On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237 kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

The generation and validation of two NKX2-5 fluorescent reporter human embryonic stem cell lines: UMANe002-A-1 and UMANe002-A-2.

The transcription factor NKX2-5 is a highly conserved master regulator of heart development which is widely expressed in cardiac progenitors and cardiomyocytes. Fluorescent reporters of NKX2-5 that minimally perturb normal protein expression can enable the identification, quantification and isolation of NKX2-5-expressing cells in a normal physiological state. Here we report the generation of two new hESC lines with eGFP inserted upstream (5') or downstream (3') of NKX2-5, linked by a cleavable T2A peptide. These complementary reporters produce a robust fluorescent signal in cardiac cells and have wide utility particularly for research on developmental biology and disease modelling.

Transverse tubules are a common feature in large mammalian atrial myocytes including human.

Transverse (t) tubules are surface membrane invaginations that are present in all mammalian cardiac ventricular cells. The apposition of L-type Ca(2+) channels on t tubules with the sarcoplasmic reticulum (SR) constitutes a "calcium release unit" and allows close coupling of excitation to the rise in systolic Ca(2+). T tubules are virtually absent in the atria of small mammals, and therefore Ca(2+) release from the SR occurs initially at the periphery of the cell and then propagates into the interior. Recent work has, however, shown the occurrence of t tubules in atrial myocytes from sheep. As in the ventricle, Ca(2+) release in these cells occurs simultaneously in central and peripheral regions. T tubules in both the atria and the ventricle are lost in disease, contributing to cellular dysfunction. The aim of this study was to determine if the occurrence of t tubules in the atrium is restricted to sheep or is a more general property of larger mammals including humans. In atrial tissue sections from human, horse, cow, and sheep, membranes were labeled using wheat germ agglutinin. As previously shown in sheep, extensive t-tubule networks were present in horse, cow, and human atrial myocytes. Analysis shows half the volume of the cell lies within 0.64 ± 0.03, 0.77 ± 0.03, 0.84 ± 0.03, and 1.56 ± 0.19 µm of t-tubule membrane in horse, cow, sheep, and human atrial myocytes, respectively. The presence of t tubules in the human atria may play an important role in determining the spatio-temporal properties of the systolic Ca(2+) transient and how this is perturbed in disease.

The mechanism and significance of the slow changes of ventricular action potential duration following a change of heart rate.

This article reviews the effects of changes of heart rate on the ventricular action potential duration. These can be divided into short term (fractions of a second), resulting from the kinetics of recovery of membrane currents, through to long term (up to days), resulting from changes of protein expression. We concentrate on the medium-term changes (time course of the order of 100 s). These medium-term changes occur in isolated tissues and in the intact human heart. They may protect against cardiac arrhythmias. Finally, we discuss the cellular mechanisms responsible for these changes.

Differences in intracellular calcium homeostasis between atrial and ventricular myocytes.

The role that Ca(2+) plays in ventricular excitation contraction coupling is well defined and much is known about the marked differences in the spatiotemporal properties of the systolic Ca(2+) transient between atrial and ventricular myocytes. However, to date there has been no systematic appraisal of the Ca(2+) homeostatic mechanisms employed by atrial cells and how these compare to the ventricle. In the present study we sought to determine the fractional contributions made to the systolic Ca(2+) transient and the decay of [Ca(2+)](i) by the sarcoplasmic reticulum and sarcolemmal mechanisms. Experiments were performed on single myocytes isolated from the atria and ventricles of the rat. Intracellular Ca(2+) concentration, membrane currents, SR Ca(2+) content and cellular Ca(2+) buffering capacity were measured at 23 degrees C. Atrial cells had smaller systolic Ca(2+) transients (251+/-39 vs. 376+/-41 nmol x L(-1)) that decayed more rapidly (7.4+/-0.6 vs. 5.45+/-0.3 s(-1)). This was due primarily to an increased rate of SR mediated Ca(2+) uptake (k(SR), 6.88+/-0.6 vs. 4.57+/-0.3 s(-1)). SR Ca(2+) content was 289% greater and Ca(2+) buffering capacity was increased approximately 3-fold in atrial cells (B(max) 371.9+/-32.4 vs. 121.8+/-8 micromol x L(-1), all differences P<0.05). The fractional release of Ca(2+) from the SR was greater in atrial cells, although the gain of excitation contraction coupling was the same in both cell types. In summary our data demonstrate fundamental differences in Ca(2+) homeostasis between atrial and ventricular cells and we speculate that the increased SR Ca(2+) content may be significant in determining the increased prevalence of arrhythmias in the atria.

