Analysis of Antiangiogenic Potential and Cell Death Mechanism of a Kinetically Inert Platinum Antitumor Agent.

Cancer is a multifaceted disease involving various pathological processes, including uncontrolled proliferation, development of resistance, angiogenesis, metastasis, etc. Therefore, chemotherapeutic agents capable of simultaneously inhibiting proliferation, circumventing chemoresistance, and inhibiting angiogenesis can address multiple aspects of cancer progression. We recently identified a highly promising kinetically inert platinum antitumor agent, namely, Pt-1, that can circumvent cisplatin resistance and showed negligible nephrotoxicity. In this study, we explored the antiangiogenic potential and elucidated the detailed mechanism of cell death through which it exerts its antitumor activity. Pt-1 strongly inhibited angiogenesis in a zebrafish in vivo model at its therapeutically relevant nontoxic dose. Further, Pt-1 exerted antitumor activity through necroptosis- and paraptosis-mediated cell death. Taken together, the combination of antitumor activity with antiangiogenic property in Pt-1 makes it a highly promising antitumor candidate.

Development of a Highly In Vivo Efficacious Dual Antitumor and Antiangiogenic Organoiridium Complex as a Potential Anti-Lung Cancer Agent.

While the phenomenal clinical success of blockbuster platinum (Pt) drugs is highly encouraging, the inherent and acquired resistance and dose-limiting side effects severely limit their clinical application. To find a better alternative with translational potential, we synthesized a library of six organo-IrIII half-sandwich [(η5-CpX)Ir(N∧N)Cl]+-type complexes. In vitro screening identified two lead candidates [(η5-CpXPh)Ir(Ph2Phen)Cl]+ (5, CpXPh = tetramethyl-phenyl-cyclopentadienyl and Ph2Phen = 4,7-diphenyl-1,10-phenanthroline) and [(η5-CpXBiPh)Ir(Ph2Phen)Cl]+ (6, CpXBiPh = tetramethyl-biphenyl-cyclopentadienyl) with nanomolar IC50 values. Both 5 and 6 efficiently overcame Pt resistance and presented excellent cancer cell selectivity in vitro. Potent antiangiogenic properties of 6 were demonstrated in the zebrafish model. Satisfyingly, 6 and its nanoliposome Lipo-6 presented considerably higher in vivo antitumor efficacy as compared to cisplatin, as well as earlier reported IrIII half-sandwich complexes in mice bearing the A549 non-small lung cancer xenograft. In particular, complex 6 is the first example of this class that exerted dual in vivo antiangiogenic and antitumor properties.

Xenograft of human pluripotent stem cell-derived cardiac lineage cells on zebrafish embryo heart.

Cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) are a promising cell source for regenerative medicine and drug discovery. However, the use of animal models for studying human cardiomyocytes derived from hiPSCs in vivo is limited and challenging. Given the shared properties between humans and zebrafish, their ethical advantages over mammalian models, and their immature immune system that is rejection-free against xenografted human cells, zebrafish provide a suitable alternative model for xenograft studies. We microinjected fluorescence-labeled cardiac lineage cells derived from hiPSCs, specifically mesoderm or cardiac mesoderm cells, into the yolk and the area proximal to the outflow tract of the linear heart at 24 hours post-fertilization (hpf). The cells injected into the yolk survived and did not migrate to other tissues. In contrast, the cells injected contiguous with the outflow tract of the linear heart migrated into the pericardial cavity and heart. After 1 day post injection (1 dpi, 22-24 hpi), the injected cells migrated into the pericardial cavity and heart. Importantly, we observed heartbeat-like movements of some injected cells in the zebrafish heart after 1 dpi. These results suggested successful xenografting of hiPSC-derived cardiac lineage cells into the zebrafish embryo heart. Thus, we developed a valuable tool using zebrafish embryos as a model organism for investigating the molecular and cellular mechanisms involved in the grafting process. This is essential in developing cell transplantation-based cardiac therapeutics as well as for drug testing, notably contributing to advancements in the field of cardio-medicine.

