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.     

Propagation of the apoptotic signal by mitochondrial waves

Generation of mitochondrial signals is believed to be important in the commitment to apoptosis, but the mechanisms coordinating the output of individual mitochondria remain elusive. We show that in cardiac myotubes exposed to apoptotic agents, Ca2+ spikes initiate depolarization of mitochondria in discrete subcellular regions, and these mitochondria initiate slow waves of depolarization and Ca2+ release propagating through the cell. Traveling mitochondrial waves are prevented by Bcl-x(L), involve permeability transition pore (PTP) opening, and yield cytochrome c release, caspase activation and nuclear apoptosis. Mitochondrial Ca2+ uptake is critical for wave propagation, and mitochondria at the origin of waves take up Ca2+ particularly effectively, providing a mechanism that may underlie selection of the initiation sites. Thus, apoptotic agents transform the mitochondria into an excitable state by sensitizing PTP to Ca2+. Expansion of the local excitation by mitochondrial waves propagating through the whole cell can be especially important in activation of the apoptotic machinery in large cells.     

Calcium signalling in glial cells

Calcium signals are the universal way of glial responses to the various types of stimulation. Glial cells express numerous receptors and ion channels linked to the generation of complex cytoplasmic calcium responses. The glial calcium signals are able to propagate within glial cells and to create a spreading intercellular Ca2+ wave which allow information exchange within the glial networks. These propagating Ca2+ waves are primarily mediated by intracellular excitable media formed by intracellular calcium storage organelles. The glial calcium signals could be evoked by neuronal activity and vice versa they may initiate electrical and Ca2+ responses in adjacent neurones. Thus glial calcium signals could integrate glial and neuronal compartments being therefore involved in the information processing in the brain.     

Spatial Organization of Calcium Signaling Involved in Cell Volume Control in the Fucus Rhizoid

Subprotoplasts prepared from different regions of rhizoid and thallus cells of Fucus zygotes displayed mechanosensitive plasma membrane channels in cell-attached patch-clamp experiments by using laser microsurgery. In excised patches, this channel was found to be voltage gated, carrying K+ outward and Ca2+ inward, with a relative permeability of Ca2+/K+ of 0.35 to 0.5, and an increased open probability at membrane potentials more positive than -80 mV. No significant difference was found in the density of this channel type from different regions of rhizoid or thallus cells. Hypoosmotic treatment of intact zygotes induced dramatic transient elevations of cytoplasmic Ca2+, initiating at the rhizoid apex and propagating in a wavelike manner to subapical regions. Localized initiation of the Ca2+ transient correlated with greater osmotic swelling at the rhizoid apex compared with other regions of the zygote. Ca2+ transients exhibited a refractory period between successive hypoosmotic shocks, during which additional transients could not be elicited and the ability to osmoregulate was impaired. Buffering the Ca2+ transients with microinjected Br2BAPTA similarly reduced the ability of rhizoid cells to osmoregulate. Ca2+ influx was associated with the initiation of the Ca2+ transient in apical regions, whereas intracellular sources contributed to its propagation. Thus, localized signal transduction is patterned by interactions of the cell wall, plasma membrane, and intracellular Ca2+ stores.     

Interplay of ryanodine receptor distribution and calcium dynamics

Spontaneously generated calcium (Ca2+) waves can trigger arrhythmias in ventricular and atrial myocytes. Yet, Ca2+ waves also serve the physiological function of mediating global Ca2+ increase and muscle contraction in atrial myocytes. We examine the factors that influence Ca2+ wave initiation by mathematical modeling and large-scale computational (supercomputer) simulations. An important finding is the existence of a strong coupling between the ryanodine receptor distribution and Ca2+ dynamics. Even modest changes in the ryanodine receptor spacing profoundly affect the probability of Ca2+ wave initiation. As a consequence of this finding, we suggest that there is information flow from the contractile system to the Ca2+ control system and this dynamical interplay could contribute to the increased incidence of arrhythmias during heart failure.     

