Ryanodine receptors as leak channels.

European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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Review

Ryanodine receptors as leak channels Agustín Guerrero-Hernández n, Guillermo Ávila, Angélica Rueda Departamento de Bioquímica, Cinvestav, Mexico city, México

art ic l e i nf o

a b s t r a c t

Article history: Accepted 21 November 2013

Ryanodine receptors are Ca2 þ release channels of internal stores. This review focuses on those situations and conditions that transform RyRs from a finely regulated ion channel to an unregulated Ca2 þ leak channel and the pathological consequences of this alteration. In skeletal muscle, mutations in either CaV1.1 channel or RyR1 results in a leaky behavior of the latter. In heart cells, RyR2 functions normally as a Ca2 þ leak channel during diastole within certain limits, the enhancement of this activity leads to arrhythmogenic situations that are tackled with different pharmacological strategies. In smooth muscle, RyRs are involved more in reducing excitability than in stimulating contraction so the leak activity of RyRs in the form of Ca2 þ sparks, locally activates Ca2 þ -dependent potassium channels to reduce excitability. In neurons the enhanced activity of RyRs is associated with the development of different neurodegenerative disorders such as Alzheimer and Huntington diseases. It appears then that the activity of RyRs as leak channels can have both physiological and pathological consequences depending on the cell type and the metabolic condition. & 2013 Elsevier B.V. All rights reserved.

Keywords: Ryanodine receptors Leak channels Skeletal muscle Heart muscle Smooth muscle Neurons

Contents 1. 2.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 RyR as leak channels in skeletal muscle. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.1. RyR1s are not “intrinsically” leaky . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.2. Leak in RyR1 mutant channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2.3. Leak in RyR1 as a consequence of MH mutations in CaV1.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2.4. Novel therapeutic strategies for MH and CCD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 2.5. FKBP12-deficient RyR1s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3. RyR as Ca2 þ leak channels in cardiac cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1. Physiological role of RyR2-mediated Ca2 þ leak in the heart. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.2. Abnormal RyR2-mediated Ca2 þ leak in cardiac diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 3.2.1. Leaky RyR2 channels in heart failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2.2. RyR2 leakiness in diabetic cardiomyopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.2.3. Inherited arrhythmias and leaky RyR2 channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3.3. Is the “stabilization” of RyR a way to stop abnormal diastolic Ca2 þ leak in the heart? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 4. Ryanodine receptors as leak channels in smooth muscle cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.1. Role of RyRs in excitation–contraction coupling in smooth muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.2. Smooth muscle expresses all three RYRs in the same cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 4.3. Leak activity of RyRs in smooth muscle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 5. RyRs as leak channels in neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5.1. Ca2 þ release by neuronal RyRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 5.2. RyRs as leak channels in neurodegenerative disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

n Corresponding author. Departamento de Bioquímica, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, Apdo. Postal 14-740, México D. F. 0700, México. Tel.: þ 52 55 5747 3950 E-mail address: [email protected] (A. Guerrero-Hernández).

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.11.016

Please cite this article as: Guerrero-Hernández, A., et al., Ryanodine receptors as leak channels. Eur J Pharmacol (2013), http://dx.doi. org/10.1016/j.ejphar.2013.11.016i

A. Guerrero-Hernández et al. / European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

1. Introduction Ryanodine receptor (RyR) is a Ca2 þ permeable non-selective cation channel that releases Ca2 þ stored in the sarco-endoplasmic reticulum of excitable and non-excitable cells. This ion channel is a tetramer and each subunit contains approximately 5000 aa, so one single ion channel weights around 2 M Da. However, the part of RyRs forming the ion pore is located at the carboxy terminus and represents approximately 10% of the protein. In mammals, there are three different genes encoding for RyR1, RyR2 and RyR3 isoforms. RyR1 is abundantly expressed in skeletal muscle, particularly in fast twitch muscle; RyR2 is greatly expressed in heart cells and RyR3 was identified as an inducible protein in lung epithelial cells in response to TGF-β although it is constitutively expressed in brain, skeletal and smooth muscles and in nonexcitable cells. Both RyR1 and RyR2 play a central role in the excitation contraction coupling in striated muscles. All three isoforms of RyRs are expressed in smooth muscle cells and yet, they play a minor role in excitation–contraction coupling in this type of muscle (Guerrero-Hernández et al., 2002; Iino et al., 1988). RyRs are also large ion pores, with an estimated single unitary current of 0.5 pA under quasi physiological conditions (Mejía-Alvarez et al., 1999), this is quite large, particularly for a Ca2 þ permeable channel. It is clear then that the number and the activity of these large ion channels need to be under tight control. Moreover, it has been found that cells can express truncated RyRs comprised by the ion channel part only (Lee et al., 2002; Takeshima et al., 1993); although we do not know their role in cell physiology. A number of reviews on biochemical, physiological and genomic characteristics of RyRs have been published (Fill and Copello, 2002; Fleischer, 2008; Zalk et al., 2007). This review focuses on the physiological and non-physiological conditions that turn RyRs from tightly regulated channels into leaky pathways, which in turn alter cell physiology and associate with different pathological states both in muscle and non-muscle cells.

2. RyR as leak channels in skeletal muscle 2.1. RyR1s are not “intrinsically” leaky The main role of RyR in striated muscle is to couple excitation with contraction by releasing Ca2 þ from SR. However, skeletal and cardiac muscles express different subunits of either voltage-gated Ca2 þ channels or ryanodine receptors (CaV1.1 and RyR1 in skeletal vs. CaV1.2 and RyR2 in cardiac). So, this leads to distinct excitationcontraction coupling (ECC) mechanisms. In skeletal muscle, a direct physical interaction between CaV1.1 and RyR1 rapidly transduces electrical depolarization of the plasma membrane in activation of RyR1 and the ensued SR Ca2 þ release (known as voltage-gated Ca2 þ release). In contrast, in cardiac muscle ECC depends on Ca2 þ entry via CaV1.2 which in turn, binds to and activates RyR2, resulting in SR Ca2 þ release (for reviews see Bannister (2007); Dulhunty (2006)). Another interesting difference is that in contrast to cardiac muscle localized Ca2 þ release events or sparks (which might be seen as manifestations of Ca2 þ leak through RyRs) are rarely seen in mammalian skeletal muscle. In fact, in this tissue a leaky behavior of RyR1 is more commonly inferred from a reduction in steady-state levels of SR Ca2 þ , which exclusively occurs in pathological situations. The absence of spontaneous opening of RyR1s

