Genetically encoded Ca2+ indicators in cardiac myocytes.

Genetically encoded Ca(2+) indicators constitute a powerful set of tools to investigate functional aspects of Ca(2+) signaling in isolated cardiomyocytes, cardiac tissue, and whole hearts. Here, we provide an overview of the concepts, experiences, state of the art, and ongoing developments in the use of genetically encoded Ca(2+) indicators for cardiac cells and heart tissue. This review is supplemented with in vivo viral gene transfer experiments and comparisons of available genetically encoded Ca(2+) indicators with each other and with the small molecule dye Fura-2. In the context of cardiac myocytes, we provide guidelines for selecting a genetically encoded Ca(2+) indicator. For future developments, we discuss improvements of a broad range of properties, including photophysical properties such as spectral spread and biocompatibility, as well as cellular and in vivo applications.

Conceptual and technical aspects of transfection and gene delivery.

Genetically modified animals are state of the art in biomedical research as gene therapy is a promising perspective in the attempt to cure hereditary diseases. Both approaches have in common that modified or corrected genetic information must be transferred into cells in general or into particular cell types of an organism. Here we give an overview of established and emerging methods of transfection and gene delivery and provide conceptual and technical advantages and drawbacks of their particular use. Additionally, based on a flow chart, we compiled a rough guideline to choose a gene transfer method for a particular field of application.

Mutation of the calmodulin binding motif IQ of the L-type Ca(v)1.2 Ca2+ channel to EQ induces dilated cardiomyopathy and death.

Cardiac excitation-contraction coupling (EC coupling) links the electrical excitation of the cell membrane to the mechanical contractile machinery of the heart. Calcium channels are major players of EC coupling and are regulated by voltage and Ca(2+)/calmodulin (CaM). CaM binds to the IQ motif located in the C terminus of the Ca(v)1.2 channel and induces Ca(2+)-dependent inactivation (CDI) and facilitation (CDF). Mutation of Ile to Glu (Ile1624Glu) in the IQ motif abolished regulation of the channel by CDI and CDF. Here, we addressed the physiological consequences of such a mutation in the heart. Murine hearts expressing the Ca(v)1.2(I1624E) mutation were generated in adult heterozygous mice through inactivation of the floxed WT Ca(v)1.2(L2) allele by tamoxifen-induced cardiac-specific activation of the MerCreMer Cre recombinase. Within 10 days after the first tamoxifen injection these mice developed dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes (CM) and fibrosis. In Ca(v)1.2(I1624E) hearts, the activity of phospho-CaM kinase II and phospho-MAPK was increased. CMs expressed reduced levels of Ca(v)1.2(I1624E) channel protein and I(Ca). The Ca(v)1.2(I1624E) channel showed "CDI" kinetics. Despite a lower sarcoplasmic reticulum Ca(2+) content, cellular contractility and global Ca(2+) transients remained unchanged because the EC coupling gain was up-regulated by an increased neuroendocrine activity. Treatment of mice with metoprolol and captopril reduced DCM in Ca(v)1.2(I1624E) hearts at day 10. We conclude that mutation of the IQ motif to IE leads to dilated cardiomyopathy and death.

Functional and morphological preservation of adult ventricular myocytes in culture by sub-micromolar cytochalasin D supplement.

In cardiac myocytes, cytochalasin D (CytoD) was reported to act as an actin disruptor and mechanical uncoupler. Using confocal and super-resolution STED microscopy, we show that CytoD preserves the actin filament architecture of adult rat ventricular myocytes in culture. Five hundred nanomolar CytoD was the optimal concentration to achieve both preservation of the T-tubular structure during culture periods of 3 days and conservation of major functional characteristics such as action potentials, calcium transients and, importantly, the contractile properties of single myocytes. Therefore, we conclude that the addition of CytoD to the culture of adult cardiac myocytes can indeed be used to generate a solid single-cell model that preserves both morphology and function of freshly isolated cells. Moreover, we reveal a putative link between cytoskeletal and T-tubular remodeling. In the absence of CytoD, we observed a loss of T-tubules that led to significant dyssynchronous Ca(2+)-induced Ca(2+) release (CICR), while in the presence of 0.5 µM CytoD, T-tubules and homogeneous CICR were majorly preserved. Such data suggested a possible link between the actin cytoskeleton, T-tubules and synchronous, reliable excitation-contraction-coupling. Thus, T-tubular re-organization in cell culture sheds some additional light onto similar processes found during many cardiac diseases and might link cytoskeletal alterations to changes in subcellular Ca(2+) signaling revealed under such pathophysiological conditions.

Optical action potential screening on adult ventricular myocytes as an alternative QT-screen.

