Global identification of modular cullin-RING ligase substrates.

Cullin-RING ligases (CRLs) represent the largest E3 ubiquitin ligase family in eukaryotes, and the identification of their substrates is critical to understanding regulation of the proteome. Using genetic and pharmacologic Cullin inactivation coupled with genetic (GPS) and proteomic (QUAINT) assays, we have identified hundreds of proteins whose stabilities or ubiquitylation status are regulated by CRLs. Together, these approaches yielded many known CRL substrates as well as a multitude of previously unknown putative substrates. We demonstrate that one substrate, NUSAP1, is an SCF(Cyclin F) substrate during S and G2 phases of the cell cycle and is also degraded in response to DNA damage. This collection of regulated substrates is highly enriched for nodes in protein interaction networks, representing critical connections between regulatory pathways. This demonstrates the broad role of CRL ubiquitylation in all aspects of cellular biology and provides a set of proteins likely to be key indicators of cellular physiology.

Dynamics of cullin-RING ubiquitin ligase network revealed by systematic quantitative proteomics.

Dynamic reorganization of signaling systems frequently accompanies pathway perturbations, yet quantitative studies of network remodeling by pathway stimuli are lacking. Here, we report the development of a quantitative proteomics platform centered on multiplex absolute quantification (AQUA) technology to elucidate the architecture of the cullin-RING ubiquitin ligase (CRL) network and to evaluate current models of dynamic CRL remodeling. Current models suggest that CRL complexes are controlled by cycles of CRL deneddylation and CAND1 binding. Contrary to expectations, acute CRL inhibition with MLN4924, an inhibitor of the NEDD8-activating enzyme, does not result in a global reorganization of the CRL network. Examination of CRL complex stoichiometry reveals that, independent of cullin neddylation, a large fraction of cullins are assembled with adaptor modules, whereas only a small fraction are associated with CAND1. These studies suggest an alternative model of CRL dynamicity where the abundance of adaptor modules, rather than cycles of neddylation and CAND1 binding, drives CRL network organization.

Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation.

All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here, we report that the unconventional linkages are abundant in vivo and that all non-K63 linkages may target proteins for degradation. Ubiquitin with K48 as the single lysine cannot support yeast viability, and different linkages have partially redundant functions. By profiling both the entire yeast proteome and ubiquitinated proteins in wild-type and ubiquitin K11R mutant strains using mass spectrometry, we identified K11 linkage-specific substrates, including Ubc6, a ubiquitin-conjugating enzyme involved in endoplasmic reticulum-associated degradation (ERAD). Ubc6 primarily synthesizes K11-linked chains, and K11 linkages function in the ERAD pathway. Thus, unconventional polyubiquitin chains are critical for ubiquitin-proteasome system function.

Systematic and quantitative assessment of the ubiquitin-modified proteome.

Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ~19,000 diGly-modified lysine residues within ~5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantitative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.

Vasopressor use in 41 critically ill cats (2007-2016).

This study describes the use of vasopressors in critically ill cats. Records of 41 cats hospitalized in the ICU were evaluated. Signalment, blood pressure, underlying conditions, evidence of sepsis, type of treatment (surgical versus non-surgical), vasopressor type and duration, adverse events attributed to vasopressors, and survival were recorded. Twenty-one cats (51%) had an underlying disease considered amenable to surgical treatment while 20 (49%) cats did not. Evidence of sepsis was present in 24 (59%) cats. Thirty-four cats developed a Doppler blood pressure (DBP) > 80 mmHg during therapy, and 29 cats became normotensive (DBP > 90 mmHg). Seven cats did not increase their DBP to > 80 mmHg. All cats received dopamine and/or norepinephrine and 6 cats also received other vasopressors. Sixteen cats survived (39%). Surgical intervention was associated with a higher survival (P = 0.004). Critically ill hypotensive cats may benefit from administration of vasopressors.

