W. J. Lederer

Relaxation of Arterial Smooth Muscle by Calcium Sparks

Local increases in intracellular calcium ion concentration ([Ca2+]i) resulting from activation of the ryanodine-sensitive calcium-release channel in the sarcoplasmic reticulum (SR) of smooth muscle cause arterial dilation. Ryanodine-sensitive, spontaneous local increases in [Ca2+]i (Ca2+ sparks) from the SR were observed just under the surface membrane of single smooth muscle cells from myogenic cerebral arteries. Ryanodine and thapsigargin inhibited Ca2+ sparks and Ca2+-dependent potassium (KCa) currents, suggesting that Ca2+ sparks activate KCa channels. Furthermore, KCa channels activated by Ca2+ sparks appeared to hyperpolarize and dilate pressurized myogenic arteries because ryanodine and thapsigargin depolarized and constricted these arteries to an extent similar to that produced by blockers of KCa channels. Ca2+ sparks indirectly cause vasodilation through activation of KCa channels, but have little direct effect on spatially averaged [Ca2+]i, which regulates contraction.

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

X-ROS Signaling: Rapid Mechano-Chemo Transduction in Heart

Reactive oxygen species produced by NADPH oxidase link mechanical stretching to calcium signaling in mammalian myocytes. We report that in heart cells, physiologic stretch rapidly activates reduced-form nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) to produce reactive oxygen species (ROS) in a process dependent on microtubules (X-ROS signaling). ROS production occurs in the sarcolemmal and t-tubule membranes where NOX2 is located and sensitizes nearby ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). This triggers a burst of Ca2+ sparks, the elementary Ca2+ release events in heart. Although this stretch-dependent “tuning” of RyRs increases Ca2+ signaling sensitivity in healthy cardiomyocytes, in disease it enables Ca2+ sparks to trigger arrhythmogenic Ca2+ waves. In the mouse model of Duchenne muscular dystrophy, hyperactive X-ROS signaling contributes to cardiomyopathy through aberrant Ca2+ release from the SR. X-ROS signaling thus provides a mechanistic explanation for the mechanotransduction of Ca2+ release in the heart and offers fresh therapeutic possibilities.

Phorbol ester increases calcium current and simulates the effects of angiotensin II on cultured neonatal rat heart myocytes.

The effects of increased protein kinase C activity were studied in neonatal rat myocytes grown in primary culture. The changes in mechanical and electrical behavior, as well as protein phosphorylation, that followed the apparent activation of protein kinase C by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were examined. As spontaneous beating frequency was increased minimally by 10 nM TPA and by 100% with 85 nM TPA, shortening amplitude, shortening velocity, and relaxation velocity decreased concomltantly. In contrast, 4-α-phorbol-12, 13-didecanoate (α-PDD), which does not activate protein kinase C, had no effect on beating behavior at 800 nM. In voltage-clamped single myocytes, both steady-state and transient components of the cadmium-sensitive calcium current were increased by the addition of TPA (65 nM). Neither the time constant for the inactivation of the transient component of this calcium current nor the reversal potential was altered by TPA. The phosphorylation state of a discrete set of proteins, with apparent molecular weights of 32 and 83 kDa, was enhanced when TPA was added to intact myocytes. Angiotensin n enhances the phosphorylation state of the same set of proteins as observed with TPA. We conclude that activation of protein kinase C can modify mechanical behavior and increase L-type Ca2+ channel activity in cultured neonatal rat ventricular myocytes. The remarkable similarity in mechanical, electrical, and protein phosphorylation responses of cultured neonatal myocytes following TPA or angiotensin II application indicate that protein kinase C may mediate the action of angiotensin II.

Cellular origins of the transient inward current in cardiac myocytes. Role of fluctuations and waves of elevated intracellular calcium.

Activation of the transient inward current (In) by a rise in intracellular calcium concentration ([Ca 2+]i) is believed to be responsible for generating triggered cardiac arrhythmias. In this study, the cellular basis of the rise in [Ca2+]i that activates I-n and aftercontractions in single rat ventricular myocytes was examined. [Ca2+]i was measured both indirectly by cell contraction and directly with fura-2. Under conditions that caused steady-state [Ca2+]i to increase (i.e., calcium overload) membrane repolarization after a voltage-clamp depolarization resulted in the appearance of In that was similar in many respects to that observed in muiticellular preparations. This In occurred at the same time that [Ca2+]; spontaneously increased and preceded the aftercontraction by 60–90 msec. However, IT, recorded from a single cell was variable in time course and amplitude (unlike that observed in muiticellular preparations). Examination of cell contraction and digital imaging of fura-2 fluorescence showed that In was often associated with propagating regions of increased [Ca2+]i, which arose from discrete sites of origin within the cell. Apparently synchronous aftercontractions could also be associated with multiple propagating waves of [Ca2+]i. The variation in the time course and amplitude of In in single cells appeared to be due to changes in the location and number of sites of origin for the waves of [Ca2+]i. After the first aftercontraction and I-n, desynchronization of the sites of origin of increased [Ca2+]i occurred, and this resulted in a decrease in the amplitude of In and an increase in its duration. We conclude that the variability seen in single cells arises from changes in the pattern of spontaneous Ca release. Such phenomena will seriously complicate interpretation of muIticellular data, even when [Ca2+], is measured directly.

