Natural Hydrogels Support Kidney Organoid Generation and Promote In Vitro Angiogenesis.

To date, strategies aiming to modulate cell to extracellular matrix (ECM) interactions during organoid derivation remain largely unexplored. Here renal decellularized ECM (dECM) hydrogels are fabricated from porcine and human renal cortex as biomaterials to enrich cell-to-ECM crosstalk during the onset of kidney organoid differentiation from human pluripotent stem cells (hPSCs). Renal dECM-derived hydrogels are used in combination with hPSC-derived renal progenitor cells to define new approaches for 2D and 3D kidney organoid differentiation, demonstrating that in the presence of these biomaterials the resulting kidney organoids exhibit renal differentiation features and the formation of an endogenous vascular component. Based on these observations, a new method to produce kidney organoids with vascular-like structures is achieved through the assembly of hPSC-derived endothelial-like organoids with kidney organoids in 3D. Major readouts of kidney differentiation and renal cell morphology are assessed exploiting these culture platforms as new models of nephrogenesis. Overall, this work shows that exploiting cell-to-ECM interactions during the onset of kidney differentiation from hPSCs facilitates and optimizes current approaches for kidney organoid derivation thereby increasing the utility of these unique cell culture platforms for personalized medicine.

Experimental Evaluation of a New Tissue Factor-Based Topical Hemostat (TT-173) for Treatment of Hepatic Bleeding.

Background: Excessive blood loss is a relevant complication of partial liver resection. Topical hemostatic agents have proven useful to improve the control of the bleeding in this among other surgical indications. Until now all of these products have been based on the action of thrombin. In contrast TT-173 is a new topical hemostatic agent based on recombinant tissue factor naturally incorporated into membrane vesicles. This work sought to assess the efficacy and toxicity of TT-173 in an animal model of liver resection.Materials and Methods: Procoagulant activity of 0.15, 0.41, and 1 mg of TT-173 was evaluated in pigs subjected the resection of hepatic lobe margins. The most effective of these doses was also compared against thrombin. In addition, the toxicity, local tolerance, systemic absorption, and immunogenicity of the product were investigated in rats subjected to liver biopsy lesion.Results: The three doses of TT-173 evaluated significantly reduced the bleeding time in liver lesions. The highest dose of product was significantly more effective than the others and thrombin. Application of high doses of TT-173 in rats did not cause any local or systemic alterations. Absorption into blood stream was negligible and no immunogenic reaction against the product was detected.Conclusions: TT-173 shows favorable pharmacodynamic properties for improving hemostasis in partial liver resection which support further investigation of the product in this surgical indication.

A new topical hemostatic agent TT-173 reduces blood loss in a sheep model of total knee arthroplasty.

BACKGROUND: Total knee arthroplasty is associated with blood loss during the intervention and may require allogenic blood transfusion. Treatments such as tranexamic acid and fibrin sealants improved the bleeding control in several clinical trials, but the hemorrhage associated with the intervention is still significant. Thus far, very few studies have evaluated hemostatic treatments in animal models of total knee arthroplasty. This work describes a sheep model of bleeding associated with total knee arthroplasty and investigates a new class of hemostatic treatment based on recombinant tissue factor. METHODS: Sheep were treated with the anticoagulant heparin, and the joint was accessed by a paramedial incision. Ligaments and menisci were eliminated and femoral condyles and tibia plateau were sectioned exposing the trabecular bone. An intra-articular drain was used to recover and quantify the blood loss during the 90-min period after treatment. The efficacy of one milligram and three milligrams of TT-173 was evaluated and compared with tranexamic acid. The occurrence of analytical alterations and systemic absorption was also investigated. RESULTS: Treatment with TT-173 reduced the blood loss in comparison with control or tranexamic acid. No significant differences were observed between the two doses evaluated. Moreover, a dose of six milligrams of TT-173 did not induce any clinical or analytical alteration, and significant systemic absorption was not observed. CONCLUSION: Data obtained strongly suggest that TT-173 could be useful in reducing the blood loss associated with total knee arthroplasty and without safety concerns derived from the systemic absorption of the product.

The mitochondrial inhibitor oligomycin induces an inflammatory response in the rat knee joint.