may be due to an inhibitory action of CaV1.1 (Eltit et al., 2011; Zhou et al., 2006b). Additionally, it has been proposed that the Cterminal region of RyR1s makes them poorly sensitive to activation by luminal SR Ca2 þ as opposed to RyR2s; (Kong et al., 2007). 2.2. Leak in RyR1 mutant channels Mutations in the gene encoding RyR1 have been linked to various human diseases (for a recent update see Løseth et al. (2013)). Malignant hyperthermia susceptibility (MH) and central core disease (CCD) were the first in being associated with a mutated RyR1 gene, so these modifications are well characterized (Fujii et al., 1991; Quane et al., 1993; Zhang et al., 1993). CCD is a congenital myopathy that leads to lower limb skeletal muscle weakness, atrophy, hypotonia, and skeletal deformities. The diagnosis is based on histopathological observation of “central cores”, that are amorphous areas devoid of oxidative enzymatic activity and mitochondria. CCD patients often test positive for MH as well, which is a pharmacologically triggered and life threatening disease. The MH episodes are characterized by sudden rise in body temperature, hyper-metabolism, acidosis, tachycardia and skeletal muscle rigidity. In general, they are triggered by exposure to inhalant anesthetics and muscle relaxants, but can also be elicited by high temperature and stress. Dantrolene, which is thought to act by inhibiting the activity of RyR1s, represents currently the only antidote and is very effective in preventing the attacks (if applied intravenous promptly), when the initial symptoms appear (Betzenhauser and Marks, 2010; Lanner et al., 2010). Functional studies of disease-linked mutations in RyR1 have resulted in models that attempt to explain the most conspicuous symptoms (muscle rigidity in MH and weakness in CCD) (i) Mutations located near to the C-terminal that leave intact gating properties but impair Ca2 þ conductance of RyR1 (Avila and Dirksen, 2001; Avila et al., 2003b; Zvaritch et al., 2007). The reduced Ca2 þ conductance is thought to result only in CCD (CCD-only mutants), since it does not involve increased sensitivity to activation by either pharmacological compounds or CaV1.1. However, it causes decreased amplitude of Ca2 þ transients from a normal SR Ca2 þ content, likely contributing to muscle weakness in CCD. The corresponding functional phenotype, termed “EC uncoupling” (Avila et al., 2001), results also from mutations that promote a reduction in ECC units due to a drastic decline in RyR1 protein content; (Zhou et al., 2006a). (ii) Mutations that produce an extremely leaky behavior of RyR1 and therefore decrease the SR Ca2 þ content and increase cytosolic Ca2 þ concentration (“decompensated leak”; (Avila and Dirksen, 2001; Tong et al., 1999)). These alterations in turn result in reduced amplitude of Ca2 þ transients elicited by voltage activation of CaV1.1, which might contribute to muscle weakness in CCD. Individuals expressing these mutant proteins are also MH susceptible (i.e. MHþ CCD mutants), probably due to an exacerbated response to MH triggering agents, which mobilizes Ca2 þ excessively even from a partially depleted store (Dirksen and Avila, 2004). (iii) Mutations that result in defective RyR1s that are moderately leaky and do not alter the steady-state distribution of Ca2 þ across the SR lumen and cytoplasm (“compensated leak”; (Dirksen and Avila, 2004; Tong et al., 1999). These mutants do



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CGRP

Ca2+-impermeable SERCA gene expression RyR1 (I4897T) (down-regulation)

CGRP1

Inefficient SR Ca2+ uptake (low activity of SR Ca 2+ pump)

cAMP PKA pPLB-Ser 16

SR depletion

Normal SR Ca2+ content

Enhanced SR Ca2+ uptake

SERCA

Inefficient SR Ca2+ uptake (uncompensated leak) Overactive RyR1 (Y523S)

SERCA gene expression (up-regulation)

Fig. 1. Mechanisms involved in SR depletion by CCD mutants and its adjustment by CGRP. Transfecting C2C12 myoblasts with leaky CCD mutants (Y523S, Overactive RyR1) results in higher transcriptional activity of the SERCA2a gene; which on the other hand, is down-regulated by Ca2 þ -impermeable CCD mutants (I4897T). As a result, the SERCA gene expression is increased and decreased, respectively. In the case of Y523S, the leak predominates over higher SERCA activity (Uncompensated leak); and thereby in both cases (leaky and Ca2 þ -impermeable) a reduced net efficiency of SR Ca2 þ uptake is observed. This explains why both leaky and Ca2 þ -impermeable RyR1 mutants result in decreased levels of releasable SR Ca2 þ content (SR depletion). The diagram also illustrates a signaling pathway whereby the calcitonin gene-related peptide, or CGRP, restores SR Ca2 þ load in myotubes expressing each of the CCD RyR1 mutants. It is about the classical activation of G-protein coupled receptors (GPCR, in particular CGRP1), which increases levels of cAMP, activates PKA, promotes phosphorylation of phospholamban (p-PLB-Ser 16), activates SERCA, enhances SR Ca2 þ uptake, and finally restore SR Ca2 þ content. Although not shown, through these effects CGRP is able to restore normal levels of voltage-gated Ca2 þ release. Scheme based on Avila et al. (2007) and Vega et al. (2011).

not alter the amplitude of voltage-gated Ca2 þ release and thus should not produce muscle weakness, which conforms to its association with only MH (i.e. MH-only mutants).

Although these mechanisms have been accepted in general, the presence of contradictory experimental results and unsolved questions call for future work. An important question that remains open is: what is the primary defect responsible for increased Ca2 þ fluxes in MH mutants? At least three fundamentally different defects have been proposed: (i) alterations in the sensitivity to cytosolic modulators (Mickelson and Louis, 1996), (ii) disruption of inter-domain interactions (termed “unzipping”; (Murayama et al., 2007)), and (iii) enhanced sensitivity to luminal Ca2 þ activation (Jiang et al., 2008). Additionally, while certain results show that MH-only mutants leave intact the steady-state level of cytosolic Ca2 þ (Censier et al., 1998; Dirksen and Avila, 2004; Iaizzo et al., 1988; Tong et al., 1999), others have found this to be increased (Feng et al., 2011; López et al., 1985; Wehner et al., 2002). The reasons for this discrepancy are not evident. It is possible that the following differences in experimental procedures may have contributed: the use of Ca2 þ sensitive fluorescent dyes vs. Ca2 þ -sensitive electrodes, intranuclear microinjection of cDNAs vs. viral transfection, and finally transient expression systems vs. cells from knock-in animals. As discussed by Avila and Dirksen (2001), an increase in steady-state levels of cytosolic Ca2 þ should be the consequence of changes in the activity of sarcolemmal Ca2 þ transport mechanisms. This represents an additional degree of freedom that may possible contribute to explain the above-mentioned conflicting reports. In fact, it has been suggested (Eltit et al., 2013) that overexpression of Ca2 þ entry channels (TRPC3 and TRPC6) induced by an MH RyR1 mutant represents the basis for elevated [Ca2 þ ] which already had been associated with enhanced Ca2 þ entry in both MH and CCD mutants; (Duke et al., 2010; Treves et al., 2011; Yang et al., 2007). Clearly, these data move the spotlight to a different scenario; that is, the number and variability of secondary or compensatory effects that need to be considered. For instance, see Tong et al. (1999), Treves et al. (2011) and Vega et al. (2011).

Accordingly, secondary effects might explain contradictory results obtained with RyR1 mutants of defective Ca2 þ conductance (heretofore called “Ca2 þ -impermeable”), which show either decrease (Brini et al., 2005; Lynch et al., 1999; Tilgen et al., 2001; Vega et al., 2011; Zorzato et al., 2003) or exert no effect (Avila et al., 2001, 2003b; Lefebvre et al., 2013; Loy et al., 2011) on SR Ca2 þ content. This is explained next. Tong et al. (1999) showed that leaky RyR1 mutants increase the expression of SERCA2b in HEK-293 cells, suggesting a compensatory mechanism that enhances ER Ca2 þ storage capacity. In support of this, Vega et al. (2011) found that leaky and Ca2 þ impermeable RyR1 mutants exert opposite effects in the transcriptional activity of the SERCA2a gene, stimulation by the former and inhibition by the latter. Thus, the puzzling observation that Ca2 þ impermeable RyR1 mutants often result in depleted SR Ca2 þ store may simply reflect a reverse version of the aforementioned compensatory mechanism (Fig. 1, left). But then the question is why these mutants often, but not always, decrease SERCA expression and SR load? Although this is not clear yet; it is possible that depletion be associated with the “reverse compensatory mechanism” (i.e., lower SERCA expression and reduced SR Ca2 þ storage capacity) occurring only in the earliest stages of development (Vega et al., 2011). In keeping with this, adult muscle fibers expressing Ca2 þ -impermeable RyR1 mutants exhibit unaltered levels of SERCA1 immunocytochemistry (Lefebvre et al., 2013). 2.3. Leak in RyR1 as a consequence of MH mutations in CaV1.1 If CaV1.1 contributes to keeping low activity of RyR1 (as suggested by Zhou et al. (2006b) and Eltit et al. (2011); then one may expect that mutations in this voltage-gated channel would interfere with such inhibitory action. In fact, evidence exists in favor of this expectation. Although most of the MH mutations lie in the gene encoding RyR1, four mutations have been located in CaV1.1 (R174W, (Carpenter et al., 2009); R1086H, (Monnier et al., 1997); R1086C, (Toppin et al., 2010); and T1354S, (Pirone et al., 2010)). Except for R1086C, all of them have been functionally characterized (Eltit et al., 2012; Pirone et al., 2010; Weiss et al., 2004). Thus far, no major alterations have been observed on the


