Exploring Syndecan-4 and MLP and Their Interaction in Primary Cardiomyocytes and H9c2 Cells.

The transmembrane proteoglycan syndecan-4 is known to be involved in the hypertrophic response to pressure overload. Although multiple downstream signaling pathways have been found to be involved in this response in a syndecan-4-dependent manner, there are likely more signaling components involved. As part of a larger syndecan-4 interactome screening, we have previously identified MLP as a binding partner to the cytoplasmic tail of syndecan-4. Interestingly, many human MLP mutations have been found in patients with hypertrophic (HCM) and dilated cardiomyopathy (DCM). To gain deeper insight into the role of the syndecan-4-MLP interaction and its potential involvement in MLP-associated cardiomyopathy, we have here investigated the syndecan-4-MLP interaction in primary adult rat cardiomyocytes and the H9c2 cell line. The binding of syndecan-4 and MLP was analyzed in total lysates and subcellular fractions of primary adult rat cardiomyocytes, and baseline and differentiated H9c2 cells by immunoprecipitation. MLP and syndecan-4 localization were determined by confocal microscopy, and MLP oligomerization was determined by immunoblotting under native conditions. Syndecan-4-MLP binding, as well as MLP self-association, were also analyzed by ELISA and peptide arrays. Our results showed that MLP-WT and syndecan-4 co-localized in many subcellular compartments; however, their binding was only detected in nuclear-enriched fractions of isolated adult cardiomyocytes. In vitro, syndecan-4 bound to MLP at three sites, and this binding was reduced in some HCM-associated MLP mutations. While MLP and syndecan-4 also co-localized in many subcellular fractions of H9c2 cells, these proteins did not bind at baseline or after differentiation into cardiomyocyte-resembling cells. Independently of syndecan-4, mutated MLP proteins had an altered subcellular localization in H9c2 cells, compared to MLP-WT. The DCM- and HCM-associated MLP mutations, W4R, L44P, C58G, R64C, Y66C, K69R, G72R, and Q91L, affected the oligomerization of MLP with an increase in monomeric at the expense of trimeric and tetrameric recombinant MLP protein. Lastly, two crucial sites for MLP self-association were identified, which were reduced in most MLP mutations. Our data indicate that the syndecan-4-MLP interaction was present in nuclear-enriched fractions of isolated adult cardiomyocytes and that this interaction was disrupted by some HCM-associated MLP mutations. MLP mutations were also linked to changes in MLP oligomerization and self-association, which may be essential for its interaction with syndecan-4 and a critical molecular mechanism of MLP-associated cardiomyopathy.

Myocardial SERCA2 Protects Against Cardiac Damage and Dysfunction Caused by Inhaled Bromine.

Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen-induced myocardial damage, we used bromine-exposed cardiac-specific SERCA2 knockout (KO) mice [tamoxifen-administered SERCA2 (flox/flox) Tg (αMHC-MerCreMer) mice] and compared them to the oil-administered controls. We performed echocardiography and hemodynamic analysis to investigate cardiac function 24 hours after bromine (600 ppm for 30 minutes) exposure and measured cardiac injury markers in plasma and proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac-specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine-exposed KO mice revealed significantly enhanced mean arterial pressure and sphericity index and decreased LV end diastolic diameter and LV end systolic pressure when compared with the bromine-exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures. SIGNIFICANCE STATEMENT: Due to their increased industrial production and transportation, halogens such as chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac-specific SERCA2 KO mice, these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.

Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Enhanced Ca2+-Driven Arrhythmias in Female Patients with Atrial Fibrillation: Insights from Computational Modeling.

