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Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

New Findings • What is the topic of this review? Heart failure is a progressive disease syndrome that, in the early stages, involves subtle tissue and cellullar changes. This review highlights novel imaging techniques that allow quantitative investigation of the underlying pathophysiological mechanisms in vivo. • What advances does it highlight? High-content voltage imaging of the left ventricle showed significant conduction slowing in the mdx mouse model of heart failure, corresponding to selective loss of Na+ channels and dystrophin in the lateral cardiac myocyte membrane. Super-resolution STED imaging after myocardial infarction uncovered proliferative, differential remodelling of individual T-tubules and network properties leading to intracellular Ca2+ release heterogeneity. These techniques reveal potential mechanisms of arrhythmia susceptibility, tissue degeneration and contractile dysfunction. Heart failure (HF) is a complex disease syndrome, which affects physiology at all levels, from the molecule to the whole organism. Following a causative insult, a maladaptive response occurs, which sustains cardiac remodelling and leads to a final common pathway of debilitating HF symptoms. In terms of mechanisms, distinct defects of excitation–contraction coupling compartments and organelles have been identified in cardiac samples of patients and animal models, which include changes in Ca2+ transport proteins and T-tubules. From a physiological standpoint, the source of regulatory intracellular Ca2+ is defined by ∼20,000 Ca2+ release units per cardiac myocyte, which jointly modulate contractile force production. We and others have characterized key changes in protein and membrane components of Ca2+ release units during HF in patient samples and transgenic models to gain insight into complex disease mechanisms. While earlier HF studies identified intracellular Ca2+ release as a major cause of contractile dysfunction, electrical dysfunction has gained attention as an important mechanism of HF mortality. In parallel, high-resolution imaging techniques have become instrumental to understand HF mechanisms in the intact cell and tissue environment, supporting translation of novel diagnostic strategies. Indeed, the increased spatial and temporal resolution of different experimental imaging techniques addresses the vastly different scales of HF pathophysiology, to correlate experimental with clinical surrogate markers, and to extend mechanisms to early, often subtle changes in HF. This last goal, in particular, will be essential to translate novel pathophysiological insight back to the growing number of asymptomatic individuals at increased risk for HF development, who may benefit most from early therapeutic interventions.

The cardiac ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR) controls intracellular Ca(2+) release and muscle contraction in the heart. Ca(2+) release via RyR2 is regulated by several physiological mediators. Protein kinase (PKA) phosphorylation dissociates the stabilizing FKBP12.6 subunit (calstabin2) from the RyR2 complex, resulting in increased contractility and cardiac output. Congestive heart failure is associated with elevated plasma catecholamine levels, and chronic stimulation of beta-adrenergic receptors leads to PKA hyperphosphorylation of RyR2 in failing hearts. PKA hyperphosphorylation results in calstabin2-depleted RyR2 that displays altered channel gating and may cause aberrant SR Ca(2+) release, depletion of SR Ca(2+) stores, and reduced myocardial contractility in heart failure. Calstabin2-depleted RyR2 may also trigger cardiac arrhythmias that cause sudden cardiac death. In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), RyR2 missense mutations cause reduced calstabin2 binding to RyR2. Increased RyR2 phosphorylation and pathologically increased calstabin2 dissociation during exercise results in aberrant diastolic calcium release, which may trigger ventricular arrhythmias and sudden cardiac death. In conclusion, heart failure and exercise-induced sudden cardiac death have been linked to defects in RyR2-calstabin2 regulation, and this may represent a novel target for the prevention and treatment of these forms of heart disease.