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BRAF is one of multiple RAF proteins responsible for the activation of the MAPK cell signalling cascade involved in cell growth, differentiation, and survival. A hotspot BRAF V600E mutation, in exon 15, was determined to be a driver in 100% hairy cell leukaemias, 50%-60% of human melanomas, 30%-50% of human thyroid carcinomas and 10%-20% of human colorectal carcinomas. The orthologous BRAF V595E mutation was seen in 67% and 80% of canine bladder transitional cell carcinomas and prostatic adenocarcinomas, respectively. Since veterinary and human cancers exploit similar pathways and BRAF is highly conserved across species, BRAF can be expected to be a driver in a feline cancer. Primers were developed to amplify exon 15 of feline BRAF. One hundred ninety-six feline tumours were analysed. Sanger sequencing of the 211 bp PCR amplicon was done. A BRAF mutation was found in one tumour, a cutaneous melanoma. The mutation was a BRAF V597E mutation, orthologous to the canine and human hotspot mutations. A common synonymous variant, BRAF T586T , was seen in 23% (47/196) of tumours. This variant was suspected to be a single nucleotide polymorphism. BRAF was not frequently mutated in common feline tumours or in tumour types that frequently harbour BRAF mutations in human and canine cancers. As is seen in canine cancer genomics, the mutational profile in feline tumours may not parallel the histologic equivalent in human oncology., (© 2024 John Wiley & Sons Ltd.)

Objective: To determine if multistrain probiotics administered to asthmatic cats treated with anti-inflammatory glucocorticoids would attenuate the asthmatic phenotype and beneficially alter respiratory, blood, and oropharyngeal (OP) microbial communities and immune parameters versus placebo., Animals: 13 client-owned asthmatic cats., Methods: A randomized, blinded, placebo-controlled clinical trial of asthmatic cats receiving anti-inflammatory glucocorticoids with oral multistrain probiotics or placebo assessed owner-perceived improvement and airway eosinophilia at baseline and after 2 weeks of treatment. Bronchoalveolar lavage fluid (BALF), blood, OP, and rectal microbial communities were compared using 16S rRNA amplicon sequencing. Real-time PCR for transcription factors, activation markers and cytokines, and IgA ELISAs were evaluated. Statistical analyses used 2-way repeated-measures ANOVA or permutational ANOVA (significance, P < .05)., Results: After treatment, there were no significant differences in owner-perceived clinical signs or mean ± SEM BALF eosinophils between groups. There was a significant decrease in rectal α-diversity but not in α- or β-diversity in BALF, blood, or OP between groups or over time. There were no significant differences in CD25, FoxP3, GATA, Helios, IL-4, IL-5, IL-10, IL-13, IL-17, IFN-γ mRNA, or serum or BALF IgA between groups or over time., Clinical Relevance: In asthmatic cats, oral multistrain probiotics failed to improve owner-perceived signs, reduce airway eosinophilia, modify microbial community composition, or alter assessed immune responses versus placebo or over time. Longer treatment, different probiotic composition or delivery (eg, aerosolized), or larger number of cats would represent the next stages of study.

Spontaneous cancers in companion dogs are robust models of human disease. Tracking tumor-specific immune responses in these models requires reagents to perform species-specific single cell T cell receptor sequencing (scTCRseq). scTCRseq and integration with scRNA data have not been demonstrated on companion dogs with cancer. Here, five healthy dogs, two dogs with T cell lymphoma and four dogs with melanoma are selected to demonstrate applicability of scTCRseq in a cancer immunotherapy setting. Single-cell suspensions of PBMCs or lymph node aspirates are profiled using scRNA and dog-specific scTCRseq primers. In total, 77,809 V(D)J-expressing cells are detected, with an average of 3498 (348 - 5,971) unique clonotypes identified per sample. In total, 29/34, 40/40, 22/22 and 9/9 known functional TRAV, TRAJ, TRBV and TRBJ gene segments are observed respectively. Pseudogene or otherwise defective gene segments are also detected supporting re-annotation of several as functional. Healthy dogs exhibit highly diverse repertoires, T cell lymphomas exhibit clonal repertoires, and vaccine-treated melanoma dogs are dominated by a small number of highly abundant clonotypes. scRNA libraries define large clusters of V(D)J-expressing CD8+ and CD4 + T cells. Dominant clonotypes observed in melanoma PBMCs are predominantly CD8 + T cells, with activated phenotypes, suggesting possible anti-tumor T cell populations., (© 2024. The Author(s).)

Introduction: Zoledronic acid (ZOL) is a third-generation bisphosphonate with a higher affinity for bone resorption areas than earlier bisphosphonates (i.e., pamidronate, PAM). In human medicine, ZOL provides improved bone pain relief and prolonged time to skeletal-related events compared to its older generational counterparts. Preclinical studies have investigated its role as an anti-neoplastic agent, both independently and synergistically, with radiation therapy (RT). ZOL and RT act synergistically in several neoplastic human cell lines: prostate, breast, osteosarcoma, and fibrosarcoma. However, the exact mechanism of ZOL's radiosensitization has not been fully elucidated., Methods: We investigated ZOL's ability to induce apoptosis in canine osteosarcoma cell lines treated with various doses of megavoltage external beam radiotherapy. Second, we evaluated cell cycle arrest in ZOL-treated cells to assess several neo-adjuvant time points. Finally, we treated 20 dogs with naturally occurring appendicular OS with 0.1 mg/kg ZOL IV 24 h before receiving 8 Gy of RT (once weekly fraction x 4 weeks)., Results: We found that apoptosis was increased in all ZOL-treated cell lines compared to controls, and the combination of ZOL and RT resulted in dissimilar apoptosis between Abrams and D-17 and HMPOS cell lines. Cell cycle arrest (G2/M phase) was minimal and variable between cell lines but perhaps greatest at 48 h post-ZOL treatment. Only 10% of dogs treated with ZOL and RT developed pathologic fractures, compared to 44% of dogs historically treated with PAM and RT ( p  = 0.027)., Discussion: ZOL and RT appear to be a well-tolerated combination treatment scheme for non-surgical candidates; future studies must elucidate the ideal timing of ZOL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Norquest, Rogic, Gimotty, Maitz, Rindt, Ashworth, Bryan, Donnelly, McCleary-Wheeler and Flesner.)

