Michal Cagalinec

Mitochondrial membrane fluidity, potential, and calcium transients in the myocardium from acute diabetic rats

In this study, we report for the first time concurrent measurements of membrane potential and dynamics and respiratory chain activities in rat heart mitochondria, as well as calcium transients in the hearts of rats in an early phase of streptozotocin diabetes, not yet accompanied with diabetes-induced complications. Quantitative relationships among these variables were assessed. The mitochondria from diabetic rats exhibited decreased fluorescence anisotropy values of diphenylhexatriene. This indicates that hydrophobic core of the membranes was more fluid compared with controls (p<0.05). We discuss the changes in fluidity as having been associated with augmented energy transduction through the diabetic membranes. Reduced ratio of JC-1 fluorescence (aggregates to monomers) in the mitochondria from diabetic hearts reflected descendent transmembrane potential. A significant negative association between membrane fluidity and potential in the diabetic group was found (p<0.05; r=0.67). Further, we observed an increase in calcium transient amplitude (CTA) in the diabetic cardiomyocytes (p=0.048). We conclude that some of the calcium-induced regulatory events that dictate fuel selection and capacity for ATP production in diabetic heart occur at the membrane level. Our findings offer new insight into acute diabetes-induced changes in cardiac mitochondria.

Comparative study of the effects of lacidipine and enalapril on the left ventricular cardiomyocyte remodeling in spontaneously hypertensive rats.

Antihypertensive medications are the most efficient drugs in achieving regression of myocardial hypertrophy in both clinical studies and animal models of hypertension. Nevertheless, there is a lack of clear and concise comparative study of their effects on the modulation of cardiomyocyte morphology and function. Here, we assessed the tissue-protective actions of 2 of these drugs, the calcium channel blocker lacidipine (3 mg/kg/day) and the angiotensin-converting enzyme-inhibitor enalapril (10 mg/kg/day) in vivo, after 8 weeks of treatment of 12-week-old spontaneously hypertensive rats, as well as in vitro, after short-term (4 min) application to isolated cardiomyocytes. Left ventricular hypertrophy (LVH) was compared at organ, tissue, and single-cell level. Our data showed that both drugs prevented the LVH of 20-week-old spontaneously hypertensive rats, but only lacidipine significantly decreased the cardiomyocyte size. Similarly, the single-cell contractility was significantly lowered in lacidipine-treated rats only. The effect of lacidipine was initiated shortly after exposure to the drug in a dose-dependent manner at 0.5 Hz, as well as at 2 Hz, with EC50 of 10−7 mol/L. These results can help in understanding the effects of these drugs on the prevention of LVH.

Calcium signaling-mediated endogenous protection of cell energetics in the acutely diabetic myocardium.

In acute diabetic myocardium, calcium signals propagated by intracellular calcium transients participate in the protection of cell energetics via upregulating the formation of mitochondrial energy transition pores (ETP). Mechanisms coupling ETP formation with an increase in membrane fluidity and a decrease in transmembrane potential of the mitochondria are discussed. Our results indicate that the amplification of calcium transients in the diabetic heart is associated with an increase in their amplitude. Moreover, the signals transferred by calcium transients also regulated ETP formation in nondiabetic myocardium. Evidence for the indispensable role of calcium in the regulation of transition pore formation is provided whereby an exchange of cadmium for calcium ions led to a rapid and dramatic decrease in the amount of ETP. Another possible regulatory factor of the mitochondrial function may be radical-induced damage to the diabetic heart. Nevertheless, our data indicate that radical-induced changes in mitochondria predominantly concern the respiratory chain and have no appreciable effect on the fluidity of the mitochondrial membranes. The residual mitochondrial production of ATP owing to its augmented transfer to the cytosol proved to be adequate to preserve sufficient levels of adenine nucleotides in the acute diabetic myocardium.