BACKGROUND AND AIMS: Substantial sex-based differences have been reported in atrial fibrillation (AF), with female patients experiencing worse symptoms, increased complications from drug side effects or ablation, and elevated risk of AF-related stroke and mortality. Recent studies revealed sex-specific alterations in AF-associated Ca2+ dysregulation, whereby female cardiomyocytes more frequently exhibit potentially proarrhythmic Ca2+-driven instabilities compared to male cardiomyocytes. In this study, we aim to gain a mechanistic understanding of the Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in males vs females and establish their responses to Ca2+-targeted interventions. METHODS AND RESULTS: We incorporated known sex differences and AF-associated changes in the expression and phosphorylation of key Ca2+-handling proteins and in ultrastructural properties and dimensions of atrial cardiomyocytes into our recently developed 3D atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Our simulations of quiescent cardiomyocytes show increased incidence of Ca2+ sparks in female vs male myocytes in AF, in agreement with previous experimental reports. Additionally, our female model exhibited elevated propensity to develop pacing-induced spontaneous Ca2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca2+ transients compared with the male model. Parameter sensitivity analysis uncovered precise arrhythmogenic contributions of each component that was implicated in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation in female AF cardiomyocytes emerged as the major SCR contributor, while reduced L-type Ca2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulations of tentative Ca2+-targeted interventions identified potential strategies to attenuate Ca2+-driven arrhythmogenic events in female atria (e.g., t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase), and revealed enhanced efficacy when applied in combination. CONCLUSIONS: Our sex-specific computational models of human atrial cardiomyocytes uncover increased propensity to Ca2+-driven arrhythmogenic events in female compared to male atrial cardiomyocytes in AF, and point to combined Ca2+-targeted interventions as promising approaches to treat AF in female patients. Our study establishes that AF treatment may benefit from sex-dependent strategies informed by sex-specific mechanisms.

Polyarginine Cell-Penetrating Peptides Bind and Inhibit SERCA2.

Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg9 and Arg11 exert off-target effects on cellular Ca2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca2+ leak remained unchanged, indicating that protracted Ca2+ removal from the cytosol stemmed from inhibition of the SR Ca2+ ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.

Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.

BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.

Augmenting workload drives T-tubule assembly in developing cardiomyocytes.

Contraction of cardiomyocytes is initiated at subcellular elements called dyads, where L-type Ca2+ channels in t-tubules are located within close proximity to ryanodine receptors in the sarcoplasmic reticulum. While evidence from small rodents indicates that dyads are assembled gradually in the developing heart, it is unclear how this process occurs in large mammals. We presently examined dyadic formation in fetal and newborn sheep (Ovis aries), and the regulation of this process by fetal cardiac workload. By employing advanced imaging methods, we demonstrated that t-tubule growth and dyadic assembly proceed gradually during fetal sheep development, from 93 days of gestational age until birth (147 days). This process parallels progressive increases in fetal systolic blood pressure, and includes step-wise colocalization of L-type Ca2+ channels and the Na+ /Ca2+ exchanger with ryanodine receptors. These proteins are upregulated together with the dyadic anchor junctophilin-2 during development, alongside changes in the expression of amphiphysin-2 (BIN1) and its partner proteins myotubularin and dynamin-2. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth. Conversely, reducing fetal systolic load with infusion of enalaprilat, an angiotensin converting enzyme inhibitor, blunted t-tubule formation. Interestingly, altered t-tubule densities did not relate to changes in dyadic junctions, or marked changes in the expression of dyadic regulatory proteins, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum. In conclusion, augmenting blood pressure and workload during normal fetal development critically promotes t-tubule growth, while additional signals contribute to dyadic assembly. KEY POINTS: T-tubule growth and dyadic assembly proceed gradually in cardiomyocytes during fetal sheep development, from 93 days of gestational age until the post-natal stage. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth and hypertrophy. In contrast, reducing fetal systolic load by enalaprilat infusion slowed t-tubule development and decreased cardiomyocyte size. Load-dependent modulation of t-tubule maturation was linked to altered expression patterns of the t-tubule regulatory proteins junctophilin-2 and amphiphysin-2 (BIN1) and its protein partners. Altered t-tubule densities did not influence dyadic formation, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum.

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Disruption of Phosphodiesterase 3A Binding to SERCA2 Increases SERCA2 Activity and Reduces Mortality in Mice With Chronic Heart Failure.

