Simple analysis of gel images with IOCBIO Gel.

BACKGROUND: Current solutions for the analysis of Western Blot images lack either transparency and reproducibility or can be tedious to use if one has to ensure the reproducibility of the analysis. RESULTS: Here, we present an open-source gel image analysis program, IOCBIO Gel. It is designed to simplify image analysis and link the analysis results with the metadata describing the measurements. The software runs on all major desktop operating systems. It allows one to use it in either a single-researcher environment with local storage of the data or in a multiple-researcher environment using a central database to facilitate data sharing within the research team and beyond. By recording the original image and all operations performed on it, such as image cropping, subtraction of background, sample lane selection, and integration boundaries, the software ensures the reproducibility of the analysis and simplifies making corrections at any stage of the research. The analysis results are available either through direct access to the database used to store it or through the export of the relevant data. CONCLUSIONS: The software is not only limited to Western Blot image analysis and can be used to analyze images obtained as a part of many other widely used biochemical techniques such as isoelectric focusing. By recording the original data and all the analysis steps, the program improves reproducibility in the analysis and contributes to the implementation of FAIR principles in the related fields.

Cardiomyocytes from female compared to male mice have larger ryanodine receptor clusters and higher calcium spark frequency.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca2+ transients due to larger Ca2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice.

Cardiac expression and location of hexokinase changes in a mouse model of pure creatine deficiency.

Creatine kinase (CK) is considered the main phosphotransfer system in the heart, important for overcoming diffusion restrictions and regulating mitochondrial respiration. It is substrate limited in creatine-deficient mice lacking l-arginine:glycine amidinotransferase (AGAT) or guanidinoacetate N-methyltranferase (GAMT). Our aim was to determine the expression, activity, and mitochondrial coupling of hexokinase (HK) and adenylate kinase (AK), as these represent alternative energy transfer systems. In permeabilized cardiomyocytes, we assessed how much endogenous ADP generated by HK, AK, or CK stimulated mitochondrial respiration and how much was channeled to mitochondria. In whole heart homogenates, and cytosolic and mitochondrial fractions, we measured the activities of AK, CK, and HK. Lastly, we assessed the expression of the major HK, AK, and CK isoforms. Overall, respiration stimulated by HK, AK, and CK was ∼25, 90, and 80%, respectively, of the maximal respiration rate, and ∼20, 0, and 25%, respectively, was channeled to the mitochondria. The activity, distribution, and expression of HK, AK, and CK did not change in GAMT knockout (KO) mice. In AGAT KO mice, we found no changes in AK, but we found a higher HK activity in the mitochondrial fraction, greater expression of HK I, but a lower stimulation of respiration by HK. Our findings suggest that mouse hearts depend less on phosphotransfer systems to facilitate ADP flux across the mitochondrial membrane. In AGAT KO mice, which are a model of pure creatine deficiency, the changes in HK may reflect changes in metabolism as well as influence mitochondrial regulation and reactive oxygen species production.NEW & NOTEWORTHY In creatine-deficient AGAT-/- and GAMT-/- mice, the myocardial creatine kinase system is substrate limited. It is unknown whether subcellular localization and mitochondrial ADP channeling by hexokinase and adenylate kinase may compensate as alternative phosphotransfer systems. Our results show no changes in adenylate kinase, which is the main alternative to creatine kinase in heart. However, we found increased expression and activity of hexokinase I in AGAT-/- cardiomyocytes. This could affect mitochondrial regulation and reactive oxygen species production.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

IOCBIO Kinetics: An open-source software solution for analysis of data traces.

Biological measurements frequently involve measuring parameters as a function of time, space, or frequency. Later, during the analysis phase of the study, the researcher splits the recorded data trace into smaller sections, analyzes each section separately by finding a mean or fitting against a specified function, and uses the analysis results in the study. Here, we present the software that allows to analyze these data traces in a manner that ensures repeatability of the analysis and simplifies the application of FAIR (findability, accessibility, interoperability, and reusability) principles in such studies. At the same time, it simplifies the routine data analysis pipeline and gives access to a fast overview of the analysis results. For that, the software supports reading the raw data, processing the data as specified in the protocol, and storing all intermediate results in the laboratory database. The software can be extended by study- or hardware-specific modules to provide the required data import and analysis facilities. To simplify the development of the data entry web interfaces, that can be used to enter data describing the experiments, we released a web framework with an example implementation of such a site. The software is covered by open-source license and is available through several online channels.