Introduction: The primary objective of this study was to assess the feasibility of a 6-week internet-delivered Mantram Repetition Program (MRP) for women recently treated for breast cancer. A secondary objective explored changes in perceived stress, psycho-spiritual measures, and cytokines in the treatment group compared to a waitlist. Methods: A feasibility study (ORBIT model Phase IIa) with a randomized controlled trial pilot was conducted. Eligible women recently treated for breast cancer were randomized to the treatment group ( n = 14) or a waitlist group ( n = 12) and participated for 12 weeks. During weeks 1-6, the treatment group received the MRP intervention while the waitlisted group was inactive. During weeks 7-12, the treatment group was inactive while the waitlisted group received the MRP intervention. The primary outcomes were feasibility and acceptability of the internet-delivered MRP intervention protocol. Participants completed pre and post-intervention psycho-spiritual health assessments. A subset of participants provided serum for cytokine analyses at enrollment and week 6, coinciding with the period in which the treatment group receiving the MRP intervention. Results: Overall study attrition was 19.2%. MRP adherence for both groups was 86% at post-intervention and 90% in the treatment group at 6-week follow-up. Pre-to-post-intervention analyses pooling both groups' data demonstrated decreased perceived stress ( p = .045) and increased spiritual well-being ( p =.004). IFN-γ and IL-17A were increased in the waitlisted group and decreased in the treatment group ( p = 0.048). Conclusion: Feasibility of a 6-week, internet-delivered MRP intervention for breast cancer survivors was established. Psycho-spiritual variables and serum cytokines are suitable clinical outcome measures for future MRP studies with breast cancer survivors. Data suggest MRP may reduce perceived stress and support spiritual well-being in women with breast cancer; however, additional studies are needed., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Purpose: Osteosarcoma (OS) is the most frequently diagnosed bone cancer in children with little improvement in overall survival in the past decades. The high surface expression of disialoganglioside GD2 on OS tumors and restricted expression in normal tissues makes it an ideal target for anti-OS radiopharmaceuticals. Since human and canine OS share many biological and molecular features, spontaneously occurring OS in canines has been an ideal model for testing new imaging and treatment modalities for human translation. In this study, we evaluated a humanized anti-GD2 antibody, hu3F8, as a potential delivery vector for targeted radiopharmaceutical imaging of human and canine OS., Methods: The cross-reactivity of hu3F8 with human and canine OS cells and tumors was examined by immunohistochemistry and flow cytometry. The hu3F8 was radiolabeled with indium-111, and the biodistribution of [ 111 In]In-hu3F8 was assessed in tumor xenograft-bearing mice. The targeting ability of [ 111 In]In-hu3F8 to metastatic OS was tested in spontaneous OS canines., Results: The hu3F8 cross reacts with human and canine OS cells and canine OS tumors with high binding affinity. Biodistribution studies revealed selective uptake of [ 111 In]In-hu3F8 in tumor tissue. SPECT/CT imaging of spontaneous OS canines demonstrated avid uptake of [ 111 In]In-hu3F8 in all metastatic lesions. Immunohistochemistry confirmed the extensive binding of radiolabeled hu3F8 within both osseous and soft lesions., Conclusion: This study demonstrates the feasibility of targeting GD2 on OS cells and spontaneous OS canine tumors using hu3F8-based radiopharmaceutical imaging. Its ability to deliver an imaging payload in a targeted manner supports the utility of hu3F8 for precision imaging of OS and potential future use in radiopharmaceutical therapy., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Deviations from a core airway microbiota have been associated with the development and progression of asthma as well as disease severity. Pet cats represent a large animal model for allergic asthma, as they spontaneously develop a disease similar to atopic childhood asthma. This study aimed to describe the lower airway microbiota of asthmatic pet cats and compare it to healthy cats to document respiratory dysbiosis occurring with airway inflammation. We hypothesized that asthmatic cats would have lower airway dysbiosis characterized by a decrease in richness, diversity, and alterations in microbial community composition including identification of possible pathobionts. In the current study, a significant difference in airway microbiota composition was documented between spontaneously asthmatic pet cats and healthy research cats mirroring the finding of dysbiosis in asthmatic humans. Filobacterium and Acinetobacter spp. were identified as predominant taxa in asthmatic cats without documented infection based on standard culture and could represent pathobionts in the lower airways of cats. Mycoplasma felis , a known lower airway pathogen of cats, was identified in 35% of asthmatic but not healthy cats. This article has been published alongside "Temporal changes of the respiratory microbiota as cats transition from health to experimental acute and chronic allergic asthma" (1)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vientós-Plotts, Ericsson, McAdams, Rindt and Reinero.)

In humans, deviation from a core airway microbiota may predispose to development, exacerbation, or progression of asthma. We proposed to describe microbiota changes using 16 rRNA sequencing in samples from the upper and lower airways, and rectal swabs of 8 cats after experimental induction of asthma using Bermuda grass allergen, in acute (6 weeks) and chronic (36 weeks) stages. We hypothesized that asthma induction would decrease richness and diversity and alter microbiota composition and structure in the lower airways, without significantly impacting other sites. After asthma induction, richness decreased in rectal ( p = 0.014) and lower airway ( p = 0.016) samples. B diversity was significantly different between health and chronic asthma in all sites, and between all time points for lower airways. In healthy lower airways Pseudomonadaceae comprised 80.4 ± 1.3% whereas Sphingobacteriaceae and Xanthobacteraceae predominated (52.4 ± 2.2% and 33.5 ± 2.1%, respectively), and Pseudomonadaceae was absent, in 6/8 cats with chronic asthma. This study provides evidence that experimental induction of asthma leads to dysbiosis in the airways and distant sites in both the acute and chronic stages of disease. This article has been published alongside "Respiratory dysbiosis in cats with spontaneous allergic asthma" (1)., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vientós-Plotts, Ericsson, McAdams, Rindt and Reinero.)

Cancer-induced bone pain, despite its frequency and severity, is a poorly understood phenomenon in people and animals. Despite excitement regarding translational osteosarcoma studies, there is a lack of attention toward examining cancer pain in dogs. In this pilot study, we used a multimodal pain assessment methodology to evaluate pain relief after therapeutic intervention in dogs with primary bone cancer. We hypothesized that intervention would cause objective evidence of pain relief. Evaluations of 8 dogs with primary bone cancer included 18 F-FDG PET/CT scans, kinetic analysis, validated owner questionnaires (Canine Brief Pain Inventory, canine BPI), and serum N-telopeptide (NTx) concentration. Dogs were routinely staged and had 18 F-FDG PET/CT scans prior to treatment with day 0, 7, 14, and 28 canine BPI, serum NTx, orthopedic exam, and kinetic analysis. Dogs treated with zoledronate and radiation underwent day 28 18 F-FDG PET scans. All clinical trial work was approved by the University of Missouri IACUC. Four dogs underwent amputation (AMP) for their appendicular bone tumors; four received neoadjuvant zoledronate and hypofractionated radiation therapy (ZOL+RT). Canine BPI revealed significant improvements in pain severity and pain interference scores compared to baseline for all dogs. Positive changes in peak vertical force (+16.7%) and vertical impulse (+29.1%) were noted at day 28 in ZOL+RT dogs. Dogs receiving ZOL+RT had a significant (at least 30%) reduction in serum NTx from baseline compared to amputated dogs ( p = 0.029). SUV max ( p = 0.11) and intensity ( p = 0.013) values from PET scans decreased while tumor uniformity ( p = 0.017) significantly increased in ZOL+RT-treated tumors; gross tumor volume did not change ( p = 0.78). Owner questionnaires, kinetic analysis, and 18 F-FDG PET/CT scans showed improved pain relief in dogs receiving ZOL+RT. Serum NTx levels likely do not directly measure pain, but rather the degree of systemic osteoclastic activity. Larger, prospective studies are warranted to identify the ideal objective indicator of pain relief; however, use of multiple assessors is presumably best. With improved assessment of pain severity and relief in dogs with cancer, we can better evaluate the efficacy of our interventions. This could directly benefit people with cancer pain, potentially decreasing the amount of subtherapeutic novel drugs entering human clinical trials., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Flesner, Torres, Hutcheson, Rindt, Zalcman and Maitz.)

