Abstract 16870: Dissociation of Mitochondrial HK-II Triggers Mitochondria Specific Autophagy

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Shigeki Miyamoto ◽  
David J Roberts ◽  
Valerie P Tan-Sah
Keyword(s):  
Energy Production ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Hexokinase Ii ◽  
Survival Pathway ◽  
Mitochondrial Membrane Depolarization ◽  
Neonatal Rat Ventricular Myocytes

Introduction: There is emerging evidence that the metabolic pathway interplays with the survival pathway to preserve cellular homeostasis. Hexokinases (HKs) catalyze the first step of glucose metabolism and hexokinase-II (HK-II) is the predominant isoform in the heart. Our recent study revealed that HK-II positively regulates general autophagy in the absence of glucose. Mitochondrial HK-II (mitoHK-II) is regulated by Akt and provides cardioprotection while it is decreased in the ischemic heart. Hypothesis: We tested the hypothesis that mitoHK-II dissociation triggers mitochondria specific autophagy (mitophagy). Results: As previously reported, mitoHK-II levels were decreased by ~40% in the perfused mouse heart subjected to global ischemia and in neonatal rat ventricular myocytes (NRVMs) subjected to simulated ischemia. To assess the role of mitoHK-II dissociation, mitoHK-II dissociating peptide (15NG) was expressed in NRVMs. MitoHK-II was decreased by 40% in NRVMs expressing 15NG which was accompanied with Parkin translocation to mitochondria and ubiquitination of mitochondrial proteins. This response was attenuated by Parkin knockdown and reversed by the recovery of mitoHK-II by co-expression of HK-II but not by that of mitochondria binding deficient mutant. 15NG expression did not induce mitochondrial membrane depolarization nor PINK1 stabilization at mitochondria, suggesting that the effects of mitoHK-II dissociation is not dependent on the previously established mitochondria depolarization/PINK1 pathway. This was confirmed by the experiments using PINK1 siRNA. Modest dissociation of mitoHK-II (by 20%) did not induce mitophagic responses but remarkably enhanced FCCP induced mitophagy, indicating that these two pathways are synergetic. We will be analyzing 15NG transgenic mice generated in our lab to determine the mitophagic role of mitoHK-II dissociation in vivo. Conclusions: These results suggest that mitoHK-II dissociation can regulate Parkin dependent mitophagy, in conjunction with depolarization dependent mechanisms and that HK-II could confer cardioprotection by switching the cell from an energy production to an energy conservation mode under ischemia.

Cytochrome P-450 metabolites in endothelin-stimulated cardiac hormone secretion

2004 ◽  
Vol 286 (5) ◽  
pp. R888-R893 ◽  
Author(s):  
Sook Jeong Lee ◽  
Carol S. Landon ◽  
Stanley J. Nazian ◽  
John R. Dietz
Keyword(s):  
Protein Kinase ◽  
Inhibitory Action ◽  
Ventricular Myocytes ◽  
Hormone Secretion ◽  
Neonatal Rat ◽  
Kinase C ◽  
Neonatal Rat Ventricular Myocytes ◽  
Cytochrome P 450 ◽  
Kinase A

We examined the role of cytochrome P-450-arachidonate (CYP450-AA) metabolites in endothelin-1 (ET-1)-stimulated atrial natriuretic peptide (ANP) and pro-ANP-(1-30) secretion from the heart. 17-Octadecynoic acid (17-ODYA, 10-5 M) significantly inhibited ANP secretion stimulated by ET-1 (10-8 M) in the isolated perfused rat atria and inhibited pro-ANP-(1-30) secretion stimulated by ET-1 (10-8 M) or 20-hydroxyeicosatetraenoic acid in cultured neonatal rat ventricular myocytes (NRVM). In NRVM, 17-ODYA significantly ( P < 0.05) increased secretion of cAMP but had no significant effect on the secretion of cGMP from NRVM. Staurosporine, an inhibitor of protein kinase C, completely blocked the inhibitory action of 17-ODYA, whereas a protein kinase A inhibitor, H-89 (5 × 10-5 M), did not significantly attenuate the effects of 17-ODYA. The results show that the inhibitory action of 17-ODYA on ET-1-augmented ANP secretion is mediated through cAMP and suggest that CYP450-AA may play an important role in ET-1-induced cardiac hormone secretion.

