Abstract
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.
Highlights
Synchronous calcium (Ca) cycling is a fundamental part of excitation contraction coupling (ECC) in cardiac myocytes
INTRACELLULAR DYSSYNCHRONY OF CYTOSOLIC Ca REMOVAL IN INTACT HEARTS Using the approach previously applied in isolated cardiac myocytes (Hohendanner et al, 2013), we analyzed subcellular Ca transients (CaT) in intact spontaneously beating Langendorff-perfused hearts using confocal line scan imaging of the epicardial layer (Figure 1)
Similar to what we have previously described in isolated cardiomyocytes, intracellular cytosolic Ca removal was not synchronous in cardiomyocytes of the intact heart
Summary
Synchronous calcium (Ca) cycling is a fundamental part of excitation contraction coupling (ECC) in cardiac myocytes. Dyssynchronous Ca release within a single myocyte has been observed in various diseased conditions in animal models as well as in human cardiac myocytes from diseased patients and has been proposed as a possible mechanism of cellular contractile dysfunction (Louch et al, 2004; Song et al, 2006; Heinzel et al, 2008). We have described increased dyssynchrony of intracellular cytosolic Ca removal which was linked to non-uniform sarcomere relengthening and cardiac malfunction in different models of heart failure and in human cardiac myocytes (Hohendanner et al, 2013). On a larger scale, mechanical dyssynchrony between different regions of the regularly beating heart—e.g., caused by bundle branch block—is a well-known promoter of HF and has led to cardiac resynchronization therapy as an established HF therapy (Yu et al, 2006; Stevenson et al, 2012)
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