Reflux and aspiration in people cause and exacerbate respiratory diseases in the absence of gastrointestinal signs. Protein biomarkers in humans detect extraesophageal reflux (EER) from oropharyngeal (OP) and bronchoalveloar lavage samples. Reflux likely contributes to respiratory disease in dogs. The objectives of this study were to analyze the canine gastric fluid (GF) proteome and compare this to the OP proteome in normal, vomiting/regurgitating, and coughing dogs to identify biomarkers for EER/aspiration. Twenty-three client-owned dogs were enrolled. Canine GF samples ( n = 5) and OP swabs in normal ( n = 6), vomiting/regurgitating ( n = 7), and coughing ( n = 5) dogs were within 2 weeks of sample collection. Protein digests were analyzed by liquid chromatography-mass spectrometry. Differential abundance (DA) of proteins between groups was evaluated by Fisher's exact test with p < 0.0004 significance level after correction for multiple comparisons. DA was found between all groups ( p < 0.0001): GF vs. normal ( n = 130 proteins), coughing vs. normal ( n = 22 proteins), and vomiting/regurgitating vs. normal ( n = 20 proteins). Protein abundance was highly variable between dogs. Gastrointestinal-specific proteins were found in OP swabs from vomiting/regurgitating and coughing dogs but not from healthy dogs. In conclusion, the proteomic composition of the OP varies between health and disease. The presence of gastrointestinal-specific proteins in OP of coughing dogs may suggest reflux and/or aspiration as contributing factors. The variable protein abundance warrants investigation into biomarker panels., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Grobman, Rindt and Reinero.)

The lungs of people and companion animals are now recognized to harbor diverse, low biomass bacterial communities. While these communities are difficult to characterize using culture-based approaches, targeted molecular methods such as 16S rRNA amplicon sequencing can do so using DNA extracted from samples such as bronchoalveolar lavage fluid (BALF). Previous studies identified a surprisingly uniform composition of the microbiota in the lungs of healthy research dogs living in a controlled environment, however there are no reports of the lung microbiota of client-owned dogs. Moreover, compositional changes in the lung microbiota depending on disease status have been reported in people, suggesting that similar events may occur in dogs, a species subject to several respiratory disease mechanisms analogous to those seen in people. To address these knowledge gaps, BALF samples from client-owned dogs presenting to the University of Missouri Veterinary Health Center for respiratory signs between 2014 and 2017 were processed for and subjected to 16S rRNA sequencing. Based on specific diagnostic criteria, dogs were categorized as Chronic Bronchitis (CB, n = 53) or non-CB (n = 11). Community structure was compared between groups, as well as to historical data from healthy research dogs (n = 16) of a uniform breed and environment. The lung microbiota detected in all client-owned dogs was markedly different in composition from that previously detected in research dogs and contained increased relative abundance of multiple canine fecal and environmental bacteria, likely due to aspiration associated with their clinical signs. While inter-sample diversity differed significantly between samples from CB and non-CB dogs, the variability within both groups made it difficult to discern reproducible bacterial classifiers of disease. During subsequent analyses to identify other sources of variability within the data however, population-wide temporal dynamics in community structure were observed, with substantial changes occurring in late 2015 and again in early 2017. A review of regional climate data indicated that the first change occurred during a historically warm and wet period, suggesting that changes in environmental conditions may be associated with changes in the respiratory microbiota in the context of respiratory disease. As the lung microbiota in humans and other animals is believed to result from repetitive micro-aspirations during health and in certain disease states associated with dyspnea and laryngeal dysfunction, these data support the increased colonization of the lower airways during compromised airway function, and the potential for temporal effects due to putative factors such as climate., Competing Interests: The authors have declared that no competing interests exist.

It is unknown how the respiratory microbiome influences and is influenced by bacterial pneumonia in dogs, as culture of lung samples and not microbial sequencing guides clinical practice. While accurate identification of pathogens are essential for treatment, not all bacteria are cultivable and the impact of respiratory dysbiosis on development of pneumonia is unclear. The study purposes were to (1) characterize the lung microbiome in canine bacterial pneumonia and compare deviations in dominant microbial populations with historical healthy controls, (2) compare bacteria identified by culture vs. 16S rDNA sequencing from bronchoalveolar lavage fluid (BALF) culture-, and (3) evaluate similarities in lung and oropharyngeal (OP) microbial communities in community-acquired and secondary bacterial pneumonia. Twenty BALF samples from 15 client-owned dogs diagnosed with bacterial pneumonia were enrolled. From a subset of dogs, OP swabs were collected. Extracted DNA underwent PCR of the 16S rRNA gene. Relative abundance of operational taxonomic units (OTUs) were determined. The relative abundance of bacterial community members found in health was decreased in dogs with pneumonia. Taxa identified via culture were not always the dominant phylotype identified with sequencing. Dogs with community-acquired pneumonia were more likely to have overgrowth of a single organism suggesting loss of dominant species associated with health. Dogs with secondary bacterial pneumonia had a greater regional continuity between the upper and lower airways. Collectively, these data suggest that dysbiosis occurs in canine bacterial pneumonia, and culture-independent techniques may provide greater depth of understanding of the changes in bacterial community composition that occur in disease., (Copyright © 2019 Vientós-Plotts, Ericsson, Rindt and Reinero.)

