Differential regulation of Krüppel-like factor family transcription factor expression in neonatal rat cardiac myocytes: effects of endothelin-1, oxidative stress and cytokines

Krüppel-like transcription factors (Klfs) modulate fundamental cell processes. Cardiac myocytes are terminally-differentiated, but hypertrophy in response to stimuli such as endothelin-1. H2O2 or cytokines promote myocyte apoptosis. Microarray studies of neonatal rat myocytes identified several Klfs as endothelin-1-responsive genes. We used quantitative PCR for further analysis of Klf expression in neonatal rat myocytes. In response to endothelin-1, Klf2 mRNA expression was rapidly increased ( approximately 9-fold; 15-30 min) with later increases in expression of Klf4 and Klf6 ( approximately 5-fold; 30-60 min). All were regulated as immediate early genes (cycloheximide did not inhibit the increases in expression). Klf5 expression was increased at 1-2 h ( approximately 13-fold) as a second phase response (cycloheximide inhibited the increase). These increases were transient and attenuated by U0126. H2O2 increased expression of Klf2, Klf4 and Klf6, but interleukin-1beta or tumor necrosis factor alpha downregulated Klf2 expression with no effect on Klf4 or Klf6. Of the Klfs which repress transcription, endothelin-1 rapidly downregulated expression of Klf3, Klf11 and Klf15. The dynamic regulation of expression of multiple Klf family members in cardiac myocytes suggests that, as a family, they are actively involved in regulating phenotypic responses (hypertrophy and apoptosis) to extracellular stimuli

Observation and interpretation of motional sideband asymmetry in a quantum electro-mechanical device

Quantum electro-mechanical systems offer a unique opportunity to probe quantum noise properties in macroscopic devices, properties which ultimately stem from the Heisenberg Uncertainty Principle. A simple example of this is expected to occur in a microwave parametric transducer, where mechanical motion generates motional sidebands corresponding to the up and down frequency-conversion of microwave photons. Due to quantum vacuum noise, the rates of these processes are expected to be unequal. We measure this fundamental imbalance in a microwave transducer coupled to a radio-frequency mechanical mode, cooled near the ground state of motion. We also discuss the subtle origin of this imbalance: depending on the measurement scheme, the imbalance is most naturally attributed to the quantum fluctuations of either the mechanical mode or of the electromagnetic field

Regulation of the cardiomyocyte transcriptome vs translatome by endothelin-1 and insulin: translational regulation of 5' terminal oligopyrimidine tract (TOP) mRNAs by insulin

Background: Changes in cellular phenotype result from underlying changes in mRNA transcription and translation. Endothelin-1 stimulates cardiomyocyte hypertrophy with associated changes in mRNA/protein expression and an increase in the rate of protein synthesis. Insulin also increases the rate of translation but does not promote overt cardiomyocyte hypertrophy. One mechanism of translational regulation is through 5' terminal oligopyrimidine tracts (TOPs) that, in response to growth stimuli, promote mRNA recruitment to polysomes for increased translation. TOP mRNAs include those encoding ribosomal proteins, but the full panoply remains to be established. Here, we used microarrays to compare the effects of endothelin-1 and insulin on the global transcriptome of neonatal rat cardiomyocytes, and on mRNA recruitment to polysomes (i.e. the translatome). Results: Globally, endothelin-1 and insulin (1 h) promoted >1.5-fold significant (false discovery rate 1.25-fold significant changes in expression in total and/or polysomal RNA induced by endothelin-1 or insulin, respectively, of which ~35% of endothelin-1-responsive and ~56% of insulin-responsive transcripts were translationally regulated. Of mRNAs for established proteins recruited to polysomes in response to insulin, 49 were known TOP mRNAs with a further 15 probable/possible TOP mRNAs, but 49 had no identifiable TOP sequences or other consistent features in the 5' untranslated region. Conclusions: Endothelin-1, rather than insulin, substantially affects global transcript expression to promote cardiomyocyte hypertrophy. Effects on RNA recruitment to polysomes are subtle, with differential effects of endothelin-1 and insulin on specific transcripts. Furthermore, although insulin promotes recruitment of TOP mRNAs to cardiomyocyte polysomes, not all recruited mRNAs are TOP mRNAs

Quantum squeezing of motion in a mechanical resonator

As a result of the quantum, wave-like nature of the physical world, a harmonic oscillator can never be completely at rest. Even in the quantum ground state, its position will always have fluctuations, called the zero-point motion. Although the zero-point fluctuations are unavoidable, they can be manipulated. In this work, using microwave frequency radiation pressure, we both prepare a micron-scale mechanical system in a state near the quantum ground state and then manipulate its thermal fluctuations to produce a stationary, quadrature-squeezed state. We deduce that the variance of one motional quadrature is 0.80 times the zero-point level, or 1 dB of sub-zero-point squeezing. This work is relevant to the quantum engineering of states of matter at large length scales, the study of decoherence of large quantum systems, and for the realization of ultra-sensitive sensing of force and motion

Mechanically Detecting and Avoiding the Quantum Fluctuations of a Microwave Field