BACKGROUND: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca2+ ATPase 2) activity is suggested to be beneficial in chronic heart failure, but no selective SERCA2-activating drugs are available. PDE3A (phosphodiesterase 3A) is proposed to be present in the SERCA2 interactome and limit SERCA2 activity. Disruption of PDE3A from SERCA2 might thus be a strategy to develop SERCA2 activators. METHODS: Confocal microscopy, 2-color direct stochastic optical reconstruction microscopy, proximity ligation assays, immunoprecipitations, peptide arrays, and surface plasmon resonance were used to investigate colocalization between SERCA2 and PDE3A in cardiomyocytes, map the SERCA2/PDE3A interaction sites, and optimize disruptor peptides that release PDE3A from SERCA2. Functional experiments assessing the effect of PDE3A-binding to SERCA2 were performed in cardiomyocytes and HEK293 vesicles. The effect of SERCA2/PDE3A disruption by the disruptor peptide OptF (optimized peptide F) on cardiac mortality and function was evaluated during 20 weeks in 2 consecutive randomized, blinded, and controlled preclinical trials in a total of 148 mice injected with recombinant adeno-associated virus 9 (rAAV9)-OptF, rAAV9-control (Ctrl), or PBS, before undergoing aortic banding (AB) or sham surgery and subsequent phenotyping with serial echocardiography, cardiac magnetic resonance imaging, histology, and functional and molecular assays. RESULTS: PDE3A colocalized with SERCA2 in human nonfailing, human failing, and rodent myocardium. Amino acids 277-402 of PDE3A bound directly to amino acids 169-216 within the actuator domain of SERCA2. Disruption of PDE3A from SERCA2 increased SERCA2 activity in normal and failing cardiomyocytes. SERCA2/PDE3A disruptor peptides increased SERCA2 activity also in the presence of protein kinase A inhibitors and in phospholamban-deficient mice, and had no effect in mice with cardiomyocyte-specific inactivation of SERCA2. Cotransfection of PDE3A reduced SERCA2 activity in HEK293 vesicles. Treatment with rAAV9-OptF reduced cardiac mortality compared with rAAV9-Ctrl (hazard ratio, 0.26 [95% CI, 0.11 to 0.63]) and PBS (hazard ratio, 0.28 [95% CI, 0.09 to 0.90]) 20 weeks after AB. Mice injected with rAAV9-OptF had improved contractility and no difference in cardiac remodeling compared with rAAV9-Ctrl after aortic banding. CONCLUSIONS: Our results suggest that PDE3A regulates SERCA2 activity through direct binding, independently of the catalytic activity of PDE3A. Targeting the SERCA2/PDE3A interaction prevented cardiac mortality after AB, most likely by improving cardiac contractility.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: II. Ca2+ -handling protein variation.

Disruption of the transverse-axial tubule system (TATS) in diseases such as heart failure and atrial fibrillation occurs in combination with changes in the expression and distribution of key Ca2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca2+ -handling protein expression and localization that occur in disease it is difficult to distinguish their individual contributions to the arrhythmogenic state. To investigate this, we applied our novel 3D human atrial myocyte model with spatially detailed Ca2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca2+ -handling proteins and variable TATS density on Ca2+ -handling abnormality driven membrane instabilities. We show that modulating the expression and distribution of the sodium-calcium exchanger, ryanodine receptors and the sarcoplasmic reticulum (SR) Ca2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca2+ leak-load and Ca2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca2+ -driven proarrhythmic behaviour varied dramatically depending on TATS density, with intermediately tubulated cells being more severely affected compared to detubulated and densely tubulated myocytes. This work provides novel mechanistic insight into the distinct and interactive consequences of TATS and Ca2+ -handling protein remodelling that underlies dysfunctional Ca2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca2+ handling with subcellular spatial details governed by the transverse-axial tubule system (TATS). Here we utilize this model to mechanistically examine the impact of TATS loss and changes in the expression and distribution of key Ca2+ -handling proteins known to be remodelled in disease on Ca2+ homeostasis and electrophysiological stability. We demonstrate that varying the expression and localization of these proteins has variable pro- and anti-arrhythmic effects with outcomes displaying dependence on TATS density. Whereas detubulated myocytes typically appear unaffected and densely tubulated cells seem protected, the arrhythmogenic effects of Ca2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca2+ -handling protein remodelling that underlies the Ca2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling.

