Differential Secretomes Of Processed Adipose Grafts, The Stromal Vascular Fraction And Adipose-derived Stem Cells.

There are multiple methods to prepare lipoaspirate for autologous fat transfer; however, graft retention remains unpredictable. The purpose of this study was to compare the cellular and protein composition of adipose grafts and the stromal vascular fraction (SVF) resulting from three common techniques to prepare adipose grafts. Adipose grafts were harvested from healthy donors and processed via three techniques: centrifugation (C), a single-filter (SF) device, a double-filtration (DF) system. Part of each graft was analyzed or further processed to isolate the SVF. Cell viability, surface markers, cytokine, and growth factors were compared between the graft and SVF as well as adipose derived stem cells (ASCs). Overall, we found variations across the three processing techniques and among the graft components (ASCs, SVF, fat). Cell viability within the grafts were similar (94.6%, 92.3%, and 93.6%; p=0.93). The trend was a greater percentage of ASCs from SF versus DF or centrifugation (6.95%, 4.63% and 1.93% respectively; p=0.06). Adipogenic markers (adiponectin, leptin) were similar among all three grafts (p=0.45). Markers of tissue remodeling were greatest in the SVF compared to fat and ASCs, regardless of processing technique. There was higher relative expression of MMP9 (2x), EMMPRIN (2.5x), endoglin (5x), and IL-8 (1.5x) in the SVF (p<0.005). Our study identified differences in cytokine expression in adipose grafts and the SVF, particularly in cytokines important in inflammation and wound healing. These secretomes may impact graft retention and fat necrosis and have potential implications in cell-assisted lipotransfer. There were no significant differences between the final products of any of the processing techniques.

OsCOL5 suppresses heading through modulation of Ghd7 and Ehd2, enhancing rice yield.

KEY MESSAGE: OsCOL5, an ortholog of Arabidopsis COL5, is involved in photoperiodic flowering and enhances rice yield through modulation of Ghd7 and Ehd2 and interactions with OsELF3-1 and OsELF3-2. Heading date, also known as flowering time, plays a crucial role in determining the adaptability and yield potential of rice (Oryza sativa L.). CONSTANS (CO)-like is one of the most critical flowering-associated gene families, members of which are evolutionarily conserved. Here, we report the molecular functional characterization of OsCOL5, an ortholog of Arabidopsis COL5, which is involved in photoperiodic flowering and influences rice yield. Structural analysis revealed that OsCOL5 is a typical member of CO-like family, containing two B-box domains and one CCT domain. Rice plants overexpressing OsCOL5 showed delayed heading and increases in plant height, main spike number, total grain number per plant, and yield per plant under both long-day (LD) and short-day (SD) conditions. Gene expression analysis indicated that OsCOL5 was primarily expressed in the leaves and stems with a diurnal rhythm expression pattern. RT-qPCR analysis of heading date genes showed that OsCOL5 suppressed flowering by up-regulating Ghd7 and down-regulating Ehd2, consequently reducing the expression of Ehd1, Hd3a, RFT1, OsMADS14, and OsMADS15. Yeast two-hybrid experiments showed direct interactions of OsCOL5 with OsELF3-1 and OsELF3-2. Further verification showed specific interactions between the zinc finger/B-box domain of OsCOL5 and the middle region of OsELF3-1 and OsELF3-2. Yeast one-hybrid assays revealed that OsCOL5 may bind to the CCACA motif. The results suggest that OsCOL5 functions as a floral repressor, playing a vital role in rice's photoperiodic flowering regulation. This gene shows potential in breeding programs aimed at improving rice yield by influencing the timing of flowering, which directly impacts crop productivity.

Pre-epithelialized cryopreserved tracheal allograft for neo-trachea flap engineering.