Abnormalities in gap junctions and Ca2+ dynamics in cardiomyocytes at the border zone of myocardial infarcts

Abnormalities in gap junction function and Ca2+ dynamics are believed to be important factors in arrhythmogenesis after myocardial infarction. To elucidate the relationship between changes in Ca2+ dynamics and gap junctions, we analyzed by real-time in situ Ca2+ imaging of fluo-3 loaded whole hearts the spatiotemporal occurrence of Ca2+ waves and the localization of connexin43 (Cx43) at the border zone of myocardial infarcts induced in the rat by coronary ligation. At early time points (2-4 hours postligation), different regions of the left ventricle showed distinct changes in cytosolic free Ca2+ concentrations [Ca2+]i. While some cardiomyocytes of infarcted regions exhibited high levels of resting fluo-3 fluorescence, at border zones frequent Ca2+ waves were observed. Some of the waves were abolished by spontaneous Ca2+ transients and others were not. Intact myocardium apart from infarcted regions exhibited homogenous Ca2+ transients. Confocal imaging of Cx43 and actin filaments in the rat heart fixed 2 hours after coronary ligation revealed that Cx43 was markedly decreased in the area of myocyte necrosis with contraction bands and in the neighboring myocardium. These results suggest that abnormal expression and function of gap junctions could be associated with Ca2+ waves at the border zone of myocardial infarcts, possibly through Ca2+ overload.     

Unloaded shortening increases peak of Ca2+ transients but accelerates their decay in rat single cardiac myocytes

It is of paramount importance to investigate the relation between the time-dependent change in intracellular Ca2+ concentration ([Ca2+]i) (Ca2+ transients) and the mechanical activity of isolated single myocytes to understand the regulatory mechanisms of heart function. However, because of technical difficulties in performing mechanical measurements with single myocytes, the simultaneous recording of Ca2+ transients and mechanical activity has mainly been performed with multicellular cardiac preparations that give conflicting results concerning Ca2+ transients during isometric twitches and during twitches with unloaded shortening. In the present study, we coupled intracellular Ca2+ measurement optics with a force measurement system using carbon fibers to examine the relation between Ca2+ transients and the mechanical activity of rat single ventricular myocytes over a wide range of load. To minimize the possible load dependence of sarcoplasmic reticulum Ca2+ loading, contraction mode was switched at every twitch from unloaded shortening to isometric contraction. During a twitch with unloaded shortening, the Ca2+ transients exhibited a higher peak and a higher rate of decay than transients during an isometric twitch. Similarly, when we changed the contraction mode in every pair of twitches, Ca2+ transients were dependent only on the mode of contraction. Mechanical uncoupling with 2,3-butanedione monoxime abolished this dependence on the mode of contraction. Our results suggest that Ca2+ transients reflect the affinity of troponin C for Ca2+, which is influenced by the change in strain on the thin filament but not by the length change per se.     

Voltage-gated calcium channels

Voltage-gated calcium (Ca(2+)) channels are key transducers of membrane potential changes into intracellular Ca(2+) transients that initiate many physiological events. There are ten members of the voltage-gated Ca(2+) channel family in mammals, and they serve distinct roles in cellular signal transduction. The Ca(V)1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The Ca(V)2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The Ca(V)3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca(2+) channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.     

Simultaneous measurements of Ca2+ currents and intracellular Ca2+ concentrations in single skeletal muscle fibers of the frog

The relationship between Ca2+ current amplitudes and myoplasmic Ca2+ transients was studied in single muscle fibers. Segments of muscle fibers were voltage-clamped in a double Vaseline gap chamber. Ca2+ transients were measured as an optical signal derived from the interaction between Ca2+ and the dye antipyrylazo III. The cells were maintained at -90 mV. Ca2+ currents were detected at pulse potentials to -50 mV, reached a maximum value at 0 mV, were reduced in size for larger depolarizations, and reversed at about 40 mV. Ca2+ transients were also detected at -50 Mv and progressively increased in size with larger pulse potentials up to 10 mV. Depolarizations to voltages greater than 10 mV did not further increase the size of the transient. The magnitude and time course of transients from 10 to 70 mV were almost identical Ca2+ fluxes into the myoplasm (Ca2+ input fluxes) were calculated from the Ca2+ transients applying a removal model. The size of the input fluxes increased with depolarization up to 0 mV. Between 0 and 70 mV the peak input flux slightly increased, while the flux measured at 200 ms remained unchanged. In conclusion, Ca2+ transients and input fluxes were not reduced during pulses to large positive potentials, even though a drastic reduction of Ca2+ current occurred at these potentials. These observations make it very unlikely that a voltage-dependent Ca2+ entry is the triggering signal for contraction.     

