Ca(2+)/calmodulin-activated phosphodiesterase 1A is highly expressed in rabbit cardiac sinoatrial nodal cells and regulates pacemaker function.

Constitutive Ca(2+)/calmodulin (CaM)-activation of adenylyl cyclases (ACs) types 1 and 8 in sinoatrial nodal cells (SANC) generates cAMP within lipid-raft-rich microdomains to initiate cAMP-protein kinase A (PKA) signaling, that regulates basal state rhythmic action potential firing of these cells. Mounting evidence in other cell types points to a balance between Ca(2+)-activated counteracting enzymes, ACs and phosphodiesterases (PDEs) within these cells. We hypothesized that the expression and activity of Ca(2+)/CaM-activated PDE Type 1A is higher in SANC than in other cardiac cell types. We found that PDE1A protein expression was 5-fold higher in sinoatrial nodal tissue than in left ventricle, and its mRNA expression was 12-fold greater in the corresponding isolated cells. PDE1 activity (nimodipine-sensitive) accounted for 39% of the total PDE activity in SANC lysates, compared to only 4% in left ventricular cardiomyocytes (LVC). Additionally, total PDE activity in SANC lysates was lowest (10%) in lipid-raft-rich and highest (76%) in lipid-raft-poor fractions (equilibrium sedimentation on a sucrose density gradient). In intact cells PDE1A immunolabeling was not localized to the cell surface membrane (structured illumination microscopy imaging), but located approximately within about 150nm inside of immunolabeling of hyperpolarization-activated cyclic nucleotide-gated potassium channels (HCN4), which reside within lipid-raft-rich microenvironments. In permeabilized SANC, in which surface membrane ion channels are not functional, nimodipine increased spontaneous SR Ca(2+) cycling. PDE1A mRNA silencing in HL-1 cells increased the spontaneous beating rate, reduced the cAMP, and increased cGMP levels in response to IBMX, a broad spectrum PDE inhibitor (detected via fluorescence resonance energy transfer microscopy). We conclude that signaling via cAMP generated by Ca(2+)/CaM-activated AC in SANC lipid raft domains is limited by cAMP degradation by Ca(2+)/CaM-activated PDE1A in non-lipid raft domains. This suggests that local gradients of [Ca(2+)]-CaM or different AC and PDE1A affinity regulate both cAMP production and its degradation, and this balance determines the intensity of Ca(2+)-AC-cAMP-PKA signaling that drives SANC pacemaker function.

CaMKII-dependent phosphorylation regulates basal cardiac pacemaker function via modulation of local Ca2+ releases.

Spontaneous beating of the heart pacemaker, the sinoatrial node, is generated by sinoatrial node cells (SANC) due to gradual change of the membrane potential called diastolic depolarization (DD). Spontaneous, submembrane local Ca(2+) releases (LCR) from ryanodine receptors (RyR) occur during late DD and activate an inward Na(+)/Ca(2+)exchange current to boost the DD rate and fire an action potential (AP). Here we studied the extent of basal Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activation and the role of basal CaMKII-dependent protein phosphorylation in generation of LCRs and regulation of normal automaticity of intact rabbit SANC. The basal level of activated (autophosphorylated) CaMKII in rabbit SANC surpassed that in ventricular myocytes (VM) by approximately twofold, and this was accompanied by high basal level of protein phosphorylation. Specifically, phosphorylation of phospholamban (PLB) at the CaMKII-dependent Thr(17) site was approximately threefold greater in SANC compared with VM, and RyR phosphorylation at CaMKII-dependent Ser(2815) site was ∼10-fold greater in the SA node, compared with that in ventricle. CaMKII inhibition reduced phosphorylation of PLB and RyR, decreased LCR size, increased LCR periods (time from AP-induced Ca(2+) transient to subsequent LCR), and suppressed spontaneous SANC firing. Graded changes in CaMKII-dependent phosphorylation (indexed by PLB phosphorylation at the Thr(17)site) produced by CaMKII inhibition, ß-AR stimulation or phosphodiesterase inhibition were highly correlated with changes in SR Ca(2+) replenishment times and LCR periods and concomitant changes in spontaneous SANC cycle lengths (R(2) = 0.96). Thus high basal CaMKII activation modifies the phosphorylation state of Ca(2+) cycling proteins PLB, RyR, L-type Ca(2+) channels (and likely others), adjusting LCR period and characteristics, and ultimately regulates both normal and reserve cardiac pacemaker function.

