Donor age effects on in vitro chondrogenic and osteogenic differentiation performance of equine bone marrow- and adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stromal cells (MSCs) have shown potential as cell-based therapies for cartilage and bone injuries and are used increasingly in human and veterinary practice to facilitate the treatment of orthopedic conditions. However, human and rodent studies have documented a sharp decline in chondrogenic and osteogenic differentiation potential with increasing donor age, which may be problematic for the important demographic of older orthopedic patients. The aim of this study was to identify the effect of donor age on the chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs in vitro. BM- and AT-MSCs and dermal fibroblasts (biological negative control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥ 22 years). Chondrogenic differentiation performance was assessed quantitatively by measuring pellet size, matrix proteoglycan levels, and gene expression of articular cartilage biomarkers. Osteogenic differentiation performance was assessed quantitatively by measuring alkaline phosphatase activity, calcium deposition, and gene expression of bone biomarkers. RESULTS: Chondrogenic and osteogenic differentiation performance of equine BM- and AT-MSCs declined with increasing donor age. BM-MSCs had a higher chondrogenic differentiation performance. AT-MSCs showed minimal chondrogenic differentiation performance in all age groups. For osteogenesis, alkaline phosphatase activity was also higher in BM-MSCs, but BM-MSCs calcium deposition was affected by donor age earlier than AT-MSCs. Chondrogenic and osteogenic differentiation performance of BM-MSCs exhibited a decline as early as between the newborn and yearling samples. Steady state levels of mRNA encoding growth factors, chondrogenic, and osteogenic biomarkers were lower with increasing donor age in both MSC types. CONCLUSIONS: The data showed that chondrogenic and osteogenic differentiation performance of equine BM-MSCs declined already in yearlings, and that AT-MSCs showed minimal chondrogenic potential, but were affected later by donor age with regards to osteogenesis (calcium deposition). The results highlight the importance of donor age considerations and MSC selection for cell-based treatment of orthopedic injuries and will help inform clinicians on when to implement or potentially cryopreserve cells. Moreover, the study provides molecular targets affected by donor age.

Transcriptional and Histochemical Signatures of Bone Marrow Mononuclear Cell-Mediated Resolution of Synovitis.

Osteoarthritis (OA) may result from impaired ability of synovial macrophages to resolve joint inflammation. Increasing macrophage counts in inflamed joints through injection with bone marrow mononuclear cells (BMNC) induces lasting resolution of synovial inflammation. To uncover mechanisms by which BMNC may affect resolution, in this study, differential transcriptional signatures of BMNC in response to normal (SF) and inflamed synovial fluid (ISF) were analyzed. We demonstrate the temporal behavior of co-expressed gene networks associated with traits from related in vivo and in vitro studies. We also identified activated and inhibited signaling pathways and upstream regulators, further determining their protein expression in the synovium of inflamed joints treated with BMNC or DPBS controls. BMNC responded to ISF with an early pro-inflammatory response characterized by a short spike in the expression of a NF-ƙB- and mitogen-related gene network. This response was associated with sustained increased expression of two gene networks comprising known drivers of resolution (IL-10, IGF-1, PPARG, isoprenoid biosynthesis). These networks were common to SF and ISF, but more highly expressed in ISF. Most highly activated pathways in ISF included the mevalonate pathway and PPAR-γ signaling, with pro-resolving functional annotations that improve mitochondrial metabolism and deactivate NF-ƙB signaling. Lower expression of mevalonate kinase and phospho-PPARγ in synovium from inflamed joints treated with BMNC, and equivalent IL-1ß staining between BMNC- and DPBS-treated joints, associates with accomplished resolution in BMNC-treated joints and emphasize the intricate balance of pro- and anti-inflammatory mechanisms required for resolution. Combined, our data suggest that BMNC-mediated resolution is characterized by constitutively expressed homeostatic mechanisms, whose expression are enhanced following inflammatory stimulus. These mechanisms translate into macrophage proliferation optimizing their capacity to counteract inflammatory damage and improving their general and mitochondrial metabolism to endure oxidative stress while driving tissue repair. Such effect is largely achieved through the synthesis of several lipids that mediate recovery of homeostasis. Our study reveals candidate mechanisms by which BMNC provide lasting improvement in patients with OA and suggests further investigation on the effects of PPAR-γ signaling enhancement for the treatment of arthritic conditions.

Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair.

Osteoarthritis (OA) is the most prevalent joint disease causing major disability and medical expenditures. Synovitis is a central feature of OA and is primarily driven by macrophages. Synovial macrophages not only drive inflammation but also its resolution, through a coordinated, simultaneous expression of pro- and anti-inflammatory mechanisms that are essential to counteract damage and recover homeostasis. Current OA therapies are largely based on anti-inflammatory principles and therefore block pro-inflammatory mechanisms such as prostaglandin E2 and Nuclear factor-kappa B signaling pathways. However, such mechanisms are also innately required for mounting a pro-resolving response, and their blockage often results in chronic low-grade inflammation. Following minor injury, macrophages shield the damaged area and drive tissue repair. If the damage is more extensive, macrophages incite inflammation recruiting more macrophages from the bone marrow to maximize tissue repair and ultimately resolve inflammation. However, sustained damage and inflammation often overwhelms pro-resolving mechanisms of synovial macrophages leading to the chronic inflammation and related tissue degeneration observed in OA. Recently, experimental and clinical studies have shown that joint injection with autologous bone marrow mononuclear cells replenishes inflamed joints with macrophage and hematopoietic progenitors, enhancing mechanisms of inflammation resolution, providing remarkable and long-lasting effects. Besides creating an ideal environment for resolution with high concentrations of interleukin-10 and anabolic growth factors, macrophage progenitors also have a direct role in tissue repair. Macrophages constitute a large part of the early granulation tissue, and further transdifferentiate from myeloid into a mesenchymal phenotype. These cells, characterized as fibrocytes, are essential for repairing osteochondral defects. Ongoing "omics" studies focused on identifying key drivers of macrophage-mediated resolution of joint inflammation and those required for efficient osteochondral repair, have the potential to uncover ways for developing engineered macrophages or off-the-shelf pro-resolving therapies that can benefit patients suffering from many types of arthropaties, not only OA.

Kinetics of Gene Expression Changes in Equine Fetal Interzone and Anlagen Cells Over 14 Days of Induced Chondrogenesis.

Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses were isolated and grown in a TGF-ß1 chondrogenic inductive medium. Total RNA was isolated at ten time points (0, 1.5, 3, 6, 12, 24, 48, 96, 168, and 336 h), and gene expression for 93 targeted gene loci was measured in a microfluidic RT-qPCR system. Differential transcriptional responses were observed as early as 1.5 h after the initiation of chondrogenesis. Genes with functional annotations that include transcription regulation responded to the chondrogenic stimulation earlier (1.5-96 h) than genes involved in signal transduction (1.5-336 h) and the extracellular matrix biology (3-336 h). Between interzone and anlagen cell cultures, expression levels of 73 out of the 93 targeted genes were not initially different at 0 h, but 47 out of the 73 genes became differentially expressed under the chondrogenic stimulation. While interzone and anlagen cells are both chondrogenic, they display clear differences in response to the same TGF-ß1 chondrogenic stimulation. This study provides new molecular insight into a timed sequence of the divergent developmental fates of interzone and anlagen cells in culture over 14 days.

Effect of Skeletal Paracrine Signals on the Proliferation of Interzone Cells.

OBJECTIVE: Articular cartilage in mammals has limited intrinsic capacity to repair structural defects, a fact that contributes to the chronic and progressive nature of osteoarthritis. In contrast, Mexican axolotl salamanders have demonstrated the remarkable ability to spontaneously and completely repair large joint cartilage lesions, a healing process that involves interzone cells in the intraarticular space. Furthermore, interzone tissue transplanted into skeletal defects in the axolotl salamander demonstrates a multi-differentiation potential. Cellular and molecular mechanisms of this repair process remain unclear. The objective of this study was to examine whether paracrine mitogenic signals are an important variable in the interaction between interzone cells and the skeletal microenvironment. DESIGN: The paracrine regulation of the proliferation of equine interzone cells was evaluated in an in vitro co-culture system. Cell viability and proliferation were measured in equine fetal interzone cells after exposure to conditioned medium from skeletal and nonskeletal primary cell lines. Steady-state expression was determined for genes encoding 37 putative mitogens secreted by cells that generated the conditioned medium. RESULTS: All experimental groups of conditioned media elicited a mitogenic response in interzone cells. Fetal anlage chondrocytes (P < 0.0001) and dermal fibroblasts (P < 0.0001) conditioned medium showed a significantly higher mitogenic potential compared with interzone cells. Conditioned medium from bone marrow-derived cells elicited a significantly higher proliferative response relative to that from young adult articular chondrocytes (P < 0.0001) or dermal fibroblasts (P < 0.0001). Sixteen genes had expression patterns consistent with the functional proliferation assays. CONCLUSIONS: The results indicate a mitogenic effect of skeletal paracrine signals on interzone cells.

Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-Derived Mesenchymal Stem Cells Decline With Increasing Donor Age.

Background: Bone marrow (BM)- and adipose tissue (AT)-derived mesenchymal stem cells (MSCs) are used increasingly for autologous cell therapy in equine practice to treat musculoskeletal and other injuries. Current recommendations often call for 10-100 million MSCs per treatment, necessitating the expansion of primary cells in culture prior to therapeutic use. Of concern, human and rodent studies have shown a decline of both MSC recovery from sampled tissue and in vitro proliferative capacity with increasing donor age. This may be problematic for applications of autologous cell-based therapies in the important equine demographic of older patients. Objectives: To investigate the effect of donor age on the cellular proliferation of equine BM- and AT-MSCs. Study Design: In vitro study. Methods: BM- and AT-MSCs and dermal fibroblasts (biological control) were harvested from horses in five different age groups (n = 4, N = 60); newborn (0 days), yearling (15-17 months), adult (5-8 years), middle-aged (12-18 years), and geriatric (≥22 years). Proliferation of the cells was tested using an EdU incorporation assay and steady state mRNA levels measured for targeted proliferation, aging, and senescence biomarkers. Results: The cellular proliferation of equine BM- and AT-MSCs declined significantly in the geriatric cohort relative to the younger age groups. Proliferation levels in the two MSC types were equally affected by donor age. Analysis of steady state mRNA levels showed an up-regulation in tumor suppressors, apoptotic genes, and multiple growth factors in MSCs from old horses, and a down-regulation of some pro-cycling genes with a few differences between cell types. Main Limitations: Potential age-dependent differences in cell function parameters relevant to cell-therapy application were not investigated. Conclusions: The cellular proliferation of equine BM- and AT-MSCs declined at advanced donor ages. High levels of in vitro proliferation were observed in both MSC types from horses in the age groups below 18 years of age.

Comparison of Poly-A+ Selection and rRNA Depletion in Detection of lncRNA in Two Equine Tissues Using RNA-seq.

Long non-coding RNAs (lncRNAs) are untranslated regulatory transcripts longer than 200 nucleotides that can play a role in transcriptional, post-translational, and epigenetic regulation. Traditionally, RNA-sequencing (RNA-seq) libraries have been created by isolating transcriptomic RNA via poly-A+ selection. In the past 10 years, methods to perform ribosomal RNA (rRNA) depletion of total RNA have been developed as an alternative, aiming for better coverage of whole transcriptomic RNA, both polyadenylated and non-polyadenylated transcripts. The purpose of this study was to determine which library preparation method is optimal for lncRNA investigations in the horse. Using liver and cerebral parietal lobe tissues from two healthy Thoroughbred mares, RNA-seq libraries were prepared using standard poly-A+ selection and rRNA-depletion methods. Averaging the two biologic replicates, poly-A+ selection yielded 327 and 773 more unique lncRNA transcripts for liver and parietal lobe, respectively. More lncRNA were found to be unique to poly-A+ selected libraries, and rRNA-depletion identified small nucleolar RNA (snoRNA) to have a higher relative expression than in the poly-A+ selected libraries. Overall, poly-A+ selection provides a more thorough identification of total lncRNA in equine tissues while rRNA-depletion may allow for easier detection of snoRNAs.

Genetics, Genomics, and Emergent Precision Medicine 12 Years After the Equine Reference Genome Was Published.

