We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca2± spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01 ± 0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca2± spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca2± spark rate (to 130.7 ± 6.4%) within 5 seconds, followed by a return to near background levels (to 104.4±5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 gmol/L GsMTx-4), perfusion with Na±/Ca2±-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca2± spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 gmol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular–sarcoplasmic reticulum complex (to within · 10−8m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca2± spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation.

This paper presents methods to build histo-anatomically detailed individualized cardiac models. The models are based on high-resolution three-dimensional anatomical and/or diffusion tensor magnetic resonance images, combined with serial histological sectioning data, and are used to investigate individualized cardiac function. The current state of the art is reviewed, and its limitations are discussed. We assess the challenges associated with the generation of histo-anatomically representative individualized in silico models of the heart. The entire processing pipeline including image acquisition, image processing, mesh generation, model set-up and execution of computer simulations, and the underlying methods are described. The multifaceted challenges associated with these goals are highlighted, suitable solutions are proposed, and an important application of developed highresolution structure-function models in elucidating the effect of individual structural heterogeneity upon wavefront dynamics is demonstrated.

Optical recording of membrane potential permits spatially resolved measurement of electrical activity in subcellular regions of single cells, which would be inaccessible to electrodes, and imaging of spatiotemporal patterns of action potential propagation in excitable tissues, such as the brain or heart. However, the available voltage-sensitive dyes (VSDs) are not always spectrally compatible with newly available optical technologies for sensing or manipulating the physiological state of a system. Here, we describe a series of 19 fluorinated VSDs based on the hemicyanine class of chromophores. Strategic placement of the fluorine atoms on the chromophores can result in either blue or red shifts in the absorbance and emission spectra. The range of one-photon excitation wavelengths afforded by these new VSDs spans 440-670 nm; the twophoton excitation range is 900-1,340 nm. The emission of each VSD is shifted by at least 100 nm to the red of its one-photon excitation spectrum. The set of VSDs, thus, affords an extended toolkit for optical recording to match a broad range of experimental requirements. We show the sensitivity to voltage and the photostability of the new VSDs in a series of experimental preparations ranging in scale from single dendritic spines to whole heart. Among the advances shown in these applications are simultaneous recording of voltage and calcium in single dendritic spines and optical electrophysiology recordings using two-photon excitation above 1,100 nm.fluorescence | microscopy O ptical recording techniques provide powerful tools for neurobiologists (1) and cardiac physiologists (2) to study detailed patterns of electrical activity over time and space in cells, tissues, and organs. Rational design methods, based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (3-5), were used to engineer dye structures. The general class of dye chromophores called hemicyanine (also referred to as styryl dyes) has emerged from this effort as a good foundation for voltage-sensitive dyes (VSDs), because they exhibit electrochromism. This mechanism, also referred to as the molecular Stark effect, involves the differential interaction of the electric field in the membrane with the ground and excited states of the dye chromophore. Several important hemicyanine dyes were produced over the years, including di-4-ANEPPS (6, 7), di-8-ANEPPS (8), di-2-ANEPEQ (also known as JPW-1114) (9, 10), RH-421 and RH-795 (11), ANNINE-6 and ANNINE-6+ (12, 13), di-3-ANEPPDHQ (14, 15), di-4-ANBDQBS, and di-4-ANBDQPQ (16,17). Because the electrochromic mechanism is a direct interaction of the electric field with the chromophore and does not require any movement of the dye molecule, all of these dyes provide rapid absorbance and fluorescence responses to membrane potential (V m ); they are, therefore, capable of recording action potentials (APs). Other mechanisms can give more sensitive voltage responses in specialized applications (18)(19)(20)(21)(22). Addit...