Normal Excitation-contraction Coupling Research Articles

Conductive polymers for cardiac tissue engineering and regeneration.

Cardiovascular diseases, such as myocardial infarction, are considered a significant global burden and the leading cause of death. Given the inability of damaged cardiac tissue to self-repair, cell-based tissue engineering and regeneration may be the only viable option for restoring normal heart function. To maintain the normal excitation-contraction coupling function of cardiac tissue, uniform electronic and ionic conductance properties are required. To transport cells to damaged cardiac tissues, several techniques, including the incorporation of cells into conductive polymers (CPs) and biomaterials, have been utilized. Due to the complexity of cardiac tissues, the success of tissue engineering for the damaged heart is highly dependent on several variables, such as the cell source, growth factors, and scaffolds. In this review, we sought to provide a comprehensive overview of the electro CPs and biomaterials used in the engineering and regeneration of heart tissue.

Role of Ca2+ in healthy and pathologic cardiac function: from normal excitation-contraction coupling to mutations that cause inherited arrhythmia.

Calcium (Ca2+) ions are a key second messenger involved in the rhythmic excitation and contraction of cardiomyocytes throughout the heart. Proper function of Ca2+-handling proteins is required for healthy cardiac function, whereas disruption in any of these can cause cardiac arrhythmias. This comprehensive review provides a broad overview of the roles of Ca2+-handling proteins and their regulators in healthy cardiac function and the mechanisms by which mutations in these proteins contribute to inherited arrhythmias. Major Ca2+ channels and Ca2+-sensitive regulatory proteins involved in cardiac excitation-contraction coupling are discussed, with special emphasis on the function of the RyR2 macromolecular complex. Inherited arrhythmia disorders including catecholaminergic polymorphic ventricular tachycardia, long QT syndrome, Brugada syndrome, short QT syndrome, and arrhythmogenic right-ventricular cardiomyopathy are discussed with particular emphasis on subtypes caused by mutations in Ca2+-handling proteins.

Critical Requirements for the Initiation of a Cardiac Arrhythmia in Rat Ventricle: How Many Myocytes?

Cardiovascular disease is the leading cause of death worldwide due in a large part to arrhythmia. In order to understand how calcium dynamics play a role in arrhythmogenesis, normal and dysfunctional Ca2+ signaling in a subcellular, cellular, and tissued level is examined using cardiac ventricular myocytes at a high temporal and spatial resolution using multiscale computational modeling. Ca2+ sparks underlie normal excitation–contraction coupling. However, under pathological conditions, Ca2+ sparks can combine to form Ca2+ waves. These propagating elevations of (Ca2+)i can activate an inward Na+–Ca2+ exchanger current (INCX) that contributes to early after-depolarization (EADs) and delayed after-depolarizations (DADs). However, how cellular currents lead to full depolarization of the myocardium and how they initiate extra systoles is still not fully understood. This study explores how many myocytes must be entrained to initiate arrhythmogenic depolarizations in biophysically detailed computational models. The model presented here suggests that only a small number of myocytes must activate in order to trigger an arrhythmogenic propagating action potential. These conditions were examined in 1-D, 2-D, and 3-D considering heart geometry. The depolarization of only a few hundred ventricular myocytes is required to trigger an ectopic depolarization. The number decreases under disease conditions such as heart failure. Furthermore, in geometrically restricted parts of the heart such as the thin muscle strands found in the trabeculae and papillary muscle, the number of cells needed to trigger a propagating depolarization falls even further to less than ten myocytes.

SPEG: a key regulator of cardiac calcium homeostasis.

Proper cardiac Ca2+ homeostasis is essential for normal excitation-contraction coupling. Perturbations in cardiac Ca2+ handling through altered kinase activity has been implicated in altered cardiac contractility and arrhythmogenesis. Thus, a better understanding of cardiac Ca2+ handling regulation is vital for a better understanding of various human disease processes. 'Striated muscle preferentially expressed protein kinase' (SPEG) is a member of the myosin light chain kinase family that is key for normal cardiac function. Work within the last 5 years has revealed that SPEG has a crucial role in maintaining normal cardiac Ca2+ handling through maintenance of transverse tubule formation and phosphorylation of junctional membrane complex proteins. Additionally, SPEG has been causally impacted in human genetic diseases such as centronuclear myopathy and dilated cardiomyopathy as well as in common acquired cardiovascular disease such as heart failure and atrial fibrillation. Given the rapidly emerging role of SPEG as a key cardiac Ca2+ regulator, we here present this review in order to summarize recent findings regarding the mechanisms of SPEG regulation of cardiac excitation-contraction coupling in both physiology and human disease. A better understanding of the roles of SPEG will be important for a more complete comprehension of cardiac Ca2+ regulation in physiology and disease.

