Search

SummaryCOVID-19, caused by SARS-CoV-2 involves multiple organs including cardiovascular, pulmonary and central nervous system. Understanding how SARS-CoV-2 infection afflicts diverse organ systems remains challenging1,2. Particularly vexing has been the problem posed by persistent organ dysfunction known as “long COVID,” which includes cognitive impairment3. Here we provide evidence linking SARS-CoV-2 infection to activation of TGF-ß signaling and oxidative overload. One consequence is oxidation of the ryanodine receptor/calcium (Ca2+) release channels (RyR) on the endo/sarcoplasmic (ER/SR) reticuli in heart, lung and brains of patients who succumbed to COVID-19. This depletes the channels of the stabilizing subunit calstabin2 causing them to leak Ca2+ which can promote heart failure4,5, pulmonary insufficiency 6 and cognitive and behavioral defects7–9. Ex-vivo treatment of heart, lung, and brain tissues from COVID-19 patients using a Rycal drug (ARM210)10 prevented calstabin2 loss and fixed the channel leak. Of particular interest is that neuropathological pathways activated downstream of leaky RyR2 channels in Alzheimer’s Disease (AD) patients were activated in COVID-19 patients. Thus, leaky RyR2 Ca2+ channels may play a role in COVID-19 pathophysiology and could be a therapeutic target for amelioration of some comorbidities associated with SARS-CoV-2 infection.

The type 1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel on the sarcoplasmic/endoplasmic reticulum that is required for skeletal muscle contraction. RyR1 channel activity is modulated by ligands, including the activators Ca2+ and ATP. Patients with inherited mutations in RyR1 may exhibit muscle weakness as part of a heterogeneous, complex disorder known as RYR1-related myopathy (RYR1-RM) or more recently termed RYR1-related disorders (RYR1-RD). Guided by high-resolution structures of skeletal muscle RyR1, obtained using cryogenic electron microscopy, we introduced mutations into putative Ca2+ and ATP binding sites and studied the function of the resulting mutant channels. These mutations confirmed the functional significance of the Ca2+ and ATP binding sites identified by structural studies based on the effects on channel regulation. Under normal conditions, Ca2+ activates RyR1 at low concentrations (µM) and inhibits it at high concentrations (mM). Mutations in the Ca2+-binding site impaired both activating and inhibitory regulation of the channel, suggesting a single site for both high and low affinity Ca2+-dependent regulation of RyR1 function. Mutation of residues that interact with the adenine ring of ATP abrogated ATP binding to the channel, whereas mutating residues that interact with the triphosphate tail only affected the degree of activation. In addition, patients with mutations at the Ca2+ or ATP binding sites suffer from muscle weakness, therefore impaired RyR1 channel regulation by either Ca2+ or ATP may contribute to the pathophysiology of RYR1-RM in some patients.

Mutations in the lamin A/C gene (LMNA), which encodes A-type lamins, cause several diseases called laminopathies, the most common of which is dilated cardiomyopathy with muscular dystrophy. The role of Ca2+ regulation in these diseases remain poorly understood. We now show biochemical remodeling of the ryanodine receptor (RyR)/intracellular Ca2+ release channel in heart samples from human subjects with LMNA mutations, including protein kinase A-catalyzed phosphorylation, oxidation and depletion of the stabilizing subunit calstabin. In the LmnaH222P/H222P murine model of Emery-Dreifuss muscular dystrophy caused by LMNA mutation, we demonstrate an age-dependent biochemical remodeling of RyR2 in the heart and RyR1 in skeletal muscle. This RyR remodeling is associated with heart and skeletal muscle dysfunction. Defective heart and muscle function are ameliorated by treatment with a novel Rycal small molecule drug (S107) that fixes ‘leaky’ RyRs. SMAD3 phosphorylation is increased in hearts and diaphragms of LmnaH222P/H222P mice, which enhances NADPH oxidase binding to RyR channels, contributing to their oxidation. There is also increased generalized protein oxidation, increased calcium/calmodulin-dependent protein kinase II-catalyzed phosphorylation of RyRs and increased protein kinase A activity in these tissues. Our data show that RyR remodeling plays a role in cardiomyopathy and skeletal muscle dysfunction caused by LMNA mutation and identify these Ca2+ channels as a potential therapeutic target.

Huntington’s disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target., This study explores the role of ryanodine receptor calcium channels in the brain, the heart, and the diaphragm and central versus peripheral pathophysiological mechanisms in Huntington’s disease.