A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells.

Induction of a pluripotent state in somatic cells through nuclear reprogramming has ushered in a new era of regenerative medicine. Heterogeneity and varied differentiation potentials among induced pluripotent stem cell (iPSC) lines are, however, complicating factors that limit their usefulness for disease modeling, drug discovery, and patient therapies. Thus, there is an urgent need to develop nonmutagenic rapid throughput methods capable of distinguishing among putative iPSC lines of variable quality. To address this issue, we have applied a highly specific chemoproteomic targeting strategy for de novo discovery of cell surface N-glycoproteins to increase the knowledge-base of surface exposed proteins and accessible epitopes of pluripotent stem cells. We report the identification of 500 cell surface proteins on four embryonic stem cell and iPSCs lines and demonstrate the biological significance of this resource on mouse fibroblasts containing an oct4-GFP expression cassette that is active in reprogrammed cells. These results together with immunophenotyping, cell sorting, and functional analyses demonstrate that these newly identified surface marker panels are useful for isolating iPSCs from heterogeneous reprogrammed cultures and for isolating functionally distinct stem cell subpopulations.

Reprogramming of cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress.

Our prior study (Tarasov et al., 2022) discovered that numerous adaptive mechanisms emerge in response to cardiac-specific overexpression of adenylyl cyclase type 8 (TGAC8) which included overexpression of a large number of proteins. Here, we conducted an unbiased phosphoproteomics analysis in order to determine the role of altered protein phosphorylation in the adaptive heart performance and protection profile of adult TGAC8 left ventricle (LV) at 3-4 months of age, and integrated the phosphoproteome with transcriptome and proteome. Based on differentially regulated phosphoproteins by genotype, numerous stress-response pathways within reprogrammed TGAC8 LV, including PKA, PI3K, and AMPK signaling pathways, predicted upstream regulators (e.g. PDPK1, PAK1, and PTK2B), and downstream functions (e.g. cell viability, protein quality control), and metabolism were enriched. In addition to PKA, numerous other kinases and phosphatases were hyper-phosphorylated in TGAC8 vs. WT. Hyper-phosphorylated transcriptional factors in TGAC8 were associated with increased mRNA transcription, immune responses, and metabolic pathways. Combination of the phosphoproteome with its proteome and with the previously published TGAC8 transcriptome enabled the elucidation of cardiac performance and adaptive protection profiles coordinately regulated at post-translational modification (PTM) (phosphorylation), translational, and transcriptional levels. Many stress-response signaling pathways, i.e., PI3K/AKT, ERK/MAPK, and ubiquitin labeling, were consistently enriched and activated in the TGAC8 LV at transcriptional, translational, and PTM levels. Thus, reprogramming of the cardiac phosphoproteome, proteome, and transcriptome confers resilience to chronic adenylyl cyclase-driven stress. We identified numerous pathways/function predictions via gene sets, phosphopeptides, and phosphoproteins, which may point to potential novel therapeutic targets to enhance heart adaptivity, maintaining heart performance while avoiding cardiac dysfunction.

Proteomic Landscape and Deduced Functions of the Cardiac 14-3-3 Protein Interactome.

Rationale: The 14-3-3 protein family is known to interact with many proteins in non-cardiac cell types to regulate multiple signaling pathways, particularly those relating to energy and protein homeostasis; and the 14-3-3 network is a therapeutic target of critical metabolic and proteostatic signaling in cancer and neurological diseases. Although the heart is critically sensitive to nutrient and energy alterations, and multiple signaling pathways coordinate to maintain the cardiac cell homeostasis, neither the structure of cardiac 14-3-3 protein interactome, nor potential functional roles of 14-3-3 protein-protein interactions (PPIs) in heart has been explored. Objective: To establish the comprehensive landscape and characterize the functional role of cardiac 14-3-3 PPIs. Methods and Results: We evaluated both RNA expression and protein abundance of 14-3-3 isoforms in mouse heart, followed by co-immunoprecipitation of 14-3-3 proteins and mass spectrometry in left ventricle. We identified 52 proteins comprising the cardiac 14-3-3 interactome. Multiple bioinformatic analyses indicated that more than half of the proteins bound to 14-3-3 are related to mitochondria; and the deduced functions of the mitochondrial 14-3-3 network are to regulate cardiac ATP production via interactions with mitochondrial inner membrane proteins, especially those in mitochondrial complex I. Binding to ribosomal proteins, 14-3-3 proteins likely coordinate protein synthesis and protein quality control. Localizations of 14-3-3 proteins to mitochondria and ribosome were validated via immunofluorescence assays. The deduced function of cardiac 14-3-3 PPIs is to regulate cardiac metabolic homeostasis and proteostasis. Conclusions: Thus, the cardiac 14-3-3 interactome may be a potential therapeutic target in cardiovascular metabolic and proteostatic disease states, as it already is in cancer therapy.

