Dr. Andrew R. Marks, Columbia University, presented work from his laboratory over the last 20 years that has led to the discovery of a new mechanism for heart failure, cardiac arrhythmias, and muscle fatigue. This discovery has resulted in development of a new therapy that will soon be tested in phase I clinical trials.

The excitation-contraction coupling mechanism in heart and skeletal muscle involves depolarization of the muscle membrane, which activates a voltage gated calcium channel, which in turn activates the ryanodine receptor/calcium release channel (RyR2) on the sarcoplasmic reticulum to open and release calcium. The released calcium is the signal that causes muscle contraction, after which the calcium is pumped back into the sarcoplasmic reticulum causing muscle relaxation. Calcium channel stabilizing binding protein-2 (calstabin2), a subunit of RyR2, stabilizes the closed state of the RyR2 channel, preventing a pathologic leak of calcium.

The ryanodine receptor is a macromolecular signaling complex comprised of four identical monomers, making it the largest of all known ion channels. Each monomer contains targeting proteins that directly bind regulatory enzymes, including phosphatases, protein kinase A (PKA), and phosphodiesterase. This complex regulates the PKA phosphorylation of serine 2808 (S2808) on the cardiac channel. When PKA phosphorylates this serine, it releases the stabilizing protein and increases the activity of the channel. This process is part of the stress response known as the “fight or flight” response.

Calstabin is also known as FK506 binding protein (FKBP12). In the 1990s, Dr. Marks found that when rapamycin enters the cell and binds to FKBP it inhibits restenosis in coronary arteries. In his work characterizing RyR, Dr. Marks learned that RyR expressed without calstabin (in insects) results in hyperactive leaky calcium channels. When RyR is coexpressed with calstabin, the channel is stabilized and the leak is “fixed.” The stabilizing role of calstabin was confirmed with calstabin knockout mice, which had defective, leaky calcium channels.

The system that regulates excitation-contraction coupling is regulated by the classic fight or flight stress response. This response involves activation of the sympathetic nervous system and release of catecholamines that bind to beta adrenergic receptors and raise the second messenger cyclic AMP (cAMP), which activates PKA. PKA activates RyR, the voltage gated calcium channel and the uptake pathway through the calcium pump. When PKA phosphorylates RyR2 in the heart, calstabin2 is dissociated, increasing the activity of the channel.

Based on these findings, Dr. Marks proposed that at rest, the RyR receptors are not phosphorylated and have a full complement of calstabin molecules, with one bound to each monomer in the tetromeric channel. Vigorous exercise causes progressive phosphorylation of the calstabin, beginning with the first monomer and possibly phosphorylating all four sites. Binding of calstabin to the channel is decreased and the channel releases more calcium, resulting in stronger heart and skeletal muscle contractions. This activation of the channel is temporary and physiologically well tolerated, stopping when the exercise ends.

Thus, calstabin has two roles in the RyR channel complex. First, it prevents leaks through the channel by stabilizing the closed state of individual RyRs. Second, PKA phosphorylation-induced release of calstabin from RyR activates the channel during stress to increase calcium release and muscle contractility.

Dr. Marks found that the RyR2 channel is PKA hyperphosphorylated in left ventricular tissue from patients with heart failure; when treated with a left ventricular assist device, the heart returns to normal and is no longer PKA hyperphosphorylated. Based on these and animal studies, Dr. Marks proposed that patients with heart failure have a maladaptive fight or flight response that chronically activates the sympathetic nervous system. This leads to chronic hyperphosphorylation and activation of RyR2, depletion of calstabin2, and a leaky calcium channel. The result is decreased contractility and decreased cardiac output. Because the leak occurs during diastole it signals inward depolarizing currents that can trigger fatal cardiac arrhythmias.

When RyR2-S2808A knock-in mice with an RyR2 channel that cannot be phosphorylated were subjected to MI they were protected from developing heart failure compared to wild type mice. Other studies revealed the presence of a phosphodiesterase (PDE4D3) in the RyR2 complex that is decreased by about 40% in heart failure patients. Therefore, low PDE4D3 activity promotes PKA hyperphosphorylation, as demonstrated in PDE4D3 deficient mice that developed severe dilated cardiomyopathy. Even when the total PDE levels were normal, PDE4D4 deficiency caused a localized increase in cAMP at the “Z-line” where RyR2 is found. The PDE4D3 deficient mice also had a higher incidence of cardiac arrhythmias compared to wild-type mice. When the PDE4D3 mice were crossed with the mice with PKA that cannot be hyperphosphorylated, they were protected from cardiac arrhythmias. Additional mouse studies demonstrated that in mice with heart failure, early skeletal muscle fatigue correlates with the degree of PKA phosphorylation of RyR1.

Human genetic studies have found RyR2 mutations that are linked to exercise-induced sudden cardiac death. These mutations cause the same calcium channel leak found in heart failure patients. Testing in calstabin2 deficient mice showed that these mice experienced exercise-induced sudden cardiac death and delayed after depolarizations (DADs) in the cardiac myocytes.

These studies suggest that therapeutically targeting the RyR could improve heart and skeletal muscle function, prevent heart failure progression, and protect against arrhythmias. Beta blockers have been shown to prevent hyperphosphorylation of RyR2, depletion of calstabin2, and leakage of calcium indirectly by blocking beta receptors. However, beta blockers are not well tolerated by many patients.

Another therapeutic approach would be to find a drug that binds to RyR2 and prevents the leak through the channel without blocking the RyR2 pore. A drug called JTV-519, which is a 1,4 benzothiazepine originally designed in the synthesis of the calcium channel blocker diltiazem was shown to be beneficial in heart failure. Dr. Marks and colleagues developed derivatives of this drug that are more potent and highly specific for RyR2.

The proposed mechanism of action of these drugs is that their binding to RyR2 causes a conformational change in the channel, allowing calstabin2 to bind to the channel even when the channel is PKA hyperphosphorylated. Studies of these calcium channel stabilizers (called rycals) in a heart failure mouse model showed that they improve cardiac function only when calstabin2 is present. The rycal drugs also improve skeletal muscle fatigue in the heart failure model. Similar results were obtained by researchers at Yamaguchi University using a canine heart failure model. Additionally, Dr. Marks demonstrated that rycal drugs prevent exercise-induced VT and sudden cardiac death in calstabin2 haploinsufficient mice by preventing the depletion of the calstabin2 from the channel.

Dr. Marks concluded that the molecular mechanism for reduced contractility is leaky RyR2 channels. These receptors also serve as a molecular trigger for arrhythmias. Beta blockers improve cardiac function indirectly by fixing leaky RYR2 channels. Impaired exercise capacity in heart failure patients that is out of proportion to the degree of cardiac dysfunction is caused by leaky RyR1 channels. Diastolic SR calcium leak via PKA hyperphosphorylated RyR2 is a molecular mechanism contributing to heart failure progression and also for cardiac arrhythmias that cause sudden cardiac death.

Calcium channel stabilizers, or rycals, inhibit the RyR2 leak by enhancing calstabin2 binding to the channel, stabilizing its closed state. Rycals improve cardiac function, slow heart failure progression, prevent sudden cardiac death, and improve exercise capacity in animal models. Rycals restore the normal function of RyR channels and are not channel blockers. Clinical trials using a novel, RyR-specific, orally available calcium channel stabilizer were scheduled to begin in April 2009.