Utilisation de vasopressine chez 41 chats en état critique (2007­2016). Cette étude décrit l'utilisation de vasopresseurs chez les chats en condition critique. Les dossiers médicaux de 41 chats hospitalisés ont été évalués. Le signalement, pression sanguine, conditions sous-jacentes, évidence de sepsis, type de traitement (chirurgical contre médical), type de vasopresseurs et durée, effets adverses reliés à l'utilisation des vasopresseurs et survie ont été comptabilisés. Vingt et un chats (51 %) avaient une condition sous-jacente susceptible au traitement chirurgical contrairement aux 20 autres chats (49 %). L'évidence de sepsis était présente dans 24 (59 %) chats. Trente-quatre chats ont développé une pression sanguine au Doppler (DBP) > 80 mmHg durant le traitement et 29 chats sont devenus normotensifs (DBP > 90 mmHg). Sept chats n'ont pas eu d'augmentation de leur DBP > 80 mmHg. Tous les chats ont reçu de la dopamine et/ou de la norépinéphrine et 6 chats ont reçus d'autres vasopresseurs. Seize chats ont survécu (39 %). Une intervention chirurgicale est associée à un plus grand taux de survie (P = 0,004). Les chats hypotensifs en condition critique peuvent bénéficier de l'utilisation de vasopresseurs.(Traduit par les auteurs).

A Novel Model for Teaching Primary Care in a Community Practice Setting: Tufts at Tech Community Veterinary Clinic.

Providing veterinary students with opportunities to develop clinical skills in a realistic, hands-on environment remains a challenge for veterinary education. We have developed a novel approach to teaching clinical medicine to fourth-year veterinary students and technical high school students via development of a primary care clinic embedded within a technical high school. The primary care clinic targets an underserved area of the community, which includes many of the participating high school students. Support from the veterinary community for the project has been strong as a result of communication, the opportunity for veterinarians to volunteer in the clinic, and the careful targeting of services. Benefits to veterinary students include the opportunity to build clinical competencies and confidence, as well as the exposure to a diverse client population. The financial model of the clinic is described and initial data on outcomes for case load, clinic income, veterinary student evaluations, and high school students' success in passing the veterinary assisting examination are reported. This clinical model, involving a partnership between a veterinary school and a technical high school, may be adoptable to other clinical teaching situations.

Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation.

Cross-talk between different types of post-translational modifications on the same protein molecule adds specificity and combinatorial logic to signal processing, but it has not been characterized on a large-scale basis. We developed two methods to identify protein isoforms that are both phosphorylated and ubiquitylated in the yeast Saccharomyces cerevisiae, identifying 466 proteins with 2,100 phosphorylation sites co-occurring with 2,189 ubiquitylation sites. We applied these methods quantitatively to identify phosphorylation sites that regulate protein degradation via the ubiquitin-proteasome system. Our results demonstrate that distinct phosphorylation sites are often used in conjunction with ubiquitylation and that these sites are more highly conserved than the entire set of phosphorylation sites. Finally, we investigated how the phosphorylation machinery can be regulated by ubiquitylation. We found evidence for novel regulatory mechanisms of kinases and 14-3-3 scaffold proteins via proteasome-independent ubiquitylation.

Combinatorial approach for large-scale identification of linked peptides from tandem mass spectrometry spectra.

The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.

Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry.

