Pulmonary Hypertension-Associated Right Ventricular Cardiomyocyte Remodelling Reduces Treprostinil Function.

(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.

Microtubule-Mediated Regulation of ß2AR Translation and Function in Failing Hearts.

BACKGROUND: ß1AR (beta-1 adrenergic receptor) and ß2AR (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 ß-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 ß-AR compartmentation in cardiomyocytes is accomplished by selective trafficking of its mRNAs and localized translation. METHODS: The localization pattern of ß-AR mRNA was investigated using single molecule fluorescence in situ hybridization and subcellular nanobiopsy in rat cardiomyocytes. The role of microtubule on ß-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 ß-AR translation sites in cardiomyocytes. The mechanism by which ß-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: ß1AR and ß2AR mRNAs show differential localization in cardiomyocytes, with ß1AR found in the perinuclear region and ß2AR showing diffuse distribution throughout the cell. Disruption of microtubules induces a shift of ß2AR transcripts toward the perinuclear region. The close proximity between ß2AR transcripts and translated proteins suggests that the translation process occurs in specialized, precisely defined cellular compartments. Redistribution of ß2AR transcripts is microtubule-dependent, as microtubule depolymerization markedly reduces the number of functional receptors on the membrane. In failing hearts, both ß1AR and ß2AR 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 ß-AR mRNA redistribution and impaired ß2AR function in failing hearts. CONCLUSIONS: Asymmetrical microtubule-dependent trafficking dictates differential ß1AR and ß2AR localization in healthy cardiomyocyte microtubules, underlying the distinctive compartmentation of the 2 ß-ARs on the plasma membrane. The localization pattern is altered post-myocardial infarction, resulting from transverse tubule remodeling, leading to distorted ß2AR-mediated cyclic adenosine monophosphate signaling.

Corrigendum: Hyperthermia as a trigger for Takotsubo syndrome in a rat model.

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

Hyperthermia as a trigger for Takotsubo syndrome in a rat model.

Takotsubo syndrome is a well-characterized cause of acute yet reversible heart failure associated with periods of intense emotional stress, often mimicking on presentation an acute coronary syndrome. Animal models of Takotsubo syndrome have been developed, either through the application of a stressor, or administration of exogenous catecholamine. We found that in a model of isoproterenol-induced Takotsubo syndrome in anesthetized rats hyperthermia (40-41°C) would occur after the administration of isoproterenol. Maintenance of this hyperthermia would result in an apical hypocontractility typical of the syndrome, whereas prevention of hyperthermia with active cooling to maintain a euthermic core body temperature prevented (but did not subsequently reverse) apical hypocontractility. In vitro experimentation with isolated cardiomyocytes showed no effect of hyperthermia on either baseline contractility or contractility change after beta-adrenoceptor stimulation. We suggest that the rise in body temperature that is characteristic of catecholamine storm may be a component in the development of Takotsubo syndrome.

Electrophysiological Remodeling: Cardiac T-Tubules and ß-Adrenoceptors.

Beta-adrenoceptors (ßAR) 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 ßAR. Despite links being assumed, relatively few studies have specifically examined the direct link between structural remodeling and electrical remodeling with a focus on ßAR. 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.

Approaches to High-Throughput Analysis of Cardiomyocyte Contractility.

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.

ß3-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes.

Cardiomyocyte ß3-adrenoceptors (ß3-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 ß3-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 ß3-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 ß3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.

Studying signal compartmentation in adult cardiomyocytes.

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.

Microtubules regulate cardiomyocyte transversal Young's modulus.

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.

Heart failure leads to altered ß2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain.

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 Förster resonance energy transfer (FRET)-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 ß2-adrenoceptors (ß2-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 ß2-AR/cAMP signals and ß2-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 ß2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted ß2-AR signalling to cAMP in the vicinity of PLM.