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ability of CaV1.1 to act as the voltage sensor for ECC. Instead, an enhanced sensitivity of RyR1 to pharmacological activation has been commonly observed (which contributes to explain the MH phenotype). In regard to possible alterations in Ca2 þ -conductance of CaV1.1, diverse observations exist. Weiss et al. (2004) and Pirone et al. (2010) found mild alterations in both maximal Ca2 þ conductance ( 30% reduction, R1086H) and Ca2 þ -current activation kinetics ( 35% faster, T1354S). In contrast, Eltit et al. (2012) reported an astonishing ablation of Ca2 þ -conductance, in addition to an increase in cytosolic Ca2 þ and a depleted SR Ca2 þ content (R174W). It will be interesting to investigate whether the increase in cytosolic Ca2 þ by this MH CaV1.1 mutant correlates also with an enhanced expression of TRPC channels, as previously reported for an MH RyR1 mutant (Eltit et al., 2013). 2.4. Novel therapeutic strategies for MH and CCD As we have mentioned above, dantrolene is very effective in controlling MH crisis, due to its well-known blocking effect on SR Ca2þ release (which on the other hand, may be due to a possible interaction with the CaV1.1-RyR1 complex; (Bannister, 2013)). Conversely, there are not currently effective therapies for CCD, only palliative procedures. Results from the following studies suggest that an effective treatment for CCD patients may be developed in the near future. Azumolene, an analog of dantrolene restores the steady-state level of cytoplasmic Ca2 þ in myotubes expressing a leaky MHþ CCD mutant (Y523S; (Lyfenko et al., 2004)). Additionally, the antioxidant Nacetylcysteine (NAC) protects against mitochondrial damage and the decline in force generation in a knock-in mice for Y523S RyR1 (Durham et al., 2008). Moreover, in this model, antioxidants and nitric oxide synthase inhibitors prevent both temperature-dependent increases in cytoplasmic Ca2þ and muscle weakness (Durham et al., 2008). These results are in line with the observation that Ca2þ leak via this mutated RyR1 results in oxidative stress, which promotes Snitrosylation of the release channels as well as higher Ca2þ release (in a vicious feed forward cycle, fueled by high temperature, which results in sudden death; Durham et al., 2008). The beneficial actions of NAC are also observed in human myotubes from patients with RyR1 mutations (Dowling et al., 2012). However, thus far only AICAR (5-aminoimidazole-4-carboxamide-1-βD-ribofuranoside, a compound that activates AMP-activated protein kinase or AMPK) has been shown to prevent heat-induced death (in a mice model; (Lanner et al., 2012)). Apparently, AICAR exert this beneficial effect not through targeting of AMPK, but by directly inhibiting the leak behavior of RyR1 mutants (probably by binding to a yet unknown nucleotide-binding domain; Lanner et al., 2012). Gene therapy approaches are also promising. In particular, the use of siRNA technology; which has been shown to effectively produce silencing of mutant allele-specific gene in muscle fibers from heterozygous knock-in mice for CCD. More precisely, in these mice preferential knock-out of the Y523S allele restores both caffeine-sensitivity and resting cytosolic Ca2 þ (Loy et al., 2012). An agonist of G-protein coupled receptors, termed calcitoningene related peptide or CGRP, may be also promising. Thus far, it represents the only compound (or maneuver) that restores voltage-gated Ca2 þ release. This was discovered in myotubes from the C2C12 cell line, expressing CCD mutants that are hallmarks of the leaky and EC uncoupling mechanisms (i.e., Y523S and I4897T; Vega et al., 2011). Basically, in myotubes expressing CCD mutants this compound was able to not only restore the SR Ca2 þ load, but also drastically enhanced the ECC gain. These actions are due to an increase in SERCA activity, provoked by PKA-dependent phosphorylation of phospholamban (Fig. 1, right). It could be argued that these beneficial effects were observed in vitro and in developing myotubes, and thus they may not necessarily occur in CCD

patients. However, Messina et al. (2004), showed that oral administration of a β-2 agonist (salbutamol) results in enhancement of muscle strength in children with CCD, following three to six weeks of treatment. Thus, there is strong evidence that activation of GPCRs is beneficial to CCD patients. 2.5. FKBP12-deficient RyR1s FK506 binding protein (FKBP12, also known as calstabin) is an accessory protein that binds to each of the four monomers of RyRs (Chelu et al., 2004). At the single channel level, RyR1s that are depleted of FKBP12 exhibit both high open probability and open primarily to subconductance states (Ahern et al., 1997; Brillantes et al., 1994; Gaburjakova et al., 2001; Shou et al., 1998). It has been proposed that PKA-dependent phosphorylation of RyR1 promotes removal of FKBP12, which in turn may result in SR Ca2 þ leak and store depletion (Betzenhauser and Marks, 2010). However, the group of Meisnner reported that mutations which disrupt PKA-dependent phosphorylation in either RyR1 or RyR2 provoke no major changes in the function of these channels. More specifically; no changes were found in regard to: FKBP12 binding, single channel conductance and activity, ryanodine binding, Ca2 þ dependence under both oxidizing and reducing conditions, and size of caffeine-sensitive Ca2 þ stores (Stange et al., 2003). Moreover, it has been reported that loss of FKBP12 binding by different approaches, such as: pharmacological intervention (Avila and Dirksen, 2005; Lamb and Stephenson, 1996), mutations in RyR1 (Avila et al., 2003a), or transgenic ablation of the accessory protein (Tang et al., 2004), all result in normal levels of both releasable SR Ca2þ content and resting cytoplasmic Ca2þ . Nevertheless, these studies did show a disruptive effect on depolarization-induced contraction and voltage-gated SR Ca2 þ release. Thus, while the FKBP12-RyR1 interaction does not seem to be required for stabilizing RyR1s in a closed state, it is important to preserve normal levels of ECC gain (Avila et al., 2003a; Avila and Dirksen, 2005). 3. RyR as Ca2 þ leak channels in cardiac cells Type 2 Ryanodine receptors (RyR2) are the main intracellular ion channels that release the Ca2 þ needed for contraction of cardiac cells during excitation-contraction coupling (ECC). Cardiac ECC is initiated by an action potential that triggers a small Ca2 þ influx (ICa) via L-type Ca2 þ channels (Cav1.2) that is not enough to activate the contractile machinery but it triggers a much larger Ca2 þ release via RyR2 by Ca2 þ induced Ca2 þ release (CICR) mechanism, enabling cell contraction (Bers, 2002; Fabiato, 1983; Fabiato and Fabiato, 1979). Thus, CICR is an amplification mechanism that should have a high degree of positive feedback and accordingly should behave in an all-or-none fashion. Nevertheless, systolic Ca2 þ release is not an all-or-none response and a small Ca2 þ influx elicits a graded Ca2 þ release indicating that CICR is under local control of trigger signal and that the activity of RyR2 is modulated by both cytoplasmic and luminal calcium concentrations ([Ca2 þ ]i and [Ca2 þ ]SR, respectively), and affected by phosphorylation and some other regulatory mechanisms, such as s-nitrosylation, redox state, and carbonylation (Bers, 2004; Capes et al., 2011; Györke and Terentyev, 2008; Laver, 2007; Shao et al., 2007). Altered RyR2-mediated Ca2 þ release occurs at both systole and diastole under pathological conditions and underlies cardiac dysfunction and arrhythmogenesis (Capes et al., 2011; FernandezVelasco et al., 2012; Kushnir and Marks, 2010). Particularly, RyR2-mediated Ca2 þ efflux from the sarcoplasmic reticulum (SR) at resting or quiescent condition is known as diastolic Ca2 þ leak (Sobie et al., 2006) and has been found in isolated cardiomyocytes mainly in the form of: (1) automatic, self-