To facilitate accurate histone variant and post-translational modification (PTM) quantification via mass spectrometry, we present a library of 93 synthetic peptides using Protein-Aqua™ technology. The library contains 55 peptides representing different modified forms from histone H3 peptides, 23 peptides representing H4 peptides, 5 peptides representing canonical H2A peptides, 8 peptides representing H2A.Z peptides, and peptides for both macroH2A and H2A.X. The PTMs on these peptides include lysine mono- (me1), di- (me2), and tri-methylation (me3); lysine acetylation; arginine me1; serine/threonine phosphorylation; and N-terminal acetylation. The library was subjected to chemical derivatization with propionic anhydride, a widely employed protocol for histone peptide quantification. Subsequently, the detection efficiencies were quantified using mass spectrometry extracted ion chromatograms. The library yields a wide spectrum of detection efficiencies, with more than 1700-fold difference between the peptides with the lowest and highest efficiencies. In this paper, we describe the impact of different modifications on peptide detection efficiencies and provide a resource to correct for detection biases among the 93 histone peptides. In brief, there is no correlation between detection efficiency and molecular weight, hydrophobicity, basicity, or modification type. The same types of modifications may have very different effects on detection efficiencies depending on their positions within a peptide. We also observed antagonistic effects between modifications. In a study of mouse trophoblast stem cells, we utilized the detection efficiencies of the peptide library to correct for histone PTM/variant quantification. For most histone peptides examined, the corrected data did not change the biological conclusions but did alter the relative abundance of these peptides. For a low-abundant histone H2A variant, macroH2A, the corrected data led to a different conclusion than the uncorrected data. The peptide library and detection efficiencies presented here may serve as a resource to facilitate studies in the epigenetics and proteomics fields.

Phosphotyrosine signaling proteins that drive oncogenesis tend to be highly interconnected.

Mutation and overexpression of receptor tyrosine kinases or the proteins they regulate serve as oncogenic drivers in diverse cancers. To better understand receptor tyrosine kinase signaling and its link to oncogenesis, we used protein microarrays to systematically and quantitatively measure interactions between virtually every SH2 or PTB domain encoded in the human genome and all known sites of tyrosine phosphorylation on 40 receptor tyrosine kinases and on most of the SH2 and PTB domain-containing adaptor proteins. We found that adaptor proteins, like RTKs, have many high affinity bindings sites for other adaptor proteins. In addition, proteins that drive cancer, including both receptors and adaptor proteins, tend to be much more highly interconnected via networks of SH2 and PTB domain-mediated interactions than nononcogenic proteins. Our results suggest that network topological properties such as connectivity can be used to prioritize new drug targets in this well-studied family of signaling proteins.

Expression Profiling of Circulating MicroRNAs in Canine Myxomatous Mitral Valve Disease.

MicroRNAs (miRNAs) are small non-coding RNAs that have shown promise as noninvasive biomarkers in cardiac disease. This study was undertaken to investigate the miRNA expression profile in dogs with myxomatous mitral valve disease (MMVD). 277 miRNAs were quantified using RT-qPCR from six normal dogs (American College of Veterinary Internal Medicine Stage A), six dogs with MMVD mild to moderate cardiac enlargement (ACVIM Stage B1/B2) and six dogs with MMVD and congestive heart failure (ACVIM Stage C/D). Eleven miRNAs were differentially expressed (False Discovery Rate < 0.05). Dogs in Stage B1/B2 or C/D had four upregulated miRNAs, including three cfa-let-7/cfa-miR-98 family members, while seven others were downregulated, compared to Stage A. Expression of six of the 11 miRNAs also were significantly different between dogs in Stage C/D and those in Stage B1/B2. The expression changes were greater as disease severity increased. These miRNAs may be candidates for novel biomarkers and may provide insights into genetic regulatory pathways in canine MMVD.

A turn-key approach for large-scale identification of complex posttranslational modifications.

The conjugation of complex post-translational modifications (PTMs) such as glycosylation and Small Ubiquitin-like Modification (SUMOylation) to a substrate protein can substantially change the resulting peptide fragmentation pattern compared to its unmodified counterpart, making current database search methods inappropriate for the identification of tandem mass (MS/MS) spectra from such modified peptides. Traditionally it has been difficult to develop new algorithms to identify these atypical peptides because of the lack of a large set of annotated spectra from which to learn the altered fragmentation pattern. Using SUMOylation as an example, we propose a novel approach to generate large MS/MS training data from modified peptides and derive an algorithm that learns properties of PTM-specific fragmentation from such training data. Benchmark tests on data sets of varying complexity show that our method is 80-300% more sensitive than current state-of-the-art approaches. The core concepts of our method are readily applicable to developing algorithms for the identifications of peptides with other complex PTMs.

