with accelerated aging. Aberrant double-strand DNA damage repair leads to genomic instability and reduced replicative lifespan of somatic cells. We observed increased autophagy in WRN knockdown cells; this was further increased by short-term rapamycin treatment. Long-term rapamycin treatment resulted in improved growth rate, reduced accumulation of DNA damage foci and improved nuclear morphology; autophagy markers were reduced to near-normal levels, possibly due to clearance of damaged proteins. These data suggest that protein aggregation plays a role in the development of WS phenotypes and that the mammalian target of rapamycin complex 1 pathway is a potential therapeutic target of WS.

Abstract P5-03-03: Effect of rapamycin on the function and transformation of aged murine mammary stem cells

Abstract Age is the number one risk fact for breast cancer with only 5% of all breast cancers occurring in women under 40 years old and age specific incidence of invasive breast cancer increases from less than 1.5% at age 40, to about 3% at age 50 and over 4% by age 70 in American women. Recent research implicated that adult mammary stem cells (MaSCs) might be responsible for the initiation and progression of certain types of breast cancer. Our preliminary study showed that aging is associated with a significant increase of MaSC frequency, but with a functional decline of self-renewal and differentiation as well as increased neoplastic transformation potential. These findings indicate that aged MaSCs might be the precursors of preneoplastic lesions and serve as the cell of origin for malignant transformation in breast tissue. Therefore, intervention of MaSC aging process could be an effective method for cancer prevention. The drug of rapamycin has been shown to extend life span and ameliorate age-related pathologies (e.g., cancer) in murine models, and a recent study suggested that rapamycin's anti-aging effect may partially act through enhancing the function of tissue-specific stem cells. It is unknown whether rapamycin treatment will also enhance the function of aged MaSCs and decrease their transformation potential. In this study, we fed C57BL/6 mice with microencapsulated rapamycin-containing food (14 mg/kg, food designed to deliver ∼2.24 mg of rapamycin per kg body weight/day to achieve about 4 ng/ml of rapamycin per kg body weight/day) or control diet with empty capsules for 2 year (starting at 2-month old) or 5-10 days (starting at 25.5-month old) and then isolated primary mammary cells at 26-month old for MaSC quantification using an in vitro mammosphere formation and 3D-ECM sphere differentiation assay as well as by the in vivo cleared mammary fat pad transplantation assay. Our findings indicate that short-term (5-10 days) or long-term (&amp;gt; 2 year) rapamycin treatment reversed phenotypic changes associated with aged MaSC, which was mainly characterized by decreased luminal-to-basal cell ratio and increased MaSC frequency. Histological analysis of regenerated glands of aged MaSCs derived from control and rapamycin-treated mice showed a significant decrease of early neoplastic transformation potential in rapamycin-treated group. Subsequent in vivo serial transplantation and mating experiments revealed that rapamycin treatment reverted aged MaSCs more resemble to young MaSCs in self-renewal/differentiation function during regeneration and improved lobulo-alveolar differentiation function for lactation. In conclusion, our findings suggest that rapamycin can rejuvenate the function of aged MaSCs as well as reduce their incidence of preneoplastic transformation. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-03-03.

Abstract B035: Preneoplastic transformation of murine mammary stem cells by aging and inhibition by rapamycin

