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Key pointsAs reactivation of the fetal gene program has been implicated in pathological remodelling during heart failure (HF), we examined whether cardiomyocyte subcellular structure and function revert to an immature phenotype during this disease. Surface and internal membrane structures appeared gradually during development, and returned to a juvenile state during HF. Similarly, dyadic junctions between the cell membrane and sarcoplasmic reticulum were progressively 'packed' with L-type Ca2+ channels and ryanodine receptors during development, and 'unpacked' during HF. Despite similarities in subcellular structure, dyads were observed to be functional from early developmental stages, but exhibited an impaired ability to release Ca2+ in failing cardiomyocytes. Thus, while immature and failing cardiomyocytes share similarities in subcellular structure, these do not fully account for the marked impairment of Ca2+ homeostasis observed in HF.AbstractReactivation of the fetal gene programme has been implicated as a driver of pathological cardiac remodelling. Here we examined whether pathological remodelling of cardiomyocyte substructure and function during heart failure (HF) reflects a reversion to an immature phenotype. Using scanning electron microscopy, we observed that Z-grooves and t-tubule openings at the cell surface appeared gradually during cardiac development, and disappeared during HF. Confocal and super-resolution imaging within the cell interior revealed similar structural parallels; disorganization of t-tubules in failing cells was strikingly reminiscent of the late stages of postnatal development, with fewer transverse elements and a high proportion of longitudinal tubules. Ryanodine receptors (RyRs) were observed to be laid down in advance of developing t-tubules and similarly 'orphaned' in HF, although RyR distribution along Z-lines was relatively sparse. Indeed, nanoscale imaging revealed coordinated packing of L-type Ca2+ channels and RyRs into dyadic junctions during development, and orderly unpacking during HF. These findings support a 'last in, first out' paradigm, as the latest stages of dyadic structural development are reversed during disease. Paired imaging of t-tubules and Ca2+ showed that the disorganized arrangement of dyads in immature and failing cells promoted desynchronized and slowed Ca2+ release in these two states. However, while developing cells exhibited efficient triggering of Ca2+ release at newly formed dyads, dyadic function was impaired in failing cells despite similar organization of Ca2+ handling proteins. Thus, pathologically deficient Ca2+ homeostasis during HF is only partly linked to the re-emergence of immature subcellular structure, and additionally reflects lost dyadic functionality.