Regulation of systolic [Ca2+]i and cellular Ca2+ flux balance in rat ventricular myocytes by SR Ca2+, L-type Ca2+ current and diastolic [Ca2+]i.

The force-frequency response is an important physiological mechanism regulating cardiac output changes and is accompanied in vivo by beta-adrenergic stimulation. We sought to determine the role of sarcoplasmic reticulum (SR) Ca2+ content and L-type current (ICa-L) in the frequency response of the systolic Ca2+ transient alone and during beta-adrenergic stimulation. Experiments (on single rat ventricular myocytes) were designed to be as physiological as possible. Under current clamp stimulation SR Ca2+ content increased in line with stimulation frequency (1-8 Hz) but the systolic Ca2+ transient was maximal at 6 Hz. Under voltage clamp, increasing frequency decreased both systolic Ca2+ transient and ICa-L. Normalizing peak ICa-L by altering the test potential decreased the Ca2+ transient amplitude less than an equivalent reduction achieved through changes in frequency. This suggests that, in addition to SR Ca2+ content and ICa-L, another factor, possibly refractoriness of Ca2+ release from the SR contributes. Under current clamp, beta-adrenergic stimulation (isoprenaline, 30 nm) increased both the Ca2+ transient and the SR Ca2+ content and removed the dependence of both on frequency. In voltage clamp experiments, beta-adrenergic stimulation still increased SR Ca2+ content yet there was an inverse relation between frequency and Ca2+ transient amplitude and ICa-L. Diastolic [Ca2+]i increased with stimulation frequency and this contributed substantially (69.3 +/- 6% at 8 Hz) to the total Ca2+ efflux from the cell. We conclude that Ca2+ flux balance is maintained by the combination of increased efflux due to elevated diastolic [Ca2+]i and a decrease of influx on IC-L) on each pulse.

Analysis of cellular calcium fluxes in cardiac muscle to understand calcium homeostasis in the heart.

Central to controlling intracellular calcium concentration ([Ca(2+)](i)) are a number of Ca(2+) transporters and channels with the L-type Ca(2+) channel, Na(+)-Ca(2+) exchanger and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) being of particular note in the heart. This review concentrates on the regulation of [Ca(2+)](i) in cardiac muscle and the homeostatic mechanisms employed to ensure that the heart can operate under steady-state conditions on a beat by beat basis. To this end we discuss the relative importance of various sources and sinks of Ca(2+) responsible for initiating contraction and relaxation in cardiac myocytes and how these can be manipulated to regulate the Ca(2+) content of the major Ca(2+) store, the sarcoplasmic reticulum (SR). We will present a simple feedback system detailing how such control can be achieved and highlight how small perturbations to the steady-state operation of the feedback loop can be both beneficial physiologically and underlie changes in systolic Ca(2+) in ageing and heart disease. In addition to manipulating the amplitude of the normal systolic Ca(2+) transient, the tight regulation of SR Ca(2+) content is also required to prevent the abnormal, spontaneous or diastolic release of Ca(2+) from the SR. Such diastolic events are a major factor contributing to the genesis of cardiac arrhythmias in disease situations and in recently identified familial mutations in the SR Ca(2+) release channel (ryanodine receptor, RyR). How such diastolic release arises and potential mechanisms for controlling this will be discussed.

Base of pore loop is important for rectification, activation, permeation, and block of Kir3.1/Kir3.4.