Revealing calcium fluxes by analyzing inhibition dynamics in action potential clamp.

In cardiac excitation-contraction coupling (ECC), calcium enters the cytosol via L-type Ca2+ channels (LTCC) and reverse Na+/Ca2+-exchange (NCXrev), or is released from the sarcoplasmic reticulum (SR) by Ca2+-induced Ca2+-release (CICR). The magnitude of Ca2+ influx via the different pathways varies with the state of the cell and is difficult to assess quantitatively, because changes in Ca2+ influx through one pathway affect the others. In rainbow trout ventricular myocytes, the role of the SR has been uncertain for decades. The aim of this work was therefore two-fold: 1) to develop a method to quantify the Ca2+ influx pathways, and 2) to determine the role of CICR from the SR in trout ventricular myocytes. The novelty of our developed method lies in the mathematical analysis of measured transsarcolemmal Ca2+ currents and their impact on the corresponding Ca2+ transient during gradual inhibition of the currents in action potential (AP) clamp. We tested the developed method using an excitation-contraction model and showed that the method was able to recover calcium fluxes from noisy synthetic data. We applied the approach to trout ventricular myocytes and quantified the relative contributions of different Ca2+ influx pathways in ECC and determined the kinetics of these fluxes. Under baseline conditions, NCXrev is the main transmembrane Ca2+ influx pathway contributing 29 ± 6% (of the Ca2+ influx), LTCC 18 ± 7%, and CICR 53 ± 10% to overall Ca2+ transient. Thus, NCXrev is an important regulator of contractility and probably plays a role in the negative force-frequency relationship of trout ventricular preparations. These results demonstrate that trout and neonatal mammalian cardiomyocytes resemble each other not only in terms of morphology and energetics but ECC as well. In summary, the developed method resolves the major problem how to separate highly interconnected fluxes in AP clamp and allows to study Ca2+ fluxes in cardiomyocytes under conditions close to in vivo.

Restricted ADP movement in cardiomyocytes: Cytosolic diffusion obstacles are complemented with a small number of open mitochondrial voltage-dependent anion channels.

Adequate intracellular energy transfer is crucial for proper cardiac function. In energy starved failing hearts, partial restoration of energy transfer can rescue mechanical performance. There are two types of diffusion obstacles that interfere with energy transfer from mitochondria to ATPases: mitochondrial outer membrane (MOM) with voltage-dependent anion channel (VDAC) permeable to small hydrophilic molecules and cytoplasmatic diffusion barriers grouping ATP-producers and -consumers. So far, there is no method developed to clearly distinguish the contributions of cytoplasmatic barriers and MOM to the overall diffusion restriction. Furthermore, the number of open VDACs in vivo remains unknown. The aim of this work was to establish the partitioning of intracellular diffusion obstacles in cardiomyocytes. We studied the response of mitochondrial oxidative phosphorylation of permeabilized rat cardiomyocytes to changes in extracellular ADP by recording 3D image stacks of NADH autofluorescence. Using cell-specific mathematical models, we determined the permeability of MOM and cytoplasmatic barriers. We found that only ~2% of VDACs are accessible to cytosolic ADP and cytoplasmatic diffusion barriers reduce the apparent diffusion coefficient by 6-10×. In cardiomyocytes, diffusion barriers in the cytoplasm and by the MOM restrict ADP/ATP diffusion to similar extents suggesting a major role of both barriers in energy transfer and other intracellular processes.

Real-time determination of sarcomere length of a single cardiomyocyte during contraction.

Sarcomere length of a cardiomyocyte is an important control parameter for physiology studies on a single cell level; for instance, its accurate determination in real time is essential for performing single cardiomyocyte contraction experiments. The aim of this work is to develop an efficient and accurate method for estimating a mean sarcomere length of a contracting cardiomyocyte using microscopy images as an input. The novelty in developed method lies in 1) using unbiased measure of similarities to eliminate systematic errors from conventional autocorrelation function (ACF)-based methods when applied to region of interest of an image, 2) using a semianalytical, seminumerical approach for evaluating the similarity measure to take into account spatial dependence of neighboring image pixels, and 3) using a detrend algorithm to extract the sarcomere striation pattern content from the microscopy images. The developed sarcomere length estimation procedure has superior computational efficiency and estimation accuracy compared with the conventional ACF and spectral analysis-based methods using fast Fourier transform. As shown by analyzing synthetic images with the known periodicity, the estimates obtained by the developed method are more accurate at the subpixel level than ones obtained using ACF analysis. When applied in practice on rat cardiomyocytes, our method was found to be robust to the choice of the region of interest that may 1) include projections of carbon fibers and nucleus, 2) have uneven background, and 3) be slightly disoriented with respect to average direction of sarcomere striation pattern. The developed method is implemented in open-source software.

Molecular dynamics simulations of creatine kinase and adenine nucleotide translocase in mitochondrial membrane patch.

Interaction between mitochondrial creatine kinase (MtCK) and adenine nucleotide translocase (ANT) can play an important role in determining energy transfer pathways in the cell. Although the functional coupling between MtCK and ANT has been demonstrated, the precise mechanism of the coupling is not clear. To study the details of the coupling, we turned to molecular dynamics simulations. We introduce a new coarse-grained molecular dynamics model of a patch of the mitochondrial inner membrane containing a transmembrane ANT and an MtCK above the membrane. The membrane model consists of three major types of lipids (phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) in a roughly 2:1:1 molar ratio. A thermodynamics-based coarse-grained force field, termed MARTINI, has been used together with the GROMACS molecular dynamics package for all simulated systems in this work. Several physical properties of the system are reproduced by the model and are in agreement with known data. This includes membrane thickness, dimension of the proteins, and diffusion constants. We have studied the binding of MtCK to the membrane and demonstrated the effect of cardiolipin on the stabilization of the binding. In addition, our simulations predict which part of the MtCK protein sequence interacts with the membrane. Taken together, the model has been verified by dynamical and structural data and can be used as the basis for further studies.

Unchanged mitochondrial organization and compartmentation of high-energy phosphates in creatine-deficient GAMT-/- mouse hearts.

Disruption of the creatine kinase (CK) system in hearts of CK-deficient mice leads to changes in the ultrastructure and regulation of mitochondrial respiration. We expected to see similar changes in creatine-deficient mice, which lack the enzyme guanidinoacetate methyltransferase (GAMT) to produce creatine. The aim of this study was to characterize the changes in cardiomyocyte mitochondrial organization, regulation of respiration, and intracellular compartmentation associated with GAMT deficiency. Three-dimensional mitochondrial organization was assessed by confocal microscopy. On populations of permeabilized cardiomyocytes, we recorded ADP and ATP kinetics of respiration, competition between mitochondria and pyruvate kinase for ADP produced by ATPases, ADP kinetics of endogenous pyruvate kinase, and ATP kinetics of ATPases. These data were analyzed by mathematical models to estimate intracellular compartmentation. Quantitative analysis of morphological and kinetic data as well as derived model fits showed no difference between GAMT-deficient and wild-type mice. We conclude that inactivation of the CK system by GAMT deficiency does not alter mitochondrial organization and intracellular compartmentation in relaxed cardiomyocytes. Thus, our results suggest that the healthy heart is able to preserve cardiac function at a basal level in the absence of CK-facilitated energy transfer without compromising intracellular organization and the regulation of mitochondrial energy homeostasis. This raises questions on the importance of the CK system as a spatial energy buffer in unstressed cardiomyocytes.

Sensitivity analysis of flux determination in heart by H2 ¹8O -provided labeling using a dynamic Isotopologue model of energy transfer pathways.

To characterize intracellular energy transfer in the heart, two organ-level methods have frequently been employed: ³¹P − NMR inversion and saturation transfer, and dynamic ¹8O labeling. Creatine kinase (CK) fluxes obtained by following oxygen labeling have been considerably smaller than the fluxes determined by ³¹P − NMR saturation transfer. It has been proposed that dynamic ¹8O labeling determines net flux through CK shuttle, whereas ³¹P − NMR saturation transfer measures total unidirectional flux. However, to our knowledge, no sensitivity analysis of flux determination by oxygen labeling has been performed, limiting our ability to compare flux distributions predicted by different methods. Here we analyze oxygen labeling in a physiological heart phosphotransfer network with active CK and adenylate kinase (AdK) shuttles and establish which fluxes determine the labeling state. A mathematical model consisting of a system of ordinary differential equations was composed describing ¹8O enrichment in each phosphoryl group and inorganic phosphate. By varying flux distributions in the model and calculating the labeling, we analyzed labeling sensitivity to different fluxes in the heart. We observed that the labeling state is predominantly sensitive to total unidirectional CK and AdK fluxes and not to net fluxes. We conclude that measuring dynamic incorporation of ¹8O into the high-energy phosphotransfer network in heart does not permit unambiguous determination of energetic fluxes with a higher magnitude than the ATP synthase rate when the bidirectionality of fluxes is taken into account. Our analysis suggests that the flux distributions obtained using dynamic ¹8O labeling, after removing the net flux assumption, are comparable with those from ³¹P − NMR inversion and saturation transfer.

Rat and mouse cardiomyocytes show subtle differences in creatine kinase expression and compartmentalization.

Creatine kinase (CK) and adenylate kinase (AK) are energy transfer systems. Different studies on permeabilized cardiomyocytes suggest that ADP-channelling from mitochondrial CK alone stimulates respiration to its maximum, VO2_max, in rat but not mouse cardiomyocytes. Results are ambiguous on ADP-channelling from AK to mitochondria. This study was undertaken to directly compare the CK and AK systems in rat and mouse hearts. In homogenates, we assessed CK- and AK-activities, and the CK isoform distribution. In permeabilized cardiomyocytes, we assessed mitochondrial respiration stimulated by ADP from CK and AK, VO2_CK and VO2_AK, respectively. The ADP-channelling from CK or AK to mitochondria was assessed by adding PEP and PK to competitively inhibit the respiration rate. We found that rat compared to mouse hearts had a lower aerobic capacity, higher VO2_CK/VO2_max, and different CK-isoform distribution. Although rat hearts had a larger fraction of mitochondrial CK, less ADP was channeled from CK to the mitochondria. This suggests different intracellular compartmentalization in rat and mouse cardiomyocytes. VO2_AK/VO2_max was similar in mouse and rat cardiomyocytes, and AK did not channel ADP to the mitochondria. In the absence of intracellular compartmentalization, the AK- and CK-activities in homogenate should have been similar to the ADP-phosphorylation rates estimated from VO2_AK and VO2_CK in permeabilized cardiomyocytes. Instead, we found that the ADP-phosphorylation rates estimated from permeabilized cardiomyocytes were 2 and 9 times lower than the activities recorded in homogenate for CK and AK, respectively. Our results highlight the importance of energetic compartmentalization in cardiac metabolic regulation and signalling.

Combined action of albumin and heparin regulates lipoprotein lipase oligomerization, stability, and ligand interactions.

Lipoprotein lipase (LPL), a crucial enzyme in the intravascular hydrolysis of triglyceride-rich lipoproteins, is a potential drug target for the treatment of hypertriglyceridemia. The activity and stability of LPL are influenced by a complex ligand network. Previous studies performed in dilute solutions suggest that LPL can appear in various oligomeric states. However, it was not known how the physiological environment, that is blood plasma, affects the action of LPL. In the current study, we demonstrate that albumin, the major protein component in blood plasma, has a significant impact on LPL stability, oligomerization, and ligand interactions. The effects induced by albumin could not solely be reproduced by the macromolecular crowding effect. Stabilization, isothermal titration calorimetry, and surface plasmon resonance studies revealed that albumin binds to LPL with affinity sufficient to form a complex in both the interstitial space and the capillaries. Negative stain transmission electron microscopy and raster image correlation spectroscopy showed that albumin, like heparin, induced reversible oligomerization of LPL. However, the albumin induced oligomers were structurally different from heparin-induced filament-like LPL oligomers. An intriguing observation was that no oligomers of either type were formed in the simultaneous presence of albumin and heparin. Our data also suggested that the oligomer formation protected LPL from the inactivation by its physiological regulator angiopoietin-like protein 4. The concentration of LPL and its environment could influence whether LPL follows irreversible inactivation and aggregation or reversible LPL oligomer formation, which might affect interactions with various ligands and drugs. In conclusion, the interplay between albumin and heparin could provide a mechanism for ensuring the dissociation of heparan sulfate-bound LPL oligomers into active LPL upon secretion into the interstitial space.

Analysis of molecular movement reveals latticelike obstructions to diffusion in heart muscle cells.

Intracellular diffusion in muscle cells is known to be restricted. Although characteristics and localization of these restrictions is yet to be elucidated, it has been established that ischemia-reperfusion injury reduces the overall diffusion restriction. Here we apply an extended version of raster image correlation spectroscopy to determine directional anisotropy and coefficients of diffusion in rat cardiomyocytes. Our experimental results indicate that diffusion of a smaller molecule (1127 MW fluorescently labeled ATTO633-ATP) is restricted more than that of a larger one (10,000 MW Alexa647-dextran), when comparing diffusion in cardiomyocytes to that in solution. We attempt to provide a resolution to this counterintuitive result by applying a quantitative stochastic model of diffusion. Modeling results suggest the presence of periodic intracellular barriers situated ∼1 µm apart having very low permeabilities and a small effect of molecular crowding in volumes between the barriers. Such intracellular structuring could restrict diffusion of molecules of energy metabolism, reactive oxygen species, and apoptotic signals, enacting a significant role in normally functioning cardiomyocytes as well as in pathological conditions of the heart.

Ontogeny of cardiomyocytes: ultrastructure optimization to meet the demand for tight communication in excitation-contraction coupling and energy transfer.

The ontogeny of the heart describes its development from the fetal to the adult stage. In newborn mammals, blood pressure and thus cardiac performance are relatively low. The cardiomyocytes are thin, and with a central core of mitochondria surrounded by a ring of myofilaments, while the sarcoplasmic reticulum (SR) is sparse. During development, as blood pressure and performance increase, the cardiomyocytes become more packed with structures involved in excitation-contraction (e-c) coupling (SR and myofilaments) and the generation of ATP (mitochondria) to fuel the contraction. In parallel, the e-c coupling relies increasingly on calcium fluxes through the SR, while metabolism relies increasingly on fatty acid oxidation. The development of transverse tubules and SR brings channels and transporters interacting via calcium closer to each other and is crucial for e-c coupling. However, for energy transfer, it may seem counterintuitive that the increased structural density restricts the overall ATP/ADP diffusion. In this review, we discuss how this is because of the organization of all these structures forming modules. Although the overall diffusion across modules is more restricted, the energy transfer within modules is fast. A few studies suggest that in failing hearts this modular design is disrupted, and this may compromise intracellular energy transfer. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Permeabilized rat cardiomyocyte response demonstrates intracellular origin of diffusion obstacles.

Intracellular diffusion restrictions for ADP and other molecules have been predicted earlier based on experiments on permeabilized fibers or cardiomyocytes. However, it is possible that the effective diffusion distance is larger than the cell dimensions due to clumping of cells and incomplete separation of cells in fiber preparations. The aim of this work was to check whether diffusion restrictions exist inside rat cardiomyocytes or are caused by large effective diffusion distance. For that, we determined the response of oxidative phosphorylation (OxPhos) to exogenous ADP and ATP stimulation in permeabilized rat cardiomyocytes using fluorescence microscopy. The state of OxPhos was monitored via NADH and flavoprotein autofluorescence. By varying the ADP or ATP concentration in flow chamber, we determined that OxPhos has a low affinity in cardiomyocytes. The experiments were repeated in a fluorometer on cardiomyocyte suspensions leading to similar autofluorescence changes induced by ADP as recorded under the microscope. ATP stimulated OxPhos more in a fluorometer than under the microscope, which was attributed to accumulation of ADP in fluorometer chamber. By calculating the flow profile around the cell in the microscope chamber and comparing model solutions to measured data, we demonstrate that intracellular structures impose significant diffusion obstacles in rat cardiomyocytes.

Modulation of energy transfer pathways between mitochondria and myofibrils by changes in performance of perfused heart.

In the heart, the energy supplied by mitochondria to myofibrils is continuously and finely tuned to the contraction requirement over a wide range of cardiac loads. This process is mediated both by the creatine kinase (CK) shuttle and by direct ATP transfer. The aim of this study was to identify the contribution of energy transfer pathways at different cardiac performance levels. For this, five protocols of (31)P NMR inversion and saturation transfer experiments were performed at different performance levels on Langendorff perfused rat hearts. The cardiac performance was changed either through variation of external calcium in the presence or absence of isoprenaline or through variation of LV balloon inflation. The recordings were analyzed by mathematical models composed on the basis of different energy transfer pathway configurations. According to our results, the total CK unidirectional flux was relatively stable when the cardiac performance was changed by increasing the calcium concentration or variation of LV balloon volume. The stability of total CK unidirectional flux is lost at extreme energy demand levels leading to a rise in inorganic phosphate, a drop of ATP and phosphocreatine, a drop of total CK unidirectional flux, and to a bypass of CK shuttle by direct ATP transfer. Our results provide experimental evidence for the existence of two pathways of energy transfer, direct ATP transfer, and PCr transfer through the CK shuttle, whose contribution may vary depending on the metabolic status of the heart.

ADP compartmentation analysis reveals coupling between pyruvate kinase and ATPases in heart muscle.

Cardiomyocytes have intracellular diffusion restrictions, which spatially compartmentalize ADP and ATP. However, the models that predict diffusion restrictions have used data sets generated in rat heart permeabilized fibers, where diffusion distances may be heterogeneous. This is avoided by using isolated, permeabilized cardiomyocytes. The aim of this work was to analyze the intracellular diffusion of ATP and ADP in rat permeabilized cardiomyocytes. To do this, we measured respiration rate, ATPase rate, and ADP concentration in the surrounding solution. The data were analyzed using mathematical models that reflect different levels of cell compartmentalization. In agreement with previous studies, we found significant diffusion restriction by the mitochondrial outer membrane and confirmed a functional coupling between mitochondria and a fraction of ATPases in the cell. In addition, our experimental data show that considerable activity of endogenous pyruvate kinase (PK) remains in the cardiomyocytes after permeabilization. A fraction of ATPases were inactive without ATP feedback by this endogenous PK. When analyzing the data, we were able to reproduce the measurements only with the mathematical models that include a tight coupling between the fraction of endogenous PK and ATPases. To our knowledge, this is the first time such a strong coupling of PK to ATPases has been demonstrated in permeabilized cardiomyocytes.

Cardiac muscle regulatory units are predicted to interact stronger than neighboring cross-bridges.

Strong interactions between cross-bridges (XB) and regulatory units (RU) lead to a steep response of cardiac muscle to an increase in intracellular calcium. We developed a model to quantitatively assess the influence of different types of interactions within the sarcomere on the properties of cardiac muscle. In the model, the ensembles consisting of cross-bridge groups connected by elastic tropomyosin are introduced, and their dynamics is described by a set of partial differential equations. Through large scans in the free energy landscape, we demonstrate the different influence of RU-RU, XB-XB, and XB-RU interactions on the cooperativity coefficient of calcium binding, developed maximal force, and calcium sensitivity. The model solution was fitted to reproduce experimental data on force development during isometric contraction, shortening in physiological contraction, and ATP consumption by acto-myosin. On the basis of the fits, we quantified the free energy change introduced through RU-RU and XB-XB interactions and showed that RU-RU interaction leads to ~ 5 times larger change in the free energy profile of the reaction than XB-XB interaction. Due to the deterministic description of muscle contraction and its thermodynamic consistency, we envision that the developed model can be used to study heart muscle biophysics on tissue and organ levels.

Marker enzyme activities in hindleg from creatine-deficient AGAT and GAMT KO mice - differences between models, muscles, and sexes.

Creatine kinase (CK) functions as an energy buffer in muscles. Its substrate, creatine, is generated by L-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Creatine deficiency has more severe consequences for AGAT than GAMT KO mice. In the present study, to characterize their muscle phenotype further, we recorded the weight of tibialis anterior (TA), extensor digitorum longus (EDL), gastrocnemius (GAS), plantaris (PLA) and soleus (SOL) from creatine-deficient AGAT and GAMT, KO and WT mice. In GAS, PLA and SOL representing glycolytic, intermediate and oxidative muscle, respectively, we recorded the activities of pyruvate kinase (PK), lactate dehydrogenase (LDH), citrate synthase (CS) and cytochrome oxidase (CO). In AGAT KO compared to WT mice, muscle atrophy and differences in marker enzyme activities were more pronounced in glycolytic than oxidative muscle. In GAMT KO compared to WT, the atrophy was modest, differences in PK and LDH activities were minor, and CS and CO activities were slightly higher in all muscles. SOL from males had higher CS and CO activities compared to females. Our results add detail to the characterization of AGAT and GAMT KO skeletal muscle phenotypes and illustrate the importance of taking into account differences between muscles, and differences between sexes.