scholarly journals Nitric oxide-induced cardioprotection in cultured rat ventricular myocytes

2000 ◽  
Vol 278 (4) ◽  
pp. H1211-H1217 ◽  
Author(s):  
Roby D. Rakhit ◽  
Richard J. Edwards ◽  
James W. Mockridge ◽  
Anwar R. Baydoun ◽  
Amanda W. Wyatt ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Ventricular Myocytes ◽  
Culture Conditions ◽  
Neonatal Rat ◽  
No Donor ◽  
Rat Ventricular Myocytes ◽  
Kinase C ◽  
Neonatal Rat Ventricular Myocytes ◽  
Oxide Synthase

The aim of this study was to investigate the role of nitric oxide (NO) in a cellular model of early preconditioning (PC) in cultured neonatal rat ventricular myocytes. Cardiomyocytes “preconditioned” with 90 min of stimulated ischemia (SI) followed by 30 min reoxygenation in normal culture conditions were protected against subsequent 6 h of SI. PC was blocked by N G-monomethyl-l-arginine monoacetate but not by dexamethasone pretreatment. Inducible nitric oxide synthase (NOS) protein expression was not detected during PC ischemia. Pretreatment (90 min) with the NO donor S-nitroso- N-acetyl-l,l-penicillamine (SNAP) mimicked PC, resulting in significant protection. SNAP-triggered protection was completely abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) but was unaffected by chelerythrine or the presence of glibenclamide and 5-hydroxydecanoate. With the use of RIA, SNAP treatment increased cGMP levels, which were blocked by ODQ. Hence, NO is implicated as a trigger in this model of early PC via activation of a constitutive NOS isoform. After exposure to SNAP, the mechanism of cardioprotection is cGMP dependent but independent of protein kinase C or ATP-sensitive K+ channels. This differs from the proposed mechanism of NO-induced cardioprotection in late PC.

scholarly journals Matrix metalloproteinase-2 in oncostatin M-induced sarcomere degeneration in cardiomyocytes

2016 ◽  
Vol 311 (1) ◽  
pp. H183-H189 ◽  
Author(s):  
Xiaohu Fan ◽  
Bryan G. Hughes ◽  
Mohammad A. M. Ali ◽  
Brandon Y. H. Chan ◽  
Katherine Launier ◽  
...  
Keyword(s):  
Matrix Metalloproteinase ◽  
Troponin I ◽  
Ventricular Myocytes ◽  
Oncostatin M ◽  
Neonatal Rat ◽  
Matrix Metalloproteinase 2 ◽  
Mmp Inhibitor ◽  
Confocal Immunofluorescence ◽  
Neonatal Rat Ventricular Myocytes

Cardiomyocyte dedifferentiation may be an important source of proliferating cardiomyocytes facilitating cardiac repair. Cardiomyocyte dedifferentiation and proliferation induced by oncostatin-M (OSM) is characterized by sarcomere degeneration. However, the mechanism underlying sarcomere degeneration remains unclear. We hypothesized that this process may involve matrix metalloproteinase-2 (MMP-2), a key protease localized at the sarcomere in cardiomyocytes. We tested the hypothesis that MMP-2 is involved in the sarcomere degeneration that characterizes cardiomyocyte dedifferentiation. Confocal immunofluorescence and biochemical methods were used to explore the role of MMP-2 in OSM-induced dedifferentiation of neonatal rat ventricular myocytes (NRVM). OSM caused a concentration- and time-dependent loss of sarcomeric α-actinin and troponin-I in NRVM. Upon OSM-treatment, the mature sarcomere transformed to a phenotype resembling a less-developed sarcomere, i.e., loss of sarcomeric proteins and Z-disk transformed into disconnected Z bodies, characteristic of immature myofibrils. OSM dose dependently increased MMP-2 activity. Both the pan-MMP inhibitor GM6001 and the selective MMP-2 inhibitor ARP 100 prevented sarcomere degeneration induced by OSM treatment. OSM also induced NRVM cell cycling and increased methyl-thiazolyl-tetrazolium (MTT) staining, preventable by MMP inhibition. These results suggest that MMP-2 mediates sarcomere degeneration in OSM-induced cardiomyocyte dedifferentiation and thus potentially contributes to cardiomyocyte regeneration.

