Calcium-induced calcium release in smooth muscle: loose coupling between the action potential and calcium release

Calcium-induced calcium release (CICR) has been observed in cardiac myocytes as elementary calcium release events (calcium sparks) associated with the opening of L-type Ca(2+) channels. In heart cells, a tight coupling between the gating of single L-type Ca(2+) channels and ryanodine receptors (RYRs) underlies calcium release. Here we demonstrate that L-type Ca(2+) channels activate RYRs to produce CICR in smooth muscle cells in the form of Ca(2+) sparks and propagated Ca(2+) waves. However, unlike CICR in cardiac muscle, RYR channel opening is not tightly linked to the gating of L-type Ca(2+) channels. L-type Ca(2+) channels can open without triggering Ca(2+) sparks and triggered Ca(2+) sparks are often observed after channel closure. CICR is a function of the net flux of Ca(2+) ions into the cytosol, rather than the single channel amplitude of L-type Ca(2+) channels. Moreover, unlike CICR in striated muscle, calcium release is completely eliminated by cytosolic calcium buffering. Thus, L-type Ca(2+) channels are loosely coupled to RYR through an increase in global [Ca(2+)] due to an increase in the effective distance between L-type Ca(2+) channels and RYR, resulting in an uncoupling of the obligate relationship that exists in striated muscle between the action potential and calcium release.     

Caffeine-activated large-conductance plasma membrane cation channels in cardiac myocytes: characteristics and significance

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca(2+) release from intracellular stores. These Ca(2+)-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of approximately 370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to Ca(2+) sparks), LCCs also showed some different characteristics. With simultaneous Ca(2+) imaging and current recording, the localized fluorescence increase due to Ca(2+) entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.     

Slow intercellular Ca(2+) signaling in wild-type and Cx43-null neonatal mouse cardiac myocytes

Focal mechanical stimulation of single neonatal mouse cardiac myocytes in culture induced intercellular Ca(2+) waves that propagated with mean velocities of approximately 14 micrometer/s, reaching approximately 80% of the cells in the field. Deletion of connexin43 (Cx43), the main cardiac gap junction channel protein, did not prevent communication of mechanically induced Ca(2+) waves, although the velocity and number of cells communicated by the Ca(2+) signal were significantly reduced. Similar effects were observed in wild-type cardiac myocytes treated with heptanol, a gap junction channel blocker. Fewer cells were involved in intercellular Ca(2+) signaling in both wild-type and Cx43-null cultures in the presence of suramin, a P(2)-receptor blocker; blockage was more effective in Cx43-null than in wild-type cells. Thus gap junction channels provide the main pathway for communication of slow intercellular Ca(2+) signals in wild-type neonatal mouse cardiac myocytes. Activation of P(2)-receptors induced by ATP release contributes a secondary, extracellular pathway for transmission of Ca(2+) signals. The importance of such ATP-mediated Ca(2+) signaling would be expected to be enhanced under ischemic conditions, when release of ATP is increased and gap junction channels conductance is significantly reduced.     

Pannexin 3 functions as an ER Ca(2+) channel, hemichannel, and gap junction to promote osteoblast differentiation

The pannexin proteins represent a new gap junction family. However, the cellular functions of pannexins remain largely unknown. Here, we demonstrate that pannexin 3 (Panx3) promotes differentiation of osteoblasts and ex vivo growth of metatarsals. Panx3 expression was induced during osteogenic differentiation of C2C12 cells and primary calvarial cells, and suppression of this endogenous expression inhibited differentiation. Panx3 functioned as a unique Ca(2+) channel in the endoplasmic reticulum (ER), which was activated by purinergic receptor/phosphoinositide 3-kinase (PI3K)/Akt signaling, followed by activation of calmodulin signaling for differentiation. Panx3 also formed hemichannels that allowed release of ATP into the extracellular space and activation of purinergic receptors with the subsequent activation of PI3K-Akt signaling. Panx3 also formed gap junctions and propagated Ca(2+) waves between cells. Blocking the Panx3 Ca(2+) channel and gap junction activities inhibited osteoblast differentiation. Thus, Panx3 appears to be a new regulator that promotes osteoblast differentiation by functioning as an ER Ca(2+) channel and a hemichannel, and by forming gap junctions.     

