119 research outputs found
Na/K Pump-Induced [Na]i Gradients in Rat Ventricular Myocytes Measured with Two-Photon Microscopy
AbstractVia the Na/Ca and Na/H exchange, intracellular Na concentration ([Na]i) is important in regulating cardiac Ca and contractility. Functional data suggest that [Na]i might be heterogeneous in myocytes that are not in steady state, but little direct spatial information is available. Here we used two-photon microscopy of SBFI to spatially resolve [Na]i in rat ventricular myocytes. In vivo calibration yielded an apparent Kd of 27±2mM Na. Similar resting [Na]i was found using two-photon or single-photon ratiometric measurements with SBFI (10.8±0.7 vs. 11.1±0.7mM). To assess longitudinal [Na]i gradients, Na/K pumps were blocked at one end of the myocyte (locally pipette-applied K-free extracellular solution) and active in the rest of the cell. This led to a marked increase in [Na]i at sites downstream of the pipette (where Na enters the myocyte and Na/K pumps are blocked). [Na]i rise was smaller at upstream sites. This resulted in sustained [Na]i gradients (up to ∼17 mM/120μm cell length). This implies that Na diffusion in cardiac myocytes is slow with respect to trans-sarcolemmal Na transport rates, although the mechanisms responsible are unclear. A simple diffusion model indicated that such gradients require a Na diffusion coefficient of 10–12μm2/s, significantly lower than in aqueous solutions
Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy
Dyssynchronous local Ca release within individual cardiac myocytes has been
linked to cellular contractile dysfunction. Differences in Ca kinetics in
adjacent cells may also provide a substrate for inefficient contraction and
arrhythmias. In a new approach we quantify variation in local Ca transients
between adjacent myocytes in the whole heart. Langendorff-perfused mouse
hearts were loaded with Fluo-8 AM to detect Ca and Di-4-ANEPPS to visualize
cell membranes. A spinning disc confocal microscope with a fast camera allowed
us to record Ca signals within an area of 465 μm by 315 μm with an acquisition
speed of 55 fps. Images from multiple transients recorded at steady state were
registered to their time point in the cardiac cycle to restore averaged local
Ca transients with a higher temporal resolution. Local Ca transients within
and between adjacent myocytes were compared with regard to amplitude, time to
peak and decay at steady state stimulation (250 ms cycle length). Image
registration from multiple sequential Ca transients allowed reconstruction of
high temporal resolution (2.4 ± 1.3 ms) local CaT in 2D image sets (N = 4
hearts, n = 8 regions). During steady state stimulation, spatial Ca gradients
were homogeneous within cells in both directions and independent of distance
between measured points. Variation in CaT amplitudes was similar across the
short and the long side of neighboring cells. Variations in TAU and TTP were
similar in both directions. Isoproterenol enhanced the CaT but not the overall
pattern of spatial heterogeneities. Here we detected and analyzed local Ca
signals in intact mouse hearts with high temporal and spatial resolution,
taking into account 2D arrangement of the cells. We observed significant
differences in the variation of CaT amplitude along the long and short axis of
cardiac myocytes. Variations of Ca signals between neighboring cells may
contribute to the substrate of cardiac remodeling
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