Skeletal muscle fibre swelling contributes to force depression in rats and humans: a mechanically-skinned fibre study.

This study investigated the effects of fibre swelling on force production in rat and human skinned muscle fibres, using osmotic compression to reverse the fibre swelling. In mechanically-skinned fibres, the sarcolemma is removed but normal excitation-contraction coupling remains functional. Force responses in mechanically-skinned fibres were examined with and without osmotic compression by polyvinylpyrrolidone 40kDa (PVP-40) or Dextran 500kDa (dextran). Fibre diameter increased to 116 ± 2% (mean ± SEM) when rat skinned type II fibres were immersed in the standard intracellular solution, but remained close to the in situ size when 3% (mass/volume) PVP-40 or 4% Dextran were present. Myofibrillar Ca2+ sensitivity, as indicated by pCa50 (-log10[Ca2+] at half-maximal force), was increased in 4% Dextran (0.072 ± 0.007 pCa50 shift), but was not significantly changed in 3% PVP-40. Maximum Ca2+-activated force increased slightly to 103 ± 1% and 104 ± 1% in PVP-40 and Dextran, respectively. Both tetanic and depolarization-induced force responses in rat skinned type II fibres, elicited by electrical stimulation and ion substitution respectively, were increased by ~ 10 to 15% when the fibres were returned to their normal in situ diameter by addition of PVP-40 or Dextran. Interestingly, the potentiation of these force responses in PVP-40 was appreciably greater than could be explained by potentiation of myofibrillar function alone. These results indicate that muscle fibre swelling, as can occur with intense exercise, decreases evoked force responses by reducing both the Ca2+-sensitivity of the contractile apparatus properties and Ca2+ release from the sarcoplasmic reticulum.

Anesthetic MS-222 eliminates nerve and muscle activity in frogs used for physiology teaching laboratories

Frogs are routinely used in physiology teaching laboratories to demonstrate important physiological processes. There have been recent directives that promote the use of the anesthetic MS-222 (tricaine methanesulfonate), rather than lowering body temperature with a cold water bath to prepare reptiles and amphibians for physiological experiments or euthanasia. Indeed, the most recent edition of the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals proclaims that chilling in water is not an appropriate method and advocates for the usage of MS-222 or other anesthetics. However, prominent researchers have responded to this position by highlighting evidence that cooling ectothermic vertebrates is, in fact, an effective and appropriate method. Furthermore, MS-222 is a known voltage-gated Na+ channel blocker, and this anesthetic's impact on the physiology of excitable tissues suggests that its use might be incompatible with experiments on nerve and muscle tissues. In the present study, I examined the effects of MS-222 at a concentration of 1.5 g/l on nerve, skeletal muscle, and cardiac muscle physiology of frogs. I found that immersion of frogs in this anesthetic blocked basic nerve and muscle physiology, making the frogs unsuitable for laboratory experiments. Applying MS-222 directly to the sciatic nerve dramatically blocked normal excitation-contraction coupling in skeletal muscle preparations, and direct application to the heart caused the organs to stop contracting. Based on these results, I conclude that MS-222 at the concentration studied may be incompatible with physiological preparations that rely on electrically excitable tissues for their normal function. Physiology educators who must use MS-222 with frogs should empirically determine an appropriate dosage and recovery time before using the anesthetic in the teaching laboratory.

Cardiac CaV1.2 channels require β subunits for β-adrenergic-mediated modulation but not trafficking.

Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant α1C subunits lacking capacity to bind CaVβ can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit-sequestering peptide sharply curtailed β-adrenergic stimulation of WT Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.

