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• W4313652014 abstract "Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation The study is overall well-planned and the amount of data presented by the authors is impressive. The work nicely incorporates animal-level physiology (echocardiography data), tests for known canonical markers of hypertrophy, and then delves into an unbiased analysis of the transcriptome and proteome of LV tissue in bulk. The techniques and analyses in the study are adequately executed and within the realm of expertise of the Lakatta laboratory. This study is a necessary and crucial first step to extensively phenotype this mouse line and generate hypotheses for further work. https://doi.org/10.7554/eLife.80949.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Adaptations have evolved in all organisms to cope with both acute and chronic internal and environmental stress. For example, during acute exercise increased autonomic sympathetic-mediated AC/cAMP/PKA/Ca2+ signaling, the quintessential mediator of both acute and chronic stress, also activates acute physiologic adaptations to moderate the exercise-induced increase in sympathetic signaling. In response to repeated bouts of acute exercise induced AC/cAMP/PKA/Ca2+ stress, chronic adaptations emerge (endurance exercise conditioning) (Vega et al., 2017; Venturaclapier et al., 2007; Lovallo, 2005). Prolonged and intense chronic cAMP-mediated stress in experimental animal models results in cardiomyopathy and death (Zhang et al., 2013; Antos et al., 2001). During chronic pathophysiologic states for example chronic heart failure (CHF) in humans, AC/cAMP/PKA/Ca2+ signaling progressively increases as the degree of heart failure progresses, leading to cardiac inflammation, mediated in part, by cyclic-AMP- induced up-regulation of renin-angiotensin system (RAS) signaling. Standard therapies for CHF include β-adrenoreceptor blockers and RAS inhibitors, (Squire and Barnett, 2000; Werner and Böhm, 2008) which although effective, are suboptimal in amelioration of heart failure progression. One strategy to devise novel and better therapies for heart failure, would be to uncover the full spectrum of concentric cardio- protective adaptations that becomes activated in response to severe, chronic AC/cAMP/PKA/Ca2+-induced cardiac stress. The young adult TGAC8 heart in which cardiac-specific over expression in mice of Adenylyl Cyclase (AC) Type 8, driven by the α myosin heavy chain promoter, markedly enhances AC activity and cAMP signaling, may be an ideal model in which to elucidate the features of a wide-spread an adaptive paradigm that must become engaged in response to incessant chronic activation of cardiac AC/cAMP/PKA/Ca2+ signaling. Specifically, concurrent with chronically increased AC activity within the young adult TGAC8 sinoatrial node (SAN) and LV, (Moen et al., 2019; Lipskaia et al., 2000) heart rate (HR), measured via telemetry in the awake, unrestrained state, increased by approximately 30%, persisting in the presence of dual autonomic blockade; Moen et al., 2019 and LV EF is also markedly increased in TGAC8 (Mougenot et al., 2019). Thus, the cardiac phenotype of the adult TGAC8 mimics cardiac responses to AC/cAMP/PKA/Ca2+ sympathetic autonomic input during strenuous, acute exercise, but this stressful state persists incessantly (Moen et al., 2019). Although this continual high cardiac load imposes a severe chronic stress on the heart, which might be expected to lead to near term heart failure and demise (Liu et al., 2020; Zhang et al., 2005), adult TGAC8 mice maintain this remarkable, hyperdynamic cardiac phenotype for up to about a one year of age, (Lipskaia et al., 2000; Georget et al., 2002; Georget et al., 2003; Mougenot et al., 2019) when signs of CHF begin to develop and cardiomyopathy ensues (Mougenot et al., 2019). We hypothesized, (1) that a panoply of intrinsic adaptive mechanisms become concurrently engaged in order