Sustained spiral calcium wave patterns in rat ventricular myocytes

Ca2+ waves, propagating in non-stimulated isolated cardiac myocytes under physiological conditions, are described as planar waves traveling along the longitudinal cell axis [1]. Other patterns of Ca2+ wave propagation – circular or spiral Ca2+ waves [2, 3]– have been observed in physiological conditions as well. Lipp & Niggli, 1993 [2], Ishida et al., 1999 [3] and Engel et al., 1994 [4] have described spiral calcium waves as individual events. We demonstrate hereby existence of repetitive spiral calcium wave patterns in adult rat ventricular cardiomyocytes. The work was done on 20-week old Wistar rats. Myocytes were isolated with retrograde perfusion of the heart with proteolytic enzymes [5] and loaded with calcium indicator Fluo-3/AM (1 μmol/l). The fluorescence emission was recorded with laser scanning confocal microscope LSM 510 Meta, Zeiss (frame rate from 8 to 14 frames/s, excitation/emission 488/515 nm), equipped with C-Apochromat 40/NA = 1.2 water immersion objective. We observed both planar, circular (Fig. 1A, B) and spiral calcium waves (Fig. 1C) in the same myocyte. Figure 1C shows four subsequent spiral waves, demonstrating repetitive propagation of the spiral wave burning from the same origin. We observed an average of 8 spiral waves per cell, ranged 1–49. The median burning time of the spiral wave was 0.9 s, ranged from 0.4 to 13.7 s (n = 133 spiral calcium waves, 12 cells). The percentage of the spiral waves duration with respect to the entire recording time (ranged from 131 to 227 seconds, n = 12 cells) was 10.8%, ranged from 0.6 to 26.5%.The repetitive spiral waves (defined as waves progressed further after reaching its initial position) were detected in 56 cases from all spiral waves observed (n = 133). The spiral wave pattern formation was a reversible process, as we observed also return to planar-type burning. No blebs or hypercontractures were observed when the recording of the cell was finished. 1 Recording of the (A) planar, (B) circular and (C) spiral calcium waves in an isolated rat left ventricular myocyte after staining with calcium indicator Fluo-3/AM imaged with confocal laser scanning microscopy. Frame interval: 113 ms. Scale bar: 20 μm. ... In contrast to the smaller amplitude of the spiral waves described by Ishida et al., 1999 [3], we observed no difference in relative amplitudes of the calcium waves: planar wave preceding the spiral wave (4.1, range 2.4–6.1), spiral (4.0, range 2.4–6.2) and subsequent planar wave (4.2, range 2.2–5.8, n = 9 waves analyzed) were not significantly different. Moreover, we observed no decrease in the amplitude of the repetitive wave when compared the starting and ending point.This contributes to the stabilization of the spiral burning and allows its long-time propagation. We suggest that the repetitive spiral waves can be considered as a temporary equilibrated state in myocyte calcium wave spreading. As calcium waves are capable of traversing to the adjacent cells via gap junctions [4], a cell with repetitive spiral bursts may negatively impact functioning of the adjacent myocytes.

Estimation of single cell volume from 3D confocal images using automatic data processing

Cardiac cells are highly structured with a non-uniform morphology. Although precise estimation of their volume is essential for correct evaluation of hypertrophic changes of the heart, simple and unified techniques that allow determination of the single cardiomyocyte volume with sufficient precision are still limited. Here, we describe a novel approach to assess the cell volume from confocal microscopy 3D images of living cardiac myocytes. We propose a fast procedure based on segementation using active deformable contours. This technique is independent on laser gain and/or pinhole settings and it is also applicable on images of cells stained with low fluorescence markers. Presented approach is a promising new tool to investigate changes in the cell volume during normal, as well as pathological growth, as we demonstrate in the case of cell enlargement during hypertension in rats.

Multimodal autofluorescence detection of cancer: from single cells to living organism

Multimodal optical imaging of suspected tissues is showing to be a promising method for distinguishing suspected cancerous tissues from healthy ones. In particular, the combination of steady-state spectroscopic methods with timeresolved fluorescence provides more precise insight into native metabolism when focused on tissue autofluorescence. Cancer is linked to specific metabolic remodelation detectable spectroscopically. In this work, we evaluate possibilities and limitations of multimodal optical cancer detection in single cells, collagen-based 3D cell cultures and in living organisms (whole mice), as a representation of gradually increasing complexity of model systems.