171 Amphiphysin ii (bin1) driven transverse tubule formation in cardiac muscle

Transverse (t)-tubules are vital for maintaining normal contractility of the heart through the tight regulation of excitation coupling. In cardiac disease, such as heart failure, t-tubule loss is closely associated...

One and Two-Photon Calcium Uncaging with Visible Light in Cardiac Myocytes

In cardiomyocytes a small amount of Ca2+ enters the cytoplasm upon depolarization and initiates Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). Spontaneous SR Ca2+ release events can remain highly localized or initiate Ca2+ waves that propagate through the cell. A powerful optical method for spatio-temporal control of [Ca2+]i is one- and two-photon uncaging. We have designed a nitroaromatic photochemical protecting group absorbing visible light very efficiently in the violet to blue range. The chromophore is a dinitro derivative of bisstyrylthiophene bearing two EGTA chelators (BIST-2EGTA). BIST-2EGTA has an absorption maximum at 440 nm, quantum yield of photolysis of 0.23, extinction coefficient at 440 nm of 66,000 M−1cm−1 and binds Ca2+ with high affinity (Kd 84 nM at pH 7.2). Fluorescence imaging with a confocal microscope in point scan mode revealed that rhod-FF showed a rapid change in signal (<200 µs) when BIST-2EGTA was photolyzed using two-photon excitation at 810 nm. Compared to DM-nitrophen the fluorescence signal from BIST-2EGTA was ∼13.7x larger when photolyzed under the same conditions and could be elicited on physiologically relevant time-scales (<1 ms). The efficiency of BIST-2EGTA to create changes in [Ca2+]i that are useful for physiological studies was examined in whole-cell patch-clamped cardiomyocytes loaded with BIST-2EGTA and rhod-2 or X-rhod-5F. Short (1 ms) two-photon excitation at 810 nm produced localized Ca2+ releases that were considerably larger than those produced by BIST-2EGTA photolysis in caffeine-treated cells, suggesting active SR Ca2+ release. Uncaging for longer periods triggered intracellular Ca2+ “mini-waves” or propagating Ca2+ waves. Cells displayed normal excitation-contraction coupling, implying that BIST-2EGTA is nontoxic. One-photon uncaging at 405 nm also produced striking Ca2+ waves. BIST-2EGTA has a set of properties that enables highly efficient and fast control of Ca2+inside living cells using visible light.

Roscovitine as the Archetypal Member of a Novel Class of Antiarrhythmics Targeting Late ICa,L

L-type calcium current (ICa,L) plays a central role in counterbalancing potassium current during the plateau phase of cardiac action potential (AP). When repolarization reserve is reduced, the aberrant reactivation of ICa,L during repolarization can cause transient depolarization events called Early Afterdepolarizations (EADs), a recognized trigger of cardiac arrhythmias. Recently we demonstrated that EADs are highly sensitive to the steady-state voltage-dependent properties of ICa,L. Using the dynamic clamp technique in isolated rabbit ventricular myocytes under EAD-favoring condition (i.e. oxidative stress or hypokalemia), we found that selectively inhibiting the non-inactivating component (late) of ICa,L potently suppressed EAD-occurrence. Roscovitine, a purine analog, has been shown to reduce the late ICa,L component while leaving peak unperturbed. Here we tested the ability of this drug to suppress EADs in isolated rabbit ventricular myocytes induced by exposure to H2O2 (600µM). This oxidative stress caused EADs in 81% [95% CI: 66-95%] of the APs, prolonging AP duration (APD90) from 276 ms [97-433 ms] to 840ms [285-1967ms] (n=4). Addition of roscovitine (20µM) completely abolished EADs and reduced the APD90 to 189 ms [155-222 ms]. Moreover, roscovitine did not significantly perturb the Ca transient, implying maintenance of normal excitation-contraction coupling. We next exposed isolated aged rat hearts to 0.1 mM H2O2 to induce EADs and spontaneous ventricular tachycardia/fibrillation (VT/VF). In all tested hearts (n=4), roscovitine terminated VT/VF and led to resumption of sinus rhythm within 10 min. Our study offers additional proof of concept supporting a pharmacological strategy to target late ICa,L to prevent EAD-mediated arrhythmias, with roscovitine as an archetypal drug to guide the development of this new class of antiarrhythmics.