Abstract 17473: GRK2 in the Mitochondria: Uncovering Novel Mechanisms in Heart Failure

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Kimberly M Ferrero ◽  
Gizem Kayki Mutlu ◽  
Jessica M Pfleger ◽  
Douglas G Tilley ◽  
Walter J Koch
Keyword(s):  
Heart Failure ◽  
Mitochondrial Dysfunction ◽  
Ventricular Myocytes ◽  
Atp Synthesis ◽  
Neonatal Rat ◽  
Negative Effects ◽  
Protein Kinase C Inhibitor ◽  
Neonatal Rat Ventricular Myocytes

Introduction: During heart failure, levels and activity of G protein-coupled receptor kinase 2 (GRK2) increase. GRK2 is canonically studied in the phosphorylation of GPCRs and β-adrenergic desensitization. Noncanonical activities of GRK2 are being uncovered, however. Our lab has recently discovered that in cardiac myocytes, GRK2 translocates to the mitochondria ( mtGRK2 ) following injury and is associated with negative effects on metabolism and cell survival. Hypothesis: GRK2 plays a role in regulating mitochondrial function following cardiac stress and contributes to HF pathogenesis in a novel manner, by interacting with a novel group of mitochondrial proteins involved in pro-death signaling, bioenergetics and substrate utilization. Methods: Mitochondrial translocation of GRK2 was validated with either protein kinase C inhibitor (chelerythine) administration or hypoxia/reoxygenation stress in primary neonatal rat ventricular myocytes or a cardiac-like cell line. Immunoprecipitation of the GRK2 interactome basally and under stress conditions was conducted endogenously in vitro, in vivo , and with purified recombinant GRK2 peptides. Proteins were separated via SDS-PAGE and potential binding partners were identified by mass spectroscopy (LCMS) and proteomics analysis conducted with Ingenuity Pathway (IPA; Qiagen) software to determine which partners in the GRK2 interactome were potentially involved in mitochondrial dysfunction. Results: Subunits of Complexes I, II, IV and V of the electron transport chain were identified as potential mtGRK2 interacting partners. Several mtGRK2-ETC interactions were increased following oxidative stress-induced translocation of GRK2. Finally, mtGRK2 appears to phosphorylate some of the interactome partners identified in mitochondrial dysfunction. Conclusions: The phosphorylation of subunits of the ATP synthesis machinery by mtGRK2, or other mechanisms of interaction between these proteins, may be regulating some of the phenotypic effects of HF previously observed by our lab, such as increased ROS production and reduced fatty acid metabolism. Further research is essential to elucidate the novel role of GRK2 in regulating mitochondrial bioenergetics and cell death in failing hearts.

Abstract 307: Phospholipase Cε Is Involved in Gq-Dependent Protein Kinase D Activation in Cardiac Myocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Alan Smrcka ◽  
Lianghui Zhang ◽  
Sundeep Malik
Keyword(s):  
Protein Kinase ◽  
G Proteins ◽  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Protein Kinase D ◽  
Dependent Protein Kinase ◽  
Dependent Protein ◽  
Neonatal Rat Ventricular Myocytes ◽  
Phospholipase Cε

