Sham Hearts Research Articles

Direct comparison of cervical and high thoracic spinal cord injury reveals distinct autonomic and cardiovascular consequences.

A wide range of spinal cord levels (cervical 8-thoracic 6) project to the stellate ganglia (which provides >90% of sympathetic supply to the heart), with a peak at the thoracic 2 (T2) level. We hypothesize that despite the proximity of the lesions, high thoracic spinal cord injuries (i.e., T2-3 SCI) do not closely mimic the hemodynamic responses recorded with cervical SCI (i.e., C6-7 SCI). To test this hypothesis, rats were instrumented with an intra-arterial telemetry device (Data Sciences International PA-C40) for recording arterial pressure, heart rate, and locomotor activity as well as a catheter within the intraperitoneal space. After recovery, rats were subjected to complete C6-7 spinal cord transection (n = 8), sham transection (n = 4), or T2-3 spinal cord transection (n = 7). After the spinal cord transection or sham transection, arterial pressure, heart rate, and activity counts were recorded in conscious animals, in a thermoneutral environment, for 20 s every minute, 24 h/day for 12 consecutive weeks. After 12 wk, chronic reflex- and stress-induced cardiovascular and hormonal responses were compared in all groups. C6-7 rats had hypotension, bradycardia, and reduced physical activity. In contrast, T2-3 rats were tachycardic. C6-7 rats compared with T2-3 and spinal intact rats also had reduced cardiac sympathetic tonus, reduced reflex- and stress induced cardiovascular responses, and reduced sympathetic support of blood pressure as well as enhanced reliance on angiotensin to maintain arterial blood pressure. Thus injuries above and below the peak level (T2) of spinal cord projections to the stellate ganglia have remarkably different outcomes.NEW & NOTEWORTHY Twelve consecutive weeks of resting hemodynamic data as well as chronic reflex- and stress-induced cardiovascular, autonomic, and hormonal responses were compared in spinal intact and C6-7 and T2-3 spinal cord-transected rats. C6-7 rats compared with T2-3 and spinal intact rats had reduced cardiac sympathetic tonus, reduced reflex- and stress-induced cardiovascular responses, and reduced sympathetic support of blood pressure as well as enhanced reliance on angiotensin to maintain arterial blood pressure. Thus injuries above and below the peak level (T2) of spinal cord projections to the stellate ganglia have remarkably different outcomes.

Analysis of mesenchymal stem cell proteomes in situ in the ischemic heart.

Rationale: Cell therapy for myocardial infarction is promising but largely unsuccessful in part due to a lack of mechanistic understanding. Techniques enabling identification of stem cell-specific proteomes in situ in the injured heart may shed light on how the administered cells respond to the injured microenvironment and exert reparative effects.Objective: To identify the proteomes of the transplanted mesenchymal stem cells (MSCs) in the infarcted myocardium, we sought to target a mutant methionyl-tRNA synthetase (MetRSL274G) in MSCs, which charges azidonorleucine (ANL), a methionine analogue and non-canonical amino acid, to tRNA and subsequently to nascent proteins, permitting isolation of ANL-labeled MSC proteomes from ischemic hearts by ANL-alkyne based click reaction.Methods and Results: Murine MSCs were transduced with lentivirus MetRSL274G and supplemented with ANL; the ANL-tagged nascent proteins were visualized by bio-orthogonal non-canonical amino-acid tagging, spanning all molecular weights and by fluorescent non-canonical amino-acid tagging, displaying strong fluorescent signal. Then, the MetRSL274G-transduced MSCs were administered to the infarcted or Sham heart in mice receiving ANL treatment. The MSC proteomes were isolated from the left ventricular protein lysates by click reaction at days 1, 3, and 7 after cell administration, identified by LC/MS. Among all identified proteins (in Sham and MI hearts, three time-points each), 648 were shared by all 6 groups, accounting for 82±5% of total proteins in each group, and enriched under mitochondrion, extracellular exosomes, oxidation-reduction process and poly(A) RNA binding. Notably, 26, 110 and 65 proteins were significantly up-regulated and 11, 28 and 19 proteins were down-regulated in the infarcted vs. Sham heart at the three time-points, respectively; these proteins are pronounced in the GO terms of extracellular matrix organization, response to stress and regulation of apoptotic process and in the KEGG pathways of complements and coagulation cascades, apoptosis, and regulators of actin cytoskeleton.Conclusions: MetRSL274G expression allows successful identification of MSC-specific nascent proteins in the infarcted hearts, which reflect the functional states, adaptive response, and reparative effects of MSCs that may be leveraged to improve cardiac repair.