The first equine reference genome was completed in 2007 and published in 2009. This major accomplishment has enabled equine science to advance in ways that broadly parallel the transformative impact that genomics has had on many animal species including humans. A conceptual overview of reference genomes, genome annotation, and the major implications for equine science is presented. The relationship between genomic sequencing and the accelerating application of precision P4 medicine is discussed in the context of human and equine patients. Emergent technologies built on the foundation of genomic sequencing and rapidly gaining traction in research and clinical settings are introduced.

Tenascin-C expression controls the maturation of articular cartilage in mice.

OBJECTIVE: Expression of the de-adhesive extracellular matrix protein tenascin-C (TNC) is associated with the early postnatal development of articular cartilage which is both load-dependent and associated with chondrocyte differentiation. We assessed morphological changes in the articular cartilage of TNC deficient mice at postnatal ages of 1, 4 and 8 weeks compared to age-matched wildtype mice. RESULTS: Cartilage integrity was assessed based on hematoxylin and eosin stained-sections from the tibial bone using a modified Mankin score. Chondrocyte density and cartilage thickness were assessed morphometrically. TNC expression was localized based on immunostaining. At 8 weeks of age, the formed tangential/transitional zone of the articular cartilage was 27% thicker and the density of chondrocytes in the articular cartilage was 55% lower in wildtype than the TNC-deficient mice. TNC protein expression was associated with chondrocytes. No relevant changes were found in mice at 1 and 4 weeks of age. The findings indicate a role of tenascin-C in the post-natal maturation of the extracellular matrix in articular cartilage. This might be a compensatory mechanism to strengthen resilience against mechanical stress.

Inflamed synovial fluid induces a homeostatic response in bone marrow mononuclear cells in vitro: Implications for joint therapy.

Synovial inflammation is a central feature of osteoarthritis (OA), elicited when local regulatory macrophages (M2-like) become overwhelmed, activating an inflammatory response (M1-like). Bone marrow mononuclear cells (BMNC) are a source of naïve macrophages capable of reducing joint inflammation and producing molecules essential for cartilage metabolism. This study investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). Equine BMNC cultured in autologous SF or ISF (n = 8 horses) developed into macrophage-rich cultures with phenotypes similar to cells native to normal SF and became more confluent in ISF (~100%) than SF (~25%). BMNC cultured in SF or ISF were neither M1- nor M2-like, but exhibited aspects of both phenotypes and a regulatory immune response, characterized by increasing counts of IL-10+ macrophages, decreasing IL-1ß concentrations and progressively increasing IL-10 and IGF-1 concentrations. Changes were more marked in ISF and suggest that homeostatic mechanisms were preserved over time and were potentially favored by progressive cell proliferation. Collectively, our data suggest that intra-articular BMNC could increase synovial macrophage counts, potentiating the macrophage- and IL-10-associated mechanisms of joint homeostasis lost during the progression of OA, preserving the production of cytokines involved in tissue repair (PGE2 , IL-10) generally impaired by frequently used corticosteroids.

Functionally Annotating Regulatory Elements in the Equine Genome Using Histone Mark ChIP-Seq.

One of the primary aims of the Functional Annotation of ANimal Genomes (FAANG) initiative is to characterize tissue-specific regulation within animal genomes. To this end, we used chromatin immunoprecipitation followed by sequencing (ChIP-Seq) to map four histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) in eight prioritized tissues collected as part of the FAANG equine biobank from two thoroughbred mares. Data were generated according to optimized experimental parameters developed during quality control testing. To ensure that we obtained sufficient ChIP and successful peak-calling, data and peak-calls were assessed using six quality metrics, replicate comparisons, and site-specific evaluations. Tissue specificity was explored by identifying binding motifs within unique active regions, and motifs were further characterized by gene ontology (GO) and protein-protein interaction analyses. The histone marks identified in this study represent some of the first resources for tissue-specific regulation within the equine genome. As such, these publicly available annotation data can be used to advance equine studies investigating health, performance, reproduction, and other traits of economic interest in the horse.

Erratum: Author Correction: Improved reference genome for the domestic horse increases assembly contiguity and composition.

[This corrects the article DOI: 10.1038/s42003-018-0199-z.].

Advances in gene ontology utilization improve statistical power of annotation enrichment.