We previously demonstrated that PLCε is a critical mediator of endothelin-1 (ET-1)- and norepinephrine (NE)-dependent hypertrophy in neonatal rat ventricular myocytes (NRVMs). Both α-adrenergic and ET-1 receptors couple to Gq as well as other G proteins. To determine if PLCε is required for Gαq-dependent hypertrophy NRVMs were infected with adenoviruses expressing wtGαq and PLCε siRNA followed by measurement of hypertrophy. PLCε-siRNA significantly inhibited Gαq-induced increases in myocyte area and atrial natriuretic factor (ANF) mRNA expression. Similarly, disruption of PLCε association with perinuclear mAKAP inhibited Gαq-dependent hypertrophy. These data suggest that ET-1 and PE signal, at least in part, through Gαq and PLCε. To explore the functional role of PLCε in ET-1/Gq dependent hypertrophy, activation of protein kinase D (PKD) in NRVMs was assessed in response to ET-1. PLCε-siRNA significantly inhibited ET-1 induced PKD activation (∼50% inhibition). Disruption of PLCε-mAKAP interactions also significantly inhibited ET-1 induced PKD activation (∼50% inhibition). We propose that PLCε scaffolded to mAKAP at the nuclear envelope responds to Gαq-dependent, as well as other hypertrophic signals, to locally regulate PKD in a process that is critical for hypertrophy development.

Abstract 4626: Optimal Dose of Adv-hHCN4 Creates Stable and Long-lasting Biopacing Activity in Cultured Ventricular Cardiomyocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Yong-Fu Xiao ◽  
Alena Nikolskaya ◽  
Lepeng Zeng ◽  
Xiaohong Qiu ◽  
Deborah A. Jaye ◽  
...  
Keyword(s):  
Channel Blocker ◽  
Ventricular Myocytes ◽  
Optimal Dose ◽  
Neonatal Rat ◽  
Normal Morphology ◽  
Rat Ventricular Myocytes ◽  
Beating Rate ◽  
Neonatal Rat Ventricular Myocytes

Purpose: Hyperpolarization-activated cyclic nucleotide-gated (HCN) genes have been successfully used as a strategy for recreating cardiac biological pacemakers in animal models. However, optimal dose of HCN and toxicity from HCN overexpression have not been investigated. Therefore, we assessed the effects of various titers of adenoviral human HCN4-GFP vector (Adv-hHCN4) on cardiomyocytes. Methods: Neonatal rat ventricular myocytes (NRVMs) were isolated, selected and cultured on microelectrode arrays to assess their automaticities. Morphology and apoptosis with and without HCN or Ca 2+ channel inhibitor were also assessed. Results: Beating rates significantly increased in NRVMs after hHCN4 infection (Fig. 1 ). For example, the rates were gradually increased to 235±11 beat/min on day 7 after hHCN4 infection with 1×10 5 PFU/array. In contrast, control cells showed low rates. NRVMs with ≥10 6 PFU/array Adv-hHCN4 reached faster rates early and subsequently stopped beating (Fig. 1 ). In addition, myocytes with ≥10 6 PFU/array Adv-hHCN4 underwent significant apoptosis (>50%) which potentially resulted from hHCN4 overexpression and was blocked by the HCN channel blocker Cs + (1 mM), but not by the Ca 2+ channel inhibitor nifedipine. In addition, myocytes infected with ≥10 6 PFU/array Adv-GFP maintained normal morphology and rate. Our data demonstrate that hHCN4 transfer significantly and dose-dependently increased beating rates of NRVMs. However, overexpression of HCN could cause apoptosis. Therefore, an optimal dose of HCN gene is important for reducing toxicity and creating stable and long-lasting biopacing activity in cardiomyocytes in vitro, and probably also in vivo. Figure 1. Effects of hHCN4 infection on automaticities of neonatal rat ventricular myocytes. Each data point represents an averaged beating rate (mean ± SE) from 8 to 10 arrays. Various titers (1×10 5 to 1×10 7 PFU/500,000 cells per array) of Adv-Hhcn4 (expect control) were added to the arrays after measurements on day 0 (see the arrow)