Background: Tracheal reconstruction presents a challenge because of the difficulty in maintaining the rigidity of the trachea to ensure an open lumen and in achieving an intact luminal lining that secretes mucus to protect against infection. Methods: On the basis of the finding that tracheal cartilage has immune privilege, researchers recently started subjecting tracheal allografts to "partial decellularization" (in which only the epithelium and its antigenicity are removed), rather than complete decellularization, to maintain the tracheal cartilage as an ideal scaffold for tracheal tissue engineering and reconstruction. In the present study, we combined a bioengineering approach and a cryopreservation technique to fabricate a neo-trachea using pre-epithelialized cryopreserved tracheal allograft (ReCTA). Results: Our findings in rat heterotopic and orthotopic implantation models confirmed that tracheal cartilage has sufficient mechanical properties to bear neck movement and compression; indicated that pre-epithelialization with respiratory epithelial cells can prevent fibrosis obliteration and maintain lumen/airway patency; and showed that a pedicled adipose tissue flap can be easily integrated with a tracheal construct to achieve neovascularization. Conclusion: ReCTA can be pre-epithelialized and pre-vascularized using a 2-stage bioengineering approach and thus provides a promising strategy for tracheal tissue engineering.

Harnessing the synergy of perfusable muscle flap matrix and adipose-derived stem cells for prevascularization and macrophage polarization to reconstruct volumetric muscle loss.

Muscle flaps must have a strong vascular network to support a large tissue volume and ensure successful engraftment. We developed porcine stomach musculofascial flap matrix (PDSF) comprising extracellular matrix (ECM) and intact vasculature. PDSF had a dominant vascular pedicle, microcirculatory vessels, a nerve network, well-retained 3-dimensional (3D) nanofibrous ECM structures, and no allo- or xenoantigenicity. In-depth proteomic analysis demonstrated that PDSF was composed of core matrisome proteins (e.g., collagens, glycoproteins, proteoglycans, and ECM regulators) that, as shown by Gene Ontology term enrichment analysis, are functionally related to musculofascial biological processes. Moreover, PDSF-human adipose-derived stem cell (hASC) synergy not only induced monocytes towards IL-10-producing M2 macrophage polarization through the enhancement of hASCs' paracrine effect but also promoted the proliferation and interconnection of both human skeletal muscle myoblasts (HSMMs) and human umbilical vein endothelial cells (HUVECs) in static triculture conditions. Furthermore, PDSF was successfully prevascularized through a dynamic perfusion coculture of hASCs and HUVECs, which integrated with PDSF and induced the maturation of vascular networks in vitro. In a xenotransplantation model, PDSF demonstrated myoconductive and immunomodulatory properties associated with the predominance of M2 macrophages and regulatory T cells. In a volumetric muscle loss (VML) model, prevascularized PDSF augmented neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant musculofascial tissue formation. These results indicate that hASCs' integration with PDSF enhances the cells' dual function in immunomodulation and angiogenesis. Owing in part to this PDSF-hASC synergy, our platform shows promise for vascularized muscle flap engineering for VML reconstruction.

Short-term influences of radiation on musculofascial healing in a laparotomy rat model.

Preoperative radiation is associated with an increased risk of wound complications. However, the influences of radiation on musculofascial wound healing remains unclear. The purpose of the study was to investigate the short-term effects of preoperative local radiation on the musculofascial healing of laparotomy incisions in a rat model. Eighteen Fischer 344 rats received radiation doses of 0, 10, or 20 Gy to the abdominal wall and underwent laparotomy 4 weeks later. Two weeks after laparotomy, samples of irradiated muscle were harvested for mechanical tests, histological (Hematoxylin & Eosin, and Masson's Trichrome) and immunohistochemical analyses using KI67, CD31, TGF-ß, and MYOD1 antibodies. The elastic modulus (EM), maximum strain (MS), and ultimate tensile strength (UTS) in the 20-Gy group were significantly weaker than those in the 0-Gy group. The EM and UTS in the 20-Gy group were significantly lower than those in the 10-Gy group. The UTS and MS in the 10-Gy group were significantly lower than those in the 0-Gy group. The mean number of inflammatory cells per mm2 in the 20-Gy group was significantly larger than those in the 10- and 0-Gy groups. The mean numbers of CD31-, KI67-, and MYOD1-positive cells, the optical density of TGF-ß, and the microvessel density in the 20-Gy group were significantly smaller than those in the 10- and 0-Gy groups. These results indicated that radiation delays musculofascial healing and decreases mechanical strength of the laparotomy incision by creating a chronic inflammatory environment, inhibiting cell proliferation, angiogenesis, granulation maturation, collagen deposition, and muscular regeneration in a dose-dependent manner. The impaired biomechanical, histological and molecular properties may be associated with the higher risk of wound complications in patients who undergo radiotherapy prior to laparotomy.

