Rationale Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

3 0 ,5 0 -cyclic adenosine monophosphate (cAMP) is an ubiquitous second messenger that regulates physiological functions by acting in distinct subcellular microdomains. Although several targeted cAMP biosensors are developed and used in single cells, it is unclear whether such biosensors can be successfully applied in vivo, especially in the context of disease. Here, we describe a transgenic mouse model expressing a targeted cAMP sensor and analyse microdomain-specific second messenger dynamics in the vicinity of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). We demonstrate the biocompatibility of this targeted sensor and its potential for real-time monitoring of compartmentalized cAMP signalling in adult cardiomyocytes isolated from a healthy mouse heart and from an in vivo cardiac disease model. In particular, we uncover the existence of a phosphodiesterase-dependent receptor-microdomain communication, which is affected in hypertrophy, resulting in reduced b-adrenergic receptor-cAMP signalling to SERCA.

Two major subtypes of cardiac β-adrenergic receptors (β−ARs) modulate the effects of catecholamines on excitation-contraction coupling. The predominantly G s -coupled β 1 -AR is evenly distributed across the sarcolemma and stimulates Brief UltraRapid Communication© 2015 American Heart Association, Inc. Objective: To directly visualize alterations in β-adrenergic receptor-associated cAMP and cGMP microdomain signaling in early cardiac disease. Methods and Results:Unexpectedly, measurements of cell shortening revealed augmented β-adrenergic receptorstimulated cardiomyocyte contractility by atrial natriuretic peptide/cGMP signaling in early cardiac hypertrophy after transverse aortic constriction, which was in sharp contrast to well-documented β-adrenergic and natriuretic peptide signaling desensitization during chronic disease. Real-time cAMP analysis in β 1 -and β 2 -adrenergic receptor-associated membrane microdomains using a novel membrane-targeted Förster resonance energy transfer-based biosensor transgenically expressed in mice revealed that this unexpected atrial natriuretic peptide effect is brought about by spatial redistribution of cGMP-sensitive phosphodiesterases 2 and 3 between both receptor compartments. Functionally, this led to a significant shift in cGMP/cAMP cross-talk and, in particular, to cGMP-driven augmentation of contractility in vitro and in vivo. The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl Conclusions:

BackgroundCardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure.Methods and ResultsMice with endothelial‐specific deletion of p53 (End.p53‐KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53‐KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53‐WT controls, whereas banded hearts of End.p53‐KO mice exhibited lower numbers of apoptotic endothelial and non‐endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53‐KO mice exhibited markedly less fibrosis compared with End.p53‐WT controls, and lower mRNA levels of p53‐regulated genes involved in extracellular matrix production and turnover (eg, Bmp‐7, Ctgf, or Pai‐1), or of transcription factors involved in controlling mesenchymal differentiation were observed.ConclusionsOur analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy.

1235 3ʹ,5ʹ-Cyclic guanosine monophosphate (cGMP) is one of the ubiquitous second messengers, which is critically involved in the regulation of cardiac contractility and pathological hypertrophy.1,2 In cardiomyocytes, 2 classes of guanylyl cyclases (GCs) are responsible for cGMP synthesis. The first class, particulate GCs (pGCs) represented by GC-A and GC-B, are plasma membrane receptors for atrial natriuretic peptide (ANP) and C-type natriuretic peptide (CNP), respectively. 3,4 Second, the so-called soluble or NO-sensitive GCs (NO-GCs) are also functionally present in cardiomyocytes.5-7 cGMP levels are negatively regulated by cGMP-hydrolyzing enzymes phosphodiesterases (PDEs) with at least 4 families (PDE1, 2, 3, and 5) expressed in cardiac myocytes, whereby the first 3 PDEs can degrade both cGMP and cAMP. [8][9][10][11] cGMP is generally considered as a cardioprotective second messenger, 12 because pharmacological elevation of cGMP levels either by inhibition of cGMP-hydrolyzing PDE5 and PDE1 or by activation of NO-GC and pGC prevents pathological cardiomyocyte growth in vitro 13 and cardiac remodeling in vivo. 2,14-16In This Issue, see p 1221Reliable cGMP measurements in adult cardiomyocytes have been challenging. 17 In adult heart, cGMP is present at much lower concentrations than cAMP and acts in a compartmentalized New Methods in Cardiovascular Biology© 2014 American Heart Association, Inc. Rationale: 3ʹ,5ʹ-Cyclic guanosine monophosphate (cGMP) is an important second messenger that regulates cardiac contractility and protects the heart from hypertrophy. However, because of the lack of real-time imaging techniques, specific subcellular mechanisms and spatiotemporal dynamics of cGMP in adult cardiomyocytes are not well understood.Objective: Our aim was to generate and characterize a novel cGMP sensor model to measure cGMP with nanomolar sensitivity in adult cardiomyocytes. Methods and Results:We generated transgenic mice with cardiomyocyte-specific expression of the highly sensitive cytosolic Förster resonance energy transfer-based cGMP biosensor red cGES-DE5 and performed the first Förster resonance energy transfer measurements of cGMP in intact adult mouse ventricular myocytes. We found very low (≈10 nmol/L) basal cytosolic cGMP levels, which can be markedly increased by natriuretic peptides (C-type natriuretic peptide >> atrial natriuretic peptide) and, to a much smaller extent, by the direct stimulation of soluble guanylyl cyclase. Constitutive activity of this cyclase contributes to basal cGMP production, which is balanced by the activity of clinically established phosphodiesterase (PDE) families. The PDE3 inhibitor, cilostamide, showed especially strong cGMP responses. In a mild model of cardiac hypertrophy after transverse aortic constriction, PDE3 effects were not affected, whereas the contribution of PDE5 was increased. In addition, after natriuretic peptide stimulation, PDE3 was also involved in cGMP/cAMP crosstalk. Conclusions:

Precise positioning of the mitotic spindle is important for specifying the plane of cell division and the subsequent partitioning of the cell's contents to the daughter cells. Studies on different organisms and cell types have suggested diverse centering mechanisms: astral microtubules grow out from the spindle and push against the cortex, cortical dynein motors pull on astral microtubules, and dynein-dependent organelle transport on astral microtubules leads to a reactive force on the spindle. The different mechanisms lead to different predictions for the precision of centering, how mutations effect the precision, and the magnitude of the forces associated with spindle centering. We used image processing to accurately track the position and orientation of the mitotic spindle during the first cell division in the C. elegans embryo. The high precision of centering, < 1% of cell diameter transverse to the anterior-posterior axis, increased after RNAi against gpr-1/2, genes encoding activators of the cortical force generators; this suggests that centering is not mediated by gpr-1/2-dependent cortical pulling forces. To measure the forces associated with spindle positioning, we built a magnetic tweezers apparatus so that forces could be exerted on the spindle via beads incorporated into the embryo: forces of approximately 20 pN were required to displace the spindle through 1 mm. These mechanical experiments constrain molecular models of the centering process.

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