Real time mitochondrial dimension measurements.

Mitochondrial volume is correlated with cell function and internal cell processes. Changes in mitochondrial volume were associated with advanced states of cardiac disease. Thus, measurements of mitochondrial dimension deformations are important to the understanding of cell function and its deterioration. Existing methods either allow measurements of the volume of isolated mitochondria, which are an inferior model to that of isolated cells, or they allow short time measurements that are toxic to the cells. Recent studies have discovered that mitochondrial deformation along a given cell axis can be measured by using the Fourier transformation on the variation in transmitted light intensity induced by the periodic lattice of myofilaments alternating with mitochondrial rows. However, this method was used only offline and in a line scan mode, making it impossible to measure both axes. We designed an open source program in LabVIEW to take advantage of the transmitted light diffraction technique and quantify mitochondrial two dimension (2D) deformation in cardiomyocytes, in situ in real time for long periods (more than several seconds). We validated the program on synthetic and on experimental images from rabbit and rat ventricular myocytes. The program can analyze offline and real time simultaneous 2D mitochondrial deformation dynamics as well as also sarcomere length dynamics. Moreover, the program can accurately analyze images acquired from different cameras. Quantification of mitochondrial 2D deformations is a powerful tool for exploring cell biophysics and bioenergetics mechanisms and will lay the foundation for a future clinical tool for quantifying mitochondrial volume changes associated with different cardiac diseases.

Bioenergetic Feedback between Heart Cell Contractile Machinery and Mitochondrial 3D Deformations.

In the heart, mitochondria are arranged in pairs sandwiched between the contractile machinery, which is the major ATP consumer. Thus, in response to the contraction-relaxation cycle of the cell, the mitochondrial membrane should deform accordingly. Membrane deformations in isolated ATP synthesis or in isolated mitochondria affect ATP production. However, it is unknown whether physiological deformation of the mitochondrial membrane in response to the contraction-relaxation cycle can act as a bioenergetic signaling mechanism between ATP demand to supply. We used both experimental and computational tools to reveal whether bioenergetic feedback exists between heart cell contractile machinery and mitochondrial three-dimensional (3D) deformations. We measured the mitochondrial 3D deformation in contracting rabbit cardiac myocytes and used published data on rat cardiac myocytes. These measurements were an input to a novel biophysics model that includes a description of ionic molecules on the mitochondrial membrane, Ca2+ cycling, and mitochondrial membrane stress. As is the case for rat cardiomyocytes, in rabbit cardiomyocytes, the mitochondrial length contracted and expanded with a similar dynamic as the sarcomere length. In contrast, the mitochondrial width expanded and then contracted with a similar dynamic as the mitochondrial length. Differences in the extent of deformation and fractional deformation between the width- and thick-axes were quantified and interpreted as the degree anisotropy between those respective axes. Finally, the model predicts that significant bioenergetic feedback between heart cell contractile machinery and mitochondrial 3D deformations does exist in unloaded rabbit and rat cells. However, this feedback is not a dominant mechanism in ATP supply to demand matching.

Semi-automated program for analysis of local Ca2+ spark release with application for classification of heart cell type.

Local Ca2+ spark releases are essential to the Ca2+ cycling process. Thus, they play an important role in ventricular and atrial cell contraction, as well as in sinoatrial cell automaticity. Characterizing their properties in healthy cells from different regions in the heart can reveal the basic biophysical differences among these regions. We designed a semi-automatic Matlab Graphical User Interface (called Sparkalyzer) to characterize parameters of Ca2+ spark release from any major cardiac tissue, as recorded in line-scan mode with a confocal laser-scanning microscope. We validated the algorithm on experimental images from rabbit sinoatrial, atrial, and ventricular cells loaded with Fluo-4 AM. The program characterizes general image parameters of Ca2+ transients and sparks: spark duration, which indicates for how long the spark provides Ca2+ to the closed intracellular mechanisms (typical value: 25±1, 23±1, 26±1ms for sinoatrial, atrial, and ventricular cells, respectively); spark amplitude, which indicates the amount of Ca2+ released by a single spark (1.6±0.1, 1.6±0.2, 1.4±0.1F/F0 for sinoatrial, atrial, and ventricular cells, respectively); spark length, which is the length of the Ca2+ wavelets fired out of a row of ryanodine receptors (5±0.1, 5±0.2, 3.4±0.3µm for sinoatrial, atrial, or ventricular cells, respectively) and number of sparks (0.14±0.02, 0.025±0.01, 0.02±0.01 for 1µm in 1s for sinoatrial, atrial, and ventricular cells, respectively). This method is reliable for Ca2+ spark analysis of sinoatrial, atrial, or ventricular cells. Moreover, by examining the average value of Ca2+ spark characteristics and their scattering around the mean, atrial, ventricular and sinoatrial cells can be differentiated.