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"Jones, Peter P."
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MINFLUX microscopy resolves subunits of the cardiac ryanodine receptor and its 3D orientation in cells
The cardiac ryanodine receptor (RyR2) constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative. Here, we present purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX microscopy. Using single-domain antibodies and DNA-PAINT we determine the location of individual RyR2 subunits with high precision (~3 nm) and resolve the 3D orientations of RyR2s in-situ. We measured labeling efficiencies of ~50%, implying RyR2 tetramer detection probability approaching 95%. In HEK293 cells, RyR2 expression was dense, with some clusters containing several hundred RyR2s. Ventricular myocytes from mice contained large clusters containing many tens of close-packed RyR2s, resolving apparent discrepancies between electron microscopy and previous super-resolution microscopy data. The methodology developed here reveals the full 3D morphological complexity of RyR2 channels and is applicable to other multi-subunit complexes in a variety of cell types.
Cardiac ryanodine receptors (RyR2) are critical for heart contraction. Here, the authors use 3D MINFLUX microscopy to image receptor subunits and RyR2 orientation with nanometre resolution, thereby providing a molecular view of the organisation and clustering of these cardiac muscle receptors.
Journal Article
The further adventures of Red Sonja
\"She lived in a savage world in an uncivilized age - a world ruled by men and governed by the sword. The[y] called her... Red Sonja - for her flame red hair, and for the smoldering fire of her pride, which gave her sword-arm a strength that few men could match, and none had ever defeated. This collection contains a variety of issues from the original Marvel Comics series 'The Savage Sword of Conan,' as well as Sonja Tales from 'Kull and the Barbarians,' with each page re-mastered for this volume. Also included is a gallery of pin-ups by Frank Thorne, Howard Chaykin, and more. These tales are where it all began, and set the stage for the current Red Sonja series from Dynamite Entertainment\"--back cover.
Book
The ryanodine receptor store-sensing gate controls Ca2+ waves and Ca2+-triggered arrhythmias
In heart muscle cells, sarcoplasmic reticulum calcium overload leads to spontaneous calcium waves that can cause arrhythmias and sudden cardiac death. S.R. Wayne Chen and his colleagues now detail the molecular mechanism by which intra–sarcoplasmic reticulum calcium ions interact with the gate of the calcium release channel RyR2, explaining how calcium waves are initiated and thereby contribute to calcium-triggered arrhythmias.
Spontaneous Ca
2+
release from intracellular stores is important for various physiological and pathological processes. In cardiac muscle cells, spontaneous store overload–induced Ca
2+
release (SOICR) can result in Ca
2+
waves, a major cause of ventricular tachyarrhythmias (VTs) and sudden death. The molecular mechanism underlying SOICR has been a mystery for decades. Here we show that a point mutation, E4872A, in the helix bundle crossing region (the proposed gate) of the cardiac ryanodine receptor (RyR2) completely abolishes luminal, but not cytosolic, Ca
2+
activation of RyR2. The introduction of metal-binding histidines at this site converts RyR2 into a luminal Ni
2+
-gated channel. Mouse hearts harboring a heterozygous RyR2 mutation at this site (E4872Q) are resistant to SOICR and are completely protected against Ca
2+
-triggered VTs. These data show that the RyR2 gate directly senses luminal (store) Ca
2+
, explaining the regulation of RyR2 by luminal Ca
2+
, the initiation of Ca
2+
waves and Ca
2+
-triggered arrhythmias. This newly identified store-sensing gate structure is conserved in all RyR and inositol 1,4,5-trisphosphate receptor isoforms.
Journal Article
Sarco/endoplasmic reticulum calcium ATPase activity is unchanged despite increased myofilament calcium sensitivity in Zucker type 2 diabetic fatty rat heart
Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca
2+
) regulation. However, there is emerging evidence that Ca
2+
mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca
2+
sensitivity and Ca
2+
handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca
2+
sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca
2+
-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca
2+
uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca
2+
mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.
