Search

First published April 11, 2008; doi:10.1152/ajpheart.91443.2007.--Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-[alpha] by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-[alpha] at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes. mitochondrial permeability transition-reactive oxygen species threshold; cardiac myocyte viability; cardiac fibroblasts; hypoxia; fibroblast-conditioned medium; fibroblast proliferation; apoptosis; necrosis; tumor necrosis factor-[alpha]

Upper aerodigestive squamous cell carcinoma (UASCC) is a common and aggressive malignancy with few effective therapeutic options. Here, we investigate amino acid metabolism in this cancer, surprisingly noting that UASCC exhibits the highest methionine level across all human cancers, driven by its transporter LAT1. We show that LAT1 is also expressed at the highest level in UASCC, transcriptionally activated by UASCC-specific promoter and enhancers, which are directly coregulated by SCC master regulators TP63/KLF5/SREBF1. Unexpectedly, unbiased bioinformatic screen identifies EZH2 as the most significant target downstream of the LAT1-methionine pathway, directly linking methionine metabolism to epigenomic reprogramming. Importantly, this cascade is indispensable for the survival and proliferation of UASCC patient-derived tumor organoids. In addition, LAT1 expression is closely associated with cellular sensitivity to inhibition of the LAT1-methionine-EZH2 axis. Notably, this unique LAT1-methionine-EZH2 cascade can be targeted effectively by either pharmacological approaches or dietary intervention in vivo. In summary, this work maps a unique mechanistic cross talk between epigenomic reprogramming with methionine metabolism, establishes its biological significance in the biology of UASCC, and identifies a unique tumor-specific vulnerability which can be exploited both pharmacologically and dietarily., Competing Interests: Competing interests statement:The authors declare no competing interest.

Squamous cell carcinomas (SCCs) are common and aggressive malignancies. Immune check point blockade (ICB) therapy using PD-1/PD-L1 antibodies has been approved in several types of advanced SCCs. However, low response rate and treatment resistance are common. Improving the efficacy of ICB therapy requires better understanding of the mechanism of immune evasion. Here, we identify that the SCC-master transcription factor TP63 suppresses interferon-γ (IFNγ) signaling. TP63 inhibition leads to increased CD8 + T cell infiltration and heighten tumor killing in in vivo syngeneic mouse model and ex vivo co-culture system, respectively. Moreover, expression of TP63 is negatively correlated with CD8 + T cell infiltration and activation in patients with SCC. Silencing of TP63 enhances the anti-tumor efficacy of PD-1 blockade by promoting CD8 + T cell infiltration and functionality. Mechanistically, TP63 and STAT1 mutually suppress each other to regulate the IFNγ signaling by co-occupying and co-regulating their own promoters and enhancers. Together, our findings elucidate a tumor-extrinsic function of TP63 in promoting immune evasion of SCC cells. Over-expression of TP63 may serve as a biomarker predicting the outcome of SCC patients treated with ICB therapy, and targeting TP63/STAT/IFNγ axis may enhance the efficacy of ICB therapy for this deadly cancer., (© 2024. The Author(s).)

Unlike most cancer types, the incidence of esophageal adenocarcinoma (EAC) has rapidly escalated in the western world over recent decades. Using whole genome bisulfite sequencing (WGBS), we identify the transcription factor (TF) FOXM1 as an important epigenetic regulator of EAC. FOXM1 plays a critical role in cellular proliferation and tumor growth in EAC patient-derived organoids and cell line models. We identify ERBB2 as an upstream regulator of the expression and transcriptional activity of FOXM1. Unexpectedly, gene set enrichment analysis (GSEA) unbiased screen reveals a prominent anti-correlation between FOXM1 and immune response pathways. Indeed, syngeneic mouse models show that FOXM1 inhibits the infiltration of CD8 + T cells into the tumor microenvironment. Consistently, FOXM1 suppresses CD8 + T cell chemotaxis in vitro and antigen-dependent CD8 + T cell killing. This study characterizes FOXM1 as a significant EAC-promoting TF and elucidates its novel function in regulating anti-tumor immune response., (© 2024. The Author(s).)