Ccn2a-FGFR1-SHH signaling is necessary for intervertebral disc homeostasis and regeneration in adult zebrafish.

Intervertebral disc (IVD) degeneration is the primary cause of back pain in humans. However, the cellular and molecular pathogenesis of IVD degeneration is poorly understood. This study shows that zebrafish IVDs possess distinct and non-overlapping zones of cell proliferation and cell death. We find that, in zebrafish, cellular communication network factor 2a (ccn2a) is expressed in notochord and IVDs. Although IVD development appears normal in ccn2a mutants, the adult mutant IVDs exhibit decreased cell proliferation and increased cell death leading to IVD degeneration. Moreover, Ccn2a overexpression promotes regeneration through accelerating cell proliferation and suppressing cell death in wild-type aged IVDs. Mechanistically, Ccn2a maintains IVD homeostasis and promotes IVD regeneration by enhancing outer annulus fibrosus cell proliferation and suppressing nucleus pulposus cell death through augmenting FGFR1-SHH signaling. These findings reveal that Ccn2a plays a central role in IVD homeostasis and regeneration, which could be exploited for therapeutic intervention in degenerated human discs.

Potent Ruthenium-Ferrocene Bimetallic Antitumor Antiangiogenic Agent That Circumvents Platinum Resistance: From Synthesis and Mechanistic Studies to In Vivo Evaluation in Zebrafish.

Emergence of resistance in cancer cells and dose-limiting side effects severely limit the widespread use of platinum (Pt) anticancer drugs. Multi-action hybrid anticancer agents that are constructed by merging two or more pharmacophores offer the prospect of circumventing issues of Pt drugs. Herein, we report the design, synthesis, and in-depth biological evaluation of a ruthenium-ferrocene (Ru-Fc) bimetallic agent [(η6-p-cymene)Ru(1,1,1-trifluoro-4-oxo-4-ferrocenyl-but-2-en-2-olate)Cl] and its five analogues. Along with aquation/anation chemistry, we evaluated the in vitro antitumor potency, Pt cross-resistance profile, and in vivo antiangiogenic properties. A structure activity analysis was performed to understand the impact of Fc, CF3, and p-cymene groups on the anticancer potency of the Ru-Fc hybrid. Finally, in addition to assessing cellular uptake and intracellular distribution, we demonstrated that the Ru-Fc hybrid binds to nucleophilic biomolecules and produces reactive oxygen species, which causes mitochondrial dysfunction and induces ER stress, leading to poly(ADP-ribose) polymerase-mediated necroptotic cell death.

Evolution, Expression and Functional Analysis of CXCR3 in Neuronal and Cardiovascular Diseases: A Narrative Review.

Chemokines form a sophisticated communication network wherein they maneuver the spatiotemporal migration of immune cells across a system. These chemical messengers are recognized by chemokine receptors, which can trigger a cascade of reactions upon binding to its respective ligand. CXC chemokine receptor 3 (CXCR3) is a transmembrane G protein-coupled receptor, which can selectively bind to CXCL9, CXCL10, and CXCL11. CXCR3 is predominantly expressed on immune cells, including activated T lymphocytes and natural killer cells. It thus plays a crucial role in immunological processes like homing of effector cells to infection sites and for pathogen clearance. Additionally, it is expressed on several cell types of the central nervous system and cardiovascular system, due to which it has been implicated in several central nervous system disorders, including Alzheimer's disease, multiple sclerosis, dengue viral disease, and glioblastoma, as well as cardiovascular diseases like atherosclerosis, Chronic Chagas cardiomyopathy, and hypertension. This review provides a narrative description of the evolution, structure, function, and expression of CXCR3 and its corresponding ligands in mammals and zebrafish and the association of CXCR3 receptors with cardiovascular and neuronal disorders. Unraveling the mechanisms underlying the connection of CXCR3 and disease could help researchers investigate the potential of CXCR3 as a biomarker for early diagnosis and as a therapeutic target for pharmacological intervention, along with developing robust zebrafish disease models.

Celsr family genes are dynamically expressed in embryonic and juvenile zebrafish.

