Single Heart Muscle Research Articles

Quantum Sensing of Free Radical Generation in Mitochondria of Single Heart Muscle Cells during Hypoxia and Reoxygenation.

Cells are damaged during hypoxia (blood supply deprivation) and reoxygenation (oxygen return). This damage occurs in conditions such as cardiovascular diseases, cancer, and organ transplantation, potentially harming the tissue and organs. The role of free radicals in cellular metabolic reprogramming under hypoxia is under debate, but their measurement is challenging due to their short lifespan and limited diffusion range. In this study, we employed a quantum sensing technique to measure the real-time production of free radicals at the subcellular level. We utilize fluorescent nanodiamonds (FNDs) that exhibit changes in their optical properties based on the surrounding magnetic noise. This way, we were able to detect the presence of free radicals. To specifically monitor radical generation near mitochondria, we coated the FNDs with an antibody targeting voltage-dependent anion channel 2 (anti-VDAC2), which is located in the outer membrane of mitochondria. We observed a significant increase in the radical load on the mitochondrial membrane when cells were exposed to hypoxia. Subsequently, during reoxygenation, the levels of radicals gradually decreased back to the normoxia state. Overall, by applying a quantum sensing technique, the connections among hypoxia, free radicals, and the cellular redox status has been revealed.

An acoustic wave sensor incorporated with a microfluidic chip for analyzing muscle cell contraction.

We report the fabrication of a microfluidic chip or lab-on-a-chip integrated with a thickness-shear mode (TSM) acoustic wave sensor for muscle cell analysis. The sensor, essentially an AT-cut quartz crystal, serves as a detector for recording changes in acoustic wave properties occurring in an attached cardiomyocyte (single heart muscle cell) during its contraction and relaxation. Presumably, the changes resulted from alterations in viscoelastic properties (e.g. stiffness) of the cells. The effects of excitation electrode size, the presence of a microfluidic channel plate, and liquid loading on the sensor were first examined. Thereafter, muscle cell contraction analysis upon chemical stimuli were described. The potential of the chip for screening of cardiovascular drugs is discussed.

Contraction bands: differences between physiologically vs. maximally activated single heart muscle cells.

High resolution interference and phase microscopy were used to inspect the striations' appearance in shortening rat heart cells. Isolated cells were treated with detergent so that shortening could be graded by addition of calcium. Upon activation sarcomeres shortened to form (a) contraction densities in the middle of the A band at 1.7 micrometer (b) disappearance of the I bands and (c) phase brightening of the A bands at 1.6 micrometer and (d) dense Cz contraction bands at shorter lengths. These changes are totally consistent with the uniform sliding of myofilaments of previously accepted fixed dimensions. However, the striated patterns differed significantly in intact cells which were electrically stimulated to shorten. Here individual A bands remained distinct, without phase brightening or contraction band formation despite sarcomere shortening to less than the length of the A band as measured in the unstimulated cell. Maximal activation of intact cells by barium contracture elicited the full sequence of striation changes (a-d) seen in the chemically skinned cells. Light diffraction analysis gave comparable interpretation, i.e., the protein within the shortened sarcomere in the physiologically activated cardiac cell is more narrowly distributed than expected for thick filaments of fixed dimensions. These optical differences may reflect the restricted presence of the globular myosin heads at the ends of the cardiac sarcomere. This situation would explain the narrow range of the cardiac length-tension relation.

Patch-voltage-clamp method for measuring fast inward current in single rat heart muscle cells.

A patch-voltage-clamp method was used to measure fast inward ionic currents in single heart muscle cells. 1. Theoretical analysis including computer simulation has shown that the method provides fast settling of membrane potential (within 10 microseconds) and reliable voltage clamp on the tested membrane patch when the area of the patch is 200-300 times smaller than the area of the whole cell membrane. 2. The transient time at the I-V converter output is increased up to 70-75 microseconds due to the stray capacity in the I-V converter feedback. When fast-response operational amplifiers are used in the set up this transient time may be decreased to 30 microseconds by partial restoration of the high-frequency components of the signal. 3. Experimental data have shown that the ionic channel population in the membrane patch of about 5 micrometers in diameter is on the one hand large enough to directly observe integral ionic current and on the other hand small enough for the fluctuations of ionic current to be appreciable. This permits the method to be applied to ionic current investigations both by classical methods and by statistical analysis.