ARID1A, a member of the chromatin remodeling SWI/SNF complex, is frequently lost in many cancer types, including esophageal adenocarcinoma (EAC). Here, we study the impact of ARID1A deficiency on the anti-tumor immune response in EAC. We find that EAC tumors with ARID1A mutations are associated with enhanced tumor-infiltrating CD8 + T cell levels. ARID1A-deficient EAC cells exhibit heightened IFN response signaling and promote CD8 + T cell recruitment and cytolytic activity. Moreover, we demonstrate that ARID1A regulates fatty acid metabolism genes in EAC, showing that fatty acid metabolism could also regulate CD8 + T cell recruitment and CD8 + T cell cytolytic activity in EAC cells. These results suggest that ARID1A deficiency shapes both tumor immunity and lipid metabolism in EAC, with significant implications for immune checkpoint blockade therapy in EAC.

Background: As one of the most common malignancies, esophageal cancer has two subtypes, squamous cell carcinoma and adenocarcinoma, arising from distinct cells-of-origin. Distinguishing cell-type-specific molecular features from cancer-specific characteristics is challenging., Results: We analyze whole-genome bisulfite sequencing data on 45 esophageal tumor and nonmalignant samples from both subtypes. We develop a novel sequence-aware method to identify large partially methylated domains (PMDs), revealing profound heterogeneity at both methylation level and genomic distribution of PMDs across tumor samples. We identify subtype-specific PMDs that are associated with repressive transcription, chromatin B compartments and high somatic mutation rate. While genomic locations of these PMDs are pre-established in normal cells, the degree of loss is significantly higher in tumors. We find that cell-type-specific deposition of H3K36me2 may underlie genomic distribution of PMDs. At a smaller genomic scale, both cell-type- and cancer-specific differentially methylated regions (DMRs) are identified for each subtype. Using binding motif analysis within these DMRs, we show that a cell-type-specific transcription factor HNF4A maintains the binding sites that it generates in normal cells, while establishing new binding sites cooperatively with novel partners such as FOSL1 in esophageal adenocarcinoma. Finally, leveraging pan-tissue single-cell and pan-cancer epigenomic datasets, we demonstrate that a substantial fraction of cell-type-specific PMDs and DMRs identified here in esophageal cancer are actually markers that co-occur in other cancers originating from related cell types., Conclusions: These findings advance our understanding of DNA methylation dynamics at various genomic scales in normal and malignant states, providing novel mechanistic insights into cell-type- and cancer-specific epigenetic regulations., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Adult (3 month) mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TG AC8 ) 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 TG AC8 , and that total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TG AC8 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 TG AC8 vs WT, and Nrf-2, Hsp90α, and ACC2 protein levels were increased. Despite increased energy demands in vivo LV ATP and phosphocreatine levels in TG AC8 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 TG AC8 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., Competing Interests: KT, KC, DR, AL, IA, MP, AS, JZ, MW, YL, JQ, MB, MJ, YT, BZ, RT, VK, MR, JL, RB, Rd, SJ, GK, AG, DY, MA, LA, CM, WO, CC, SC, NP, TH, KP, RW, LF, SS, EL No competing interests declared

Inactivation of the tumor suppressor genes tumor protein p53 ( TP53 ) and cyclin-dependent kinase inhibitor 2A ( CDKN2A ) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated TP53 and CDKN2A knockout ( TP53/CDKN2A KO ) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of TP53/CDKN2A KO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

Tumor organoid modeling has been recognized as a state-of-the-art system for in vitro research on cancer biology and precision oncology. Organoid culture technologies offer distinctive advantages, including faithful maintenance of physiological and pathological characteristics of human disease, self-organization into three-dimensional multicellular structures, and preservation of genomic and epigenomic landscapes of the originating tumor. These features effectively position organoid modeling between traditional cell line cultures in two dimensions and in vivo animal models as a valid, versatile, and robust system for cancer research. Here, we review recent advances in genomic and epigenomic characterization of tumor organoids and the novel findings obtained, highlight significant progressions achieved in organoid modeling of gene-drug interactions and genotype-phenotype associations, and offer perspectives on future opportunities for organoid modeling in basic and clinical cancer research.

