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Marks’ paper is titled, “Ryanodine Receptor Oxidation Causes Intracellular Calcium Leak and Muscle Weakness in Aging.” In addition to Andersson, his coauthors include Mathew Betzenhauser, Steven Reiken, Albano Meli, Alisa Umanskaya, Wenjun Xie, Takayuki Shiomi, Ran Zalk at CUMC and Alain Lacampagne at Universités Montpellier.

This research was supported by grants from the National Heart, Lung, and Blood Institute and the Swedish Research Council.

Source: Columbia Univ. Medical Center




Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging.




Cell Metabolism, Volume 14, Issue 2, 196-207, 3 August 2011 [Have full article]
Copyright 2011 Elsevier Inc. All rights reserved.


Daniel C. Andersson, Matthew J. Betzenhauser, Steven Reiken, Albano C. Meli, Alisa Umanskaya, Wenjun Xie, Takayuki Shiomi, Ran Zalk, Alain Lacampagne, Andrew R. Markssend emailSee Affiliations
Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA The Clyde and Helen Wu Center for Molecular Cardiology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA INSERM, U-1046, Universités Montpellier, 34295 Montpellier, France Corresponding author


Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (36 months) adults. This RyR1 channel complex remodeling resulted in leaky channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.


Monday, July 16, 2012

In-depth R&D news and innovations - Sign up now!

New research shows that exercise is a key step in building a muscle-like implant in the lab with the potential to repair muscle damage from injury or disease. In mice, these implants successfully prompt the regeneration and repair of damaged or lost muscle tissue, resulting in significant functional improvement.

"While the body has a capacity to repair small defects in skeletal muscle, the only option for larger defects is to surgically move muscle from one part of the body to another. This is like robbing Peter to pay Paul," said George Christ, Ph.D., a professor at Wake Forest Baptist Medical Center's Institute for Regenerative Medicine. "Rather than moving existing muscle, our aim is to help the body grow new muscle."

In the current issue of Tissue Engineering Part A, Christ and team build on their prior work and report their second round of experiments showing that placing cells derived from muscle tissue on a strip of biocompatible material—and then "exercising" the strip in the lab—results in a muscle-like implant that can prompt muscle regeneration and significant functional recovery. The researchers hope the treatment can one day help patients with muscle defects ranging from cleft lip and palate to those caused by traumatic injuries or surgery.

For the study, small samples of muscle tissue from rats and mice were processed to extract cells, which were then multiplied in the lab. The cells, at a rate of 1 million per square centimeter, were placed onto strips of a natural biological material. The material, derived from pig bladder with all cells removed, is known to be compatible with the body.

Next, the strips were placed in a computer-controlled device that slowly expands and contracts—essentially "educating" the implants on how to perform in the body. This cyclic stretching and relaxation occurred three times per minute for the first five minutes of each hour for about a week. In the current study, the scientists tried several different protocols, such as adding more cells to the strips during the exercise process.

The next step was implanting the strips in mice with about half of a large muscle in the back (latissimus dorsi) removed to create functional impairment. While the strips are "muscle-like" at the time of implantation, they are not yet functional. Implantation in the body—sometimes referred to as "nature's incubator"—prompts further development.

The goal of the project was to speed up the body's natural recovery process as well as prompt the development of new muscle tissue. The scientists compared four groups of mice. One group received no surgical repair. The other groups received implants prepared in one of three ways: one was not exercised before implantation, one was exercised for five to seven days, and one had extra cells added midway through the exercise process. The results showed that exercising the implants made a significant difference in both muscle development and function.

"The implant that wasn't exercised, or pre-conditioned, was able to accelerate the repair process, but recovery then stopped," said Christ. "On the other hand, when you exercise the implant, there is a more prolonged and extensive functional recovery. Through exercising the implant, you can increase both the rate and the magnitude of the recovery."

A variety of laboratory tests were used to measure results. A test of muscle force at two months, for example, showed that animals who received the implants with extra cells added had a threefold increase in absolute force compared to animals whose muscle damage was not repaired. The force-producing capacity of muscle is what determines the ability to perform everyday tasks.

"If these same results were repeated in humans, the recovery in function would clearly be considered significant," said Christ. "Within two months after implantation, the force generated by the repaired muscle is 70% that of native tissue, compared to 30% in animals that didn't receive repair."