Ryanodine Receptor Sensitivity Governs the Stability and Synchrony of Local Calcium Release during Cardiac Excitation-Contraction Coupling

Synchronous coordination of calcium-induced calcium release (CICR) across thousands of calcium release units (CRUs) within the cardiomyocyte is critical for normal excitation-contraction coupling (ECC). Here we utilize a novel “local control” mathematical model of ECC, designed to simulate a mouse cardiomyocyte, to investigate physiological and pathophysiological calcium (Ca2+) signaling. The model contains 20,000 independent CRUs each composed of 6 stochastically gated L-type Ca2+ channels (LCCs) in the transverse tubule membrane that are positioned across a local “dyadic subspace” from a junctional sarcoplasmic reticulum (JSR) compartment containing a cluster of 50 stochastically gated ryanodine receptors (RyR2s). Action potential depolarization of the sarcolemmal membrane activates LCC openings which elevate subspace [Ca2+] ([Ca2+]ds) across each of the cell's 20,000 CRUs. This rise in [Ca2+]ds promotes RyR2 opening to initiate the fundamental element of triggered Ca2+ release, the Ca2+ spark. The mechanistic design and true SR Ca2+ pump/leak balance displayed by our model allows us to quantify ECC fidelity and Ca2+ spark fidelity (the probability that a LCC opening or RyR2 opening induces a Ca2+ spark, respectively). We study the effect of excess SR Ca2+ leak in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)” caused by the R33Q mutation in calsequestrin (CASQ2). CPVT is simulated by increasing RyR2 sensitivity to [Ca2+]i and reducing JSR Ca2+ buffering capacity to levels identified by prior studies of the CASQ2-R33Q “knock-in” mice. Our model effectively reproduces both normal and arrhythmogenic CPVT RyR2 [Ca2+]i sensitivity, local Ca2+ spark activity, and global Ca2+ signals. Under CPVT conditions enhanced RyR2 open probability is critical for the development of “unstable”, quiescent Ca2+ sparks that fail to terminate robustly as well as asynchronous systolic Ca2+ spark activity and increased diastolic SR Ca2+ leak.

Novel Compounds Inhibit Calmodulin Deficient RyR2 Activity and Arrhythmias in a CPVT Mouse Model

The cardiac ryanodine receptor (RyR2) plays a key role in excitation-contraction (EC) coupling. Mutations in RyR2 are known to be linked to the arrhythmogenic disorder, catecholaminergic polymorphic ventricular tachycardia (CPVT), a deadly disease which is characterized by a leak of calcium from sarcoplasmic reticulum and a decrease in calmodulin (CaM) binding. A novel drug, 84-F2, is shown to inhibit arrhythmias in RyR2-R176Q heterozygous CPVT mouse hearts (2.5 μg/kg), decrease spark frequency in cells derived from CPVT mice (IC50 = 35nM), and inhibit RyR2 single channel activity at low nanomolar concentrations (IC50 = 11nM). When CaM is added back, 84-F2's ability to inhibit channel activity is suppressed ∼100 fold. We propose that this new drug decreases arrhythmias by binding to the CaM deficient RyR2, but does not affect normal EC coupling because of the presence of CaM. This work shows for the first time a class of drugs whose inhibitory effects are dependent upon the removal of CaM from RyR2. Supported by NIH 2R42HL114206 and R41-HL129570 to Elex Biotech, LLC.

Optical Mapping of Intra-Sarcoplasmic Reticulum Ca2+ and Transmembrane Potential in the Langendorff-perfused Rabbit Heart.

Sarcoplasmic reticulum (SR) Ca2+ handling plays a key role in normal excitation-contraction coupling and aberrant SR Ca2+ handling is known to play a significant role in certain types of arrhythmia. Because arrhythmias are spatially distinct, emergent phenomena, they must be investigated at the tissue level. However, methods for directly probing SR Ca2+ in the intact heart remain limited. This article describes the protocol for dual optical mapping of transmembrane potential (Vm) and free intra-SR [Ca2+] ([Ca2+]SR) in the Langendorff-perfused rabbit heart. This approach takes advantage of the low-affinity Ca2+ indicator Fluo-5N, which has minimal fluorescence in the cytosol where intracellular [Ca2+] ([Ca2+]i) is relatively low but exhibits significant fluorescence in the SR lumen where [Ca2+]SR is in the millimolar range. In addition to revealing SR Ca2+ characteristics spatially across the epicardial surface of the heart, this approach has the distinct advantage of simultaneous monitoring of Vm, allowing for investigations into the bidirectional relationship between Vm and SR Ca2+ and the role of SR Ca2+ in arrhythmogenic phenomena.