?Micropipet? method of recording fast inward ionic currents of single heart muscle cells

?Micropipet? method of recording fast inward ionic currents of single heart muscle cells

Sodium current in single heart muscle cells

A COMPLETE description of the sodium current, INa, in heart muscle has not been obtained despite the practical importance of such information as a rational basis for the therapy of arrhythmias. INa has been examined under voltage clamp using micropipette1 or sucrose gap methods2, but the adequacy of spatial control of voltage has been questioned and temporal resolution of the early kinetics has been unsatifactory and results therefore inconclusive3. These problems reflect the fact that heart muscle is a syncytium with restricted extracellular compartments. One of the more favourable preparations is rabbit Purkinje fibres4 and Colatsky and Tsien5 have recently performed voltage clamp studies on them with the two micro-pipettes technique. They have demonstrated the feasibility of this approach when proper precautions are taken5. Quite a different approach has been taken by other investigators such as Mehdi and Sachs6 and Powell et al.7–9, who have dispersed individual heart muscle cells and examined them with micropipette techniques. In this way the complex geometry is greatly simplified although the enzymes used in dispersion may pose another set of problems. We have followed this second approach and have isolated individual heart muscle cells from adult rats with a modified version of the dispersion method reported by Powell and Twist9. To perform electrophysiological studies we have sucked single cells into a suction pipette and used a technique which allows voltage clamp and internal perfusion10. INa has been isolated by suppression of other cardiac currents and its kinetics separated from the capacitative current transient. The success of the present approach indicates its potential usefulness in cardiac electrophysiology.

Effect of Ca2+, Mg2+, and K+ on the Zeta Potential of Single Heart-Muscle Cells Isolated from Chick Embryos

Effect of Ca<SUP>2+</SUP>, Mg<SUP>2+</SUP>, and K<SUP>+</SUP> on the Zeta Potential of Single Heart-Muscle Cells Isolated from Chick Embryos

Protein und Myoglobin in hypertrophierten und dilatierten Menschenherzen: Quantitative ultraviolett-zytophotometrische Untersuchungen