BACKGROUND/AIMS: QT-interval screens are increasingly important for cardiac safety on all new medications. So far, investigations rely on animal experiments or cell-based screens solely probing for conductance alterations in heterologously expressed hERG-channels in cell lines allowing for a high degree of automation. Adult cardiomyocytes can not be handled by automated patch-clamp setups. Therefore optical screening of primary isolated ventricular myocytes is regarded as an alternative. Several optical voltage sensors have been reported for ratiometric measurements, but they all influenced the naïve action potential. The aim of the present study was to explore the recording conditions and define settings that allow optical QT-interval screens. METHODS: Based on an improved optical design, individual action potentials could be recorded with an exceptional signal-to-noise-ratio. The sensors were validated using the patch-clamp technique, confocal microscopy and fluorescence lifetime imaging in combination with global unmixing procedures. RESULTS: We show that the small molecule dye di-8-ANEPPS and the novel genetically encoded sensor Mermaid provide quantitative action potential information. When applying such sensors we identified distinctly different pharmacological profiles of action potentials for adult and neonatal rat cardiomyocytes. CONCLUSION: Optical methods can be used for QT-interval investigations based on cellular action potentials using either the small molecule dye di-8-ANEPPS or the genetically encoded sensor Mermaid. Adult cardiomyocytes are superior to neonatal cardiomyocytes for such pharmacological investigations. Optical QT-screens may replace intricate animal experiments.

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

It is well known that for cardiomyocytes, isolation and culturing induce largely unknown remodelling processes. We analysed changes in the structure of cell compartments with optical techniques such as confocal microscopy and fluorescence redistribution after photobleaching employing adenoviral-mediated transduction of targeted fluorescent proteins and small molecule dyes. We identified characteristic remodelling processes: the T-tubular membrane system was gradually lost by a process referred to as "sequential pinching off", in an outward direction. Mitochondria fell in one of three classes, very small (0.9 microm length), medium long (1.8 microm) or extended shape (3.6 microm) organelles. Over the culturing time mitochondria gradually fused. Bleaching of individual mitochondria revealed association between apparently separated mitochondria by "tunnelling" via sub-resolution organelle-tubes. This tunnelling process was increasing over the culturing time. A gradual loss of the cross-striation arrangement in the endoplasmic/sarcoplasmic reticulum was visualised. Analysis of large populations of Ca(2+) sparks by video-rate confocal 2D-scanning revealed significant albeit small changes of these elementary SR-Ca(2+) release events in adult cardiomyocytes that could be related to changes in SR-Ca(2+) content rather than resting Ca(2+) concentration. In conclusion, primary isolated cardiomyocytes from adult hearts undergo a well-defined, but reproducible subcellular remodelling during optimised long term culture.

Cardiac remodeling in Gαq and Gα11 knockout mice.

BACKGROUND: Although both Gαq- and Gα11-protein signaling are believed to be involved in the regulation of cardiac hypertrophy, their detailed contribution to myocardial function remains elusive. METHODS AND RESULTS: We studied remodeling processes in healthy transgenic mice with genetically altered Gαq/Gα11-expression, in particular a global Gα11-knockout and a novel inducible cardiac specific Gαq-knockout, as well as a combined double knockout (dKO) mouse line. Echocardiography and telemetric ECG recordings revealed that compared with wild type mice, hearts of dKO mice showed an increased ejection fraction and a decreased heart rate, irrespective of age resulting in a maintained cardiac output. We attributed these findings to the lack of Gα11, which the absence was associated with a decreased afterload. Histological analysis of the extracellular matrix in the heart depicted a diminished presence of collagen in aging hearts of dKO mice compared to wild-type mice. The results of a transcriptome analysis on isolated ventricular cardiac myocytes revealed alterations of the activity of genes involved in the Gαq/Gα11-dependent regulation of the extracellular matrix, such as the matricellular protein Cyr61. CONCLUSIONS: From our data we conclude that Gαq/Gα11 signaling pathways play a pivotal role in maintaining gene activity patterns. For the heart we revealed their importance in modulating the properties of the extracellular matrix, a mechanism that might be an important contributor and mechanistic basis for the development of pressure-overload induced cardiac hypertrophy.

Endothelin-1-induced remodelling of murine adult ventricular myocytes.