A lack of understanding of specific immune defects underlying canine immune-mediated diseases hampers optimal therapy. Failure to tailor treatment to an individual's immune abnormality can result in lack of efficacy, secondary complications, added expense, and drug-potentiated adverse effects. We adopted a small-volume whole-blood flow cytometric assay to determine the effect of immunosuppressant drugs on T-lymphocyte proliferation. Using healthy dogs in this proof-of-principle study, we hypothesized that there would be dose-dependent suppression of T-lymphocyte proliferation in response to dexamethasone, cyclosporine, mycophenolic acid, and the active metabolite of leflunomide (A77 1726). Whole blood was collected from 6 healthy pet dogs and incubated for 4 d with or without the mitogens concanavalin A and lipopolysaccharide and with increasing concentrations of immunosuppressant. Samples were subsequently stained with viability dye and with antibodies against the pan-T-lymphocyte marker CD5 and the cell proliferation marker Ki67. Percentages of proliferating T-lymphocytes were determined by flow cytometry, and the 50% inhibitory concentration (IC 50 ) was calculated. Inhibition of T-lymphocyte proliferation by the panel of immunosuppressants was shown to be dose-dependent, with marked variability among the dogs. The mean IC 50 was 394.8 ± 871 (standard deviation) μM for dexamethasone, 18.89 ± 36.2 ng/mL for cyclosporine, 106.3 ± 157.7 nM for mycophenolic acid, and 3.746 ± 6.8 μM for A77 1726. These results support the use of this assay for detecting the efficacy of individual immunosuppressants used to diminish T-lymphocyte proliferation. In future, the assay may be applied to pet dogs with spontaneous immune-mediated disease to help tailor individual treatment., (Copyright and/or publishing rights held by the Canadian Veterinary Medical Association.)

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by deletions or mutations in survival motor neuron 1 (SMN1). The molecular mechanisms underlying motor neuron degeneration in SMA remain elusive, as global cellular dysfunction obscures the identification and characterization of disease-relevant pathways and potential therapeutic targets. Recent reports have implicated microRNA (miRNA) dysregulation as a potential contributor to the pathological mechanism in SMA. To characterize miRNAs that are differentially regulated in SMA, we profiled miRNA levels in SMA induced pluripotent stem cell (iPSC)-derived motor neurons. From this array, miR-23a downregulation was identified selectively in SMA motor neurons, consistent with previous reports where miR-23a functioned in neuroprotective and muscle atrophy-antagonizing roles. Reintroduction of miR-23a expression in SMA patient iPSC-derived motor neurons protected against degeneration, suggesting a potential miR-23a-specific disease-modifying effect. To assess this activity in vivo, miR-23a was expressed using a self-complementary adeno-associated virus serotype 9 (scAAV9) viral vector in the Smn2B/- SMA mouse model. scAAV9-miR-23a significantly reduced the pathology in SMA mice, including increased motor neuron size, reduced neuromuscular junction pathology, increased muscle fiber area, and extended survival. These experiments demonstrate that miR-23a is a novel protective modifier of SMA, warranting further characterization of miRNA dysfunction in SMA., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by a polyglutamine (polyQ) expansion in exon 1 of the Huntingtin ( HTT ) gene. We have previously demonstrated that spliceosome-mediated trans -splicing is a viable molecular strategy to specifically reduce and repair mutant HTT (mtHTT). Here, the targeted tethering efficacy of the pre-mRNA trans -splicing modules (PTM) in HTT was optimized. Various PTMs that targeted the 3' end of HTT intron 1 or the intron 1 branch point were shown trans -splice into an HTT mini-gene, as well as the endogenous HTT pre-mRNA. PTMs that specifically target the endogenous intron 1 branch point increased the trans -splicing efficacy from 1-5 to 10-15%. Furthermore, lentiviral expression of PTMs in a human HD patient iPSC-derived neural culture significantly reversed two previously established polyQ-length dependent phenotypes. These results suggest that pre-mRNA repair of mtHTT could hold therapeutic benefit and it demonstrates an alternative platform to correct the mRNA product produced by the mt HTT allele in the context of HD.

Probiotics have been advocated as a novel therapeutic approach to respiratory disease, but knowledge of how oral administration of probiotics influences the respiratory microbiota is needed. Using 16S rRNA amplicon sequencing of bacterial DNA our objective was to determine whether oral probiotics changed the composition of the upper and lower airway, rectal, and blood microbiota. We hypothesized that oral probiotics would modulate the respiratory microbiota in healthy cats, demonstrated by the detection and/or increased relative abundance of the probiotic bacterial species and altered composition of the microbial population in the respiratory tract. Six healthy young research cats had oropharyngeal (OP), bronchoalveolar lavage fluid (BALF), rectal, and blood samples collected at baseline and 4 weeks after receiving oral probiotics. 16S rRNA gene amplicon libraries were sequenced, and coverage, richness, and relative abundance of representative operational taxonomic units (OTUs) were determined. Hierarchical and principal component analyses (PCA) demonstrated relatedness of samples. Mean microbial richness significantly increased only in the upper and lower airways. The number of probiotic OTUs (out of 5 total) that significantly increased in relative abundance vs. baseline was 5 in OP, 3 in BAL and 2 in feces. Using hierarchical clustering, BALF and blood samples grouped together after probiotic administration, and PERMANOVA supported that these two sites underwent significant changes in microbial composition. PERMANOVA revealed that OP and rectal samples had microbial population compositions that did not significantly change. These findings were visualized via PCA, which revealed distinct microbiomes in each site; samples clustered more tightly at baseline and had more variation after probiotic administration. This is the first study describing the effect of oral probiotics on the respiratory microbiota via detection of probiotic species in the airways. Finding bacterial species present in the oral probiotics in the upper and lower airways provides pilot data suggesting that oral probiotics could serve as a tool to target dysbiosis occurring in inflammatory airway diseases such as feline asthma, a disease in which cats serve as an important comparative and translational model for humans.

The urinary bladder in healthy dogs has dogmatically been considered free of bacteria. This study used culture independent techniques to characterize the healthy canine urinary microbiota. Urine samples collected by antepubic cystocentesis from dogs without urinary infection were used for DNA extraction. Genital tract and rectal samples were collected simultaneously from the same dogs. The V4 hypervariable region of the 16S rRNA bacterial gene was amplified and compared against Greengenes database for OTU assignment and relative abundance for urine, genital, and rectal samples. After excluding 4 dogs with cultivable bacteria, samples from 10 male (M; 1 intact) and 10 female (F) spayed dogs remained. All samples provided adequate genetic material for analysis. Four taxa (Pseudomonas sp., Acinetobacter sp., Sphingobium sp. and Bradyrhizobiaceae) dominated the urinary microbiota in all dogs of both sexes. These taxa were also detected in the genital swabs of both sexes, while the rectal microbiota differed substantially from the other sample sites. Principal component (PC) analysis of PC1 through PC3 showed overlap of urinary and genital microbiota and a clear separation of rectal swabs from the other sample sites along PC1, which explained 44.94% variation. Surprisingly, the urinary microbiota (mean # OTU 92.6 F, 90.2 M) was significantly richer than the genital (67.8 F, 66.6 M) or rectal microbiota (68.3 F, 71.2 M) (p < 0.0001), with no difference between sexes at any sample site. The canine urinary bladder is not a sterile environment and possesses its own unique and diverse microbiota compared to the rectal and genital microbiota. There was no difference between the sexes at any microbiota sample site (urine, genital, and rectal). The predominant bacterial genus for either sex in the urine and genital tracts was Pseudomonas sp.