propagating Ca2 þ waves that may lead to aftercontractions (Belevych et al., 2011; Wier et al., 1987); (2) spontaneous Ca2 þ sparks due to the coordinated opening of an unknown number of RyR2 within a cluster either in quiescent conditions or between electrical stimulations (Biesmans et al., 2011; Cheng and Lederer, 2008; Fernandez-Velasco et al., 2012); and (3) experimentally undetectable by confocal microscopy (“invisible”) or quarky Ca2 þ release that underlies single RyR2 activity (Brochet et al., 2011; Sobie et al., 2006). Since inositol 1,4,5-trisphosphate receptor (IP3R) is also expressed in cardiac cells, its role, if any, in mediating diastolic Ca2 þ leak remains elusive (Sobie et al., 2006), though one possibility is that IP3Rs could participate in the Ca2 þ leak insensitive to ruthenium red (Bovo et al., 2011). It is thought that RyR2-mediated Ca2 þ leak is present in healthy cardiac cells and together with the SR Ca2 þ ATPase (SERCA)mediated Ca2 þ uptake, determines the extent of SR Ca2 þ load and therefore the amount of releasable Ca2 þ from the SR at each cardiac cycle (Negretti et al., 1993; Zima et al., 2010). However, abnormal RyR2-mediated Ca2 þ leak in cardiomyocytes is associated with some cardiac diseases and has profound effects on heart function as has been observed in both acquired (e.g.: heart failure, diabetic cardiomyopathy and atrial fibrillation) and inherited (e.g.: catecholaminergic polymorphic ventricular tachycardia and arrhythmogenic right ventricular cardiomyopathy type 2) arrhythmogenic disorders. One pathological consequence of an augmented RyR2 Ca2 þ leakage during diastole is that would activate the Na þ /Ca2 þ exchanger (NCX) generating a net inward current that underlies delayed after depolarization (DADs), would trigger RyR2 activation and undesired aftercontractions (Venetucci et al., 2007). Since RyR2 activity is regulated by [Ca2 þ ] SR, those conditions that produce SR Ca2 þ overload, would promote that RyR2s from one cluster ignite neighboring clusters by a fire-diffuse-fire mechanism to trigger pro-arrhythmic regenerative CICR-driven Ca 2 þ waves (Keizer et al., 1998). However, an increase of diastolic Ca2 þ leak cannot last forever, cardiac cells contain powerful autoregulatory mechanisms for controlling abnormally active RyR2 channels in long-term. Accordingly, higher RyR2 activity would produce greater Ca2 þ extrusion by NCX, which in turn results in diminished SR Ca 2 þ load and reduction of Ca 2 þ availability to be released at subsequent beats, so a leaky RyR does not necessary produce Ca2 þ waves but eventually would lead to a reduction in SR Ca2 þ content (Eisner et al., 2009). Conversely, decreasing RyR openings during diastole increases SR Ca 2 þ level and the amplitude of Ca2 þ transients (Gómez et al., 2004; Prestle et al., 2001) leading to SR Ca 2 þ overload. Thus, the importance of understanding RyR2mediated Ca2 þ leak regulation and its functional impact in the heart warrant further work. 3.1. Physiological role of RyR2-mediated Ca2 þ leak in the heart The first clear evidence that RyR2 could mediate SR Ca2 þ leak in heart came from the observation that ryanodine, applied at nanomolar concentrations to rat ventricle myocytes, caused an important Ca 2 þ release that in quiescent – diastolic – condition rapidly depleted SR Ca2 þ stores (Hansford and Lakatta, 1987). This effect of ryanodine was explained by its property of locking RyR2 in the open state resulting in a leaky SR and helped to understand its toxic actions, since in its presence the SR was unable to store Ca2 þ so muscle would not relax (Fleischer et al., 1985; Urthaler et al., 1989). This SR leakiness was overcome by combining electrical stimulation and beta-adrenergic agonists, which allowed for the replenishment of SR Ca2 þ content (duBell et al., 1993). Later on, Yamazawa and collaborators reported that RyR2 expressed in

5

cultured skeletal muscle cells evoked depolarization-independent spontaneous Ca2 þ sparks and waves supporting the idea that RyR2 indeed opens at resting [Ca2 þ ]i and could mediate diastolic Ca2 þ leak (Yamazawa et al., 1996). Apparently, the main role of RyR2 in embryonic heart is to function as a Ca2 þ leak channel to maintain the normal luminal Ca2 þ level in the developing SR. This proposition was based on the observation that Ca2 þ gradually overloaded SR stores, induced vacuole formation of the developing SR, mitochondria abnormalities, irregular Ca2 þ homeostasis, and overall dysfunction of embryonic RyR2-lacking cardiac cells (Takeshima et al., 1998). These data suggest that RyR2s by being active at diastole in response to elevation of luminal Ca2 þ levels became safety valves in the developing Ca2 þ stores. Then, diastolic Ca2 þ leak from SR of cardiomyocytes is considered to play a physiological role as this leak helped to maintain SR Ca2 þ content at certain level stabilizing the activity of RyR2 for the next beat (Takeshima et al., 1998). However, RyR-mediated SR Ca2 þ leak during diastole is not exclusive of the developing heart, it also participates in the regulation of luminal SR Ca2 þ levels in adult heart, and in modifying the amplitude of Ca2 þ transients (Negretti et al., 1993; Zima et al., 2010). Experimental data from normal cardiomyocytes have shown that elevated SR Ca2 þ load increases the frequency of Ca2 þ sparks while the opposite decreases the frequency of this type of leak (Lukyanenko et al., 2001); hence, RyR2s regulate Ca2 þ loading of SR stores through spark-mediated Ca2 þ leak.