Gene-annotation enrichment is a common method for utilizing ontology-based annotations in gene and gene-product centric knowledgebases. Effective utilization of these annotations requires inferring semantic linkages by tracing paths through edges in the ontological graph, referred to as relations. However, some relations are semantically problematic with respect to scope, necessitating their omission or modification lest erroneous term mappings occur. To address these issues, we created the Gene Ontology Categorization Suite, or GOcats-a novel tool that organizes the Gene Ontology into subgraphs representing user-defined concepts, while ensuring that all appropriate relations are congruent with respect to scoping semantics. Here, we demonstrate the improvements in annotation enrichment by re-interpreting edges that would otherwise be omitted by traditional ancestor path-tracing methods. Specifically, we show that GOcats' unique handling of relations improves enrichment over conventional methods in the analysis of two different gene-expression datasets: a breast cancer microarray dataset and several horse cartilage development RNAseq datasets. With the breast cancer microarray dataset, we observed significant improvement (one-sided binomial test p-value = 1.86E-25) in 182 of 217 significantly enriched GO terms identified from the conventional path traversal method when GOcats' path traversal was used. We also found new significantly enriched terms using GOcats, whose biological relevancy has been experimentally demonstrated elsewhere. Likewise, on the horse RNAseq datasets, we observed a significant improvement in GO term enrichment when using GOcat's path traversal: one-sided binomial test p-values range from 1.32E-03 to 2.58E-44.

Chondrogenic differentiation potential of adult and fetal equine cell types.

OBJECTIVE: To determine the chondrogenic potential of cells derived from interzone tissue, the normal progenitor of articular cartilage during fetal development, compared to that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. The objective of this study was to compare the chondrogenic potential of fetal musculoskeletal progenitor cells to adult cell types, which are currently used therapeutically to facilitate joint cartilage repair in equine clinical practice. The hypothesis tested was that cells derived from interzone tissue have a chondrogenic potential that exceeds that of adult bone marrow-derived and adipose-derived mesenchymal cell isolates. STUDY DESIGN: In vitro study. ANIMALS: Six young adult horses (15-17 months of age) and 6 equine fetuses aged 45-46 days of gestation. METHODS: Three-dimensional pellet cultures were established under chondrogenic conditions with fresh, primary cells isolated from adult (articular cartilage, bone marrow, adipose, dermis) and fetal (interzone, skeletal anlagen cartilage, dermis) tissues. Cellular morphology, pellet architecture, and proteoglycan synthesis were assessed in the pellet cultures. Steady state levels of ACAN (aggrecan core protein), COL2A1 (collagen type II), and COL1A1 (collagen type I) messenger RNA (mRNA) were compared among these cell types as pellet cultures and monolayer cultures. RESULTS: Adult articular chondrocytes, fetal interzone cells, and fetal anlage cells generated the largest pellets under these chondrogenic culture conditions. Pellets derived from adult articular chondrocytes and fetal anlage cells had the highest scores on a neocartilage grading scale. Fetal anlage and adult articular chondrocyte pellets had low steady-state levels of COL1A mRNA but high COL2A1 expression. Anlage chondrocyte pellets also had the highest expression of ACAN. CONCLUSION: Adult articular chondrocytes, fetal interzone cells, and fetal anlage chondrocytes exhibited the highest chondrogenic potential. In this study, adult adipose-derived cells exhibited very limited chondrogenesis, and bone marrow-derived cells had limited and variable chondrogenic potential. CLINICAL SIGNIFICANCE: Additional investigation of the high chondrogenic potential of fetal interzone cells and anlage chondrocytes to advance cell-based therapies in diarthrodial joints is warranted.

Improved reference genome for the domestic horse increases assembly contiguity and composition.

Recent advances in genomic sequencing technology and computational assembly methods have allowed scientists to improve reference genome assemblies in terms of contiguity and composition. EquCab2, a reference genome for the domestic horse, was released in 2007. Although of equal or better quality compared to other first-generation Sanger assemblies, it had many of the shortcomings common to them. In 2014, the equine genomics research community began a project to improve the reference sequence for the horse, building upon the solid foundation of EquCab2 and incorporating new short-read data, long-read data, and proximity ligation data. Here, we present EquCab3. The count of non-N bases in the incorporated chromosomes is improved from 2.33 Gb in EquCab2 to 2.41 Gb in EquCab3. Contiguity has also been improved nearly 40-fold with a contig N50 of 4.5 Mb and scaffold contiguity enhanced to where all but one of the 32 chromosomes is comprised of a single scaffold.

iMapSplice: Alleviating reference bias through personalized RNA-seq alignment.

