Interactions between cardiac cells enhance cardiomyocyte hypertrophy and increase fibroblast proliferation

In cardiac hypertrophy, both excessive enlargement of cardiac myocytes (CMs) and progressive fibrosis are known to occur simultaneously. To investigate the nature of interactions between ventricular CMs and cardiac fibroblasts (CFs) in these conditions, we have established a "dedifferentiated model" of adult murine CMs in coculture with CFs. In such a model, which is recognized to study cardiac cell hypertrophy in vitro, dedifferentiated CMs in culture and in coculture were characterized by immunopositive staining to ANP (atrial natriuretic peptide) and beta-myosin heavy chain (beta-MHC). The results confirm that ANP secretion by CMs was significantly increased during the cultures. The increase size of cultured CMs was significantly higher in CM/CF cocultures than in CM cultures which was also observed when CMs were cultured with fibroblast conditioned medium (FCM). In addition, fibroblast proliferation studies showed that CMs favored fibroblast adhesion and/or growth at the beginning of the coculture and fibroblast proliferation throughout the time course of the coculture. Furthermore, a significant level of interleukin-6 (IL-6) production was detected by ELISA in CM/CF cocultures. A similar higher increase was observed when CMs were cultured in the presence of FCM. These results demonstrate that CFs enhance myocyte hypertrophy and that CMs regulate fibroblast adhesion and/or proliferation, suggesting a paracrine interaction between CMs and CFs which could involve IL-6.     

Role of interleukin-6 in cardiomyocyte/cardiac fibroblast interactions during myocyte hypertrophy and fibroblast proliferation

The process of cardiac hypertrophy is considered to involve two components: that of cardiac myocyte (CM) enlargement and cardiac fibroblast (CF) proliferation. The interleukin-6 (IL-6) family cytokines have been implicated in a variety of cellular and molecular interactions between myocytes and non-myocytes (NCMs), which in turn have important roles in the development of cardiac hypertrophy. In the study of these interactions, we previously detected very high levels of IL-6 in supernatants of a "dedifferentiated model" of adult ventricular CMs cultured with CFs. In the present study, we have used this in vitro coculture system to examine how IL-6 is involved in the interactions between CMs and CFs during CM hypertrophy and CF proliferation. IL-6 and its signal transducer, 130-kDa glycoprotein (gp130), were detected by immunostaining cultured CMs and CFs with anti-IL-6 or anti-gp130 antibodies. Addition of anti-IL-6 or anti-gp130 antagonist antibodies into CM/CF cocultures induced a significant decrease in expression of atrial natriuretic peptide (ANP) and beta-myosin heavy chain (beta-MHC) in CMs. The presence of IL-6 antagonist also resulted in a decrease in the surface area of 12-day-old CMs cultured with CFs or in the presence of fibroblast conditioned medium (FCM), and decreased fibroblast proliferation in CM/CF cocultures, particularly in the presence of a gp130 antagonist. The results also show that angiotensin II (AngII) is mainly secreted by CFs and induces IL-6 secretion in CMs cultured with CFs or with FCM. In addition, the effects of IL-6 on cardiomyocyte hypertrophy and fibroblast proliferation were inhibited by addition of the AT-1 receptor antagonist, losartan. These results suggest that IL-6 contributes significantly to CM hypertrophy by an autocrine pathway and to fibroblast proliferation by a paracrine pathway and that these effects could be mediated by AngII.     

Cell culture modifies Ca2+ signaling during excitation-contraction coupling in neonate cardiac myocytes