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca2+) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ leak out of the SR is an important process for cellular Ca2+ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca2+ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca2+ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca2+ sparks and robust Ca2+ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca2+ spark and nonspark-based SR Ca2+ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca2+-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca2+ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca2+ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca2+ release properties due to variations in inter-RyR coupling via local subspace Ca2+ concentration ([Ca2+]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Overexpression of junctophilin-2 does not enhance baseline function but attenuates heart failure development after cardiac stress.

Heart failure is accompanied by a loss of the orderly disposition of transverse (T)-tubules and a decrease of their associations with the junctional sarcoplasmic reticulum (jSR). Junctophilin-2 (JP2) is a structural protein responsible for jSR/T-tubule docking. Animal models of cardiac stresses demonstrate that down-regulation of JP2 contributes to T-tubule disorganization, loss of excitation-contraction coupling, and heart failure development. Our objective was to determine whether JP2 overexpression attenuates stress-induced T-tubule disorganization and protects against heart failure progression. We therefore generated transgenic mice with cardiac-specific JP2 overexpression (JP2-OE). Baseline cardiac function and Ca(2+) handling properties were similar between JP2-OE and control mice. However, JP2-OE mice displayed a significant increase in the junctional coupling area between T-tubules and the SR and an elevated expression of the Na(+)/Ca(2+) exchanger, although other excitation-contraction coupling protein levels were not significantly changed. Despite similar cardiac function at baseline, overexpression of JP2 provided significantly protective benefits after pressure overload. This was accompanied by a decreased percentage of surviving mice that developed heart failure, as well as preservation of T-tubule network integrity in both the left and right ventricles. Taken together, these data suggest that strategies to maintain JP2 levels can prevent the progression from hypertrophy to heart failure.

Optical Mapping of Sarcoplasmic Reticulum Ca 2+ in the Intact Heart

Rationale: Sarcoplasmic reticulum (SR) Ca 2+ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia. Objective: To measure intra-SR free [Ca 2+ ] ([Ca 2+ ] SR ) changes in intact hearts during alternans and ventricular fibrillation (VF). Methods and Results: Simultaneous optical mapping of V m (with RH237) and [Ca 2+ ] SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca 2+ and action potential duration (APD) alternans occurred in-phase, but SR Ca 2+ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P &lt;0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca 2+ alternans, with SR Ca 2+ release alternans routinely occurring without changes in diastolic [Ca 2+ ] SR . Sensitizing RyR with caffeine (200 μmol/L) significantly reduced the pacing threshold for both SR Ca 2+ and APD alternans (188±15 and 173±12 ms; P &lt;0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca 2+ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca 2+ ] SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca 2+ release throughout VF. Conclusions: In intact hearts, RyR refractoriness initiates SR Ca 2+ release alternans that can be amplified by diastolic [Ca 2+ ] SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca 2+ and APD alternans. Despite increased [Ca 2+ ] SR during VF, SR Ca 2+ release was nearly continuously refractory. This novel method provides insight into SR Ca 2+ handling during cardiac alternans and arrhythmia.

Inhibition of MMP-2 expression affects metabolic enzyme expression levels: proteomic analysis of rat cardiomyocytes.