Exposure to high levels of ionizing γ radiation leads to irreversible DNA damage and cell death. Here, we establish that exogenous application of electric stimulation enables cellular plasticity and the re-establishment of stem cell activity in tissues damaged by ionizing radiation. We show that subthreshold direct current stimulation (DCS) rapidly restores pluripotent stem cell populations previously eliminated by lethally γ-irradiated tissues of the planarian flatworm Schmidtea mediterranea. Our findings reveal that DCS enhances DNA repair, transcriptional activity, and cell cycle entry in post-mitotic cells. These responses involve rapid increases in cytosolic Ca2+ concentration through the activation of L-type Cav channels and intracellular Ca2+ stores, leading to the activation of immediate early genes and ectopic expression of stem cell markers in post-mitotic cells. Overall, we show the potential of electric current stimulation to reverse the damaging effects of high-dose γ radiation in adult tissues. Furthermore, our results provide mechanistic insights describing how electric stimulation effectively translates into molecular responses capable of regulating fundamental cellular functions without the need for genetic or pharmacological intervention., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Background: The use of direct current electric stimulation (DCS) is an effective strategy to treat disease and enhance body functionality. Thus, treatment with DCS is an attractive biomedical alternative, but the molecular underpinnings remain mostly unknown. The lack of experimental models to dissect the effects of DCS from molecular to organismal levels is an important caveat. Here, we introduce the planarian flatworm Schmidtea mediterranea as a tractable organism for in vivo studies of DCS. We developed an experimental method that facilitates the application of direct current electrical stimulation to the whole planarian body (pDCS). Materials and Methods: Planarian immobilization was achieved by combining treatment with anesthesia, agar embedding, and low temperature via a dedicated thermoelectric cooling unit. Electric currents for pDCS were delivered using pulled glass microelectrodes. The electric potential was supplied through a constant voltage power supply. pDCS was administered up to six hours, and behavioral and molecular effects were measured by using video recordings, immunohistochemistry, and gene expression analysis. Results: The behavioral immobilization effects are reversible, and pDCS resulted in a redistribution of mitotic cells along the mediolateral axis of the planarian body. The pDCS effects were dependent on the polarity of the electric field, which led to either increase in reductions in mitotic densities associated with the time of pDCS. The changes in mitotic cells were consistent with apparent redistribution in gene expression of the stem cell marker smedwi-1 . Conclusion: The immobilization technique presented in this work facilitates studies aimed at dissecting the effects of exogenous electric stimulation in the adult body. Treatment with DCS can be administered for varying times, and the consequences evaluated at different levels, including animal behavior, cellular and transcriptional changes. Indeed, treatment with pDCS can alter cellular and transcriptional parameters depending on the polarity of the electric field and duration of the exposure., Competing Interests: No competing financial interests exist., (Copyright 2021, Mary Ann Liebert, Inc., publishers.)

Candida albicans is one of the most common fungal pathogens of humans. Prior work introduced the planarian Schmidtea mediterranea as a new model system to study the host response to fungal infection at the organismal level. In the current study, we analyzed host-pathogen changes that occurred in situ during early infection with C. albicans . We found that the transcription factor Bcr1 and its downstream adhesin Als3 are required for C. albicans to adhere to and colonize the planarian epithelial surface, and that adherence of C. albicans triggers a multi-system host response that is mediated by the Dectin signaling pathway. This infection response is characterized by two peaks of stem cell divisions and transcriptional changes in differentiated tissues including the nervous and the excretory systems. This response bears some resemblance to a wound-like response to physical injury; however, it takes place without visible tissue damage and it engages a distinct set of progenitor cells. Overall, we identified two C. albicans proteins that mediate epithelial infection of planarians and a comprehensive host response facilitated by diverse tissues to effectively clear the infection., Competing Interests: CN is a cofounder of BioSynesis, Inc., a company developing inhibitors and diagnostics of biofilm infections. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Maciel, Valle Arevalo, Ziman, Nobile and Oviedo.)

