BACKGROUND:Atrial fibrillation (AF) is associated by alterations in cardiomyocyte membrane microdomain organization that can modify the distribution and biophysical properties of L-type Ca2+ channels (LTCCs), contributing to downregulation of the L-type Ca2+ current (ICa,L). OBJECTIVE:This study aimed to examine the role of cellular microarchitecture and microdomain-specific changes of single LTCCs in ICa,L remodeling in chronic AF. METHODS:Right atrial (RA) and left atrial (LA) biopsies from patients in sinus rhythm (SR, n = 51) and AF (n = 61) were analyzed to assess cardiomyocyte microarchitecture and microdomain-specific remodeling of single LTCCs and ICa,L. Computational modeling estimated the contributions of different cellular components in ICa,L reduction. RESULTS:In AF, ICa,L was reduced in RA (∼32%, P < .05) and LA (∼71%, P < .01). In RA, this was associated with preserved transverse (T)-tubular LTCCs (T-LTCCs) density and decreased extra-tubular (crest, C-LTCCs) density. In LA-AF, densities of both T- and C-LTCCs were reduced. These changes paralleled T-tubule downregulation in both atria and an LA-specific decrease in caveolin-3 expression. In AF, the open probabilities of T- and C-LTCCs in both atria were 3-5-fold higher than in SR and accompanied by 2-fold increase in protein kinase A and phosphorylated Ca2+/calmodulin kinase II activities. However, computational simulations showed that enhanced LTCC open probability did not compensate for reduced channel density. Caveolin-3 overexpression in LA-AF cardiomyocytes increased CaV1.2 membrane expression, partially restoring ICa,L. CONCLUSION:ICa,L downregulation in AF is driven by chamber-specific degradation of membrane structures and loss of functional LTCCs, not offset by increased channel activity. Caveolin-3 is crucial for maintaining functional LTCCs at the sarcolemma.

(1) Pulmonary hypertension (PH)-associated right ventricular (RV) failure is linked to a reduction in pulmonary vasodilators. Treprostinil has shown effectiveness in PAH patients with cardiac decompensation, hinting at potential cardiac benefits. We investigated treprostinil’s synergy with isoprenaline in RV and LV cardiomyocytes. We hypothesised that disease-related RV structural changes in cardiomyocytes would reduce contractile responses and cAMP/PKA signalling activity. (2) We induced PH in male Sprague Dawley rats using monocrotaline and isolated their ventricular cardiomyocytes. The effect of in vitro treprostinil and isoprenaline stimulation on contraction was assessed. FRET microscopy was used to study PKA activity associated with treprostinil stimulation in AKAR3-NES FRET-based biosensor-expressing cells. (3) RV cells exhibited maladaptive remodelling with hypertrophy, impaired contractility, and calcium transients compared to control and LV cardiomyocytes. Combining treprostinil and isoprenaline failed to enhance inotropy in PH RV cardiomyocytes. PH RV cardiomyocytes displayed an aberrant contractile behaviour, which the combination treatment could not rectify. Finally, we observed decreased PKA activity in treprostinil-treated PH RV cardiomyocytes. (4) PH-associated RV cardiomyocyte remodelling reduced treprostinil sensitivity, inotropic support, and impaired relaxation. Overall, this study highlights the complexity of RV dysfunction in advanced PH and suggests the need for alternative therapeutic strategies.

Background: β 1 AR (beta-1 adrenergic receptor) and β 2 AR (beta-2 adrenergic receptor)-mediated cyclic adenosine monophosphate signaling has distinct effects on cardiac function and heart failure progression. However, the mechanism regulating spatial localization and functional compartmentation of cardiac β-ARs remains elusive. Emerging evidence suggests that microtubule-dependent trafficking of mRNP (messenger ribonucleoprotein) and localized protein translation modulates protein compartmentation in cardiomyocytes. We hypothesized that β-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. Methods: The localization pattern of β-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on β-AR mRNA localization was studied using vinblastine, and its effect on receptor localization and function was evaluated with immunofluorescent and high-throughput Förster resonance energy transfer microscopy. An mRNA protein co-detection assay identified plausible β-AR translation sites in cardiomyocytes. The mechanism by which β-AR mRNA is redistributed post–heart failure was elucidated by single molecule fluorescence in situ hybridization, nanobiopsy, and high-throughput Förster resonance energy transfer microscopy on 16 weeks post–myocardial infarction and detubulated cardiomyocytes. Results: β 1 AR and β 2 AR mRNAs show differential localization in cardiomyocytes, with β 1 AR found in the perinuclear region and β 2 AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of β 2 AR transcripts toward the perinuclear region. The close proximity between β 2 AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of β 2 AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both β 1 AR and β 2 AR mRNAs are redistributed toward the cell periphery, similar to what is seen in cardiomyocytes undergoing drug-induced detubulation. This suggests that t-tubule remodeling contributes to β-AR mRNA redistribution and impaired β 2 AR function in failing hearts. Conclusions: Asymmetrical microtubule-dependent trafficking dictates differential β 1 AR and β 2 AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 β-ARs on the plasma membrane. The localization pattern is altered post–myocardial infarction, resulting from transverse tubule remodeling, leading to distorted β 2 AR-mediated cyclic adenosine monophosphate signaling.