3.2. Abnormal RyR2-mediated Ca2 þ leak in cardiac diseases The participation of an abnormal SR Ca2 þ leak in muscle dysfunction was initially proposed by Marks′ group, when reported that the immunosuppressant drugs FK506 and rapamicin, increased skeletal RyR activity (Brillantes et al., 1994; Marks, 1996). The same group also showed that RyR2 channels from human heart failure (HF) hearts became abnormally leaky due to PKAmediated hyperphosphorylation at residue Ser2808, which induces dissociation of the tightly-bound protein FKBP12.6 from RyR2 resulting in higher activity by increased sensitivity to Ca2þ (Marx et al., 2000). However, this scenario has not always being supported (Capes et al., 2011; Eschenhagen, 2010). Moreover, a lower instead of higher [Ca2þ ]SR is found in HF models with propensity to develop cardiac arrhythmias. This is rather paradoxical because increased sparkmediated Ca2 þ leak is found at diminished SR Ca2þ load, revealing a gain-of-function RyR2 (Belevych et al., 2007; Fernandez-Velasco et al., 2012; Terentyev et al., 2008). Increased SR Ca2 þ leak via RyR2 has also been found in several pathological conditions that do not necessarily involve augmented phosphorylation of RyR2 or SR Ca2 þ overload; for instance, in experimental models of chronic HF (Terentyev et al., 2008); in the inherited dominant CPVT disorder (Fernández-Velasco et al., 2009); in aldosterone-treated cardiac cells and human mineralocorticoid receptor overexpressing hearts (Gómez et al., 2009), in type-1 diabetic ventricular myocytes (Tian et al., 2011), and in those cells adjacent to myocardial infarction (Biesmans et al., 2011) just to mention a few. On the contrary, several heart diseases are associated with a reduction in RyR2 activity, so spark-mediated Ca2 þ leak is diminished. Examples are myocardial dysfunctions associated with septic shock (Patel et al., 2000) type-2 diabetes (Pereira et al., 2006) and metabolic syndrome (Barrera-Lechuga et al., 2010). Next, it will be reviewed the participation of RyR2mediated Ca2 þ leak in some acquired and inherited cardiac diseases.


6


3.2.1. Leaky RyR2 channels in heart failure HF remains as a large health problem in Western countries. The molecular basis of HF appear to be due to altered Ca2 þ handling by the presence of “leaky” RyR2 (Kushnir and Marks, 2010; Marx et al., 2000). It has been proposed that the amount of FKBP12.6 bound to the RyR2 in HF is diminished due to PKA-mediated hyperphosphorylation of Ser2808 (the term “hyperphosphorylation” refers to RyR2 with 3 or 4 out of 4 monomers that are chronically phosphorylated (Kushnir and Marks, 2010)) promoting elevated activity of RyR2, increased diastolic Ca2 þ leak and triggered arrhythmias (Marx et al., 2000). Part of the dysfunction is the consequence of exacerbated ß-adrenergic receptor activity, that has been found in HF patients, and that should lead to increased RyR phosphorylation. However, there are also data showing that PKA-mediated RyR2 phosphorylation has little functional relevance for RyR-mediated Ca2 þ leak when the level of [Ca2 þ ]SR remains constant (Li et al., 2002) arguing against PKAinduced Ser2808 hyperphosphorylation being the major cause of abnormal Ca2 þ handling in HF hearts. In fact, different research groups have reported that PKA phosphorylation of RyR2 at Ser2808 neither dissociated FKBP12.6 nor substantially modified channel gating (Capes et al., 2011; Stange et al., 2003). Moreover, cAMP does indeed increase Ca2 þ spark frequency in streptolysinO-permeabilized ventricular myocytes but was due to the ensuing increase in SR Ca2 þ load more than an intrinsic effect on the activity of RyR2 (Li et al., 2002). Another issue that needs to be considered is the redox state of RyR2 that could account for the increased Ca2 þ leak in ventricular cells from chronic HF hearts (Terentyev et al., 2008). RyR2s undergo phosphorylation during ß-adrenergic stimulation by protein kinase A (PKA) and Ca2 þ /calmodulin-dependent protein kinase II (CaMKII). However, the functional effects of these modifications remain being the subject of intense debate (Capes et al., 2011; Li et al., 2002). RyR2 contains different phosphorylation sites that, depending on their state of phosphorylation may induce distinctive behavior of this receptor. Actually, three RyR2phosphorylation out of several sites have been shown physiological relevance: Ser 2808 that is directly phosphorylated by both PKA and CaMKII, although some researchers suggest that is only a PKA site (Benkusky et al., 2007; Wehrens et al., 2006; Witcher et al., 1991); Ser2814 that has been argued to be exclusively phosphorylated by CaMKII (Pereira et al., 2013) and Ser2030 that is uniquely phosphorylated by PKA (Xiao et al., 2006). However, there is no consensus which of these, if any, is responsible for promoting the leaky phenotype. This affirmation is particularly true for Ser2808 because this site has found no augmented phosphorylation in several experimental models of HF (Benkusky et al., 2007; Jiang et al., 2002). Other data have brought to light S2814 as a potential generator of RyR2-mediated calcium leak that leads to arrhythmias and exacerbates other forms of HF (Valdivia, 2012). Under ß-adrenergic stimulation, CaMKII-dependent RyR2 phosphorylation resulted in higher RyR activity by involvement of Epac (exchange protein activated by cAMP). Particularly, Epac 2 isoform increased RyR2mediated diastolic Ca2 þ leak via CaMKIIδ activation and Ser2814 phosphorylation, reducing SR Ca2 þ load (Pereira et al., 2013; Pereira et al., 2007). Hence evidence is piling up that CaMKII, and not PKA, increases diastolic RyR2-mediated Ca2 þ leak and arrhythmogenic activity in HF.

there is a decreased number of functional RyR2 channels and diminished SR Ca2 þ load probably by decreased activity of SERCA pump (Barrera-Lechuga et al., 2010; Pereira et al., 2006), tough this is not universal for all experimental models of diabetes (Shao et al., 2012; Tian et al., 2011), so more exhaustive studies are needed. 3.2.3. Inherited arrhythmias and leaky RyR2 channels Some types of arrhythmias are attributed to automatic Ca2 þ waves due to increased activity of RyR channels. Point mutations in the gene encoding RyR2 channels are associated with arrhythmogenic syndromes such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and arrhythmogenic right ventricular cardiomyopathy type 2 (ARDV) (Priori et al., 2001, 2002). The dominant inherited CPVT phenotype is characterized by syncope and sudden death of children and young adults, which have been subjected to physical stress or increased ß-adrenergic stimulation. One of the first RyR2 mutations identified that is related to this disorder was R4497C (Priori et al., 2001) and a knock-in mouse model harboring this mutation was generated (Cerrone et al., 2005). In RyR2R4497C cardiomyocytes, diastolic Ca2 þ leak is found in the form of increased Ca2 þ spark frequency in quiescent conditions which is exacerbated after isoproterenol exposure, promoting automatic and spontaneous Ca2 þ waves or triggered activity in the absence of SR Ca2 þ overload (Fernández-Velasco et al., 2009). RyR2R4497C that are expressed in HEK cells exhibit increased activity and sensitivity to luminal Ca2 þ , which apparently reduced the threshold for store-overload-induced Ca2 þ release (SOICR)(Jiang et al., 2004). Interestingly, RyR2R4497C cardiomyocytes have reduced caffeine-induced Ca2 þ transients and in vitro experiments have shown that RyR2R4497C channels have increased sensitivity to cytoplasmic Ca2 þ which might help to explain why in an environment with decreased SR Ca2 þ load the RyR2R4497C channel is more eager to be activated, hence showing a gain-of-function phenomenon (Fernandez-Velasco et al., 2012; Fernández-Velasco et al., 2009). Another point mutation of RyR2 that is linked to CPVT and highly arrhythmogenic is RyR2V2475F. The recombinant RyR2V2475F channel displays augmented cytoplasmic Ca2 þ sensitivity, altered luminal Ca2 þ regulation and augmented response to PKA phosphorylation; these alterations produce a significant diastolic Ca2 þ leak in cardiomyocytes expressing the mutated channel (Loaiza et al., 2013). Almost all well-characterized RyR2 point mutations related to CPVT phenotype make the channel more active even at reduced SR Ca2 þ load displaying a gain-of-function phenomenon (Fernández-Velasco et al., 2011). Finally, ARVD2 constitutes a rare form of arrhythmogenic right ventricular dysplasia and presents exercise-induced bi-directional tachycardia very similar to those of CPVT (http://www.fsm.it/ cardmoc/). ARVD2-linked RyR2 mutation (N2386I and R176Q/ T2504M) induced gain-of-function of RyR2 channels. However, it was only L433P mutation that was found associated with loss-offunction channels (George et al., 2005). In an acquired form of ARVD in boxer dogs, decreased expression of FKBP12.6 was associated with a significantly increased activity of RyR2 channels suggesting an augmented Ca2 þ leak (Oyama et al., 2008). However, data about RyR2-mediated diastolic Ca2 þ leak in this animal model is still lacking. 3.3. Is the “stabilization” of RyR a way to stop abnormal diastolic Ca2 þ leak in the heart?