In the planarian field, two techniques are mostly used for protein detection: immunohistochemistry (IHC) and western blotting. While IHC is great for visualizing the spatial distribution of proteins in whole organisms, it has limitations in antibody availability and issues related to nonspecific expression. The use of western blotting can circumvent nonspecific expression, providing a dependable way to quantify proteins of interest. Here, we present a standardized, easily reproducible protocol with details on protein extractions of whole planarians and western blotting. For complete details on the use and execution of this protocol, please refer to Ziman et al. (2020a)., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Tissue homeostasis relies on the timely renewal of cells that have been damaged or have surpassed their biological age. Nonetheless, the underlying molecular mechanism coordinating tissue renewal is unknown. The planarian Schmidtea mediterranea harbors a large population of stem cells that continuously divide to support the restoration of tissues throughout the body. Here, we identify that TNF Receptor Associated Factors (TRAFs) play critical roles in cellular survival during tissue repair in S . mediterranea . Disruption with RNA-interference of TRAF signaling results in rapid morphological defects and lethality within 2 weeks. The TRAF phenotype is accompanied by an increased number of mitoses and cell death. Our results also reveal TRAF signaling is required for proper regeneration of the nervous system. Taken together, we find functional conservation of TRAF-like proteins in S . mediterranea as they act as crucial regulators of cellular survival during tissue homeostasis and regeneration., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Nutrient availability upon feeding leads to an increase in body size in the planarian Schmidtea mediterranea However, it remains unclear how food consumption integrates with cell division at the organismal level. Here, we show that the NAD-dependent protein deacetylases sirtuins are evolutionarily conserved in planarians, and specifically demonstrate that the homolog of human sirtuin-1 (SIRT1) (encoded by Smed-Sirt-1 ), regulates organismal growth by impairing both feeding behavior and intestinal morphology. Disruption of Smed-Sirt-1 with RNAi or pharmacological inhibition of Sirtuin-1 leads to reduced animal growth. Conversely, enhancement of Sirtuin-1 activity with resveratrol accelerates growth. Differences in growth rates were associated with changes in the amount of time taken to locate food and overall food consumption. Furthermore, Smed-Sirt-1(RNAi) animals displayed reduced cell death and increased stem cell proliferation accompanied by impaired expression of intestinal lineage progenitors and reduced branching of the gut. Taken together, our findings indicate that Sirtuin-1 is a crucial metabolic hub capable of controlling animal behavior, tissue renewal and morphogenesis of the adult intestine., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

Background: Spontaneous firing of sinoatrial node cells (SANCs) is regulated by cAMP-mediated, PKA (protein kinase A)-dependent (cAMP/PKA) local subsarcolemmal Ca 2+ releases (LCRs) from RyRs (ryanodine receptors). LCRs occur during diastolic depolarization and activate an inward Na + /Ca 2+ exchange current that accelerates diastolic depolarization rate prompting the next action potential. PDEs (phosphodiesterases) regulate cAMP-mediated signaling; PDE3/PDE4 represent major PDE activities in SANC, but how they modulate LCRs and basal spontaneous SANC firing remains unknown., Methods: Real-time polymerase chain reaction, Western blot, immunostaining, cellular perforated patch clamping, and confocal microscopy were used to elucidate mechanisms of PDE-dependent regulation of cardiac pacemaking., Results: PDE3A, PDE4B, and PDE4D were the major PDE subtypes expressed in rabbit SANC, and PDE3A was colocalized with α-actinin, PDE4D, SERCA (sarcoplasmic reticulum Ca 2+ ATP-ase), and PLB (phospholamban) in Z-lines. Inhibition of PDE3 (cilostamide) or PDE4 (rolipram) alone increased spontaneous SANC firing by ≈20% ( P <0.05) and ≈5% ( P >0.05), respectively, but concurrent PDE3+PDE4 inhibition increased spontaneous firing by ≈45% ( P <0.01), indicating synergistic effect. Inhibition of PDE3 or PDE4 alone increased L-type Ca 2+ current (I Ca,L ) by ≈60% ( P <0.01) or ≈5% ( P >0.05), respectively, and PLB phosphorylation by ≈20% ( P >0.05) each, but dual PDE3+PDE4 inhibition increased I Ca,L by ≈100% ( P <0.01) and PLB phosphorylation by ≈110% ( P <0.05). Dual PDE3+PDE4 inhibition increased the LCR number and size ( P <0.01) and reduced the SR (sarcoplasmic reticulum) Ca 2+ refilling time ( P <0.01) and the LCR period (time from action potential-induced Ca 2+ transient to subsequent LCR; P <0.01), leading to decrease in spontaneous SANC cycle length ( P <0.01). When RyRs were disabled by ryanodine and LCRs ceased, dual PDE3+PDE4 inhibition failed to increase spontaneous SANC firing., Conclusions: Basal cardiac pacemaker function is regulated by concurrent PDE3+PDE4 activation which operates in a synergistic manner via decrease in cAMP/PKA phosphorylation, suppression of LCR parameters, and prolongation of the LCR period and spontaneous SANC cycle length., (© 2018 American Heart Association, Inc.)