Regional variations in ex-vivo diffusion tensor anisotropy are associated with cardiomyocyte remodeling in rats after left ventricular pressure overload

BackgroundPressure overload left ventricular (LV) hypertrophy is characterized by increased cardiomyocyte width and ventricle wall thickness, however the regional variation of this remodeling is unclear. Cardiovascular magnetic resonance (CMR) diffusion tensor imaging (DTI) may provide a non-invasive, comprehensive, and geometrically accurate method to detect regional differences in structural remodeling in hypertrophy. We hypothesized that DTI parameters, such as fractional and planar anisotropy, would reflect myocyte remodeling due to pressure overload in a regionally-dependent manner.MethodsWe investigated the regional distributions of myocyte remodeling in rats with or without transverse aortic constriction (TAC) via direct measurement of myocyte dimensions with confocal imaging of thick tissue sections, and correlated myocyte cross-sectional area and other geometric features with parameters of diffusivity from ex-vivo DTI in the same regions of the same hearts.ResultsWe observed regional differences in several parameters from DTI between TAC hearts and SHAM controls. Consistent with previous studies, helix angles from DTI correlated strongly with those measured directly from histological sections (p < 0.001, R2 = 0.71). There was a transmural gradient in myocyte cross-sectional area in SHAM hearts that was diminished in the TAC group. We also found several regions of significantly altered DTI parameters in TAC LV compared to SHAM, especially in myocyte sheet angle dispersion and planar anisotropy. Among others, these parameters correlated significantly with directly measured myocyte aspect ratios.ConclusionsThese results show that structural remodeling in pressure overload LV hypertrophy is regionally heterogeneous, especially transmurally, with a greater degree of remodeling in the sub-endocardium compared to the sub-epicardium. Additionally, several parameters derived from DTI correlated significantly with measurements of myocyte geometry from direct measurement in histological sections. We suggest that DTI may provide a non-invasive, comprehensive method to detect regional structural myocyte LV remodeling during disease.

266Loss of pumilio 1 phosphorylation turns off the regulation of miRNA-221 on gene p27kip1 in the heart

Abstract Background MicroRNAs regulate gene expression post-transcriptionally via complementary sequence pairing of mRNA 3'UTR and miRNA seed region. MiR-221 downregulates p27kip1 Cyclin-dependent kinase inhibitor 1B (p27), a cell cycle inhibitor, through direct targeting. The down-regulation of p27 caused by miR-221 upregulation induces cancer cell proliferation and has been reported to be closely associated with human cancer metastasis. This regulation is dependent on the existence of pumilio 1 (PUM1). Meanwhile miR-221 mimics protect cardiac myocytes against ischemic injury. It is a potential therapeutic target for cardioprotection. We investigated whether miR-221 targeted p27 in the heart. Purpose To test the hypothesis that the regulation of miR-221 on p27 is abolished in the heart due to lack of RNA binding protein (RBP) PMU1 phosphorylation but not due to p27 3'UTR shortening. Methods Primary cardiac fibroblasts (cFB) were isolated and cultured under normoxia and hypoxia. After transfections of miR-221 mimics and mimic control (MC), cell proliferation was assessed by cell number, CCk-8 and Ki67 staining. In vivo rats undergoing left coronary artery ligation (MI) and Sham were treated with intravenous miR-221 mimics. Cells or cardiac tissue were collected to measure p27 and PUM1 by RT-qPCR and Western blot (WB). Full length p27 (coding plus 3'UTR sequence) and 3'UTR p27 sequence were quantified using specific primers. The ratio of 3'UTR/Full length was compared to indicate changes between groups. The ratios of 3'UTR/Full length were compared to indicate changes between groups. Results MiR-221 mimics significantly downregulated the expression of p27 mRNA and protein in cFB in both normoxia and hypoxia. P27 downregulation led to cFB proliferation, which could be abrogated by p27 overexpression. miR-221 mimic treatment increased cardiac miR-221 by ∼2–5 fold in Sham and ∼15 fold in MI. Whilst p27 expression was not affected in either Sham or MI hearts. Simultaneously α-SMA+ cells, an indication of cFB number and activation, were reduced by miR-221 mimic in Sham and MI heart. The ratios of p27 3'UTR/full length did not vary significantly between cultured cFB and heart (Sham and MI), indicating the 3'UTR length did not vary between cFB and the whole heart. PUM1 mRNA expression was stable in cFB and the heart. However, phosphorylated PUM1 was highly expressed in cFB but completely diminished in Sham and MI hearts. Conclusion MiR-221 downregulated p27 in primary cFB and induced cell proliferation in vitro. miR-221 could not regulate cardiac p27 expression nor increase cFB proliferation in vivo. MiR-221 induced p27 regulation was correlated with the presence or absence of phosphorylated PUM1. For the first time, we demonstrated that loss of phosphorylated PUM1 turns off the regulation of miRNA-221 on gene p27 in the heart. The mechanism of PUM1 phosphorylation warrant future investigation.