The Kir3.1/Kir3.4 channel is an inward rectifier, agonist-activated K(+) channel. The location of the binding site within the channel pore that coordinates polyamines (and is thus responsible for inward rectification) and the location of the gate that opens the channel in response to agonist activation is unclear. In this study, we show, not surprisingly, that mutation of residues at the base of the selectivity filter in the pore loop and second transmembrane domain weakens Cs(+) block and decreases selectivity (as measured by Rb(+) and spermine permeation). However, unexpectedly, the mutations also weaken inward rectification and abolish agonist activation of the channel. In the wild-type channel and 34 mutant channels, there are significant (p < 0.05) correlations among the K(D) for Cs(+) block, Rb(+) and spermine permeation, inward rectification, and agonist activation. The significance of these findings is discussed. One possible conclusion is that the selectivity filter is responsible for inward rectification and agonist activation as well as permeation and block.

Photoperiod-dependent modulation of cardiac excitation contraction coupling in the Siberian hamster.

In mammals, changes in photoperiod regulate a diverse array of physiological and behavioral processes, an example of which in the Siberian hamster (Phodopus sungorus) is the expression of bouts of daily torpor following prolonged exposure to a short photoperiod. During torpor, body temperature drops dramatically; however, unlike in nonhibernating or nontorpid species, the myocardium retains the ability to contract and is resistant to the development of arrhythmias. In the present study, we sought to determine whether exposure to a short photoperiod results in alterations to cardiac excitation-contraction coupling, thus potentially enabling the heart to survive periods of low temperature during torpor. Experiments were performed on single ventricular myocytes freshly isolated from the hearts of Siberian hamsters that had been exposed to either 12 wk of short-day lengths (SD) or 12 wk of long-day lengths (LD). In SD-acclimated animals, the amplitude of the systolic Ca(2+) transient was increased (e.g., from 142 +/- 17 nmol/l in LD to 229 +/- 31 nmol/l in SD at 4 Hz; P < 0.001). The increased Ca(2+) transient amplitude in the SD-acclimated animals was not associated with any change in the shape or duration of the action potential. However, sarcoplasmic reticulum Ca(2+) content measured after current-clamp stimulation was increased in the SD-acclimated animals (at 4 Hz, 110 +/- 5 vs. 141 +/- 15 mumol/l, P < 0.05). We propose that short photoperiods reprogram the function of the cardiac sarcoplasmic reticulum, resulting in an increased Ca(2+) content, and that this may be a necessary precursor for maintenance of cardiac function during winter torpor.

Mechanisms underlying enhanced cardiac excitation contraction coupling observed in the senescent sheep myocardium.

Ageing related stiffening of the vascular system is believed to be in part responsible for a number of clinical outcomes including hypertension and heart failure. In the present study, we sought to determine whether there are alterations in cardiac excitation contraction coupling that may help compensate for the increased vessel stiffness. Experiments were performed on single cardiac myocytes isolated from young (18 months) and aged (>8 years) sheep. Intracellular Ca(2+) concentration, action potentials, L-type Ca(2+) currents and SR Ca(2+) content were measured at 23 degrees C. With ageing, cell capacitance increased by 26% indicating cellular hypertrophy. Action potential duration (APD90) (590 +/- 21 vs. 726 +/- 36 ms), Ca(2+) transient amplitude (112 +/- 15 vs. 202 +/- 25 nmol l(-1)) and fractional cell shortening (by 37%) also increased in the aged hearts (all values P < 0.05). The larger Ca(2+) transient amplitude observed under current clamp conditions was maintained under voltage clamp control; however, SR Ca(2+) content was identical. Both the peak L-type Ca(2+) current (2.8 +/- 0.3 vs. 4.9 +/- 0.5 pA pF(-1)) and integrated Ca(2+) entry (5.1 +/- 0.7 vs. 7.9 +/- 0.8 micromol l(-1), all P < 0.01) were greater in aged cells. In this study we show that in the ageing ovine myocardium, the amplitude of the systolic Ca(2+) transient is increased. The larger Ca(2+) transients cannot simply be explained by changes in APD and we suggest that the greater inward L-type Ca(2+) current provides a more effective trigger for calcium-induced-calcium release from the SR whilst maintaining a stable SR Ca(2+) content. These changes in cardiac excitation contraction coupling may help maintain cardiac output in the face of increased great vessel stiffness.

K+ activation of kir3.1/kir3.4 and kv1.4 K+ channels is regulated by extracellular charges.

