Subcellular localization of hippocampal ryanodine receptor 2 and its role in neuronal excitability and memory.

Ryanodine receptor 2 (RyR2) is abundantly expressed in the heart and brain. Mutations in RyR2 are associated with both cardiac arrhythmias and intellectual disability. While the mechanisms of RyR2-linked arrhythmias are well characterized, little is known about the mechanism underlying RyR2-associated intellectual disability. Here, we employed a mouse model expressing a green fluorescent protein (GFP)-tagged RyR2 and a specific GFP probe to determine the subcellular localization of RyR2 in hippocampus. GFP-RyR2 was predominantly detected in the soma and dendrites, but not the dendritic spines of CA1 pyramidal neurons or dentate gyrus granular neurons. GFP-RyR2 was also detected within the mossy fibers in the stratum lucidum of CA3, but not in the presynaptic terminals of CA1 neurons. An arrhythmogenic RyR2-R4496C+/- mutation downregulated the A-type K+ current and increased membrane excitability, but had little effect on the afterhyperpolarization current or presynaptic facilitation of CA1 neurons. The RyR2-R4496C+/- mutation also impaired hippocampal long-term potentiation, learning, and memory. These data reveal the precise subcellular distribution of hippocampal RyR2 and its important role in neuronal excitability, learning, and memory.

Impaired myosin isoform shift and calcium transients contribute to cellular pathogenesis of rat cirrhotic cardiomyopathy.

BACKGROUND & AIMS: Cirrhotic cardiomyopathy is a recently recognized entity, but detailed cellular and molecular mechanisms remain unclarified. We aimed to elucidate the role of myosin heavy chain isoform shifts and their relation to calcium transients in the contractile kinetics of cirrhotic rats. METHODS: Cirrhosis was induced in male Lewis Brown-Norway rats by bile duct ligation (BDL). Myosin heavy chain (MHC) isoform distribution was evaluated by gel electrophoresis. Contractile force, Ca2+ transients and cell shortening were studied at varied frequency and extracellular [Ca2+ ]. T-tubular integrity was analysed by power spectrum analysis of images of myocytes stained with di-8-ANEPPS. RESULTS: Compared with sham controls, the phenotypes of cirrhotic rats were as follows: (a) alpha-myosin heavy chain shifted to beta-MHC isoform; (b) mild loss of T-tubular integrity in myocytes; (c) a reduced maximum and rate of rise of the Ca2+ transient (max F/Fo ); (d) a reduction in both the rate of rise and fall of contraction; (e) decreased maximal force-generating capacity; (f) loss of the inotropic effect of increased stimulus frequency; (g) unchanged sensitivity of force development to varied extracellular [Ca2+ ] and (h) increased spontaneous diastolic sarcomere length fluctuations. CONCLUSION: Cardiomyocytes and ventricular trabeculae in a cirrhotic rat model showed features of typical heart failure including systolic and diastolic prolongation, impaired force-frequency relation and decreased force-generating capacity. Impaired myosin isoform shift and calcium transients are important contributory mechanisms underlying the pathogenesis of the heart failure phenotype seen in cirrhosis.

Alternative Approaches to the Assessment of the Systemic Circulation and Left Ventricular Performance: A Proof-of-Concept Study.

BACKGROUND: The purpose of this article is to examine the systemic circulation and left ventricular (LV) performance by alternative, nonconventional approaches: systemic vascular conductance (G SV ) and the head-capacity relation (ie, the relation between LV pressure and cardiac output), respectively; in so doing, we aspired to present a novel and improved interpretation of integrated cardiovascular function. METHODS: In 16 open-chest, anaesthetized pigs, we measured LV pressure (P LV ), central aortic pressure (P Ao ), and central venous pressure (P CV ) and aortic flow (Q Ao ). We calculated heart rate (HR), stroke volume, cardiac index (CI = cardiac output/body weight), mean PLV ( P ¯ LV ) , and the average arteriovenous pressure difference ( Δ P = P ¯ Ao - P ¯ CV ); G SV  = CI/( P ¯ Ao - P ¯ CV ). We studied the effects of changing loading conditions with the administration of phenylephrine (Δ P ¯ Ao ≥ +25 mm Hg), isoproterenol (ΔHR ∼+25%), sodium nitroprusside (Δ P ¯ Ao ≥ -25 mm Hg), and proximal aortic constriction (to maximize developed P LV and minimize Q Ao ). RESULTS: Sodium nitroprusside and isoproterenol increased G SV compared with phenylephrine and constriction. A maximum head-capacity curve was derived from pooled data using nonlinear regression on the maximum P ¯ LV values in Q Ao bins 12.5 mL/min/kg wide. The head-capacity relation and the plots of conductance were combined using CI as a common axis, which illustrated that CI is the output of the heart and the input of the circulation. CONCLUSIONS: Thus, at a given CI, G SV determines the driving pressure and, thereby, P Ao . We also demonstrated how decreases in G SV compensate for arterial hypotension by restoring the arteriovenous pressure difference and arterial pressure.