Cardiac pacemaker cells, including cells of the sinoatrial node, are heterogeneous in size, morphology, and electrophysiological characteristics. The exact extent to which these cells differ electrophysiologically is unclear yet is critical to understanding their functioning. We examined major ionic currents in individual intercaval pacemaker cells (IPCs) sampled from the paracristal, intercaval region (including the sinoatrial node) that were spontaneously beating after enzymatic isolation from rabbit hearts. The beating rate was measured at baseline and after inhibition of the Ca 2+ pump with cyclopiazonic acid. Thereafter, in each cell, we consecutively measured the density of funny current ( I f ), delayed rectifier K + current ( I K ) (a surrogate of repolarization capacity), and L-type Ca 2+ current ( I Ca,L ) using whole cell patch clamp . The ionic current densities varied to a greater extent than previously appreciated, with some IPCs demonstrating very small or zero I f . The density of none of the currents was correlated with cell size, while I Ca,L and I f densities were related to baseline beating rates. I f density was correlated with I K density but not with that of I Ca,L . Inhibition of Ca 2+ cycling had a greater beating rate slowing effect in IPCs with lower I f densities. Our numerical model simulation indicated that 1) IPCs with small (or zero) I f or small I Ca,L can operate via a major contribution of Ca 2+ clock, 2) I f -Ca 2+ -clock interplay could be important for robust pacemaking function, and 3) coupled I f - I K function could regulate maximum diastolic potential. Thus, we have demonstrated marked electrophysiological heterogeneity of IPCs. This heterogeneity is manifested in basal beating rate and response to interference of Ca 2+ cycling, which is linked to I f . NEW & NOTEWORTHY In the present study, a hitherto unrecognized range of heterogeneity of ion currents in pacemaker cells from the intercaval region is demonstrated. Relationships between basal beating rate and L-type Ca 2+ current and funny current ( I f ) density are uncovered, along with a positive relationship between I f and delayed rectifier K + current. Links are shown between the response to Ca 2+ cycling blockade and I f density.

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., (Copyright © 2016. Published by Elsevier Ltd.)

A recurring theme of a host of gerontologic studies conducted in either experimental animals or in humans is related to documenting the functional decline with age. We hypothesize that elevated circulating levels of a powerful antiangiogenic peptide, endostatin, represent one of the potent systemic causes for multiorgan microvascular rarefaction and functional decline due to fibrosis. It is possible that during the life span of an organism there is an accumulation of dormant transformed cells producing antiangiogenic substances (endostatin) that maintain the dormancy of such scattered malignant cells. The proof of this postulate cannot be obtained by physically documenting these scattered cells, and it rests exclusively on the detection of sequelae of shifted pro- and antiangiogenic balance toward the latter. Here we compared circulating levels of endostatin in young and aging mice of two different strains and showed that endostatin levels are elevated in the latter. Renal expression of endostatin increased ~5.6-fold in aging animals. This was associated with microvascular rarefaction and progressive tubulointerstitial fibrosis. In parallel, the levels of sirtuins 1 and 3 were significantly suppressed in aging mice in conjunction with the expression of markers of senescence. Treating young mice with endostatin for 28 days showed delayed recovery of circulation after femoral artery ligation and reduced patency of renal microvasculature but no fibrosis. In conclusion, the findings are consistent with the hypothesis on elevation of endostatin levels and parallel microvascular rarefaction and induction of renal fibrosis in aging mice., (Copyright © 2014 the American Physiological Society.)

Recent perspectives on sinoatrial nodal cell (SANC)(*) function indicate that spontaneous sarcoplasmic reticulum (SR) Ca(2+) cycling, i.e. an intracellular "Ca(2+) clock," driven by cAMP-mediated, PKA-dependent phosphorylation, interacts with an ensemble of surface membrane electrogenic molecules ("surface membrane clock") to drive SANC normal automaticity. The role of AC-cAMP-PKA-Ca(2+) signaling cascade in mouse, the species most often utilized for genetic manipulations, however, has not been systematically tested. Here we show that Ca(2+) cycling proteins (e.g. RyR2, NCX1, and SERCA2) are abundantly expressed in mouse SAN and that spontaneous, rhythmic SR generated local Ca(2+) releases (LCRs) occur in skinned mouse SANC, clamped at constant physiologic [Ca(2+)]. Mouse SANC also exhibits a high basal level of phospholamban (PLB) phosphorylation at the PKA-dependent site, Serine16. Inhibition of intrinsic PKA activity or inhibition of PDE in SANC, respectively: reduces or increases PLB phosphorylation, and markedly prolongs or reduces the LCR period; and markedly reduces or accelerates SAN spontaneous firing rate. Additionally, the increase in AP firing rate by PKA-dependent phosphorylation by β-adrenergic receptor (β-AR) stimulation requires normal intracellular Ca(2+) cycling, because the β-AR chronotropic effect is markedly blunted when SR Ca(2+) cycling is disrupted. Thus, AC-cAMP-PKA-Ca(2+) signaling cascade is a major mechanism of normal automaticity in mouse SANC., (Published by Elsevier Ltd.)