The Cadherin EGF LAG seven-pass G-type receptor (Celsr) family belongs to the adhesion G-protein coupled receptor superfamily. In most vertebrates, the Celsr family has three members (CELSR1-3), whereas zebrafish display four paralogues (celsr1a, 1b, 2, 3). Although studies have shown the importance of the Celsr family in planar cell polarity, axonal guidance, and dendritic growth, the molecular mechanisms of the Celsr family regulating these cellular processes in vertebrates remain elusive. Zebrafish is an experimentally more amenable model to study vertebrate development, as zebrafish embryos develop externally, optically transparent, remain alive with malformed organs, and zebrafish is genetically similar to humans. Understanding the detailed expression pattern is the first step of exploring the functional mechanisms of the genes involved in development. Thus, we report the spatiotemporal expression pattern of Celsr family members in zebrafish nervous tissues. Our analysis shows that celsr1b and celsr2 are expressed maternally. In embryos, celsr1a, celsr1b, and celsr2 are expressed in the neural progenitors, and celsr3 is expressed in all five primary neural clusters of the brain and mantle layer of the spinal cord. In juvenile zebrafish, celsr1a, celsr1b, and celsr2 are presumably expressed in the neural progenitor enriched regions of the CNS. Therefore, the expression pattern of zebrafish Celsr family members is reminiscent of patterns described in other vertebrates or mammalian speciate. This indicates the conserved role of Celsr family genes in nervous system development and suggests zebrafish as an excellent model to explore the cellular and molecular mechanisms of Celsr family genes in vertebrate neurogenesis.

Zebrafish Model to Study Angiotensin II-Mediated Pathophysiology.

Hypertension, a common chronic condition, may damage multiple organs, including the kidney, heart, and brain. Thus, it is essential to understand the pathology upon ectopic activation of the molecular pathways involved in mammalian hypertension to develop strategies to manage hypertension. Animal models play a crucial role in unraveling the disease pathophysiology by allowing incisive experimental procedures impossible in humans. Zebrafish, a small freshwater fish, have emerged as an important model system to study human diseases. The primary effector, Angiotensin II of the RAS pathway, regulates hemodynamic pressure overload mediated cardiovascular pathogenesis in mammals. There are various established mammalian models available to study pathophysiology in Angiotensin II-induced hypertension. Here, we have developed a zebrafish model to study pathogenesis by Angiotensin II. We find that intradermal Angiotensin II injection every 12 h can induce cardiac remodeling in seven days. We show that Angiotensin II injection in adult zebrafish causes cardiomyocyte hypertrophy and enhances cardiac cell proliferation. In addition, Angiotensin II induces ECM protein-coding gene expression and fibrosis in the cardiac ventricles. Thus, this study can conclude that Angiotensin II injection in zebrafish has similar implications as mammals, and zebrafish can be a model to study pathophysiology associated with AngII-RAS signaling.

Ccn2a is an injury-induced matricellular factor that promotes cardiac regeneration in zebrafish.

The ability of zebrafish to heal their heart after injury makes them an attractive model for investigating the mechanisms governing the regenerative process. In this study, we show that the gene cellular communication network factor 2a (ccn2a), previously known as ctgfa, is induced in endocardial cells in the injured tissue and regulates CM proliferation and repopulation of the damaged tissue. We find that, whereas in wild-type animals, CMs track along the newly formed blood vessels that revascularize the injured tissue, in ccn2a mutants CM proliferation and repopulation are disrupted, despite apparently unaffected revascularization. In addition, we find that ccn2a overexpression enhances CM proliferation and improves the resolution of transient collagen deposition. Through loss- and gain-of-function as well as pharmacological approaches, we provide evidence that Ccn2a is necessary for and promotes heart regeneration by enhancing the expression of pro-regenerative extracellular matrix genes, and by inhibiting the chemokine receptor gene cxcr3.1 through a mechanism involving Tgfß/pSmad3 signaling. Thus, Ccn2a positively modulates the innate regenerative response of the adult zebrafish heart.

EF-hand domain containing 2 (Efhc2) is crucial for distal segmentation of pronephros in zebrafish.