During the theoretical investigation of the ultimate sensitivity of gravitational wave detectors through the 1970's and '80's, it was debated whether quantum fluctuations of the light field used for detection, also known as photon shot noise, would ultimately produce a force noise which would disturb the detector and limit the sensitivity. Carlton Caves famously answered this question with "They do." With this understanding came ideas how to avoid this limitation by giving up complete knowledge of the detector's motion. In these back-action evading (BAE) or quantum non-demolition (QND) schemes, one manipulates the required quantum measurement back-action by placing it into a component of the motion which is unobserved and dynamically isolated. Using a superconducting, electro-mechanical device, we realize a sensitive measurement of a single motional quadrature with imprecision below the zero-point fluctuations of motion, detect both the classical and quantum measurement back-action, and demonstrate BAE avoiding the quantum back-action from the microwave photons by 9 dB. Further improvements of these techniques are expected to provide a practical route to manipulate and prepare a squeezed state of motion with mechanical fluctuations below the quantum zero-point level, which is of interest both fundamentally and for the detection of very weak forces

ERK1/2 signaling dominates over RhoA signaling in regulating early changes in RNA expression induced by endothelin-1 in neonatal rat cardiomyocytes

Cardiomyocyte hypertrophy is associated with changes in gene expression. Extracellular signal-regulated kinases 1/2 (ERK1/2) and RhoA [activated by hypertrophic agonists (e.g. endothelin-1)] regulate gene expression and are implicated in the response, but their relative significance in regulating the cardiomyocyte transcriptome is unknown. Our aim was to establish the significance of ERK1/2 and/or RhoA in the early cardiomyocyte transcriptomic response to endothelin-1.Cardiomyocytes were exposed to endothelin-1 (1 h) with/without PD184352 (to inhibit ERK1/2) or C3 transferase (C3T, to inhibit RhoA). RNA expression was analyzed using microarrays and qPCR. ERK1/2 signaling positively regulated approximately 65% of the early gene expression response to ET-1 with a small (approximately 2%) negative effect, whereas RhoA signaling positively regulated approximately 10% of the early gene expression response to ET-1 with a greater (approximately 14%) negative contribution. Of RNAs non-responsive to endothelin-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by endothelin-1 encoded a number of receptor ligands (e.g. Ereg, Areg, Hbegf) and transcription factors (e.g. Abra/Srf) that potentially propagate the response.ERK1/2 dominates over RhoA in the early transcriptomic response to endothelin-1. RhoA plays a major role in maintaining baseline RNA expression but, with upregulation of Abra/Srf by endothelin-1, RhoA may regulate changes in RNA expression over longer times. Our data identify ERK1/2 as a more significant node than RhoA in regulating the early stages of cardiomyocyte hypertrophy

Loss of Pi-Junction Behaviour in an Interacting Impurity Josephson Junction

Using a generalization of the non-crossing approximation which incorporates Andreev reflection, we study the properties of an infinite-U Anderson impurity coupled to two superconducting leads. In the regime where $\Delta$ and $T_K$ are comparable, we find that the position of the sub-gap resonance in the impurity spectral function develops a strong anomalous phase dependence-- its energy is a minimum when the phase difference between the superconductors is equal to $\pi$. Calculating the Josephson current through the impurity, we find that $\pi$-junction behaviour is lost as the position of the bound-state moves above the Fermi energy.Comment: 4 pages, 4 figures; labelling of Fig. 3 corrected; final published form, only trivial change

Glycogen synthase kinases 3α and 3β in cardiac myocytes: regulation and consequences of their inhibition

Inhibition of glycogen synthase kinase 3β (GSK3β) as a consequence of its phosphorylation by protein kinase B/Akt (PKB/Akt) has been implicated in cardiac myocyte hypertrophy in response to endothelin-1 or phenylephrine. We examined the regulation of GSK3α (which we show to constitute a significant proportion of the myocyte GSK3 pool) and GSK3β in cardiac myocytes. Although endothelin increases phosphorylation of GSK3 and decreases its activity, the response is less than that induced by insulin (which does not promote cardiac myocyte hypertrophy). GSK3 phosphorylation induced by endothelin requires signalling through the extracellular signal-regulated kinase 1/2 (ERK1/2) cascade and not the PKB/Akt pathway, whereas the reverse is true for insulin. Cardiac myocyte hypertrophy involves changes in morphology, and in gene and protein expression. The potent GSK3 inhibitor 1-azakenpaullone increases myocyte area as a consequence of increased cell length whereas phenylephrine increases both length and width. Azakenpaullone or insulin promotes AP1 transcription factor binding to an AP1 consensus oligonucleotide, but this was significantly less than that induced by endothelin and derived principally from increased binding of JunB protein, the expression of which was increased. Azakenpaullone promotes significant changes in gene expression (assessed by Affymetrix microarrays), but the overall response is less than with endothelin and there is little overlap between the genes identified. Thus, although GSK3 may contribute to cardiac myocyte hypertrophy in some respects (and presumably plays an important role in myocyte metabolism), it does not appear to contribute as significantly to the response induced by endothelin as has been maintained