The results also showed that new muscle tissue developed both in the implant, as well as in the area where the implant and native tissue met, suggesting that the implant works by accelerating the body's natural healing response, as well as by prompting the growth of new muscle tissue.

The researchers hope to evaluate the treatment in patients who need additional surgery for cleft lip and palate, a relatively common birth defect where there is a gap in muscle tissue required for normal facial development. These children commonly undergo multiple surgeries that involve moving muscle from one location to another or stretching existing muscle tissue to cover the tissue gap. The implant used in the current research is almost exactly the size required for these surgeries.

"As a surgeon I am excited about the advances in tissue-engineered muscle repair, which have been very promising and exciting potential in the surgical correction of both functional and cosmetic deformities in cleft lip and cleft palate" said Phillip N. Freeman, M.D., D.M.D., associate professor of Oral and Maxillofacial Surgery at the University of Texas Health Science Center at Houston. "Current technology does not address the inadequate muscle volume or function that is necessary for complete correction in 20 to 30% of cases. With this innovative technology there is the potential to make significant advances in more complete corrections of cleft lip and cleft palate patients."

The technology was originally developed under the Armed Forces Institute of Regenerative Medicine (AFIRM) with funding from the Department of Defense and the National Institutes of Health. The sponsor of the current research was the Telemedicine & Advanced Technology Research Center. A longer-term goal is to use the implant—in combination with other tissue-engineered implants and technologies being developed as part of AFIRM—to treat the severe head and facial injuries sustained by military personnel. For example, AFIRM-sponsored projects under way to engineer bone, skin and nerve may one day be combined to make a "composite" tissue.



2011 Aug 3;14(2):196-207. doi: 10.1016/j.cmet.2011.05.014.

Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging.

Andersson DC, Betzenhauser MJ, Reiken S, Meli AC, Umanskaya A, Xie W, Shiomi T, Zalk R, Lacampagne A, Marks AR.


Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA.


Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for xxmuscle contraction. RyR1 from aged (24 months) rodents was oxidized, cysteine-nitrosylated, and depleted of the channel-stabilizing subunit calstabin1, compared to RyR1 from younger (3-6 months) adults. This RyR1 channel complex remodeling resulted in "leaky" channels with increased open probability, leading to intracellular calcium leak in skeletal muscle. Similarly, 6-month-old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month-old wild-type mice. Treating aged mice with S107 stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca(2+) release, muscle-specific force, and exercise capacity. Taken together, these data indicate that leaky RyR1 contributes to age-related loss of muscle function.

Copyright © 2011 Elsevier Inc. All rights reserved.



2010 Nov;1211:25-36. doi: 10.1111/j.1749-6632.2010.05809.x.

Recent advances in the biology and therapy of muscle wasting.

Glass D, Roubenoff R.


Muscle Disease Group Musculoskeletal Translational Medicine, Novartis Institutes for Biomedical Research, Cambridge, Massachusetts, USA.


The recent advances in our understanding of the biology of muscle, and how anabolic and catabolic stimuli interact to control muscle mass and function, have led to new interest in pharmacological treatment of muscle wasting. Loss of muscle occurs as a consequence of many chronic diseases (cachexia), as well as normal aging (sarcopenia). Although anabolic effects of exercise on muscle have been know for many years, the development of pharmacological treatment for muscle loss is in its infancy. However, there is growing excitement among researchers in this field that developments may yield new treatments for muscle wasting in the future.


2011 Apr;7(4):426-8. doi: 10.4161/auto.7.4.14392.

Autophagy induction rescues muscular dystrophy. [Have paper]

Grumati P, Coletto L, Sandri M, Bonaldo P.


Department of Histology, Microbiology & Medical Biotechnology, University of Padova, Padova, Italy.


Collagen VI is an extracellular matrix protein forming a microfibrillar network in the endomysium of skeletal muscles. In humans, mutations in any of the three genes coding for collagen VI cause several skeletal muscle diseases, including Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). Collagen VI null (Col6a1(-/-)) mice display a myopathic phenotype resembling that of BM and UCMD patients. Muscles lacking collagen VI are characterized by the presence of dilated sarcoplasmic reticulum and dysfunctional mitochondria, which triggers apoptosis and leads to muscle wasting. We have found that accumulation of abnormal organelles is due to an impairment of autophagy. Reactivation of the autophagic flux by either nutritional approaches or by pharmacological and genetics tools removes dysfunctional organelles and greatly ameliorates the dystrophic phenotype.