The precise role of hormones binding to Gαq protein-coupled receptors (H-GαqPCRs) in chronic heart diseases remains poorly understood. To address this, we used a model of cultured adult rat ventricular myocytes stimulated with endothelin-1 (ET-1) or phenylephrine (PE) over a period of 8 days in vitro (DIV). Chronically treated cells showed an increased number of arrhythmogenic Ca(2+) transients when electrically paced at 0.5 Hz. While their post-rest behaviour was preserved, from DIV6 onwards the amplitude of caffeine-evoked Ca(2+) transients was increased in hormone-treated cells, suggesting an elevated sarcoplasmic reticulum Ca(2+) load. The duration of electrically evoked global Ca(2+) transients gradually increased over the culturing time indicating decreased activity of processes removing cytosolic Ca(2+). In treated cells, spontaneous Ca(2+) sparks displayed smaller amplitudes from DIV6 onwards, and a slower decay period for PE (from DIV3) and for ET-1 (from DIV6). This cellular functional remodelling was associated with changes in gene expression: chronic ET-1 treatment decreased PKCγ transcripts, whereas PE increased PKCγ and SERCA2a transcripts as probed by qPCR. Western blot analysis confirmed the upregulation of PKCγ with PE. To study ET-1 receptor desensitization in vivo, osmotic minipumps containing either NaCl or ET-1 were implanted in mice and Ca(2+) signalling was studied in acutely isolated ventricular myocytes after 2 weeks of chronic treatment. Interestingly, while cellular responses to isoproterenol stimulation were preserved in ET-1 treated animals, the inotropic response of myocytes to ET-1 stimulation was abrogated. We therefore conclude that chronic stimulation of cardiac myocytes by H-GαqPCRs induces cellular remodelling of Ca(2+) cycling with altered PKCγ expression and promotion of arrhythmogenic cellular responses.

Gαq and Gα11 contribute to the maintenance of cellular electrophysiology and Ca2+ handling in ventricular cardiomyocytes.

AIMS: Gα(q) and Gα(11) signalling pathways contribute to cardiac diseases such as hypertrophy and arrhythmia, but their role in cardiac myocytes from healthy hearts has remained unclear. We aimed to investigate the contribution of Gα(q) and Gα(11) signalling to the basal properties of ventricular myocytes. METHODS AND RESULTS: We created a conditional Gα(q) knockout (KO) after tamoxifen injection into gnaq(flox/flox) gna11(-/-) α-MHC Cre(tg/0) mice and found alterations in the electrophysiological and Ca(2+) handling properties of ventricular myocytes using patch-clamp and Fura-2 video imaging. To reveal the genuine effects of protein KO, we investigated the individual contributions of (i) tamoxifen injection, (ii) Cre recombinase expression, (iii) Gα(11) KO, and (iv) Gα(q) KO. Profound and persistent alterations in myocyte properties occurred following the tamoxifen injection alone. Consequently, we used the presence or absence of Cre recombinase expression as the determinant for the Gα(q) KO. Myocytes from the Gα(q) and/or Gα(11) KO mice displayed genuine alterations in the action potentials, membrane capacitance, membrane currents, and Ca(2+) handling (amplitude, post-rest behaviour, and Ca(2+) removal processes). CONCLUSIONS: We conclude that, in a transgenic model, the role of Gα(q) can be best studied using Cre recombinase expression as the molecular determinant for Gα(q) KO rather than tamoxifen/miglyol injection. While excessive hormonal stimulation of the Gα(q)/Gα(11) signalling pathways plays an essential role in cardiac diseases, we propose that the persistent low-level stimulation of these pathways by Gα(q)/Gα(11) activation is instrumental in the physiological behaviour of ventricular myocytes.

Isolation and genetic manipulation of adult cardiac myocytes for confocal imaging.

Cardiac myocytes isolated from adult hearts are widely accepted as a model somewhere half way between embryonic and neonatal muscle cells on one side and a working heart on the other. Thus, cardiomyocytes serve as good models for cardiac cellular physiology and pathophysiology, for pharmaceutical investigations as well as for the exploration of transgenic animal models. Here we describe a method of isolating the cells from the heart. Furthermore we show how a genetic manipulation on cardiac myocytes can be performed without breeding a transgenic animal: This is the combination of long term culture (1 week) and adenoviral gene transfer. The latter one is described from the construction of the virus to the transduction of the cells. It can be used for the expression of genetically encoded biosensors (GEBs), fluorescent fusion proteins, but also for protein over expression and down regulation, e.g. using RNAi. Here we provide an example for the expression of a fusion protein staining a subcellular structure (Golgi). Such protein expression can be visualized by confocal imaging of z-stacks for a 3D-reconstruction of subcellular structures. The protocol comprises state-of-the-art in cell culture, molecular biology and biophysics and thus provides an approach for exploring new horizons in cellular cardiology.

Phosphorylation of sucrose synthase at serine 170: occurrence and possible role as a signal for proteolysis.

Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.