Stabilization of cardiac ryanodine receptor prevents intracellular calcium leak and arrhythmias

Catecholaminergic polymorphic ventricular tachycardia is a form of exercise-induced sudden cardiac death that has been linked to mutations in the cardiac Ca2+ release channel/ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR). We have shown that catecholaminergic polymorphic ventricular tachycardia-linked RyR2 mutations significantly decrease the binding affinity for calstabin-2 (FKBP12.6), a subunit that stabilizes the closed state of the channel. We have proposed that RyR2-mediated diastolic SR Ca2+ leak triggers ventricular tachycardia (VT) and sudden cardiac death. In calstabin-2-deficient mice, we have now documented diastolic SR Ca2+ leak, monophasic action potential alternans, and bidirectional VT. Calstabin-deficient cardiomyocytes exhibited SR Ca2+ leak-induced aberrant transient inward currents in diastole consistent with delayed after-depolarizations. The 1,4-benzothiazepine JTV519, which increases the binding affinity of calstabin-2 for RyR2, inhibited the diastolic SR Ca2+ leak, monophasic action potential alternans and triggered arrhythmias. Our data suggest that calstabin-2 deficiency is as a critical mediator of triggers that initiate cardiac arrhythmias.

Voltage-independent calcium release in heart muscle

The Ca2+ that activates contraction in heart muscle is regulated as in skeletal muscle by processes that depend on voltage and intracellular Ca2+ and involve a positive feedback system. How the initial electrical signal is amplified in heart muscle has remained controversial, however. Analogous protein structures from skeletal muscle and heart muscle have been identified physiologically and sequenced; these include the Ca2+ channel of the sarcolemma and the Ca2+ release channel of the sarcoplasmic reticulum. Although the parallels found in cardiac and skeletal muscles have provoked valuable experiments in both tissues, separation of the effects of voltage and intracellular Ca2+ on sarcoplasmic reticulum Ca2+ release in heart muscle has been imperfect. With the use of caged Ca2+ and flash photolysis in voltage-clamped heart myocytes, effects of membrane potential in heart muscle cells on Ca2+ release from intracellular stores have been studied. Unlike the response in skeletal muscle, voltage across the sarcolemma of heart muscle does not affect the release of Ca2+ from the sarcoplasmic reticulum, suggesting that other regulatory processes are needed to control Ca2(+)-induced Ca2+ release.

Mitochondrial calcium uptake

Calcium (Ca2+) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca2+ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca2+ ([Ca2+]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca2+ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca2+ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca2+ uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca2+ influx into the mitochondria via the MCU is small relative to other cytosolic Ca2+ extrusion pathways; (ii) single MCU conductance is ∼6–7 pS (105 mM [Ca2+]), and MCU flux appears to be modulated by [Ca2+]i, suggesting Ca2+ regulation of MCU open probability (PO); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca2+ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca2+ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca2+]i, mitochondrial Ca2+ uptake can increase 10- to 1,000-fold and begin to shape [Ca2+]i dynamics.

Ca2+ and voltage inactivate Ca2+ channels in guinea-pig ventricular myocytes through independent mechanisms.

1. L‐type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea‐pig ventricular heart cells using the whole‐cell patch‐clamp technique, in order to investigate the mechanism of Ca(2+)‐dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM‐nitrophen and nitr‐5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L‐type Ca2+ current. Specifically, Ca2+ photorelease from DM‐nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr‐5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)‐dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)‐dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM‐nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)‐dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)‐dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L‐type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)‐dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage‐sensing region of the channel. Furthermore, we found that Ca(2+)‐dependent inactivation is entirely sensitive to beta‐adrenergic stimulation. These facts suggest that either Ca(2+)‐dependent inactivation results from Ca(2+)‐dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)‐dependent inactivation is modulated by protein kinase A.

Ca2+ diffusion and sarcoplasmic reticulum transport both contribute to [Ca2+]i decline during Ca2+ sparks in rat ventricular myocytes.

1. We sought to evaluate the contribution of the sarcoplasmic reticulum (SR) Ca2+ pump (vs. diffusion) to the kinetics of [Ca24]i decline during Ca2+ sparks, which are due to spontaneous local SR Ca2+ release, in isolated rat ventricular myocytes measured using fluo‐3 and laser scanning confocal microscopy. 2. Resting Ca2+ sparks were compared before (control) and after the SR Ca2(+)‐ATPase was either completely blocked by 5 microM thapsigargin (TG) or stimulated by isoprenaline. Na(+)‐Ca2+ exchange was blocked using Na(+)‐free, Ca(2+)‐free solution (0 Na+, O Ca2+) and conditions were arranged so that the SR Ca2+ content was the same under all conditions when Ca2+ sparks were measured. 3. The control Ca2+ spark amplitude (281 +/‐ 13 nM) was not changed by TG (270 +/‐ 21 nM) or isoprenaline (302 +/‐ 10 nM). However, the time constant of [Ca2+]i decline was significantly slower in the presence of TG (29.3 +/‐ 4.3 ms) compared with control (21.6 +/‐ 1.5 ms) and faster with isoprenaline (14.5 +/‐ 0.9 ms), but in all cases was much faster than the global [Ca2+]i decline during a control twitch (177 +/‐ 10 ms). 4. The spatial spread of Ca2+ during the Ca2+ spark was also influenced by the SR Ca2+ pump. The apparent ‘space constant’ of the Ca2+ sparks was longest when the SR Ca2+ pump was blocked, intermediate in control and shortest with isoprenaline. 5. We conclude that while Ca2+ diffusion from the source of Ca2+ release is the dominant process in local [Ca2+]i decline during the Ca2+ spark, Ca2+ transport by the SR contributes significantly to both the kinetics and spatial distribution of [Ca2+]i during the Ca2+ spark.