P3487Pre-ischemic succinate dehydrogenase inhibition is protective against ischemia-reperfusion injury in rat and human myocardium with and without diabetes mellitus

Abstract Background Ischemia-reperfusion (IR) injury can be attenuated through modulation of mitochondrial metabolism with succinate dehydrogenase (SDH) inhibition by dimethyl malonate (DiMAL). However, it is unknown whether SDH inhibition yields protection in aged, diabetic individuals and whether protection translate into human cardiac tissue. Purpose We wished to evaluate if SDH inhibition can be protective in aged, diabetic rat hearts and could be translated to the human myocardium. Methods We studied infarct size in aged non-diabetic and diabetic rat hearts subjected to isolated, retrograde perfusion and global ischemia and reperfusion. The hearts were randomized to: Sham hearts, IR injured hearts and IR injured hearts co-perfused with 0.1 mM or 0.6 mM DiMAL. Infarct size and mitochondrial respiratory capacity were evaluated post-ischemic. To translate our findings into human cardiac tissue, we tested the efficacy of DiMAL treatment in human atrial trabeculae from patients with and without diabetes mellitus undergoing cardiac surgery. We randomized atrial trabeculae to: IR injury, IR injury treated with ischemic preconditioning (IPC) and IR injury treated with 5 mM DiMAL. Contractile force recovery and mitochondrial respiratory capacity were evaluated post-ischemic. Results In non-diabetic rat hearts, DiMAL 0.1 mM reduced infarct size/area-at-risk (IS/AAR) compared to IR hearts (53±7% vs. 69±6% of IS/AAR; p&lt;0.05). In diabetic hearts, an increased concentration of 0.6 mM DiMAL was required to confer protection (62±13% vs. 80±8% of IS/AAR; p&lt;0.05). Mitochondrial complex I linked respiratory capacity was higher in both Sham and DiMAL 0.1 mM groups than with DiMAL 0.6 mM in non-diabetic hearts (157.4±26.5 and 140.0±35.3 vs. 81.8±42.4 pmol O2/(s*mg); p&lt;0.01 and p&lt;0.05). In trabeculae from humans without diabetes mellitus, IPC and DiMAL improved contractile force recovery compared to IR (43±12% and 43±13% vs. 23±13%, p&lt;0.05). In trabeculae from patients with diabetes, IPC but not DiMAL administration improved contractile force recovery compared to IR (48±16% and 19±7% vs. 25±13%, p&lt;0.01 and p&gt;0.99). Mitochondrial complex I linked respiration capacity did not improve by IPC or DiMAL compared to IR in non-diabetic trabeculae (p&gt;0.99). Conclusion Inhibition of the SDH by DiMAL protects the aged non-diabetic and diabetic heart from IR injury but the threshold for cardioprotection is increased in diabetic rat hearts. DiMAL provides cardioprotection similar to IPC in non-diabetic trabeculae but does not protect the human diabetic myocardium when given in identical concentrations. SDH inhibition can provide protection in both animal and human tissue and may provide a new target for pharmacologic conditioning in patients.

Abstract 823: The Extracellular Matrix Component, Hyaluronan, Provokes a Pro-Inflammatory Phenotype in Macrophages

Background: The extracellular matrix (ECM) provides structural and functional support for the myocardium, but myocardial infarction (MI) changes the composition of the ECM. One of the chief components of the ECM, hyaluronan (HA), is elevated after MI; however, specific biological actions of HA—particularly at the level of infiltrating immune cells and implications of such interactions on ventricular remodeling—have not been explored. Goals: Because upregulation of HA coincides with macrophage infiltration after MI, we determined whether hyaluronan interacts with macrophages and investigated the implication of such interactions on macrophage function. Methods: WT mice were subjected to non-reperfused MI to determine changes in hyaluronan synthases (HAS), hyaluronidases (HYAL), and HA levels in the heart. Interaction of HA with macrophages was studied by polarizing bone marrow derived macrophages and analyzing cells by flow cytometry. Next, we characterized the ability of macrophages to metabolize HA by profiling polarized macrophages for HA-metabolizing enzymes, HA receptors, HA-binding proteins, hyaluronidase activity, and phagocytosis. Results: Compared to Sham hearts, MI (n=5/group) augmented the expression of HAS-2 (10-fold, p=0.002) and HYAL-2 (2 to 4-fold, p=0.0004) in the infarct and remote regions of the heart at 5 d post-MI. HA levels (n=8/group) were elevated in the infarct (1 to 2-fold, p=0.0200) and remote (2 to 3-fold, p=0.0007) regions of the heart compared to sham hearts. Polarizing macrophages (n=3/group) in the presence fluorescein-conjugated HA (HA-FL) showed that naïve (M0), pro-inflammatory (M1), and pro-resolving (M2) macrophages interact with HA-FL; M1 showed the highest FITC intensity. Interestingly, exposing macrophages (n=5/group) to HA provoked an inflammatory phenotype, as reflected by enhanced expression of TNFα (4-fold, p=0.0001) and IL-1β (7-fold, p=0.0094) mRNA; HA also enhanced macrophage phagocytosis (0.5-fold, p= 0.0476). Conclusion: Hyaluronan is elevated following MI and can influence macrophage function. Because of the accumulation of hyaluronan and macrophages in the post-MI heart, macrophage-hyaluronan interactions may be a nexus regulating ventricular remodeling.