Thermodynamically irreversible gating of ryanodine receptors in situ revealed by stereotyped duration of release in Ca(2+) sparks

For a single or a group of Markov channels gating reversibly, distributions of open and closed times should be the sum of positively weighted decaying exponentials. Violation of this microscopic reversibility has been demonstrated previously on a number of occasions at the single channel level, and has been attributed to possible channel coupling to external sources of free energy. Here we show that distribution of durations of Ca(2+) release underlying Ca(2+) sparks in intact cardiac myocytes exhibits a prominent mode at approximately 8 ms. Analysis of the cycle time for repetitive sparks at hyperactive sites revealed no intervals briefer than approximately 35 ms and a mode at approximately 90 ms. These results indicate that, regardless of whether Ca(2+) sparks are single-channel or multi-channel in origin, they are generated by thermodynamically irreversible stochastic processes. In contrast, data from planar lipid bilayer experiments were consistent with reversible gating of RyR under asymmetric cis (4 microM) and trans Ca(2+) (10 mM), suggesting that the irreversibility for Ca(2+) spark genesis may reside at a supramolecular level. Modeling suggests that Ca(2+)-induced Ca(2+) release among adjacent RyRs may couple the external energy derived from Ca(2+) gradients across the SR to RyR gating in situ, and drive the irreversible generation of Ca(2+) sparks.     

On the role of junctin in cardiac Ca2+ handling, contractility, and heart failure

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the Ca(2+) release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the Ca(2+) sensitivity of the Ca(2+) release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal Ca(2+) transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of Ca(2+) homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. Ca(2+) transients showed a decreased maximum Ca(2+) amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired Ca(2+) homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism.     

Reverse engineering the L-type Ca2+ channel alpha1c subunit in adult cardiac myocytes using novel adenoviral vectors

The alpha(1c) subunit of the cardiac L-type Ca(2+) channel, which contains the channel pore, voltage- and Ca(2+)-dependent gating structures, and drug binding sites, has been well studied in heterologous expression systems, but many aspects of L-type Ca(2+) channel behavior in intact cardiomyocytes remain poorly characterized. Here, we develop adenoviral constructs with E1, E3 and fiber gene deletions, to allow incorporation of full-length alpha(1c) gene cassettes into the adenovirus backbone. Wild-type (alpha(1c-wt)) and mutant (alpha(1c-D-)) Ca(2+) channel adenoviruses were constructed. The alpha(1c-D-) contained four point substitutions at amino acid residues known to be critical for dihydropyridine binding. Both alpha(1c-wt) and alpha(1c-D-) expressed robustly in A549 cells (peak L-type Ca(2+) current (I(CaL)) at 0 mV: alpha(1c-wt) -9.94+/-1.00pA/pF, n=9; alpha(1c-D-) -10.30pA/pF, n=12). I(CaL) carried by alpha(1c-D-) was markedly less sensitive to nitrendipine (IC(50) 17.1 microM) than alpha(1c-wt) (IC(50) 88 nM); a feature exploited to discriminate between engineered and native currents in transduced guinea-pig myocytes. 10 microM nitrendipine blocked only 51+/-5% (n=9) of I(CaL) in alpha(1c-D-)-expressing myocytes, in comparison to 86+/-8% (n=9) of I(CaL) in control myocytes. Moreover, in 20 microM nitrendipine, calcium transients could still be evoked in alpha(1c-D-)-transduced cells, but were largely blocked in control myocytes, indicating that the engineered channels were coupled to sarcoplasmic reticular Ca(2+) release. These alpha(1c) adenoviruses provide an unprecedented tool for structure-function studies of cardiac excitation-contraction coupling and L-type Ca(2+) channel regulation in the native myocyte background.     

Molecular identification of a TTX-sensitive Ca(2+) current

The TTX-sensitive Ca(2+) current [I(Ca(TTX))] observed in cardiac myocytes under Na(+)-free conditions was investigated using patch-clamp and Ca(2+)-imaging methods. Cs(+) and Ca(2+) were found to contribute to I(Ca(TTX)), but TEA(+) and N-methyl-D-glucamine (NMDG(+)) did not. HEK-293 cells transfected with cardiac Na(+) channels exhibited a current that resembled I(Ca(TTX)) in cardiac myocytes with regard to voltage dependence, inactivation kinetics, and ion selectivity, suggesting that the cardiac Na(+) channel itself gives rise to I(Ca(TTX)). Furthermore, repeated activation of I(Ca(TTX)) led to a 60% increase in intracellular Ca(2+) concentration, confirming Ca(2+) entry through this current. Ba(2+) permeation of I(Ca(TTX)), reported by others, did not occur in rat myocytes or in HEK-293 cells expressing cardiac Na(+) channels under our experimental conditions. The report of block of I(Ca(TTX)) in guinea pig heart by mibefradil (10 microM) was supported in transfected HEK-293 cells, but Na(+) current was also blocked (half-block at 0.45 microM). We conclude that I(Ca(TTX)) reflects current through cardiac Na(+) channels in Na(+)-free (or "null") conditions. We suggest that the current be renamed I(Na(null)) to more accurately reflect the molecular identity of the channel and the conditions needed for its activation. The relationship between I(Na(null)) and Ca(2+) flux through slip-mode conductance of cardiac Na(+) channels is discussed in the context of ion channel biophysics and "permeation plasticity."     

