In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.     

Theoretical support for the heart phosphocreatine energy transport shuttle based on the intracellular diffusion limited mobility of ADP

Flux rates for phosphate metabolites were calculated using the equation for radial diffusion, assuming heart intracellular conditions and a 5% concentration gradient. The data show that while the flux of phosphocreatine is about 3 times faster than ATP, both are more than two orders of magnitude greater than the known maximum rate of ATP utilization. In contrast, since the concentration of free ADP is very low, its flux is below the maximum rate of ATP turnover, while the flux of creatine is almost 3 orders of magnitude greater than ADP. The data suggest that the rate of high-energy phosphate production could be limited by ADP diffusion, with creatine thus substituting as the primary cytoplasmic-mitochondrial phosphate acceptor.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Requirement of intracellular free thiols for hydrogen peroxide-induced hypertrophy in cardiomyocytes

Reactive oxygen species (ROS) are by-products of aerobic metabolism and are implicated in the pathogenesis of several diseases. H(2)O(2) produces oxidative stress and acts as a second messenger in several cell types. We tested whether the effect of H(2)O(2) on cellular events could be altered by changes in the intracellular redox status in a cardiomyocyte cell line. Using flow cytometric measurements, we found that adding H(2)O(2) induced hypertrophy in control cells in a time-dependent manner. Pre-incubation of the cells with buthionine sulfoximine (BSO), an inhibitor of de novo GSH synthesis, induced increase in the number of cells of small sizes by the addition of H(2)O(2) as compared to non-BSO pre-incubated control cells, and exacerbated the decrease in viability. Total thiol and GSH levels in H9c2 cells pre-incubated with BSO were about 75 and 30% of control, respectively, and GSH levels fell to below the limitation of detection after the addition of H(2)O(2), although total thiol levels were not markedly decreased. In the cells pre-incubated with BSO, hypertrophy was not observed by the addition of H(2)O(2) at any level of concentration. N-acetyl-L-cysteine and cysteine not only prevented increase in the number of cells of small sizes caused by H(2)O(2) but also induced hypertrophy in cells pre-incubated with BSO. These results suggest that the intracellular free thiol levels determine whether cell death or hypertrophy occurs in cardiomyocytes in the presence of H(2)O(2). On the other hand, the hypertrophied cells did not become larger by adding H(2)O(2), but had high levels of cellular GSH, suggesting the possibility that the hypertrophied cells have tolerance to oxidative stress.     

Maturation strategies and limitations of induced pluripotent stem cell-derived cardiomyocytes

Induced pluripotent stem cells (iPSCs) have the ability to differentiate into cardiomyocytes (CMs). They are not only widely used in cardiac pharmacology screening, human heart disease modeling, and cell transplantation-based treatments, but also the most promising source of CMs for experimental and clinical applications. However, their use is largely restricted by the immature phenotype of structure and function, which is similar to embryonic or fetal CMs and has certain differences from adult CMs. In order to overcome this critical issue, many studies have explored and revealed new strategies to induce the maturity of iPSC-CMs. Therefore, this article aims to review recent induction methods of mature iPSC-CMs, related mechanisms, and limitations.     

Testing for diffusion limitations in salt-activated enzyme catalysts operating in organic solvents

The dramatic activation of serine proteases in nonaqueous media resulting from lyophilization in the presence of KCl is shown to be unrelated to relaxation of potential substrate diffusional limitations. Specifically, lyophilizing subtilisin Carlsberg in the presence of KCl and phosphate buffer in different proportions, ranging from 99% (w/w) enzyme to 1% (w/w) enzyme in the final lyophilized solids, resulted in biocatalyst preparations that were not influenced by substrate diffusion. This result was made evident through use of a classical analysis whereby initial catalytic rates, normalized per weight of total enzyme in the catalyst material, were measured as a function of active enzyme for biocatalyst preparations containing different ratios of active to inactive enzyme. The active enzyme content of a given biocatalyst preparation was controlled by mixing native subtilisin with subtilisin preinactivated with PMSF, a serine protease inhibitor, and lyophilizing the enzyme mixture in the presence of different fractions of KCl and phosphate buffer. Plots of initial reaction rates as a function of percent active subtilisin in the biocatalyst were linear for all biocatalyst preparations. Thus, enzyme activation (reported elsewhere to be as high as 3750-fold in hexane for the transesterification of N-Ac-L-Phe-OEt with n-PrOH) is a manifestation of intrinsic enzyme activation and not relaxation of diffusional limitations resulting from diluted enzyme preparations. Similar activation is reported herein for thermolysin, a nonserine protease, thereby demonstrating that enzyme activation due to lyophilization in the presence of KCl may be a general phenomenon for proteolytic enzymes.     

Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells

Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.     

Intracellular diffusion restrictions in isolated cardiomyocytes from rainbow trout

Background  

Restriction of intracellular diffusion of adenine nucleotides has been studied intensively on adult rat cardiomyocytes. However, their cause and role in vivo is still uncertain. Intracellular membrane structures have been suggested to play a role. We therefore chose to study cardiomyocytes from rainbow trout (Oncorhynchus mykiss), which are thinner and have fewer intracellular membrane structures than adult rat cardiomyocytes. Previous studies suggest that trout permeabilized cardiac fibers also have diffusion restrictions. However, results from fibers may be affected by incomplete separation of the cells. This is avoided when studying permeabilized, isolated cardiomyocytes. The aim of this study was to verify the existence of diffusion restrictions in trout cardiomyocytes by comparing ADP-kinetics of mitochondrial respiration in permeabilized fibers, permeabilized cardiomyocytes and isolated mitochondria from rainbow trout heart. Experiments were performed at 10, 15 and 20°C in the absence and presence of creatine.     

Calcium inhibits muscle FBPase and affects its intracellular localization in cardiomyocytes

As our recent investigation revealed, in mammalian heart muscle, fructose 1,6-bisphosphatase (FBPase)--a key enzyme of glyconeogenesis--is located around the Z-line, inside cells' nuclei and, as we demonstrate here for the first time, it associates with intercalated discs. Since the degree of association of numerous enzymes with subcellular structures depends on the metabolic state of the cell, we studied the effect of elevated Ca2+ concentration on localization of FBPase in cardiomyocytes. In such conditions, FBPase dissociated from the Z-line, but no visible effect on FBPase associated with intercalated discs or on the nuclear localization of the enzyme was observed. Additionally, Ca2+ appeared to be a strong inhibitor of muscle FBPase.     

Modelling intracellular H(+) ion diffusion

Intracellular pH, an important modulator of cell function, is regulated by plasmalemmal proteins that transport H(+), or its equivalent, into or out of the cell. The pH(i) is also stabilised by high-capacity, intrinsic buffering on cytoplasmic proteins, oligopeptides and other solutes, and by the extrinsic CO(2)/HCO(3)(-) (carbonic) buffer. As mobility of these buffers is lower than for the H(+) ion, they restrict proton diffusion. In this paper we use computational approaches, based on the finite difference and finite element methods (FDM and FEM, respectively), for analysing the spatio-temporal behaviour of [H(+)] when it is locally perturbed. We analyse experimental data obtained for various cell-types (cardiac myocytes, duodenal enterocytes, molluscan neurons) where pH(i) has been imaged confocally using intracellular pH-sensitive dyes. We design mathematical algorithms to generate solutions for two-dimensional diffusion that fit data in terms of an apparent intracellular H(+) diffusion coefficient, D(H)(app). The models are used to explore how the spatial distribution of [H(+)](i) is affected by membrane H(+)-equivalent transport and by cell geometry. We then develop a mechanistic model, describing spatio-temporal changes of [H(+)](i) in a cardiac ventricular myocyte in terms of H(+)-shuttling on mobile buffers and H(+)-anchoring on fixed buffers. We also discuss how modelling may include the effects of extrinsic carbonic-buffering. Overall, our computational approach provides a framework for future analyses of the physiological consequences of pH(i) non-uniformity.     