Cell-autonomous lipid-handling defects in Stargardt iPSC-derived retinal pigment epithelium cells.

Stargardt retinopathy is an inherited form of macular degeneration caused by mutations in gene ABCA4 and characterized by the accumulation of lipid-rich deposits in the retinal pigment epithelium (RPE), RPE atrophy, and photoreceptor cell death. Inadequate mechanistic insights into pathophysiological changes occurring in Stargardt RPE have hindered disease treatments. Here, we show that ABCA4 knockout and induced pluripotent stem cell-derived RPE (STGD1-iRPE) from patients with Stargardt differentiate normally but display intracellular lipid and ceramide deposits reminiscent of the disease phenotype. STGD1-iRPE also shows defective photoreceptor outer segment (POS) processing and reduced cathepsin B activity-indicating higher lysosomal pH. Lipid deposits in STGD1-iRPE are lowered by increasing the activity of ABCA1, a lipid transporter, and ABCA4 ortholog. Our work suggests that ABCA4 is involved in POS and lipid handling in RPE cells and provides guidance for ongoing gene therapy approaches to target both RPE and photoreceptor cells for an effective treatment.

A small erythropoietin derived non-hematopoietic peptide reduces cardiac inflammation, attenuates age associated declines in heart function and prolongs healthspan.

Background: Aging is associated with increased levels of reactive oxygen species and inflammation that disrupt proteostasis and mitochondrial function and leads to organism-wide frailty later in life. ARA290 (cibinetide), an 11-aa non-hematopoietic peptide sequence within the cardioprotective domain of erythropoietin, mediates tissue protection by reducing inflammation and fibrosis. Age-associated cardiac inflammation is linked to structural and functional changes in the heart, including mitochondrial dysfunction, impaired proteostasis, hypertrophic cardiac remodeling, and contractile dysfunction. Can ARA290 ameliorate these age-associated cardiac changes and the severity of frailty in advanced age? Methods: We conducted an integrated longitudinal (n = 48) and cross-sectional (n = 144) 15 months randomized controlled trial in which 18-month-old Fischer 344 x Brown Norway rats were randomly assigned to either receive chronic ARA290 treatment or saline. Serial echocardiography, tail blood pressure and body weight were evaluated repeatedly at 4-month intervals. A frailty index was calculated at the final timepoint (33 months of age). Tissues were harvested at 4-month intervals to define inflammatory markers and left ventricular tissue remodeling. Mitochondrial and myocardial cell health was assessed in isolated left ventricular myocytes. Kaplan-Meier survival curves were established. Mixed ANOVA tests and linear mixed regression analysis were employed to determine the effects of age, treatment, and age-treatment interactions. Results: Chronic ARA290 treatment mitigated age-related increases in the cardiac non-myocyte to myocyte ratio, infiltrating leukocytes and monocytes, pro-inflammatory cytokines, total NF-κB, and p-NF-κB. Additionally, ARA290 treatment enhanced cardiomyocyte autophagy flux and reduced cellular accumulation of lipofuscin. The cardiomyocyte mitochondrial permeability transition pore response to oxidant stress was desensitized following chronic ARA290 treatment. Concurrently, ARA290 significantly blunted the age-associated elevation in blood pressure and preserved the LV ejection fraction. Finally, ARA290 preserved body weight and significantly reduced other markers of organism-wide frailty at the end of life. Conclusion: Administration of ARA290 reduces cell and tissue inflammation, mitigates structural and functional changes within the cardiovascular system leading to amelioration of frailty and preserved healthspan.