TSLP signaling network revealed by SILAC-based phosphoproteomics.

Thymic stromal lymphopoietin (TSLP) is a cytokine that plays diverse roles in the regulation of immune responses. TSLP requires a heterodimeric receptor complex consisting of IL-7 receptor α subunit and its unique TSLP receptor (gene symbol CRLF2) to transmit signals in cells. Abnormal TSLP signaling (e.g. overexpression of TSLP or its unique receptor TSLPR) contributes to the development of a number of diseases including asthma and leukemia. However, a detailed understanding of the signaling pathways activated by TSLP remains elusive. In this study, we performed a global quantitative phosphoproteomic analysis of the TSLP signaling network using stable isotope labeling by amino acids in cell culture. By employing titanium dioxide in addition to antiphosphotyrosine antibodies as enrichment methods, we identified 4164 phosphopeptides on 1670 phosphoproteins. Using stable isotope labeling by amino acids in cell culture-based quantitation, we determined that the phosphorylation status of 226 proteins was modulated by TSLP stimulation. Our analysis identified activation of several members of the Src and Tec families of kinases including Btk, Lyn, and Tec by TSLP for the first time. In addition, we report TSLP-induced phosphorylation of protein phosphatases such as Ptpn6 (SHP-1) and Ptpn11 (Shp2), which has also not been reported previously. Co-immunoprecipitation assays showed that Shp2 binds to the adapter protein Gab2 in a TSLP-dependent manner. This is the first demonstration of an inducible protein complex in TSLP signaling. A kinase inhibitor screen revealed that pharmacological inhibition of PI-3 kinase, Jak family kinases, Src family kinases or Btk suppressed TSLP-dependent cellular proliferation making them candidate therapeutic targets in diseases resulting from aberrant TSLP signaling. Our study is the first phosphoproteomic analysis of the TSLP signaling pathway that greatly expands our understanding of TSLP signaling and provides novel therapeutic targets for TSLP/TSLPR-associated diseases in humans.

A proof-of-concept study evaluating cardiac compression techniques for cardiopulmonary resuscitation in laying hens (Gallus gallus).

OBJECTIVE: To determine in adult chickens which of 3 CPR techniques, sternal compressions (SC), SC with interposed caudal coelomic compressions (ICCC), or lateral compressions (LC), results in the highest mean systolic (SAP), diastolic (DAP), and mean arterial pressure (MAP) as measured directly from the carotid artery. DESIGN: Prospective, nonblinded, experimental crossover study. SETTING: University teaching hospital laboratory. ANIMALS: Ten retired laying hens. INTERVENTIONS: Birds were sedated, anesthetized, and placed in dorsal recumbency. A carotid artery catheter was placed to directly measure arterial pressure. Ventricular fibrillation was induced with direct cardiac stimulation using a 9-Volt battery. Each bird then received 2 minutes of the 3 different cardiac compression techniques in a random order by 3 different compressors, with the compressor order also randomized. Birds were subsequently administered IV epinephrine, and transthoracic defibrillation was attempted. At the end of experimentation, each bird was euthanized, and simple gross necropsies were performed. Linear mixed models followed by pairwise paired t-tests were performed to evaluate differences in pressures generated by each technique. MEASUREMENTS AND MAIN RESULTS: The primary study outcomes were SAP, DAP, and MAP over 2 minutes of compressions for each compression technique. Pressures from ICCC (SAP: 27.6 ± 5.3 mm Hg, DAP: 18.7 ± 5.2 mm Hg, MAP: 21.7 ± 5.2 mm Hg) were significantly higher than those from LC (SAP: 18.9 ± 5.4 mm Hg, DAP: 11.6 ± 4.1 mm Hg, MAP: 14.1 ± 4.5 mm Hg). Pressures from SC (SAP: 24.5 ± 6.4 mm Hg, DAP: 15.2 ± 4.3 mm Hg, MAP: 18.3 ± 5.0 mm Hg) were not significantly different from ICCC or LC. CONCLUSIONS: External compressions can generate detectable increases in arterial pressure in chickens with ventricular fibrillation. SC with ICCC generated significantly higher arterial pressures than LC. SC alone generated blood pressures that were not significantly different from those generated by SC with ICCC or LC.