Responses of intracellular cofactors to single and dual substrate limitations

The highly systematic responses of cellular cofactors to controlled substrate limitations of electron donor, electron acceptor, and both (dual limitation) were quantified using continuous-flow cultures of Pseudomonas putida. The results showed that the NADH concentration in the cells decreased gradually as the specific rate of electron-donor utilization (-q(d)) fell or increased systematically as oxygen limitation became more severe for fixed -q(d), while the NAD concentration was invariant. The NAD(H) responses demonstrated a common strategy; compensation for a low concentration of an externally supplied substrate by increasing (or decreasing) the concentration of its internal cosubstrate (or coproduct). The compensation was dramatic, as the NAD/NADH ratio showed a 24-fold modulation in response to depletion of dissolved oxygen (DO) or acetate. In the dual-limitation region, the compensating effects toward depletion of one substrate were damped, because the other substrate was simultaneously at low concentration. However, the NAD(H) responses minimized the adverse impact from substrate depletion on overall cell metabolism. Cellular contents of ATP, ADP, and P(i) were mostly affected by -q(d), such that the phosphorylation potential, ATP/ADP . P(i), increased as -q(d) fell due to depletion of acetate, DO, or both. Since the respiration rate should be slowed by high ATP/ADP . P(i), the cellular response seems to amplify an unfavorable environmental condition when oxygen is depleted. The likely reason for this apparent disadvantageous response is that the response of phosphorylation potential is more keenly associated with other aspects of metabolic control, such as for synthesis, which requires P(i) for production of phospholipids and nucleotides. (c) 1996 John Wiley & Sons, Inc.     

Increased intracellular [dATP] enhances cardiac contraction in embryonic chick cardiomyocytes

Although ATP is the physiological substrate for cardiac contraction, cardiac contractility is significantly enhanced in vitro when only 10% of ATP substrate is replaced with 2'-deoxy-ATP (dATP). To determine the functional effects of increased intracellular [dATP] ([dATP](i)) within living cardiac cells, we used hypertonic loading with varying exogenous dATP/ATP ratios, but constant total nucleotide concentration, to elevate [dATP](i) in contractile monolayers of embryonic chick cardiomyocytes. The increase in [dATP](i) was estimated from dilution of dye added in parallel with dATP. Cell viability, average contractile amplitude, rates of contraction/relaxation, spontaneous beat frequency, and Ca2+ transient amplitude and kinetics were examined. At total [dATP](i) above approximately 70 microM, spontaneous contractions ceased, and above approximately 100 microM [dATP](i), membrane blebbing was also observed, consistent with apoptosis. Interestingly, [dATP](i) of approximately 60 microM ( approximately 40% increase over basal [dATP](i) levels) enhanced both amplitude of contraction and the rates of contraction and relaxation without affecting beat frequency. With total [dATP](i) of approximately 60 microM or less, we found no significant change in Ca2+ transients. These data indicate that there is an "optimal" concentration of exogenously loaded [dATP](i) that under controlled conditions can enhance contractility in living cardiomyocytes without affecting beat frequency or Ca2+ transients.     

Taurine prevents intracellular calcium overload during calcium paradox of cultured cardiomyocytes

Summary The effect of taurine on the cellular distribution of [Ca²⁺]_i, during the calcium paradox was examined by digital imaging of a single fura-2-loaded cell. Cardiomyocytes superfused with control medium containing 2mM Ca²⁺ exhibited typical transients associated with spontaneous beating. When the cells were exposed to Ca²⁺-free buffer, immediate cessation of both spontaneous contractions and calcium transients was observed as [Ca²⁺]; rapidly fell to a level of 3–6 × 10^–8M. Subsequent restoration of medium calcium increased [Ca²⁺]_i to level 4–7 times normal. Large increases in [Ca²⁺]_i were observed in most cells and were associated with the development of contracture and bleb formation.Taurine pretreatment (20mM) caused no significant effect on [Ca²⁺]_i during Ca²⁺ depletion. However, it inhibited excessive accumulation of [Ca²⁺]_i during the Ca²⁺ repletion. Moreover, taurine treated cells recovered their Ca²⁺-transients and beating pattern earlier than non-treated cells. Finally morphological abnormalities commonly associated with calcium overload were attenuated by taurine treatment.     