BACKGROUND: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints. METHODS: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated. RESULTS: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue. CONCLUSIONS: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Nonclinical Evaluation of the New Topical Hemostatic Agent TT-173 for Skin Grafting Procedures.

Blood loss during grafting surgery represents a major concern of this procedure and the development of hemostatic agents for this indication is highly desirable. TT-173 is the first biologically active treatment based on tissue factor instead of thrombin. This study sought to investigate the efficacy, systemic absorption, and toxicology of TT-173 in animal models to support clinical evaluation of the product in donor sites of patients subjected to skin grafting. Procoagulant efficacy of 148 µg of TT-173 was evaluated in pigs in presence and absence of anticoagulant treatment with unfractioned heparin. Systemic absorption was quantified and characterized in rats subjected to severe skin lesions with affectation of muscular plane using TT-173 radiolabeled with I. The same animal model was used to test the toxicology of a dose of 80 µg of the product. Application of TT-173 significantly reduced the bleeding time of donor sites, even under anticoagulant treatment. Systemic absorption was low; it was excreted through urine and did not concentrate in organs such as liver, lung, or spleen suggesting that the absorbed dose could correspond to degradation fragments without procoagulant activity. Finally, a dose of 80 µg of TT-173 did not cause analytical disturbances suggestive of intravascular coagulation or any other adverse reaction. Nonclinical data obtained suggest that TT-173 could be useful to reduce the blood loss associated to burns treatment and support the clinical evaluation of the product in donor sites of patients subjected to skin grafting.

A MicroRNA-Transcription Factor Blueprint for Early Atrial Arrhythmogenic Remodeling.

Spontaneous self-terminating atrial fibrillation (AF) is one of the most common heart rhythm disorders, yet the regulatory molecular mechanisms underlying this syndrome are rather unclear. MicroRNA (miRNA) transcriptome and expression of candidate transcription factors (TFs) with potential roles in arrhythmogenesis, such as Pitx2, Tbx5, and myocardin (Myocd), were analyzed by microarray, qRT-PCR, and Western blotting in left atrial (LA) samples from pigs with transitory AF established by right atrial tachypacing. Induced ectopic tachyarrhythmia caused rapid and substantial miRNA remodeling associated with a marked downregulation of Pitx2, Tbx5, and Myocd expression in atrial myocardium. The downregulation of Pitx2, Tbx5, and Myocd was inversely correlated with upregulation of the corresponding targeting miRNAs (miR-21, miR-10a/10b, and miR-1, resp.) in the LA of paced animals. Through in vitro transient transfections of HL-1 atrial myocytes, we further showed that upregulation of miR-21 did result in downregulation of Pitx2 in cardiomyocyte background. The results suggest that immediate-early miRNA remodeling coupled with deregulation of TF expression underlies the onset of AF.

Uniportal video-assisted thoracoscopic lobectomy in the animal model.

We introduce the training on uniportal video-assisted thoracoscopic (VATS) lobectomy in sheep. This animal model is helpful to learn the different view, the importance of lung exposure and the key points of the instrumentation. In this article we present three videos with the left upper lobectomy, the left lower lobectomy and the right upper lobectomy in the sheep.

A sheep model for endoscopic treatment of mandible subcondylar fractures.

BACKGROUND: Mandible subcondylar fractures may be treated via a traditional visible access incision; however, with the advances in surgical endoscopy surgeons are transitioning to a minimally invasive approach in an effort to reduce surgical morbidity and external facial scarring. We sought to design a clinically applicable teaching tool in a large animal model that would allow the operator to gain experience treating mandible subcondylar fractures via an endoscopic approach. METHODS: A large animal model was developed using the Churra sheep. Subcondylar fractures were created, reduced, and internally plated in ten specimens via an extraoral, two-port endoscopic approach. Animals were monitored for surgical success during the intraoperative and immediate postoperative periods. RESULTS: Mandibles were reduced and fixated successfully in each of the animals. Operative time was reduced from 70 to 40 min as the surgeons became more familiar with the surgical procedure. Each of the ten Churra sheep used in the study tolerated the surgeries without postoperative complications. CONCLUSIONS: Capitalizing on a mandibular anatomy similar to humans, the Churra sheep successfully demonstrated utility for the extraoral, endoscopic approach in treating mandibular condyle fractures. This model offers surgeons the opportunity to gain surgical endoscopic experience before treating clinical patients.