Microscopic spiral waves reveal positive feedback in subcellular calcium signaling.

The regenerative Ca(2+)-induced Ca2+ release mechanism is an important amplifier of signal transduction in diverse cells. In heart muscle cells, this mechanism contributes to the Ca2+ transient activating the mechanical contraction, but it is also believed to drive Ca2+ waves propagating within the cytosol. We investigated the subcellular Ca2+ distribution in heart muscle cells during spontaneous Ca2+ release using laser scanning confocal microscopy with a ratiometric fluorescent indicator technique. Besides planar Ca2+ waves with linear propagation, sequences of confocal optical sections also revealed spiral Ca2+ waves spinning around a subcellular core at approximately 1 Hz. Although the Ca2+ spirals were continuous processes they frequently exhibited an apparently oscillatory output function into the elongated cell body. These oscillatory waves emanating from the spiral at regular intervals were formally considered to be short outer segments of the spiral but could not be distinguished from planar Ca2+ waves propagating along the longitudinal cell axis. The complex spatiotemporal pattern of spiral Ca2+ waves implies the participation of an active process exhibiting a large degree of positive feedback, most likely the Ca(2+)-induced Ca2+ release mechanism.     

Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.     

Arrhythmogenic Ca(2+) release from cardiac myofilaments

We investigated the initiation of Ca²⁺waves underlying triggered propagated contractions (TPCs) occurring in rat cardiac trabeculae under conditions that simulate the functional non-uniformity caused by mechanical or ischemic local damage of the myocardium. A mechanical discontinuity along the trabeculae was created by exposing the preparation to a small constant flow jet of solution with a composition that reduces excitation–contraction coupling in myocytes within that segment. Force was measured and sarcomere length as well as [Ca²⁺]_i were measured regionally. When the jet-contained Caffeine, BDM or Low-[Ca²⁺], muscle-twitch force decreased and the sarcomeres in the exposed segment were stretched by shortening of the normal regions outside the jet. During relaxation the sarcomeres in the exposed segment shortened rapidly. Short trains of stimulation at 2.5 Hz reproducibly caused Ca²⁺-waves to rise from the borders exposed to the jet. Ca²⁺-waves started during force relaxation of the last stimulated twitch and propagated into segments both inside and outside of the jet. Arrhythmias, in the form of non-driven rhythmic activity, were triggered when the amplitude of the Ca²⁺-wave increased by raising [Ca²⁺]_o. The arrhythmias disappeared when the muscle uniformity was restored by turning the jet off. We have used the four state model of the cardiac cross bridge (Xb) with feedback of force development to Ca²⁺ binding by Troponin-C (TnC) and observed that the force–Ca²⁺ relationship as well as the force–sarcomere length relationship and the time course of the force and Ca²⁺ transients in cardiac muscle can be reproduced faithfully by a single effect of force on deformation of the TnC·Ca complex and thereby on the dissociation rate of Ca²⁺. Importantly, this feedback predicts that rapid decline of force in the activated sarcomere causes release of Ca²⁺ from TnC.Ca²⁺,which is sufficient to initiate arrhythmogenic Ca²⁺ release from the sarcoplasmic reticulum. These results show that non-uniform contraction can cause Ca²⁺-waves underlying TPCs, and suggest that Ca²⁺ dissociated from myofilaments plays an important role in the initiation of arrhythmogenic Ca²⁺-waves.     