Functional consequence of protein kinase A-dependent phosphorylation of the cardiac ryanodine receptor: sensitization of store overload-induced Ca2+ release

The phosphorylation of the cardiac Ca(2+)-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.     

Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.     

alpha(1)-adrenergic stimulation mediates Ca(2+)-dependent inositol phosphate formation through the alpha(1B)-like adrenoceptor subtype in adult rat cardiac myocytes.

We studied the effects of increased Ca(2+) influx on alpha(1)-adrenoceptor-stimulated InsP formation in adult rat cardiac myocytes. We further examined if such effects could be mediated through a specific alpha(1)-adrenoceptor subtype. [(3)H]InsP responses to adrenaline were dependent on extracellular Ca(2+) concentration, from 0.1 microM to 2 mM, and were completely blocked by Ca(2+) removal. However, in cardiac myocytes preloaded with BAPTA, a highly selective calcium chelating agent, Ca(2+) concentrations higher than 1 microM had no effect on adrenaline-stimulated [(3)H]InsP formation. Taken together these results suggest that [(3)H]InsP formation induced by alpha(1)-adrenergic stimulation is in part mediated by increased Ca(2+) influx. Consistent with this, ionomycin, a calcium ionophore, stimulated [(3)H]InsP formation. This response was additive with the response to adrenaline stimulation implying that different signaling mechanisms may be involved. In cardiac myocytes treated with the alpha(1B)-adrenoceptor alkylating agent, CEC, [(3)H]InsP formation remained unaffected by increased Ca(2+) concentrations, a pattern similar to that observed when intracellular Ca(2+) was chelated with BAPTA. In contrast, addition of the alpha(1A)-subtype antagonist, 5'-methyl urapidil, did not affect the Ca(2+) dependence of [(3)H]InsP formation. Neither nifedipine, a voltage-dependent Ca(2+) channel blocker nor the inorganic Ca(2+) channel blockers, Ni(2+) and Co(2+), had any effect on adrenaline stimulated [(3)H]InsP, at concentrations that inhibit Ca(2+) channels. The results suggest that in adult rat cardiac myocytes, in addition to G protein-mediated response, alpha(1)-adrenergic-stimulated [(3)H]InsP formation is activated by increased Ca(2+) influx mediated by the alpha(1B)-subtype.     

T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion

T-type Ca(2+) channels have been suggested to play a role in cardiac automaticity, cell growth, and cardiovascular remodeling. Although three genes encoding for a T-type Ca(2+) channel have been identified, the nature of the isoform(s) supporting the cardiac T-type Ca(2+) current (I(Ca,T)) has not yet been determined. We describe the postnatal evolution of I(Ca,T) density in freshly dissociated rat atrial and ventricular myocytes and its functional properties at peak current density in young atrial myocytes. I(Ca,T) displays a classical low activation threshold, rapid inactivation kinetics, negative steady-state inactivation, slow deactivation, and the presence of a window current. Interestingly, I(Ca,T) is poorly sensitive to Ni(2+) and insensitive to R-type current toxin SNX-482. RT-PCR experiments and comparison of functional properties with recombinant Ca(2+) channel subtypes suggest that neonatal I(Ca,T) is related to the alpha(1G)-subunit. Atrial natriuretic factor (ANF) secretion was measured using peptide radioimmunoassays in atrial tissue. Pharmacological dissection of ANF secretion indicates an important contribution of I(Ca,T) to Ca(2+) signaling during the neonatal period.     

A simplified local control model of calcium-induced calcium release in cardiac ventricular myocytes

Calcium (Ca2+)-induced Ca2+ release (CICR) in cardiac myocytes exhibits high gain and is graded. These properties result from local control of Ca2+ release. Existing local control models of Ca2+ release in which interactions between L-Type Ca2+ channels (LCCs) and ryanodine-sensitive Ca2+ release channels (RyRs) are simulated stochastically are able to reconstruct these properties, but only at high computational cost. Here we present a general analytical approach for deriving simplified models of local control of CICR, consisting of low-dimensional systems of coupled ordinary differential equations, from these more complex local control models in which LCC-RyR interactions are simulated stochastically. The resulting model, referred to as the coupled LCC-RyR gating model, successfully reproduces a range of experimental data, including L-Type Ca2+ current in response to voltage-clamp stimuli, inactivation of LCC current with and without Ca2+ release from the sarcoplasmic reticulum, voltage-dependence of excitation-contraction coupling gain, graded release, and the force-frequency relationship. The model does so with low computational cost.     