Abstract 336: The Effect Of Phosphodiesterase 9a Inhibition In 2 Mouse Models Of Diastolic Dysfunction

Low myocardial cGMP-PKG activity is associated with increased cardiomyocyte stiffness in HFpEF. cGMP is mainly hydrolyzed by PDE (phosphodiesterases)5a and PDE9a. Importantly, PDE9a protein expression was reported to be upregulated in human HFpEF myocardium and chronic administration of PDE9a inhibitor reversed pre-established cardiac hypertrophy and systolic dysfunction in mice subjected to TAC (Transverse Aortic Constriction). We evaluated whether PDE9a inhibition had a beneficial effect on diastolic dysfunction (DD). We investigated two mouse models of DD: 1) TAC-DOCA (TAC with deoxycorticosterone acetate (DOCA) supplementation) and 2) LepR db/db , a metabolically induced DD models. Mice were subjected to subcutaneous implantation with osmotic minipumps containing either PD9a inhibitor (PF-4449613) or vehicle for 4 wks. PDE9a transcript was not upregulated in myocardium of either TAC-DOCA or LepR db/db mice. PV analysis revealed that in TAC-DOCA mice, inhibition of PDE9a resulted in reduced LV diastolic stiffness, but impairment of systolic function observed as ventricular-vascular decoupling. In LepR db/db mice, cardiomyocyte stiffness was slightly but significantly reduced in PDE9a inhibitor treated animals. No differences in myocardial fibrosis or cardiac morphometry were observed. The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from LVs of TAC-DOCA mice. Cellular work loop force measurement was conducted before and after administration of PDE9a inhibitor combined with ANP (Atrial Natriuretic Peptide) for 15 mins. Cardiomyocytes from TAC-DOCA had high diastolic stiffness compared to sham hearts, however, no stiffness reduction was observed after acute inhibitor treatment. Conclusion: Chronic inhibition of PDE9a ameliorates LV diastolic stiffness, but suppresses systolic function in TAC-DOCA induced DD mice. The LV stiffness reduction is in part due to cardiomyocyte stiffness reduction. The advantage of PDE9a inhibition on diastolic function might be more evident, with less systolic suppression, in hearts with PDE9a upregulation.

Single-cell expression profiling reveals dynamic flux of cardiac stromal, vascular and immune cells in health and injury.

Besides cardiomyocytes (CM), the heart contains numerous interstitial cell types which play key roles in heart repair, regeneration and disease, including fibroblast, vascular and immune cells. However, a comprehensive understanding of this interactive cell community is lacking. We performed single-cell RNA-sequencing of the total non-CM fraction and enriched (Pdgfra-GFP+) fibroblast lineage cells from murine hearts at days 3 and 7 post-sham or myocardial infarction (MI) surgery. Clustering of >30,000 single cells identified >30 populations representing nine cell lineages, including a previously undescribed fibroblast lineage trajectory present in both sham and MI hearts leading to a uniquely activated cell state defined in part by a strong anti-WNT transcriptome signature. We also uncovered novel myofibroblast subtypes expressing either pro-fibrotic or anti-fibrotic signatures. Our data highlight non-linear dynamics in myeloid and fibroblast lineages after cardiac injury, and provide an entry point for deeper analysis of cardiac homeostasis, inflammation, fibrosis, repair and regeneration.

PKA phosphorylation underlies functional recruitment of sarcolemmal SK2 channels in ventricular myocytes from hypertrophic hearts.

Key points Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.SK channels are voltage independent and their gating is controlled by intracellular [Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.Using a rat model of cardiac hypertrophy induced by thoracic aortic banding, we found that functional upregulation of SK2 channels in hypertrophic rat ventricular cardiomyocytes is driven by protein kinase A (PKA) phosphorylation. Using site‐directed mutagenesis, we identified serine‐465 as the site conferring PKA‐dependent effects on SK2 channel function.PKA phosphorylation attenuates I SK rectification by reducing the Ca2+/voltage‐dependent inhibition of SK channels without changing their sensitivity to activating submicromolar [Ca2+]i.This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. Small‐conductance Ca2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation (isoproterenol, 100 nmol L–1) evoked I SK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of I SK via reduction Ca2+/voltage‐dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.

Increased ketone body oxidation provides additional energy for the failing heart without improving cardiac efficiency.