In heart, the excitation-contraction coupling (ECC) mechanism changes during development. Primary cell culture has been used to study Ca(2+) signaling in newborn (NB) rat heart. In this work, the effects of cell culture on the action potential (AP) and ECC Ca(2+) signaling during development were investigated. Specifically, AP, Ca(2+) currents (I(Ca)), and ryanodine receptor (RyR) properties (i.e. density, distribution, and contribution to Ca(2+) transients and Ca(2+) sparks) were defined in cultured myocytes (CM) from 0-day-old NB rat at different times in culture (1-4 days). Compared with acutely dissociated myocytes (ADM) from NB of equivalent ages (1-4 days), CM showed lower RyR density (50% at 1 day, 25% at 4 days), but larger RyR contribution to the Ca(2+) transient (25% at 1 day, 57% at 4 days). Additionally, Ca(2+) sparks were larger, longer, wider, and more frequent in CM than in ADM. RyR cellular distribution also showed different arrangement. While in CM, RyRs were located peripherally, in ADM of equivalent ages a sarcomeric arrangement was predominant. Finally, CM showed a two-fold increase in sarcolemmal Ca(2+) entry during the AP. These results indicated that primary culture is a feasible model to study Ca(2+) signaling in heart; however, it does not precisely reproduce what occurs in ECC during development.     

Lipopolysaccharide improves cardiomyocyte survival and function after serum deprivation

Toll-like receptor-4 (TLR4) and its signaling molecule interleukin-1 receptor-associated kinase (IRAK-1) play an important role in host defense and tissue inflammation. Intriguingly, systemic administration of lipopolysaccharide (LPS), the agonist for TLR4, confers a cardio-protective effect against ischemic injury. However, the mechanisms leading to the cardiac protection remain largely unknown. The present study was designed to investigate the role of TLR4 activation by LPS in protecting cardiomyocytes (CM) against apoptosis in an in vitro model of ischemia and to explore the downstream mechanisms leading to the protective effect. Incubation with LPS led to activation of IRAK-1 and protected CMs against serum deprivation (SD)-induced apoptosis as demonstrated by DNA laddering, histone-DNA fragment enzyme-linked immunosorbent assay, and activation of caspase-3. Phosphatidylinositol 3-kinase/Akt, extracellular signal-regulated kinase 1/2, and IkappaB kinase beta appear to contribute to the anti-apoptotic effect of LPS since the specific inhibitors, wortmannin, PD98059, and dominant negative IKKbeta transgene expression reversed the LPS effect. To assess whether LPS improves CM function, we examined intracellular Ca(2+) transients and cell shortening in single adult rat CMs. SD for 6 h dramatically inhibited Ca(2+) transients and CM contractility. LPS at 500 ng/ml significantly improved the [Ca(2+)](i) transients and enhanced contractility in control CMs as well as in CMs subjected to SD. Importantly, transient ischemia led to rapid activation of IRAK-1 in cultured CMs and in adult rat myocardium. Adenovirus-mediated transgene expression of IRAK-1 but not its kinase-deficient mutant IRAK-1(K239S) protected CMs against SD-induced apoptosis. Taken together, these data suggest an important role of TLR4 signaling via IRAK-1 in protecting against SD-induced apoptosis.     

Maintenance and characterization of spontaneous contraction rhythm in cultured cardiac myocytes fused with cardiac fibroblasts

Cardiomyocytes (CMs) fuse with various cells including endothelial cells, cardiac fibroblasts (CFs). In addition, recent studies have shown that stem cells fuse spontaneously with cells remaining in the damaged tissues, and restore tissue functions after myocardial infarction. In this study, we investigated whether cultured cardiomyocytes fused with proliferative cardiac fibroblasts maintained the phenotype of functional myocytes by analyzing the spontaneous contraction rhythm after fusion with CFs lacking a beating capability. CMs and CFs cultured for 4 days in vitro were used in this study. The fusion of cultured CMs and CFs was achieved with polyethylene glycol (PEG) and hemagglutinating virus of Japan (HVJ). Analyses of CMs fused with CFs by using either PEG or HVJ to imitate spontaneous fusion in vivo demonstrated that CMs and CFs actually fused together and fused cells expressed lineage marker proteins of both CMs and CFs. In addition, fused cells reentered the G2-M phase of the cell cycle. Furthermore, fused cells retained the spontaneous contraction activity. The present study demonstrated that CMs fused with proliferative CFs showed the phenotype of both CMs and CFs and spontaneous rhythmic contraction.     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.     