2012 Oct 22. [Epub ahead of print]

Ryanodine Receptor Patents.

Kushnir A, Marks AR.


Clyde and Helen Wu Center for Molecular Cardiology, Departments of Physiology and Cellular Biophysics, and Medicine, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.


Research over the past two decades has implicated dysfunction of the ryanodine receptmolmor (RyR), a Ca2+ release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction (EC) coupling, in the pathogenesis of cardiac and skeletal myopathies. These discoveries have led to the development of novel drugs, screening tools, and research methods. The patents associated with these advances tell the story of the initial discovery of RyRs as a target for plant alkaloids, to their central role in cardiac and skeletal muscle excitation-contraction coupling, and ongoing clinical trials with a novel class of drugs called RycalsTM that inhibit pathological intracellular Ca2+ leak. Additionally, these patents highlight questions, controversies, and future directions of the RyR field.


2010 Mar-Apr;3(2):199-204. doi: 10.3892/mmr_00000240. [Free e-journal]

Modulation of ryanodine receptor Ca2+ channels (Review).

Ozawa T.


Department of Physiology, Tohoku University Graduate School of Medicine, Sendai 980-8575, Japan.


Ryanodine-sensitive Ca2+ release channels (ryanodine receptors, RyRs) play a crucial role in the mobilization of Ca2+ from the sarcoplasmic reticulum (SR) during the excitation-contraction coupling of muscle cells. In skeletal muscle, depolarization of transverse tubules activates the RyR, whereas in cardiac muscle, a Ca2+ influx through an L-type Ca2+ channel activates the RyR. The RyR is also activated by caffeine, a low concentration (<10 µM) of ryanodine or cyclic ADP-ribose. RyR activity is inhibited by Mg2+, ruthenium red, or higher concentrations (≥100 µM) of ryanodine. The activity of RyR channels is modulated by phosphorylation and by associated proteins, including calmodulin (CaM), calsequestrin (CSQ) and FK506-binding proteins (FKBPs). In muscle cells, apoCaM (Ca2+-free CaM) activates the RyR channel, and Ca2+ CaM (Ca2+-bound CaM) inhibits the channel. CSQ can bind approximately 40 moles of Ca2+/mole of CSQ in the SR lumen of muscle cells, and interacts functionally with RyR protein. When the RyR is stimulated, Ca2+ released from the lumen is dissociated from the CSQ- Ca2+ complex. A 12-kDa or 12.6-kDa FK506-binding protein (FKBP12 or FKBP12.6, respectively) is associated with RyR protein. When FKBP12 or FKBP12.6 is dissociated from the FKBP-RyR complex, the RyR is modulated (activated). Phosphorylation of the RyR by cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase II modulates the channel. PKA phosphorylation of the RyR on the skeletal and cardiac muscle SR dissociates FKBP12 or FKBP12.6 from the RyR complex. This review deals with the modulation mechanisms of RyR proteins by associated proteins and phosphorylation.

[PubMed - in process]


1997 Jul;77(3):699-729. [Free]

Ryanodine receptors of striated muscles: a complex channel capable of multiple interactions.

Franzini-Armstrong C, Protasi F.


Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, USA.


The ryanodine receptor (RyR) is a high-conductance Ca2+ channel of the sarcoplasmic reticulum in muscle and of the endoplasmic reticulum in other cells. In striated muscle fibers, RyRs are responsible for the rapid release of Ca2+ that activates contraction. Ryanodine receptors are complex molecules, with unusually large cytoplasmic domains containing numerous binding sites for agents that control the state of activity of the channel-forming domain of the molecule. Structural considerations indicate that long-range interactions between cytoplasmic and intramembrane domains control channel function. Ryanodine receptors are located in specialized regions of the SR, where they are structurally and functionally associated with other intrinsic proteins and, indirectly, also with the luminal Ca2(+)-binding protein calsequestrin. Activation of RyRs during the early part of the excitation-contraction coupling cascade is initiated by the activity of surface-membrane Ca2+ channels, the dihydropyridine receptors (DHPRs). Skeletal and cardiac muscles contain different RyR and DHPR isoforms and both contribute to the diversity in cardiac and skeletal excitation-contraction coupling mechanisms. The architecture of the sarcoplasmic reticulum-surface junctions determines the types of RyR-DHPR interactions in the two muscle types.