Journal Article
Direct regulation of the cardiac ryanodine receptor (RyR2) by O-GlcNAcylation
Background
O-GlcNAcylation is the enzymatic addition of a sugar, O-linked β-N-Acetylglucosamine, to the serine and threonine residues of proteins, and is abundant in diabetic conditions. We have previously shown that O-GlcNAcylation can trigger arrhythmias by indirectly increasing pathological Ca
2+
leak through the cardiac ryanodine receptor (RyR2) via Ca
2+
/calmodulin-dependent kinase II (CaMKII). However, RyR2 is well known to be directly regulated by other forms of serine and threonine modification, therefore, this study aimed to determine whether RyR2 is directly modified by O-GlcNAcylation and if this also alters the function of RyR2 and Ca
2+
leak.
Methods
O-GlcNAcylation of RyR2 in diabetic human and animal hearts was determined using western blotting. O-GlcNAcylation of RyR2 was pharmacologically controlled and the propensity for Ca
2+
leak was determined using single cell imaging. The site of O-GlcNAcylation within RyR2 was determined using site-directed mutagenesis of RyR2.
Results
We found that RyR2 is modified by O-GlcNAcylation in human, animal and HEK293 cell models. Under hyperglycaemic conditions O-GlcNAcylation was associated with an increase in Ca
2+
leak through RyR2 which persisted after CaMKII inhibition. Conversion of serine-2808 to alanine prevented an O-GlcNAcylation induced increase in Ca
2+
leak.
Conclusions
These data suggest that the function of RyR2 can be directly regulated by O-GlcNAcylation and requires the presence of serine-2808.
Journal Article
Carvedilol and its new analogs suppress arrhythmogenic store overload–induced Ca2+ release
The widely prescribed beta blocker carvedilol has strong antiarrhythmic effects on the heart, but the underlying mechanisms have been unclear. Qiang Zhou
et al
. now show that carvedilol, unlike other beta blockers, is also able to block proarrhythmogenic spontaneous calcium waves by directly inhibiting the calcium release channel RyR2. By generating new carvedilol analogs that inhibit RyR2 but not β-adrenergic receptors, the authors provide evidence that antiarrhythmic therapy might be optimized by combining agents that are individually selective for these two targets.
Carvedilol is one of the most effective beta blockers for preventing ventricular tachyarrhythmias in heart failure, but the mechanisms underlying its favorable antiarrhythmic benefits remain unclear. Spontaneous Ca
2+
waves, also called store overload–induced Ca
2+
release (SOICR), evoke ventricular tachyarrhythmias in individuals with heart failure. Here we show that carvedilol is the only beta blocker tested that effectively suppresses SOICR by directly reducing the open duration of the cardiac ryanodine receptor (RyR2). This unique anti-SOICR activity of carvedilol, combined with its beta-blocking activity, probably contributes to its favorable antiarrhythmic effect. To enable optimal titration of carvedilol's actions as a beta blocker and as a suppressor of SOICR separately, we developed a new SOICR-inhibiting, minimally beta-blocking carvedilol analog, VK-II-86. VK-II-86 prevented stress-induced ventricular tachyarrhythmias in RyR2-mutant mice and did so more effectively when combined with either of the selective beta blockers metoprolol or bisoprolol. Combining SOICR inhibition with optimal beta blockade has the potential to provide antiarrhythmic therapy that can be tailored to individual patients.
Journal Article
Inhibition of calcium/calmodulin-dependent kinase II restores contraction and relaxation in isolated cardiac muscle from type 2 diabetic rats
Background
Calcium/calmodulin-dependent kinase II-delta (CaMKIIδ) activity is enhanced during hyperglycemia and has been shown to alter intracellular calcium handling in cardiomyocytes, ultimately leading to reduced cardiac performance. However, the effects of CaMKIIδ on cardiac contractility during type 2 diabetes are undefined.
