Anetta Wronska

RyR1, an enormous, approximately 2.2 MDa, tetrameric calcium-release channel in the sarcoplasmic reticulum (SR) of skeletal muscle, is essential for excitation-contraction coupling. Pathogenic mutations in RyR1 cause RyR1-related myopathies. ARM210 (S48168), a Rycal-class drug, has shown promise in treating RyR1-related myopathies in a recently concluded phase 1b clinical trial. Structural studies revealed that ARM210 binds to the peripheral RY1&2 domain of RyR1, stabilizing it in a closed state to prevent aberrant SR Ca ²⁺ leak. Here, we investigate ARM210’s mechanism of action at the atomistic level using extensive all-atom (~2.5 million atoms) explicit-solvent molecular dynamics simulations totaling 36.6 μs. Simulations revealed a long-range allosteric pathway in the wild-type RyR1 that propagates over 200 Å, linking the RY1&2 domain to the pore-forming S6 helices of adjacent protomers and enabling pore opening. Subsequently, we demonstrated that ARM210 binding stabilizes the RY1&2 domain in a closed conformation, rigidifying the cytosolic shell and activation core and disrupting the allosteric network necessary for pore dilation. These findings describe a plausible mode of action for how ARM210 stabilizes RyR1 in a closed state by modulating long-range allostery. Furthermore, we show that this allosteric regulatory network is perturbed by the oxidative stress-mimicking C3892D mutation near the Ca ²⁺ activator site, identified via mass spectrometry, live-cell fluorescence microscopy, and electrophysiology.

Statin therapies, used to lower high blood cholesterol, have been proposed to cause skeletal muscle side effects including myalgia and rhabdomyolysis. The ryanodine receptor 1 (RyR1)/Ca²⁺ release channel in skeletal muscle has recently been identified as an off-target binder of statins. Here, we report the cryo-electron microscopy structure of RyR1 in complex with rosuvastatin. Rosuvastatin occupies a single binding site in the pore region of RyR1 stabilizing the open conformation. In the closed conformation, the rosuvastatin binding site is occupied by the RyR1 auxiliary transmembrane helices (TMx1-2). Structural comparison indicates steric overlap of rosuvastatin with simvastatin (at Sim-1) but not with atorvastatin on RyR1, while all these statins may interfere with the conformational stability of the TMx1-2 helices. In contrast to simvastatin and atorvastatin, rosuvastatin does not exhibit multiple binding sites on RyR1 while similarly promoting RyR1 activation, indicating its potential as a preferable lead structure for developing statins that minimizes adverse side effects on RyR1.

Heart failure, the leading cause of mortality and morbidity in the developed world, is characterized by cardiac ryanodine receptor 2 channels that are hyperphosphorylated, oxidized, and depleted of the stabilizing subunit calstabin-2. This results in a diastolic sarcoplasmic reticulum Ca²⁺ leak that impairs cardiac contractility and triggers arrhythmias. Genetic mutations in ryanodine receptor 2 can also cause Ca²⁺ leak, leading to arrhythmias and sudden cardiac death. Here, we solved the cryogenic electron microscopy structures of ryanodine receptor 2 variants linked either to heart failure or inherited sudden cardiac death. All are in the primed state, part way between closed and open. Binding of Rycal drugs to ryanodine receptor 2 channels reverts the primed state back towards the closed state, decreasing Ca²⁺ leak, improving cardiac function, and preventing arrhythmias. We propose a structural-physiological mechanism whereby the ryanodine receptor 2 channel primed state underlies the arrhythmias in heart failure and arrhythmogenic disorders.

S100A1, a small homodimeric EF-hand Ca²⁺-binding protein (~21 kDa), plays an important regulatory role in Ca²⁺signaling pathways involved in various biological functions including Ca²⁺cycling and contractile performance in skeletal and cardiac myocytes. One key target of the S100A1 interactome is the ryanodine receptor (RyR), a huge homotetrameric Ca²⁺release channel (~2.3 MDa) of the sarcoplasmic reticulum. Here, we report cryoelectron microscopy structures of S100A1 bound to RyR1, the skeletal muscle isoform, in absence and presence of Ca²⁺. Ca²⁺-free apo-S100A1 binds beneath the bridging solenoid (BSol) and forms contacts with the junctional solenoid and the shell-core linker of RyR1. Upon Ca²⁺-binding, S100A1 undergoes a conformational change resulting in the exposure of the hydrophobic pocket known to serve as a major interaction site of S100A1. Through interactions of the hydrophobic pocket with RyR1, Ca²⁺-bound S100A1 intrudes deeper into the RyR1 structure beneath BSol than the apo-form and induces sideways motions of the C-terminal BSol region toward the adjacent RyR1 protomer resulting in tighter interprotomer contacts. Interestingly, the second hydrophobic pocket of the S100A1-dimer is largely exposed at the hydrophilic surface making it prone to interactions with the local environment, suggesting that S100A1 could be involved in forming larger heterocomplexes of RyRs with other protein partners. Since S100A1 interactions stabilizing BSol are implicated in the regulation of RyR-mediated Ca²⁺release, the characterization of the S100A1 binding site conserved between RyR isoforms may provide the structural basis for the development of therapeutic strategies regarding treatments of RyR-related disorders.