K+ activates many inward rectifier and voltage-gated K+ channels. In each case, an increase in K+ current through the channel can occur despite a reduced driving force. We have investigated the molecular mechanism of K+ activation of the inward rectifier K+ channel, Kir3.1/Kir3.4, and the voltage-gated K+ channel, Kv1.4. In the Kir3.1/Kir3.4 channel, mutation of an extracellular arginine residue, R155, in the Kir3.4 subunit markedly reduced K+ activation of the channel. The same mutation also abolished Mg2+ block of the channel. Mutation of the equivalent residue in Kv1.4 (K532) abolished K+ activation as well as C-type inactivation of the Kv1.4 channel. Thus, whereas C-type inactivation is a collapse of the selectivity filter, K+ activation could be an opening of the selectivity filter. K+ activation of the Kv1.4 channel was enhanced by acidic pH. Mutation of an extracellular histidine residue, H508, that mediates the inhibitory effect of protons on Kv1.4 current, abolished both K+ activation and the enhancement of K+ activation at acidic pH. These results suggest that the extracellular positive charges in both the Kir3.1/Kir3.4 and the Kv1.4 channels act as "guards" and regulate access of K+ to the selectivity filter and, thus, the open probability of the selectivity filter. Furthermore, these data suggest that, at acidic pH, protonation of H508 inhibits current through the Kv1.4 channel by decreasing K+ access to the selectivity filter, thus favoring the collapse of the selectivity filter.

Cs+ block of the cardiac muscarinic K+ channel, GIRK1/GIRK4, is not dependent on the aspartate residue at position 173.

Cs+ block of GIRK1/GIRK4 expressed in Xenopus oocytes has been investigated. It has been reported that a negatively charged aspartate residue at position 172 in IRK1 is responsible for Cs+ block of the channel. IRK1, a homotetramer, has four aspartate residues at this position. GIRK1/GIRK4 is a heterotetramer and has two aspartate residues at the equivalent position (GIRK1-D173) and, consequently, it should be less sensitive to Cs+. Cs+ caused voltage-dependent block of GIRK1/GIRK4 current (measured with the two-microelectrode voltage-clamp technique). The apparent fraction of the electrical field through which Cs+ moves in order to reach its site of block (delta approximately equals 1.66) is comparable to that in IRK1, suggesting that Cs+ binds to a similar site in the two channels. GIRK1/GIRK4 was less sensitive than IRK1 to Cs+ -the Kd was 3.0-8.5 times greater and at potentials more negative than approximately or = to 130 mV there was voltage-dependent relief of block of GIRK1/GIRK4 (not the case with IRK1). However, the mutations GIRK1-D173A and GIRK1-D173Q increased the sensitivity of the channel to Cs+, while adding a negatively charged aspartate residue to GIRK4 at the equivalent position (GIRK4-N 79D) decreased Cs+ sensitivity. GIRK1-D173 cannot be the site of Cs+ block of GIRK1/GIRK4.

Residues and mechanisms for slow activation and Ba2+ block of the cardiac muscarinic K+ channel, Kir3.1/Kir3.4.

Mechanisms and residues responsible for slow activation and Ba(2+) block of the cardiac muscarinic K(+) channel, Kir3.1/Kir3.4, were investigated using site-directed mutagenesis. Mutagenesis of negatively charged residues located throughout the pore of the channel (in H5, M2, and proximal C terminus) reduced or abolished slow activation. The strongest effects resulted from mutagenesis of residues in H5 close to the selectivity filter; mutagenesis of residues in M2 and proximal C terminus equivalent to those identified as important determinants of the activation kinetics of Kir2.1 was less effective. In giant patches, slow activation was present in cell-attached patches, lost on excision of the patch, and restored on perfusion with polyamine. Mutagenesis of residues in H5 and M2 close to the selectivity filter also decreased Ba(2+) block of the channel. A critical residue for Ba(2+) block was identified in Kir3.4. Mutagenesis of the equivalent residue in Kir3.1 failed to have as pronounced an effect on Ba(2+) block, suggesting an asymmetry of the channel pore. It is concluded that slow activation is principally the result of unbinding of polyamines from negatively charged residues close to the selectivity filter of the channel and not an intrinsic gating mechanism. Ba(2+) block involves an interaction with the same residues.