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.

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.

The cardiac sodium-calcium exchanger NCX1 is a key player in the initiation and maintenance of a stable heart rhythm.

AIMS: The complex molecular mechanisms underlying spontaneous cardiac pacemaking are not fully understood. Recent findings point to a co-ordinated interplay between intracellular Ca(2+) cycling and plasma membrane-localized cation transport determining the origin and periodicity of pacemaker potentials. The sodium-calcium exchanger (NCX1) is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. Here, we investigated the contribution of NCX1 to cardiac pacemaking. METHODS AND RESULTS: We used an inducible and sinoatrial node-specific Cre transgene to create micelacking NCX1 selectively in cells of the cardiac pacemaking and conduction system (cpNCX1KO). RT-PCR and immunolabeling experiments confirmed the precise tissue-specific and temporally controlled deletion. Ablation of NCX1 resulted in a progressive slowing of heart rate accompanied by severe arrhythmias. Isolated sinoatrial tissue strips displayed a significantly decreased and irregular contraction rate underpinning a disturbed intrinsic pacemaker activity. Mutant animals displayed a gradual increase in the heart-to-body weight ratio and developed ventricular dilatation; however, their ventricular contractile performance was not significantly affected. Pacemaker cells from cpNCX1KO showed no NCX1 activity in response to caffeine-induced Ca(2+) release, determined by Ca(2+) imaging. Regular spontaneous Ca(2+) discharges were frequently seen in control, but only sporadically in knockout (KO) cells. The majority of NCX1KO cells displayed an irregular and a significantly reduced frequency of spontaneous Ca(2+) signals. Furthermore, Ca(2+) transients measured during electrical field stimulation were of smaller magnitude and decelerated kinetics in KO cells. CONCLUSIONS: Our results establish NCX1 as a critical target for the proper function of cardiac pacemaking.

Morphologically homogeneous red blood cells present a heterogeneous response to hormonal stimulation.

Red blood cells (RBCs) are among the most intensively studied cells in natural history, elucidating numerous principles and ground-breaking knowledge in cell biology. Morphologically, RBCs are largely homogeneous, and most of the functional studies have been performed on large populations of cells, masking putative cellular variations. We studied human and mouse RBCs by live-cell video imaging, which allowed single cells to be followed over time. In particular we analysed functional responses to hormonal stimulation with lysophosphatidic acid (LPA), a signalling molecule occurring in blood plasma, with the Ca(2+) sensor Fluo-4. Additionally, we developed an approach for analysing the Ca(2+) responses of RBCs that allowed the quantitative characterization of single-cell signals. In RBCs, the LPA-induced Ca(2+) influx showed substantial diversity in both kinetics and amplitude. Also the age-classification was determined for each particular RBC and consecutively analysed. While reticulocytes lack a Ca(2+) response to LPA stimulation, old RBCs approaching clearance generated robust LPA-induced signals, which still displayed broad heterogeneity. Observing phospatidylserine exposure as an effector mechanism of intracellular Ca(2+) revealed an even increased heterogeneity of RBC responses. The functional diversity of RBCs needs to be taken into account in future studies, which will increasingly require single-cell analysis approaches. The identified heterogeneity in RBC responses is important for the basic understanding of RBC signalling and their contribution to numerous diseases, especially with respect to Ca(2+) influx and the associated pro-thrombotic activity.