A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8.

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an increased cAMP-induced cardiac workload (~30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here, we show classical cardiac hypertrophy markers were absent in TGAC8, and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes, and a network of small interstitial proliferative non-cardiac myocytes compared to wild type (WT) littermates; Protein synthesis, proteosome activity, and autophagy were enhanced in TGAC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TGAC8 did not differ from WT. Unbiased omics analyses identified more than 2,000 transcripts and proteins, comprising a broad array of biological processes across multiple cellular compartments, which differed by genotype; compared to WT, in TGAC8 there was a shift from fatty acid oxidation to aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, marked overexpression of AC8 engages complex, coordinate adaptation "circuity" that has evolved in mammalian cells to defend against stress that threatens health or life (elements of which have already been shown to be central to cardiac ischemic pre-conditioning and exercise endurance cardiac conditioning) that may be of biological significance to allow for proper healing in disease states such as infarction or failure of the heart.

A small nonerythropoietic helix B surface peptide based upon erythropoietin structure is cardioprotective against ischemic myocardial damage.

Strong cardioprotective properties of erythropoietin (EPO) reported over the last 10 years have been difficult to translate to clinical applications for ischemic cardioprotection owing to undesirable parallel activation of erythropoiesis and thrombogenesis. A pyroglutamate helix B surface peptide (pHBP), recently engineered to include only a part of the EPO molecule that does not bind to EPO receptor and thus, is not erythropoietic, retains tissue protective properties of EPO. Here we compared the ability of pHBP and EPO to protect cardiac myocytes from oxidative stress in vitro and cardiac tissue from ischemic damage in vivo. HBP, similar to EPO, increased the reactive oxygen species (ROS) threshold for induction of the mitochondrial permeability transition by 40%. In an experimental model of myocardial infarction induced by permanent ligation of a coronary artery in rats, a single bolus injection of 60 µg/kg of pHBP immediately after coronary ligation, similar to EPO, reduced apoptosis in the myocardial area at risk, examined 24 h later, by 80% and inflammation by 34%. Myocardial infarction (MI) measured 24 h after coronary ligation was similarly reduced by 50% in both pHBP- and EPO-treated rats. Two wks after surgery, left ventricular remodeling (ventricular dilation) and functional decline (fall in ejection fraction) assessed by echocardiography were significantly and similarly attenuated in pHBP- and EPO-treated rats, and MI size was reduced by 25%. The effect was retained during the 6-wk follow-up. A single bolus injection of pHBP immediately after coronary ligation was effective in reduction of MI size in a dose as low as 1 µg/kg, but was ineffective at a 60 µg/kg dose if administered 24 h after MI induction. We conclude that pHBP is equally cardioprotective with EPO and deserves further consideration as a safer alternative to rhEPO in the search for therapeutic options to reduce myocardial damage following blockade of the coronary circulation.

Molecular mechanisms of cardiomyocyte aging.

Western societies are rapidly aging, and cardiovascular diseases are the leading cause of death. In fact, age and cardiovascular diseases are positively correlated, and disease syndromes affecting the heart reach epidemic proportions in the very old. Genetic variations and molecular adaptations are the primary contributors to the onset of cardiovascular disease; however, molecular links between age and heart syndromes are complex and involve much more than the passage of time. Changes in CM (cardiomyocyte) structure and function occur with age and precede anatomical and functional changes in the heart. Concomitant with or preceding some of these cellular changes are alterations in gene expression often linked to signalling cascades that may lead to a loss of CMs or reduced function. An understanding of the intrinsic molecular mechanisms underlying these cascading events has been instrumental in forming our current understanding of how CMs adapt with age. In the present review, we describe the molecular mechanisms underlying CM aging and how these changes may contribute to the development of cardiovascular diseases.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3',5'-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2-3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR's characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Linkage of pluripotent stem cell-associated transcripts to regulatory gene networks.