Published in final edited form as:
Drug Discov Today Dis Mech. 2010 SUMMER; 7(2): e151–e157.  [Have full paper]
doi: 10.1016/j.ddmec.2010.09.009
PMCID: PMC2989530

Fixing ryanodine receptor Ca2+ leak - a novel therapeutic strategy for contractile failure in heart and skeletal muscle

1 Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA
2 Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, NY 10032, USA
Address correspondence to: Andrew R. Marks, Department of Physiology and Cellular Biophysics, Russ Berrie Medical Sciences Pavilion, Room 520, 1150, St. Nicholas Ave, NY, NY 10032 Phone (212) 851-5340, Fax (212) 851-5346. Email:


A critical component in regulating cardiac and skeletal muscle contractility is the release of Ca2+ via ryanodine receptor (RyR) Ca2+ release channels in the sarcoplasmic reticulum (SR). In heart failure and myopathy, the RyR has been found to be excessively phosphorylated or nitrosylated and depleted of the RyR-stabilizing protein calstabin (FK506 binding protein 12/12.6). This remodeling of the RyR channel complex results in an intracellular SR Ca2+ leak and impaired contractility. Despite recent advances in heart failure treatment, there are still devastatingly high mortality rates with this disease. Moreover, pharmacological treatment for muscle weakness and myopathy is nearly nonexistent. A novel class of RyR-stabilizing drugs, rycals, which reduce Ca2+ leak by stabilizing the RyR channels due to preservation of the RyR-calstabin interaction, have recently been shown to improve contractile function in both heart and skeletal muscle. This opens up a novel therapeutic strategy for the treatment of contractile failure in the cardiac and skeletal muscle.


Contractility is the central property of all types of muscle. This feature enables the heart to produce the power necessary for its pump function and the skeletal muscles to cause movement. Impaired contractility is the key phenomenon in muscular diseases such as heart failure, cardiac arrhythmias and myopathies. Heart failure is a gravely debilitating condition with a mortality rate higher than many forms of cancer[]; a majority of patients with advanced cardiac failure die within the first year after diagnosis[]. The incidence of heart failure increases progressively with age and approaches 10 per 1000 after 65 years of age[]. With a growing proportion of elderly in Western societies, the future problems associated with failing heart function are expected to increase. Cardiac arrhythmias are a common cause of death in heart failure and ventricular tachycardia underlies half of the deaths in heart failure. Moreover, skeletal muscle function is also weakened in chronic heart failure and the impaired contractility seen in these two organs have common mechanisms, centered around defective cellular Ca2+ handling[]. Interestingly, myopathies such as Duchenne muscular dystrophy, have been shown to share similar Ca2+ handling defects[]. Despite progress in the therapeutic management of heart failure, major clinical problems and high mortality rates persist. Moreover, the absence of an effective treatment for myopathies shows that novel pharmacological regimens for contractile disorders are needed.

Stress signals in health and disease

The body is regularly exposed to different types of stress that can result in physiological responses leading to enhanced muscle function. For example, the evolutionary conserved neuro-hormonal-mediated fight-or-flight response activates many organs as manifested by increased heart rate and contractility and enhanced survival of the organism in acutely threatening situations. Furthermore, stress signals in endurance training improve heart and skeletal muscle function and this constitutes an example of beneficial adaptations to stress. Prolonged increases in the stress level, on the other hand, have harmful effects on cardiac and skeletal muscle. For example, sustained increase in catecholamine levels and adrenergic stimulation can manifest as deleterious stress and lead to impaired myocyte function. Chronically increased sympathetic activity and over-activation of the cardiac β-adrenergic receptor is linked to the deterioration of heart function and development of heart failure[]. This has prompted the use of β-adrenergic receptor blockers in the management of cardiovascular disease, including heart failure and arrhythmias[,]. Moreover, skeletal muscle function is interlaced with beta-adrenergic signaling. Activation of the sympathetic nervous system and elevated plasma catecholamine levels during exercise constitutes the key adaptive signal that couples skeletal and cardiac muscle work in vivo. Moreover, both patients and animal models of heart failure suffer from reduced muscle fatigue resistance[]. In fact, quality of life and prognosis of patients with heart failure is severely decreased due to skeletal muscle dysfunction (e.g. shortness of breath due to diaphragmatic weakness, and exercise intolerance due to limb skeletal muscle fatigue)[].