CONTEXTE: Le présent article examine l'efficacité de la circulation générale et la fonction ventriculaire gauche à l'aide de paramètres de rechange non conventionnels, soit la conductance vasculaire systémique (G VS ) pour l'une et la relation pression-volume (c.-à-d. la relation entre la pression ventriculaire gauche et le débit cardiaque) pour l'autre, dans le but de présenter une interprétation nouvelle et améliorée de la fonction cardiovasculaire intégrée. MÉTHODOLOGIE: Chez 16 porcs anesthésiés, nous avons mesuré à thorax ouvert la pression ventriculaire gauche (P VG ), la pression aortique centrale (P AC ), la pression veineuse centrale (P VC ) et le flux aortique (Q A ). Nous avons établi la fréquence cardiaque (FC), le volume d'éjection systolique, l'index cardiaque (IC; rapport entre le débit cardiaque et le poids corporel), la P VG moyenne ( P ¯ VG ) et la différence de pression artérioveineuse moyenne ( Δ P = P ¯ A C − P ¯ V C ); G VS  = IC/( P ¯ AC − P ¯ VC ). Nous avons aussi étudié les effets d'une modification des conditions de charge cardiaque provoquée par l'administration de phényléphrine (Δ P ¯ AC ≥ + 25 mmHg), d'isoprotérénol (ΔFC d'environ + 25 %) ou de nitroprussiate de sodium (Δ P ¯ AC ≥ − 25 mmHg) et par la constriction de l'aorte proximale (pour maximiser la P VG développée et réduire le plus possible le Q A ). RÉSULTATS: Le nitroprussiate de sodium et l'isoprotérénol ont augmenté la G VS comparativement à la phényléphrine et à la constriction. Une courbe de la relation pression-volume maximale a été dérivée à partir des données groupées, au moyen d'une régression non linéaire sur les valeurs maximales de la P ¯ VG réparties dans des classes de Q A de 12,5 ml/min/kg d'amplitude. La courbe de la relation pression-volume et le tracé de la conductance ont été superposés en utilisant l'IC comme axe commun, ce qui a permis de constater que l'IC correspond au débit cardiaque et au volume entrant dans la circulation. CONCLUSIONS: Pour un IC donné, la G VS détermine la pression motrice et donc, la P AC . Nous avons aussi démontré comment une diminution de la G VS compense l'hypotension artérielle en rétablissant la différence de pression artérioveineuse et la pression artérielle.

Dynamic and Irregular Distribution of RyR2 Clusters in the Periphery of Live Ventricular Myocytes.

Cardiac ryanodine receptors (RyR2s) are Ca2+ release channels clustering in the sarcoplasmic reticulum membrane. These clusters are believed to be the elementary units of Ca2+ release. The distribution of these Ca2+ release units plays a critical role in determining the spatio-temporal profile and stability of sarcoplasmic reticulum Ca2+ release. RyR2 clusters located in the interior of cardiomyocytes are arranged in highly ordered arrays. However, little is known about the distribution and function of RyR2 clusters in the periphery of cardiomyocytes. Here, we used a knock-in mouse model expressing a green fluorescence protein (GFP)-tagged RyR2 to localize RyR2 clusters in live ventricular myocytes by virtue of their GFP fluorescence. Confocal imaging and total internal reflection fluorescence microscopy was employed to determine and compare the distribution of GFP-RyR2 in the interior and periphery of isolated live ventricular myocytes and in intact hearts. We found tightly ordered arrays of GFP-RyR2 clusters in the interior, as previously described. In contrast, irregular distribution of GFP-RyR2 clusters was observed in the periphery. Time-lapse total internal reflection fluorescence imaging revealed dynamic movements of GFP-RyR2 clusters in the periphery, which were affected by external Ca2+ and RyR2 activator (caffeine) and inhibitor (tetracaine), but little detectable movement of GFP-RyR2 clusters in the interior. Furthermore, simultaneous Ca2+- and GFP-imaging demonstrated that peripheral RyR2 clusters with an irregular distribution pattern are functional with a Ca2+ release profile similar to that in the interior. These results indicate that the distribution of RyR2 clusters in the periphery of live ventricular myocytes is irregular and dynamic, which is different from that of RyR2 clusters in the interior.