to protect the TGAC8 heart during the incessant, high level of cardiac work in several months, and (2) that, some of these mechanisms are those that become activated in the endurance, trained heart, including shifts in mechanisms of energy generation, enhanced protein synthesis and quality control, and increased defenses against reactive O2 species (ROS) and cell death (Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011). We reasoned that a discovery bioinformatics approach, in conjunction with deeper phenotypic characterization of the TGAC8 LV, would generate a number of testable hypotheses about the characteristics of some of these mechanisms utilized to sustain this adaptive paradigm of heart performance and protection in response to severe, chronic, adenylyl cyclase-induced cardiac stress. To this end, we performed unbiased, RNASEQ and proteomic analyses of adult TGAC8 and WT LVs, and selectively validated genotypic differences in numerous transcripts and proteins. Our results delineate a pattern of consilient adaptations that becomes activated within the TGAC8 heart in response to chronically increased AC/cAMP/PKA/Ca2+-signaling and offers numerous testable hypotheses to further define the details of mechanisms that underlie what we will show here to be a remarkable adaptive heart paradigm. Results Cardiac structure and performance We performed echocardiography and cardiac histology for in-depth characterization of the TGAC8 heart structure and function. Representative echocardiograms of TGAC8 and WT are illustrated in Figure 1—figure supplement 1, selected Echo parameters are shown in Figure 1, (and a complete listing of parameters is provided in Supplementary file 1a). Both EF and HR were higher in TGAC8 than in WT (Figure 1A and B), confirming prior reports (Moen et al., 2019; Mougenot et al., 2019). Because stroke volume did not differ by genotype (Figure 1C), cardiac output was elevated by 30% in TGAC8 (Figure 1D) on the basis of its 30% increase in HR. Arterial blood pressure was only mildly increased in TGAC8 averaging 3.5 mmHg higher than in WT (Figure 1E). Figure 1 with 1 supplement see all Download asset Open asset Echocardiographic parameters and body weight of TGAC8 and WT mice. (A–K), Echocardiographic parameters (N=28 for TGAC8; WT = 21); (L) heart weight/body weight at sacrifice (N=75 for TGAC8 and N=85 for WT). See Supplementary file 1a for additional Echo parameters. Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, ** p<0.01, **** p<0.0001 (t test). A sustained high cardiac workload is usually expected to result in an increase of a total LV mass, Vega et al., 2017; Dorn, 2007; Maillet et al., 2013 that is, cardiac hypertrophy. Although both the LV posterior wall and inter-ventricular septum were thicker in TGAC8 vs WT (indicative of an increased LV wall biomass; Figure 1F and G) the LV cavity volume was markedly reduced (Figure 1H, I), and the echo derived total LV mass did not differ by genotype (Figure 1J). Postmortem measurements indicated that although the body weight did not differ between TGAC8 and WT (Figure 1K), the heart weight/body weight (HW/BW) was actually modestly reduced in TGAC8 vs WT (Figure 1L). Pathological hypertrophy markers, for example, β-myosin heavy chain (MYH7), ANP (NPPA) or BNP (NPPB) were not increased in TGAC8 vs WT by Western Blot (WB) (Figure 2A); however, α skeletal actin, commonly considered to be a pathologic hypertrophy marker, was increased in TGAC8 vs WT (Figure 2A). Calcineurin (PP2B), which activates the hypertrophic response by dephosphorylating nuclear factor of activated T cells (NFAT), Wilkins and Molkentin, 2004 did not differ in WB of TGAC8 vs WT (Figure 2A). Figure 2 with 1 supplement see all Download asset Open asset Expression of pathologic hypertrophy markers and cardiac myocyte sizes in LV of TGAC8 and WT mice. (A) WB of Pathologic hypertrophy markers, (B, C) cardiac myocyte cross-sectional areas and distributions of cardiac