Abstract The number one risk fact for breast cancer is aging. Recent research implicated that adult mammary stem cells (MaSCs) might be responsible for the initiation and progression of certain types of breast cancer. We tested the hypothesis that aging-associated mammary tumorigenesis may be due to altered number and function of MaSCs using mouse models. MaSC-enriched basal cells, characterized by their immunophenotype (Lin-CD49fhighCD24med), were utilized for the evaluation of MaSC frequency and function during aging by in vitro mammosphere formation and 3D-ECM sphere differentiation assays and by in vivo cleared mammary fat pad transplantation (IVT) as we reported recently (Stem Cell Res. 2013). We found that the basal-to-luminal cell ratios analyzed by FACS and the frequency of MaSCs analyzed with in vitro assays increased steadily with increasing age in various strains of mice. Subsequent IVT using mammosphers or 3D-ECM structures formed by young (4 months) or old (28 months) MaSCs derived from C57BL/6 mice showed similar in vivo functional mammary gland regenerative capacity indicating similar self-renewal/differentiation ability between young and old MaSCs. However, we found that the regenerated glands from old MaSCs had significantly higher number of spontaneous hyperplastic and atypical ductal hyperplastic lesions than those from young MaSCs. These findings indicate that aged MaSCs can serve as the cell of origin for early neoplastic transformation in breast tissue. Because rapamycin treatment has been shown to ameliorate age-related pathologies including cancer in murine models, we fed old C57BL/6 mice with microencapsulated rapamycin-containing food or control diet to determine whether rapamycin treatment can inhibit the aging-associated alteration of MaSC number and function. Our findings indicate that short-term (5-10 days) or long-term (&amp;gt; 2 year) rapamycin treatment reversed phenotypic changes associated with aged mammary gland, which were mainly characterized by decreased luminal-to-basal cell ratio and increased MaSC frequency. Histological analysis of regenerated glands by aged MaSCs derived from control and rapamycin-treated mice showed a significant decrease of early neoplastic lesions in rapamycin-treated group. Subsequent whole genome transcriptome analysis with the second generation sequencing revealed age-associated differential expression of genes involved in immune, inflammatory, and wounding responses in both mammosphere-forming cells and stromal cells suggesting that these may be the main cellular processes contributing to the altered MaSC phenotypes and inhibited by rapamycin. In conclusion, our findings suggest that aging causes MaSC to increase its pool and to form early neoplastic lesions, which can be inhibited by rapamycin treatment. Citation Format: Lu-Zhe Sun, Abhik Bandyopadhyay, Qiaoxiang Dong, Hui Gao, Xiang Gu, Danhan Wang, Anqi Wu, Changjiang Huang. Preneoplastic transformation of murine mammary stem cells by aging and inhibition by rapamycin. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B035.

Expansion of Memory-Type CD8+ T Cells Correlates With the Failure of Early Immunosuppression Withdrawal After Cadaver Liver Transplantation Using High-Dose ATG Induction and Rapamycin

We report on a pilot study investigating the feasibility of early immunosuppression withdrawal after liver transplantation (LT) using antithymocyte globulin (ATG) induction and rapamycin. LT recipients received 3.75 mg/kg per day ATG from days 0 to 5 followed by rapamycin-based immunosuppression. In the absence of acute rejection (AR), rapamycin was withdrawn after month 4. Immunomonitoring included analysis of peripheral T-cell phenotypes and clonality, cytokine production in mixed lymphocyte reaction, and characterization of intragraft infiltrating cells. Ten patients were enrolled between October 2009 and July 2010. In the first three patients, complete withdrawal of immunosuppression after month 4 led to AR. No further withdrawals of immunosuppressive were attempted. Two AR occurred in the remaining seven patients. ATG induced profound T-cell depletion followed by CD8(+) T-cell reexpansion exhibiting memory/effector-like phenotype associated with progressive oligoclonal T-cell expansion (Vβ/HPRT ratio) and gradually enhanced anti-cytomegalovirus and anti-Epstein-Barr virus T-cell frequencies. Patients developing AR were characterized by decreased TCAIM expression. AR were associated with increased donor-specific production of interferon (IFN)-γ and interleukin (IL)-17, increased intragraft expression of IFN-γ mRNA, and significant CD8(+) T-cell infiltrates colocalizing with IL-17(+) cells. High-dose ATG followed by short-term rapamycin treatment failed to promote early operational tolerance to LT. AR correlates with expansion of memory-type CD8(+) T cells and increased levels of IFN-γ and IL-17 in mixed lymphocyte reaction and in the graft. This suggests that resistance and preferential expansion of effector memory T-cell in lymphopenic environment could represent the major barrier for establishment of tolerance to LT in approaches using T-cell-depleting induction.