[VEGF overespression promotes the proliferation and differentiation of human adipose-derived stem cells].

Objective To investigate the effect of vascular endothelial growth factor (VEGF) overexpression on the phenotype, proliferation and multilineage differentiation ability of human adipose-derived stem cells (hADSCs). Methods Passage-2 hADSCs were transfected with pIRES2-EGFP-VEGF plasmid using DOTAP liposomal technique, whereas hADSCs in control group were transfected in absence of plasmid. The success of transfection was confirmed by immunofluorescent cytochemistry. Phenotype differences between the two groups were detected using flow cytometry. Proliferation ability was assessed using MTT assay. Adipogenic and osteogenic differentiation were induced and confirmed using Oil Red O staining and alizarin red staining. Results The transfected hADSCs expressed EGFP and VEGF, whereas no EGFP was observed in the untransfected hADSCs. Both groups of hADSCs showed high expressions of CD29, CD44, CD90, and low expressions of CD31 and CD45. Compared with the untransfected hADSCs, the transfected hADSCs exhibited significantly increased proliferation ability as well as the number of lipid droplets, calcium nodules, and the stained area. Conclusion VEGF overexpression promotes proliferation and multilineage differentiation potential of hADSCs without evident phenotype changes.

Decellularized skin/adipose tissue flap matrix for engineering vascularized composite soft tissue flaps.

Using a perfusion decellularization protocol, we developed a decellularized skin/adipose tissue flap (DSAF) comprising extracellular matrix (ECM) and intact vasculature. Our DSAF had a dominant vascular pedicle, microcirculatory vascularity, and a sensory nerve network and retained three-dimensional (3D) nanofibrous structures well. DSAF, which was composed of collagen and laminin with well-preserved growth factors (e.g., vascular endothelial growth factor, basic fibroblast growth factor), was successfully repopulated with human adipose-derived stem cells (hASCs) and human umbilical vein endothelial cells (HUVECs), which integrated with DSAF and formed 3D aggregates and vessel-like structures in vitro. We used microsurgery techniques to re-anastomose the recellularized DSAF into nude rats. In vivo, the engineered flap construct underwent neovascularization and constructive remodeling, which was characterized by the predominant infiltration of M2 macrophages and significant adipose tissue formation at 3months postoperatively. Our results indicate that DSAF co-cultured with hASCs and HUVECs is a promising platform for vascularized soft tissue flap engineering. This platform is not limited by the flap size, as the entire construct can be immediately perfused by the recellularized vascular network following simple re-integration into the host using conventional microsurgical techniques. STATEMENT OF SIGNIFICANCE: Significant soft tissue loss resulting from traumatic injury or tumor resection often requires surgical reconstruction using autologous soft tissue flaps. However, the limited availability of qualitative autologous flaps as well as the donor site morbidity significantly limits this approach. Engineered soft tissue flap grafts may offer a clinically relevant alternative to the autologous flap tissue. In this study, we engineered vascularized soft tissue free flap by using skin/adipose flap extracellular matrix scaffold (DSAF) in combination with multiple types of human cells. Following vascular reanastomosis in the recipient site, the engineered products successful regenerated large-scale fat tissue in vivo. This approach may provide a translatable platform for composite soft tissue free flap engineering for microsurgical reconstruction.

Inhibition of IL-6 signaling: A novel therapeutic approach to treating spinal cord injury pain.