myocyte sizes in TGAC8 and WT hearts (cardiomyocytes were counted on left free wall at the middle level sections from four animals in each group; 37 high power fields from WT mice and 33 high power fields from TGAC8 mice; 77 cells from WT mice and 70 cells from TGAC8 mice were analyzed) Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, ** p<0.01, (two-tailed t test)., (D) Representative LV sections depicting cardiac myocyte diameters (scale bar 100 µm). Figure 2—source data 1 Raw unedited blot images and uncropped blot images of MYH7, α Sk. Actin, ANP, BNP, and Calcineurin. https://cdn.elifesciences.org/articles/80949/elife-80949-fig2-data1-v2.zip Download elife-80949-fig2-data1-v2.zip LV histologic analysis The average LV cardiac myocyte size was smaller in TGAC8 compared to WT (Figure 2B and C), and LV myocyte size distribution was different by genotype (Figure 2D). LV collagen content in TGAC8 was not increased vs WT (Figure 2—figure supplement 1). To address the issue of DNA synthesis within LV cardiac myocyte we loaded EdU for 28 days. EdU-labeled nuclei, detected in both TGAC8 and WT whole mount ventricular preparations were randomly scattered throughout the LV from the mitral annulus to the apex. LV myocyte nuclei, however, were rarely EdU labeled. Rather nearly all EdU labeling was detected in small interstitial cells that expressed vimentin or in cells that enclosed the capillary lumina (Figure 3A–G), suggesting that EdU was also incorporated within the DNA of endothelial cells in both WT and TGAC8. However, total nuclear EdU labeling was 3-fold higher in TGAC8 than in WT Figure 3H. Figure 3 Download asset Open asset EdU labeling of cardiac tissue. (A–G) representative examples of confocal images (400 x) of LV WGA (red), vimentin (Cyan), EdU (yellow), DAPI (blue), (H) average number of EdU-labeled nuclei positive field counts in LV TGAC8 vs WT (N=3 mice in each genotype). Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, (t test). Detection of cardiomyocyte S-phase activity. (I) Section from the heart of a TGAC8, MHC-nLAC double-transgenic mouse subjected to 12 days of BrdU treatment. The section was processed for β-galactosidase (to identify cardiomyocyte nuclei, red signal) and BrdU (to identify DNA synthesis, green signal) immune reactivity, and then counterstained with Hoechst (which stains all nuclei, blue signal). (J) Example of an S-phase cardiomyocyte nucleus detected with this assay. The upper panel shows β-galactosidase immune reactivity (red channel), the middle panel shows BrdU immune reactivity (green channel), and the lower panel shows a red and green color combined image of the same field. The arrow identifies an S-phase cardiomyocyte nucleus, as evidenced by the overlay of β-galactosidase and BrdU immune reactivity, which appears yellow in the color combined image. (H) Graph representing S-phase activity in the TGAC8, MHC-nLAC double-transgenic vs. the MHC-nLAC single transgenic animals (mean +/- SEM, p=0.315; 5 mice per genotype and 3 sections per mouse were analyzed). To monitor cardiomyocyte S-phase activity, TGAC8 mice were crossed with MHC-nLAC mice (which express a nuclear-localized β-galactosidase reporter under the transcriptional regulation of the mouse α-cardiac MHC promoter; these mice are useful to identify cardiomyocyte nuclei in histologic sections) (Soonpaa et al., 1994). The resulting TGAC8, MHC-nLAC double-transgenic mice and MHC-nLAC single-transgenic mice were identified and sequestered. At 28-to-30 days of age, the mice were administered BrdU via drinking water (0.5 mg/ml, changed every 2nd day) for a total of 12 days. There was no difference in the level of ventricular cardiomyocyte S-phase activity in the TGAC8, MHC-nLAC double-transgenic vs. the MHC-nLAC single transgenic animals (Figure 3H–K). Thus, the adult TGAC8 heart has a hyper-dynamic LV with thicker walls, harboring not only an increased number of small cardiac myocytes, but also an increased number