Distribution and Function of Cardiac Ryanodine Receptor Clusters in Live Ventricular Myocytes.

The cardiac Ca(2+) release channel (ryanodine receptor, RyR2) plays an essential role in excitation-contraction coupling in cardiac muscle cells. Effective and stable excitation-contraction coupling critically depends not only on the expression of RyR2, but also on its distribution. Despite its importance, little is known about the distribution and organization of RyR2 in living cells. To study the distribution of RyR2 in living cardiomyocytes, we generated a knock-in mouse model expressing a GFP-tagged RyR2 (GFP-RyR2). Confocal imaging of live ventricular myocytes isolated from the GFP-RyR2 mouse heart revealed clusters of GFP-RyR2 organized in rows with a striated pattern. Similar organization of GFP-RyR2 clusters was observed in fixed ventricular myocytes. Immunofluorescence staining with the anti-α-actinin antibody (a z-line marker) showed that nearly all GFP-RyR2 clusters were localized in the z-line zone. There were small regions with dislocated GFP-RyR2 clusters. Interestingly, these same regions also displayed dislocated z-lines. Staining with di-8-ANEPPS revealed that nearly all GFP-RyR2 clusters were co-localized with transverse but not longitudinal tubules, whereas staining with MitoTracker Red showed that GFP-RyR2 clusters were not co-localized with mitochondria in live ventricular myocytes. We also found GFP-RyR2 clusters interspersed between z-lines only at the periphery of live ventricular myocytes. Simultaneous detection of GFP-RyR2 clusters and Ca(2+) sparks showed that Ca(2+) sparks originated exclusively from RyR2 clusters. Ca(2+) sparks from RyR2 clusters induced no detectable changes in mitochondrial Ca(2+) level. These results reveal, for the first time, the distribution of RyR2 clusters and its functional correlation in living ventricular myocytes.

Impact of detubulation on force and kinetics of cardiac muscle contraction.

Action potential-driven Ca(2+) currents from the transverse tubules (t-tubules) trigger synchronous Ca(2+) release from the sarcoplasmic reticulum of cardiomyocytes. Loss of t-tubules has been reported in cardiac diseases, including heart failure, but the effect of uncoupling t-tubules from the sarcolemma on cardiac muscle mechanics remains largely unknown. We dissected intact rat right ventricular trabeculae and compared force, sarcomere length, and intracellular Ca(2+) in control trabeculae with trabeculae in which the t-tubules were uncoupled from the plasma membrane by formamide-induced osmotic shock (detubulation). We verified disconnection of a consistent fraction of t-tubules from the sarcolemma by two-photon fluorescence imaging of FM4-64-labeled membranes and by the absence of tubular action potential, which was recorded by random access multiphoton microscopy in combination with a voltage-sensitive dye (Di-4-AN(F)EPPTEA). Detubulation reduced the amplitude and prolonged the duration of Ca(2+) transients, leading to slower kinetics of force generation and relaxation and reduced twitch tension (1 Hz, 30°C, 1.5 mM [Ca(2+)]o). No mechanical changes were observed in rat left atrial trabeculae after formamide shock, consistent with the lack of t-tubules in rodent atrial myocytes. Detubulation diminished the rate-dependent increase of Ca(2+)-transient amplitude and twitch force. However, maximal twitch tension at high [Ca(2+)]o or in post-rest potentiated beats was unaffected, although contraction kinetics were slower. The ryanodine receptor (RyR)2 Ca-sensitizing agent caffeine (200 µM), which increases the velocity of transverse Ca(2+) release propagation in detubulated cardiomyocytes, rescued the depressed contractile force and the slower twitch kinetics of detubulated trabeculae, with negligible effects in controls. We conclude that partial loss of t-tubules leads to myocardial contractile abnormalities that can be rescued by enhancing and accelerating the propagation of Ca(2+)-induced Ca(2+) release to orphan RyR2 clusters.