To characterize the contribution of interleukin-6 (IL-6) to spinal cord injury pain (SCIP), we employed a clinically relevant rat contusion model of SCIP. Using Western blots, we measured IL-6 levels in lumbar segments (L1-L5), at the lesion site (T10), and in the corresponding lumbar and thoracic dorsal root ganglia (DRG) in 2 groups of similarly injured rats: (a) SCI rats that developed hind-limb mechanical allodynia (SCIP), and (b) SCI rats that did not develop SCIP. Only in SCIP rats did we find significantly increased IL-6 levels. Immunocytochemistry showed elevated IL-6 predominantly in reactive astrocytes. Our data also showed that increased production of IL-6 in hyperreactive astrocytes in SCIP rats may explain still-poorly understood astrocytic contribution to SCIP. To test the hypothesis that IL-6 contributes to mechanical allodynia, we treated SCIP rats with neutralizing IL-6 receptor antibody (IL-6-R Ab), and found that one systemic injection abolished allodynia and associated weight loss; in contrast to gabapentin, the analgesic effect lasted for at least 2weeks after the injection, despite the shorter presence of the Ab in the circulation. We also showed that IL-6-R Ab partially reversed SCI-induced decreases in the protein levels of the glutamate transporter GLT-1 12hours and 8days after Ab injection, which may explain the lasting analgesic effect of the Ab in SCIP rats. A link between reactive astrocytes IL-6-GLT-1 has not been previously shown. Given that the humanized IL-6-R Ab tocilizumab is Food and Drug Administration-approved for rheumatoid arthritis, we are proposing tocilizumab as a novel and potentially effective treatment for SCIP.

Delta-opioid augments cardiac contraction through ß-adrenergic and CGRP-receptor co-signaling.

Cardiac epinephrine and calcitonin gene-related peptide (CGRP) are produced by intrinsic cardiac adrenergic cells (ICA cells) residing in human and animal hearts. ICA cells are neuroparicine cells expressing δ-opioid receptors (DOR). We hypothesized that δ-opioid stimulation of ICA cells enhances epinephrine and CGRP release, which results in the augmentation of heart contraction. Rats were injected with DOR-agonist DPDPE (100 µg/kg) with or without 10-min pretreatment with either ß-adrenergic receptor (ß-AR) blocker propranolol (2mg/kg) or CGRP-receptor (CGRPR) blocker CGRP(8-37) (300 µg/kg), or their combination. Hemodynamics were monitored with echocardiogram and systolic blood pressure (SBP) was monitored via a tail arterial catheter. Changes in left ventricular fraction-shortening (LVFS) and heart rate (HR) were observed at 5-min after DPDPE infusion. At 5-min DPDPE induced a 36 ± 18% (p<0.001) increase of the LVFS, which continues to increase to 51 ± 24% (p<0.0001) by 10 min, and 68 ± 19% (p<0.001) by 20 min. The increase in LVFS was accompanied by the decrease of HR by 9±5% (p<0.01) by 5 min and 11 ± 6% (p<0.001) by 15 min post DPDPE infusion. This magnitude of HR reduction was observed for the remainder of the 20 min. Despite the HR-reduction, cardiac output was increased by 17 ± 8% (p<0.05) and 28±5% (p<0.001) by 5- and 20-min post DPDPE administration, respectively. There was a modest (9 ± 9%, p=0.03) decrease in SBP that was not apparent until 20 min post DPDPE infusion. The positive inotropism of DPDPE was abrogated in animals pretreated with propranolol, CGRP(8-37), or combined propranolol+CGRP(8-37). Furthermore, in whole animal and cardiomyocyte cell culture preparations, DPDPE induced myocardial protein-kinase A (PKA) activation which was abrogated in the animals pretreated with propranolol+CGRP(8-37). DOR agonists augment myocardial contraction through enhanced ß-AR and CGRPR co-signaling.

Heart protection by combination therapy with esmolol and milrinone at late-ischemia and early reperfusion.