Data sharing is not applicable to this article as no new data were created or analysed in this study.

[This corrects the article DOI: 10.3389/fcvm.2022.869585.].

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and in the absence of culprit coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS. Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal), cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4), and G-protein subunit Gβ (GNB1) as underlying these effects. Conclusion miR-16 and miR-26a sensitize the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future.

HomeCirculation ResearchVol. 130, No. 9Junctophillin-2: Coupling Hopes for Cardiac Gene Therapy to Gene Transcription Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBJunctophillin-2: Coupling Hopes for Cardiac Gene Therapy to Gene Transcription Peter Wright and Julia Gorelik Peter WrightPeter Wright School of Life and Health Sciences, University of Roehampton, London, United Kingdom (P.W.). National Heart, and Lung Institute (NHLI), Faculty of Medicine, Imperial College London, United Kingdom (P.W., J.G.). and Julia GorelikJulia Gorelik Correspondence to: Julia Gorelik, PhD, Cardiac Section, National Heart, and Lung Institute (NHLI), Faculty of Medicine, Imperial College London, Hammersmith Campus, Du Cane Rd, London W12 0NN, United Kingdom. Email E-mail Address: [email protected] https://orcid.org/0000-0003-1148-9158 National Heart, and Lung Institute (NHLI), Faculty of Medicine, Imperial College London, United Kingdom (P.W., J.G.). Originally published28 Apr 2022https://doi.org/10.1161/CIRCRESAHA.122.321066Circulation Research. 2022;130:1318–1320This article is a commentary on the followingGene Therapy With the N-Terminus of Junctophilin-2 Improves Heart Failure in MiceHeart failure continues to have devasting societal and economic impacts. Current gold-standard pharmacological treatments only provide symptomatic relief, meaning that surgical replacement of failing hearts (through transplant) remains the sole curative intervention.1 In the past decade, hopes were raised that therapies using (adeno and adeno-associated [AAV]) viral vectors—targeting aberrant cellular genetic programs, would restore a normal cardiac phenotype by modulating the expression of crucial proteins.2 The 2 sets of human clinical trials which attempted these gene therapies, sought to modulate the expression of sarcoplasmic reticulum calcium ATPase3and adenyl cyclase 6.4 These proteins are crucial components of cardiomyocyte excitation-contraction coupling machinery modulating the abbreviation of cellular calcium-induced calcium release and the phosphorylation/activation of calcium-handling proteins, respectively. In the aftermath of these trials which had neutral results, researchers began to question whether directly targeting excitation-contraction coupling was the correct approach. The pathological remodeling of the myocardium during heart failure is driven by the activation of a fetal gene program. This hypertrophic program modulates a panel of genes causing significant changes to cardiomyocyte structure, which are initially compensatory, but deleterious in the medium to long-term. Thus, targeting the transcriptional machinery which controls hypertrophic signaling, has been suggested as an alternative approach for therapies.5Article, see p 1306In this issue, Wang et al6 describe an approach that may bridge the two approaches through the singular behavior of the structural protein JPH-2 (junctophillin 2).The plasma membrane of the adult cardiomyocyte is penetrated at regular intervals by tubular elements, which form part of a wider transverse-axial tubular system. The transverse-axial tubular system permits the transduction of ion currents rapidly and synchronously throughout the cardiomyocyte, where diffusion will not suffice. L-type calcium channels (LTCCs) modulate calcium-induced calcium release at junctional membrane complexes within the transverse-axial tubular system. JPH-2, a scaffolding protein, recruits functional LTCCs to the junctional membrane complexes, associating them with the ryanodine receptors which drive the release of calcium ions through calcium-induced calcium release.7 JPH-2 supports the structural integrity of the transverse-axial tubular system through a cholesterol dependent mechanism and interaction with the cytoskeleton through microtubules.8,9 Alongside, these crucial functions JPH-2 was discovered to have, potentially salutary, transcription factor activity.10 In situations of cellular stress JPH-2 can be cleaved by calpain-1. This process yields an N-terminal peptide (JPH-2NT) which can move to the nucleus and bind to TATA-box elements which has the effect of repressing the prohypertrophic gene program induced by cardiac pathology. This unusual, multifaceted behavior provides an important homeostatic mechanism linking the structures which control excitation-contraction coupling and the processes which govern the induction of prohypertrophic signaling.10Wang et al6 have demonstrated that mice overexpressing JPH-2NT have a significantly milder cardiac phenotype following the induction of cardiac hypertrophy following transverse aortic constriction (TAC). The hearts of mice overexpressing JPH-2NT display much less fibrosis and a preserved cellular TAT network. An investigation of the genetic changes induced by JPH-2NT