Advances in the field of metagenomics using culture-independent methods of microbial identification have allowed characterization of rich and diverse communities of bacteria in the lungs of healthy humans, mice, dogs, sheep and pigs. These data challenge the long held belief that the lungs are sterile and microbial colonization is synonymous with pathology. Studies in humans and animals demonstrate differences in the composition of airway microbiota in health versus disease suggesting respiratory dysbiosis occurs. Using 16S rRNA amplicon sequencing of DNA extracted from rectal and oropharyngeal (OP) swabs, bronchoalveolar lavage fluid (BALF), and blood, our objective was to characterize the fecal, OP, blood, and lower airway microbiota over time in healthy cats. This work in healthy cats, a species in which a respiratory microbiota has not yet been characterized, sets the stage for future studies in feline asthma in which cats serve as a comparative and translational model for humans. Fecal, OP and BALF samples were collected from six healthy research cats at day 0, week 2, and week 10; blood was collected at week 10. DNA was extracted, amplified via PCR, and sequenced using the Illumina MiSeq platform. Representative operational taxonomic units (OTUs) were identified and microbial richness and diversity were assessed. Principal component analysis (PCA) was used to visualize relatedness of samples and PERMANOVA was used to test for significant differences in microbial community composition. Fecal and OP swabs provided abundant DNA yielding a mean±SEM of 65,653±6,145 and 20,6323±4,360 sequences per sample, respectively while BALF and blood samples had lower coverage (1,489±430 and 269±18 sequences per sample, respectively). Oropharyngeal and fecal swabs were significantly richer than BALF (mean number OTUs 93, 88 and 36, respectively; p < 0.001) with no significant difference (p = 0.180) in richness between time points. PCA revealed site-specific microbial communities in the feces, and upper and lower airways. In comparison, blood had an apparent compositional similarity with BALF with regard to a few dominant taxa, but shared more OTUs with feces. Samples clustered more by time than by individual, with OP swabs having subjectively greater variation than other samples. In summary, healthy cats have a rich and distinct lower airway microbiome with dynamic bacterial populations. The microbiome is likely to be altered by factors such as age, environmental influences, and disease states. Further data are necessary to determine how the distinct feline microbiomes from the upper and lower airways, feces and blood are established and evolve. These data are relevant for comparisons between healthy cats and cats with respiratory disease.

Asthma is prevalent in children and cats, and needs means of noninvasive diagnosis. We sought to distinguish noninvasively the differences in 53 cats before and soon after induction of allergic asthma, using NMR spectra of exhaled breath condensate (EBC). Statistical pattern recognition was improved considerably by preprocessing the spectra with probabilistic quotient normalization and glog transformation. Classification of the 106 preprocessed spectra by principal component analysis and partial least squares with discriminant analysis (PLS-DA) appears to be impaired by variances unrelated to eosinophilic asthma. By filtering out confounding variances, orthogonal signal correction (OSC) PLS-DA greatly improved the separation of the healthy and early asthmatic states, attaining 94% specificity and 94% sensitivity in predictions. OSC enhancement of multi-level PLS-DA boosted the specificity of the prediction to 100%. OSC-PLS-DA of the normalized spectra suggest the most promising biomarkers of allergic asthma in cats to include increased acetone, metabolite(s) with overlapped NMR peaks near 5.8 ppm, and a hydroxyphenyl-containing metabolite, as well as decreased phthalate. Acetone is elevated in the EBC of 74% of the cats with early asthma. The noninvasive detection of early experimental asthma, biomarkers in EBC, and metabolic perturbation invite further investigation of the diagnostic potential in humans., Competing Interests: The authors have declared that no competing interests exist.

Table of Contents: A1 One health advances and successes in comparative medicine and translational researchCheryl StroudA2 Dendritic cell-targeted gorilla adenoviral vector for cancer vaccination for canine melanomaIgor Dmitriev, Elena Kashentseva, Jeffrey N. Bryan, David T. CurielA3 Viroimmunotherapy for malignant melanoma in the companion dog modelJeffrey N. Bryan, David Curiel, Igor Dmitriev, Elena Kashentseva, Hans Rindt, Carol Reinero, Carolyn J. HenryA4 Of mice and men (and dogs!): development of a commercially licensed xenogeneic DNA vaccine for companion animals with malignant melanomaPhilip J. BergmanA5 Successful immunotherapy with a recombinant HER2-expressing Listeria monocytogenes in dogs with spontaneous osteosarcoma paves the way for advances in pediatric osteosarcomaNicola J. Mason, Josephine S. Gnanandarajah, Julie B. Engiles, Falon Gray, Danielle Laughlin, Anita Gaurnier-Hausser, Anu Wallecha, Margie Huebner, Yvonne PatersonA6 Human clinical development of ADXS-HER2Daniel O'ConnorA7 Leveraging use of data for both human and veterinary benefitLaura S. TremlA8 Biologic replacement of the knee: innovations and early clinical resultsJames P. StannardA9 Mizzou BioJoint Center: a translational success storyJames L. CookA10 University and industry translational partnership: from the lab to commercializationMarc JacobsA11 Beyond docking: an evolutionarily guided OneHealth approach to drug discoveryGerald J. Wyckoff, Lee Likins, Ubadah Sabbagh, Andrew SkaffA12 Challenges and opportunities for data applications in animal health: from precision medicine to precision husbandryAmado S. GuloyA13 A cloud-based programmable platform for healthHarlen D. HaysA14 Comparative oncology: One Health in actionAmy K. LeBlancA15 Companion animal diseases bridge the translational gap for human neurodegenerative diseaseJoan R. Coates, Martin L. Katz, Leslie A. Lyons, Gayle C. Johnson, Gary S. Johnson, Dennis P. O'BrienA16 Duchenne muscular dystrophy gene therapyDongsheng DuanA17 Polycystic kidney disease: cellular mechanisms to emerging therapiesJames P. CalvetA18 The domestic cat as a large animal model for polycystic kidney diseaseLeslie A. Lyons, Barbara GandolfiA19 The support of basic and clinical research by the Polycystic Kidney Disease FoundationDavid A. BaronA20 Using naturally occurring large animal models of human disease to enable clinical translation: treatment of arthritis using autologous stromal vascular fraction in dogsMark L. WeissA21 Regulatory requirements regarding clinical use of human cells, tissues, and tissue-based productsDebra A. WebsterA22 Regenerative medicine approaches to Type 1 diabetes treatmentFrancis N. KaranuA23 The zoobiquity of canine diabetes mellitus, man's best friend is a friend indeed-islet transplantationEdward J. RobbA24 One Medicine: a development model for cellular therapy of diabetesRobert J. Harman.