Structural adaptation in adult rabbit ventricular myocytes

The mechanism of induction of cardiomyocyte (CM) dedifferentiation, as seen in chronic hibernating myocardium, is largely unknown. Recently, a cellular model was proposed consisting of long-term cocultures of adult rabbit CMs and cardiac fibroblasts in which typical structural characteristics of hibernation-like dedifferentiation could be induced. Only CMs in close contact with fibroblasts underwent these changes. In this study, we further investigated the characteristics of the fibroblast-CM interaction to seek for triggers and phenomena involved in CM dedifferentiation. Adult rabbit CMs were cocultured with cardiac or 3T3 fibroblasts. Heterocellular interactions and the structural adaptation of the CMs were quantified and studied with vital microscopy and electron microscopy. Immunocytochemical analysis of several adhesion molecules, i.e., N-cadherin, vinculin, β1-integrin, and desmoplakin, were examined. Upon contact with CMs, fibroblasts attached firmly and pulled the former cells, resulting in anisotropic stretch. Quantification of the attachment sites revealed a predominant binding of the fibroblast to the distal ends of the CM in d 1 cocultures and a shift towards the lateral sides of the CMs on d 2 of coculture, suggesting a redistribution of CM membrane proteins. Immunocytochemical analysis of cell adhesion proteins showed that these were upregulated at the heterocellular contact sites. Addition of autologous and nonautologous fibroblasts to the CM culture similarly induced a progressive and accelerated structural adaptation of the CM. Dynamic passive stretch invoked by the fibroblasts and/or intercellular communication involving cell adhesion molecule expression at the interaction sites may play an important role in the induction of hibernation-like dedifferentiation of the cocultured adult rabbit CMs. These authors contributed equally to this study.     

Cell transplantation for treatment of acute myocardial infarction: unique capacity for repair by skeletal muscle satellite cells

An adult heart injured by an ischemic episode has a limited capacity to regenerate. We administered three types of adult guinea pig cells [cardiomyocytes (CMs), cardiac fibroblasts (CFs), and skeletal myoblasts (Mbs)] to compare their suitability for repair of acute myocardial infarction. We used confocal fluorescent microscopy and a variety of specific immunomarkers and echocardiography to provide anatomic evidence for the viability of such cells and their possible functional beneficial effects. All cells were transfected with adenovirus-containing beta-galactosidase gene so that migration from the injection sites could be traced. Both freshly isolated CMs as well as CFs were found concentrated in the infarcted zone; these cells survived for at least 2 wk posttransplantation. Transplanted CMs were regularly striated and grew long projections that could form gap junctions with native CMs, which was evidenced by connexin43 labeling. In addition, CM transplantation resulted in increased angiogenesis in the infarcted areas. In contrast, transplanted CFs did not appear to make any gap junctional contacts with native CMs nor did they enhance local angiogenesis. Mbs cultured for 7 days and transfected Mbs were identified 7 days posttransplantation in the infarcted area. During that time and thereafter, Mbs proliferated and differentiated into myotubes that formed new, regularly striated myofibers that occupied most (50-70%) of the infarcted area by 2-3 wk. These newly formed myofibers maintained their Mb skeletal muscle origin as evidenced by their capacity to express myogenin and fast skeletal myosin. This skeletal phenotype appeared to downregulate with time, and Mbs partially transdifferentiated into a cardiac phenotype as indicated by labeling for cardiac-specific troponin T and cardiac myosin heavy chain. By the third week posttransplantation, new myofibers formed apparent contacts with the native CMs via putative gap junctions that expressed connexin43. Myocardial performance of animals that were successfully transplanted with Mbs was improved.     