The failing heart is energy-starved and inefficient due to perturbations in energy metabolism. Although ketone oxidation has been shown recently to increase in the failing heart, it remains unknown whether this improves cardiac energy production or efficiency. We therefore assessed cardiac metabolism in failing hearts and determined whether increasing ketone oxidation improves cardiac energy production and efficiency. C57BL/6J mice underwent sham or transverse aortic constriction (TAC) surgery to induce pressure overload hypertrophy over 4-weeks. Isolated working hearts from these mice were perfused with radiolabelled β-hydroxybutyrate (βOHB), glucose, or palmitate to assess cardiac metabolism. Ejection fraction decreased by 45% in TAC mice. Failing hearts had decreased glucose oxidation while palmitate oxidation remained unchanged, resulting in a 35% decrease in energy production. Increasing βOHB levels from 0.2 to 0.6 mM increased ketone oxidation rates from 251 ± 24 to 834 ± 116 nmol·g dry wt-1 · min-1 in TAC hearts, rates which were significantly increased compared to sham hearts and occurred without decreasing glycolysis, glucose, or palmitate oxidation rates. Therefore, the contribution of ketones to energy production in TAC hearts increased to 18% and total energy production increased by 23%. Interestingly, glucose oxidation, in parallel with total ATP production, was also significantly upregulated in hearts upon increasing βOHB levels. However, while overall energy production increased, cardiac efficiency was not improved. Increasing ketone oxidation rates in failing hearts increases overall energy production without compromising glucose or fatty acid metabolism, albeit without increasing cardiac efficiency.

Role of the cytoskeleton in the development of a hypofibrotic cardiac fibroblast phenotype in volume overload heart failure.

Hemodynamic load regulates cardiac remodeling. In contrast to pressure overload (increased afterload), hearts subjected to volume overload (VO; preload) undergo a distinct pattern of eccentric remodeling, chamber dilation, and decreased extracellular matrix content. Critical profibrotic roles of cardiac fibroblasts (CFs) in postinfarct remodeling and in response to pressure overload have been well established. Little is known about the CF phenotype in response to VO. The present study characterized the phenotype of primary cultures of CFs isolated from hearts subjected to 4 wk of VO induced by an aortocaval fistula. Compared with CFs isolated from sham hearts, VO CFs displayed a "hypofibrotic" phenotype, characterized by a ~50% decrease in the profibrotic phenotypic markers α-smooth muscle actin, connective tissue growth factor, and collagen type I, despite increased levels of profibrotic transforming growth factor-β1 and an intact canonical transforming growth factor-β signaling pathway. Actin filament dynamics were characterized, which regulate the CF phenotype in response to biomechanical signals. Actin polymerization was determined by the relative amounts of G-actin monomers versus F-actin. Compared with sham CFs, VO CFs displayed ~78% less F-actin and an increased G-actin-to-F-actin ratio (G/F ratio). In sham CFs, treatment with the Rho kinase inhibitor Y-27632 to increase the G/F ratio resulted in recapitulation of the hypofibrotic CF phenotype observed in VO CFs. Conversely, treatment of VO CFs with jasplakinolide to decrease the G/F ratio restored a more profibrotic response (>2.5-fold increase in α-smooth muscle actin, connective tissue growth factor, and collagen type I). NEW & NOTEWORTHY The present study is the first to describe a "hypofibrotic" phenotype of cardiac fibroblasts isolated from a volume overload model. Our results suggest that biomechanical regulation of actin microfilament stability and assembly is a critical mediator of cardiac fibroblast phenotypic modulation.

Abstract 275: Quantification of the Mitochondrial Protein Interactome in Failing Hearts

Mitochondrial dysfunction is a maladaptive mechanism in the progression of heart failure. However, the cause of mitochondrial dysfunction during cardiac stress remains elusive. To understand the pathogenic role of mitochondria in heart failure, a systems biology approach was used to quantify the protein interaction network by comparing mitochondria isolated from healthy and failing mouse heart. This is achieved by Protein Interaction Reporter (PIR) technology consisting of a cleavable, MS-identifiable, isotope labelled cross-linker, which allows identification and quantification of interacting peptide pairs. Heart failure was induced in three month old, wild-type C57Bl6/NCrl mice using chronic pressure overload by transverse aortic constriction (TAC, n=8) surgery. Age-matched Sham-operated (Sham, n=8) mice were used as controls. Four weeks post-surgery, cardiac function was measured by echocardiography. TAC hearts showed significantly lower contractile function than Sham (fractional shortening 49±2.1% vs 13±4.0%), and were dilated (LVID 2.7±0.34mm vs 3.4±0.46mm). Cardiac hypertrophy of TAC hearts (HW/TL 5.0±0.35 vs 9.1±0.58) was associated with greater lung edema (LW wet/dry 3.9±0.49 vs 4.9±0.24). Freshly isolated mitochondria from TAC and Sham hearts were cross-linked with heavy- or light-isotope labelled crosslinkers, digested, enriched, and analyzed by LC/MS 3 . Released peptide spectra were identified and categorized by known molecular functions using a MitoCarta 2.0 constructed database. We identified 3502 non-redundant cross-linked peptide pairs from 297 proteins, involving 1758 lysine residues linked in 2571 lysine-lysine interactions. From 3502 total links, we detected 2304 intra- and 1016 inter-protein links. Inter-protein changes between TAC/Sham were most prevalent in the electron transport chain and ATP synthasome networks. Intra-protein changes were evident in TCA cycle, fatty acid oxidation, and nucleotide transport, suggesting conformational remodeling. Interactome changes in amino acid metabolism, mitochondrial transcription and translation pathways were undetected. A complete picture of mitochondrial protein landscape in the failing heart will lead us to novel mechanisms and therapy.