Evoked centripetal Ca(2+) mobilization in cardiac Purkinje cells: insight from a model of three Ca(2+) release regions.

Despite strong suspicion that abnormal Ca(2+) handling in Purkinje cells (P-cells) is implicated in life-threatening forms of ventricular tachycardias, the mechanism underlying the Ca(2+) cycling of these cells under normal conditions is still unclear. There is mounting evidence that P-cells have a unique Ca(2+) handling system. Notably complex spontaneous Ca(2+) activity was previously recorded in canine P-cells and was explained by a mechanistic hypothesis involving a triple layered system of Ca(2+) release channels. Here we examined the validity of this hypothesis for the electrically evoked Ca(2+) transient which was shown, in the dog and rabbit, to occur progressively from the periphery to the interior of the cell. To do so, the hypothesis was incorporated in a model of intracellular Ca(2+) dynamics which was then used to reproduce numerically the Ca(2+) activity of P-cells under stimulated conditions. The modelling was thus performed through a 2D computational array that encompassed three distinct Ca(2+) release nodes arranged, respectively, into three consecutive adjacent regions. A system of partial differential equations (PDEs) expressed numerically the principal cellular functions that modulate the local cytosolic Ca(2+) concentration (Cai). The apparent node-to-node progression of elevated Cai was obtained by combining Ca(2+) diffusion and 'Ca(2+)-induced Ca(2+) release'. To provide the modelling with a reliable experimental reference, we first re-examined the Ca(2+) mobilization in swine stimulated P-cells by 2D confocal microscopy. As reported earlier for the dog and rabbit, a centripetal Ca(2+) transient was readily visible in 22 stimulated P-cells from six adult Yucatan swine hearts (pacing rate: 0.1 Hz; pulse duration: 25 ms, pulse amplitude: 10% above threshold; 1 mm Ca(2+); 35°C; pH 7.3). An accurate replication of the observed centripetal Ca(2+) propagation was generated by the model for four representative cell examples and confirmed by statistical comparisons of simulations against cell data. Selective inactivation of Ca(2+) release regions of the computational array showed that an intermediate layer of Ca(2+) release nodes with an ~30-40% lower Ca(2+) activation threshold was required to reproduce the phenomenon. Our computational analysis was therefore fully consistent with the activation of a triple layered system of Ca(2+) release channels as a mechanism of centripetal Ca(2+) signalling in P-cells. Moreover, the model clearly indicated that the intermediate Ca(2+) release layer with increased sensitivity for Ca(2+) plays an important role in the specific intracellular Ca(2+) mobilization of Purkinje fibres and could therefore be a relevant determinant of cardiac conduction.

The velocity of cardiac sarcomere shortening: mechanisms and implications.

A classical paper published by Michael Barany almost 50 years ago demonstrated a tight correlation between the mechanical parameter of maximal velocity of shortening and the biochemical parameter of myosin ATPase activity in a wide spectrum of species. Here, we review the determinants of muscle dynamics by mechanical load and the relation between sarcomere shortening velocity and cross-bridge dynamics in rat myocardium containing a range of fast and slow myosin. Observations from molecular level to mechanics of the intact human heart suggest that cardiac actin-myosin kinetic properties are matched so as to optimize myocardial strain rate and allow for the maximum rate of hydraulic energy output observed during ejection in the whole ventricle.

Role of cardiac myofilament proteins titin and collagen in the pathogenesis of diastolic dysfunction in cirrhotic rats.