Mice over-expressing the alpha(1)&#95;subunit (pore) of the L-type Ca2+ channel (alpha(1C)TG) by 4 months (mo) of age exhibit an enlarged heart, hypertrophied myocytes, increased Ca2+ current and Ca2+ transient amplitude, but a normal SR Ca2+ load. With advancing age (8-11 mo), some mice demonstrate advanced hypertrophy but are not in congestive heart failure (NFTG),while others evolve to frank dilated congestive heart failure (FTG). We demonstrate that older NFTG myocytes exhibit a hypercontractile state over a wide range of stimulation frequencies, but maintain a normal SR Ca2+ load compared to age matched non-transgenic (NTG) myocytes. However, at high stimulation rates (2-4 Hz) signs of diastolic contractile failure appear in NFTG cells. The evolution of frank congestive failure in FTG is accompanied by a further increase in heart mass and myocyte size, and phospholamban and ryanodine receptor protein levels and phosphorylation become reduced. In FTG, the SR Ca2+ load increases and Ca2+ release following excitation, increases further. An enhanced NCX function in FTG, as reflected by an accelerated relaxation of the caffeine-induced Ca2+ transient, is insufficient to maintain a normal diastolic Ca2+ during high rates of stimulation. Although a high SR Ca2+ release following excitation is maintained, the hypercontractile state is not maintained at high rates of stimulation, and signs of both systolic and diastolic contractile failure appear. Thus, the dilated cardiomyopathy that evolves in this mouse model exhibits signs of both systolic and diastolic failure, but not a deficient SR Ca2+ loading or release, as occurs in some other cardiomyopathic models.

Pleiotrophin is a development-regulated cytokine and growth factor that can promote angiogenesis, cell proliferation, or differentiation, and it has been reported to have neovasculogenic effects in damaged heart. Developmentally, it is prominently expressed in fetal and neonatal hearts, but it is minimally expressed in normal adult heart. Conversely, we show in a rat model of myocardial infarction and in human dilated cardiomyopathy that pleiotrophin is markedly up-regulated. To elucidate the effects of pleiotrophin on cardiac contractile cells, we employed primary cultures of rat neonatal and adult cardiomyocytes. We show that pleiotrophin is released from cardiomyocytes in vitro in response to hypoxia and that the addition of recombinant pleiotrophin promotes caspase-mediated genomic DNA fragmentation in a dose- and time-dependent manner. Functionally, it potentiates the apoptotic response of neonatal cardiomyocytes to hypoxic stress and to ultraviolet irradiation and of adult cardiomyocytes to hypoxia-reoxygenation. Moreover, UV-induced apoptosis in neonatal cardiomyocytes can be partially inhibited by small interfering RNA-mediated knockdown of endogenous pleiotrophin. Mechanistically, pleiotrophin antagonizes IGF-1 associated Ser-473 phosphorylation of AKT/PKB, and it concomitantly decreases both BAD and GSK3beta phosphorylation. Adenoviral expression of constitutively active AKT and lithium chloride-mediated inhibition of GSK3beta reduce the potentiated programmed cell death elicited by pleiotrophin. These latter data indicate that pleiotrophin potentiates cardiomyocyte cell death, at least partially, through inhibition of AKT signaling. In conclusion, we have uncovered a novel function for pleiotrophin on heart cells following injury. It fosters cardiomyocyte programmed cell death in response to pro-apoptotic stress, which may be critical to myocardial injury repair.