3.2.2. RyR2 leakiness in diabetic cardiomyopathy Apparently not all RyR2-mediated Ca2 þ leak alterations are due to elevated activity of RyR2 channels. For instance, spark-mediated Ca2 þ leak is reduced in myocardial dysfunction associated with type-2 diabetes and sugar-induced metabolic syndrome, because

Since the idea is that abnormally active or leaky RyR2s contribute to the pathological manifestation of both acquired and inherited cardiac diseases, several groups have developed therapeutic strategies for reducing the activity of leaky RyRs



7

Table 1 Main pharmacological tools to control abnormal RyR2-mediated SR Ca2 þ leak in heart; d, dog; g, guinea pig; h, human; m, mouse; rb, rabbit; rt, rat. Heart model

Key findings

Reference

Metoprolol ß-blocker

Heart failure (rb)

Z et al. (2012)

Carvediol

Dantrolene Hydantoin derivative

Pacing-induced failing heart (d) Pacing-induced failing heart (d)

Prevents SR Ca2 þ leak by diminishing both PKA-and CaMKII-dependent phosphorylation of RyR2 Prevents SR Ca2 þ leak by modifying RyR2 redox state

JTV-519

Heterozygous RyR2R2474S cardiomyocytes (m) Pressure-induced HF cardiomyocytes (rb) Heterologous RyRP2328S (h)

Drug

Type

ß-blocker

1,4-benzothiazepine derivative

FKBP12.6

/þ

cardiomyocytes (m)

Intact cardiomyocytes (rt) Ouabain-treated cardiomyocytes (m) Flecainide Class Ic antiarrhythmic Csq2 / cardiomyocytes (m) agent (fluor-etoxybenzamide) Intact cardiomyocytes (rt) Polydatin

Resveratrol glucoside

Burn-generated cardiac dysfunction (rt)

Suppresses RyR2 domain unzipping and spark-mediated Ca

2þ

Decreases Ca2 þ spark frequency after isoproterenol treatment Decreases Ca2 þ spark frequency and increased SR Ca2 þ load Diminishes the activity of PKA-phosphorylated RyR channels and reduces the dissociation of FKPB12.6 Decreases aberrant Ca2 þ oscillations Suppresses SOICR irrespective of FKBP12.6 association Decreases SR Ca2 þ leak irrespective of S2814 phosphorylation Reduces spontaneous SR Ca2 þ release events Reduces spark mass, but increases spark frequency Hence, spark-mediated Ca2 þ leak is unchanged Reduces spark and wave frequency due to INa reduction rather than direct RyR inhibition Corrects abnormal spark mediated Ca2 þ leak by decreasing ROS levels and restoring free thiols in RyR2

(McCauley and Wehrens, 2011). Several drugs have been used for reducing abnormal RyR2 activity with the idea of conferring protection against triggered arrhythmias through decreasing RyR2-mediated Ca2 þ leak. Such drugs are also called “RyR2 stabilizers” and a review about this topic has been published recently (McCauley and Wehrens, 2011). Table 1 summarizes the actual knowledge about the use and main outcomes of some pharmacological tools for stabilizing RyR2 activity, some of them still controversial on its mechanism of action. It is important to keep in mind that not all the diastolic Ca2 þ leak in cardiac cells is mediated by RyR2 channels, since other type of channels might be involved as well. This scenario should be considered when developing therapeutical approaches for decreasing abnormal Ca2 þ leak. For instance, it has been documented recently that phosphorylated phospholamban can construct pentameric channels that may mediate passive Ca2 þ leak from cardiac SR vesicles since the PKA-mediated Ca2 þ leak was not inhibited by ruthenium red (Aschar-Sobbi et al., 2012). Nevertheless, we cannot overlook the large amount of evidence showing that Ca2 þ leak via RyR plays a physiological role during diastole at regulating SR Ca2 þ content and avoiding SR Ca2 þ overload. Hence, whether these therapies directed to stop diastolic Ca2 þ leak by targeting RyR2 would succeed, only time will tell.

4. Ryanodine receptors as leak channels in smooth muscle cells 4.1. Role of RyRs in excitation–contraction coupling in smooth muscle Similarly to striated muscles, an increase in the [Ca2 þ ]i triggers smooth muscle cell contraction. However, smooth muscle cells are much smaller in volume than striated muscle cells, so the Ca2 þ involved in contraction can come from two main sources, the outside of the cell or from internal stores (Somlyo and Somlyo, 1994). Ca2 þ ions can enter smooth muscle cells via a large collection of Ca2 þ permeable channels such as voltage dependent

leak

Mochizuki et al. (2007) Kobayashi et al. (2009) Kobayashi et al. (2010) Maxwell et al. (2012) Lehnart et al. (2004) Lehnart et al. (2006) Hunt et al. (2007) Sacherer et al. (2012) Watanabe et al. (2009) Hilliard et al. (2010) Sikkel et al. (2013) Jiang et al. (2013)

Ca2 þ channels (VDCCs) and receptor- and second messengergated Ca2 þ channels. The main internal store that supplies Ca2 þ for contraction is the sarcoplasmic reticulum via two main release channels, IP3Rs and RyRs (Somlyo and Somlyo, 1994). Although RyRs of smooth muscle release enough Ca2 þ to induce contraction when stimulated by caffeine (Iino et al., 1988; Xu et al., 1994), it appears that their participation in agonist-induced contraction is very limited (Guerrero-Hernández et al., 2002; Iino et al., 1988; Rueda et al., 2002). Although smooth muscle cells express RyR type 2 (Xu et al., 1994) as cardiac cells do and these receptors colocalize with VDCCs in smooth muscle cells as well (Moore et al., 2004), apparently calcium-induced calcium release (CICR) process is of very low efficiency since it takes around 100 msec for VDCCs to recruit RyRs and the contribution of CICR to the [Ca2 þ ]i transient is of only 20% (Kirber et al., 2000). This can be contrasted with cardiac cells where VDCCs recruit RyR2s in only 5–10 msec and release represents up to 90% of the [Ca2 þ ]i transient (Callewaert et al., 1988; Zhou et al., 2009). For this reason CICR is called loose coupling in smooth muscle cells (Kotlikoff, 2003). Interestingly, the increase in the [Ca2 þ ]i induced by activation of stretch channels is strongly amplified by Ca2 þ release from SR, but the time frame for this process is much slower that the activation of VDCCs so the participation of second messengers activating other release channels different to RyRs cannot be discarded that easily (Kirber et al., 2000). 4.2. Smooth muscle expresses all three RYRs in the same cell Smooth muscle cells express all three RyRs isoforms in the same cell, but their role in excitation–contraction coupling in smooth muscle is very limited (Kotlikoff, 2003). Under the microscope, sarcoplasmic reticulum of smooth muscle cells has been separated arbitrarily in three different sections, the superficial or subplasmalemmal, the cytoplasmic and the perinuclear regions (Gómez-Viquez et al., 2010). SERCA pump appears to be present only in the superficial and perinuclear regions of SR using fluorescent thapsigargin (Gómez-Viquez et al., 2010). Subcellular SERCA pump distribution was corroborated with antibodies,