The upper and lower airways of healthy humans are reported to harbor stable and consistent bacterial populations, and the composition of these communities is altered in individuals affected with several respiratory diseases. Data regarding the presence of airway microbiota in other animals are scant and a better understanding of the composition and metabolic function of such bacterial populations is essential for the development of novel therapeutic and diagnostic modalities for use in both veterinary and human medicine. Based on targeted next-generation sequencing of feces and samples collected at multiple levels of the airways from 16 healthy female dogs, we demonstrate that canine airways harbor a topographically continuous microbiota with increasing relative abundance of proteobacterial species from the upper to lower airways. The lung-associated microbiota, as assessed via bronchoalveolar lavage fluid (BALF), was the most consistent between dogs and was dominated by three distinct taxa, two of which were resolved to the species level and one to the level of family. The gene content of the nasal, oropharyngeal, and lung-associated microbiota, predicted using the Phylogenetic Investigations into Communities by Reconstruction of Unobserved States (PICRUSt) software, provided information regarding the glyoxylate and citrate cycle metabolic pathways utilized by these bacterial populations to colonize such nutrient-poor, low-throughput environments. These data generated in healthy subjects provide context for future analysis of diseased canine airways. Moreover, as dogs have similar respiratory anatomy, physiology, and immune systems as humans, are exposed to many of the same environmental stimuli, and spontaneously develop similar respiratory diseases, these data support the use of dogs as a model species for prospective studies of the airway microbiota, with findings translatable to the human condition.

Systemically low levels of survival motor neuron-1 (SMN1) protein cause spinal muscular atrophy (SMA). α-Motor neurons of the spinal cord are considered particularly vulnerable in this genetic disorder and their dysfunction and loss cause progressive muscle weakness, paralysis and eventually premature death of afflicted individuals. Historically, SMA was therefore considered a motor neuron-autonomous disease. However, depletion of SMN in motor neurons of normal mice elicited only a very mild phenotype. Conversely, restoration of SMN to motor neurons in an SMA mouse model had only modest effects on the SMA phenotype and survival. Collectively, these results suggested that additional cell types contribute to the pathogenesis of SMA, and understanding the non-autonomous requirements is crucial for developing effective therapies. Astrocytes are critical for regulating synapse formation and function as well as metabolic support for neurons. We hypothesized that astrocyte functions are disrupted in SMA, exacerbating disease progression. Using viral-based restoration of SMN specifically to astrocytes, survival in severe and intermediate SMA mice was observed. In addition, neuromuscular circuitry was improved. Astrogliosis was prominent in end-stage SMA mice and in post-mortem patient spinal cords. Increased expression of proinflammatory cytokines was partially normalized in treated mice, suggesting that astrocytes contribute to the pathogenesis of SMA., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Spinal Muscular Atrophy (SMA) is due to the loss of the survival motor neuron gene 1 (SMN1), resulting in motor neuron (MN) degeneration, muscle atrophy and loss of motor function. While SMN2 encodes a protein identical to SMN1, a single nucleotide difference in exon 7 causes most of the SMN2-derived transcripts to be alternatively spliced resulting in a truncated and unstable protein (SMNΔ7). SMA patients retain at least one SMN2 copy, making it an important target for therapeutics. Many of the existing SMA models are very severe, with animals typically living less than 2 weeks. Here, we present a novel intermediate mouse model of SMA based upon the human genomic SMN2 gene. Genetically, this model is similar to the well-characterized SMNΔ7 model; however, we have manipulated the SMNΔ7 transgene to encode a modestly more functional protein referred to as SMN read-through (SMN(RT)). By introducing the SMN(RT) transgene onto the background of a severe mouse model of SMA (SMN2(+/+);Smn(-/-)), disease severity was significantly decreased based upon a battery of phenotypic parameters, including MN pathology and a significant extension in survival. Importantly, there is not a full phenotypic correction, allowing for the examination of a broad range of therapeutics, including SMN2-dependent and SMN-independent pathways. This novel animal model serves as an important biological and therapeutic model for less severe forms of SMA and provides an in vivo validation of the SMN(RT) protein.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder caused by polyglutamine expansion in the amino-terminus of huntingtin (HTT). HD offers unique opportunities for promising RNA-based therapeutic approaches aimed at reducing mutant HTT expression, since the HD mutation is considered to be a "gain-of-function" mutation. Allele-specific strategies that preserve expression from the wild-type allele and reduce the levels of mutant protein would be of particular interest. Here, we have conducted proof-of-concept studies to demonstrate that spliceosome-mediated trans-splicing is a viable molecular strategy to specifically repair the HTT allele. We employed a dual plasmid transfection system consisting of a pre-mRNA trans-splicing module (PTM) containing HTT exon 1 and a HTT minigene to demonstrate that HTT exon 1 can be replaced in trans. We detected the presence of the trans-spliced RNA in which PTM exon 1 was correctly joined to minigene exons 2 and 3. Furthermore, exon 1 from the PTM was trans-spliced to the endogenous HTT pre-mRNA in cultured cells as well as disease-relevant models, including HD patient fibroblasts and primary neurons from a previously described HD mouse model. These results suggest that the repeat expansion of HTT can be repaired successfully not only in the context of synthetic minigenes but also within the context of HD neurons. Therefore, pre-mRNA trans-splicing may be a promising approach for the treatment of HD and other dominant genetic disorders.

Spinal Muscular Atrophy (SMA) is a devastating neurodegenerative disease and is a leading genetic cause of infantile death. SMA is caused by the homozygous loss of Survival Motor Neuron-1 (SMN1). The presence of a nearly identical copy gene called SMN2 has led to the development of several strategies that are designed to elevate SMN levels, and it is clear that SMN2 is an important modifier gene. However, the possibility exists that SMN-independent strategies to lessen the severity of the SMA phenotype could provide insight into disease development as well as aid in the identification of potential therapeutic targets. Muscle enhancement has been considered an interesting target for a variety of neurodegenerative diseases, including SMA. Previously we have shown in SMA mice that delivery of recombinant follistatin resulted in an extension in survival and a general lessening of disease severity. Follistatin is known to functionally block myostatin (MSTN), a potent inhibitor of muscle development. However, follistatin is a multifaceted protein involved in a variety of cellular pathways. To determine whether MSTN inhibition was the primary pathway associated with the previously reported follistatin results, we generated an animal model of SMA in which Mstn was genetically inactivated. In this report we characterize the novel SMA/Mstn model and demonstrate that Mstn inactivation does not significantly enhance muscle development in neonatal animals, nor does it result in an amelioration of the SMA phenotype., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Spinal muscular atrophy (SMA) is the most common genetic cause of infant mortality. SMA is caused by loss of functional survival motor neuron 1 (SMN1), resulting in death of spinal motor neurons. Current therapeutic research focuses on modulating the expression of a partially functioning copy gene, SMN2, which is retained in SMA patients. However, a treatment strategy that improves the SMA phenotype by slowing or reversing the skeletal muscle atrophy may also be beneficial. Myostatin, a member of the TGF-beta super-family, is a potent negative regulator of skeletal muscle mass. Follistatin is a natural antagonist of myostatin, and over-expression of follistatin in mouse muscle leads to profound increases in skeletal muscle mass. To determine whether enhanced muscle mass impacts SMA, we administered recombinant follistatin to an SMA mouse model. Treated animals exhibited increased mass in several muscle groups, elevation in the number and cross-sectional area of ventral horn cells, gross motor function improvement and mean lifespan extension by 30%, by preventing some of the early deaths, when compared with control animals. SMN protein levels in spinal cord and muscle were unchanged in follistatin-treated SMA mice, suggesting that follistatin exerts its effect in an SMN-independent manner. Reversing muscle atrophy associated with SMA may represent an unexploited therapeutic target for the treatment of SMA.

