Remodelling of Ca2+ handling organelles in adult rat ventricular myocytes during long term culture

Hammer, K; Ruppenthal, Sandra; Viero, Cédric; Scholz, Anke; Edelmann, L; Kaestner, Lars; Lipp, Peter

doi:10.1016/j.yjmcc.2010.05.010

Cited by 28 publications

(19 citation statements)

References 40 publications

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“…When using gene transfer with adenoviruses, protein expression of the genetically encoded fusion proteins or biosensors reached optically detectable levels within 24 hours. 30,31 Adenovirus-mediated expression is rather stable during the time course of 1 week, and we did not observe adverse effects caused by the viral transduction or induced gene expression during that period. 32,33 Therefore, adenovirus-mediated gene transfer seems to be the most suitable method for the expression of proteins in cultured adult cardiomyocytes.…”

Section: Delivery Of Genetically Encoded Ca 2+ Sensors To Cardiac Myomentioning

confidence: 52%

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Kaestner

Scholz

Tian

et al. 2014

Circulation Research

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Section: Delivery Of Genetically Encoded Ca 2+ Sensors To Cardiac Myomentioning

confidence: 52%

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Kaestner

Scholz

Tian

et al. 2014

Circulation Research

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“…The endoplasmic reticulum (ER)-targeting sequence of calreticulin at the 5= end of DsRed2 and the KDEL ER retention sequence at the 3= end of DsRed2 were cloned from the commercial pDsRed2-ER (Clontech, USA) vector and inserted into the pCR259 vector (21). As a Golgi body target, we used the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase C-terminally fused to monomoric red fluorescent protein (mRFP) (in pCR259).…”

Section: Methodsmentioning

confidence: 99%

“…As a Golgi body target, we used the N-terminal 81 amino acids of human beta 1,4-galactosyltransferase C-terminally fused to monomoric red fluorescent protein (mRFP) (in pCR259). As a mitochondrial target, we used subunit VIII of human cytochrome c oxidase C-terminally fused to mRFP (in pCR259) (21).…”

Section: Methodsmentioning

confidence: 99%

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Hui

Reither

Kaestner

et al. 2014

Molecular and Cellular Biology

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Activation of the two ubiquitous families of protein kinases, protein kinase A (PKA) and protein kinase C (PKC), is thought to be independently coupled to stimulation of G␣ s and G␣ q , respectively. Live-cell confocal imaging of protein kinase C fluorescent protein fusion constructs revealed that simultaneous activation of G␣ s and G␣ q resulted in a differential translocation of the conventional PKC␣ to the plasma membrane while the novel PKC␦ was recruited to the membrane of the endoplasmic reticulum (ER). We demonstrate that the PKC␦ translocation was driven by a novel G␣ s -cyclic AMP-EPAC-RAP-PLC pathway resulting in specific diacylglycerol production at the membrane of the ER. Membrane-specific phosphorylation sensors revealed that directed translocation resulted in phosphorylation activity confined to the target membrane. Specific stimulation of PKC␦ caused phosphorylation of the inositol-1,4,5-trisphosphate receptor and dampening of global Ca 2؉ signaling revealed by graded flash photolysis of caged inositol-1,4,5-trisphosphate. Our data demonstrate a novel signaling pathway enabling differential decoding of incoming stimuli into PKC isoform-specific membrane targeting, significantly enhancing the versatility of cyclic AMP signaling, thus demonstrating the possible interconnection between the PKA and PKC pathways traditionally treated independently. We thus provide novel and elementary understanding and insights into intracellular signaling events.

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“…A movie comparing the raw data and the data after pixel-wise fitting is included in Online Video I. system. 15 We simultaneously visualized the plasma membrane by Di-8-ANEPPS staining ( Figure 3A). From Ca 2ϩ transients obtained from this cell, we extracted noise-free input data ( Figure 3B).…”

Section: Graded Noise Analysis Algorithm Robustness and Reproducibimentioning

confidence: 99%

“…Although analysis of spatially isolated Ca 2ϩ signals (eg, Ca 2ϩ sparks) 15,28 or atrial ECC 29 has been possible, the beating ventricular myocyte represents a particular challenge.…”

Section: Microscopic Versus Macroscopic Alternansmentioning

confidence: 99%

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

Tian

Kaestner

Lipp

2012

Circulation Research

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Rationale: Our insights into physiological and pathophysiological cardiac excitation-contraction coupling has greatly benefited from significant advancement in optical technologies such as high-speed confocal microscopy. This has pushed pixel dwell times into the time domain of nanoseconds, resulting in low signal-to-noise ratios, which have limited data analysis and interpretation.Objective: Line scan imaging has been and still is dominant in high speed confocal recording. It allows analysis only of a small fraction of a cell's cross section (1.5%), but the appreciation of spatiotemporal fine details of excitation-contraction coupling is instrumental for the further understanding of pathological mechanisms. We aim to provide a novel analysis tool to extract otherwise hidden fine details in cardiac excitation-contraction coupling from high-speed 2-dimensional confocal image series. Methods and Results:We demonstrate that high-speed 2-dimensional confocal data (150 frames/s) can be analyzed quantitatively by a pixel-wise fitting approach, using a mathematical formalism to phenomenologically describe local calcium transients. Such an approach produces virtually noise-free fluorescence data originating from minute volumes (0.025 femtoliter) and allows extraction of detailed and most importantly quantitative and mechanistically novel information on microscopic calcium signaling and excitation-contraction coupling in a robust manner. Conclusions:Pixel-wise fitting provides novel insights into cardiac excitation-contraction coupling. Specifically, it revealed microscopic calcium alternans on the level of individual coupling sites. Microscopic calcium alternans is an early precursor of cellular alternans and as such will shed more light onto this mechanism leading to cardiac arrhythmia. (Circ Res. 2012;111:17-27.) Key Words: excitation-contraction coupling Ⅲ calcium-induced calcium release Ⅲ noise-free image sequences Ⅲ alternans Ⅲ arrhythmogenic precursor Ⅲ Ca 2ϩ transients H igh-resolution live-cell imaging of subcellular signaling events has greatly fostered our understanding of cardiac physiology and pathophysiology. 1 In this, a major driving force was the advancement in laser-scanning technology that enabled ultrafast confocal imaging (Ͼ100 frames/s) without compromising the optical resolution. 2 Although researchers are technically able to follow fast subcellular signaling, such as excitation-contraction coupling (ECC) in cardiac myocytes, 3 decreasing the single pixel dwell times and concomitant decreases in the signal-to-noise ratio (SNR) have limited the progress. However, scientific interest is not limited to the occurrence of Ca 2ϩ induced Ca 2ϩ release (CICR) per se but also focuses on where, how much, and how fast the Ca 2ϩ increases occur because the signaling information is encoded in all of these properties. 4 Although cardiac Ca 2ϩ transients appear homogeneous under physiological conditions, they arise from the coordinated activity of many microscopic ECC units, 3 comprising voltage operated Ca 2ϩ ch...

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Remodelling of Ca2+ handling organelles in adult rat ventricular myocytes during long term culture

Cited by 28 publications

References 40 publications

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

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Remodelling of Ca2+ handling organelles in adult rat ventricular myocytes during long term culture

Cited by 28 publications

References 40 publications

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Genetically Encoded Ca 2+ Indicators in Cardiac Myocytes

Targeted Activation of Conventional and Novel Protein Kinases C through Differential Translocation Patterns

Noise-Free Visualization of Microscopic Calcium Signaling by Pixel-Wise Fitting

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Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes

Genetically Encoded Ca ²⁺ Indicators in Cardiac Myocytes