Viscoelastic coagulation monitoring parameters in cats with acute arterial thromboembolism.

BACKGROUND: Hypercoagulability has been documented in cats with cardiac disease. However, hemostatic parameters, including viscoelastic coagulation monitoring (VCM) have not been reported in cats with arterial thromboembolism (ATE). HYPOTHESIS/OBJECTIVES: Compare VCM parameters in cats with acute cardiogenic ATE and in control cats. ANIMALS: Sixteen cats with ATE and 30 control cats. METHODS: Multicenter university-based prospective study. Cardiogenic ATE was diagnosed based on physical examination and by ultrasonographically-diagnosed left atrial enlargement. Viscoelastic coagulation monitor analysis, CBC, serum biochemistry profile and coagulation profile were performed at admission in cats with ATE. Analysis from healthy control cats was performed using blood collected by direct venipuncture. Our objective was comparison of VCM parameters clot time (CT), clot formation time (CFT), alpha angle (Angle), maximum clot formation (MCF), amplitude at 10 and 20 minutes (A10 and A20, respectively) and clot lysis index at 30 and 45 minutes (LI30 and LI45, respectively) between ATE and control cats. RESULTS: Cats with ATE had a decreased angle compared to control cats, with a median (range) of 43° (30-48°) compared to 47° (14-59°; P = .01). The parameters A10, A20 and MCF were decreased in ATE cats compared to control cats with a median (range) of 19 units (8-32) compared to 22 units (6-38), 24.5 units (11-40) compared to 29 units (10-47) and 29.5 units (13-44) compared to 33.5 units (14-53), respectively (P = .01, .01 and .01, respectively). The parameters CT, CFT, LI30 and LI45 were similar between groups (P = .22, .09, .62 and .34, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Cats with cardiogenic ATE cats have VCM parameters consistent with hypocoagulability compared with healthy cats.

Echocardiographic and electrocardiographic findings in Irish Wolfhounds eating high-pulse or low-pulse diets.

BACKGROUND: Apparently healthy dogs of various breeds eating nontraditional, high-pulse diets can have larger left ventricular diameter, lower systolic function, and more ventricular premature complexes (VPCs) compared with dogs eating traditional, low-pulse diets. It is unknown whether Irish Wolfhounds eating high-pulse diets have similar cardiac abnormalities. HYPOTHESIS/OBJECTIVES: To compare electrocardiographic and echocardiographic findings between Irish Wolfhounds eating high- or low-pulse diets. ANIMALS: Ninety-seven Irish Wolfhounds. METHODS: Retrospective study of Irish Wolfhounds that had echocardiography performed at dog shows between October 2018 and May 2021. Demographic information, echocardiographic measurements, cardiac rhythm (1-minute lead II rhythm strip), and main diet were recorded retrospectively. Diets were classified as high-pulse or low-pulse based on the presence and location of pulses (peas, lentils, chickpeas, or dry beans) on the ingredient list. RESULTS: Thirty-five of 97 Irish Wolfhounds (36%) were eating high-pulse diets and 62 of 97 (64%) were eating low-pulse diets. There were no significant differences between diet groups in echocardiographic measurements. A significantly higher percentage of dogs in the high-pulse diet group (6/35 [17%]) had VPCs compared with those in the low-pulse diet group (1/62 [2%]; effect size = 0.15 [95% confidence interval: 0.004-0.31]; P = .005). CONCLUSIONS AND CLINICAL IMPORTANCE: In this retrospective study of apparently healthy Irish Wolfhounds, high-pulse diets were associated with a higher prevalence of VPCs which could represent early cardiac abnormalities.

Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology.

Protein ubiquitination regulates many cellular processes, including protein degradation, signal transduction, DNA repair and cell division. In the classical model, a uniform polyubiquitin chain that is linked through Lys 48 is required for recognition and degradation by the 26S proteasome. Here, we used a reconstituted system and quantitative mass spectrometry to demonstrate that cyclin B1 is modified by ubiquitin chains of complex topology, rather than by homogeneous Lys 48-linked chains. The anaphase-promoting complex was found to attach monoubiquitin to multiple lysine residues on cyclin B1, followed by poly-ubiquitin chain extensions linked through multiple lysine residues of ubiquitin (Lys 63, Lys 11 and Lys 48). These heterogeneous ubiquitin chains were sufficient for binding to ubiquitin receptors, as well as for degradation by the 26S proteasome, even when they were synthesized with mutant ubiquitin that lacked Lys 48. Together, our observations expand the context of what can be considered to be a sufficient degradation signal and provide unique insights into the mechanisms of substrate ubiquitination.

RSK phosphorylates SOS1 creating 14-3-3-docking sites and negatively regulating MAPK activation.

The extent and duration of MAPK (mitogen-activated protein kinase) signalling govern a diversity of normal and aberrant cellular outcomes. Genetic and pharmacological disruption of the MAPK-activated kinase RSK (ribosomal S6 kinase) leads to elevated MAPK activity indicative of a RSK-dependent negative feedback loop. Using biochemical, pharmacological and quantitative MS approaches we show that RSK phosphorylates the Ras activator SOS1 (Son of Sevenless homologue 1) in cultured cells on two C-terminal residues, Ser(1134) and Ser(1161). Furthermore, we find that RSK-dependent SOS1 phosphorylation creates 14-3-3-binding sites. We show that mutating Ser(1134) and Ser(1161) disrupts 14-3-3 binding and modestly increases and extends MAPK activation. Together these data suggest that one mechanism whereby RSK negatively regulates MAPK activation is via site-specific SOS1 phosphorylation.

Pit bull-type breeds with dilated cardiomyopathy eating nontraditional diets improve after diet change (2015-2022).

OBJECTIVE: To compare signalment, clinical signs, diet, echocardiographic findings, and outcome for pit bull-type breeds diagnosed between 2015 and 2022 with dilated cardiomyopathy (DCM) or with DCM diagnosed by a cardiologist but that did not meet all study echocardiographic criteria (DCM-C). ANIMALS: 91 dogs with DCM and 11 dogs with DCM-C. PROCEDURES: Data were collected on clinical findings, echocardiographic measurements, and diet at the time of diagnosis (for 76/91 dogs); echocardiographic changes; and survival. RESULTS: For dogs with diet information available for time of diagnosis, 64/76 (84%) dogs were eating nontraditional commercial diets, while 12/76 (16%) were eating traditional commercial diets. There were few differences between diet groups at baseline, with congestive heart failure and arrhythmias common in both groups. Thirty-four dogs with known baseline diet and diet change status had follow-up echocardiograms between 60 and 1,076 days later (traditional diet, n = 7; nontraditional diet that changed diets, 27; and nontraditional diet group without diet change, 0). Dogs in the nontraditional diet group that changed diets had a significantly greater decrease in normalized left ventricular diameter (diastolic, P = .02; systolic, P = .048) and the left atrium-to-aorta ratio (P = .002) and a significantly greater increase in fractional shortening (P = .02) compared to dogs eating traditional diets. Dogs eating nontraditional diets with diet change (n = 45; P < .001) and dogs eating traditional diets (12; P < .001) had a significantly longer survival time compared to dogs eating nontraditional diets without diet change (4). Dogs with DCM-C also had significant echocardiographic improvements after diet change. CLINICAL RELEVANCE: Congestive heart failure and arrhythmias were common in pit bull-type breeds with DCM. Those eating nontraditional diets that changed diets had significant improvements in echocardiographic measurements after diet change.