Targeted gene-silencing reveals the functional significance of myocardin signaling in the failing heart.

BACKGROUND: Myocardin (MYOCD), a potent transcriptional coactivator of smooth muscle (SM) and cardiac genes, is upregulated in failing myocardium in animal models and human end-stage heart failure (HF). However, the molecular and functional consequences of myocd upregulation in HF are still unclear. METHODOLOGY/PRINCIPAL FINDINGS: The goal of the present study was to investigate if targeted inhibition of upregulated expression of myocd could influence failing heart gene expression and function. To this end, we used the doxorubicin (Dox)-induced diastolic HF (DHF) model in neonatal piglets, in which, as we show, not only myocd but also myocd-dependent SM-marker genes are highly activated in failing left ventricular (LV) myocardium. In this model, intra-myocardial delivery of short-hairpin RNAs, designed to target myocd variants expressed in porcine heart, leads on day 2 post-delivery to: (1) a decrease in the activated expression of myocd and myocd-dependent SM-marker genes in failing myocardium to levels seen in healthy control animals, (2) amelioration of impaired diastolic dysfunction, and (3) higher survival rates of DHF piglets. The posterior restoration of elevated myocd expression (on day 7 post-delivery) led to overexpression of myocd-dependent SM-marker genes in failing LV-myocardium that was associated with a return to altered diastolic function. CONCLUSIONS/SIGNIFICANCE: These data provide the first evidence that a moderate inhibition (e.g., normalization) of the activated MYOCD signaling in the diseased heart may be promising from a therapeutic point of view.

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction.

BACKGROUND AIMS: We evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model. METHODS AND RESULTS: Myocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10(6) 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo group. CONCLUSIONS: Infarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.

Exon-skipping brain natriuretic peptide variant is overexpressed in failing myocardium and attenuates brain natriuretic peptide production in vitro.

Brain natriuretic peptide/natriuretic peptide precursor B (NPPB) is one of the most studied genes in relation to heart failure (HF) conditions. However, it is still unclear as to whether alternative splicing could create NPPB mRNA variants, which may be expressed in normal and diseased myocardium. We aimed to identify and characterize a novel alternatively spliced variant of porcine and human NPPB resulting from exon 2 skipping (designated as DeltaE2-NPPB). A variety of conventional molecular, biochemical and immunochemical methods were used to examine the expression and functional consequences of DeltaE2-NPPB in vitro and in vivo. The pig DeltaE2-NPPB mRNA is effectively translated into stable protein in cell-based assays but, in contrast to normally spliced NPPB, the DeltaE2-NPPB protein is not secreted into the media. Co-transfection assays demonstrate that DeltaE2-NPPB attenuates production and secretion of normally spliced NPPB, suggesting a negative feedback loop of NPPB signaling through generation of DeltaE2-NPPB. The inhibitory effects of DeltaE2-NPPB on the expression of NPPB are associated with sequence elements residing in exon 3 of DeltaE2-NPPB. In piglets, DeltaE2-NPPB gene expression is downregulated in both ventricles after birth, but it is markedly re-activated in the postnatal myocardium in experimental diastolic heart failure. In addition, we demonstrate that the exon-skipped NPPB variants are expressed in the postnatal and adult human myocardium and upregulated at end-stage HF due to dilated cardiomyopathy. Our work uncovers an important role of alternative exon skipping in the regulation of NPPB gene expression, thereby pinpointing a putative new mechanism for post-transcriptional regulation of NPPB production and secretion.

Intron retention generates ANKRD1 splice variants that are co-regulated with the main transcript in normal and failing myocardium.