BACKGROUND & AIMS: Significance of diastolic dysfunction in cirrhotic cardiomyopathy has been brought to the forefront with several reports of unexpected heart failure following liver transplantation and transjugular intrahepatic portosystemic stent-shunt, but the etiology remains unclear. The present study investigated the role of passive tension regulators - titin and collagen - in the pathogenesis of this condition. METHODS: Cirrhosis was induced by bile duct ligation (BDL) in rats, while controls underwent bile duct inspection with no ligation. Four weeks after operation, cardiac mRNA and protein levels of titin, collagen, and protein kinase A (PKA) were determined. Diastolic function was examined in isolated right ventricular cardiomyocytes, while passive tension was examined in right ventricular trabeculae muscles. RESULTS: In BDL animals, diastolic return velocity was significantly decreased, relaxation time increased and passive tension increased. However, no significant difference in mRNA and protein levels of titin was observed. PKA mRNA and protein levels were significantly decreased in BDL animals. Collagen levels were also significantly altered in the BDL group. CONCLUSIONS: Therefore, diastolic dysfunction exists in cirrhosis with alterations in titin modulation, PKA levels, and collagen configuration contributing to the pathogenesis of this condition.

Electromechanical coupling in the cardiac myocyte; stretch-arrhythmia feedback.

The macroscopic hallmarks of the normal heartbeat are rapid onset of contraction and rapid relaxation and an inotropic response to both increased end diastolic volume and increased heart rate. At the microscopic level, the calcium ion (Ca(2+)) plays a crucial role in normal cardiac contraction. This paper reviews the cycle of Ca(2+) fluxes during the normal heartbeat, which underlie the coupling between excitation and contraction (ECC) and permit a highly synchronized action of cardiac sarcomeres. Length dependence of the response of the regulatory sarcomeric proteins mediates the Frank-Starling Law of the heart. However, Ca(2+) transport may go astray in heart disease and both jeopardize the exquisite mechanism of systole and diastole and triggering arrhythmias. The interplay between weakened and strong segments in nonuniform cardiac muscle may further lead to mechanoelectric feedback-or reverse excitation contraction coupling (RECC) mediating an early diastolic Ca(2+) transient caused by the rapid force decrease during the relaxation phase. These rapid force changes in nonuniform muscle may cause arrhythmogenic Ca(2+) waves to propagate by activation of neighbouring SR by diffusing Ca(2+) ions.

Nonuniform activation and the mechanics of myocardial trabeculae with fast or slow myosin.

Left ventricular (LV) wall motion abnormalities reflect regional nonuniform contraction which may be arrhythmogenic. We studied sarcomere mechanics and force development (F) in uniform and nonuniform trabeculae using a model in which half of the muscle can be rendered weak by exposure to low [Ca2+]o. Stretch allowed the weak muscle segment to generate a force that was four-fold higher than force when the whole muscle was exposed to low [Ca2+]o. The sarcomere force-velocity relationships (FSVR) and the force-sarcomere-length relationships (FSLR) explained the force increase in the weak segment and the decrease of force in the strong segment such that both carried the same force. Correction for muscle stiffness converted the FSVR into a [Ca2+]o-independent linear FVRXB for "the single cross-bridge (XB)." Stretch increased XB force<10% above FXB-max, but recruited more XBs by feedback of V to the rate of XB, weakening (g=g0+g1V). The g1 here was indistinguishable from g1 of XBs in slow myosin of aged animals. The mechanics of nonuniform muscle can be explained by a linear FVRXB combined with the effect of V on the XB weakening rate.

Effects of therapy using the Celacade system on structural and functional cardiac remodelling in rats following myocardial infarction.