Ankyrin-B targets beta2-spectrin to an intracellular compartment in neonatal cardiomyocytes

Ankyrin-B is a spectrin-binding protein that is required for localization of inositol 1,4,5-trisphosphate receptor and ryanodine receptor in neonatal cardiomyocytes. This work addresses the interaction between ankyrin-B and beta(2)-spectrin in these cells. Ankyrin-B and beta(2)-spectrin are colocalized in an intracellular striated compartment overlying the M-line and distinct from T-tubules, sarcoplasmic reticulum, Golgi, endoplasmic reticulum, lysosomes, and endosomes. Beta(2)-Spectrin is absent in ankyrin-B-null cardiomyocytes and is restored to a normal striated pattern by rescue with green fluorescent protein-220-kDa ankyrin-B. We identified two mutants (A1000P and DAR976AAA) located in the ZU5 domain which eliminate spectrin binding activity of ankyrin-B. Ankyrin-B mutants lacking spectrin binding activity are normally targeted but do not reestablish beta(2)-spectrin in ankyrin-B(+/-) cardiomyocytes. However, both mutant forms of ankyrin-B are still capable of restoring inositol 1,4,5-trisphosphate receptor localization and normal contraction frequency of cardiomyocytes. Therefore, direct binding of beta(2)-spectrin to ankyrin-B is required for the normal targeting of beta(2)-spectrin in neonatal cardiomyocytes. In contrast, ankyrin-B localization and function are independent of beta(2)-spectrin. In summary, this work demonstrates that interaction between members of the ankyrin and beta-spectrin families previously established in erythrocytes and axon initial segments also occurs in neonatal cardiomyocytes with ankyrin-B and beta(2)-spectrin. This work also establishes a functional hierarchy in which ankyrin-B determines the localization of beta(2)-spectrin and operates independently of beta(2)-spectrin in its role in organizing membrane-spanning proteins.     

Effects of extremely low frequency electromagnetic fields on intracellular calcium transients in cardiomyocytes

Abstract

Calcium transients play an essential role in cardiomyocytes and electromagnetic fields (EMF) and affect intracellular calcium levels in many types of cells. Effects of EMF on intracellular calcium transients in cardiomyocytes are not well studied. The aim of this study was to assess whether extremely low frequency electromagnetic fields (ELF-EMF) could affect intracellular calcium transients in cardiomyocytes. Cardiomyocytes isolated from neonatal Sprague-Dawley rats were exposed to rectangular-wave pulsed ELF-EMF at four different frequencies (15?Hz, 50?Hz, 75?Hz and 100?Hz) and at a flux density of 2?mT. Intracellular calcium concentration ([Ca²⁺]_i) was measured using Fura-2/AM and spectrofluorometry. Perfusion of cardiomyocytes with a high concentration of caffeine (10?mM) was carried out to verify the function of the cardiac Na⁺/Ca²⁺ exchanger (NCX) and the activity of sarco(endo)-plasmic reticulum Ca²⁺-ATPase (SERCA2a). The results showed that ELF-EMF enhanced the activities of NCX and SERCA2a, increased [Ca²⁺]_i baseline level and frequency of calcium transients in cardiomyocytes and decreased the amplitude of calcium transients and calcium level in sarcoplasmic reticulum. These results indicated that ELF-EMF can regulate calcium-associated activities in cardiomyocytes.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na+ is known to increase the intracellular Ca2+ concentration ([Ca2+]i) in cardiac muscle, the exact mechanisms of low Na+ -induced increases in [Ca2+]i are not completely defined. To gain information in this regard, we examined the effects of low Na+ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca2+]i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na+ -induced increase in [Ca2+], was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na+ did not alter the ATP-induced increase in [Ca2+]i in the cardiomyocytes. This increase in [Ca2+]i due to low Na+ and elevated KCl was dependent on the extracellular concentration of Ca2+ (0.25-2.0 mM). The L-type Ca2+ -channel blockers, verapamil and diltiazem, at low concentrations (1 microM) depressed the low Na+, KCl-induced increase in [Ca2+]i without significantly affecting the response to low Na+ alone. The low Na+, high KCl-induced increase in [Ca2+]i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 microM), and diltiazem (5 and 10 microM) as well as with amiloride (5-20 microM), nickel (1.25-5.0 mM), cyclopiazonic acid (25 and 50 microM) and thapsigargin (10 and 20 microM). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 microM). These data suggest that in addition to the sarcolemmal Na+ - Ca2+ exchanger, both sarcolemmal Na+ - K+ ATPase, as well as the sarcoplasmic reticulum Ca2+ -pump play prominent roles in the low Na+ -induced increase in [Ca2+]i.     