TRPV4 channel deletion or pharmacological inhibition protects heart against adverse remodeling post‐myocardial infarction

Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. Cardiac remodeling following myocardial infarction involves scar formation by synthesis and reorganization of ECM which is mediated by myofibroblasts. We have previously shown that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts via integration of soluble and mechanical signaling. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To determine this, we have subjected WT and TRPV4KO mice to MI (permanent LAD ligation). 2D‐echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post‐MI in TRPV4KO mice compared to WT mice. Further, we found reduced fibrosis at infarcted and remote zones in TRPV4KO‐MI hearts compared to WT‐MI and sham hearts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post‐MI compared to WT hearts. To explore the translational significance of these findings, in separate experiments, we have given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. Cardiac function analysis revealed that both ejection fraction and fractional shortening were preserved in GSK2193874‐treated WT mice compared to either WT or vehicle treated mice. Our results thus suggest that targeting TRPV4 protects the heart from myocardial infarction‐induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The contribution of fatty acid and ketone body oxidation to energy production increases in the failing heart and is associated with a decrease in cardiac efficiency

Background: The failing heart is energy-starved and inefficient due to perturbations in energy metabolism. Since recent evidence suggests that ketone body oxidation increases in the failing heart as an adaptive mechanism to counteract reductions in fatty acid oxidation, our aim was to assess overall cardiac metabolism in heart failure, to establish what metabolic alterations contribute to reduced cardiac efficiency. Methods: C57BL/6J male mice (12 wk old) underwent either sham surgery, or transverse aortic constriction (TAC) surgery to induce pressure overload hypertrophy over a 4 wk period. Isolated working hearts from these mice were then perfused with appropriately 3H or 14C labelled glucose (5 mM), palmitate (0.8 mM), and ß-hydroxybutyrate (0.6 mM) to assess oxidative metabolism and glycolysis. Results: A 45% reduction in %EF was seen in intact TAC mice and a 54% decrease in cardiac work was seen in isolated working hearts from TAC mice. However, normal cardiac Krebs Cycle acetyl CoA production was seen compared to sham mice, reflecting a reduction in cardiac efficiency. Correspondingly, absolute glucose oxidation rates decreased in TAC compared to sham hearts, whereas absolute rates of fatty acid and ketone body oxidation were similar. However, normalization to cardiac work revealed that glucose oxidation was not depressed in failing hearts, although glycolysis was increased. Conversely, both fatty acid and ketone body oxidation increased in TAC hearts when normalized to cardiac work. This increased reliance on fatty acid oxidation challenges the current dogma suggesting that fatty acid oxidation is depressed in the failing heart. Conclusion: In the failing heart, a decreased cardiac efficiency is associated with increases in the contributions of ketone body and fatty acid oxidation to energy production. The latter observation suggests that normalizing excessive fatty acid oxidation in the failing heart may be a novel approach to improve cardiac efficiency.

Abstract 171: Promoter Negative Elongation Factor a (NelfA) Occupancy Required for Gene Transcription in Heart During Hypertrophy

Adaptive change in gene expression is one of the earliest responses of heart to hypertrophy. Gene transcription is tightly regulated by systematic recruitment of factors including positive and negative elongation complexes of RNA Pol II. Recently we embarked on the task of deconstructing the transcriptional machinery in the heart, by examining Pol II and TFIIB dynamics across the genome in sham and hypertrophied hearts. In this study, we continue examining the active transcriptional code by measuring occupancy of two interdependent factors that belong to positive (PTEFb) and negative (NELF) elongation complexes, Cdk9 and NelfA, respectively. Studies in non-cardiac cells have implicated these two factors in the regulation of promoter (prom) clearance of pol II, where Cdk9 mediated phosphorylation of NelfA results in its release from paused pol II and initiation of productive elongation. As expected, we observed increase Cdk9 (&gt;1.5) association with proms in 59.7% of expressed genes (8257 of 13816), while 6% show decrease in prom Cdk9 (&lt;0.75) in TAC vs. sham hearts. In contrast to previous reports, we did not see any correlation between Cdk9 and NelfA occupancy, where 93.2% and 68% of genes did not show change in NelfA with increased or decreased Cdk9 levels in TAC hearts, respectively. Interestingly, highest enrichment of NelfA (av. density 53.5) was observed on genes regulated by prom clearance of pol II (av. prom den 9.5 vs. 7.4; av. Ingene den 3.0 vs. 3.6, sham vs. TAC; housekeeping genes) with no change in NelfA (89.8% of genes). On other hand, 3.3% of genes showed increase in NelfA (av. den 4.6 vs. 8.4, sham vs. TAC), along with increase in pol II recruitment (av. prom den 3.3 vs. 4.04; av. ingene den 1.8 vs. 2.4, sham vs. TAC; cytoskeletal, cell cycle genes). We confirmed increase in mRNA abundance of these genes, (eg. Acta 1) in TAC vs. sham hearts. Intriguingly, genes that were associated with reduced NelfA (av. den 21.7 vs. 14.8, sham vs. TAC) were mostly cardiac specific genes (e.g. cardiac muscle contraction), which show increase in pol II, but no change in mRNA levels with TAC (eg. Actc). While we are investigating the role of NelfA in heart, ChIP- Seq data suggest that NelfA might be required for productive elongation and increase in global gene expression during hypertrophy