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additionally it was found that SERCA pump isoforms have a differential location, SERCA 2a is mainly expressed at the perinuclear region while the subplasmalemmal region is enriched in SERCA2b in pulmonary artery smooth muscle (Clark et al., 2010), whether this is the case for other smooth muscle cells, we do not know yet. The distribution of the three isoforms of RyRs has been studied in different types of smooth muscles. In all cases RyR3 is preferentially localized in the perinuclear region (Clark et al., 2010; Du et al., 2005; Fritz et al., 2007; Vaithianathan et al., 2010; Yang et al., 2005) while the subplasmalemmal region may express either RyR1 (Du et al., 2005; Fritz et al., 2007) or RyR2 (Vaithianathan et al., 2010; Yang et al., 2005) or both channels equally well (Clark et al., 2010). Interestingly, the superficial SR, unlike the perinuclear SR, showed that SERCA pump and RYRs did not always colocalize, it is not clear the reason for this nonhomogeneous distribution of these two proteins (Gómez-Viquez et al., 2010). 4.3. Leak activity of RyRs in smooth muscle Ryanodine has the property of recognizing and binding only to the open conformation of RyRs locking them in an open state (Iino et al., 1988). This characteristic makes the use of ryanodine an excellent approach to assess whether RYRs are open at resting conditions, to verify whether RyRs are acting as Ca2 þ leak channels. Incubation of small mesenteric artery strips with ryanodine showed that all RyRs go through the open state during the incubation time; but in aorta the situation was different, since only a small fraction of RyRs opened for the same incubation time (Shima and Blaustein, 1992). These data imply that RyRs can open at rest in smooth muscle under tension, but the number of opened RyRs varies depending on the type of smooth muscle. However, freshly isolated cells that are not stimulated, can be incubated for tens of minutes with ryanodine (10 μM) without decreasing the response to caffeine, suggesting that the number of active RyRs is very small under these recording conditions (Guerrero-Hernández et al., 2002; Gómez-Viquez et al., 2005; Rueda et al., 2002). Moreover, the application of IP3-producing agonist in the presence of ryanodine does not recruit RyRs since the caffeine-induced [Ca2 þ ]i response is similar to control (Guerrero-Hernández et al., 2002; Rueda et al., 2002). Elimination of internal Ca2 þ stores with the combination of ryanodine plus caffeine increases smooth muscle contraction induced by either high K þ or IP3-producing agonists (Shima and Blaustein, 1992). Collectively these data suggest that the main role of SR during agonist- or membrane depolarization-induced contraction is more as Ca2 þ buffer than source for Ca2 þ . Actually, this role of SR was discovered by vanBreemen and given the name of superficial buffer barrier (van Breemen et al., 1995). Elimination of this buffer barrier with either SERCA pump inhibitor (CPA) or prolonged incubation in ryanodine (30 μM) increased smooth muscle contractions; particularly those induced by small membrane depolarization (Janssen et al., 1999). Besides SERCA pump, RyR2 is the other essential component for the superficial buffer barrier to be functional, because RyR2 capability to increase its ion channel activity in response to the elevation of the luminal [Ca2 þ ] in the SR; a characteristic known as SOICR or Store-Overload Induced Calcium Release (Jiang et al., 2004; Kong et al., 2008). This capability appears to be responsible for the production of localized, rapid, short-lived Ca2 þ transients known as Ca2 þ sparks that are involved in the activation of clusters of BKCa channels that generate Spontaneous Transient Outward Currents or STOCs (Benham and Bolton, 1986), which in turn hyperpolarize and deactivate Ca2 þ influx via VDCCs reducing the increase of [Ca2 þ ]i and the ensuing contraction (Jaggar et al., 1998; Nelson et al., 1995). Whether this Ca2 þ release via Ca2 þ sparks represents a substantial Ca2 þ leak for SR has not been assessed in smooth muscle as it has been done in

Fig. 2. Sub plasma membrane sparks are not the Ca2 þ leak balancing SERCA pump activity. Freshly isolated smooth muscle cells loaded with Mag-fluo 4 to determine the SR Ca2 þ level (Dagnino-Acosta and Guerrero-Hernández, 2009) were voltage clamped at 0 mV and exposed to either caffeine or thapsigargin at the times indicated in the bottom trace. Spontaneous transient outward currents (STOCs, upper trace), which represent activation of Ca2 þ sparks close to the plasma membrane, were evident before the application of caffeine. Once Ca2 þ was released, STOCs disappeared and did not return even when luminal SR Ca2 þ level has gone above the resting level (dash line). Addition of thapsigargin to block SERCA pumps revealed the absence of a strong Ca2 þ leak because luminal SR Ca2 þ level was not reduced. Note that thapsigargin did inhibit SERCA pumps because the luminal SR Ca2 þ level did not return after caffeine application.

heart cells where it is clear that not all Ca2 þ leak occurs by sparks, but there is also a substantial sparkless leak via RyR2 (Zima et al., 2010). What we know is that the luminal SR Ca2 þ level can be regulated in the absence of Ca2 þ sparks at the cell surface (Fig. 2). In this case the presence of Ca2 þ sparks was indirectly assessed by recording STOCs in smooth muscle cells from guinea pig urinary bladder (Fig. 2). Note that before application of caffeine there is clear evidence of Ca2 þ sparks as STOCs, while 30–45 s after the application of caffeine the luminal Ca2 þ level has transiently climbed above resting levels (indicated by the dash line) but the STOCs have not recovered yet (Fig. 2). These data suggest that those Ca2 þ sparks that are involved in generating STOCs are not the Ca2 þ leak required by the SR to balance out the activity of SERCA pump in this particular experimental condition. Actually, it appears that the SR prevents overloading with Ca2 þ more by decreasing the activity of SERCA pump than by having a strong Ca2 þ leak activity. This can be deduced from the observation that thapsigargin application did not reveal any substantial Ca2 þ leak (Fig. 2), (Dagnino-Acosta and Guerrero-Hernández, 2009; Gómez-Viquez et al., 2003, 2005). In heart, PKA-mediated phosphorylation of RyR2s increases its channel activity as leak channels (Marx et al., 2000). Actually, it has been found that phosphorylation of RyRs by either PKA or CamKII reduces the inhibitory effect that Mg2 þ has on the ion channel activity in both RyR1 (Hain et al., 1994) and RyR2 (Hain et al., 1995; Uehara et al., 2002). We do not know the exact molecular mechanism for the increase in leak activity in conditions that promote phosphorylation, mainly because the substitution of Ser 2808 or Ser 2843 for aspartic residue to simulate a permanently phosphorylated RyR2 or RyR1 at the specified positions cannot reproduce the characteristics found for these RyRs in conditions of phosphorylation (Stange et al., 2003). So at the present time we do not know whether the reduction in Mg2 þ sensitivity is due to a direct phosphorylation of RyRs or alteration of an accessory protein. Alternatively, it has been proposed that phosphorylation at Ser 2030 increases RyRs activity by increasing SOICR sensitivity (Xiao et al., 2007; Xiao et al., 2006). Activation of PKA in smooth muscle cells