Regulation of muscle contractility

At the cellular level muscle contraction occurs through a process referred to as excitation-contraction (E-C) coupling. This process starts with an action potential (AP) in the cell membrane (sarcolemma). Then, a series of steps occur that couple the initial excitation to commencement of contractile work. Central to the E-C coupling process is an increase in cytoplasmic free Ca2+. Cellular Ca2+ handling is a highly controlled process that involves ion exchange systems, ion pumps and specialized compartments for Ca2+ storage within the cell (Fig 1A–B).

Figure 1Figure 1
Figure 1
Excitation-contraction coupling in cardiac and skeletal muscle

In the heart, generation of an AP normally occurs in the specialized pacemaker cells of the sino-atrial (sinus) node, from which a depolarization wave propagates cell to cell via gap junctions (intercalated discs) and thereby linking the cardiac cells electrically with each other. On the cardiomyocyte level, the AP propagates over the plasma membrane and into the cell via the transverse (t)-tubule system, where it activates voltage gated L-type Ca2+ channels (also referred to as dihydropyridine receptors, DHPRs; Cav1.2). These channels allow Ca2+ to pass through the sarcolemma and enter the cell. The trans-sarcolemmal Ca2+ influx occurs down a ~10,000 fold concentration gradient, with extracellular [Ca2+] being ~3–5 mM and resting cytoplasmic [Ca2+] ~100 nM. A narrow junction between the t-tubules and the SR places the cardiac L-type Ca2+ channels in close proximity to the SR Ca2+ release channels (RyRs). At rest, the SR lumen contains ~1–2 mM Ca2+. The L-type Ca2+ channel-mediated trans-sarcolemmal Ca2+-influx triggers RyR2 opening, which then permits SR Ca2+ release. This process is referred to as Ca2+-induced Ca2+ release[]. Importantly, the Ca2+ released from the SR contributes substantially more to the total increase in cytoplasmic Ca2+ than the L-type Ca2+ influx. In humans, ~70% of the total increase in cytoplasmic Ca2+ can be attributed to SR Ca2+ release[]. The increase in cytosolic [Ca2+] permits binding of Ca2+ to troponin C, a myofilament regulatory protein which in turn facilitates interaction of myosin and actin filaments leading to sarcomere shortening which generates contraction (systole). To allow relaxation of the heart (diastole), cytoplasmic Ca2+ is reduced mainly by uptake into the SR via an energy-requiring process dependent on the cardiac SR Ca2+-ATPase (SERCA2a) []. However in the steady state condition, an efflux of Ca2+ over the sarcolemma must also be present to balance the L-type Ca2+ influx (otherwise there would be a net build-up of total cellular Ca2+). This efflux is mainly effectuated by the Na/Ca exchanger (NCX)[].

The general principles of E-C coupling in cardiac and skeletal muscle are similar. However, there are some important differences regarding the regulation of cytoplasmic Ca2+. Similar to E-C coupling in the heart, action potential-mediated depolarization of the skeletal myocyte plasma membrane causes activation of L-type Ca2+ channels (Cav1.1). However in the skeletal muscle, the L-type Ca2+ channel does not conduct any Ca2+ current that is of importance to E-C coupling. Upon activation, the skeletal muscle L-type channel interacts directly with the juxtaposed ryanodine receptors (RyR1) that open and allow Ca2+ to be released of from the SR into the cytoplasm so that contraction can occur. To relax the muscle, Ca2+ is pumped back into the SR by the skeletal muscle SERCAs. Thus, in skeletal muscle, cellular Ca2+ is cycled between the SR and cytoplasm with little or no exchange with the extracellular environment[].