Increasing evidence shows that stimulation of beta-adrenergic receptor (AR) activates mitogen-activated protein kinases (MAPKs), in addition to the classical G(s)-adenylyl cyclase-cAMP-dependent protein kinase (PKA) signaling cascade. In the present study, we demonstrate a novel beta(2)-AR-mediated cross-talk between PKA and p38 MAPK in adult mouse cardiac myocytes expressing beta(2)-AR, with a null background of beta(1)beta(2)-AR double knockout. beta(2)-AR stimulation by isoproterenol increased p38 MAPK activity in a time- and dose-dependent manner. Inhibiting G(i) with pertussis toxin or scavenging Gbetagamma with betaARK-ct overexpression could not prevent beta(2)-AR-induced p38 MAPK activation. In contrast, a specific peptide inhibitor of PKA, PKI (5 microm), completely abolished the stimulatory effect of beta(2)-AR, suggesting that beta(2)-AR-induced p38 MAPK activation is mediated via a PKA-dependent mechanism, rather than by G(i) or Gbetagamma. This conclusion was further supported by the ability of forskolin (10 microm), an adenylyl cyclase activator, to elevate p38 MAPK activity in a PKI-sensitive manner. Furthermore, inhibition of p38 MAPK with SB203580 (10 microm) markedly enhanced the beta(2)-AR-mediated contractile response, without altering base-line contractility. These results provide the first evidence that cardiac beta(2)-AR activates p38 MAPK via a PKA-dependent signaling pathway, rather than by G(i) or Gbetagamma, and reveal a novel role of p38 MAPK in regulating cardiac contractility.

Cardiac-specific overexpression of the human beta(2)-adrenergic receptor (AR) in transgenic mice (TG4) enhances basal cardiac function due to ligand-independent spontaneous beta(2)-AR activation. However, agonist-mediated stimulation of either beta(1)-AR or beta(2)-AR fails to further enhance contractility in TG4 ventricular myocytes. Although the lack of beta(2)-AR response has been ascribed to an efficient coupling of the receptor to pertussis toxin-sensitive G(i) proteins in addition to G(s), the contractile response to beta(1)-AR stimulation by norepinephrine and an alpha(1)-adrenergic antagonist prazosin is not restored by pertussis toxin treatment despite a G(i) protein elevation of 1.7-fold in TG4 hearts. Since beta-adrenergic receptor kinase, betaARK1, activity remains unaltered, the unresponsiveness of beta(1)-AR is not caused by betaARK1-mediated receptor desensitization. In contrast, pre-incubation of cells with anti-adrenergic reagents such as muscarinic receptor agonist, carbachol (10(-5)m), or a beta(2)-AR inverse agonist, ICI 118,551 (5 x 10(-7)m), to abolish spontaneous beta(2)-AR signaling, both reduce the base-line cAMP and contractility and, surprisingly, restore the beta(1)-AR contractile response. The "rescued" contractile response is completely reversed by a beta(1)-AR antagonist, CGP 20712A. Furthermore, these results from the transgenic animals are corroborated by in vitro acute gene manipulation in cultured wild type adult mouse ventricular myocytes. Adenovirus-directed overexpression of the human beta(2)-AR results in elevated base-line cAMP and contraction associated with a marked attenuation of beta(1)-AR response; carbachol pretreatment fully revives the diminished beta(1)-AR contractile response. Thus, we conclude that constitutive beta(2)-AR activation induces a heterologous desensitization of beta(1)-ARs independent of betaARK1 and G(i) proteins; suppression of the constitutive beta(2)-AR signaling by either a beta(2)-AR inverse agonist or stimulation of the muscarinic receptor rescues the beta(1)-ARs from desensitization, permitting agonist-induced contractile response.

Rapid development of transgenic and gene-targeted mice and acute genetic manipulation via gene transfer vector systems have provided powerful tools for cardiovascular research. To facilitate the phenotyping of genetically engineered murine models at the cellular and subcellular levels and to implement acute gene transfer techniques in single mouse cardiomyocytes, we have modified and improved current enzymatic methods to isolate a high yield of high-quality adult mouse myocytes (5.3 +/- 0.5 x 10(5) cells/left ventricle, 83.8 +/- 2.5% rod shaped). We have also developed a technique to culture these isolated myocytes while maintaining their morphological integrity for 2-3 days. The high percentage of viable myocytes after 1 day in culture (72.5 +/- 2.3%) permitted both physiological and biochemical characterization. The major functional aspects of these cells, including excitation-contraction coupling and receptor-mediated signaling, remained intact, but the contraction kinetics were significantly slowed. Furthermore, gene delivery via recombinant adenoviral infection was highly efficient and reproducible. In adult beta(1)/beta(2)-adrenergic receptor (AR) double-knockout mouse myocytes, adenovirus-directed expression of either beta(1)- or beta(2)-AR, which occurred in 100% of cells, rescued the functional response to beta-AR agonist stimulation. These techniques will permit novel experimental settings for cellular genetic physiology.