suggests that this is, in part, due to the reversal of deleterious prohypertrophic signaling programs. Overexpression of JPH-2NT is a promising strategy for cardiac gene therapy as it retains the useful properties of earlier gene therapy approaches (maintaining inotropy and preserving cellular ultrastructure) but also offers the broadband reversal of hypertrophic signaling.Neonatal mice were infected with AAV (2/9) vectors expressing JPH-2NT or a mutant peptide incapable of binding DNA (ΔbNLS/ARR). After 9 weeks, the mice were subjected to transaortic constriction to induce cardiac stress and failure due to hypertrophy. After 5 weeks of TAC, mice that received the bona fide JPH-2NT retained a significant degree of cardiac functionality, exhibited far less cardiac remodeling and fibrosis in comparison to mice that received eGFP (enhanced green fluorescent protein) or ΔbNLS/ARR. None of the vectors induced aberrant effects in the SHAM control animals. The positive functional effects of JPH-2NT overexpression in TAC animals appeared to correlate with changes in the cardiomyocytes at the ultrastructural level. TAT regularity was preserved in mice that received JPH-2NT. Importantly, the presence of AAV was confirmed in cardiomyocytes infected by the 3 vectors. Only cells infected with AAV-expressing JPH-2NT had preserved TAT integrity (at levels similar to SHAM animals). The phenotype of mice subjected to TAC continued to worsen past 8 weeks in animals infected with AAV-eGFP but animals which received AAV-JPH-2NT escaped the phenotype. An RNA-Seq investigation demonstrated that JPH-2NT infection prevented the induction of hypertrophic and heart failure related gene programs.As discussed, human clinical trials of cardiac gene therapy have so far only targeted 2 genes, with indifferent results. A significant number of other preclinical trials (in mice and large animal species) have shown efficacy of a gene targeting approach, across different phenotypes, but failed to make the transition to human trials.2 Numerous potential reasons for the inability of human trials to meet their primary goals have been posited, including issues with the vector, gene, delivery, human heterogeneity, and the designs of studies themselves. The purview of this study was to investigate a better genetic target. A lack of beneficial effect of protein overexpression or immunogenicity have been speculated to be the reasons for nonefficacy stemming from the target selection. In this instance, JPH-2NT undoubtedly has efficacy in this preclinical model and there were no indications of adverse immune effects. JPH-2NT based therapy offers the potential benefits indicated by early investigations of gene therapy with sarcoplasmic reticulum calcium ATPase in preclinical models. These include the restoration of cardiac function and the maintenance of normal cardiomyocyte ultrastructure.11 As a therapeutic strategy JPH-2NT also offers a broadband modification of the induction of prohypertrophic transcription. Numerous transcriptional regulator targets, such as BRD4, CDK8/9, and HDAC, have been identified for pharmacological therapies but targeting these processes with genetic manipulation offers a more sustainable approach.5This study prepares mice by infecting them with AAV before inducing cardiac damage. Therefore, the phenomena that the article presents is the escape from the cardiac phenotype induced by TAC after JPH-2NT delivery. Clearly, this approach is not currently viable (or perhaps even desirable) in human patients. Therefore, the focus of further studies should be to evaluate whether JPH-2NT application to wild-type mice with preexisting heart failure, has a therapeutic effect. One important finding of the study relates the choice of vector. AAV2 remains the only rationally designed AAV-vector with excellent tropism and transduction efficiency for cardiomyocytes. The liver tropism which seems to ruin the efficacy of other vectors is not apparent in this study.12 A significant amount of the viral vector seems to collect in the skeletal muscle of infected animals. JPH-2 performs extensive functions within skeletal muscle (another type of striated muscle) which are extremely similar to those performed in cardiac muscle.13 The significance of the overexpression of JPH-2 in skeletal muscle induced by this vector should, therefore, be explored. Unlike, cardiac muscle, skeletal muscle hypertrophy seems to invariably associate with improved health outcomes, especially in senescent patients. But the specific effect of JPH-2NT on skeletal muscle hypertrophy is unclear.14 Finally, it must be mentioned that the potential utility of the JPH-2 molecule, with respect to transcription factor manipulation, is not limited to the N terminus, given that a fragment of the C terminus has also been demonstrated to have transcriptional activity.15In summary, promoting beneficial anti-hypertrophic signaling and the preservation of cell structure by overexpressing the JPH-2NT fragment is a promising avenue for cardiac therapy. Targeting JPH-2 related mechanisms recapitulates the useful effects of past gene therapy approaches linking maintenance of excitation-contraction-coupling with a novel broader effect on prohypertrophic gene programs.Article InformationSources of FundingThis work was supported by British Heart Foundation Grant (RG/17/13/33173 to Dr Gorelik).Disclosures None.