Our RESEARCH

Structure and Function of Calcium Release Channels: Role in Human Diseases

Molecular mechanisms of cardiac and skeletal muscle function, heart failure, arrhythmias, muscular dystrophy, neurodegenerative disease, and aging

When animals crawled out from the oceans onto land they encountered the toxicities of UV radiation from the sun and oxidative overload from breathing 21% O2. In return they evolved over millions of years to become humans. In 1940 the average life span in the United States was about 35-40 years of age. Now it’s nearly 80. The cumulative impacts of radiation, O2 and other environmental stresses form the basis for chronic disorders including the leading causes of death and disability, heart failure, diabetes, cancer, neurodegenerative diseases.  Even rare genetic disorders share have commonality with the most prevalent diseases, because genetic mutations stress biological systems which also leads to oxidative overload.

We are interested in the molecular physiology of stress responses that underlie human disorders. In organisms that model human diseases we dissect the molecular signals that cause human diseases using a combination of structural biology (cryo-EM), biophysics (recording single channel behavior and cellular imaging), genetics, behavior and physiology to discover what causes human disorders. We use these discoveries to identify new therapeutic druggable targets, develop drugs. These discoveries are ultimately translated into novel therapeutics and tested in patients. Examples include development of the first drug (rapamycin) eluting coronary artery stent for treatment of heart disease and a new class of drugs, Rycals, that fix leaky ryanodine receptor/calcium release channels and are being tested in patients.

A major focus of the laboratory is the study of mechanisms that regulate muscle contraction. In particular we use a variety of techniques including molecular biology, biophysics, cell biology, imaging (Live5 Zeiss Confocal), and structural biology to gain better understandings of the regulation of calcium release channels on the sarcoplasmic reticulum that control excitation-contraction (EC) coupling in cardiac and skeletal muscle. There are opportunities for graduate students and postdoctoral fellows to head their own projects using any of the various techniques that we employ to examine the regulation of calcium signaling and muscle function in normal and diseased states. In addition the laboratory has developed numerous genetic mouse models (primarily knock-ins and knock-outs) that are available to address specific questions concerning the regulation of key signaling pathways that control muscle contraction - in both cardiac and skeletal systems.

Much of the work in the laboratory is "translational" in that it leads directly to understanding the molecular basis of human diseases including heart failure and sudden cardiac death. In addition, novel therapeutic approaches are being tested including those that fix the "leak" in the RyR2 calcium release channel that causes heart failure and sudden cardiac death. timeline of RyR

In addition, there are projects focusing on gaining better understandings of cardiac muscle growth and excitability, T cell and B cell activation, as well as vascular smooth muscle proliferation. The latter project has lead directly to the development of the drug eluting stents that are currently used for patients with coronaryartery disease.

Bio Bytes 19: Molecular Cardiology with Andy Marks Interview

Leaky ryanodine receptors linked to human diseases.

Our research has shown that ryanodine receptor (RyR) calcium release channels can become leaky either due to inherited mutations or under stress conditions or both (1, 2). There are three forms of RyR. We have used cryogenic electron microscopy to solve the high resolution structures of RyR channels showing that they are members of the six-transmembrane cation channel family (3, 4). RyR1 (5, 6) is the major form in skeletal muscle where it is required for muscle contraction, and is also found in other organs including brain, RyR2 is the major form in cardiac muscle where it is required for heart muscle contraction, and is also found in brain and other organs, RyR3 is a minor form found in muscle, brain and other organs. RyR1, RyR2, and RyR3 share ~65% homology, with divergence predominantly localized to three major divergent regions (7, 8). Depending on which organ the leaky RyR channels are located they can cause diverse diseases including: heart failure (9-12); cardiac arrhythmias including atrial fibrillation (2, 13, 14) and a form of exercise induced sudden cardiac death known as catecholaminergic polymorphic ventricular tacchycardia (CPVT) (15); diabetes (16); muscular dystrophy (17, 18); age-dependent loss of muscle function (19); cancer-associated muscle weakness (20); ventilator-induced diaphragmatic dysfunction (VIDD)(21, 22); cognitive dysfunction and behavioral abnormalities associated with neuronal disorders including post-traumatic stress disorder (23); Alzheimer’s Disease(24, 25); and Huntington’s Disease (26). The ECG is from a CPVT patient with an RyR2-F2483I mutation [adapted from (27)]. Histology is from patients with RYR1-related myopathy [central core disease (CCD) and multi-minicore disease (MmD) adapted from (28) and (29)].

1.   S. O. Marx, A. R. Marks, Dysfunctional ryanodine receptors in the heart: new insights into complex cardiovascular diseases. J Mol Cell Cardiol 58, 225-231 (2013).