Genomic variants in both coding and non-coding sequences can have functionally important and sometimes deleterious effects on exon splicing of gene transcripts. For transcriptome profiling using RNA-seq, the accurate alignment of reads across exon junctions is a critical step. Existing algorithms that utilize a standard reference genome as a template sometimes have difficulty in mapping reads that carry genomic variants. These problems can lead to allelic ratio biases and the failure to detect splice variants created by splice site polymorphisms. To improve RNA-seq read alignment, we have developed a novel approach called iMapSplice that enables personalized mRNA transcriptome profiling. The algorithm makes use of personal genomic information and performs an unbiased alignment towards genome indices carrying both reference and alternative bases. Importantly, this breaks the dependency on reference genome splice site dinucleotide motifs and enables iMapSplice to discover personal splice junctions created through splice site polymorphisms. We report comparative analyses using a number of simulated and real datasets. Besides general improvements in read alignment and splice junction discovery, iMapSplice greatly alleviates allelic ratio biases and unravels many previously uncharacterized splice junctions created by splice site polymorphisms, with minimal overhead in computation time and storage. Software download URL: https://github.com/LiuBioinfo/iMapSplice.

Changes in the appendicular skeleton during metamorphosis in the axolotl salamander (Ambystoma mexicanum).

Axolotl salamanders (Ambystoma mexicanum) remain aquatic in their natural state, during which biomechanical forces on their diarthrodial limb joints are likely reduced relative to salamanders living on land. However, even as sexually mature adults, these amphibians can be induced to metamorphose into a weight-bearing terrestrial stage by environmental stress or the exogenous administration of thyroxine hormone. In some respects, this aquatic to terrestrial transition of axolotl salamanders through metamorphosis may model developmental and changing biomechanical skeletal forces in mammals during the prenatal to postnatal transition at birth and in the early postnatal period. To assess differences in the appendicular skeleton as a function of metamorphosis, anatomical and gene expression parameters were compared in skeletal tissues between aquatic and terrestrial axolotls that were the same age and genetically full siblings. The length of long bones and area of cuboidal bones in the appendicular skeleton, as well as the cellularity of cartilaginous and interzone tissues of femorotibial joints were generally higher in aquatic axolotls compared with their metamorphosed terrestrial siblings. A comparison of steady-state mRNA transcripts encoding aggrecan core protein (ACAN), type II collagen (COL2A1), and growth and differentiation factor 5 (GDF5) in femorotibial cartilaginous and interzone tissues did not reveal any significant differences between aquatic and terrestrial axolotls.

Molecular cloning and functional expression of the K+ channel KV7.1 and the regulatory subunit KCNE1 from equine myocardium.

BACKGROUND: The voltage-gated K+-channel KV7.1 and the subunit KCNE1, encoded by the KCNQ1 and KCNE1 genes, respectively, are responsible for termination of the cardiac action potential. In humans, mutations in these genes can predispose patients to arrhythmias and sudden cardiac death (SCD). AIM: To characterize equine KV7.1/KCNE1 currents and compare them to human KV7.1/KCNE1 currents to determine whether KV7.1/KCNE1 plays a similar role in equine and human hearts. METHODS: mRNA encoding KV7.1 and KCNE1 was isolated from equine hearts, sequenced, and cloned into expression vectors. The channel subunits were heterologously expressed in Xenopus laevis oocytes or CHO-K1 cells and characterized using voltage-clamp techniques. RESULTS: Equine KV7.1/KCNE1 expressed in CHO-K1 cells exhibited electrophysiological properties that are overall similar to the human orthologs; however, a slower deactivation was found which could result in more open channels at fast rates. CONCLUSION: The results suggest that the equine KV7.1/KCNE1 channel may be important for cardiac repolarization and this could indicate that horses are susceptible to SCD caused by mutations in KCNQ1 and KCNE1.