Ryanodine receptor type 2 (RyR2) mutations have been linked to an inherited form of exercise-induced sudden cardiac death called catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT results from stress-induced sarcoplasmic reticular Ca ²⁺ leak via the mutant RyR2 channels during diastole. We present atomic models of human wild-type (WT) RyR2 and the CPVT mutant RyR2-R2474S determined by cryo–electron microscopy with overall resolutions in the range of 2.6 to 3.6 Å, and reaching local resolutions of 2.25 Å, unprecedented for RyR2 channels. Under nonactivating conditions, the RyR2-R2474S channel is in a “primed” state between the closed and open states of WT RyR2, rendering it more sensitive to activation that results in stress-induced Ca ²⁺ leak. The Rycal drug ARM210 binds to RyR2-R2474S, reverting the primed state toward the closed state. Together, these studies provide a mechanism for CPVT and for the therapeutic actions of ARM210.

Since their discovery in 1993, microRNAs (miRNAs, miRs) have emerged as important regulators of many crucial cellular processes and their dysregulation have been shown to contribute to multiple pathological conditions, including cardiovascular disease (CVD). miRNAs have been found to regulate the expression of various genes involved in cardiac development and function, and in the development and progression of CVD. Many miRNAs are master regulators fine-tuning the expression of multiple, often interrelated, genes involved in inflammation, apoptosis, fibrosis, senescence, and other processes crucial for the development of different forms of CVD. This article presents a review of recent developments in our understanding of the role of miRNAs in the development of CVD and surveys their potential applicability as therapeutic targets and biomarkers to facilitate CVD diagnosis, prognosis, and treatment. There are currently multiple potential miRNA-based therapeutic agents in different stages of development, which can be grouped into two classes: miRNA mimics (replicating the sequence and activity of their corresponding miRNAs) and antagomiRs (antisense inhibitors of specific miRNAs). However, in spite of promising preliminary data and our ever-increasing knowledge about the mechanisms of action of specific miRNAs, miRNA-based therapeutics and biomarkers have yet to be approved for clinical applications. Significance Statement Over the last few years microRNAs have emerged as crucial, specific regulators of cardiovascular system and in the development of cardiovascular disease, by posttranscriptional regulation of their target genes. The minireview presents the most recent developments in this area of research, including the progress in diagnostic and therapeutic applications of microRNAs. microRNAs seem very promising candidates for biomarkers and therapeutic agents, though some challenges, such as efficient delivery and unwanted effects, need to be resolved.

Patients with heart failure (HF) have augmented vascular tone, which increases cardiac workload, impairing ventricular output and promoting further myocardial dysfunction. The molecular mechanisms underlying the maladaptive vascular responses observed in HF are not fully understood. Vascular smooth muscle cells (VSMCs) control vasoconstriction via a Ca²⁺-dependent process, in which the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) on the sarcoplasmic reticulum (SR) plays a major role. To dissect the mechanistic contribution of intracellular Ca²⁺ release to the increased vascular tone observed in HF, we analyzed the remodeling of IP3R1 in aortic tissues from patients with HF and from controls. VSMC IP3R1 channels from patients with HF and HF mice were hyperphosphorylated by both serine and tyrosine kinases. VSMCs isolated from IP3R1^VSMC–/– mice exhibited blunted Ca²⁺ responses to angiotensin II (ATII) and norepinephrine compared with control VSMCs. IP3R1^VSMC–/– mice displayed significantly reduced responses to ATII, both in vivo and ex vivo. HF IP3R^1VSMC–/– mice developed significantly less afterload compared with HF IP3R1^fl/fl mice and exhibited significantly attenuated progression toward decompensated HF and reduced interstitial fibrosis. Ca²⁺-dependent phosphorylation of the MLC by MLCK activated VSMC contraction. MLC phosphorylation was markedly increased in VSMCs from patients with HF and HF mice but reduced in VSMCs from HF IP3R1^VSMC–/– mice and HF WT mice treated with ML-7. Taken together, our data indicate that VSMC IP3R1 is a major effector of increased vascular tone, which contributes to increased cardiac afterload and decompensation in HF.

Cardiovascular disease (CVD) is a major cause of morbidity and mortality worldwide. Innovative, more stringent diagnostic and prognostic biomarkers and effective treatment options are needed to lessen its burden. In recent years, microRNAs have emerged as master regulators of gene expression – they bind to complementary sequences within the mRNAs of their target genes and inhibit their expression by either mRNA degradation or translational repression. microRNAs have been implicated in all major cellular processes, including cell cycle, differentiation and metabolism. Their unique mode of action, fine‐tuning gene expression rather than turning genes on/off, and their ability to simultaneously regulate multiple elements of relevant pathways makes them enticing potential biomarkers and therapeutic targets. Indeed, cardiovascular patients have specific patterns of circulating microRNA levels, often early in the disease process. This article provides a systematic overview of the role of microRNAs in the pathophysiology, diagnosis and treatment of CVD.