Abstract 33: Contractile Dysfunction In The Mouse Heart Caused By Phospholipase C beta1b Mediated Activation Of Protein Kinase Calpha

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
David R Grubb ◽  
Yi Ma ◽  
Jieting Luo ◽  
Bryony Crook ◽  
Nicola Cooley ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ventricular Myocytes ◽  
Contractile Function ◽  
Pressure Overload ◽  
Neonatal Rat ◽  
Mouse Heart ◽  
Contractile Dysfunction ◽  
Functional Responses ◽  
Rat Cardiomyocytes

The activity of the early signaling enzyme, phospholipase Cβ1b (PLCβ1b), is elevated in diseased myocardium and activity increases with disease progression. PLCβ1b and the alternative splice variant, PLCβ1a, were expressed in mouse hearts using adeno-associated viral constructs (rAAV6-FLAG-PLCβ1b, rAAV6-FLAG- PLCβ1a) delivered intravenously. Functional responses were assessed in vivo and confirmatory mechanistic studies were conducted in neonatal rat ventricular myocytes (NRVM). FLAG-PLCβ1b was expressed in all of the chambers of the mouse heart, but was highest in left ventricle, where expression was observed in >90% of the cells and was localized to the sarcolemma and T-tubules. Heightened PLCβ1b expression caused a rapid loss of contractility and down-regulation of Phospholamban expression. The loss of contractility induced by PLCβ1b was reversed by inhibition of protein kinase Cα (PKCα). PLCβ1a did not affect contractile function or phospholamban expression. Mechanistic analysis performed in neonatal rat cardiomyocytes confirmed PLCβ1b increased the membrane association of PKCα as well as downstream dephosphorylation of phospholamban and depletion of the Ca2+ stores of the sarcoplasmic reticulum, both of which were mediated by PKCα. Trans-aortic constriction (TAC) resulted in progressive hypertrophy together with reduced contractility in PLCβ1a expressing mice. In PLCβ1b-expressing mice, TAC induced a similar hypertrophic response, but did not cause further contractile depression above that due to PLCβ1b expression alone, suggesting that PLCβ1b is responsible for lowering contractility in response to pressure overload. We conclude that heightened PLCβ1b activity observed in diseased myocardium contributes to pathology by PKCα-mediated contractile dysfunction. PLCβ1b is a cardiac-specific signaling system, and thus provides an ideal therapeutic target for the development of well-tolerated inotropic agents for use in failing myocardium.

scholarly journals KLF15 Regulates Oxidative Stress Response in Cardiomyocytes through NAD+

Metabolites ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 620
Author(s):  
Le Li ◽  
Weiyi Xu ◽  
Lilei Zhang
Keyword(s):  
Oxidative Stress ◽  
Ventricular Myocytes ◽  
Cardiac Metabolism ◽  
Neonatal Rat ◽  
Oxygen Species ◽  
Oxidative Stresses ◽  
Neonatal Rat Ventricular Myocytes ◽  
Rate Limiting ◽  
Defective Clearance

KLF15 has recently emerged as a central regulator of metabolism. Although its connection to oxidative stress has been suspected, there has not been any study to date that directly demonstrates the molecular link. In this study, we sought to determine the role of KLF15 in cardiac oxidative stress. We found that KLF15 deficiency in the heart is associated with increased oxidative stress. Acute deficiency of KLF15 in neonatal rat ventricular myocytes (NRVMs) leads to the defective clearance of reactive oxygen species (ROS) and an exaggerated cell death following a variety of oxidative stresses. Mechanistically, we found that KLF15 deficiency leads to reduced amounts of the rate-limiting NAD+ salvage enzyme NAMPT and to NAD+ deficiency. The resultant SIRT3-dependent hyperacetylation and the inactivation of mitochondrial antioxidants can be rescued by MnSOD mimetics or NAD+ precursors. Collectively, these findings suggest that KLF15 regulates cardiac ROS clearance through the regulation of NAD+ levels. Our findings establish KLF15 as a central coordinator of cardiac metabolism and ROS clearance.

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