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.For Sources of Funding and Disclosures, see page 1319 & 1320.Correspondence to: Julia Gorelik, PhD, Cardiac Section, National Heart, and Lung Institute (NHLI), Faculty of Medicine, Imperial College London, Hammersmith Campus, Du Cane Rd, London W12 0NN, United Kingdom. Email j.[email protected]ac.ukReferences1. Virani SS, Alonso A, Aparicio HJ, Benjamin EJ, Bittencourt MS, Callaway CW, Carson AP, Chamberlain AM, Cheng S, Delling FN, et al; American Heart Association Council on Epidemiology and Prevention Statistics Committee and Stroke Statistics Subcommittee. Heart disease and stroke statistics-2021 update: a report from the American Heart Association.Circulation. 2021; 143:e254–e743. doi: 10.1161/CIR.0000000000000950LinkGoogle Scholar2. Ishikawa K, Weber T, Hajjar RJ. Human cardiac gene therapy.Circ Res. 2018; 123:601–613. doi: 10.1161/CIRCRESAHA.118.311587LinkGoogle Scholar3. Greenberg B, Butler J, Felker GM, Ponikowski P, Voors AA, Desai AS, Barnard D, Bouchard A, Jaski B, Lyon AR, et al. Calcium upregulation by percutaneous administration of gene therapy in patients with cardiac disease (CUPID 2): a randomised, multinational, double-blind, placebo-controlled, phase 2b trial.Lancet. 2016; 387:1178–1186. doi: 10.1016/S0140-6736(16)00082-9CrossrefMedlineGoogle Scholar4. Hammond HK, Penny WF, Traverse JH, Henry TD, Watkins MW, Yancy CW, Sweis RN, Adler ED, Patel AN, Murray DR, et al. Intracoronary gene transfer of adenylyl cyclase 6 in patients with heart failure: a randomized clinical trial.JAMA Cardiol. 2016; 1:163–171. doi: 10.1001/jamacardio.2016.0008CrossrefMedlineGoogle Scholar5. Minerath RA, Hall DD, Grueter CE. Targeting transcriptional machinery to inhibit enhancer-driven gene expression in heart failure.Heart Fail Rev. 2019; 24:725–741. doi: 10.1007/s10741-019-09792-3CrossrefMedlineGoogle Scholar6. Wang J, Shi Q, Wang Y, Dawson LW, Ciampa G, Zhao W, Zhang G, Chen B, Weiss RM, Grueter CE, et al. Gene therapy with the N-terminus of Junctophilin-2 improves heart failure in mice.Circ Res. 2022; 130:1306–1317. doi: 10.1161/CIRCRESAHA.121.320680LinkGoogle Scholar7. Gross P, Johnson J, Romero CM, Eaton DM, Poulet C, Sanchez-Alonso J, Lucarelli C, Ross J, Gibb AA, Garbincius JF, et al. Interaction of the joining region in Junctophilin-2 With the L-Type Ca2+ Channel Is pivotal for cardiac dyad assembly and intracellular Ca2+ dynamics.Circ Res. 2021; 128:92–114. doi: 10.1161/CIRCRESAHA.119.315715LinkGoogle Scholar8. Poulet C, Sanchez-Alonso J, Swiatlowska P, Mouy F, Lucarelli C, Alvarez-Laviada A, Gross P, Terracciano C, Houser S, Gorelik J. Junctophilin-2 tethers T-tubules and recruits functional L-type calcium channels to lipid rafts in adult cardiomyocytes.Cardiovasc Res. 2021; 117:149–161. doi: 10.1093/cvr/cvaa033CrossrefMedlineGoogle Scholar9. Zhang C, Chen B, Guo A, Zhu Y, Miller JD, Gao S, Yuan C, Kutschke W, Zimmerman K, Weiss RM, et al. Microtubule-mediated defects in junctophilin-2 trafficking contribute to myocyte transverse-tubule remodeling and Ca2+ handling dysfunction in heart failure.Circulation. 2014; 129:1742–1750. doi: 10.1161/CIRCULATIONAHA.113.008452LinkGoogle Scholar10. Ang G, Yihui W, Biyi C, Yunhao W, Jinxiang Y, Liyang Z, Duane H, Jennifer W, Yun S, Qi Z, et al. E-C coupling structural protein junctophilin-2 encodes a stress-adaptive transcription regulator.Science (80-). 2018; 362:eaan3303. doi: 10.1126/science.aan3303CrossrefMedlineGoogle Scholar11. Lyon AR, Nikolaev VO, Miragoli M, Sikkel MB, Paur H, Benard L, Hulot J-S, Kohlbrenner E, Hajjar RJ, Peters NS, et al. Plasticity of surface structures and β2-adrenergic receptor localization in failing ventricular cardiomyocytes during recovery from heart failure.Circ Hear Fail. 2012; 5:357–365. doi: 10.1161/CIRCHEARTFAILURE.111.964692LinkGoogle Scholar12. Asokan A, Conway JC, Phillips JL, Li C, Hegge J, Sinnott R, Yadav S, DiPrimio N, Nam HJ, Agbandje-McKenna M, et al. Reengineering a receptor footprint of adeno-associated virus enables selective and systemic gene transfer to muscle.Nat Biotechnol. 2010; 28:79–82. doi: 10.1038/nbt.1599CrossrefMedlineGoogle Scholar13. Perni S. The Builders of the junction: roles of Junctophilin1 and Junctophilin2 in the assembly of the sarcoplasmic reticulum–plasma membrane junctions in striated muscle.Biomolecules. 2022; 12:109. doi: 10.3390/biom12010109CrossrefMedlineGoogle Scholar14. Springer J, Springer JI, Anker SD. Muscle wasting and sarcopenia in heart failure and beyond: update 2017.ESC Heart Fail. 2017; 4:492–498. doi: 10.1002/ehf2.12237CrossrefMedlineGoogle Scholar15. Lahiri SK, Quick AP, Samson-Couterie B, Hulsurkar M, Elzenaar I, van Oort RJ, Wehrens XHT. Nuclear localization of a novel calpain-2 mediated junctophilin-2 C-terminal cleavage peptide promotes cardiomyocyte remodeling.Basic Res Cardiol. 2020; 115:49. doi: 10.1007/s00395-020-0807-1CrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesGene Therapy With the N-Terminus of Junctophilin-2 Improves Heart Failure in MiceJinxi Wang, et al. Circulation Research. 2022;130:1306-1317 April 29, 2022Vol 130, Issue 9 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCRESAHA.122.321066PMID: 35482830 Originally publishedApril 28, 2022 Keywordsheart failuresarcoplasmic reticulumphosphorylationgenetic therapyEditorialsphenotypePDF download Advertisement