INTRODUCTION: The present study determined whether late-ischemia/early reperfusion therapy with the ß(1)-adrenergic receptor (AR) blocker esmolol and phosphodiesterase III inhibitor milrinone reduced left ventricular (LV) myocardial infarct size (IS). METHODS AND RESULTS: In an ischemia/reperfusion rat model (30-min ischemia/4-hr reperfusion), esmolol, milrinone or esmolol + milrinone were intravenous (IV) infused over 10 min (from the last 5 min of ischemia to the first 5 min of reperfusion). LV-IS were 48.9 ± 8.9%, 41.5 ± 5.4%, 25.8 ± 7.7% and 16.8 ± 7.3% for saline, esmolol, milrinone, and esmolol + milrinone, respectively (n = 12/group). Esmolol + milrinone further reduced LV-IS compared with esmolol or milrinone alone (p < 0.05). LV-IS-reduction induced by esmolol + milrinone was eliminated in the presence of protein kinase A-(PKA)-inhibitor (Rp-cAMPS) or Akt-inhibitor (AKT 1/2 kinase inhibitor). In mixed rat ventricular cardiomyocyte cultures, intra-ischemic application of esmolol, milrinone or esmolol + milrinone reduced myocyte death rates by 5.5%, 13.3%, and 16.8%, respectively, compared with saline (p < 0.01). This cell protective effect by esmolol + milrinone was abrogated in the presence of PKA-inhibitor or Akt-inhibitor. Esmolol, milrinone or esmolol + milrinone increased myocardial PKA activity by 22%, 28% and 59%, respectively, compared with saline (n = 6, p < 0.01). No non-specific adverse effect of Rp-cAMPS on myocytes was identified in a purified myocyte preparation during hypoxia/re-oxygenation. Antiapoptotic pathways were assessed by measuring myocardial phosphorylated Akt (pAkt) levels combined with terminal dUTP nick-end labelling staining analysis. Ten minutes following infusion of esmolol, milrinone or esmolol + milrinone, there were 1.7-, 2.7-, and 6-fold increase in tissue pAkt levels, respectively. This esmolol + milrinone induced pAkt activation was abolished in the presence of PKA inhibitor. Esmolol, milrinone and esmolol + milrinone reduced myocyte apoptosis rates by 22%, 37% and 60%, respectively, compared with saline (p < 0.01). CONCLUSIONS: Late-ischemia/early reperfusion therapy with esmolol + milrinone additively reduces LV-IS associated with robust activation of myocardial PKA and subsequent Akt-antiapoptotic pathway.

Acute myocardial infarction in rats.

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes.

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in a mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL), or Tc secreted proteins (TcSP) for 0-72 h, and ROS were measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production for 2-48 h postinfection (max 18-fold increase), which was further enhanced by recombinant cytokines (IL-1beta, TNF-alpha, and IFN-gamma). We observed no increase in NADPH oxidase, xanthine oxidase, or myeloperoxidase activity, and specific inhibitors of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed and resulted in inefficient electron transport chain (ETC) activity and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (rho) cardiomyocytes lacked a functional ETC and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and an inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.

Reducing ischaemia/reperfusion injury through delta-opioid-regulated intrinsic cardiac adrenergic cells: adrenopeptidergic co-signalling.