The cardiac ankyrin repeat domain 1 protein (ANKRD1, also known as CARP) has been extensively characterized with regard to its proposed functions as a cardio-enriched transcriptional co-factor and stress-inducible myofibrillar protein. The present results show the occurrence of alternative splicing by intron retention events in the pig and human ankrd1 gene. In pig heart, ankrd1 is expressed as four alternatively spliced transcripts, three of which have non-excised introns: ankrd1-contained introns 6, 7 and 8 (i.e., ankrd1-i6,7,8), ankrd1-contained introns 7 and 8 (i.e., ankrd1-i7,8), and ankrd1 retained only intron 8 (i.e., ankrd1-i8). In the human heart, two orthologues of porcine intron-retaining ankrd1 variants (i.e., ankrd1-i8 and ankrd1-i7,8) are detected. We demonstrate that these newly-identified intron-retaining ankrd1 transcripts are functionally intact, efficiently translated into protein in vitro and exported to the cytoplasm in cardiomyocytes in vivo. In the piglet heart, both the intronless and intron-retaining ankrd1 mRNAs are co-expressed in a chamber-dependent manner being more abundant in the left as compared to the right myocardium. Our data further indicate co-upregulation of the ankrd1 spliced variants in myocardium in the porcine model of diastolic heart failure. Most significantly, we demonstrate that in vivo forced expression of recombinant intronless ankrd1 markedly increases the levels of intron-retaining ankrd1 variants (but not of the endogenous main transcript) in piglet myocardium, suggesting that ANKRD1 may positively regulate the expression of its own intron-containing RNAs in response to cardiac stress. Overall, our findings demonstrate that in cardiomyocytes ANKRD1 can exist in multiple isoforms which may contribute to the functional diversity of this factor in heart development and disease.

In vivo forced expression of myocardin in ventricular myocardium transiently impairs systolic performance in early neonatal pig heart.

The aim of this study was to determine the effects of forced expression of myocd-A in the left ventricular (LV) myocardium on cardiac performance in early neonatal piglets. LV transfection with the gene for homeodomain only protein (hop), an antagonist of myocd-mediated activities, was also performed. Gene delivery was performed in 6-day-old piglets using a low-traumatic, catheter-based, video-assisted procedure developed by us for direct intra-myocardial injections of plasmid DNA into 3-4 target areas of the ventral LV free wall (LVFW). Two isoforms of porcine myocd were identified, cloned and characterized: the exon 11-lacking myocd-A and its larger exon 11-containig variant, myocd-B. In neonatal piglets, myocd-A seems to be a cardio-predominant isoform enriched in the LVFW/septum, whereas the myocd-B isoform is detected not only in the heart but also in various smooth muscle cell-containing tissues. Intramyocardial myocd-A gene delivery resulted in forced transgene expression in the target areas of the LVFW as compared to controls. On day 2 post-delivery, a marked decrease of LV-end systolic pressure values (an accepted marker for impaired LV function) was observed in myocd-A-transfected piglets as compared to hop-transfected and control groups. In addition, forced myocd-A expression in the LVFW caused abnormal ECG. A significant up-regulation of the gene for fetal-predominant muscle light chain 3F myosin was detected in myocd-A-transfected LVFWs harvested on day 2 post-delivery. Extended analysis on day 7 post-delivery revealed a drop decrease in myocd-A transgene expression in target LVFW regions which was correlated with normalization of the LV systolic parameters in experimented piglets.

Segmental heterogeneity in Bcl-2, Bcl-xL and Bax expression in rat tubular epithelium after ischemia-reperfusion.

AIMS: Studies in rats with bilateral clamping of renal arteries showed transient Bcl-2, Bcl-xL and Bax expression in renal tubular epithelium following ischemia-reperfusion. However, current data on the preferential localization of specific mRNAs or proteins are limited because gene expression was not analysed at segmental level. This study analyses the mRNA expression of Bcl-2, Bcl-xL and Bax in four segments of proximal and distal tubules localized in the renal cortex and outer medulla in rat kidneys with bilateral renal clamping for 30 min and seven reperfusion times versus control animals without clamp. METHODS: Proximal convoluted tubule (PCT), distal convoluted tubule (DCT), proximal straight tubule (PST) and medullary thick ascending limb (MTAL) were obtained by manual microdissection. RT-PCR was used to analyse mRNA expression at segmental level. RESULTS: Proximal convoluted tubule and MTAL showed early, persistent and balanced up-regulation of Bcl-2, Bcl-xL and Bax, while PST and DCT revealed only Bcl-2 and Bcl-xL, when only Bax was detected in PST. DCT expressed Bcl-xL initially, and persistent Bcl-2 later. CONCLUSION: These patterns suggest a heterogeneous apoptosis regulatory response in rat renal tubules after ischemia-reperfusion, independently of cortical or medullary location. This heterogeneity of the expression patterns of Bcl-2 genes could explain the different susceptibility to undergo apoptosis, the different threshold to ischemic damage and the different adaptive capacity to injury among these tubular segments.