Beta-adrenoceptors (βAR) are often viewed as archetypal G-protein coupled receptors. Over the past fifteen years, investigations in cardiovascular biology have provided remarkable insights into this receptor family. These studies have shifted pharmacological dogma, from one which centralized the receptor to a new focus on structural micro-domains such as caveolae and t-tubules. Important studies have examined, separately, the structural compartmentation of ion channels and βAR. Despite links being assumed, relatively few studies have specifically examined the direct link between structural remodeling and electrical remodeling with a focus on βAR. In this review, we will examine the nature of receptor and ion channel dysfunction on a substrate of cardiomyocyte microdomain remodeling, as well as the likely ramifications for cardiac electrophysiology. We will then discuss the advances in methodologies in this area with a specific focus on super-resolution microscopy, fluorescent imaging, and new approaches involving microdomain specific, polymer-based agonists. The advent of powerful computational modelling approaches has allowed the science to shift from purely empirical work, and may allow future investigations based on prediction. Issues such as the cross-reactivity of receptors and cellular heterogeneity will also be discussed. Finally, we will speculate as to the potential developments within this field over the next ten years.

Cardiomyocyte β3-adrenoceptors (β3-ARs) coupled to soluble guanylyl cyclase (sGC)-dependent production of the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) have been shown to protect from heart failure. However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points in contrast to the clustering problems associated with isolated cell studies. The greater productivity and sampling power provided by CC-HTS may help to reestablish the utility of isolated cell studies, while preserving the unique insights provided by studying the contribution of the fundamental, cellular unit of myocardial contractility.