Mechanisms of spontaneous Ca2+ release-mediated arrhythmia in a novel 3D human atrial myocyte model: I. Transverse-axial tubule variation.

Intracellular calcium (Ca2+ ) cycling is tightly regulated in the healthy heart ensuring effective contraction. This is achieved by transverse (t)-tubule membrane invaginations that facilitate close coupling of key Ca2+ -handling proteins such as the L-type Ca2+ channel and Na+ -Ca2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca2+ store. Although less abundant and regular than in the ventricle, t-tubules also exist in atrial myocytes as a network of transverse invaginations with axial extensions known as the transverse-axial tubule system (TATS). In heart failure and atrial fibrillation, there is TATS remodelling that is associated with aberrant Ca2+ -handling and Ca2+ -induced arrhythmic activity; however, the mechanism underlying this is not fully understood. To address this, we developed a novel 3D human atrial myocyte model that couples electrophysiology and Ca2+ -handling with variable TATS organization and density. We extensively parameterized and validated our model against experimental data to build a robust tool examining TATS regulation of subcellular Ca2+ release. We found that varying TATS density and thus the localization of key Ca2+ -handling proteins has profound effects on Ca2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca2+ concentration through decreased Ca2+ extrusion. This elevated Ca2+ increases RyR open probability causing spontaneous Ca2+ releases and the promotion of arrhythmogenic waves (especially in the cell interior) leading to voltage instabilities through delayed afterdepolarizations. In summary, the present study demonstrates a mechanistic link between TATS remodelling and Ca2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca2+ and voltage instabilities through reduced Na+ -Ca2+ exchanger-mediated Ca2+ removal, cleft Ca2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca2+ releases are larger and more frequent in the cell interior than at the periphery. Our work provides mechanistic insight into how atrial TATS remodelling can lead to Ca2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease.

Live-cell photo-activated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes.

Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photo-activated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution Photo-Activated Localization Microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and "travelling" events that spread between neighbouring RyR clusters. Travelling sparks exhibited up to 8 distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute ß-adrenergic stimulation augments intra-cluster RyR recruitment to generate larger events. In contrast, RyR "dispersion" during heart failure facilitates the generation of travelling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

ADAMTSL3 knock-out mice develop cardiac dysfunction and dilatation with increased TGFß signalling after pressure overload.

Heart failure is a major cause of morbidity and mortality worldwide, and can result from pressure overload, where cardiac remodelling is characterized by cardiomyocyte hypertrophy and death, fibrosis, and inflammation. In failing hearts, transforming growth factor (TGF)ß drives cardiac fibroblast (CFB) to myofibroblast differentiation causing excessive extracellular matrix production and cardiac remodelling. New strategies to target pathological TGFß signalling in heart failure are needed. Here we show that the secreted glycoprotein ADAMTSL3 regulates TGFß in the heart. We found that Adamtsl3 knock-out mice develop exacerbated cardiac dysfunction and dilatation with increased mortality, and hearts show increased TGFß activity and CFB activation after pressure overload by aortic banding. Further, ADAMTSL3 overexpression in cultured CFBs inhibits TGFß signalling, myofibroblast differentiation and collagen synthesis, suggesting a cardioprotective role for ADAMTSL3 by regulating TGFß activity and CFB phenotype. These results warrant future investigation of the potential beneficial effects of ADAMTSL3 in heart failure.

Tubulator: an automated approach to analysis of t-tubule and dyadic organization in cardiomyocytes.

During cardiac disease, t-tubules and dyads are remodelled and disrupted within cardiomyocytes, thereby reducing cardiac performance. Given the pathological implications of such dyadic remodelling, robust and versatile tools for characterizing these sub-cellular structures are needed. While analysis programs for continuous and regular structures such as rodent ventricular t-tubules are available, at least in two dimensions, these approaches are less appropriate for assessment of more irregular structures, such as dyadic proteins and non-rodent t-tubules. Here, we demonstrate versatile, easy-to-use software that performs such analyses. This software, called Tubulator, enables automated analysis of t-tubules and dyadic proteins alike, in both tissue sections and isolated myocytes. The program measures densities of subcellular structures and proteins in individual cells, quantifies their distribution into transversely and longitudinally oriented elements, and supports detailed co-localization analyses. Importantly, Tubulator provides tools for three-dimensional assessment and rendering of image stacks, extending examinations from the single plane to the whole-myocyte level. To provide insight into the consequences of dyadic organization for synchrony of Ca2+ handling, Tubulator also creates 'distance maps', by calculating the distance from all cytosolic positions to the nearest t-tubule and/or dyad. In conclusion, this freely accessible program provides detailed automated analysis of the three-dimensional nature of dyadic and t-tubular structures. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Prolonged ß-adrenergic stimulation disperses ryanodine receptor clusters in cardiomyocytes and has implications for heart failure.