Abstract 269: Epicardial Deletion of Myocardin-related Transcription Factors Prevent Pathological Cardiac Remodeling After Ischemic Injury

Epicardium-derived progenitor cell (EPDC) activation after injury is a cardio-restorative mechanism in the zebrafish heart, but this response is limited in adult mammals. Identification of mechanisms that regulate adult EPDC function is critical to enhance epicardium-mediated cardiac repair. We previously found that myocardin-related transcription factors (MRTFs) regulate EPDC mobilization and coronary vessel maturation during development. Adult mice lacking both MRTF-A and -B in the epicardium (DKO) were evaluated at baseline and after myocardial infarction (MI) in this study. In non-injured DKO hearts, MRTF expression is reduced in epicardium-derived lineages such as fibroblasts and pericytes, but is unchanged in cardiomyocytes and endothelial cells. DKO mice display increased cellular retention in the epicardium as compared to wildtype mice (WT), consistent with observations during embryonic development. While WT mice demonstrate a decline in cardiac function after MI, ejection fraction is preserved in DKO mice, which also display reduced fibrotic scarring and cardiomyocyte hypertrophy. In order to determine the expression levels of pro-fibrotic genes in epicardial-derived lineages, we isolated CD31-NG2+PDGFRβ+ (pericytes), CD31-NG2-PDGFRβ- (fibroblasts) and CD31+ (endothelial cells) populations from sham and 7-day post-MI hearts. Postn (11-fold), Col1a1 (4-fold), Col3a1 (4.8-fold) and Acta2 (5-fold) are similarly activated in pericyte and fibroblast populations after MI. Of note, pericytes express abundant levels of fibronectin (38-fold vs. 16-fold in fibroblasts) and pro-inflammatory interleukin-1β (41-fold vs 12-fold in fibroblasts) suggesting that pericytes may also contribute to inflammation and fibrosis after cardiac injury. Indeed, NG2+ pericytes are dramatically reduced in remote (5-fold) and damaged regions (3.3-fold) of DKO hearts compared to WT. From this data, we hypothesize that MRTF deletion alters pro-migratory and fibrotic gene programs and reduces inflammatory signals in EPDCs decreasing the presence of scar and improving cardiac function after ischemic injury.

Abstract 436: Targeting Fatty Acid Oxidation by Acetyl-CoA Carboxylase 2 Deletion in Pathological Hypertrophy

We have previously shown that preserving fatty acid oxidation (FAO) by cardiac-specific deletion of Acetyl-CoA Carboxylase 2 (ACC2) prevents the shift of substrate preference towards glucose, reduces cardiomyocyte hypertrophy and preserves cardiac function during chronic pressure overload. To determine whether maintaining FAO specifically prevented cardiomyocyte hypertrophy, we treated adult rat cardiomyocytes (CMs) with and without adenoviral-mediated ACC2 knock-down (KD) with phenylephrine (PE, 10 μM). ACC2 KD effectively prevented CM hypertrophy after PE stimulation compared to control CMs (+9±6% vs. 42±6%) in medium supplemented with fatty acids (FA) (5.5 mM glucose, 0.4 mM mixed long-chain FAs and 0.1 mU/ml insulin). Whereas PE stimulation in control CMs increased glucose uptake (+28±8%) this was normalized after ACC2 KD. Inhibiting FAO by etomoxir or increasing glucose utilization by dichloroacetate abolished the beneficial effects of ACC2 KD after PE stimulation. When cultured in glucose-free medium supplemented with FA, ACC2 KD was incapable of preventing cardiomyocyte hypertrophy. However, replacing glucose with pyruvate or propionate restored the anti-hypertrophic effect of ACC2 KD. To determine the therapeutic effects of increasing FAO in vivo , male mice were subjected to transverse aortic constriction (TAC) and sham surgery. Three weeks after surgery, TAC mice had a significant increase in left ventricular (LV) mass as determined by echocardiography compared to sham operated mice (130 vs. 92 mg). At this time point, cardiac-specific ACC2 deletion (iKO) was induced by tamoxifen (tam) administration. ACC2 protein was effectively deleted in iKO sham and TAC hearts compared to controls (CON) 2 weeks post tam injection. FAO was 2-fold higher in iKO TAC vs. CON TAC hearts as assessed by isolated heart perfusion and 13C NMR spectroscopy. CON and iKO mice will be followed until 12 weeks after TAC to determine cardiac function and assess hypertrophy. Together, these data indicate that increasing FAO via inactivation of ACC2 exerts anti-hypertrophic effect in adult cardiomyocytes. Deleting ACC2 in the hypertrophic heart in vivo can increase FAO and represents a valid target to treat pathological cardiac hypertrophy.