Design and creation of an experimental program of advanced training in reconstructive microsurgery.

In this study, we design an experimental protocol for the purpose of enhancing performance in training in microsurgery. It is based on five free tissue transfer exercises in rat (epigastric cutaneous flap, saphenous fasciocutaneous flap, epigastric neurovascular flap, saphenous muscular flap, and hindlimb replantation), which simulate the principal clinical procedures of reconstructive microsurgery. The first part of the study consists of an anatomical review of the flaps of 5 rats and in the second part we have carried out the free transfer of flaps on 25 rats divided into 5 groups. To differentiate between them, we have created a mathematical function, referred to as difficulty in a microsurgical exercise, which has enabled us to establish a scale of progression for training, ranging form the easiest to the most difficult. As a conclusion, we believe that this protocol is a useful instrument as it allows for a more precise assessment of microsurgical capacity due to enhanced accuracy in the reproduction of global procedures and the fact that the quantification of progress in training is based on clinical monitoring after 7 days.

Human DAF on pig cells protects against human and non-human primate sera cytotoxicity mediated by exogenous or endogenous complement, as determined by flow cytometry.

Expression of human complement regulatory proteins (CRP) in pig cells through transgenesis was proposed to prevent complement activation and the ensuing rejection of pig tissues and organs following pig-to-primate transplantation. Transplantation in non-human primates of organs from transgenic pigs for human decay accelerating factor (hDAF) did not undergo hyperacute rejection, but hDAF could not prevent humoral xenograft rejection (AHXR). A possible explanation for the lack of efficacy of the expression of human complement regulatory proteins in pig cells to prevent AHXR may be interspecies differences between human and non-human complement regulatory system. We assayed the efficacy of transgenic hDAF expressed on porcine cells to inhibit the in vitro complement activity of primate sera. The individual cytotoxicity of sera from seven untreated baboons and of pools of normal human and baboon sera was assayed with endogenous and exogenous complement using a flow-cytometry complement-mediated cytotoxicity assay (FCCA) against peripheral blood lymphocytes (PBL) from hDAF and non-transgenic pigs. We also analyzed the anti-Galalpha1-3Gal (alphaGal) antibody titre of the baboon sera by ELISA and the expression of hDAF on the PBL surface by immunofluorescence. Transgenic hDAF expression was capable of protecting pig cells against injury produced by both baboon and human serum. Cellular expression of hDAF reduced cytotoxicity mediated by endogenous and exogenous complement, although the former was slightly higher. Humoral cytotoxicity was not related to a particular antibody but was inversely related to hDAF expression. The presence of hDAF protected pig cells against lysis by NHS more effectively than against NBS. These results confirm in vitro the protective role of hDAF in pig cells to heterologous complement mediated damage, but they also suggest that the extent of hDAF protection decreases, however, if cells express low levels of hDAF.

Differential atrial versus ventricular ANKRD1 gene expression is oppositely regulated at diastolic heart failure.

Diastolic heart failure (DHF) was produced in 6-day-old piglets by intravenous administration of Doxorubicin, and ANKRD1 protein and mRNA levels were determined in atrial (A) and ventricular (V) chambers of failing vs control hearts. In controls, ANKRD1 showed a left-right (L-R) asymmetric distribution with protein levels 2-fold higher in the LA as compared to the RA, and 8-fold higher in the LV than the RV. In failing hearts, ANKRD1 levels were augmented about 2-fold in each ventricle but equally reduced in both atria as compared to controls. ANKRD1 downregulation in atria is discussed as a process associated with advanced DHF.

Lack of cross-species transmission of porcine endogenous retrovirus in pig-to-baboon xenotransplantation with sustained depletion of anti-alphagal antibodies.