During ischemia and reperfusion of cardiomyocytes, abnormality in excitation-contraction coupling and decreased energy metabolism often lead to myocardial infarction, but the cellular mechanisms are not fully elucidated. We show for the first time that intracellular inhibition of MMP-2 in cardiomyocytes increases contractility of aerobically perfused myocytes, which was accompanied by increased expression of contractile proteins (e.g., MLC-1). We also showed that MMP-2 inhibition produced a cardiomyocyte proteome that is consistent with improved mitochondrial energy metabolism (e.g., increased expression and activity of mitochondrial beta ATP synthase). Thus, MMP-2 appears to be involved in homeostatic regulation of protein turnover. Our results are significant since they point to targeting MMP-2 activity as a novel therapeutic option to limit myocardial damage by decreasing proteolytic degradation of mitochondrial metabolic enzymes and myocardial contractile proteins during ischemia. In addition, the development of novel pharmacological agents that selectively targets cardiac MMP-2 represents a novel approach to treat and prevent other heart diseases.

Exploring SR Calcium and Cytosolic Calcium Wave Dynamics using a 3D Stochastic Myocyte Model

The local regulation of intracellular calcium has been widely known to play an important role in the normal excitation-contraction coupling in the cardiac myocytes. While Ca2+ sparks do not normally trigger regenerative SR Ca2+ release (i.e. Ca2+ waves), under calcium overload conditions calcium sparks can trigger spontaneous calcium waves. While experimental imaging using confocal microscopy with fluorescent dyes is the major technique used to study calcium dynamics inside a cardiac myocyte, the dynamics of SR calcium during calcium overload is poorly understood and controversial. In the study presented here, we present a new temporal-spatial model of calcium sparks to examine this issue. The resolution is sufficiently high (100nm) so that all critical local fluxes can be properly considered. The model also takes into account the different expression levels of the sodium-calcium exchanger (NCX) and SERCA pumps near the SR Ca2+ release sites. From that, we want to demonstrate different factors contributing to the characteristics of Ca2+ sparks. This work includes an examination of the extent of SR calcium elevation, and how much calcium must be released to support the triggering of a sustained propagating Ca2+ wave within the cell. The findings presented here will be compared to published work in other studies.

Ca2+ Nanosparks: Junctional Ca2+ Dynamics Probed with a New Targeted Biosensor

In cardiac dyads, junctional Ca2+ directly controls the gating of the ryanodine receptors (RyRs), and is itself dominated by RyR-mediated Ca2+ release from the sarcoplasmic reticulum. Being able to directly probe junctional Ca2+ dynamics should increase our understanding of cardiac excitation-contraction coupling and its modification in disease states. We developed a new targeted calcium biosensor based on the latest generation of fluorescent protein probes, GCaMP6f by fusing it to triadin and/or junctin. This probe targeted the dyad as shown by colocalization with RyRs after adenovirus-mediated transfection in single cardiac myocytes from adult rat. This biosensor had faster kinetics and slightly elevated Kd compared to native GCaMP6f. Confocal imaging revealed high-contrast biosensor fluorescent transients (“Ca2+ nanosparks”) that occupied a ∼50-times smaller volume than conventional Ca2+ spark records obtained with diffusible indicators. These spatially discrete signals showed no diffusive component outside the labeled region, allowing high-contrast recordings of dyad activation. The maintenance of high dyad-to-background contrast amidst whole cell Ca2+ transients allowed us to detect the latency and duration of individual dyad activation during normal excitation-contraction coupling. Furthermore, the high fidelity of the signal enabled a critical examination of non-spark Ca2+ leak that is currently thought to play a key role in heart diseases. In summay, this new, targeted biosensor permits the first selective detection of nanodomain Ca2+ dynamics and may be extended to whole tissues by transfection or transgenic approaches.

Do t‐tubules play a role in arrhythmogenesis in cardiac ventricular myocytes?

The transverse (t-) tubules of mammalian ventricular myocytes are invaginations of the surface membrane. The function of many of the key proteins involved in excitation-contraction coupling is located predominantly at the t-tubules, which thus form a Ca(2+)-handling micro-environment that is central to the normal rapid activation and relaxation of the ventricular myocyte. Although cellular arrhythmogenesis shares many ion flux pathways with normal excitation-contraction coupling, the role of the t-tubules in such arrhythmogenesis has not previously been considered. In this brief review we consider how the location and co-location of proteins at the t-tubules may contribute to the generation of arrhythmogenic delayed and early afterdepolarisations, and how the loss of t-tubules that occurs during heart failure may alter the generation of such arrhythmias, as well as contributing to other types of arrhythmia as a result of changes of electrical heterogeneity within the whole heart.