BACKGROUND: The blood filtering organ in zebrafish embryos is the pronephros, which consists of two functional nephrons. Segmentation of a nephron into different domains is essential for its function and is well conserved among vertebrates. Zebrafish has been extensively used as a model to understand nephron segmentation during development. Here, we have identified EF-hand domain containing 2 (Efhc2) as a novel component of genetic programme regulating nephron segmentation in zebrafish. Human EFHC2 is a protein with one predicted calcium-binding EF-hand motif and three DM10 domains, whose function is unknown. EFHC2 has been implicated in several brain-related genetic diseases like Turner syndrome and juvenile myoclonic epilepsy. However, there is limited information on its normal physiological function. RESULTS: efhc2 mRNA is primarily expressed in the pronephros of zebrafish embryos. Other sites of expression include olfactory placode, notochord, otic vesicle, epiphysis and neuromast cells. Morpholino antisense oligonucleotide-mediated knock-down of Efhc2 resulted in defects in pronephros development and function in zebrafish embryos. Efhc2 knock-down leads to expansion of distal early segment of pronephros, whereas, the corpuscle of stannius and distal late segments were reduced. The number of multi-ciliated cells (MCC) that are present in a salt-and-pepper fashion throughout the middle of each nephron and vital for fluid flow were also reduced. It is known that retinoic acid (RA) signaling regulates pronephros segmentation in vertebrates and we show that Efhc2 function is crucial for nephron segmentation in zebrafish. Our data suggests that RA and Efhc2 function independent of each other in pronephros segmentation. However, Efhc2 and RA synergistically regulate MCC development. CONCLUSION: In this study, we have identified Efhc2 as a regulator of segmentation of the distal part of nephron and pronephros function during zebrafish development.

The zebrafish ventricle: A hub of cardiac endothelial cells for in vitro cell behavior studies.

Despite our increasing understanding of zebrafish heart development and regeneration, there is limited information about the distribution of endothelial cells (ECs) in the adult zebrafish heart. Here, we investigate and compare the distribution of cardiac ECs (cECs) in adult mouse and zebrafish ventricles. Surprisingly, we find that (i) active coronary vessel growth is present in adult zebrafish, (ii) ~37 and ~39% of cells in the zebrafish heart are ECs and cardiomyocytes, respectively, a composition similar to that seen in mouse. However, we find that in zebrafish, ~36% of the ventricular tissue is covered with ECs, i.e., a substantially larger proportion than in mouse. Capitalising on the high abundance of cECs in zebrafish, we established a protocol to isolate them with high purity using fluorescent transgenic lines. Our approach eliminates side-effects due to antibody utilisation. Moreover, the isolated cECs maintained a high proliferation index even after three passages and were amenable to pharmacological treatments to study cEC migration in vitro. Such primary cultures will be a useful tool for supplementary in vitro studies on the accumulating zebrafish mutant lines as well as the screening of small molecule libraries on cardiac specific endothelial cells.

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish.

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration. We performed a genome-wide profiling screen for secreted factors that are up-regulated during zebrafish spinal cord regeneration. We found that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupted spinal cord repair, and transgenic ctgfa overexpression or local delivery of human CTGF recombinant protein accelerated bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration.

Vascularisation for cardiac tissue engineering: the extracellular matrix.

Cardiovascular diseases present a major socio-economic burden. One major problem underlying most cardiovascular and congenital heart diseases is the irreversible loss of contractile heart muscle cells, the cardiomyocytes. To reverse damage incurred by myocardial infarction or by surgical correction of cardiac malformations, the loss of cardiac tissue with a thickness of a few millimetres needs to be compensated. A promising approach to this issue is cardiac tissue engineering. In this review we focus on the problem of in vitro vascularisation as implantation of cardiac patches consisting of more than three layers of cardiomyocytes (> 100 µm thick) already results in necrosis. We explain the need for vascularisation and elaborate on the importance to include non-myocytes in order to generate functional vascularised cardiac tissue. We discuss the potential of extracellular matrix molecules in promoting vascularisation and introduce nephronectin as an example of a new promising candidate. Finally, we discuss current biomaterial-based approaches including micropatterning, electrospinning, 3D micro-manufacturing technology and porogens. Collectively, the current literature supports the notion that cardiac tissue engineering is a realistic option for future treatment of paediatric and adult patients with cardiac disease.

