OBJECTIVE To characterize expression profiles of circulating microRNAs via genome-wide sequencing for dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve degeneration (MMVD). ANIMALS 9 healthy client-owned dogs and 8 age-matched client-owned dogs with CHF secondary to MMVD. PROCEDURES Blood samples were collected before administering cardiac medications for the management of CHF. Isolated microRNAs from plasma were classified into microRNA libraries and subjected to next-generation sequencing (NGS) for genome-wide sequencing analysis and quantification of circulating microRNAs. Quantitative reverse transcription PCR (qRT-PCR) assays were used to validate expression profiles of differentially expressed circulating microRNAs identified from NGS analysis of dogs with CHF. RESULTS 326 microRNAs were identified with NGS analysis. Hierarchical analysis revealed distinct expression patterns of circulating microRNAs between healthy dogs and dogs with CHF. Results of qRT-PCR assays confirmed upregulation of 4 microRNAs (miR-133, miR-1, miR-let-7e, and miR-125) and downregulation of 4 selected microRNAs (miR-30c, miR-128, miR-142, and miR-423). Results of qRT-PCR assays were highly correlated with NGS data and supported the specificity of circulating microRNA expression profiles in dogs with CHF secondary to MMVD. CONCLUSIONS AND CLINICAL RELEVANCE These results suggested that circulating microRNA expression patterns were unique and could serve as molecular biomarkers of CHF in dogs with MMVD.

Given similarities in metabolic parameters and cardiovascular physiology, the pig is well positioned as a biomedical model for metabolic disease and obesity in humans. Better understanding molecular mechanisms governing porcine adipocyte hyperplasia may provide insight into the regulation of adipose tissue development that is useful both when considering the pig as a commodity and when extrapolating porcine data to human disease. Primary cultures of pig stromal-vascular cells have served as a useful tool for investigating factors that regulate preadipocyte proliferation and differentiation. However, such cultures have generally been maintained at 37°C in vitro despite euthermia being 39°C in pigs. To address potential concerns about the physiological relevance of culturing primary pig preadipocytes under what would be hypothermic conditions in vivo, the objective of this study was to investigate the effect of culture temperature on the proliferation and differentiation of pig preadipocytes in primary culture. Culturing primary preadipocytes at 37 rather than 39°C decreases their proliferation rates based upon cleavage of the tetrazolium salt, MTT (P < 0.001), reduction of resazurin (P < 0.001), and daily cell counts (P < 0.001). Likewise, culturing primary porcine preadipocytes at 37°C suppressed their adipogenic potential based upon monitoring adipogenesis morphologically, biochemically, and via the expression of mRNA encoding adipogenic marker genes. Collectively, these data indicate the proliferation and differentiation of primary pig preadipocytes is suppressed when cultures are incubated at 37°C compared to normal body temperature of pigs. This may confound investigation of factors that impact adipocyte hyperplasia in the pig.

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Myxomatous mitral valvular degeneration (MMVD) is the most common heart disease in dogs, accounting for more than 70% of canine heart disease cases. It primarily affects middle‐aged small dogs, though, any dog can develop MMVD. It can affect all heart valves but is usually detected in mitral valves. Degenerative processes cause mitral valves to thicken and retract, creating an unwanted hole which blood can flow through. Tearing of mitral valves can lead to blood overflow and later, CHF in the heart’s left‐side. Currently, there are no medications proven to prevent, slow, or reverse the development of MMVD. Because of limited knowledge on MMVD, heart murmurs and coughing are the earliest means of detection. A microRNA (miRNA) is a small non‐coding RNA molecule, about 22 nucleotides long. We would test miRNA‐21, miRNA‐29a‐3p, miRNA‐210, and miRNA‐133a. If said miRNA significantly upregulated in CHF‐positive dog plasma, then upregulation of those miRNA could indicate development of MMVD. Plasma would be collected from dogs at an animal shelter. Plasma RNA would be purified using QIAzol Lysis Reagent. RT‐PCR would be used to reverse transcribe RNA to cDNA. SYBR Green (fluorescent dye) would detect miRNA amplification. If we can determine which miRNAs have a significantly high concentration in CHF‐positive dog plasma, we would be able to detect MMVD in pre‐developmental stages. For example, if a dog’s blood screening results show abnormal upregulation of miRNA precursor to MMVD, inhibitors of said miRNA could administered as a preventative measure.