BACKGROUND: Nonhuman primates are potential permissive animals for studying the risk of in vivo infection with porcine endogenous retrovirus (PERV). Anti-alphaGal natural antibodies are considered one of the barriers for preventing PERV infection, and it has been postulated that reduction of these antibodies could increase the risk of this infection. The aim of this study was to investigate the role of GAS 914, which depletes anti-alphaGal antibodies, in the potential in vivo transfer of PERV after pig-to-baboon organ xenotransplantation. METHODS: Twenty-seven baboons underwent xenotransplantation with hDAF or hMCP/hDAF transgenic pig organs, including heterotopic heart (n = 14) and kidney (n = 13) transplants. All of them received GAS 914 along with different immunosuppression protocols. PERV sequences were investigated by reverse-transcriptase polymerase chain reaction and by polymerase chain reaction assays in samples obtained at autopsy. The presence of PERV-specific antibodies and/or pig xenomicrochimerism was also evaluated. RESULTS: PERV RNA was not detected in any baboon plasma sample. In addition, all plasma samples were negative for PERV antibodies. However, PERV DNA sequences were detected in peripheral blood mononuclear cells from 6 of 14 (43%) animals investigated. Porcine mitochondrial DNA was also found in all of these positive samples and in six of the eight (75%) samples with negative PERV DNA, indicating that the detection of PERV sequences was attributable to xenochimerism. PERV-positive cells as a result of xenochimerism were also found in eight of nine (89%) spleen and lymph node tissue samples tested. CONCLUSIONS: Sustained depletion of anti-alphaGal antibodies does not augment the risk of PERV infection in pig-to-baboon organ transplantation.

ANKRD1 specifically binds CASQ2 in heart extracts and both proteins are co-enriched in piglet cardiac Purkinje cells.

It has been suggested that the cardiac ankyrin repeat domain 1 protein (ANKRD1), also known as CARP, can play a pathophysiological role in the contractile responsiveness of myocardium. Here, we study the potential functional roles of ANKRD1 by searching for endogenous cardiac proteins that interact preferentially with ANKRD1 in the heart-tissue extract from neonatal piglets, using non-biased pull-down approaches. These approaches identified, for the first time, a selective interaction between ANKRD1 and endogenous cardiac calsequestrin-2 (CASQ2) that is important for Ca2+ release and excitation-contraction coupling. Blot-overlay and co-immunoprecipitation assays provided further confirmation of the direct and specific interaction between the two proteins. Mapping of the peptides involved in the interaction revealed five non-overlapping binding sequences for CASQ2 on ANKRD1, as well as, three binding peptides for ANKRD1 in CASQ2. For the first time, we show by immunohistochemistry that endogenous ANKRD1 and CASQ2 are co-enriched in piglet cardiac Purkinje cells. Collectively, the results provide the first sing of a possible functional interaction between ANKRD1 and CASQ2 and suggest a potentially novel role for both proteins in cardiac Purkinje fibers.

FDA/PI flow cytometry assay of complement-mediated cytotoxicity of antibodies generated during xenotransplantation.

BACKGROUND: A flow cytometry complement-mediated cytotoxicity assay (FCCA) using fluorescein diacetate (FDA) and propidium iodide (PI) to measure antibody-dependent toxicity is useful to determine the success of xenotransplant organs. We evaluated the validity of different mathematical models as a measure of cytotoxicity in FCCA. METHODS: Sera from untreated baboons (n = 7) and from immunosuppressed animals (n = 5) undergoing different xenotransplantation protocols with pig organs were tested by endogenous FCCA and a similar assay also using exogenous complement, and the results were compared with those of a complement-dependent hemolytic assay to detect anti-pig antibodies (APHA). The influence of PI/FDA staining and the use of several mathematical models were analyzed. RESULTS: For both groups of animals, we observed high correlations between the endogenous and exogenous FCCA pathways and between calculations based on PI and FDA staining. Of the four mathematical models tested--the Von Krogh equation, two exponential models, and area under the curve--the Von Krogh equation was the most appropriate in terms of goodness of fit and concordance with APHA. CONCLUSIONS: FDA/PI FCCA is useful to measure endogenous and exogenous complement-mediated cytotoxicities, and it has advantages related to identification of potential new xenoantibodies. Although all four mathematical models produced acceptable solutions, the Von Krogh equation was the best option.