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 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.

A three-dimensional in vitro culture model for primary neonatal rat ventricular myocytes

2012 ◽  
Vol 12 (3) ◽  
pp. 826-833 ◽  
Author(s):  
Hong-xia Zheng ◽  
Shan-shan Liu ◽  
Wei-ming Tian ◽  
Hong-ji Yan ◽  
Yao Zhang ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Ventricular Myocytes ◽  
Three Dimensional ◽  
Neonatal Rat ◽  
Rat Ventricular Myocytes ◽  
Culture Model ◽  
Neonatal Rat Ventricular Myocytes

CARP Plays a Key Role in Cellular Growth Under Hypertrophic Stimuli in Neonatal Rat Ventricular Myocytes

2013 ◽  
Vol 9 ◽  
Author(s):  
Tara A Shrout
Keyword(s):  
Gene Expression ◽  
Ventricular Myocytes ◽  
Cellular Growth ◽  
Neonatal Rat ◽  
Transcriptional Level ◽  
Dual Nature ◽  
Hypertrophic Growth ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes ◽  
Over Time

Cardiac hypertrophy is a growth process that occurs in response to stress stimuli or injury, and leads to the induction of several pathways to alter gene expression. Under hypertrophic stimuli, sarcomeric structure is disrupted, both as a consequence of gene expression and local changes in sarcomeric proteins. Cardiac-restricted ankyrin repeat protein (CARP) is one such protein that function both in cardiac sarcomeres and at the transcriptional level. We postulate that due to this dual nature, CARP plays a key role in maintaining the cardiac sarcomere. GATA4 is another protein detected in cardiomyocytes as important in hypertrophy, as it is activated by hypertrophic stimuli, and directly binds to DNA to alter gene expression. Results of GATA4 activation over time were inconclusive; however, the role of CARP in mediating hypertrophic growth in cardiomyocytes was clearly demonstrated. In this study, Neonatal Rat Ventricular Myocytes were used as a model to detect changes over time in CARP and GATA4 under hypertrophic stimulation by phenylephrine and high serum media. Results were detected by analysis of immunoblotting. The specific role that CARP plays in mediating cellular growth under hypertrophic stimuli was studied through immunofluorescence, which demonstrated that cardiomyocyte growth with hypertrophic stimulation was significantly blunted when NRVMs were co-treated with CARP siRNA. These data suggest that CARP plays an important role in the hypertrophic response in cardiomyocytes.

Abstract 15280: Comparisons of Action Potentials and Ionic Currents Between Neonatal Rat Ventricular Myocytes and Adult Zebrafish Ventricular Myocytes

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Adonis Z Wu ◽  
Shien-Fong Lin ◽  
Sheng-Nan Wu
Keyword(s):  
Electrical Properties ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Firing Frequency ◽  
Neonatal Rat ◽  
Adult Zebrafish ◽  
Patch Clamp Technique ◽  
Action Current ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

Introduction: Zebrafish heart is established as a model to investigate cardiac electrical abnormalities. However, electrical properties of adult zebrafish cardiomyocytes are not sufficiently characterized. Hypothesis: In this study, by comparing the electrical properties between neonatal rat ventricular myocytes (NRVMs) and adult zebrafish ventricular myocytes (AZVMs), we intended to characterize the action potential (AP), action current (AC) and the properties of Na + current ( I Na ) in AZVMs. Methods: We used patch-clamp technique to characterize the electrical properties, including AP, AC and I Na , in cultured NRVMs and freshly isolated AZVMs. Results: NVRMs showed larger AP amplitude (119±6 vs. 79±4mV, p<.05) but shorter AP duration (APD 90 , 136±11 vs. 213±19 ms, p<.05) than those of AZVMs. The AP duration exhibited marked frequency-dependent alterations in AZVMs. Under the slow pacing rate, early after-depolarizations (EAD) emerged under slow pacing rate with 0.05 Hz. In cell-attached voltage-clamp recordings made from AZVMs, ACs could be elicited by +10 mV steps. As the depolarization step increased to +70 mV, the latency for appearance of ACs was progressively reduced from >123 ms to 9.8 ms. The presence of spontaneous ACs was monitored in spontaneously beating NRVMs and AZVMs. The AC amplitude in NRVMs was larger compared to that in AZVMs (17.3±2.1 vs. 11.6±1.1 pA, p<.05), although firing frequency of AC in NRVMs is higher than in AZVMs (1.13±0.09 vs. 0.38±0.03 Hz, p<.05). The lowering effect of ranolazine, a I Na antagonist, on firing frequency was significantly larger in NRVMs (1.13±0.09 to 0.31±0.02 Hz, p<.05) than in AZVMs (0.38±0.03 to 0.27±0.02 Hz). There was a hyperpolarizing shift of peak I Na in AZVM compared to NRVM. Conclusions: Our results demonstrated major differences in the cellular electrical behavior between AZVMs and NRVMs.

scholarly journals Chronotropic Response of Cultured Neonatal Rat Ventricular Myocytes to Short-Term Fluid Shear

2006 ◽  
Vol 46 (2) ◽  
pp. 113-122 ◽  
Author(s):  
Ilka Lorenzen-Schmidt ◽  
Geert W. Schmid-Schönbein ◽  
Wayne R. Giles ◽  
Andrew D. McCulloch ◽  
Shu Chien ◽  
...  
Keyword(s):  
Ventricular Myocytes ◽  
Neonatal Rat ◽  
Fluid Shear ◽  
Short Term ◽  
Chronotropic Response ◽  
Rat Ventricular Myocytes ◽  
Neonatal Rat Ventricular Myocytes

scholarly journals Hexokinase–mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes

2013 ◽  
Vol 142 (4) ◽  
pp. 425-436 ◽  
Author(s):  
Guillaume Calmettes ◽  
Scott A. John ◽  
James N. Weiss ◽  
Bernard Ribalet
Keyword(s):  
Cardiac Tissue ◽  
Ventricular Myocytes ◽  
Glycogen Synthesis ◽  
Neonatal Rat ◽  
Metabolic Profiles ◽  
Rat Ventricular Myocytes ◽  
Glycolytic Activity ◽  
Extracellular Glucose ◽  
Functional Consequences ◽  
Neonatal Rat Ventricular Myocytes

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

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