Chronic inflammation and apoptosis propagate in ischemic cerebellum and heart of non-human primates.

The major pathological consequences of cerebral ischemia are characterized by neurological deficits commonly ascribed to the infarcted tissue and its surrounding region, however, brain areas, as well as peripheral organs, distal from the original injury may manifest as subtle disease sequelae that can increase the risks of co-morbidities complicating the disease symptoms. To evaluate the vulnerability of the cerebellum and the heart to secondary injuries in the late stage of transient global ischemia (TGI) model in non-human primates (NHP), brain and heart tissues were collected at six months post-TGI. Unbiased stereological analyses of immunostained tissues showed significant Purkinje cells loss in lobule III and lobule IX of the TGI cerebellum relative to sham cerebellum, with corresponding upregulation of inflammatory and apoptotic cells. Similarly, TGI hearts revealed significant activation of inflammatory and apoptotic cells relative to sham hearts. Aberrant inflammation and apoptosis in the cerebellum and the heart of chronic TGI-exposed NHPs suggest distal secondary injuries manifesting both centrally and peripherally. These results advance our understanding on the sustained propagation of chronic secondary injuries after TGI, highlighting the need to develop therapeutic interventions targeting the brain, as well as the heart, in order to abrogate cerebral ischemia and its related co-morbidities.

Label-free imaging of healthy and infarcted fetal sheep hearts by two-photon microscopy.

Coronary heart disease is one of the largest causes of death worldwide, making this a significant health care issue. A critical problem for the adult human heart is that it does not undergo effective repair in response to damage, leaving patients with a poor prognosis. Unlike the adult, fetal hearts have the ability to repair after myocardial damage. Using two-photon microscopy, we have visualised the morphological and metabolic changes following myocardial infarction in sheep fetuses, to characterise response to cardiac injury in a mammalian model. Following myocardial infarction, fetal hearts showed no significant increase in collagen deposition in the region of the infarction, when compared to either the surrounding tissue or shams. In contrast, metabolic activity (i. e. NAD(P)H and FAD) was significantly reduced in the region of myocardial infarction, when compared to either the surrounding tissue or sham hearts. For comparison, we also imaged two hearts from preadolescent sheep (sham and myocardial infarction) and showed highly ordered collagen deposition with decreased metabolic activity within the infarcted area. Therefore, two-photon imaging had the capacity to image both morphological and metabolic changes in response to myocardial infarction and showed differences in the response with age. Picture: Two-photon imaging of myocardial infarction (b and d) enabled the visualisation of increased collagen (blue; Em=431 nm) and changes in other tissue autofluorescence (green; Em=489-606 nm) in fetal (a and b) and preadolescent (c and d) hearts, compared to shams (a and c). The excitation wavelength was 840 nm. Scale bars: 10 μm.

Nestin expression is upregulated in the fibrotic rat heart and is localized in collagen-expressing mesenchymal cells and interstitial CD31(+)- cells.

Renal and lung fibrosis was characterized by the accumulation of collagen-immunoreactive mesenchymal cells expressing the intermediate filament protein nestin. The present study tested the hypothesis that nestin expression was increased in the hypertrophied/fibrotic left ventricle of suprarenal abdominal aorta constricted adult male Sprague-Dawley rats and induced in ventricular fibroblasts by pro-fibrotic peptide growth factors. Nestin protein levels were upregulated in the pressure-overloaded left ventricle and expression positively correlated with the rise of mean arterial pressure. In sham and pressure-overloaded hearts, nestin immunoreactivity was detected in collagen type I(+)-and CD31(+)-cells identified in the interstitium and perivascular region whereas staining was absent in smooth muscle α-actin(+)-cells. A significantly greater number of collagen type I(+)-cells co-expressing nestin was identified in the left ventricle of pressure-overloaded rats. Moreover, an accumulation of nestin(+)-cells lacking collagen, CD31 and smooth muscle α-actin staining was selectively observed at the adventitial region of predominantly large calibre blood vessels in the hypertrophied/fibrotic left ventricle. Angiotensin II and TGF-β1 stimulation of ventricular fibroblasts increased nestin protein levels via phosphatidylinositol 3-kinase- and protein kinase C/SMAD3-dependent pathways, respectively. CD31/eNOS(+)-rat cardiac microvascular endothelial cells synthesized/secreted collagen type I, expressed prolyl 4-hydroxylase and TGF-β1 induced nestin expression. The selective accumulation of adventitial nestin(+)-cells highlighted a novel feature of large vessel remodelling in the pressure-overloaded heart and increased appearance of collagen type I/nestin(+)-cells may reflect an activated phenotype of ventricular fibroblasts. CD31/collagen/nestin(+)-interstitial cells could represent displaced endothelial cells displaying an unmasked mesenchymal phenotype, albeit contribution to the reactive fibrotic response of the pressure-overloaded heart remains unknown.