of small interstitial cells and endothelial cells with increased EdU labeling vs WT. The LV cavity volumes at both end-diastole and end-systole were markedly reduced, and LV EF was markedly increased in TGAC8 vs WT. But, neither total LV mass, nor collagen content were increased in TGAC8 vs WT and the profile of pathologic cardiac hypertrophy markers in TGAC8 was absent. AC/cAMP/PKA/Ca2+ signaling Given that the transgene in our study was an AC, we next focused on expected differences in AC/cAMP/PKA/Ca2+ signaling in TGAC8 vs WT. Immunolabeling of single LV myocytes showed that AC8 expression was markedly increased (by 8–9-fold in TGAC8 vs WT), and AC activity (measured in membranes isolated from LV tissue) in TGAC8 was 50% higher than that in WT (Figure 4A–C). Expression of the PKA catalytic subunit was increased by 65.6%, and expression of the regulatory subunit was decreased by 26.7% in TGAC8 by WB (Figure 4D and E). PKA activity was increased by 57.8% in TGAC8 vs WT (Figure 4F). Figure 4 Download asset Open asset Expression of molecules involved in AC/cAMP/PKA/Ca2+ signaling in LV of TGAC8 and WT mice. (A) Representative examples of ADCY8 immunolabeling in TGAC8 and WT LV cardiomyocytes (scale bar 10 µm), (B) Average AC8 fluorescence in LV cardiomyocytes (N=3 animals per group; 60 cells for each genotype were analyzed. Data are presented as Mean± SEM. The statistical significance is indicated by **** p<0.0001 (two-tailed t test).) and (C) Average AC activity in TGAC8 vs WT (N=3 per group), (D, E) Expression levels of PKA catalytic and regulatory subunits, and (F) PKA activity in TGAC8 vs WT (N=8 per group), (G–M) Western Blot analysis of selected proteins involved in excitation - Ca release – contraction-relaxation coupling TGAC8 vs WT LV. (L) RyR2 immunolabeling. Antibodies employed are listed in supplemental methods. (N=199 WT cells and 195 TGAC8 cells each from 3 mice). Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, ** p<0.01, *** p<0.001,**** p<0.0001 (two-tailed t test). Figure 4—source data 1 Raw unedited blot images and uncropped blot images of PKAca, PKAr, MYH6, SERCA2, LTCC, NCX1, CamKII. https://cdn.elifesciences.org/articles/80949/elife-80949-fig4-data1-v2.zip Download elife-80949-fig4-data1-v2.zip To search for mechanisms that increase contractility within cardiac myocytes, we examined the expression of selected proteins downstream of PKA signaling that are involved in excitation/Ca2+ release/contraction/relaxation. Western blot analysis revealed that a number of proteins that determine increase cardiac myocyte performance were upregulated in TGAC8 vs WT, including αMHC (MYH6 by 18.7%), SERCA2 (ATP2A2 by 62.0%), L-type Ca Channel (Cav1.2, by 61.8%), and NCX1 (AKA SLC8A1) by 117.6%, and CaMKII by 35.0% (Figure 4G–K). Immunolabeling of total RyR2 was increased by 66% in TGAC8 vs WT (Figure 4L). This pattern of increased protein expression is consistent with increased Ca2+ flux into and out of cardiac myocytes, an increased Ca2+ cycling between SR and cytosol, and increased myosin cross-bridge kinetics during heart contraction in TGAC8 vs WT. Thus, as would be expected in the context of markedly increased transcription of AC type VIII, AC and PKA protein levels and activities, and levels of proteins downstream of PKA signaling, were markedly increased and associated with a chronic, marked increase in LV performance. Protein synthesis, degradation, and quality control Because PKA signaling-driven increased cardiac work is known to be associated with increased protein synthesis, Pinson et al., 1993; Yamazaki et al., 1997 we next compared the rate of protein synthesis in TGAC8 and WT LV lysates. Despite the absence of increase of total LV mass, the rate of protein synthesis was 40% higher in TGAC8 than in WT (Figure 5A). WB analysis indicated that expression or activation of p21Ras, p-c-Raf, MEK1/2, molecules downstream of PKA signaling that are implicated in protein synthesis, were increased