AIMS: The purpose of this study was to determine whether intrinsic cardiac adrenergic (ICA) cells release calcitonin gene-related peptide (CGRP), exerting synergistic adrenopeptidergic cardioprotection. METHODS AND RESULTS: In situ hybridization coupled with immunostaining demonstrated that ICA cells exclusively expressed CGRP mRNA and co-expressed CGRP and delta-opioid receptor in human and rat left ventricular (LV) myocardium. Radioimmunoassay detected constitutive CGRP release from ICA cells in human and rat hearts. The delta-opioid agonist [D-Pen(25)]-enkephalin (DPDPE) increased CGRP release from ICA cells in denervated rat heart. In an ischaemia/reperfusion rat model, pre-ischaemic treatment with DPDPE reduced infarct size (IS) by 51 +/- 16% (P < 0.01). Co-infusion of beta(2)-adrenergic receptor (beta(2)-AR) and CGRP receptor (CGRP-R) antagonists increased IS by 62 +/- 23% (P < 0.01) compared with saline and abolished DPDPE-initiated IS reduction. Pre-treatment of ICA cell-myocyte co-culture with the beta(2)-AR/CGRP-R antagonists increased myocyte death rate by 24 +/- 4% (P < 0.01) and abolished DPDPE-initiated myocyte protection against hypoxia/reoxygenation (re-O(2)). In the ICA cell-depleted myocyte culture, DPDPE did not confer myocyte protection. Supplementing ICA cell-depleted myocyte culture with beta(2)-AR/CGRP-R agonists reduced hypoxia/re-O(2)-induced myocyte death by 24 +/- 5% (P < 0.01), simulating endogenous neurohormonal effects of ICA cells. Western blot analysis showed that DPDPE markedly increased phosphorylated myocardial Akt levels. This effect was abolished in the presence of beta(2)-AR/CGRP-R blockade. Terminal dUTP nick-end labelling staining analysis of the LV infarct zone demonstrated that DPDPE reduced myocyte apoptosis by 58 +/- 19% (P < 0.05), an effect that was eliminated in the presence of beta(2)-AR/CGRP-R blockade. Finally, echocardiography showed that DPDPE increased LV contractility in a manner dependent on beta-AR/CGRP-R stimulation. CONCLUSION: ICA cells constitute a delta-opioid-regulated adrenopeptidergic paracrine system conferring robust cardioprotection through beta(2)-AR/CGRP-R co-signalling, resulting in the activation of an anti-apoptotic pathway during ischaemia/reperfusion.

Mediating delta-opioid-initiated heart protection via the beta2-adrenergic receptor: role of the intrinsic cardiac adrenergic cell.

Stimulation of cardiac beta(2)-adrenergic receptor (beta(2)-AR) or delta-opioid receptor (DOR) exerts a similar degree of cardioprotection against myocardial ischemia in experimental models. We hypothesized that delta-opioid-initiated cardioprotection is mediated by the intrinsic cardiac adrenergic (ICA) cell via enhanced epinephrine release. Using immunohistochemical and in situ hybridization methods, we detected in situ tyrosine hydroxylase (TH) mRNA and TH immunoreactivity that was colocalized with DOR immunoreactivity in ICA cells in human and rat hearts. Western blot analysis detected DOR protein in ICA cells isolated from rat ventricular myocytes. The physiology of DOR expression was examined by determining changes of cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients in isolated rat ICA cells using fluorescence spectrophotometry. Exposing the selective delta-opioid agonist D-[Pen(2,5)]enkephalin (DPDPE) to ICA cells increased [Ca(2+)](i) transients in a concentration-dependent manner. Such an effect was abolished by the Ca(2+) channel blocker nifedipine. HPLC-electrochemical detection demonstrated a 2.4-fold increase in epinephrine release from ICA cells following DPDPE application. The significance of the ICA cell and its epinephrine release in delta-opioid-initiated cardioprotection was demonstrated in the rat myocardial infarction model and ICA cell-ventricular myocyte coculture. DPDPE administered before coronary artery occlusion or simulated ischemia-reperfusion reduced left ventricular infarct size by 54 +/- 15% or myocyte death by 26 +/- 4%, respectively. beta(2)-AR blockade markedly attenuated delta-opioid-initiated infarct size-limiting effect and abolished delta-opioid-initiated myocyte survival protection in rat ICA cell-myocyte coculture. Furthermore, delta-opioid agonist exerted no myocyte survival protection in the absence of cocultured ICA cells during ischemia-reperfusion. We conclude that delta-opioid-initiated myocardial infarct size reduction is primarily mediated via endogenous epinephrine/beta(2)-AR signaling pathway as a result of ICA cell activation.