Improving production efficiency while enhancing pork quality is pivotal for strengthening sustainable pork production. Being able to study both gene expression and indices of pork quality from the same anatomical location of an individual animal would better enable research conducted to study relationships between animal growth and carcass merit. To facilitate gene expression studies, adipose and muscle tissue samples are often collected immediately following exsanguination to maximize RNA integrity, which is a primary determinant of the sensitivity of RNA-based assays, such as real-time PCR. However, collecting soft tissue samples requires cutting through the hide or skin. This leaves the underlying tissue exposed during scalding, poses possible food safety issues, and potentially confounds pork quality measures. To overcome these limitations, the effect of tissue sample timing post-harvest on RNA integrity, real-time PCR results, and pork quality measurements was investigated by sampling subcutaneous adipose tissue and longissimus thoracis et lumborum muscle immediately following either exsanguination, scalding, or chilling. Sampling time did not affect RNA quality, as determined by the RNA integrity number of RNA samples purified from either adipose (RIN; p > 0.54) or muscle tissue (p > 0.43). Likewise, the sampling time did not influence the results of real-time PCR analysis of gene expression when comparing RNA samples prepared from adipose or muscle tissue immediately following either exsanguination or scalding (p > 0.92). However, sampling tissue prior to scalding resulted in a greater visual color score (p < 0.001) and lesser L* (p < 0.001) and b* (p < 0.001) values without impacting the 24 h pH (p < 0.41). These results suggested that if both RNA-based assays and meat quality endpoints are to be performed at the same anatomical location on an animal, tissue sampling to facilitate RNA-based assays should occur at a time point immediately following scalding. These findings demonstrated that sampling of adipose and muscle tissue can be delayed until after scalding/dehairing without decreasing the RNA integrity or altering the results of real-time PCR assays, while doing so was associated with little impact on measures of pork quality.

By influencing protein translation, microRNAs (miRNAs) have emerged as powerful regulators of a wide range of biological processes. miRNAs can be found freely circulating in blood as well as in circulating exosomes. It has been speculated that evaluating the quantity of miRNAs contained in exosomes may serve as a better diagnostic and prognostic tool than those found freely circulating. A good test case for this relationship is to look at miRNA dysregulation effecting Canine Congestive Heart Failure, resulting from Myxomatous Mitral Valve Disease (MMVD), in these two contexts. In order to do this, we are assessing levels of miR‐1, miR‐126, and miR‐133a, in nine small breed, older dogs with this disorder, compared to healthy dogs, using Realtime qPCR analysis. We hypothesize that our results will confirm the assertion that exosomes reveal a more reliable indication of MMVD than by quantifying freely circulating miRNAs.

By influencing protein translation, microRNAs (miRNAs) have emerged as powerful regulators of a wide range of biological processes. In recent years, extraordinary progress has been made in terms of identifying the origin and exact functions of miRNAs. An important correlation has recently been made between the role of miRNAs and Cardiorenal Syndrome (CRS). A good candidate for investigating this has been determined to be dogs with Congestive Heart Failure (CHF) and those with CRS, using plasma samples. We intend to investigate the role of miRNAs in cell‐to‐cell communication by paracrine interactions resulting in these dysfunctions by analyzing levels of the passenger strand miR‐21*. Animals and Procedures We collected blood samples from 9 healthy small breed client‐owned dogs and 9 small breed client‐owned dogs with CHF and CRS, ages 9 years and up. Blood samples were collected from affected dogs before administering medications for the management of CHF and CRS. Isolated microRNAs from plasma were obtained by processing the samples through the reverse transcription process, using Bio‐Rad Lab's Reverse Transcription Kits. The resulting assays were then processed by the CFX Connect qPCR analysis machine, using the real‐time quantitative PCR (polymerase chain reaction) process. This resulted in expression profiles of our samples. Discussion Exosome mediated communication has emerged as an important factor in interactions that lead to Cardiorenal Syndrome. The heart distributes nutrients and oxygen to other organs, including the kidney. The kidney is the major organ responsible for regulating salt and water balance. Impaired cardiac function leads to low cardiac output which lessens the efficiency of the kidney. This renal dysfunction then results in increased fluid retention, which impairs heart function further. miR‐21 is highly expressed in the heart and kidneys and it has been shown that elevated levels of miR‐21 lead to poor outcomes in most primary organ dysfunctions. Cardiac fibroblasts secrete miR‐enriched exosomes, which are subsequently taken up by cardiomyocytes, in which they alter gene expression. In particular, a passenger strand miR, miR‐21*, has been identified as a potent paracrine factor that induces cardiomyocyte hypertrophy when shuttled through exosomes.

ObjectiveTo characterize expression profiles of circulating microRNAs via miRNA isolation from plasma (Qiagen) for dogs with congestive heart failure (CHF) secondary to myxomatous mitral valve degeneration (MMVD).Materials and Methods9 healthy small breed client‐owned dogs and 9 small breed client‐owned dogs (ages 9 and up) with CHF secondary to MMVD. Blood samples were collected from both healthy and affected dogs. Isolated microRNAs from plasma were subjected to quantitative reverse transcription PCR (qRT‐PCR) used to examine expression profiles of differentially expressed circulating microRNAs of dogs with CHF.ResultsResults of qRT‐PCR assays confirmed up‐regulation of several microRNAs as well as downregulation of multiple microRNAs. Results of qRT‐PCR assays were highly correlated with NGS data from a prior study done and supported our results.ConclusionThese results suggested that miRNAs serve as novel biomarkers for congestive heart failure (CHF) in small breed canines.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.