in TGAC8 vs WT (Figure 5B–C). The transcription factor CREB1, involved in PKA signaling directed protein synthesis was increased by 58.1% in TGAC8 vs WT in WB analysis (Figure 5D). Expression of CITED4 (family of transcriptional coactivators that bind to several proteins, including CREB-binding protein [CBP]) was increased by 51% in TGAC8 vs WT in WB analysis (Figure 5E). Expression of protein kinases, that are required for stress-induced activation of CREB1, MSK1 and MNK1, direct substrates of MAPK, were increased by 40% and 48% in TGAC8 vs WT, respectively (Figure 5F and G). Figure 5 Download asset Open asset Expression of proteins,involved in protein synthesis, degradation, and quality control in LV of TGAC8 and WT mice. (A) Rate of global protein synthesis and (B–G) mechanisms downstream of PKA signaling involved in protein synthesis in the TGAC8 and WT. Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, ** p<0.01,(two-tailed t test). Figure 5—source data 1 Raw unedited blot images and uncropped blot images of Puromycin, CITED4, CREB1, MNK1, MSK1, GAPDH, pMEK1/2, p21 Ras, p-c-Raf, total Raf, total Mek 1/2, p-ERK 1/2 and total ERK 1/2. https://cdn.elifesciences.org/articles/80949/elife-80949-fig5-data1-v2.zip Download elife-80949-fig5-data1-v2.zip Autophagy Proteasome activity within LV lysates was increased in TGAC8 vs WT (Figure 6A). However, although there was a significant increase in the amount of soluble misfolded proteins in TGAC8 vs WT (Figure 6B), insoluble protein aggregates did not accumulate within the TGAC8 LV (Figure 6C). This suggest that an increase in micro-autophagy of the TGAC8 LV circumvents the potential proteotoxic stress of aggregated protein accumulation, which can negatively impact cardiac cell health and function. A 24% increase in the expression of HSP90α (Figure 6D) in TGAC8 suggested that chaperone-mediated autophagy was also involved in preventing an accumulation of insoluble protein aggregates. Figure 6 Download asset Open asset Proteosome activity assay, accumulated proteins in soluble and insoluble fractions and expression levels of selected proteins involved in the autophagy process in LV of TGAC8 and WT mice. (A) Proteosome activity assay and (B, C) accumulated proteins in soluble and insoluble fractions of LV lysates in TGAC8 vs WT. (D) WB of HSP90 in TGAC8 and WT, (E–H) Expression levels of selected proteins involved in the autophagy process (Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05, **** p<0.0001 (two-tailed t test).). ;(I, J) Autophagolysosome accumulation is enhanced in AC8 mice (46 cells for each group; 3 aminals per genotype). Data expressed as Mean± SEM. The statistical significance is indicated by **p<0.01, and t (p<0.01 in one-tailed t test). Figure 6—source data 1 Raw unedited blot images and uncropped blot images of HSP90, ATG13, PARKIN, ATG4B, and PI3Kc3. https://cdn.elifesciences.org/articles/80949/elife-80949-fig6-data1-v2.zip Download elife-80949-fig6-data1-v2.zip We reasoned that another type of protein quality control, macro-autophagy, might also be activated in TGAC8 vs WT. Indeed, protein levels of several members of the autophagy machinery were increased in TGAC8 vs WT: both ATG13, a factor required for autophagosome formation and mitophagy, and the ubiquitin-protein ligase PARKIN, which protects against mitochondrial dysfunction during cellular stress, by coordinating mitochondrial quality control mechanisms to remove/replace dysfunctional mitochondrial components, were significantly increased in TGAC8 vs WT (Figure 6E and F). Furthermore, protein levels of the cysteine protease ATG4B, involved in processing, lipidation/delipidation (conjugation/removal of phosphatidylethanolamine) and insertion of MAP1LC3, GABARAPL1, GABARAPL2 and GABARAP into membranes during autophagy and endocytosis, were also increased (Figure 6G). In addition, the catalytic subunit of the PI3Kc3, involved in initiation and maturation of autophagosomes and