The field of cardiomyocyte mechanobiology is gaining significant attention, due to accumulating evidence concerning the significant role of cellular mechanical effects on the integrated function of the heart. To date, the protein titin has been demonstrated as a major contributor to the cardiomyocytes Young's modulus (YM). The microtubular network represents another potential regulator of cardiac mechanics. However, the contribution of microtubules (MTs) to the membrane YM is still understudied and has not been interrogated in the context of myocardial infarction (MI) or mechanical loading and unloading. Using nanoscale mechanoscanning ion conductance microscopy, we demonstrate that MTs contribute to cardiomyocyte transverse YM in healthy and pathological states with different mechanical loading. Specifically, we show that posttranslational modifications of MTs have differing effects on cardiomyocyte YM: Acetylation provides flexibility, whereas detyrosination imparts rigidity. Further studies demonstrate that there is no correlation between the total protein amount of acetylated and detyrosinated MT. Yet, in the polymerized-only populations, an increased level of acetylation results in a decline of detyrosinated MTs in an MI model.

Multiple intra-cellular signalling pathways rely on calcium and 3′–5′ cyclic adenosine monophosphate (cAMP) to act as secondary messengers. This is especially true in cardiomyocytes which act as the force-producing units of the cardiac muscle and are required to react rapidly to environmental stimuli. The specificity of functional responses within cardiomyocytes and other cell types is produced by the organellar compartmentation of both calcium and cAMP. In this review, we assess the role of molecular localisation and relative contribution of active and passive processes in producing compartmentation. Active processes comprise the creation and destruction of signals, whereas passive processes comprise the release or sequestration of signals. Cardiomyocytes display a highly articulated membrane structure which displays significant cell-to-cell variability. Special attention is paid to the way in which cell membrane caveolae and the transverse-axial tubule system allow molecular localisation. We explore the effects of cell maturation, pathology and regional differences in the organisation of these processes. The subject of signal compartmentation has had a significant amount of attention within the cardiovascular field and has undergone a revolution over the past two decades. Advances in the area have been driven by molecular imaging using fluorescent dyes and genetically encoded constructs based upon fluorescent proteins. We also explore the use of scanning probe microscopy in the area. These techniques allow the analysis of molecular compartmentation within specific organellar compartments which gives researchers an entirely new perspective.

Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time CAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium- potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of CAMP compartmentation in heart failure. Methods and results A novel Forster resonance energy transfer (FRED-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by beta(2)-adrenoceptors (beta(2)-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific beta(2)-AR/cAMP signals and beta(2)-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct CAMP microdomain which is directly regulated by beta(2)-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted beta(2)-AR signalling to cAMP in the vicinity of PLM.

Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (βAR-cAMP) microdomains. Previous studies have investigated PMU using a model of heterotopic-heart and lung transplantation (HTHAL). These studies have demonstrated that PMU disrupts the structure of cardiomyocytes and calcium handling. We sought to understand these processes by studying L-Type Calcium Channel (LTCC) activity and sub-type-specific βAR-cAMP signaling within cardiomyocyte membrane microdomains.Method: We utilized an 8-week model of HTHAL, whereby the hearts of syngeneic Lewis rats were transplanted into the abdomens of randomly assigned cage mates. A pronounced atrophy was observed in hearts after HTHAL. Cardiomyocytes were isolated via enzymatic perfusion. We utilized Förster Resonance Energy Transfer (FRET) based cAMP-biosensors and scanning ion conductance microscopy (SICM) based methodologies to study localization of LTCC and βAR-cAMP signaling.Results: β2AR-cAMP responses measured by FRET in the cardiomyocyte cytosol were reduced by PMU (loaded 28.51 ± 7.18% vs. unloaded 10.84 ± 3.27% N,n 4/10-13 mean ± SEM ∗p < 0.05). There was no effect of PMU on β2AR-cAMP signaling in RII_Protein Kinase A domains. β1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that β2AR-cAMP was specifically reduced in t-tubules (TTs) after PMU (loaded TT 0.721 ± 0.106% vs. loaded crest 0.104 ± 0.062%, unloaded TT 0.112 ± 0.072% vs. unloaded crest 0.219 ± 0.084% N,n 5/6-9 mean ± SEM ∗∗p < 0.01, ∗∗∗p < 0.001 vs. loaded TT). By comparison β1AR-cAMP responses in either TT or sarcolemmal crests were unaffected by the PMU. LTCC occurrence and open probability (Po) were reduced by PMU (loaded TT Po 0.073 ± 0.011% vs. loaded crest Po 0.027 ± 0.006% N,n 5/18-26 mean ± SEM ∗p < 0.05) (unloaded TT 0.0350 ± 0.003% vs. unloaded crest Po 0.025 N,n 5/20-30 mean ± SEM NS #p < 0.05 unloaded vs. loaded TT). We discovered that PMU had reduced the association between Caveolin-3, Junctophilin-2, and Cav1.2.Discussion: PMU suppresses’ β2AR-cAMP and LTCC activity. When activated, the signaling of β2AR-cAMP and LTCC become more far-reaching after PMU. We suggest that a situation of ‘suppression/decompartmentation’ is elicited by the loss of refined cardiomyocyte structure following PMU. As PMU is a component of modern device therapy for heart failure this study has clinical ramifications and raises important questions for regenerative medicine.

Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to β2-adrenoceptor (β2AR) stimulation, which may underlie the development of Takotsubo cardiomyopathy. However, both cell types produce comparable cytosolic amounts of the second messenger cAMP. We investigated this discrepancy using nanoscale imaging techniques and found that, structurally, basal cardiomyocytes have more organized membranes (higher T-tubular and caveolar densities). Local membrane microdomain responses measured in isolated basal cardiomyocytes or in whole hearts revealed significantly smaller and more short-lived β2AR/cAMP signals. Inhibition of PDE4, caveolar disruption by removing cholesterol or genetic deletion of Cav3 eliminated differences in local cAMP production and equilibrated the contractile response to β2AR. We conclude that basal cells possess tighter control of cAMP because of a higher degree of signaling microdomain organization. This provides varying levels of nanostructural control for cAMP-mediated functional effects that orchestrate macroscopic, regional physiological differences within the heart.

INTRODUCTION:To measure the range of T1 values of synovitis using three Tesla (3 T) magnetic resonance imaging (MRI) in patients with osteoarthritis to assess the potential of T1 mapping for identifying synovitis from other features in the knee on unenhanced magnetic resonance scans. METHODS:After receiving institutional ethical approval, 83 patients who met the American College of Rheumatology criteria for diagnosis of osteoarthritis of the knee were scanned using a 3 T Magnetic Resonance Imaging scanner. T1 maps were calculated from spoiled gradient echo images acquired with five different flip angles of 5°-25°. Mean values for the T1 measurements were calculated and compared to existing data from the published literature for anatomical and pathological structures of the knee. RESULTS:T1 values recorded in patients suffering from osteoarthritis demonstrated that T1 values for synovitis (confirmed on gadolinium enhanced images) fall in a narrow range (849-1277 ms, mean 1005 ms SD 91) delineating this from other structures of the knee such as muscle (T1 value range 1305-2638 ms, mean 1785 ms SD 304) and synovial fluid (T1 value range 3867-4129 ms, mean 3915 SD 899) at 3 T. CONCLUSION:T1 values measured in synovitis in patients with osteoarthritis of the knee demonstrated a range of values distinct to those measured in muscle and synovial fluid at 3 T. This offers potential for the use of T1 maps to delineate or quantify synovitis in patients who are unwilling or unable to receive injectable contrast agents.

Heart failure leads to altered 2-adrenoceptor/cAMP dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain