Contractility of ventricular myocytes is well preserved despite altered mechanisms of Ca2+ transport and a changing pattern of mRNA in aged type 2 Zucker diabetic fatty rat heart

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The objective of the study was to investigate ventricular myocyte shortening, intracellular Ca(2+) signalling and expression of genes encoding cardiac muscle proteins in the aged Zucker diabetic fatty (ZDF) rat. There was a fourfold elevation in non-fasting blood glucose in ZDF rats (478.43 ± 29.22 mg/dl) compared to controls (108.22 ± 2.52 mg/dl). Amplitude of shortening, time to peak (TPK) and time to half (THALF) relaxation of shortening were unaltered in ZDF myocytes compared to age-matched controls. Amplitude and THALF decay of the Ca(2+) transient were unaltered; however, TPK Ca(2+) transient was prolonged in ZDF myocytes (70.0 ± 3.2 ms) compared to controls (58.4 ± 2.3 ms). Amplitude of the L-type Ca(2+) current was reduced across a wide range of test potentials (-30 to +40 mV) in ZDF myocytes compared to controls. Sarcoplasmic reticulum Ca(2+) content was unaltered in ZDF myocytes compared to controls. Expression of genes encoding cardiac muscle proteins, membrane Ca(2+) channels, and cell membrane ion transport and intracellular Ca(2+) transport proteins were variously altered. Myh6, Tnnt2, Cacna2d3, Slc9a1, and Atp2a2 were downregulated while Myl2, Cacna1g, Cacna1h, and Atp2a1 were upregulated in ZDF ventricle compared to controls. The results of this study have demonstrated that preserved ventricular myocyte shortening is associated with altered mechanisms of Ca(2+) transport and a changing pattern of genes encoding a variety of Ca(2+) signalling and cardiac muscle proteins in aged ZDF rat.     

Phenomenological models of the dynamics of muscle during isotonic shortening

We investigated the effectiveness of simple, Hill-type, phenomenological models of the force-length-velocity relationship for simulating measured length trajectories during muscle shortening, and, if so, what forms of the model are most useful. Using isotonic shortening data from mouse soleus and toad depressor mandibulae muscles, we showed that Hill-type models can indeed simulate the shortening trajectories with sufficiently good accuracy. However, we found that the standard form of the Hill-type muscle model, called the force-scaling model, is not a satisfactory choice. Instead, the results support the use of less frequently used models, the f-max scaling model and force-scaling with parallel spring, to simulate the shortening dynamics of muscle.     

Calcium-independent activation of skeletal muscle fibers by a modified form of cardiac troponin C.

A conformational change accompanying Ca2+ binding to troponin C (TnC) constitutes the initial event in contractile regulation of vertebrate striated muscle. We replaced endogenous TnC in single skinned fibers from rabbit psoas muscle with a modified form of cardiac TnC (cTnC) which, unlike native cTnC, probably contains an intramolecular disulfide bond. We found that such activating TnC (aTnC) enables force generation and shortening in the absence of calcium. With aTnC, both force and shortening velocity were the same at pCa 9.2 and pCa 4.0. aTnc could not be extracted under conditions which resulted in extraction of endogenous TnC. Thus, aTnC provides a stable model for structural studies of a calcium binding protein in the active conformation as well as a useful tool for physiological studies on the primary and secondary effects of Ca2+ on the molecular kinetics of muscle contraction.     

Mathematical models for the study of electromechanical and mechanoelectrical coupling in the myocardium

Mathematical models have been developed to describe interactions of electrical, mechanical and chemical processes in cardiomyocytes. The models simulate wide range of experimental data on excitation-contraction coupling and, more importantly, on mechanoelectric feedback in heart muscle. The model results clearly show that mechano-dependence of intracellular calcium handling due to cooperative effects of contractile proteins activation plays a key role in cardiac mechanoelectric coupling. At the same time, mechanosensitive currents can also contribute to action potential responses to mechanical perturbations. Using this model to study the heterogeneous myocardium we have shown that temporal and functional electromechanical heterogeneity of coupled cardiomyocytes can essentially determine the myocardium contractility. Optimization of the electromechanical function of contractile system emerges from the fine coordination between the activation sequence of cardiomyocytes, their local electromechanical properties and the mechanical interaction during contraction.     

A cell-based framework for modeling cardiac mechanics

Cardiomyocytes are the functional building blocks of the heart—yet most models developed to simulate cardiac mechanics do not represent the individual cells and their surrounding matrix. Instead, they work on a homogenized tissue level, assuming that cellular and subcellular structures and processes scale uniformly. Here we present a mathematical and numerical framework for exploring tissue-level cardiac mechanics on a microscale given an explicit three-dimensional geometrical representation of cells embedded in a matrix. We defined a mathematical model over such a geometry and parametrized our model using publicly available data from tissue stretching and shearing experiments. We then used the model to explore mechanical differences between the extracellular and the intracellular space. Through sensitivity analysis, we found the stiffness in the extracellular matrix to be most important for the intracellular stress values under contraction. Strain and stress values were observed to follow a normal-tangential pattern concentrated along the membrane, with substantial spatial variations both under contraction and stretching. We also examined how it scales to larger size simulations, considering multicellular domains. Our work extends existing continuum models, providing a new geometrical-based framework for exploring complex cell–cell and cell–matrix interactions.

     

Stochastic Simulations of Normal Aging and Werner’s Syndrome

Human cells typically consist of 23 pairs of chromosomes. Telomeres are repetitive sequences of DNA located at the ends of chromosomes. During cell replication, a number of basepairs are lost from the end of the chromosome and this shortening restricts the number of divisions that a cell can complete before it becomes senescent, or non-replicative. In this paper, we use Monte Carlo simulations to form a stochastic model of telomere shortening to investigate how telomere shortening affects normal aging. Using this model, we study various hypotheses for the way in which shortening occurs by comparing their impact on aging at the chromosome and cell levels. We consider different types of length-dependent loss and replication probabilities to describe these processes. After analyzing a simple model for a population of independent chromosomes, we simulate a population of cells in which each cell has 46 chromosomes and the shortest telomere governs the replicative potential of the cell. We generalize these simulations to Werner’s syndrome, a condition in which large sections of DNA are removed during cell division and, amongst other conditions, results in rapid aging. Since the mechanisms governing the loss of additional basepairs are not known, we use our model to simulate a variety of possible forms for the rate at which additional telomeres are lost per replication and several expressions for how the probability of cell division depends on telomere length. As well as the evolution of the mean telomere length, we consider the standard deviation and the shape of the distribution. We compare our results with a variety of data from the literature, covering both experimental data and previous models. We find good agreement for the evolution of telomere length when plotted against population doubling.     

Dynamical description of sinoatrial node pacemaking: improved mathematical model for primary pacemaker cell

We developed an improved mathematical model for a single primary pacemaker cell of the rabbit sinoatrial node. Original features of our model include 1) incorporation of the sustained inward current (I(st)) recently identified in primary pacemaker cells, 2) reformulation of voltage- and Ca(2+)-dependent inactivation of the L-type Ca(2+) channel current (I(Ca,L)), 3) new expressions for activation kinetics of the rapidly activating delayed rectifier K(+) channel current (I(Kr)), and 4) incorporation of the subsarcolemmal space as a diffusion barrier for Ca(2+). We compared the simulated dynamics of our model with those of previous models, as well as with experimental data, and examined whether the models could accurately simulate the effects of modulating sarcolemmal ionic currents or intracellular Ca(2+) dynamics on pacemaker activity. Our model represents significant improvements over the previous models, because it can 1) simulate whole cell voltage-clamp data for I(Ca,L), I(Kr), and I(st); 2) reproduce the waveshapes of spontaneous action potentials and ionic currents during action potential clamp recordings; and 3) mimic the effects of channel blockers or Ca(2+) buffers on pacemaker activity more accurately than the previous models.     

Engineering virtual cardiac tissue

The kinetics of proteins involved in ion transfer, sequestration and binding in cardiac cells can be modelled to construct a model of the electrical activity of isolated cardiac cells as a system of ordinary differential equations. These cell models may be incorporated into tissue models, which, when combined with histology and anatomy, form virtual tissues. The effects of changes in specific protein expression, or changes in protein kinetics, produced by mutations or pharmacological agents, can be simulated using these tissue models and used to account for the whole organ effects of changes in specific ion-transport protein activity.     

Anion channels influence ECC by modulating L-type Ca(2+) channel in ventricular myocytes.

Anion channels are extensively expressed in the heart, but their roles in cardiac excitation-contraction coupling (ECC) are poorly understood. We, therefore, investigated the effects of anion channels on cardiac ventricular ECC. Edge detection, fura 2 fluorescence measurements, and whole cell patch-clamp techniques were used to measure cell shortening, the intracellular Ca(2+) transient, and the L-type Ca(2+) current (I(Ca,L)) in single rat ventricular myocytes. The anion channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid reversibly inhibited the Ca(2+) transients and cell shortening in a dose-dependent manner. Comparable results were observed when the majority of the extracellular Cl(-) was replaced with the relatively impermeant anions glutamate (Glt(-)) and aspartate (Asp(-)). NPPB and niflumic acid or the Cl(-) substitutes did not affect the resting intracellular Ca(2+) concentration but significantly inhibited I(Ca,L). In contrast, replacement of extracellular Cl(-) with the permeant anions NO, SCN(-), and Br(-) supported the ECC and I(Ca,L), which were still sensitive to blockade by NPPB. Exposure of cardiac ventricular myocytes to a hypotonic bath solution enhanced the amplitude of cell shortening and supported I(Ca,L), whereas hypertonic stress depressed the contraction and I(Ca,L). Moreover, cardiac contraction was completely abolished by NPPB (50 microM) under hypotonic conditions. It is concluded that a swelling-activated anion channel may be involved in the regulation of cardiac ECC through modulating L-type Ca(2+) channel activity.     

Influence of viscosity on myocardium mechanical activity: a mathematical model

We have previously proposed and validated a mathematical model of myocardium contraction-relaxation cycle based on current knowledge of regulatory role of Ca2+ and cross-bridge kinetics in cardiac cell. That model did not include viscous elements. Here we propose a modification of the model, in which two viscous elements are added, one in parallel to the contractile element, and one more in parallel to the series elastic element. The modified model allowed us to simulate and explain some subtle experimental data on relaxation velocity in isotonic twitches and on a mismatch between the time course of sarcomere shortening/lengthening and the time course of active force generation in isometric twitches. Model results were compared with experimental data obtained from 28 rat LV papillary muscles contracting and relaxing against various loads. Additional model analysis suggested contribution of viscosity to main inotropic and lusitropic characteristics of myocardium performance.     

Sensing and refilling calcium stores in an excitable cell.

Inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ mobilization leads to depletion of the endoplasmic reticulum (ER) and an increase in Ca2+ entry. We show here for the gonadotroph, an excitable endocrine cell, that sensing of ER Ca2+ content can occur without the Ca2+ release-activated Ca2+ current (Icrac), but rather through the coupling of IP3-induced Ca2+ oscillations to plasma membrane voltage spikes that gate Ca2+ entry. Thus we demonstrate that capacitative Ca2+ entry is accomplished through Ca(2+)-controlled Ca2+ entry. We develop a comprehensive model, with parameter values constrained by available experimental data, to simulate the spatiotemporal behavior of agonist-induced Ca2+ signals in both the cytosol and ER lumen of gonadotrophs. The model combines two previously developed models, one for ER-mediated Ca2+ oscillations and another for plasma membrane potential-driven Ca2+ oscillations. Simulations show agreement with existing experimental records of store content, cytosolic Ca2+ concentration ([Ca2+]i), and electrical activity, and make a variety of new, experimentally testable predictions. In particular, computations with the model suggest that [Ca2+]i in the vicinity of the plasma membrane acts as a messenger for ER content via Ca(2+)-activated K+ channels and Ca2+ pumps in the plasma membrane. We conclude that, in excitable cells that do not express Icrac, [Ca2+]i profiles provide a sensitive mechanism for regulating net calcium flux through the plasma membrane during both store depletion and refilling.     

Negative inotropic effects of high-mobility group box 1 protein in isolated contracting cardiac myocytes

High-mobility group box 1 (HMGB1) released from necrotic cells or macrophages functions as a late inflammatory mediator and has been shown to induce cardiovascular collapse during sepsis. Thus far, however, the effect(s) of HMGB1 in the heart are not known. We determined the effects of HMGB1 on isolated feline cardiac myocytes by measuring sarcomere shortening in contracting cardiac myocytes, intracellular Ca2+ transients by using fluo-3, and L-type calcium currents by using whole cell perforate configuration of the patch-clamp technique. Treatment of isolated myocytes with HMGB1 (100 ng/ml) resulted in a 70% decrease in sarcomere shortening and a 50% decrease in the height of the peak Ca2+ transient within 5 min (P < 0.01). The immediate negative inotropic effects of HMGB1 on cell contractility and calcium homeostasis were partially reversible upon washout of HMGB1. A significant inhibition of the inward l-type calcium currents was also documented by the patch-clamp technique. HMGB1 induced the PKC-epsilon translocation, and a PKC inhibitor significantly attenuated the negative inotropic effects of HMGB1. These studies show for the first time that HMGB1 impairs sarcomere shortening by decreasing calcium availability in cardiac myocytes through modulating membrane calcium influx and suggest that HMGB1 maybe acts as a novel myocardial depressant factor during cardiac injury.     

Influence of motoneuron firing synchronization on SEMG characteristics in dependence of electrode position.

The frequency content of the surface electromyography (SEMG) signal, expressed as median frequency (MF), is often assumed to reflect the decline of muscle fiber conduction velocity in fatigue. MF also decreases when motor unit firings synchronize, and we hypothesized that this effect can explain the electrode-dependent pattern in our previous recordings from the trapezius muscle. An existing motoneuron (MN) model describes the afterhyperpolarization following a spike as an exponential function on which membrane noise is superimposed. Splitting the noise into a common and an individual component extended the model to a MN pool with a tunable level of firing synchrony. An analytical volume conduction model was used to generate motor unit action potentials to simulate SEMG. A realistic level of synchrony decreased the MF of the simulated bipolar SEMG by approximately 30% midway between endplate position and tendon but not above the endplate. This is in accordance with experimental data from the biceps brachii muscle. It was concluded that the pattern of decrease of MF during sustained contractions indeed reflects MN synchronization.     

Sarcomere mechanics in uniform and non-uniform cardiac muscle: a link between pump function and arrhythmias

Starling's Law and the well-known end-systolic pressure-volume relationship (ESPVR) of the left ventricle reflect the effect of sarcomere length (SL) on stress (sigma) development and shortening by myocytes in the uniform ventricle. We show here that tetanic contractions of rat cardiac trabeculae exhibit a sigma-SL relationship at saturating [Ca2+] that depends on sarcomere geometry in a manner similar to skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL-[Ca2+]free relationships (sigma-SL-CaR) at submaximal [Ca2+] in intact and skinned trabeculae were similar, albeit that the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-CaR using a kinetic model where we assumed that the rates of Ca2+ binding by Troponin-C (Tn-C) and/or cross-bridge (XB) cycling are determined by SL, [Ca2+] or stress. We analyzed the correlation between the model results and steady state stress measurements at varied SL and [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that: (i) the dominant feedback mechanism is SL, stress or [Ca2+]-dependent; and (ii) the feedback mechanism regulates: Tn-C-Ca2+ affinity, XB kinetics or, unitary XB-force. The analysis strongly suggests that feedback of the number of strong XBs to cardiac Tn-C-Ca2+ affinity is the dominant mechanism that regulates XB recruitment. Application of this concept in a mathematical model of twitch-stress accurately reproduced the sigma-SL-CaR and the time course of twitch-stress as well as the time course of intracellular [Ca2+]i. Modeling of the response of the cardiac twitch to rapid stress changes using the above feedback model uniquely predicted the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. The above concept has important repercussions for the non-uniformly contracting heart in which arrhythmogenic Ca2+ waves arise from weakened areas in cardiac muscle. These Ca2+ waves can reversibly be induced in muscle with non-uniform excitation contraction coupling (ECC) by the cycle of stretch and release in the border zone between the damaged and intact regions. Stimulus trains induced propagating Ca2+ waves and reversibly induced arrhythmias. We hypothesize that rapid force loss by sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). These observations suggest the unifying hypothesis that force feedback to Ca2+ binding by Tn-C is responsible for Starling's Law and the ESPVR in uniform myocardium and leads in non-uniform myocardium to a surge of Ca2+ released by the myofilaments during relaxation, which initiates arrhythmogenic propagating Ca2+ release by the SR.     

Leptin-induced suppression of cardiomyocyte contraction is amplified by ceramide

Uncorrected obesity is often accompanied by ventricular contractile dysfunction, elevation of the lipotoxic mediator ceramide and the obesity gene product leptin. Both ceramide and leptin participate in the regulation of cardiac function and are speculated to play roles in obesity-related cardiac dysfunctions. The purpose of this study was to examine the effect of ceramide on leptin-elicited cardiac contractile response. Adult rat left ventricular myocytes were incubated for 24 h with low (5 nM) or high (50 nM) concentration of leptin in the absence or presence of the active ceramide analog C2-dihydroceramide (25 microM). Contractile and intracellular Ca2+ properties were evaluated using an IonOptix MyoCam system including peak shortening (PS), maximal velocity of shortening/relengthening (+/-dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR90), intracellular Ca2+ rise (Delta[Ca2+]) and intracellular Ca2+ decay. While ceramide did not elicit any effect on cell mechanics and intracellular Ca2+ transients, it sensitized leptin-induced effects on myocyte shortening and intracellular Ca2+ transients. In the absence of ceramide, 5 nM leptin had no effect on cell mechanics while 50 nM depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90. With ceramide co-incubation, 5 nM leptin depressed PS, +/-dL/dt, Delta[Ca2+] and prolonged TR90 whereas 50 nM leptin-elicited effects on PS, +/-dL/dt, Delta[Ca2+] and TR90 were significantly potentiated in addition to slowing intracellular Ca2+ decay. In summary, our data demonstrated that ceramide sensitizes cardiac depressive effects of leptin and may contribute to hyperleptinemia-related cardiac contractile dysfunction.     

TNFR1 and TNFR2 signaling interplay in cardiac myocytes

Tumor necrosis factor alpha (TNFalpha) plays a major role in chronic heart failure, signaling through two different receptor subtypes, TNFR1 and TNFR2. Our aim was to further delineate the functional role and signaling pathways related to TNFR1 and TNFR2 in cardiac myocytes. In cardiac myocytes isolated from control rats, TNFalpha induced ROS production, exerted a dual positive and negative action on [Ca(2+)] transient and cell fractional shortening, and altered cell survival. Neutralizing anti-TNFR2 antibodies exacerbated TNFalpha responses on ROS production and cell death, arguing for a major protective role of the TNFR2 pathway. Treatment with either neutralizing anti-TNFR1 antibodies or the glutathione precursor, N-acetylcysteine (NAC), favored the emergence of TNFR2 signaling that mediated a positive effect of TNFalpha on [Ca(2+)] transient and cell fractional shortening. The positive effect of TNFalpha relied on TNFR2-dependent activation of the cPLA(2) activity, independently of serine 505 phosphorylation of the enzyme. Together with cPLA(2) redistribution and AA release, TNFalpha induced a time-dependent phosphorylation of ERK, MSK1, PKCzeta, CaMKII, and phospholamban on the threonine 17 residue. Taken together, our results characterized a TNFR2-dependent signaling and illustrated the close interplay between TNFR1 and TNFR2 pathways in cardiac myocytes. Although apparently predominant, TNFR1-dependent responses were under the yoke of TNFR2, acting as a critical limiting factor. In vivo NAC treatment proved to be a unique tool to selectively neutralize TNFR1-mediated effects of TNFalpha while releasing TNFR2 pathways.     

Biological effects of 6 mT static magnetic fields: a comparative study in different cell types

The present work was a comparative study of the bio-effects induced by exposure to 6 mT static magnetic field (MF) on several primary cultures and cell lines. Particular attention was dedicated to apoptosis. Cell viability, proliferation, intracellular Ca(2+) concentration and morphology were also examined. Primary cultures of human lymphocytes, mice thymocytes and cultures of 3DO, U937, HeLa, HepG2 and FRTL-5 cells were grown in the presence of 6 mT static MF and different apoptosis-inducing agents (cycloheximide, H(2)O(2), puromycin, heat shock, etoposide). Biological effects of static MF exposure were found in all the different cells examined. They were cell type-dependent but apoptotic inducer-independent. A common effect of the exposure to static MF was the promotion of apoptosis and mitosis, but not of necrosis or modifications of the cell shape. Increase of the intracellular levels of Ca(2+) ions were also observed. When pro-apoptotic drugs were combined with static MF, the majority of cell types rescued from apoptosis. To the contrary, apoptosis of 3DO cells was significantly increased under simultaneous exposure to static MF and incubation with pro-apoptotic drugs. From these data we conclude that 6 mT static MF exposure interfered with apoptosis in a cell type- and exposure time-dependent manner, while the effects of static MF exposure on the apoptotic program were independent of the drugs used.     

Chronic coexistence of two troponin T isoforms in adult transgenic mouse cardiomyocytes decreased contractile kinetics and caused dilatative remodeling

Our previous in vivo and ex vivo studies suggested that coexistence of two or more troponin T (TnT) isoforms in adult cardiac muscle decreased cardiac function and efficiency (Huang QQ, Feng HZ, Liu J, Du J, Stull LB, Moravec CS, Huang X, Jin JP, Am J Physiol Cell Physiol 294: C213-C22, 2008; Feng HZ, Jin JP, Am J Physiol Heart Circ Physiol 299: H97-H105, 2010). Here we characterized Ca(2+)-regulated contractility of isolated adult cardiomyocytes from transgenic mice coexpressing a fast skeletal muscle TnT together with the endogenous cardiac TnT. Without the influence of extracellular matrix, coexistence of the two TnT isoforms resulted in lower shortening amplitude, slower shortening and relengthening velocities, and longer relengthening time. The level of resting cytosolic Ca(2+) was unchanged, but the peak Ca(2+) transient was lowered and the durations of Ca(2+) rising and decaying were longer in the transgenic mouse cardiomyocytes vs. the wild-type controls. Isoproterenol treatment diminished the differences in shortening amplitude and shortening and relengthening velocities, whereas the prolonged durations of relengthening and Ca(2+) transient in the transgenic cardiomyocytes remained. At rigor state, a result from depletion of Ca(2+), resting sarcomere length of the transgenic cardiomyocytes became shorter than that in wild-type cells. Inhibition of myosin motor diminished this effect of TnT function on cross bridges. The length but not width of transgenic cardiomyocytes was significantly increased compared with the wild-type controls, corresponding to longitudinal addition of sarcomeres and dilatative remodeling at the cellular level. These dominantly negative effects of normal fast TnT demonstrated that chronic coexistence of functionally distinct variants of TnT in adult cardiomyocytes reduces contractile performance with pathological consequences.