Methods
We examined the expression and activation of CaMKIIδ in right atrial appendages from non-diabetic and type 2 diabetic patients (n = 7 patients per group) with preserved ejection fraction, and also in right ventricular tissue from Zucker Diabetic Fatty rats (ZDF) (n = 5–10 animals per group) during early diabetic cardiac dysfunction, using immunoblot. We also measured whole heart function of ZDF and control rats using echocardiography. Then we measured contraction and relaxation parameters of isolated trabeculae from ZDF to control rats in the presence and absence of CaMKII inhibitors.
Results
CaMKIIδ phosphorylation (at Thr287) was increased in both the diabetic human and animal tissue, indicating increased CaMKIIδ activation in the type 2 diabetic heart. Basal cardiac contractility and relaxation were impaired in the cardiac muscles from the diabetic rats, and CaMKII inhibition with KN93 partially restored contractility and relaxation. Autocamtide-2-related-inhibitor peptide (AIP), another CaMKII inhibitor that acts via a different mechanism than KN93, fully restored cardiac contractility and relaxation.
Conclusions
Our results indicate that CaMKIIδ plays a key role in modulating performance of the diabetic heart, and moreover, suggest a potential therapeutic role for CaMKII inhibitors in improving myocardial function during type 2 diabetes.
Journal Article
Altered membrane properties but unchanged intrinsic excitability and spontaneous postsynaptic currents in an aged APPswe/PS1dE9 model of Alzheimer’s disease
Neuronal hyperexcitability in Alzheimer’s disease (AD) models is thought to either contribute to the formation of amyloid beta plaques or result from their formation. Neuronal hyperexcitability has been shown in the cerebral cortex of the widely used young APPswe/PS1dE9 mice, which have accelerated plaque formation. However, it is currently unclear if hyperexcitability also occurs in CA1 hippocampal neurons of aged animals in this model. In the present work, we have compared intrinsic excitability and spontaneous synaptic inputs from CA1 pyramidal cells of 8-month-old APPswe/PS1dE9 and wildtype control mice. We find no change in intrinsic excitability or spontaneous postsynaptic currents between groups. We did, however, find a reduced input resistance and an increase in hyperpolarization-activated sag current. These results are consistent with findings from other aged AD model mice, including the widely used 5 x FAD and 3 x Tg. Together these results suggest that neuronal hyperexcitability is not a consistent feature of all AD mouse models, particularly at advanced ages.
Journal Article
Stage-specific regulation of signalling pathways to differentiate pluripotent stem cells to cardiomyocytes with ventricular lineage
Background
Pluripotent stem cells (PSCs) can be an ideal source of differentiation of cardiomyocytes in vitro and during transplantation to induce cardiac regeneration. However, differentiation of PSCs into a heterogeneous population is associated with an increased incidence of arrhythmia following transplantation. We aimed to design a protocol to drive PSCs to a ventricular lineage by regulating Wnt and retinoic acid (RA) signalling pathways.
Methods
Mouse embryonic stem cells were cultured either in monolayers or three-dimensional hanging drop method to form embryonic bodies (EBs) and exposed to different treatments acting on Wnt and retinoic acid signalling. Samples were collected at different time points to analyse cardiomyocyte-specific markers by RT-PCR, flow cytometry and immunofluorescence.
Results
Treatment of monolayer and EBs with Wnt and RA signalling pathways and ascorbic acid, as a cardiac programming enhancer, resulted in the formation of an immature non-contractile cardiac population that expressed many of the putative markers of cardiac differentiation. The population exhibited upregulation of ventricular specific markers while suppressing the expression of pro-atrial and pro-sinoatrial markers. Differentiation of EBs resulted in early foetal like non-contractile ventricular cardiomyocytes with an inherent propensity to contract when stimulated.
Conclusion
Our results provide the first evidence of in vitro differentiation that mimics the embryonic morphogenesis towards ventricular specific cardiomyocytes through regulation of Wnt and RA signalling pathways.
Journal Article