The multiple signaling modalities of adhesion G protein-coupled receptor GPR126 in development.

The G protein-coupled receptor (GPCR) superfamily is the largest known receptor family in the human genome. Although the family of adhesion GPCRs comprises the second largest sub-family, their function is poorly understood. Here, we review the current knowledge about the adhesion GPCR family member GPR126. GPR126 possesses a signal peptide, a 7TM domain homologous to secretin-like GPCRs, a GPS motif and an extended N-terminus containing a CUB (Complement, Uegf, Bmp1) domain, a PTX (Pentraxin) domain, a hormone binding domain and 27 putative N-glycosylation sites. Knockdown and knockout experiments in zebrafish and mice have demonstrated that Gpr126 plays an essential role in neural, cardiac and ear development. In addition, genome-wide association studies have implicated variations at the GPR126 locus in obstructive pulmonary dysfunction, in scoliosis and as a determinant of trunk length and body height. Gpr126 appears to exert its function depending on the organ system via G protein- and/or N-terminus-dependent signaling. Here, we review the current knowledge about Gpr126, which, due to the variety of its functions and its multiple signaling modalities, provides a model adhesion GPCR to understand general functional concepts utilized by adhesion GPCRs.

FGF1-mediated cardiomyocyte cell cycle reentry depends on the interaction of FGFR-1 and Fn14.

Fibroblast growth factors (FGFs) signal through FGF receptors (FGFRs) mediating a broad range of cellular functions during embryonic development, as well as disease and regeneration during adulthood. Thus, it is important to understand the underlying molecular mechanisms that modulate this system. Here, we show that FGFR-1 can interact with the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) resulting in cardiomyocyte cell cycle reentry. FGF1-induced cell cycle reentry in neonatal cardiomyocytes could be blocked by Fn14 inhibition, while TWEAK-induced cell cycle activation was inhibited by blocking FGFR-1 signaling. In addition, costimulation experiments revealed a synergistic effect of FGF1 and TWEAK in regard to cardiomyocyte cell cycle induction via PI3K/Akt signaling. Overexpression of Fn14 with either FGFR-1 long [FGFR-1(L)] or FGFR-1 short [FGFR-1(S)] isoforms resulted after FGF1/TWEAK stimulation in cell cycle reentry of >40% adult cardiomyocytes. Finally, coimmunoprecipitation and proximity ligation assays indicated that endogenous FGFR-1 and Fn14 interact with each other in cardiomyocytes. This interaction was strongly enhanced in the presence of their corresponding ligands, FGF1 and TWEAK. Taken together, our data suggest that FGFR-1/Fn14 interaction may represent a novel endogenous mechanism to modulate the action of these receptors and their ligands and to control cardiomyocyte cell cycle reentry.

Gpr126 functions in Schwann cells to control differentiation and myelination via G-protein activation.

The myelin sheath surrounding axons ensures that nerve impulses travel quickly and efficiently, allowing for the proper function of the vertebrate nervous system. We previously showed that the adhesion G-protein-coupled receptor (aGPCR) Gpr126 is essential for peripheral nervous system myelination, although the molecular mechanisms by which Gpr126 functions were incompletely understood. aGPCRs are a significantly understudied protein class, and it was unknown whether Gpr126 couples to G-proteins. Here, we analyze Dhh(Cre);Gpr126(fl/fl) conditional mutants, and show that Gpr126 functions in Schwann cells (SCs) for radial sorting of axons and myelination. Furthermore, we demonstrate that elevation of cAMP levels or protein kinase A activation suppresses myelin defects in Gpr126 mouse mutants and that cAMP levels are reduced in conditional Gpr126 mutant peripheral nerve. Finally, we show that GPR126 directly increases cAMP by coupling to heterotrimeric G-proteins. Together, these data support a model in which Gpr126 functions in SCs for proper development and myelination and provide evidence that these functions are mediated via G-protein-signaling pathways.