The magnitude of cardiomyocyte contractility is in large part graded by the amount of Ca2+ released into the cytosol. Thus, increased contractility is associated with mechanisms leading to increased [Ca2+]cyt. Cardiac Ca2+ handling can be influenced by a multitude of factors, including redox dependent modifications and phosphorylation of proteins involved in E-C coupling [,]. An important stimulus that influences Ca2+ handling is mediated by catecholamines (adrenalin and noradrenalin) from the sympathetic adrenergic system. In the heart, catecholamines bind to the β-adrenergic receptors (β-receptor). Activation of these receptors increase heart rate (chronotropy), relaxation speed (lusitropy), and contractility (inotropy). The β-receptor-induced increase in relaxation speed and contractility are considered to be mainly the effect of cAMP-dependent protein kinase (PKA)-mediated phosphorylation of proteins involved in cardiomyocyte Ca2+ handling []. Three important Ca2+ handling proteins that are targeted by PKA-mediated phosphorylation are the L-type Ca2+ channel, phospholamban and the RyR2. When phosphorylated, the influx of Ca2+ through the L-type Ca2+ channel is increased[,]. Phosphorylation of phospholamban leads to increased activity of SERCA2a, which accelerates SR Ca2+ uptake[]. Moreover, the RyR2 can be PKA-phosphorylated which leads to increased channel open probability and facilitated SR Ca2+ release[,]. β-adrenergic signaling is therefore of central importance in the acute regulation of heart function and cardiovascular homeostasis. Nevertheless, chronically sustained adrenergic stress can be deleterious for cardiac function and promotes the development of heart failure and cardiac arrhythmias[,,].

RyR Ca2+ release channel

The key cellular signal that controls muscle contractility is cytoplasmic Ca2+ released from the SR. Regulation of SR Ca2+ release channel RyR is therefore of pivotal interest in the control of muscle cell performance[]. Three isoforms of RyR (RyR1, RyR2, RyR3) exists in mammals. Although the RyRs are found in an array of tissue types (e.g. muscle, neurons, testis, thymus, pancreas and ovaries)[], RyR1 and RyR2 are predominantly expressed in striated muscle. In skeletal muscle, RyR1 is the isoform responsible for ECC, whereas RyR2 mediates Ca2+ release in the heart[]. The RyRs are homotetrameric channels that are located in the SR membrane. Each monomer of the RyR consists of a ~560-kDa molecule, with a massive N-terminal domain protruding into the cytosol, and a smaller C-terminal domain that contains the transmembrane segments and the pore region[].

In its intracellular environment, the RyR forms the centerpiece of a large macromolecular complex[]. The cytoplasmic N-terminal domain of the RyR serves as a scaffold for allosteric modulators which regulate the function of the C-terminal Ca2+-conducting pore region. These modulators include cyclic AMP dependent protein kinase A (PKA)[], phosphatases such as protein phosphatase 1 and 2A[], calcium dependent calmodulin kinase II (CaMKII)[,]. Small molecules and ions such as ATP, Ca2+, Mg2+ and the pharmacological substance caffeine are other modulators of RyR function[]. The peptidyl-propyl-cis-trans isomerases calstabin1 (FK506 binding protein 12 or FKBP12) and calstabin2 (FKBP12.6) are proteins with important regulatory function on SR Ca2+ release. Calstabins bind to the RyR via amphiphilic β-sheet structures and effectuate their modulatory function through protein-protein interactions with the SR Ca2+ release channel. Each RyR monomer binds one calstabin and under normal conditions an RyR tetramer is associated with four calstabin proteins[]. Calstabin1 associates predominantly with the skeletal muscle RyR1. In contrast, the cardiac muscle RyR2 have the highest affinity for calstabin2[]. The calstabins function as RyR channel-stabilizing proteins. In a resting myocyte, the calstabins increase the probability of the channel to be in its closed state[,]. This is an important mechanism to prevent spontaneous SR Ca2+ release due to a pathologic leak via RyR channels [].