also appears to activate RyRs as leak channels because the application of forskolin to smooth muscle cells with overloaded Ca2 þ stores (by removing phospholamban) produces a smaller caffeineinduced [Ca2 þ ]i response (Wellman et al., 2001), suggesting that phosphorylation of RyRs increases its leak activity and reduces the amount of Ca2 þ stored in SR. Actually, forskolin increases the frequency of both Ca2 þ sparks and STOCs that are totally inhibited by the incubation with ryanodine (Porter et al., 1998). Whether this increase in spark frequency is enough to explain the increased Ca2 þ leakage by PKA or there is also an increase in sparkless activity of RyRs is an issue that has not been addressed in smooth muscle cells. These data suggest that although RyRs do not seem to be working as leak channels in smooth muscle, they are safety valves to avoid overloading of SR with Ca2 þ . Indeed, reducing the activity of RyRs would lead to overexcitability of smooth muscle, which in turn will develop different illness depending on the type of muscle involved. For instance in vascular smooth muscle would lead to hypertension (Jaggar et al., 1998; Porter et al., 1998) and in urinary bladder to incontinence (Herrera et al., 2000; Hotta et al., 2007; Huang et al., 2007; Meredith et al., 2004).

5. RyRs as leak channels in neurons 5.1. Ca2 þ release by neuronal RyRs The simultaneous measurement of [Ca2 þ ]i and the endoplasmic reticulum (ER) luminal [Ca2 þ ] in response to either caffeine or membrane depolarization has demonstrated that RyRs expressed in cultured dorsal root ganglion (DRG) neurons display a low efficiency CICR, because activation of VDCCs induced a larger [Ca2 þ ]i response with a much smaller and slower reduction in the ER luminal [Ca2 þ ] than those induced by the application of caffeine to fully open neuronal RyRs (Solovyova et al., 2002). The inhibition of SERCA pump with either thapsigargin or CPA induced an immediate reduction of the ER luminal [Ca2 þ ] in cultured DRGs; however this velocity in the reduction of luminal [Ca2 þ ] was barely increased by locking RyRs in the open state with 500 nM ryanodine, suggesting that only a small fraction of RyRs is behaving as leak channels. We have reached this conclusion based on the observation that application of 20 mM caffeine, to open all RyRs, produced a much faster reduction in the luminal ER [Ca2 þ ] than 500 nM ryanodine (Solovyova and Verkhratsky, 2003). It appears that although neurons express RyRs, these ion channels are not working as leak channels, at least under normal conditions. 5.2. RyRs as leak channels in neurodegenerative disorders It has been reported that different pathological conditions alter the ER luminal Ca2 þ homeostasis in neurons. DRGs neurons from diabetic rats display a lowered ER luminal [Ca2 þ ], probably due to a reduced activity of SERCA pump more than an increased leak activity of ER Ca2 þ channels, this lowered activity of SERCA pumps is partially compensated by a reduced activity of ER leak channels (Zherebitskaya et al., 2012), it is important to highlight that an increase in the leak activity of ER Ca2 þ permeable channels would lead to a further reduction in the luminal ER [Ca2 þ ], the triggering of ER stress and the ensuing cell death. Diabetes is associated with an increase in oxidative stress (Evans et al., 2002) and the latter has been found to generate a leak activity of RyR1(Andersson et al., 2011) that could facilitate generation of ER stress and neuronal cell death. Alzheimer is another ailment associated with alterations in the ER Ca2 þ homeostasis (Green and LaFerla, 2008). Mutations in presenilin 1, responsible for the induction of familial Alzheimer disease, have been shown to produce an overloaded internal Ca2 þ

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stores in different cell models (LaFerla, 2002). It was proposed that presenilins might function as ER Ca2 þ leak channels and mutations of these proteins disrupted their leak activity leading to an overloaded Ca2 þ stores (Supnet and Bezprozvanny, 2011). This pathological mechanism has been disputed and alternatively proposed that presenilin mutations enhance IP3R channel activity (Cheung et al., 2008). However, this does not seem to be the whole story, because Alzheimer disease is also associated with an increased activity of RyRs as well (Stutzmann et al., 2007). Interestingly, these data suggest that ER Ca2 þ stores are overloaded, but it turned out that the free luminal ER [Ca2 þ ], detected with Ca2 þ indicators directed to the ER, is reduced instead of increased (Kipanyula et al., 2012; Oulès et al., 2012). This lowered ER [Ca2þ ] is due to a much larger Ca2þ leak and the expression of RyRs is also increased, particularly RyR2 (Oulès et al., 2012). However, whether this increased ER Ca2 þ leak was due to a higher activity of RyR at rest, it was not tested. Moreover, it appears that higher expression of RyRs is part of the pathology since their inhibition with dantrolene produced some improvement both at the cellular and whole organism level (Oulès et al., 2012). Huntington is another degenerative disease that has been associated with a higher activity of RyRs and its inhibition or stabilization with FKBP12 reduced the lethal effect of the polyglutamine extension (Suzuki et al., 2012). These data suggest that a higher activity of RyRs, particularly as leak channels, is responsible for the development of these degenerative disorders.

6. Conclusion Ryanodine receptors have a clear role in excitation–contraction coupling in striated muscles, while in smooth muscle RyRs are involved in reducing excitability for most cases. At the same time RyRs, particularly RyR2, can work as safety valves for avoiding Ca2 þ overload of internal stores. In neuronal cells, RyRs are also loosely coupled to VGCCs, and it appears that an increased leak activity of these release channels is associated with cell death in neurodegenerative diseases. It is important then to understand under which circumstances and to know the factors involved in triggering an increased leak activity of RyRs to reduce the morbidity and mortality of patients with degenerative diseases.

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Cell type-specific spatial and functional coupling between mammalian brain Kv2.1 K+ channels and ryanodine receptors.

Abnormal ryanodine receptor channels in malignant hyperthermia.

CaMKII regulation of cardiac ryanodine receptors and inositol triphosphate receptors.

Ryanodine receptors: dual contribution to Alzheimer disease?

Ryanodine receptors: allosteric ion channel giants.

Tissue RNA as source of ion channels and receptors.

Voltage-gated calcium channels function as Ca2+-activated signaling receptors.

Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors.

Bcl-2 binds to and inhibits ryanodine receptors.

Immunohistochemical localization of ryanodine receptors in mouse central nervous system.

Ryanodine receptors: physiological function and deregulation in Alzheimer disease.

Functional ryanodine receptors in the membranes of neurohypophysial secretory granules.

Planar bilayer recording of ryanodine receptors of sarcoplasmic reticulum.

Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation.

Fluorescent probes for insect ryanodine receptors: candidate anthranilic diamides.

Eudistomin D and penaresin derivatives as modulators of ryanodine receptor channels and sarcoplasmic reticulum Ca2+ ATPase in striated muscle.

Cerebrospinal Fluid Leak Presenting as Epiphora.

Synthesis and biological activities of 2,3-dihydro-1,3,4-oxadiazole compounds and its derivatives as potential activator of ryanodine receptors.

Ryanodine-sensitive intracellular Ca2+ channels are involved in the output from the SCN circadian clock.

Intracellular calcium channels: inositol-1,4,5-trisphosphate receptors.

CGRP receptors and TRP channels in migraine.

Opposing roles of smooth muscle BK channels and ryanodine receptors in the regulation of nerve-evoked constriction of mesenteric resistance arteries.

Dampened activity of ryanodine receptor channels in mutant skeletal muscle lacking TRIC-A.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.