BACKGROUND: Immune modulation by the Celacade system (Vasogen Inc, Canada) decreases mortality and hospitalization in human heart failure. OBJECTIVES: To study the effects of Celacade in rats on acute cytokine expression after coronary artery ligation, cardiac dimensions following myocardial infarction (MI), and systolic and diastolic function of cardiac muscle in MI. METHODS: Celacade treatment was administered 14 days before coronary artery ligation and monthly after the surgery. Cytokine expression in cardiac tissue was measured on days 1 and 7 by ELISA in sham rats and in rats with MI (with or without Celacade treatment). Echocardiograms were obtained serially for 16 weeks. Force and sarcomere length (SL) were measured by strain gauge and laser diffraction in isolated right ventricle trabeculas at 16 weeks. The inotropic effect of pacing on force was quantified as F5 Hz/0.5 Hz. Diastolic dysfunction was quantified as the root mean square of spontaneous SL fluctuations. RESULTS: Celacade inhibited transforming growth factor beta-1 production in the infarct area on day 7 (191.6+/-22.6 pg/mg versus 275.4+/-30.1 pg/mg; P<0.05), but did not attenuate cardiac dilation in MI. Celacade restored positive inotropism of pacing in MI (F5 Hz/0.5 Hz in Celacade, 219.1+/-46.7%; MI, 148.1+/-27.1% [P<0.05 compared with 211.4+/-37.9% in sham]). Celacade reduced diastolic dysfunction in MI (root mean square of spontaneous SL fluctuations: 121+/-15% and 143+/-19% with Celacade versus 184+/-19% and 190+/-26% without Celacade at 26 degrees C and 36 degrees C, respectively) compared with sham (100%; P<0.05). CONCLUSIONS: Celacade reduces the increase of transforming growth factor beta-1 expression during the acute stage of MI in rats, but does not prevent chronic cardiac dilation. Celacade restores the positive inotropic effect of increased pacing rate in trabeculas from rat right ventricles with large MIs and reduces diastolic dysfunction.

Contribution of Na+/Ca2+ exchange current to the formation of delayed afterdepolarizations in intact rat ventricular muscle.

AIM: To evaluate the role of the Na+-Ca2+ exchange current in the induction of arrhythmias during Ca2+ waves, we investigated the relationship between Ca2+ waves and delayed afterdepolarizations (DADs) and further investigated the effect of KB-R7943, an Na+-Ca2+ exchange inhibitor, on such relationship in multicellular muscle. METHODS: Force, sarcomere length, membrane potential, and [Ca2+]i dynamics were measured in 32 ventricular trabeculae from rat hearts. After the induction of Ca2+ waves by trains of electrical stimuli (400, 500, or 600 ms intervals) for 7.5 seconds, 23 Ca2+ waves in the absence of KB-R7943 and cilnidipine ([Ca2+]o = 2.3 +/- 0.2 mmol/L), 11 Ca2+ waves in the presence of 10 micromol/L KB-R7943 ([Ca2+]o = 2.5 +/- 0.5 mmol/L), and 8 Ca2+ waves in the presence of 1 micromol/L cilnidipine ([Ca]o = 4.1 +/- 0.3 mmol/L) were measured at a sarcomere length of 2.1 microm (23.9 +/- 0.8 degrees C). RESULTS: The amplitude of DADs correlated with the velocity (r = 0.90) and the amplitude (r = 0.90) of Ca2+ waves. The amplitude of DADs was significantly decreased to approximately 40% of the initial value by 10 micromol/L KB-R7943. CONCLUSIONS: These results suggest that the velocity and the amplitude of Ca2+ waves determine the formation of DADs principally through the activation of the Na+-Ca2+ exchange current, thereby inducing triggered arrhythmias in multicellular ventricular muscle.

Spatial non-uniformity of excitation-contraction coupling can enhance arrhythmogenic-delayed afterdepolarizations in rat cardiac muscle.

AIMS: We examined whether non-uniform muscle contraction affects delayed afterdepolarizations (DADs) by dissociating Ca(2+) from myofilaments within the border zone (BZ) between contracting and stretched regions. METHODS AND RESULTS: Force, sarcomere length (SL), membrane potential, and [Ca(2+)](i) dynamics were measured in 31 ventricular trabeculae from rat hearts. Non-uniform muscle contraction was produced by exposing a restricted region of muscle to a jet of solution containing 20 mmol/L 2,3-butanedione monoxime (BDM). DADs were induced by 7.5 s-2 Hz stimulus trains at an SL of 2.0 microm (24 degrees C, [Ca(2+)](o) 2.0 mmol/L). The BDM jet enhanced DADs (n = 6, P < 0.05) and aftercontractions (n = 6, P < 0.05) with or without 100 micromol/L streptomycin and occasionally elicited an action potential. A stretch pulse from an SL of 2.0 microm to 2.1 or 2.2 microm during the last stimulated twitch of the trains accelerated Ca(2+) waves in proportion to the increment of force by the stretch (P < 0.01) with or without streptomycin. In the presence of 1 mmol/L caffeine, rapid shortening of the muscle after the stretch pulse increased [Ca(2+)](i) within the BZ, whose amplitude correlated with the increment of force by the stretch (n = 15, P < 0.01). CONCLUSION: These results suggest that non-uniform muscle contraction can enhance DADs by dissociating Ca(2+) from myofilaments within the BZ and thereby cause triggered arrhythmias.