Organ-specific function of adhesion G protein-coupled receptor GPR126 is domain-dependent.

Despite their abundance and multiple functions in a variety of organ systems, the function and signaling mechanisms of adhesion G protein-coupled receptors (GPCRs) are poorly understood. Adhesion GPCRs possess large N termini containing various functional domains. In addition, many of them are autoproteolytically cleaved at their GPS sites into an N-terminal fragment (NTF) and C-terminal fragment. Here we demonstrate that Gpr126 is expressed in the endocardium during early mouse heart development. Gpr126 knockout in mice and knockdown in zebrafish caused hypotrabeculation and affected mitochondrial function. Ectopic expression of Gpr126-NTF that lacks the GPS motif (NTF(ΔGPS)) in zebrafish rescued the trabeculation but not the previously described myelination phenotype in the peripheral nervous system. These data support a model in which the NTF of Gpr126, in contrast to the C-terminal fragment, plays an important role in heart development. Collectively, our analysis provides a unique example of the versatile function and signaling properties of adhesion GPCRs in vertebrates.

EGFL7 ligates αvß3 integrin to enhance vessel formation.

Angiogenesis, defined as blood vessel formation from a preexisting vasculature, is governed by multiple signal cascades including integrin receptors, in particular integrin αVß3. Here we identify the endothelial cell (EC)-secreted factor epidermal growth factor-like protein 7 (EGFL7) as a novel specific ligand of integrin αVß3, thus providing mechanistic insight into its proangiogenic actions in vitro and in vivo. Specifically, EGFL7 attaches to the extracellular matrix and by its interaction with integrin αVß3 increases the motility of EC, which allows EC to move on a sticky underground during vessel remodeling. We provide evidence that the deregulation of EGFL7 in zebrafish embryos leads to a severe integrin-dependent malformation of the caudal venous plexus, pointing toward the significance of EGFL7 in vessel development. In biopsy specimens of patients with neurologic diseases, vascular EGFL7 expression rose with increasing EC proliferation. Further, EGFL7 became upregulated in vessels of the stroke penumbra using a mouse model of reversible middle cerebral artery occlusion. Our data suggest that EGFL7 expression depends on the remodeling state of the existing vasculature rather than on the phenotype of neurologic disease analyzed. In sum, our work sheds a novel light on the molecular mechanism EGFL7 engages to govern physiological and pathological angiogenesis.

Deletion of Fn14 receptor protects from right heart fibrosis and dysfunction.

Pulmonary arterial hypertension (PAH) is a fatal disease for which no cure is yet available. The leading cause of death in PAH is right ventricular (RV) failure. Previously, the TNF receptor superfamily member fibroblast growth factor-inducible molecule 14 (Fn14) has been associated with different fibrotic diseases. However, so far there is no study demonstrating a causal role for endogenous Fn14 signaling in RV or LV heart disease. The purpose of this study was to determine whether global ablation of Fn14 prevents RV fibrosis and remodeling improving heart function. Here, we provide evidence for a causative role of Fn14 in pulmonary artery banding (PAB)-induced RV fibrosis and dysfunction in mice. Fn14 expression was increased in the RV after PAB. Mice lacking Fn14 (Fn14(-/-)) displayed substantially reduced RV fibrosis and dysfunction following PAB compared to wild-type littermates. Cell culture experiments demonstrated that activation of Fn14 induces collagen expression via RhoA-dependent nuclear translocation of myocardin-related transcription factor-A (MRTF-A)/MAL. Furthermore, activation of Fn14 in vitro caused fibroblast proliferation and myofibroblast differentiation, which corresponds to suppression of PAB-induced RV fibrosis in Fn14(-/-) mice. Moreover, our findings suggest that Fn14 expression is regulated by endothelin-1 (ET-1) in cardiac fibroblasts. We conclude that Fn14 is an endogenous key regulator in cardiac fibrosis and suggest this receptor as potential new target for therapeutic interventions in heart failure.

The functional properties of nephronectin: an adhesion molecule for cardiac tissue engineering.

Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.