Knowledge of the transcriptional circuitry responsible for pluripotentiality and self-renewal in embryonic stem cells is tantamount to understanding early mammalian development and a prerequisite to determining their therapeutic potential. Various techniques have employed genomics to identify transcripts that were abundant in stem cells, in an attempt to define the molecular basis of 'stemness'. In this study, we have extended traditional genomic analyses to identify cis-elements that might be implicated in the control of embryonic stem cell-restricted gene promoters. The strategy relied on the generation of a problem-specific list from serial analysis of gene expression profiles and subsequent promoter analyses to identify frameworks of multiple cis-elements conserved in space and orientation among genes from the problem-specific list. Subsequent experimental data suggest that 2 novel transcription factors, B-Myb and Maz, predicted from these models, are implicated either in the maintenance of the undifferentiated stem cell state or in early steps of differentiation.

Immunophenotyping of Live Human Pluripotent Stem Cells by Flow Cytometry.

Human pluripotent stem cells (hPSCs) have great potential for use in regenerative medicine and cell replacement therapies; however, prior to clinical application, cultured cell populations need to be screened to ensure the quality of the culture, as well as the capacity of these pluripotent cells to differentiate into desired cell types. Flow cytometry, utilizing antibodies recognizing targets restricted to the hPSC surfaceome, offers an invaluable tool for high-throughput validation of hPSC lines. Here we describe the immunophenotyping of live human embryonic stem cell (hESC, H9) and human induced pluripotent stem cell (hiPSC, KB3) lines by flow cytometry using a panel of antibodies identified as either stem cell reference markers (CD90, EpCam) or reported as being prevalent or restricted (c-Kit, HPI-1, Integrin α6, Semaphorin-6A) to these cells. The protocols described here with hPSCs are also applicable to differentiated hPSC progeny and should be instrumental in the immunophenotyping and isolation of well-defined homogeneous cell populations useful in regenerative medicine.

Heterogeneity of calcium clock functions in dormant, dysrhythmically and rhythmically firing single pacemaker cells isolated from SA node.

Current understanding of how cardiac pacemaker cells operate is based mainly on studies in isolated single sinoatrial node cells (SANC), specifically those that rhythmically fire action potentials similar to the in vivo behavior of the intact sinoatrial node. However, only a small fraction of SANC exhibit rhythmic firing after isolation. Other SANC behaviors have not been studied. Here, for the first time, we studied all single cells isolated from the sinoatrial node of the guinea pig, including traditionally studied rhythmically firing cells ('rhythmic SANC'), dysrhythmically firing cells ('dysrhythmic SANC') and cells without any apparent spontaneous firing activity ('dormant SANC'). Action potential-induced cytosolic Ca2+ transients and spontaneous local Ca2+ releases (LCRs) were measured with a 2D camera. LCRs were present not only in rhythmically firing SANC, but also in dormant and dysrhythmic SANC. While rhythmic SANC were characterized by large LCRs synchronized in space and time towards late diastole, dysrhythmic and dormant SANC exhibited smaller LCRs that appeared stochastically and were widely distributed in time. ß-adrenergic receptor (ßAR) stimulation increased LCR size and synchronized LCR occurrences in all dysrhythmic and a third of dormant cells (25 of 75 cells tested). In response to ßAR stimulation, these dormant SANC developed automaticity, and LCRs became coupled to spontaneous action potential-induced cytosolic Ca2+ transients. Conversely, dormant SANC that did not develop automaticity showed no significant change in average LCR characteristics. The majority of dysrhythmic cells became rhythmic in response to ßAR stimulation, with the rate of action potential-induced cytosolic Ca2+ transients substantially increasing. In summary, isolated SANC can be broadly categorized into three major populations: dormant, dysrhythmic, and rhythmic. We interpret our results based on simulations of a numerical model of SANC operating as a coupled-clock system. On this basis, the two previously unstudied dysrhythmic and dormant cell populations have intrinsically partially or completely uncoupled clocks. Such cells can be recruited to fire rhythmically in response to ßAR stimulation via increased rhythmic LCR activity and ameliorated coupling between the Ca2+ and membrane clocks.

Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca2+ transients and contraction in cardiomyocytes derived from embryonic stem cells.