Factors involved in the susceptibility of spontaneously hypertensive rats to low K+-induced arrhythmias

Disorders of intracellular Ca2+ homeostasis and intercellular coupling are thought to be crucial in the initiation and maintenance of malignant arrhythmias. The aim of this study was to investigate possible arrhythmogenic factors in spontaneously hypertensive rats (SHR) as well as their susceptibility to low K+-related arrhythmias. The experiments were performed on isolated hearts of 13 weeks-old SHR and age-matched Wistar Kyoto rats (WKY). Equilibration of the heart by Langendorff perfusion with oxygenated, 37 degrees C warm, standard Krebs solution at a constant pressure was followed by perfusion with low K+ solution for 60 min, unless sustained ventricular fibrillation occurred earlier. Electrocardiogram and epicardial monophasic action potentials (MAPs) were continuously monitored for incidence of arrhythmias and action potential changes. Myocardial tissue was taken for ultrastructural analysis and immunodetection of the main gap junction protein, connexin-43. The results showed that hypertrophic hearts of SHR exhibited prolongation of MAPs and a decrease in phosphorylation of connexin-43. Moreover, they were more prone to low K+-induced early after-depolarisations and ventricular premature beats as well as to connexin-43 and ultrastructural alterations than WKY rats. Consequently, the incidence of ventricular tachycardia (70% vs. 50%) and both transient (50% vs. 25%) and sustained (60% vs. 25%) ventricular fibrillation was higher in SHR than WKY rats. The results suggest that both prolongation of MAP and connexin-43 alterations are important arrhythmogenic factors facilitating arrhythmias in the setting of Ca2+ disorders due to hypokalaemia.     

Interstitial cell of Cajal-like cells in the upper urinary tract

Autorhythmicity in the upper urinary tract (UUT) has long been considered to arise in specialized atypical smooth muscle cells (SMC) predominately situated in the most proximal regions of the pyeloureteric system. These atypical SMC pacemakers have been thought to trigger adjacent electrically-quiescent typical SMC to fire action potentials which allow an influx of Ca2+ and the generation of muscle contraction. More recently, the presence of cells with many of the morphological, electrical and immunohistochemical characteristics of interstitial cells of Cajal (ICC), the pacemaker cells of the gastrointestinal tract, have been located in many regions of both the upper and lower urinary tract. This article reviews the evidence from the literature and from our laboratory supporting a role of both atypical SMC and ICC-like cells in the initiation and propagation of pyeloureteric peristalsis in the UUT. We propose a new model in which there are 2 populations of pacemaker cells, high frequency atypical SMC and lower frequency ICC-like cells, both of which can drive electrically-quiescent typical SMC. The relative presence of these 2 populations of pacemaker cells and the relatively-long refractoriness of typical SMC determines the decreasing frequency of contraction with distance from the renal fornix. In the absence of the proximal pacemaker drive from atypical SMC after pyeloureteral/ureteral obstruction or surgery, ICC-like cell pacemaking provides a compensatory mechanism allowing the ureter to maintain rudimentary peristaltic waves and movement of urine from the pyelon towards the bladder.     

Atrial contractile dysfunction, fibrosis, and arrhythmias in a mouse model of cardiomyopathy secondary to cardiac-specific overexpression of tumor necrosis factor-{alpha}

Transgenic mice overexpressing the inflammatory cytokine TNF-alpha in the heart develop a progressive heart failure syndrome characterized by biventricular dilatation, decreased ejection fraction, decreased survival compared with non-transgenic littermates, and earlier pathology in males. TNF-alpha mice (TNF1.6) develop atrial arrhythmias on ambulatory telemetry monitoring that worsen with age and are more severe in males. We performed in vivo electrophysiological testing in transgenic and control mice, ex vivo optical mapping of voltage in the atria of isolated perfused TNF1.6 hearts, and in vitro studies on isolated atrial muscle and cells to study the mechanisms that lead to the spontaneous arrhythmias. Programmed stimulation induces atrial arrhythmias (n = 8/32) in TNF1.6 but not in control mice (n = 0/37), with a higher inducibility in males. In the isolated perfused hearts, programmed stimulation with single extra beats elicits reentrant atrial arrhythmias (n = 6/6) in TNF1.6 but not control hearts due to slow heterogeneous conduction of the premature beats. Lowering extracellular Ca(2+) normalizes conduction and prevents the arrhythmias. Atrial muscle and cells from TNF1.6 compared with control mice exhibit increased collagen deposition, decreased contractile function, and abnormal systolic and diastolic Ca(2+) handling. Thus abnormalities in action potential propagation and Ca(2+) handling contribute to the initiation of atrial arrhythmias in this mouse model of heart failure.     