Introduction In hypertrophied human hearts the volume of heart muscle fibres increases only up to 500 g heart weight. As heart weight increases further the number of heart muscle cells and connective tissue cells rises. In addition, there is a change in intracellular constituents promoting the insufficiency of the hypertrophied myocardium. In this communication protein and myoglobin was determined by U. V. cytophotometry in isolated muscle fibres of hypertrophied and dilated human hearts. The data were correlated to the different degrees of heart hypertrophy. Material and Methods Six different tissue samples were taken at autopsy from each of 6 human hearts weighing between 404 g and 855g. The aliquots were teased and single fibres were separated, under a stereomicroscope (Fa. C. Zeiss), by dissectionneedles on quartz slides in waterfree glycerine medium. The unstained single fibres were measured by an UMSP I (Fa. C. Zeiss) over a wave range from 500 nm to 260 nm. The measurements were carried out: a) over the whole width of the fibres covering a length of 10 μ, b) over an area of 5 × 10 (μ 2) inside the fibres. Regarding light scattering and loss of substances by glycerine-preparation, the results were expressed in arbitrary units (AE = extinction × area) and compared with each other. The arbitrary units (AE) at λ 420 nm were taken to represent myoglobin and at λ 280 nm tyrosine and tryptophane as an index of the protein content. Results and Discussion By preparing single fibres in glycerine medium some soluble proteins are extracted. The amount of tyrosine and tryptophane (λ 280 nm) in our measurements essentially corresponds to the amount of contractile proteins (actinomyosin). Insoluble proteins (i.e. intracellular membranes) are also included but constitute only about 20% of the total protein. The quantity of the protein in 10 μ long heart muscle fibres increases from 40 AE to 80 AE as the heart weight rises from 405 g to 855 g. Also the protein concentration per unit area shows a significant elevation from 12.05 AE to 26.55 AE. A slight swelling of muscle fibres in glycerine medium causes relatively larger volumes to be measured in the hypertrophied hearts than are usually observed. The mean diameter nevertheless remains constant at nearly 30 μ at heart weights of more than 500 g. From our measurements it appears that there is, contrary to the situation with contractile proteins, no increase in the concentration of myoglobin in hearts weighing more than 500 g. The mean amount of myoglobin in the muscle fibres of the 405 g heart was found to be 6.17 AE, in hearts above 500 g it ranged between 10.41 AE and 13.95 AE, and no significant increase with growing heart weight was recognized. Thus it is indicated that the concentration of contractile proteins in heart muscle fibres of chronically insufficient human hearts increases with increasing heart weights also beyond 500 g. Since the mean diameter of the muscle fibres remains practically constant, the increase of contractile proteins has to correspond to a decrease of other intracellular substances, of which the myoglobin remains constant. Therefore, the oxygen, i.e. energy supplying system of single heart muscle fibres is obviously involved and is expected to progressively deteriorate in hearts the weight of which exceeds 500 g.

Lipofuscin in Cardiac Hypertrophy

Summary Quantitative investigations on the lipofuscin content in human hearts from various weight classes should allow us to conclude whether or not a correlation exists between pigment content and the degree of cardiac hypertrophy. The investigations were performed on 7 μ thick tissue sections from 12 autopsied hearts, and included microscopic determinations of nuclear size, fiber width, and pigment content. Nuclear volume was calculated according to the formula of a simple circular cylinder. Lipofuscin granules, stored at the nuclear poles in the form of two circular cones, were counted directly in an optical plane and their total quantity in single heart muscle cells was calculated on the basis of nuclear volume. In addition, the chemical composition of lipofuscin was investigated by means of absorption measurements on 3 μ thick sections from the myocardium of five hearts. This led to the following results : 1. A correlation between granule content and width of heart muscle fibers was established. The pigment granule count increases with increasing fiber width and reaches a maximum of 43 granules per measuring plane among 17 μ wide fibers, in which 100-114 granules per fiber are present. No further increase in the pigment content was found as the fiber width increased still further. 2. Pigment granule count per measuring plane increased with increasing nuclear size. Maximal pigment accumulation was found among 17 μ thick heart muscle cells with nuclei 250 μ3 in size. 3. Lipofuscin content increases with increasing heart weight up to 500 g and decreases with further cardiac hypertrophy. 4. Cytophotometric measurements of intracellular and isolated lipofuscin resulted in a clear absorption peak at 280 nm, evidence for the presence of tyrosine and tryptophan, and a shoulder in the 300–400 nm region, related to the presence of conjugated fatty acids. These results indicate that lipofuscin content in heart muscle is correlated to the degree of cardiac hypertrophy (fiber width, nuclear size). Lipofuscin content, which probably represents a degradation product of cellular components (membranes, mitochondria), is related to an increased functional output of the hypertrophied heart.

Membrane resting and action potentials of single cardiac muscle fibers.

An initial report is made on the electrocardiogram of a single heart muscle cell in vivo. The potential variations obtained by electrodes placed on opposite sides of the membrane of a heart muscle fibre are 50 to 100 times as large as those recorded by standard limb leads. The observations support the assumption that during activation the cell interior becomes positive with respect to its surrounding (depolarization, followed by polarization reversal). Induced alterations in shape and form of the action current of a single heart muscle fiber should provide further insight into the nature of the normal and abnormal electrocardiogram.