Cardiac-specific overexpression of wild-type alpha(1B)-adrenergic receptors (alpha(1B)-AR) in mice predisposes to dilated cardiomyopathy and sudden death. Although alpha-adrenergic stimulation is thought to contribute to induction of arrhythmias in heart failure, the electrophysiological consequences of chronic alpha(1)-adrenergic activation have not been clearly defined. Thus we characterized ventricular repolarization and monitored incidence of spontaneous arrhythmias in end-stage heart failure alpha(1B)-AR mice (9-12 mo) and younger alpha(1B)-AR mice (2-3 mo) that do not present signs of heart failure. Compared with aged-matched controls, the corrected QT interval was 34% longer in the 9- to 12-mo alpha(1B)-AR mice, and the action potential durations were also significantly prolonged in these mice. These changes were associated with a decrease in the density of the outward K(+) currents, Ca(2+)-independent transient, ultrarapid delayed rectifier, and steady state (at +30 mV, reduction of 68, 64, and 41%, respectively), and underlying K(+) channel expression. Electrocardiogram (ECG) recordings revealed that older alpha(1B)-AR mice exhibited spontaneous ventricular arrhythmias. The alterations in repolarization can contribute to these rhythm abnormalities and are likely caused by chronic alpha(1B)-AR activity. Additional data obtained in 2- to 3-mo alpha(1B)-AR mice clearly showed that electrical remodeling was already observed in younger transgenic animals. However, it appeared to be slightly less pronounced than in older mice. These results suggest that there are two waves of remodeling: one due to chronic alpha(1B)-AR activity, and a second due to heart failure. Taken together, these data provide strong evidence for a pathological role of chronic alpha(1B)-AR activity in the development of repolarization defects and ventricular arrhythmias.

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased beta-myosin heavy chain (betaMyHC) gene expression. Our previous work showed that Sp3 contributes to decreased betaMyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Puralpha, and Purbeta proteins mediate repression of betaMyHC expression under NWB conditions. Binding of Puralpha or Purbeta to the single-stranded betaMyHC distal negative regulatory element-sense strand (dbetaNRE-S) element is markedly increased under NWB conditions. Ectopic expression of Puralpha and Purbeta decreased betaMyHC reporter gene expression, while mutation of the dbetaNRE-S element increased expression in C2C12 myotubes. The dbetaNRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Puralpha and Purbeta decreased endogenous Sp3 and Pur protein levels and increased betaMyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Puralpha, Purbeta, and Sp3, while chromatin immunoprecipitation assays demonstrated Puralpha, Purbeta, and Sp3 binding to the betaMyHC proximal promoter region harboring the dbetaNRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.

The function of beta-adrenergic receptor (betaAR) is modulated by the activity status of alpha1-adrenergic receptors (alpha1ARs) via molecular crosstalk, and this becomes evident when measuring cardiac contractile responses to adrenergic stimulation. The molecular mechanism underlying this crosstalk is unknown. We have previously demonstrated that overexpression of alpha1B-adrenergic receptor (alpha1BAR) in transgenic mice leads to a marked desensitization of betaAR-mediated adenylyl cyclase stimulation which is correlated with increased levels of activated protein kinase C (PKC) beta, delta and [J. Mol. Cell. Cardiol. 30 (1998) 1827]. Therefore, we wished to determine which PKC isoforms play a role in heterologous betaAR desensitization and also which isoforms of the betaAR were the molecular target(s) for PKC. In experiments using constitutively activated PKC expression constructs transfected into HEK 293 cells also expressing the beta2AR, constitutively active (CA)-PKC overexpression was first confirmed by immunoblots using specific anti-PKC antibodies. We then demonstrated that the different PKC subtypes lead to a decreased maximal cAMP accumulation following isoproterenol stimulation with a rank order of PKCalpha > or = PKCzeta>PKC>PKCbetaII. However, a much more dramatic desensitization of adenylyl cyclase stimulation was observed in cells co-transfected with different PKC isoforms and beta1AR. Further, the modulation of beta1AR by PKC isoforms had a different rank order than for the beta2AR: PKCbetaII>PKCalpha>PKC>PKCzeta. PKC-mediated desensitization was reduced by mutating consensus cAMP-dependent protein kinase (PKA)/PKC sites in the third intracellular loop and/or the carboxy-terminal tail of either receptor. Our results demonstrate therefore that the beta1AR is the most likely molecular target for PKC-mediated heterologous desensitization in the mammalian heart and that modulation of adrenergic receptor activity in any given cell type will depend on the complement of PKC isoforms present., (Copyright 2004 Elsevier Inc.)

Cardiomyocyte-specific overexpression of the wild-type alpha(1B)-adrenergic receptor (alpha(1B)-AR) produces a slowly progressing cardiomyopathy associated with clinical signs of heart failure and premature death around middle age (Lemire et al. 2001). In the heart, alpha(1)-AR activate the extracellular signal-regulated kinase (ERK) MAPK cascade. The aim of this project was to determine if cardiac-specific overexpression of the wild-type alpha(1B)-AR results in sustained activation of the ERK pathway. At 3 and 9 months, ERK activity was increased in alpha(1B)-AR overexpressing hearts relative to non-transgenic animals. Similarly, phosphorylation of MEK and p90(rsk) were also elevated. MAP kinase phosphatases (MKPs), which inactivate MAP kinases, are transcriptionally regulated. MKP2 mRNA levels were reduced at 3 months in alpha(1B)-AR overexpressing hearts. Interestingly, there was a general trend for reduced expression of MKP-1, -2, and -3 with increased age. In addition, expression of the modulatory calcineurin-interacting protein (MCIP) 1, an indicator of calcineurin activity, was elevated 3-fold in alpha(1B)-AR overexpressing hearts at both 3 and 9 months. These results indicate that the overexpression of the wild-type alpha(1B)-AR leads to chronic changes in the activation of signalling pathways previously shown to be associated with the hypertrophic response.