We examined the effect of alpha 1-adrenergic receptor (AR) subtypes on contraction, cytosolic Ca2+ concentration ([Ca2+]i), and cytosolic pH (pHi) of rat ventricular myocytes loaded with the Ca2+ indicator indo 1 or the pH indicator carboxyseminaphthorhodafluor-1. Nonselective alpha 1-AR stimulation was effected with phenylephrine plus nadolol. alpha 1-AR subtype stimulation was achieved with alpha 1-AR and chloroethylclonidine (CEC) or with alpha 1-AR and WB-4101. Cells were in bicarbonate buffer with 0.5 mM Ca2+ and were electrically stimulated at 0.5 Hz. Results show that 1) nonselective alpha 1-AR stimulation increased twitch and [Ca2+]i transient amplitudes, myofilament response to Ca2+, and pHi; 2) alpha 1-AR plus CEC increased twitch and [Ca2+]i transient amplitudes and also enhanced myofilament response to Ca2+ via cytosolic alkalinization; 3) alpha 1-AR plus WB-4101 decreased twitch and [Ca2+]i transient amplitudes and also pHi; and 4) cytosolic acidification due to alpha 1-AR plus WB-4101 was abolished by protein kinase C inhibition (staurosporine pretreatment) or downregulation (prolonged exposure to phorbol esters). In summary, the net effects of alpha 1-adrenergic stimulation on contraction, [Ca2+]i, and pHi are due to opposing WB-4101- and CEC-sensitive alpha 1-AR subtype signaling pathways.

We compared L-type Ca current (ICaL) and T-type Ca current (ICaT) in finch and rat myocytes, using whole-cell patch clamp techniques. Cell capacitance averaged 50 +/- 4 pF in finch (n = 25) v 145 +/- 8 pF in rat (n = 38) cells, P < 0.001. In cells bathed in 1 mM Cao at 22 degrees C, peak ICaL amplitude, during a voltage clamp step (10 mM EGTA in pipette) from -45 mV to -5 mV, averaged 10.5 +/- 0.3 pA/pF in finch v 6.9 +/- 0.6 pA/pF, P < 0.001 in rat cells. ICaL inactivation kinetics were faster in finch (4.6 +/- 0.3 ms) than in rat (13.4 +/- 1.3 ms) cells. P < 0.001. ICaT was not detectable in rat cells (2 mM bathing [Ca]); but in finch cells, a large ICaT which averaged 6.8 +/- 1.4 pA/pF was activated at -30 mV and was relatively insensitive to nitrendipine (0.1 microM). The distinctive features of ICaL and ICaT in finch cells may have a role in the ability of the finch to achieve a very rapid heart rate. They may also facilitate excitation-Ca2+ release coupling in finch ventricular cells which are devoid of T tubules and have relatively few junctions between the sarcolemma and the sarcoplasmic reticulum.

We examined the effects of a selective kappa opioid receptor agonist (U-50,488H) on the contractile properties of single ventricular myocytes from 127 day old control (F1B) and cardiomyopathic (BIO 14.6) hamsters. Myocytes in bicarbonate buffered solution with 1.5 mM [Ca2+] were electrically stimulated with field electrodes in the bath. Length changes were monitored via myocyte edge tracking. Twitch amplitude and the velocity of cell shortening were less in the cardiomyopathic hamster myocytes than in age-matched hamsters (P less than or equal to 0.05). There was a concentration-dependent effect of U-50,488H (0.1-20 microM) to decrease twitch amplitude and shortening velocity in both control and cardiomyopathic myocytes (P less than or equal to 0.001). In cells loaded with the Ca2+ indicator indo-1 the negative inotropic action of U-50,488H was associated with a decreased indo-1 fluorescence transient amplitude. There was no difference in the negative inotropic effect of U-50,488H on control and cardiomyopathic cells. Thus, the CM hamster does not demonstrate a different contractile response to U-50,488H.