Sarcomere mechanics in uniform and nonuniform cardiac muscle: a link between pump function and arrhythmias.

Starling's law and the end-systolic pressure-volume relationship (ESPVR) reflect the effect of sarcomere length (SL) on the development of stress (sigma) 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 that of skeletal sarcomeres and the existence of opposing forces in cardiac muscle shortened below slack length. The sigma-SL -[Ca2+](free) relationships (sigma-SL-Ca relationships) at submaximal [Ca2+] in intact and skinned trabeculae were similar, although the sensitivity for Ca2+ of intact muscle was higher. We analyzed the mechanisms underlying the sigma-SL-Ca relationship by using a kinetic model assuming that the rates of Tn-C Ca2+ binding and/or cross-bridge (XB) cycling are determined by either the SL, [Ca2+], or sigma. We analyzed the correlation between the model results and steady-state sigma measurements at varied SL at [Ca2+] from skinned rat cardiac trabeculae to test the hypotheses that the dominant feedback mechanism is SL-, sigma-, or [Ca2+]-dependent, and that the feedback mechanism regulates Tn-C Ca2+ affinity, XB kinetics, or the unitary XB force. The analysis strongly suggests that the feedback of the number of strong XBs to cardiac Tn-C Ca2+ affinity is the dominant mechanism regulating XB recruitment. Using this concept in a model of twitch-sigma accurately reproduced the sigma-SL-Ca relationship and the time courses of twitch sigma and the intracellular [Ca2+]i. The foregoing concept has equally important repercussions for the nonuniformly contracting heart, in which arrhythmogenic Ca2+ waves arise from weakened areas in the cardiac muscle. These Ca2+ waves can reversibly be induced with nonuniform 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 the sarcomeres in the border zone during relaxation causes Ca2+ release from Tn-C and initiates Ca2+ waves propagated by the sarcoplasmic reticulum (SR). Modeling of the response of the cardiac twitch to rapid force changes using the feedback concept uniquely predicts the occurrence of [Ca2+]i transients as a result of accelerated Ca2+ dissociation from Tn-C. These results are consistent with the hypothesis that a force feedback to Ca2+ binding by Tn-C is responsible for Starling's law and the ESPVR in the uniform myocardium and leads to a surge of Ca2+ released by the myofilaments during relaxation in the nonuniform myocardium, which initiates arrhythmogenic propagating Ca2+ release by the SR.

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.

Function of Ca(2+) release channels in Purkinje cells that survive in the infarcted canine heart: a mechanism for triggered Purkinje ectopy.

BACKGROUND: Triggered Purkinje ectopy can lead to the initiation of serious ventricular arrhythmias in post-myocardial infarction patients. In the canine model, Purkinje cells from the subendocardial border of the healing infarcted heart can initiate ventricular arrhythmias. Intracellular Ca(2+) abnormalities underlie these arrhythmias, yet the subcellular reasons for these abnormalities remain unknown. METHODS AND RESULTS: Using 2D confocal microscopy, we directly quantify and compare typical spontaneous Ca(2+) events in specific subcellular regions of normal Purkinje cells with those Purkinje cells from the subendocardium of the 48-hour infarcted canine heart (IZPCs). The Ca(2+) event rate was higher in the subsarcolemmal region of IZPCs when compared with normal Purkinje cells; IZPC amplitudes were higher, yet the spatial extents of these events were similar. The amplitude of caffeine-releasable Ca(2+) in either the subsarcolemmal or core regions of IZPCs did not differ from normal Purkinje cells, suggesting that Ca(2+) overload was not related to the frequency change. In permeabilized Purkinje cells from both groups, the event rate was related to free [Ca(2+)] in both subsarcolemmal and core, but in IZPCs, this event rate was significantly increased at each free Ca(2+), suggesting an enhanced sensitivity to Ca(2+) release. Furthermore, decays of wide long lasting Ca(2+) release events in IZPC's core were significantly accelerated compared with those in normal Purkinje cells. JTV519 (K201) suppressed IZPC cell wide Ca(2+) waves as well as normalized the enhanced event rate and its response to free Ca(2+). CONCLUSIONS: Increased spontaneous Ca(2+) release events in IZPCs are due to uniform regionally increased Ca(2+) release channel sensitivity to Ca(2+) without a change in sarcoplasmic reticulum content. In addition, Ca(2+) reuptake in IZPCs is accelerated. These properties would lower the threshold of Ca(2+) release channels, setting the stage for the highly frequent arrhythmogenic cell wide Ca(2+) waves observed in IZPCs.