Mechanisms of low Na+-induced increase in intracellular calcium in KCl-depolarized rat cardiomyocytes

Although low Na⁺ is known to increase the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cardiac muscle, the exact mechanisms of low Na⁺-induced increases in [Ca²⁺]_i are not completely defined. To gain information in this regard, we examined the effects of low Na⁺ (35 mM) on freshly isolated cardiomyocytes from rat heart in the absence and presence of different interventions. The [Ca²⁺]_i in cardiomyocytes was measured fluorometrically with Fura-2 AM. Following a 10 min incubation, the low Na⁺-induced increase in [Ca²⁺]_i was only observed in cardiomyocytes depolarized with 30 mM KCl, but not in quiescent cardiomyocytes. In contrast, low Na⁺ did not alter the ATP-induced increase in [Ca²⁺]_i in the cardiomyocytes. This increase in [Ca²⁺]_i due to low Na⁺ and elevated KCl was dependent on the extracellular concentration of Ca²⁺ (0.25–2.0 mM). The L-type Ca²⁺-channel blockers, verapamil and diltiazem, at low concentrations (1 M) depressed the low Na⁺, KCl-induced increase in [Ca²⁺]_i without significantly affecting the response to low Na⁺ alone. The low Na⁺, high KCl-induced increase in [Ca²⁺]_i was attenuated by treatments of cardiomyocytes with high concentrations of both verapamil (5 and 10 M), and diltiazem (5 and 10 M) as well as with amiloride (5–20 M), nickel (1.25–5.0 mM), cyclopiazonic acid (25 and 50 M) and thapsigargin (10 and 20 M). On the other hand, this response was augmented by ouabain (1 and 2 mM) and unaltered by 5-(N-methyl-N-isobutyl) amiloride (5 and 10 M). These data suggest that in addition to the sarcolemmal Na⁺–Ca²⁺ exchanger, both sarcolemmal Na⁺–K⁺ATPase, as well as the sarcoplasmic reticulum Ca²⁺-pump play prominent roles in the low Na⁺-induced increase in [Ca²⁺]_i. (Mol Cell Biochem 263: 151–162, 2004)     

Slow diffusion of K+ in the T tubules of rat cardiomyocytes.

Cardiomyocyte contractility is regulated by the extracellular K(+) concentration ([K(+)](o)). Potassium dynamics in the T tubules during the excitation-contraction cycle depends on the diffusion rate of K(+), but this rate is not known. Detubulation of rat cardiomyocytes was induced by osmotic shock using formamide, which separated the surface membrane from the T tubules. Changes in current and membrane potential in voltage-clamped (-80 mV) and current-clamped control and detubulated cardiomyocytes were compared during rapid switches between 5.4 and 8.1 mM [K(+)](o), and the results were simulated in a mathematical model. In the voltage-clamp experiments, the current changed significantly slower in control than in detubulated cardiomyocytes during the switch from 5.4 to 8.1 mM [K(+)](o), as indicated by the times to achieve 25, 50, 90, and 95% of the new steady-state current [control (ms) t(25) = 98 +/- 12, t(50) = 206 +/- 20, t(90) = 570 +/- 72, t(95) = 666 +/- 92; detubulated t(25) = 61 +/- 11, t(50) = 142 +/- 17, t(90) = 352 +/- 52, t(95) = 420 +/- 69]. These time points were not significantly different either during the 8.1 to 5.4 mM [K(+)](o) switch or in current-clamped cardiomyocytes switching from 5.4 to 8.1 mM [K(+)](o). Mathematical simulation of the difference current between control and detubulated cardiomyocytes gave a t-tubular diffusion rate for K(+) of approximately 85 mum(2)/s. We conclude that the diffusion of K(+) in the T tubules is so slow that they constitute a functional compartment. This might play a key role in local regulation of the action potential, and thus in the regulation of cardiomyocyte contractility.