Refractoriness of sarcoplasmic reticulum Ca2+ release determines Ca2+ alternans in atrial myocytes

Cardiac alternans is a recognized risk factor for cardiac arrhythmia and sudden cardiac death. At the cellular level, Ca(2+) alternans appears as cytosolic Ca(2+) transients of alternating amplitude at regular beating frequency. Cardiac alternans is a multifactorial process but has been linked to disturbances in intracellular Ca(2+) regulation. In atrial myocytes, we tested the role of voltage-gated Ca(2+) current, sarcoplasmic reticulum (SR) Ca(2+) load, and restitution properties of SR Ca(2+) release for the occurrence of pacing-induced Ca(2+) alternans. Voltage-clamp experiments revealed that peak Ca(2+) current was not affected during alternans, and alternans of end-diastolic SR Ca(2+) load, evaluated by application of caffeine or measured directly with an intra-SR fluorescent Ca(2+) indicator (fluo-5N), were not a requirement for cytosolic Ca(2+) alternans. Restitution properties and kinetics of refractoriness of Ca(2+) release after activation during alternans were evaluated by four different approaches: measurements of 1) the delay (latency) of occurrence of spontaneous global Ca(2+) releases and 2) Ca(2+) spark frequency, both during rest after a large and small alternans Ca(2+) transient; 3) the magnitude of premature action potential-induced Ca(2+) transients after a large and small beat; and 4) the efficacy of a photolytically induced Ca(2+) signal (Ca(2+) uncaging from DM-nitrophen) to trigger additional Ca(2+) release during alternans. The results showed that the latency of global spontaneous Ca(2+) release was prolonged and Ca(2+) spark frequency was decreased after the large Ca(2+) transient during alternans. Furthermore, the restitution curve of the Ca(2+) transient elicited by premature action potentials or by photolysis-induced Ca(2+) release from the SR lagged behind after a large-amplitude transient during alternans compared with the small-amplitude transient. The data demonstrate that beat-to-beat alternation of the time-dependent restitution properties and refractory kinetics of the SR Ca(2+) release mechanism represents a key mechanism underlying cardiac alternans.     

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.     

Inositol 1,4,5-trisphosphate receptor expression in cardiac myocytes

Calcium release from intracellular stores is the signal generated by numerous regulatory pathways including those mediated by hormones, neurotransmitters and electrical activation of muscle. Recently two forms of intracellular calcium release channels (CRCs) have been identified. One, the inositol 1,4,5-trisphosphate receptors (IP3Rs) mediate IP3-induced Ca2+ release and are believed to be present on the ER of most cell types. A second form, the ryanodine receptors (RYRs) of the sarcoplasmic reticulum, have evolved specialized functions relevant to muscle contraction and are the major CRCs found in striated muscles. Though structurally related, IP3Rs and RYRs have distinct physiologic and pharmacologic profiles. In the heart, where the dominant mechanism of intracellular calcium release during excitation-contraction coupling is Ca(2+)-induced Ca2+ release via the RYR, a role for IP3-mediated Ca2+ release has also been proposed. It has been assumed that IP3Rs are expressed in the heart as in most other tissues, however, it has not been possible to state whether cardiac IP3Rs were present in cardiac myocytes (which already express abundant amounts of RYR) or only in non- muscle cells within the heart. This lack of information regarding the expression and structure of an IP3R within cardiac myocytes has hampered the elucidation of the significance of IP3 signaling in the heart. In the present study we have used combined in situ hybridization to IP3R mRNA and immunocytochemistry to demonstrate that, in addition to the RYR, an IP3R is also expressed in rat cardiac myocytes. Immunoreactivity and RNAse protection have shown that the IP3R expressed in cardiac myocytes is structurally similar to the IP3R in brain and vascular smooth muscle. Within cardiac myocytes, IP3R mRNA levels were approximately 50-fold lower than that of the cardiac RYR mRNA. Identification of an IP3R in cardiac myocytes provides the basis for future studies designed to elucidate its functional role both as a mediator of pharmacologic and hormonal influences on the heart, and in terms of its possible interaction with the RYR during excitation- contraction coupling in the heart.