Calcium and arrhythmogenesis.

Triggered activity in cardiac muscle and intracellular Ca2+ have been linked in the past. However, today not only are there a number of cellular proteins that show clear Ca2+ dependence but also there are a number of arrhythmias whose mechanism appears to be linked to Ca2+-dependent processes. Thus we present a systematic review of the mechanisms of Ca2+ transport (forward excitation-contraction coupling) in the ventricular cell as well as what is known for other cardiac cell types. Second, we review the molecular nature of the proteins that are involved in this process as well as the functional consequences of both normal and abnormal Ca2+ cycling (e.g., Ca2+ waves). Finally, we review what we understand to be the role of Ca2+ cycling in various forms of arrhythmias, that is, those associated with inherited mutations and those that are acquired and resulting from reentrant excitation and/or abnormal impulse generation (e.g., triggered activity). Further solving the nature of these intricate and dynamic interactions promises to be an important area of research for a better recognition and understanding of the nature of Ca2+ and arrhythmias. Our solutions will provide a more complete understanding of the molecular basis for the targeted control of cellular calcium in the treatment and prevention of such.

Role of sarcomere mechanics and Ca2+ overload in Ca2+ waves and arrhythmias in rat cardiac muscle.

Ca(2+) release from the sarcoplasmic reticulum (SR) depends on the sarcoplasmic reticulum (SR) Ca(2+) load and the cytosolic Ca(2+) level. Arrhythmogenic Ca(2+) waves underlying triggered propagated contractions arise from Ca(2+) overloaded regions near damaged areas in the cardiac muscle. Ca(2+) waves can also be induced in undamaged muscle, in regions with nonuniform excitation-contraction (EC) coupling by the cycle of stretch and release in the border zone between the damaged and intact regions. We hypothesize that rapid shortening of sarcomeres in the border zone during relaxation causes Ca(2+) release from troponin C (TnC) on thin filaments and initiates Ca(2+) waves. Elimination of this shortening will inhibit the initiation of Ca(2+) waves, while SR Ca(2+) overload will enhance the waves. Force, sarcomere length (SL), and [Ca(2+)](i) were measured and muscle length was controlled. A small jet of Hepes solution with an extracellular [Ca(2+)] 10 mM (HC), or HC containing BDM, was used to weaken a 300 mum long muscle segment. Trains of electrical stimuli were used to induce Ca(2+) waves. The effects of small exponential stretches on triggered propagatory contraction (TPC) amplitude and propagation velocity of Ca(2+) waves (V(prop)) were studied. Sarcomere shortening was uniform prior to activation. HC induced spontaneous diastolic sarcomere contractions in the jet region and attenuated twitch sarcomere shortening; HC+ butanedione monoxime (BDM) caused stretch only in the jet region. Stimulus trains induced Ca(2+) waves, which started inside the HC jet region during twitch relaxation. Ca(2+) waves started in the border zone of the BDM jet. The initial local [Ca(2+)](i) rise of the waves by HC was twice that by BDM. The waves propagated at a V(prop) of 2.0 +/- 0.2 mm/sec. Arrhythmias occurred frequently in trabeculae following exposure to the HC jet. Stretch early during relaxation, which reduced sarcomere shortening in the weakened regions, substantially decreased force of the TPC (F(TPC)) and delayed Ca(2+) waves, and reduced V(prop) commensurate with the reduction F(TPC). These results are consistent with the hypothesis that Ca(2+) release from the myofilaments initiates arrhythmogenic propagating Ca(2+) release. Prevention of sarcomere shortening, by itself, did not inhibit Ca(2+) wave generation. SR Ca(2+) overload potentiated initiation and propagation of Ca(2+) waves.