Three-Dimensional Scaffold-Free Fusion Culture: the Way to Enhanced Chondrogenesis of in vitro Propagated Human Articular Chondrocytes

Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D) cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-β2) and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-β2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.Key words: in vitro cartilage, 3-D cell culture, fusion culture technique, tissue engineering, cell differentiation, extracellular matrix, immunohistochemistry     

MCF-7 Human Breast Cancer Cells Form Differentiated Microtissues in Scaffold-Free Hydrogels

Three-dimensional (3D) cultures are increasing in use because of their ability to represent in vivo human physiology when compared to monolayer two-dimensional (2D) cultures. When grown in 3D using scaffold-free agarose hydrogels, MCF-7 human breast cancer cells self-organize to form directionally-oriented microtissues that contain a luminal space, reminiscent of the in vivo structure of the mammary gland. When compared to MCF-7 cells cultured in 2D monolayer culture, MCF-7 microtissues exhibit increased mRNA expression of luminal epithelial markers keratin 8 and keratin 19 and decreased expression of basal marker keratin 14 and the mesenchymal marker vimentin. These 3D MCF-7 microtissues remain responsive to estrogens, as demonstrated by induction of known estrogen target mRNAs following exposure to 17β-estradiol. Culture of MCF-7 cells in scaffold-free conditions allows for the formation of more differentiated, estrogen-responsive structures that are a more relevant system for evaluation of estrogenic compounds than traditional 2D models.     

Construction of multi-layered cardiomyocyte sheets using magnetite nanoparticles and magnetic force

Heart tissue engineering requires construction of three-dimensional (3-D) tissues composed of cardiomyocytes (CMs) that are tightly connected to each other. The aim of this study was to construct "scaffold-less" multi-layered 3-D CM sheets using magnetic force-based tissue engineering (Mag-TE) and to evaluate the cell-to-cell functional connections within the CM sheets. Original magnetite cationic liposomes (MCLs) with a positive surface charge (which facilitate adsorption to the target cell surface) were taken up by CMs that were isolated from 2-day-old Wistar rats. When MCLs were added to the medium of CMs at magnetite concentrations of 25, 50, and 100 pg per cell, subsequent measurements showed that 7.2, 13.2, and 27.3 pg of magnetite were taken up per cell, respectively, after 4 h incubation at 37 degrees C. Further, no toxicity was observed after a 24 h incubation period. Using magnetically labeled CMs (magnetite concentration, 100 pg/cell), multi-layered CM sheets were constructed. Immunofluorescent staining of connexin43 demonstrated the presence of gap junctions within the CM sheets that were constructed by Mag-TE. Moreover, electrical connections within the CM sheets constructed by Mag-TE were confirmed using extracellular potential mapping. These results indicate that Mag-TE is a viable methodology for heart tissue engineering.     

Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33

To study PLB (phospholamban) inhibition of the cardiac Ca(2+) pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a. When expressed in insect cells, SER-20G-PLB produced active Ca(2+) uptake, which was stimulated by the anti-PLB antibody, both similar to that which occurred with the control sample co-expressing WT (wild-type)-SERCA2a and WT-PLB. The K(Ca) values of Ca(2+)-dependent ATPase were similar for SER-20G-PLB (0.29±0.02 μM) and for the control sample (0.30±0.02 μM), both greater than 0.17±0.01 μM for WT-SERCA2a expressed alone. Thus SER-20G-PLB retains a fully active Ca(2+) pump, but its apparent Ca(2+) affinity was decreased intrinsically by tethered PLB at a 1:1 molar stoichiometry. Like WT-PLB, SER-20G-PLB ran as both monomers and homo-pentamers on SDS/PAGE. As Ca(2+) concentrations increase from 0 to the micromolar range, the proportion of non-inhibiting pentamers increased from 32% to 52%, suggesting that Ca(2+) activation of the pump completely dissociates the PLB tether from the inhibition site on SERCA2a, with concurrent association of PLB pentamers. Collectively, the regulation of SERCA2a is achieved through the Ca(2+)-dependent equilibria involving PLB association and dissociation from SERCA2a, and assembling and disassembling of SER-20G-PLB pentamers.     