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged ß-adrenergic stimulation, a hallmark of HF, promotes RyR cluster dispersion and examined the underlying mechanisms. We observed that treatment of healthy rat cardiomyocytes with isoproterenol for 1 hr triggered progressive fragmentation of RyR clusters. Pharmacological inhibition of Ca2+/calmodulin-dependent protein kinase II (CaMKII) reversed these effects, while cluster dispersion was reproduced by specific activation of CaMKII, and in mice with constitutively active Ser2814-RyR. A similar role of protein kinase A (PKA) in promoting RyR cluster fragmentation was established by employing PKA activation or inhibition. Progressive cluster dispersion was linked to declining Ca2+ spark fidelity and magnitude, and slowed release kinetics from Ca2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of ß-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca2+ waves. However, during HF, RyR dispersion was linked to impaired Ca2+ release. Thus, RyR localization and function are intimately linked via channel phosphorylation by both CaMKII and PKA, which, while finely tuned in healthy cardiomyocytes, underlies impaired cardiac function during pathology.

Nanoscale organization of ryanodine receptor distribution and phosphorylation pattern determines the dynamics of calcium sparks.

Super-resolution imaging techniques have provided a better understanding of the relationship between the nanoscale organization and function of ryanodine receptors (RyRs) in cardiomyocytes. Recent data have indicated that this relationship is disrupted in heart failure (HF), as RyRs are dispersed into smaller and more numerous clusters. However, RyRs are also hyperphosphorylated in this condition, and this is reported to occur preferentially within the cluster centre. Thus, the combined impact of RyR relocalization and sensitization on Ca2+ spark generation in failing cardiomyocytes is likely complex and these observations suggest that both the nanoscale organization of RyRs and the pattern of phosphorylated RyRs within clusters could be critical determinants of Ca2+ spark dynamics. To test this hypothesis, we used computational modeling to quantify the relationships between RyR cluster geometry, phosphorylation patterns, and sarcoplasmic reticulum (SR) Ca2+ release. We found that RyR cluster disruption results in a decrease in spark fidelity and longer sparks with a lower amplitude. Phosphorylation of some RyRs within the cluster can play a compensatory role, recovering healthy spark dynamics. Interestingly, our model predicts that such compensation is critically dependent on the phosphorylation pattern, as phosphorylation localized within the cluster center resulted in longer Ca2+ sparks and higher spark fidelity compared to a uniformly distributed phosphorylation pattern. Our results strongly suggest that both the phosphorylation pattern and nanoscale RyR reorganization are critical determinants of Ca2+ dynamics in HF.

Blocking phospholamban with VHH intrabodies enhances contractility and relaxation in heart failure.

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.

Discordant Ca2+ release in cardiac myocytes: characterization and susceptibility to pharmacological RyR2 modulation.

Systolic Ca2+ transients are shaped by the concerted summation of Ca2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca2+ release across the cell, although the triggers and consequences of this phenomenon remain unclear. Rat ventricular cardiomyocytes were paced at increasing rates. A discordance index (SD of local alternans ratios) was developed to quantify discordance in confocal recordings of Ca2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca2+ transient-frequency relationship. In addition, the largest local calcium transient in two consecutive transients was measured to build a potential "best release" profile, which quantitatively confirmed discordance-induced Ca2+ release impairment (DICRI). Diastolic Ca2+ homeostasis was also observed to be disrupted by discordance, as late Ca2+ release events elicited instability of resting Ca2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use.