Partial Mechanical Unloading of the Heart Disrupts L-Type Calcium Channel and Beta-Adrenoceptor Signaling Microdomains.

Introduction: We investigated the effect of partial mechanical unloading (PMU) of the heart on the physiology of calcium and beta-adrenoceptor-cAMP (ßAR-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 ßAR-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 ßAR-cAMP signaling. Results: ß2AR-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 ß2AR-cAMP signaling in RII_Protein Kinase A domains. ß1AR-cAMP was unaffected by PMU in either microdomain. Consistent with this SICM/FRET analysis demonstrated that ß2AR-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 ß1AR-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' ß2AR-cAMP and LTCC activity. When activated, the signaling of ß2AR-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.

Cardiomyocyte Membrane Structure and cAMP Compartmentation Produce Anatomical Variation in ß2AR-cAMP Responsiveness in Murine Hearts.

Cardiomyocytes from the apex but not the base of the heart increase their contractility in response to ß2-adrenoceptor (ß2AR) 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 ß2AR/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 ß2AR. 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.

Microtubule-Dependent Mitochondria Alignment Regulates Calcium Release in Response to Nanomechanical Stimulus in Heart Myocytes.

Arrhythmogenesis during heart failure is a major clinical problem. Regional electrical gradients produce arrhythmias, and cellular ionic transmembrane gradients are its originators. We investigated whether the nanoscale mechanosensitive properties of cardiomyocytes from failing hearts have a bearing upon the initiation of abnormal electrical activity. Hydrojets through a nanopipette indent specific locations on the sarcolemma and initiate intracellular calcium release in both healthy and heart failure cardiomyocytes, as well as in human failing cardiomyocytes. In healthy cells, calcium is locally confined, whereas in failing cardiomyocytes, calcium propagates. Heart failure progressively stiffens the membrane and displaces sub-sarcolemmal mitochondria. Colchicine in healthy cells mimics the failing condition by stiffening the cells, disrupting microtubules, shifting mitochondria, and causing calcium release. Uncoupling the mitochondrial proton gradient abolished calcium initiation in both failing and colchicine-treated cells. We propose the disruption of microtubule-dependent mitochondrial mechanosensor microdomains as a mechanism for abnormal calcium release in failing heart.

Studying GPCR/cAMP pharmacology from the perspective of cellular structure.

Signal transduction via G-protein coupled receptors (GPCRs) relies upon the production of cAMP and other signaling cascades. A given receptor and agonist pair, produce multiple effects upon cellular physiology which can be opposite in different cell types. The production of variable cellular effects via the signaling of the same GPCR in different cell types is a result of signal organization in space and time (compartmentation). This organization is usually based upon the physical and chemical properties of the membranes in which the GPCRs reside and the repertoire of downstream effectors and co-factors that are available at that location. In this review we explore mechanisms of GPCR signal compartmentation and broadly review the state-of-the-art methodologies which can be utilized to study them. We provide a clear rationale for a "localized" approach to the study of the pharmacology and physiology of GPCRs and particularly the secondary messenger cAMP.

Pathophysiology of takotsubo syndrome: temporal phases of cardiovascular responses to extreme stress.

Takotsubo syndrome (TTS), also known as takotsubo cardiomyopathy, is an acute heart failure syndrome that typically occurs after a period of great emotional stress. The archetypal patient is a postmenopausal woman who presents with chest pain, ST-segment elevation and acute hypokinesia of the apical and middle segment of the left ventricle that extends beyond the territory of a single coronary artery, coupled with hyperkinesia of the basal myocardium. Recent preclinical and clinical studies have shown the importance of high catecholamine levels in precipitating TTS. We propose that this is caused by activation of ß-adrenoceptors and the subsequent activation of a negatively-inotropic pathway, perhaps to protect the heart from catecholamine overload. We explore the pathophysiology of TTS according to its "phases", both preclinically and clinically. This will show that the condition is not one of static apical hypokinesia that simply improves, but rather a dynamic condition that changes as the disease progresses. We hope that further exploration of TTS using its "phases" will aid in its characterization, diagnosis and treatment.