in endocytosis, was also significantly higher in the TGAC8 vs WT (Figure 6H). Finally, direct measurements in single LV myocytes showed that autophagolysosomes were appreciably increased in TGAC8 vs WT, indicating that autophagy (mitophagy) was activated to a greater extent in the TGAC8 vs WT. (Figure 6I and J). Mitochondrial structure We employed transmission electron microscopy (TEM) to directly visualize ultrastructural details of the mitochondria and cardiac myofibers within the LV of TGAC8 and WT. Representative panoramic electron micrographs of LV cardiac muscle fibers and mitochondria in TGAC8 and WT are illustrated in Figure 7A and B. Cardiac myocytes, presenting a very distinctive morphology with high content of myofibrils, a large number of high-electron dense mitochondria and several capillaries surrounding cardiac myocytes, are depicted (see white arrows). Cardiac myocyte ultrastructure is depicted in Figure 7 panels C and E, for WT mice, and in panels D and F, for TGAC8. Asterisks show swollen, disrupted mitochondria with lighter cristae compared to the surrounding healthy mitochondria. Figure 7 Panels G-J present quantitative stereological analyses of normal and damaged mitochondria. Although there was a mild increase in the number of damaged mitochondria (0.3 %), and in the percent of cell volume occupied by damaged mitochondria (0.4%) in TGAC8, the numbers of healthy mitochondria and the percent of cell volume occupied by healthy mitochondria did not differ between TGAC8 and WT. Nevertheless, the presence of mitochondrial deterioration at a young age is uncommon and may be further evidence for enhanced cleaning and recycling mechanisms such as autophagic signaling, (Figures 6 and 7). Figure 7 Download asset Open asset Mitochondrial structure and function. (A, B) Representative panoramic electron micrographs (white arrows depict capillaries surrounding cardiac myocytes) and (C–F) higher resolution images of LV cardiac muscle fibers and mitochondria in TGAC8 and WT. White arrows depict lipid droplets; asterisks show swollen, disrupted mitochondria with lighter cristae compared to the surrounding, healthy mitochondria. (G, H) Average mitochondrial number of quantitative stereological analyses of normal mitochondrial number and volume, (I, J) damaged mitochondria, (K, L) number of lipid droplets per cell area and volume of lipid droplets. (M, N) mPTP-ROS threshold, measured in a single LV cardiac myocyte, did not differ between TGAC8 and WT mice. Insulin was employed as a positive control. (3 animals in each genotype; 29 cells - WT Control; 26 cells - WT insuline; 30 cells - TGAC8 control; 25 cells - TGAC8 insulin) (** p<0.01, *** p<0.001, one-way anova; Mean± SEM). Mitochondrial fitness The healthy functioning and survival of cardiac myocytes during severe, chronic myocardial stress requires close coordination of survival mechanisms and numerous mitochondrial functions that require a high level of mitochondrial fitness (Zorov et al., 2000; Juhaszova et al., 2004; Zorov et al., 2014; Aon et al., 2021). The mitochondrial permeability transition pore (mPTP) is a key regulator of mitochondrial functions, including energy metabolism (e.g. with the pore performing as a ‘safety valve’, opening transiently and reversibly, to prevent: (1) the excessive accumulation of certain regulatory species, such as Ca2+; and (2) bioenergetic byproducts/damaging reactive species, such as free radicals, from achieving toxic levels). The mPTP also regulates cell fate: enduring and irreversible pore opening, plays decisive mechanistic roles in mitochondrial and cell life vs death decisions during normal development or pathological stress (e.g. involving excess and damaging free radical exposure). Measurement of the pore susceptibility or resistance to being induced/opened can serve as a biomarker of mitochondrial fitness. Figure 7 panels M and N shows that the mPTP ROS threshold did not differ in TGAC8 vs WT, suggesting a