In adult myocardium, excitation-contraction coupling is critically regulated by sarcoplasmic reticulum (SR) Ca2+ release via type 2 ryanodine receptor (RyR2), but generally, it is believed that SR-function is rudimentary in the fetal heart and in embryonic stem (ES) cell-derived cardiomyocytes (ESCMs), a possible source for cell replacement therapies. This study used wild-type (RyR2+/+) and RyR2 null (RyR2-/-) ESCMs as an in vitro model of cardiomyogenesis, together with pharmacological approaches and expression profiles of genes relevant for SR function, to elucidate the functional importance of RyR2 and SR on the regulation of Ca2+ transients and contraction during early cardiomyocyte development. During differentiation of RyR2+/+ ESCMs, SR function developed progressively with increased basal cytosolic free Ca2+ concentration ([Ca2+]i), enhanced frequency and amplitude, and decreased duration of Ca2+ transients that were inhibited by ryanodine and thapsigargin. These functional traits correlated with SR Ca2+ load and the expression of RyR2, SERCA2a, and phospholamban. RyR2-/- ESCMs, comparatively, demonstrated a significantly prolonged time-to-peak and reduced frequency of Ca2+ transients and contractions. Beta-adrenergic stimulation of RyR2+/+ ESCMs increased the frequency and amplitude of Ca2+ transients with differentiation but was much weaker in RyR2-/- ESCMs. We conclude that functional SR and control of RyR2-mediated SR Ca2+ release directly contribute to the spontaneous and beta-adrenergic receptor-stimulated contraction of ESCMs, even at very immature stages of development.

Ascorbic acid promotes cardiomyogenesis through SMAD1 signaling in differentiating mouse embryonic stem cells.

Numerous groups have documented that Ascorbic Acid (AA) promotes cardiomyocyte differentiation from both mouse and human ESCs and iPSCs. AA is now considered indispensable for the routine production of hPSC-cardiomyocytes (CMs) using defined media; however, the mechanisms involved with the inductive process are poorly understood. Using a genetically modified mouse embryonic stem cell (mESC) line containing a dsRED transgene driven by the cardiac-restricted portion of the ncx1 promoter, we show that AA promoted differentiation of mESCs to CMs in a dose- and time-dependent manner. Treatment of mPSCs with AA did not modulate total SMAD content; however, the phosphorylated/active forms of SMAD2 and SMAD1/5/8 were significantly elevated. Co-administration of the SMAD2/3 activator Activin A with AA had no significant effect, but the addition of the nodal co-receptor TDGF1 (Cripto) antagonized AA's cardiomyogenic-promoting ability. AA could also reverse some of the inhibitory effects on cardiomyogenesis of ALK/SMAD2 inhibition by SB431542, a TGFß pathway inhibitor. Treatment with BMP2 and AA strongly amplified the positive cardiomyogenic effects of SMAD1/5/8 in a dose-dependent manner. AA could not, however, rescue dorsomorphin-mediated inhibition of ALK/SMAD1 activity. Using an inducible model system, we found that SMAD1, but not SMAD2, was essential for AA to promote the formation of TNNT2+-CMs. These data firmly demonstrate that BMP receptor-activated SMADs, preferential to TGFß receptor-activated SMADs, are necessary to promote AA stimulated cardiomyogenesis. AA-enhanced cardiomyogenesis thus relies on the ability of AA to modulate the ratio of SMAD signaling among the TGFß-superfamily receptor signaling pathways.

Mammalian γ2 AMPK regulates intrinsic heart rate.

AMPK is a conserved serine/threonine kinase whose activity maintains cellular energy homeostasis. Eukaryotic AMPK exists as αßγ complexes, whose regulatory γ subunit confers energy sensor function by binding adenine nucleotides. Humans bearing activating mutations in the γ2 subunit exhibit a phenotype including unexplained slowing of heart rate (bradycardia). Here, we show that γ2 AMPK activation downregulates fundamental sinoatrial cell pacemaker mechanisms to lower heart rate, including sarcolemmal hyperpolarization-activated current (I f) and ryanodine receptor-derived diastolic local subsarcolemmal Ca2+ release. In contrast, loss of γ2 AMPK induces a reciprocal phenotype of increased heart rate, and prevents the adaptive intrinsic bradycardia of endurance training. Our results reveal that in mammals, for which heart rate is a key determinant of cardiac energy demand, AMPK functions in an organ-specific manner to maintain cardiac energy homeostasis and determines cardiac physiological adaptation to exercise by modulating intrinsic sinoatrial cell behavior.

A human pluripotent stem cell surface N-glycoproteome resource reveals markers, extracellular epitopes, and drug targets.