Caveolin-3 regulates compartmentation of cardiomyocyte beta2-adrenergic receptor-mediated cAMP signaling.

The purpose of this study was to investigate whether caveolin-3 (Cav3) regulates localization of ß2-adrenergic receptor (ß2AR) and its cAMP signaling in healthy or failing cardiomyocytes. We co-expressed wildtype Cav3 or its dominant-negative mutant (Cav3DN) together with the Förster resonance energy transfer (FRET)-based cAMP sensor Epac2-camps in adult rat ventricular myocytes (ARVMs). FRET and scanning ion conductance microscopy were used to locally stimulate ß2AR and to measure cytosolic cAMP. Cav3 overexpression increased the number of caveolae and decreased the magnitude of ß2AR-cAMP signal. Conversely, Cav3DN expression resulted in an increased ß2AR-cAMP response without altering the whole-cell L-type calcium current. Following local stimulation of Cav3DN-expressing ARVMs, ß2AR response could only be generated in T-tubules. However, the normally compartmentalized ß2AR-cAMP signal became diffuse, similar to the situation observed in heart failure. Finally, overexpression of Cav3 in failing myocytes led to partial ß2AR redistribution back into the T-tubules. In conclusion, Cav3 plays a crucial role for the localization of ß2AR and compartmentation of ß2AR-cAMP signaling to the T-tubules of healthy ARVMs, and overexpression of Cav3 in failing myocytes can partially restore the disrupted localization of these receptors.

Takotsubo cardiomyopathy: the pathophysiology.

Takotsubo cardiomyopathy (TTC) is an acute heart failure syndrome classically characterized by hypocontractile apical and midventricular regions of the left ventricle, with a compensatory hypercontractile base. Available data support the hypothesis that TTC and atypical TTC-like disorders are primarily induced by catecholaminergic overstimulation, with epinephrine playing a crucial role. Knowledge from the available preclinical models should be used to guide the development of potential clinical trials in the most severe cases, where rates of acute morbidity and mortality are highest, and also to prevent recurrence in susceptible individuals.

Spatial control of the ßAR system in heart failure: the transverse tubule and beyond.

The beta1-adrenoceptors (ß(1)AR) and beta-2 (ß(2)AR) adrenoceptors represent the predominant pathway for sympathetic control of myocardial function. Diverse mechanisms have evolved to translate signalling via these two molecules into differential effects on physiology. In this review, we discuss how the functions of the ßAR are organized from the level of secondary messengers to the whole heart to achieve this. Using novel microscopy and bio-imaging methods researchers have uncovered subtle organization of the control of cyclic adenosine monophosphate (cAMP), the predominant positively inotropic pathway for the ßAR. The ß(2)AR in particular is demonstrated to give rise to highly compartmentalized, spatially confined cAMP signals. Organization of ß(2)AR within the T-tubule and caveolae of cardiomyocytes concentrates this receptor with molecules which buffer and shape its cAMP signal to give fine control. This situation is undermined in various forms of heart failure. Human and animal models of heart failure demonstrate disruption of cellular micro-architecture which contributes to the change in response to cardiac ßARs. Loss of cellular structure has proved key to the observed loss of confined ß(2)AR signalling. Some pharmacological and genetic treatments have been successful in returning failing cells to a more structured phenotype. Within these cells it has been possible to observe the partial restoration of normal ß(2)AR signalling. At the level of the organ, the expression of the two ßAR subtypes varies between regions with the ß(2)AR forming a greater proportion of the ßAR population at the apex. This distribution may contribute to regional wall motion abnormalities in Takotsubo cardiomyopathy, a syndrome of high sympathetic activity, where the phosphorylated ß(2)AR can signal via Gi protein to produce negatively inotropic effects.