Maladaptations of RyR and Ca2+ leak– a mechanism of contractile failure

Heart failure is characterized by an inadequate cardiac pump function that does not meet the metabolic requirements of the organs for blood flow. This feature can be a consequence of several cardiovascular pathologies that directly and indirectly compromise cardiac function, e.g. cardiac infarction, hypertension, inherited and acquired cardiomyopathies. Initially, the failing cardiac function can be offset by increased sympathetic and catecholaminergic signaling which will trigger cell surface β-adrenergic receptors and cause downstream activation of PKA that phosphorylates important Ca2+ handling proteins, including the RyR2. This leads to increased SR Ca2+ release, hence increased cytoplasmic [Ca2+] and stronger contractions. However chronic adrenergic activation, as seen in heart failure or during prolonged strenuous exercise, is associated with maladaptations in the SR Ca2+ release function. One such adaptation, shown by our laboratory, is PKA hyperphosphorylation of the RyR[,,]. This term refers to a situation where the RyR is chronically PKA phosphorylated at 3 to 4 of the 4 PKA sites per tetrameric channel (1 per RyR monomer) and depleted of 3 to 4 of the 4 calstabins bound to each channel [,]. Under these circumstances the RyRs from either heart or skeletal muscle exhibit a pathologic increase in the open probability under resting conditions (i.e. low activating cytosolic calcium). The RyR channels are leaky and Ca2+ leaks out from the SR. Over time, this can lead to reduction in SR Ca2+ content, with less Ca2+ available for release and consequently weaker muscle contractions (Fig. 2).

Figure 2
Figure 2
Dissociation of calstabin (FKBP12/FKBP12.6) from the RyR macromolecular complex underlies SR Ca2+ leak. A–B)

About half the deaths in heart failure are due to sudden cardiac death and spontaneous openings of the RyR2 in cardiac diastole can cause triggered ventricular arrhythmias. The increase in stochastic openings of the calstabin2 depleted RyR2, e.g. as seen in heart failure, links cardiac contractile failure with the increased propensity for ventricular arrhythmias to a common mechanism. Spontaneous release of SR Ca2+ during diastole activates the forward mode of the NCX which provides a depolarizing transient inward current. If the threshold to activate Na+ channels is reached an action potential will be triggered and an aberrant contraction will occur[].

In skeletal muscle, sustained β-adrenergic signaling and depletion of calstabin1 from RyR1 plays a role in the mechanism of contractile failure and muscle fatigue during strenuous exercise[]. This has recently been shown in both an animal model as well as in exercising humans[]. More recently it was shown that impaired Ca2+ regulation is also present in the severely debilitating human myopathy, Duchenne muscular dystrophy (DMD). In DMD a null mutation in the dystrophin gene leads to disruption of the important extracellular-cytoskeleton spanning dystroglycan complex and patients present with overt muscle weakness and cardiomyopathy[,]. RyR1 from the DMD mouse model (the mdx mouse) was shown to be excessively cysteine nitrosylated and this was coupled to depletion of calstabin1 from RyR1, increased spontaneous RyR1 openings (calcium sparks), and reduced specific muscle force[]. Moreover in the mdx mouse, RyR2 in the heart is also nitrosylated, depleted from calstabin2 and these mice display increased frequency of calcium sparks and ventricular arrhythmias []. In a mouse model of the inherited arrhythmogenic disorder catecholaminergic polymorphic ventricular tachycardia (CPVT) that harbors a heterozygous RyR2-R2474S mutation, RyR2 are calstabin2 depleted and both cardiac and neuronal cells display Ca2+ leak. These mice present a global phenotype with cardiac arrhythmias and seizures[]. This identifies calstabin dissociation and SR Ca2+ leak as mechanisms of importance, not only in diseases characterized by contractile dysfunction, but also in a more general pathophysiological setting.

Reducing RyR leak as a treatment for contractile dysfunction

Reducing SR Ca2+ leak by restoring RyR-calstabin interaction is a promising therapeutic strategy for conditions with failing contractile function (e.g. heart failure and myopathies)[,]. The 1,4-benzothiazepine derivatives JTV519 and S107 are novel small molecule drugs that have the effectively enhance RyR-calstabin binding and stabilizing the closed state of the RyR. These drugs are often referred to as Rycals due to their stabilizing effect on the RyR Ca2+ release function. Haploinsufficient calstabin-2+/−, but not calstabin-2−/− knockout mice that were treated with JTV519 were rescued from pacing-induced ventricular arrhythmias [], indicating that JTV519 exerts its effects through facilitating RyR-calstabin binding. In dogs with pacing-induced heart failure, where cardiac RyR2 was hyperphosphorylated and depleted from calstabin2, JTV519 restored the RyR2/calstabin2 interaction to normal levels, reduced Ca2+ leak and improved left ventricular function[]. In addition, JTV519 was shown to enhance RyR1-calstabin1 binding, restore skeletal muscle RyR1 channel function and decrease muscle fatigue in mice after myocardial infarction[]. These results suggest that rycals might be an effective treatment not only to prevent heart failure and ventricular arrhythmias after myocardial infarction but also to reduce concomitant skeletal muscle symptoms such as muscle weakness and fatigability.