Detailed knowledge of cell-surface proteins for isolating well-defined populations of human pluripotent stem cells (hPSCs) would significantly enhance their characterization and translational potential. Through a chemoproteomic approach, we developed a cell-surface proteome inventory containing 496 N-linked glycoproteins on human embryonic (hESCs) and induced PSCs (hiPSCs). Against a backdrop of human fibroblasts and 50 other cell types, >100 surface proteins of interest for hPSCs were revealed. The >30 positive and negative markers verified here by orthogonal approaches provide experimental justification for the rational selection of pluripotency and lineage markers, epitopes for cell isolation, and reagents for the characterization of putative hiPSC lines. Comparative differences between the chemoproteomic-defined surfaceome and the transcriptome-predicted surfaceome directly led to the discovery that STF-31, a reported GLUT-1 inhibitor, is toxic to hPSCs and efficient for selective elimination of hPSCs from mixed cultures.

The B-MYB transcriptional network guides cell cycle progression and fate decisions to sustain self-renewal and the identity of pluripotent stem cells.

Embryonic stem cells (ESCs) are pluripotent and have unlimited self-renewal capacity. Although pluripotency and differentiation have been examined extensively, the mechanisms responsible for self-renewal are poorly understood and are believed to involve an unusual cell cycle, epigenetic regulators and pluripotency-promoting transcription factors. Here we show that B-MYB, a cell cycle regulated phosphoprotein and transcription factor critical to the formation of inner cell mass, is central to the transcriptional and co-regulatory networks that sustain normal cell cycle progression and self-renewal properties of ESCs. Phenotypically, B-MYB is robustly expressed in ESCs and induced pluripotent stem cells (iPSCs), and it is present predominantly in a hypo-phosphorylated state. Knockdown of B-MYB results in functional cell cycle abnormalities that involve S, G2 and M phases, and reduced expression of critical cell cycle regulators like ccnb1 and plk1. By conducting gene expression profiling on control and B-MYB deficient cells, ChIP-chip experiments, and integrative computational analyses, we unraveled a highly complex B-MYB-mediated transcriptional network that guides ESC self-renewal. The network encompasses critical regulators of all cell cycle phases and epigenetic regulators, pluripotency transcription factors, and differentiation determinants. B-MYB along with E2F1 and c-MYC preferentially co-regulate cell cycle target genes. B-MYB also co-targets genes regulated by OCT4, SOX2 and NANOG that are significantly associated with stem cell differentiation, embryonic development, and epigenetic control. Moreover, loss of B-MYB leads to a breakdown of the transcriptional hierarchy present in ESCs. These results coupled with functional studies demonstrate that B-MYB not only controls and accelerates cell cycle progression in ESCs it contributes to fate decisions and maintenance of pluripotent stem cell identity.

B-MYB is essential for normal cell cycle progression and chromosomal stability of embryonic stem cells.

BACKGROUND: The transcription factor B-Myb is present in all proliferating cells, and in mice engineered to remove this gene, embryos die in utero just after implantation due to inner cell mass defects. This lethal phenotype has generally been attributed to a proliferation defect in the cell cycle phase of G1. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we show that the major cell cycle defect in murine embryonic stem (mES) cells occurs in G2/M. Specifically, knockdown of B-Myb by short-hairpin RNAs results in delayed transit through G2/M, severe mitotic spindle and centrosome defects, and in polyploidy. Moreover, many euploid mES cells that are transiently deficient in B-Myb become aneuploid and can no longer be considered viable. Knockdown of B-Myb in mES cells also decreases Oct4 RNA and protein abundance, while over-expression of B-MYB modestly up-regulates pou5f1 gene expression. The coordinated changes in B-Myb and Oct4 expression are due, at least partly, to the ability of B-Myb to directly modulate pou5f1 gene promoter activity in vitro. Ultimately, the loss of B-Myb and associated loss of Oct4 lead to an increase in early markers of differentiation prior to the activation of caspase-mediated programmed cell death. CONCLUSIONS/SIGNIFICANCE: Appropriate B-Myb expression is critical to the maintenance of chromosomally stable and pluripotent ES cells, but its absence promotes chromosomal instability that results in either aneuploidy or differentiation-associated cell death.