Calcium transients and neural induction in vertebrates

Evidence indicates that a variety of different types of Ca2+ transients (i.e., standing gradients, pulses and propagating waves) may be occurring both simultaneously and sequentially during neural induction in vertebrate embryos. Transients have been observed in the dorsal marginal zone and in the presumptive neural ectoderm, suggesting that they may be generated within two distinct germ layers at separate embryological locations. It has been proposed that the Ca2+ transients might have multiple roles during the period of neural induction, ranging from: activating the expression of early neural genes; contributing to the inhibition of BMP-4 signalling; generating secretion gradients of morphogens; regulating and co-ordinating convergent extension; and establishing and reinforcing dorsoventral axis specification. Both intra- and extracellular stores (either acting separately or in combination) have been shown to generate the neuralizing Ca2+ transients via well-established release mechanisms, and transients have been shown to propagate between connected cells, suggesting an intercellular signalling dimension. Thus, good evidence is accumulating to suggest that Ca2+ might be a key central regulator in the process of neural induction.     

Formation of planar and spiral Ca2+ waves in isolated cardiac myocytes.

A novel Nipkow-type confocal microscope was applied to image spontaneously propagating Ca2+ waves in isolated rat ventricular myocytes by means of fluo-3. The sarcolemma was imaged with di-8-ANEPPS and the nucleus with SYTO 11. Full frame images in different vertical sections were obtained at video frame rate by means of an intensified CCD camera. Three types of Ca2+ waves were identified: spherical waves, planar waves, and spiral waves. Both spherical waves and spiral waves could initiate a planar wave, and planar waves were not influenced by the presence of a nucleus. Spiral waves, however, were consistently found adjacent to a nucleus and displayed a slower propagation rate and slower rate of increase in Ca2+ concentration in the wave front than did spherical and planar waves. The planar waves were apparent throughout the vertical axis of the cell, whereas spiral waves appeared to have a vertical height of approximately 3 microm, less than the maximum thickness of the nucleus (5.0 +/- 0.3 microm). These results provide experimental confirmation of previous modeling studies which predicted an influence of the nucleus on spiral-type Ca2+ waves. When a spontaneous Ca2+ wave is small relative to the size of the nucleus, it appears that the Ca2+ buffering by the nucleus is sufficient to slow the rate of spontaneous propagation of the Ca2+ wave in close proximity to the nucleus. These findings thus support the idea that the nucleus can influence complex behavior of Ca2+ waves in isolated cardiac myocytes.     

Calcium-dependent arrhythmias in transgenic mice with heart failure

Transgenic mice overexpressing the inflammatory cytokine tumor necrosis factor (TNF)-alpha (TNF-alpha mice) in the heart develop a progressive heart failure syndrome characterized by biventricular dilatation, decreased ejection fraction, atrial and ventricular arrhythmias on ambulatory telemetry monitoring, and decreased survival compared with nontransgenic littermates. Programmed stimulation in vitro with single extra beats elicits reentrant ventricular arrhythmias in TNF-alpha (n = 12 of 13 hearts) but not in control hearts. We performed optical mapping of voltage and Ca(2+) in isolated perfused ventricles of TNF-alpha mice to study the mechanisms that lead to the initiation and maintenance of the arrhythmias. When compared with controls, hearts from TNF-alpha mice have prolonged of action potential durations (action potential duration at 90% repolarization: 23 +/- 2 ms, n = 7, vs. 18 +/- 1 ms, n = 5; P < 0.05), no increased dispersion of refractoriness between apex and base, elevated diastolic and depressed systolic [Ca(2+)], and prolonged Ca(2+) transients (72 +/- 6 ms, n = 10, vs. 54 +/- 5 ms, n = 8; P < 0.01). Premature beats have diminished action potential amplitudes and conduct in a slow, heterogeneous manner. Lowering extracellular [Ca(2+)] normalizes conduction and prevents inducible arrhythmias. Thus both action potential prolongation and abnormal Ca(2+) handling may contribute to the initiation of reentrant arrhythmias in this heart failure model by mechanisms distinct from enhanced dispersion of refractoriness or triggered activity.     