Prolonged Rapamycin treatment led to beneficial metabolic switch

Prolonged Rapamycin treatment led to beneficial metabolic switch

The mTOR Inhibitor Rapamycin Has Limited Acute Anticonvulsant Effects in Mice

ObjectiveThe mammalian target of rapamycin (mTOR) pathway integrates signals from different nutrient sources, including amino acids and glucose. Compounds that inhibit mTOR kinase activity such as rapamycin and everolimus can suppress seizures in some chronic animal models and in patients with tuberous sclerosis. However, it is not known whether mTOR inhibitors exert acute anticonvulsant effects in addition to their longer term antiepileptogenic effects. To gain insights into how rapamycin suppresses seizures, we investigated the anticonvulsant activity of rapamycin using acute seizure tests in mice.MethodsFollowing intraperitoneal injection of rapamycin, normal four-week-old male NIH Swiss mice were evaluated for susceptibility to a battery of acute seizure tests similar to those currently used to screen potential therapeutics by the US NIH Anticonvulsant Screening Program. To assess the short term effects of rapamycin, mice were seizure tested in ≤6 hours of a single dose of rapamycin, and for longer term effects of rapamycin, mice were tested after 3 or more daily doses of rapamycin.ResultsThe only seizure test where short-term rapamycin treatment protected mice was against tonic hindlimb extension in the MES threshold test, though this protection waned with longer rapamycin treatment. Longer term rapamycin treatment protected against kainic acid-induced seizure activity, but only at late times after seizure onset. Rapamycin was not protective in the 6 Hz or PTZ seizure tests after short or longer rapamycin treatment times. In contrast to other metabolism-based therapies that protect in acute seizure tests, rapamycin has limited acute anticonvulsant effects in normal mice.SignificanceThe efficacy of rapamycin as an acute anticonvulsant agent may be limited. Furthermore, the combined pattern of acute seizure test results places rapamycin in a third category distinct from both fasting and the ketogenic diet, and which is more similar to drugs acting on sodium channels.

Rapamycin Generates Graft-Homing Murine Suppressor CD8+T Cells That Confer Donor-Specific Graft Protection

It has been reported that rapamycin (RPM) can induce de novo conversion of the conventional CD4(+)Foxp3(-) T cells into CD4(+)Foxp3(+) regulatory T cells (iTregs) in transplantation setting. It is not clear whether RPM can similarly generate suppressor CD8(+) T cells to facilitate graft acceptance. In this study, we investigated the ability of short-term RPM treatment in promoting long-term acceptance (LTA) of MHC-mismatched skin allografts by generating a CD8(+) suppressor T-cell population. We found that CD4 knockout (KO) mice (in C57BL/6 background, H-2(b)) can promptly reject DBA/2 (H-2(d)) skin allografts with mean survival time (MST) being 13 days (p < 0.01). However, a short course RPM treatment in these animals induced LTA with graft MST longer than 100 days. Adoptive transfer of CD8(+) T cells from LTA group into recombination-activating gene 1 (Rag-1)-deficient mice provided donor-specific protection of DBA/2 skin grafts against cotransferred conventional CD8(+) T cells. Functionally active immunoregulatory CD8(+) T cells also resided in donor skin allografts. Eighteen percent of CD8(+) suppressor T cells expressed CD28 as measured by flow cytometry, and produced reduced levels of IFN-γ, IL-2, and IL-10 in comparison to CD8(+) effector T cells as measured by ELISA. It is unlikely that CD8(+) suppressor T cells mediated graft protection via IL-10, as IL-10/Fc fusion protein impaired RPM-induced LTA in CD4 KO mice. Our data supported the notion that RPM-induced suppressor CD8(+) T cells home to the allograft and exert donor-specific graft protection.

Haploinsufficiency of Target of Rapamycin Attenuates Cardiomyopathies in Adult Zebrafish

Although a cardioprotective function of target of rapamycin (TOR) signaling inhibition has been suggested by pharmacological studies using rapamycin, genetic evidences are still lacking. We explored adult zebrafish as a novel vertebrate model for dissecting signaling pathways in cardiomyopathy. We generated the second adult zebrafish cardiomyopathy model induced by doxorubicin. By genetically analyzing both the doxorubicin and our previous established anemia-induced cardiomyopathy models, we decipher the functions of TOR signaling in cardiomyopathies of different etiology. Along the progression of both cardiomyopathy models, we detected dynamic TOR activity at different stages of pathogenesis as well as distinct effects of TOR signaling inhibition. Nevertheless, cardiac enlargement in both models can be effectively attenuated by inhibition of TOR signaling through short-term rapamycin treatment. To assess the long-term effects of TOR reduction, we used a zebrafish target of rapamycin (ztor) mutant identified from an insertional mutagenesis screen. We show that TOR haploinsufficiency in the ztor heterozygous fish improved cardiac function, prevented pathological remodeling events, and ultimately reduced mortality in both adult fish models of cardiomyopathy. Mechanistically, these cardioprotective effects are conveyed by the antihypertrophy, antiapoptosis, and proautophagy function of TOR signaling inhibition. Our results prove adult zebrafish as a conserved novel vertebrate model for human cardiomyopathies. Moreover, we provide the first genetic evidence to demonstrate a long-term cardioprotective effect of TOR signaling inhibition on at least 2 cardiomyopathies of distinct etiology, despite dynamic TOR activities during their pathogenesis.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4+Foxp3+ Treg cell accumulation

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ+CD3+CD4−CD8−NK1.1− (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1–9) induced long-term heart allograft survival (101±31 vs. 39±13days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4+ Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12days vs. 101±31days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4+Foxp3+ Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.