2.   H. Dridi et al., Intracellular calcium leak in heart failure and atrial fibrillation: a unifying mechanism and therapeutic target. Nat Rev Cardiol 17, 732-747 (2020).

3.   A. des Georges et al., Structural Basis for Gating and Activation of RyR1. Cell 167, 145-157 e117 (2016).

4.   R. Zalk et al., Structure of a mammalian ryanodine receptor. Nature 517, 44-49 (2015).

5.   A. B. Brillantes et al., Stabilization of calcium release channel (ryanodine receptor) function by FK506-binding protein. Cell 77, 513-523 (1994).

6.   A. R. Marks et al., Molecular cloning and characterization of the ryanodine receptor/junctional channel complex cDNA from skeletal muscle sarcoplasmic reticulum. Proc Natl Acad Sci U S A 86, 8683-8687 (1989).

7.   G. Santulli, D. Lewis, A. des Georges, A. R. Marks, J. Frank, Ryanodine Receptor Structure and Function in Health and Disease. Subcell Biochem 87, 329-352 (2018).

8.   A. Kushnir, B. Wajsberg, A. R. Marks, Ryanodine receptor dysfunction in human disorders. Biochim Biophys Acta Mol Cell Res 1865, 1687-1697 (2018).

9.   S. O. Marx et al., PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365-376 (2000).

10.  A. R. Marks, A guide for the perplexed: towards an understanding of the molecular basis of heart failure. Circulation 107, 1456-1459 (2003).

11.  A. R. Marks, Ryanodine receptors, FKBP12, and heart failure. Front Biosci 7, d970-977 (2002).

12.  F. Huang, J. Shan, S. Reiken, X. H. Wehrens, A. R. Marks, Analysis of calstabin2 (FKBP12.6)-ryanodine receptor interactions: rescue of heart failure by calstabin2 in mice. Proc Natl Acad Sci U S A 103, 3456-3461 (2006).

13.  J. Shan et al., Calcium leak through ryanodine receptors leads to atrial fibrillation in 3 mouse models of catecholaminergic polymorphic ventricular tachycardia. Circ Res 111, 708-717 (2012).

14.  J. A. Vest et al., Defective cardiac ryanodine receptor regulation during atrial fibrillation. Circulation 111, 2025-2032 (2005).

15.  X. H. Wehrens et al., FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell 113, 829-840 (2003).

16.  G. Santulli et al., Calcium release channel RyR2 regulates insulin release and glucose homeostasis. J Clin Invest 125, 4316 (2015).

17.  A. M. Bellinger et al., Hypernitrosylated ryanodine receptor calcium release channels are leaky in dystrophic muscle. Nat Med 15, 325-330 (2009).

18.  D. C. Andersson et al., Leaky ryanodine receptors in beta-sarcoglycan deficient mice: a potential common defect in muscular dystrophy. Skelet Muscle 2, 9 (2012).

19.  D. C. Andersson et al., Ryanodine receptor oxidation causes intracellular calcium leak and muscle weakness in aging. Cell Metab 14, 196-207 (2011).

20.  D. L. Waning et al., Excess TGF-beta mediates muscle weakness associated with bone metastases in mice. Nature medicine 21, 1262-1271 (2015).

21.   H. Dridi et al., Late Ventilator-Induced Diaphragmatic Dysfunction After Extubation. Crit Care Med 48, e1300-e1305 (2020).

22.   S. Matecki et al., Leaky ryanodine receptors contribute to diaphragmatic weakness during mechanical ventilation. Proc Natl Acad Sci U S A 113, 9069-9074 (2016).

23.   X. Liu et al., Role of leaky neuronal ryanodine receptors in stress-induced cognitive dysfunction. Cell 150, 1055-1067 (2012).

24.  A. Lacampagne et al., Post-translational remodeling of ryanodine receptor induces calcium leak leading to Alzheimer's disease-like pathologies and cognitive deficits. Acta Neuropathol 10.1007/s00401-017-1733-7 (2017).

25.  R. Bussiere et al., Amyloid beta production is regulated by beta2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor. J Biol Chem 292, 10153-10168 (2017).

26.  H. Dridi et al., Role of defective calcium regulation in cardiorespiratory dysfunction in Huntington's disease. JCI Insight 5 (2020).

27.  A. Fatima et al., In vitro modeling of ryanodine receptor 2 dysfunction using human induced pluripotent stem cells. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology 28, 579-592 (2011).

28.  H. Jungbluth et al., Autosomal recessive inheritance of RYR1 mutations in a congenital myopathy with cores. Neurology 59, 284-287 (2002).

29.  R. S. Laughlin, Z. Niu, E. Wieben, M. Milone, RYR1 causing distal myopathy. Mol Genet Genomic Med 5, 800-804 (2017).

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