Three-dimensional co-culture facilitates the differentiation of embryonic stem cells into mature cardiomyocytes

The cardiomyocyte (CM) differentiation of embryonic stem cells (ESCs) is routinely cultured as two-dimensional (2D) monolayer, which doesn't mimic in vivo physiological environment and may lead to low differentiated level of ESCs. Here, we develop a novel strategy that enhances CM differentiation of ESCs in collagen matrix three-dimensional (3D) culture combined with indirect cardiac fibroblasts co-culture. ESCs were cultured in hanging drops to form embryoid bodies (EBs) and then applied on collagen matrix. The EBs were indirectly co-cultured with cardiac fibroblasts by the hanging cell culture inserts (PET 1 μm). The molecular expressions and ultrastructural characteristics of ESC-derived CMs (ESCMs) were analyzed by real time RT-PCR, immunocytochemistry, and Transmission Electron Microscopy (TEM). We found that the percentage of beating EBs with cardiac fibroblasts co-culture was significantly higher than that without co-culture after differentiation period of 8 days. Type I collagen used as 3D substrates enhanced the late-stage CM differentiation of ESCs and had effect on ultrastructural mature of ESCMs in late-stage development. The combined effects of 3D and co-culture that mimic in vivo physiological environment further improved the efficiency of CM differentiation from ESCs, resulting in fiber-like structures of cardiac cells with organized sarcomeric structure in ESCMs. This novel 3D co-culture system emphasizes the fact that the ESC differentiation is actively responding to cues from their environment and those cues can drive phenotypic control, which provides a useful in vitro model to investigate CM differentiation of stem cells.     

Spatial Ca(2+) distribution in contracting skeletal and cardiac muscle cells

The spatiotemporal distribution of intracellular Ca(2+) release in contracting skeletal and cardiac muscle cells was defined using a snapshot imaging technique. Calcium imaging was performed on intact skeletal and cardiac muscle cells during contractions induced by an action potential (AP). The sarcomere length of the skeletal and cardiac cells was approximately 2 micrometer. Imaging Rhod-2 fluorescence only during a very brief (7 ns) snapshot of excitation light minimized potential image-blurring artifacts due to movement and/or diffusion. In skeletal muscle cells, the AP triggered a large fast Ca(2+) transient that peaked in less than 3 ms. Distinct subsarcomeric Ca(2+) gradients were evident during the first 4 ms of the skeletal Ca(2+) transient. In cardiac muscle, the AP-triggered Ca(2+) transient was much slower and peaked in approximately 100 ms. In contrast to the skeletal case, there were no detectable subsarcomeric Ca(2+) gradients during the cardiac Ca(2+) transient. Theoretical simulations suggest that the subsarcomeric Ca(2+) gradients seen in skeletal muscle were detectable because of the high speed and synchrony of local Ca(2+) release. Slower asynchronous recruitment of local Ca(2+) release units may account for the absence of detectable subsarcomeric Ca(2+) gradients in cardiac muscle. The speed and synchrony of local Ca(2+) gradients are quite different in AP-activated contracting cardiac and skeletal muscle cells at normal resting sarcomere lengths.     

A smooth muscle Cav1.2 calcium channel splice variant underlies hyperpolarized window current and enhanced state-dependent inhibition by nifedipine

Native smooth muscle L-type Ca(v)1.2 calcium channels have been shown to support a fraction of Ca(2+) currents with a window current that is close to resting potential. The smooth muscle L-type Ca(2+) channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca(v)1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca(v)1.2 channels. Here, we report a smooth muscle L-type Ca(v)1.2 calcium channel splice variant, Ca(v)1.2SM (1/8/9(*)/32/Delta33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca(v)1.2b clone (1/8/9(*)/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca(v)1.2CM clone (1a/8a/Delta9(*)/32/33) that is different from the established Ca(v)1.2a (1a/8a/Delta9(*)/31/33). Importantly, Ca(v)1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca(v)1.2b and cardiac Ca(v)1.2CM channels when currents were recorded in either 5 mM Ba(2+) or 1.8 mM Ca(2+) external solutions. This is the first time that a smooth muscle Ca(v)1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.     

Coculture of Endothelial Cells with Human Pluripotent Stem Cell‐Derived Cardiac Progenitors Reveals a Differentiation Stage‐Specific Enhancement of Cardiomyocyte Maturation

Cardiomyocytes (CMs) generated from human pluripotent stem cells (hPSCs) are immature in their structure and function, limiting their potential in disease modeling, drug screening, and cardiac cellular therapies. Prior studies have demonstrated that coculture of hPSC‐derived CMs with other cardiac cell types, including endothelial cells (ECs), can accelerate CM maturation. To address whether the CM differentiation stage at which ECs are introduced affects CM maturation, the authors coculture hPSC‐derived ECs with hPSC‐derived cardiac progenitor cells (CPCs) and CMs and analyze the molecular and functional attributes of maturation. ECs have a more significant effect on acceleration of maturation when cocultured with CPCs than with CMs. EC coculture with CPCs increases CM size, expression of sarcomere, and ion channel genes and proteins, the presence of intracellular membranous extensions, and chronotropic response compared to monoculture. Maturation is accelerated with an increasing EC:CPC ratio. This study demonstrates that EC incorporation at the CPC stage of CM differentiation expedites CM maturation, leading to cells that may be better suited for in vitro and in vivo applications of hPSC‐derived CMs.     

Label-free enrichment of functional cardiomyocytes using microfluidic deterministic lateral flow displacement

Progress in cardiac cell replacement therapies and tissue engineering critically depends on our ability to isolate functional cardiomyocytes (CMs) from heterogeneous cell mixtures. Label-free enrichment of cardiomyocytes is desirable for future clinical application of cell based products. Taking advantage of the physical properties of CMs, a microfluidic system was designed to separate CMs from neonatal rat heart tissue digest based on size using the principles of deterministic lateral displacement (DLD). For the first time, we demonstrate enrichment of functional CMs up to 91 ± 2.4% directly from the digested heart tissue without any pre-treatment or labeling. Enriched cardiomyocytes remained viable after sorting and formed contractile cardiac patches in 3-dimensional culture. The broad significance of this work lies in demonstrating functional cell enrichment from the primary tissue digest leading directly to the creation of the engineered tissue.     

Proton-sensing Ca2+ binding domains regulate the cardiac Na+/Ca2+ exchanger

The cardiac Na(+)/Ca(2+) exchanger (NCX) regulates cellular [Ca(2+)](i) and plays a central role in health and disease, but its molecular regulation is poorly understood. Here we report on how protons affect this electrogenic transporter by modulating two critically important NCX C(2) regulatory domains, Ca(2+) binding domain-1 (CBD1) and CBD2. The NCX transport rate in intact cardiac ventricular myocytes was measured as a membrane current, I(NCX), whereas [H(+)](i) was varied using an ammonium chloride "rebound" method at constant extracellular pH 7.4. At pH(i) = 7.2 and [Ca(2+)](i) < 120 nM, I(NCX) was less than 4% that of its maximally Ca(2+)-activated value. I(NCX) increases steeply at [Ca(2+)](i) between 130-150 nM with a Hill coefficient (n(H)) of 8.0 ± 0.7 and K(0.5) = 310 ± 5 nM. At pH(i) = 6.87, the threshold of Ca(2+)-dependent activation of I(NCX) was shifted to much higher [Ca(2+)](i) (600-700 nM), and the relationship was similarly steep (n(H) = 8.0±0.8) with K(0.5) = 1042 ± 15 nM. The V(max) of Ca(2+)-dependent activation of I(NCX) was not significantly altered by low pH(i). The Ca(2+) affinities for CBD1 (0.39 ± 0.06 μM) and CBD2 (K(d) = 18.4 ± 6 μM) were exquisitely sensitive to [H(+)], decreasing 1.3-2.3-fold as pH(i) decreased from 7.2 to 6.9. This work reveals for the first time that NCX can be switched off by physiologically relevant intracellular acidification and that this depends on the competitive binding of protons to its C(2) regulatory domains CBD1 and CBD2.