Overexpression of human beta2-adrenergic receptors increases gain of excitation-contraction coupling in mouse ventricular myocytes

This study investigated cardiac excitation-contraction coupling at 37 degrees C in transgenic mice with cardiac-specific overexpression of human beta2-adrenergic receptors (TG4 mice). In field-stimulated myocytes, contraction was significantly greater in TG4 compared with wild-type (WT) ventricular myocytes. In contrast, when duration of depolarization was controlled with rectangular voltage clamp steps, contraction amplitudes initiated by test steps were the same in WT and TG4 myocytes. When cells were voltage clamped with action potentials simulating TG4 and WT action potential configurations, contractions were greater with long TG4 action potentials and smaller with shorter WT action potentials, which suggests an important role for action potential configuration. Interestingly, peak amplitude of L-type Ca2+ current (I(Ca-L)) initiated by rectangular test steps was reduced, although the voltage dependencies of contractions and currents were not altered. To explore the basis for the altered relation between contraction and I(Ca-L), Ca2+ concentrations were measured in myocytes loaded with fura 2. Diastolic concentrations of free Ca2+ and amplitudes of Ca2+ transients were similar in voltage-clamped myocytes from WT and TG4 mice. However, sarcoplasmic reticulum (SR) Ca2+ content assessed with the rapid application of caffeine was elevated in TG4 cells. Increased SR Ca2+ was accompanied by increased frequency and amplitudes of spontaneous Ca2+ sparks measured at 37 degrees C with fluo 3. These observations suggest that the gain of Ca(2+)-induced Ca2+ release is increased in TG4 myocytes. Increased gain counteracts the effects of decreased amplitude of I(Ca-L) in voltage-clamped myocytes and likely contributes to increased contraction amplitudes in field-stimulated TG4 myocytes.     

Transmural heterogeneity of calcium activity and mechanical function in the canine left ventricle

Although electrical heterogeneity within the ventricular myocardium has been the focus of numerous studies, little attention has been directed to the mechanical correlates. This study examines unloaded cell shortening, Ca(2+) transients, and inward L-type Ca(2+) current (I(Ca,L)) characteristics of epicardial, endocardial, and midmyocardial cells isolated from the canine left ventricle. Unloaded cell shortening was recorded using a video edge detector, Ca(2+) transients were measured in cells loaded with 15 microM fluo-3 AM and voltage and current-clamp recordings were obtained using patch-clamp techniques. Time to peak and latency to onset of contraction were shortest in epicardial and longest in endocardial cells; midmyocardial cells displayed an intermediate time to peak. When contraction was elicited using uniform voltage-clamp square waves, epicardial versus endocardial distinctions persisted and midmyocardial cells displayed a time to peak comparable to that of epicardium. The current-voltage relationship for I(Ca,L) and fluorescence-voltage relationship were similar in the three cell types when quantitated using square pulses. However, peak I(Ca,L) and total charge were significantly larger when an epicardial versus endocardial action potential waveform was used to elicit the current under voltage-clamp conditions. Sarcoplasmic reticulum Ca(2+) content, assessed by rapid application of caffeine, was largest in epicardial cells and contributed to a faster time to peak. Our data point to important differences in calcium homeostasis and mechanical function among the three ventricular cell types. These differences serve to synchronize contraction across the ventricular wall. Although these distinctions are conferred in part by differences in electrical characteristics of the three cell types, intrinsic differences in excitation-contraction coupling are evident.