Because JTV519 is also known to affect other ion channels (e.g. L-type Ca2+ and Na+ channels)[] and thereby potentially interfere with cardiac electrophysiological properties, another rycal, S107, with more specific RyR stabilizing properties has been developed[,,]. In a screening procedure, S107 was found to have no activity against hundreds of enzymes, channels and signal transduction molecules[]. Nonetheless, S107 facilitates the binding of calstabin1/2 to RyR1/2. Via this mechanism, S107 prevented depletion of calstabin1 from RyR1 and improved skeletal muscle fatigue resistance in mice subjected to severe exercise[]. In the Duchenne muscular dystrophy model, the mdx mouse, treatment with S107 reduced signs of muscle damage and improved skeletal muscle function[]. Interestingly, the mdx mice also develop cardiomyopathy with a Ca2+ leak phenotype and S107 treatment inhibited the Ca2+ leak and prevented arrhythmias in these mice[]. Moreover, CPVT mice that harbor a heterozygous RyR2-R2474S mutation were protected against stress-induced ventricular arrhythmias and displayed an increased threshold for seizures after treatment with S107[]. Thus, several lines of experimental evidence show that rycals can be used to stabilize RyR Ca2+ release function in pathological conditions where SR Ca2+ leak is present.

Summary and conclusions

Stress-induced maladaptations of the RyR and dissociation of calstabin are linked to defective Ca2+ handling and impaired contractility in diseases affecting both cardiac and skeletal muscle. In heart failure, impaired Ca2+ homeostasis is a key cellular mechanism that contributes to reduced cardiac output as seen in these patients. Despite improved pharmacological and device-based therapies of heart failure in recent years, a devastating mortality rate is still apparent in heart failure patients. Moreover, skeletal muscle dysfunction, as seen in heart failure or inherited muscular dystrophies, is virtually without effective pharmacological treatment. Thus, there is a strong need for novel therapeutic regimens that combat contractile dysfunction. Drugs that restore RyR Ca2+ release function, like the rycals JTV519 and S107, are therefore promising candidates. Rycal treatment would be ideal in conditions with co-morbidity between cardiac and skeletal muscle, as seen in cardiomyopathy and muscular dystrophy. Speculatively, there could also be benefits of rycal treatment in other conditions where cardiac and/or skeletal function is impaired. With a growing proportion of elderly in the society, aging-dependent dysfunction of the heart and skeletal muscle would be particularly important to study with respect to RyR Ca2+ leak and potential benefits of rycal treatment.

In summary, restoring RyR-calstabin binding and inhibiting Ca2+ leak through rycal treatment holds promise as a novel pharmacological tool in combating contractile failure of the heart and skeletal muscle.


We thank Dr. Maria Armiento for valuable assistance with figure editing.


Conflict of interest: A.R. Marks is a consultant for a start-up company, ARMGO Pharma Inc., that is targeting RyR channels to treat heart disease and to improve exercise capacity in muscle diseases

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2007 Apr;7(2):225-32. Epub 2007 Feb 15.

Novel targets for treating heart and muscle disease: stabilizing ryanodine receptors and preventing intracellular calcium leak.

Lehnart SE.


Department of Physiology and Cellular Biophysics, Clyde and Helen Wu Center for Molecular Cardiology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.


Ryanodine receptors (RyRs) function as intracellular Ca(2+) release channels on the endoplasmic and sarcoplasmic reticulum membranes. In striated muscles, Ca(2+) release through RyRs controls muscle excitation-contraction coupling. RyR channel function is regulated by a cytoplasmic scaffold domain that forms a macromolecular signaling complex including calstabin (formerly known as FK506-binding protein), calmodulin, phosphodiesterase, kinase and phosphatase proteins. An increasing number of genetic and acquired diseases has been associated with intracellular Ca(2+) leak. In heart failure, for instance, the RyR complex becomes altered, resulting in chronic channel dysfunction and chronic sarcoplasmic reticulum Ca(2+) leak. Recently, the efficacy of novel Ca(2+) release channel-stabilizing drugs has been demonstrated in cardiac and skeletal muscle disease models.