This study was done to identify the mechanism of the alpha1-adrenoceptor (AR) mediated negative inotropic effects of phenylephrine (PE) on adult mouse myocardium. As reported by others, we also found that the nonselective alpha1AR agonist PE produced a negative inotropic effect on ventricular strips from adult mice that was inhibited by the alpha1AAR antagonist 5-methylurapidil (5MU) but not by the alpha1BAR antagonist chloroethylclonidine (CEC) or the alpha1DAR antagonist BMY 7378. The selective alpha1AAR agonist A61603 also produced a negative inotropic effect, which was antagonized by 5MU. Phorbol 12,13-dibutyrate (activator of all PKC isoforms) mimicked the negative inotropic responses to PE and A61603. The negative inotropic effects of PE were inhibited by bisindolylmaleimide (inhibitor of all PKC isoforms) but not by Gö 6976 (inhibitor of Ca2+-dependent PKC). Rottlerin, an inhibitor of Ca2+-independent PKCdelta, antagonized the negative inotropic effects of PE and A61603. PE and A61603 increased the translocation of PKCdelta, which was prevented by rottlerin. These data suggest that the alpha1AR-mediated negative inotropy on adult mouse myocardium is signaled by Ca2+-independent PKCdelta.

Postnatal cardiac myocytes respond to stress signals by hypertrophic growth and activation of a fetal gene program. Recently, we showed that class II histone deacetylases (HDACs) suppress cardiac hypertrophy, and mice lacking the class II HDAC, HDAC9, are sensitized to hypertrophic signals. To further define the roles of HDACs in cardiac hypertrophy, we analyzed the effects of HDAC inhibitors on the responsiveness of primary cardiomyocytes to hypertrophic agonists. Paradoxically, HDAC inhibitors imposed a dose-dependent blockade to hypertrophy and fetal gene activation. We conclude that distinct HDACs play positive or negative roles in the control of cardiomyocyte hypertrophy. HDAC inhibitors are currently being tested in clinical trials as anti-cancer agents. Our results suggest that these inhibitors may also hold promising clinical value as therapeutics for cardiac hypertrophy and heart failure.

The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart.

Using transgenesis as a paradigm, we show here that alpha1-adrenergic receptors (alpha1AR) play an important role in cardiac homeostasis. Cardiomyocyte-specific overexpression of the alpha(1B)AR subtype resulted in the development of dilated cardiomyopathy and death at ~9 mo of age with typical signs of heart failure. Histological analyses showed the enlargement of all four cardiac chambers and cardiomyocyte disarray in the failing hearts. Transgenic animals showed increased left ventricular areas, as assessed by echocardiography. In addition, a progressive decrease in left ventricular systolic function was revealed. The abundance and activity of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) were reduced, and the ratio of phospholamban to SERCA2 was increased. alpha-Myosin heavy chain (MHC) mRNA was less abundant in older transgenic ventricles, whereas beta-MHC was induced in the failing hearts. Titin mRNA abundance was decreased at 9 mo, whereas atrial natriuretic factor mRNA was elevated at all times. This model mimics structural and functional features of idiopathic dilated cardiomyopathy. The results of this study suggest that chronic alpha1AR activity is deleterious for cardiac function.

We previously described an ultrarapid delayed rectifier current in dog atrial myocytes (IKur,d) with properties resembling currents reported for Kv3.1 channels in neural tissue; however, there was no direct molecular evidence for Shaw subfamily (Kv3) subunit expression in the heart. To identify the molecular basis of IKur,d, we cloned a full-length cDNA (dKv3.1) from canine atrium with homology-based reverse transcription (RT)- polymerase chain reaction (PCR) cloning techniques. A 1755 bp full-length cDNA (dKv3.1) was obtained, with 94.2 % homology to rat brain Kv3.1 (rbKv3.1). The deduced amino acid sequence had 99.3 % homology with rbKv3.1. Heterologous expression of dKv3.1 in Xenopus oocytes produced currents with activation voltage dependence, rectification, and activation and deactivation kinetics that strongly resemble native IKur,d. Like IKur,d, dKv3.1 was found to be highly sensitive to extracellular 4-aminopyridine (4-AP) and tetraethylammonium (TEA). RNase protection assays, Western blots and immunohistochemical studies demonstrated the presence of dKv3.1 transcripts and proteins in dog atrial preparations and isolated canine atrial myocytes. Protein corresponding to the Kv1.5 subunit, which can also carry ultrarapid delayed rectifier current, was absent. Unlike neural tissues, which express two splice variants (Kv3.1a and Kv3.1b), canine atrium showed only Kv3.1b transcripts. Whole-cell patch-clamp studies showed that IKur,d is absent in canine ventricular myocytes, and immunohistochemical and Western blot analysis demonstrated the absence of dKv3.1 protein in canine ventricle. We conclude that the Shaw-type channel dKv3.1 is present in dog atrium, but not ventricle, and is the likely molecular basis of canine atrial IKur,d.

The overexpression of either oncogenic ras or calmodulin in cardiac myocytes can elicit a hypertrophic response, albeit their recruitment by physiologically relevant stimuli remains unresolved. The present study utilized a pharmacological approach to examine the role of ras and calmodulin in norepinephrine- and endothelin-1-stimulated hypertrophy of neonatal rat cardiac myocytes. The pretreatment of cardiac myocytes with the farnesyltransferase inhibitor BMS-191563 (25 microM) increased the level of unfarnesylated ras in the cytosolic fraction, and caused a concomitant 42 +/- 2% decrease in immunodetectable farnesylated ras in the particulate fraction. In parallel, BMS-191563 pretreatment inhibited norepinephrine-mediated 3H-leucine uptake (80 +/- 10% decrease: n = 6; P<0.01), whereas a significant but less pronounced effect on the endothelin-1 response (46 +/- 6% decrease: n = 6; P<0.05) was observed. The calmodulin inhibitor W7 caused a 50 +/- 10% decrease (n = 8; P<0.05) of norepinephrine stimulated protein synthesis, whereas the endothelin-1 response was unaffected. Consistent with the recruitment of ras, BMS-191563 pretreatment attenuated norepinephrine and endothelin-1-stimulated extracellular signal-regulated kinase (ERK) activity. However, PD098059-mediated inhibition of MEK-dependent stimulation of ERK did not alter the hypertrophic response of either agonist. At the molecular level, the pretreatment with either BMS-191563 or W7 attenuated the norepinephrine-mediated increase of prepro-ANP and -BNP mRNA. Likewise, BMS-191563 caused a significant decrease of endothelin-1-mediated expression of the natriuretic peptide mRNAs, but to a lesser extent, as compared to norepinephrine. Thus, the present study has shown the treatment of neonatal rat cardiac myocytes with a farnesyltransferase inhibitor can attenuate the hypertrophic phenotype in response to physiologically relevant stimuli, thereby supporting a role of the small GTP-binding protein ras. Moreover, these data further suggest alternative ras-independent signaling pathways are also implicated in the hypertrophic response, albeit, there appears to exist a stimulus-specific heterogeneity in their recruitment.