comparable degree of mitochondrial fitness in both genotypes. ROS levels and NRF signaling Given the incessantly elevated myocardial contractility and heart rate, and increased protein synthesis and quality control mechanisms in TGAC8 vs WT, it might be expected that ROS levels are increased in TGAC8. To this end, we measured superoxide radical accumulation in isolated, perfused, isometrically contracting TGAC8 hearts that maintained markedly enhanced cardiac workload observed in vivo (Figure 8A). A marked increased heart rate pressure product in TGAC8, suggests that myocardial oxygen consumption was increased vs WT, possibly due to increased coronary flow or increased oxygen extraction from the perfusate. Superoxide radical accumulation did not differ by genotype, suggesting that mechanisms to scavenge ROS are increased in TGAC8. Indeed, the level of NRF2 protein, a key regulator of ROS defense signaling was increased by 24% in TGAC8 vs WT (Figure 8B), suggesting that increased NRF signaling in the TGAC8 LV may be a factor that prevents the accumulation of superoxide ROS. Interestingly, NRF2 signaling, was one of the top enriched and activated signaling pathway in transcriptome and proteome bioinformatic analysis (see below and Supplementary file 1i). Figure 8 Download asset Open asset Detection of ROS levels and NRF signaling in LV of TGAC8 and WT mice. (A) LV performance and the rate of superoxide (ROS) generation in isolated working TGAC8 and WT hearts. (B) WB analysis of Nrf2 expression in LV TGAC8 vs WT. Differences between two groups were assessed by a t-test, and reported as Mean± SEM; * p<0.05; ** p<0.01. Figure 8—source data 1 Raw unedited blot images and uncropped blot images of NRF2. https://cdn.elifesciences.org/articles/80949/elife-80949-fig8-data1-v2.zip Download elife-80949-fig8-data1-v2.zip High-energy phosphates Given the fact that increased protein synthesis and quality control mechanisms (Figure 6), maintenance of normal ROS levels (Figure 8) and the incessant high cardiac performance of the TGAC8 (Figure 1) require increased energy production, it is reasonable to assume that the total energy requirements of the TGAC8 LV are probably considerably higher than those in WT. It was important, therefore, to assess high-energy phosphate levels in TGAC8 and WT. A schematic of ATP-creatine energy system is depicted in Figure 9A. Steady state levels of ATP, phosphocreatine and the ATP: phosphocreatine assessed in vivo were maintained at the same level in TGAC8 as in WT (Figure 9B–E), suggesting that the rate of ATP production in the TGAC8 LV is adequate to meet its increased energy demands, at least when animals rest. Figure 9 Download asset Open asset High-energy phosphates in TGAC8 and WT hearts. (A) A schematic of ATP creatine energy system, (B) Representative P31 NMR spectra of TGAC8 and WT hearts. (C–E) Average levels of ATP, PCr, and ATP/PCr in TGAC8 and WT hearts derived from NMR spectra. Data are presented as Mean± SEM. The statistical significance is indicated by * p<0.05 (two-tailed t test). Unbiased RNASEQ and proteome analyses We performed unbiased, RNASEQ and proteome analyses of adult TGAC8 and WT left ventricles (LV) in order to realize facets of stress circuitry that are known to become activated in response to environmental stress, Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011 beyond those identified in our experiments illustrated in Figures 1—9. We reasoned that taking advantage of the knowledge base within bioinformatic tools would also generate a number of testable hypotheses regarding the components of adaptive paradigm of heart performance and protection in respon" @default.
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• W4313652014 title "Editor's evaluation: A remarkable adaptive paradigm of heart performance and protection emerges in response to marked cardiac-specific overexpression of ADCY8" @default.
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