The co-ordinated electrical activity of ∼2 billion cardiac cells ensures stability of the heartbeat. Indeed, the remarkably low incidence (<1%) of ventricular arrhythmias in the healthy heart is only possible when the electrical event across this syncytium is closely controlled. In contrast, the diseased myocardium is associated with increased electrophysiological heterogeneity, unstable rhythm, and increased incidence of lethal arrhythmias. But what is the link between cellular and tissue level heterogeneity? Recent research has shown the existence of considerable cellular heterogeneity even in the healthy heart, suggesting that cell-to-cell variability in electrical (e.g. action potential duration) and mechanical performance (e.g. twitch amplitude) is a normal property. This observation has been previously unappreciated because the aggregated function in the form of QT-interval and cardiac output varies <1% on a beat-to-beat basis. This article describes the underlying cellular variability that is tolerated-and perhaps needed-by different regions of the heart for normal function and indicates why this variability is not apparent in function at the chamber and organ level. Thus, in contrast to the current dominant view, this article postulates that heterogeneity is normal and potentially endows various functional benefits. This new view of how the component parts of the heart come together to function also suggests novel mechanisms for cardiac pathologies, namely that dysfunction may emerge from changes in the extent and/or nature of heterogeneity. Once understood, restoring normal forms of heterogeneity could be a novel approach to treatment., Competing Interests: Conflict of interest: none declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background: Substantial sex-based differences have been reported in atrial fibrillation (AF), but the underlying mechanisms are poorly understood., Objectives: This study sought to gain a mechanistic understanding of Ca 2+ -handling disturbances and Ca 2+ -driven arrhythmogenic events in male vs female atrial cardiomyocytes and establish their responses to Ca 2+ -targeted interventions., Methods: We integrated reported sex differences and AF-associated changes (ie, expression and phosphorylation of Ca 2+ -handling proteins, cardiomyocyte ultrastructural characteristics, and dimensions) into our human atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca 2+ -handling processes. Sex-specific responses of atrial cardiomyocytes to arrhythmia-provoking protocols and Ca 2+ -targeted interventions were evaluated., Results: Simulated quiescent cardiomyocytes showed increased incidence of Ca 2+ 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 Ca 2+ releases (SCRs) and augmented beat-to-beat variability in action potential (AP)-elicited Ca 2+ transients compared with the male model. Sensitivity analysis uncovered distinct arrhythmogenic contributions of each component involved in sex and/or AF alterations. Specifically, increased ryanodine receptor phosphorylation emerged as the major SCR contributor in female AF cardiomyocytes, whereas reduced L-type Ca 2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulated Ca 2+ -targeted interventions identified potential strategies (eg, t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca 2 ⁺-ATPase) to attenuate Ca 2+ -driven arrhythmogenic events in women, and revealed enhanced efficacy when applied in combination., Conclusions: Sex-specific modeling uncovers increased Ca 2+ -driven arrhythmogenic events in female vs male atria in AF, and suggests combined Ca 2+ -targeted interventions are promising therapeutic approaches in women., Competing Interests: Funding Support and Author Disclosures This work was supported by American Heart Association Career Development Award 24CDA1258695 (to Dr Ni), Postdoctoral Fellowships 20POST35120462 (to Dr Ni), 24POST1195229 (to Dr Wu), and Predoctoral Fellowship 20PRE35120465 (to Dr Zhang); National Institutes of Health/National Heart, Lung, and Blood Institute grants R01HL131517 (to Drs Grandi, Louch, and Dobrev), R01HL170521 (to Drs Grandi and Ni), R01HL141214 and P01HL141084 (to Dr Grandi), R01HL136389 (to Dr Dobrev), R01HL089598 (to Dr Dobrev), R01HL163277 (to Dr Dobrev), R01HL160992 (to Dr Dobrev), R01HL165704 (to Dr Dobrev), and R00HL138160 (to Dr Morotti); National Institutes of Health Stimulating Peripheral Activity to Relieve Conditions Grant 1OT2OD026580-01 (to Dr Grandi); the Research Council of Norway (Grant No. 287395 to Dr Louch); UC Davis School of Medicine Dean’s Fellow Award (to Dr Grandi); the European Union (large-scale integrative project MAESTRIA, No. 965286, to Dr Dobrev); and the Burroughs Wellcome Fund–Doris Duke Charitable Foundation "COVID-19 Fund to Retain Clinical Scientists" Award (to Dr Morotti). All authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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 Ca 2+ 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 Ca 2+ transients, linked to upregulation of L-type Ca 2+ channels, Na + -Ca 2+ 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 Ca 2+ 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 Ca 2+ transients, with upregulation of the L-type Ca 2+ channel, Na + -Ca 2+ 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., (© 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Contraction of cardiomyocytes is initiated at subcellular elements called dyads, where L-type Ca 2+ 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 Ca 2+ channels and the Na + /Ca 2+ 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., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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 ( α MHC-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., (Copyright © 2024 by The Author(s).)

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.

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 Arg 9 and Arg 11 exert off-target effects on cellular Ca 2+ homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca 2+ recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca 2+ leak remained unchanged, indicating that protracted Ca 2+ removal from the cytosol stemmed from inhibition of the SR Ca 2+ 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.

Sex differences in cardiac physiology are receiving increased attention as it has become clear that men and women have different aetiologies of cardiac disease and require different treatments. There are experimental data suggesting that male cardiomyocytes exhibit larger Ca 2+ transients due to larger Ca 2+ sparks and a higher excitation-contraction coupling gain; in addition, they exhibit a larger response to adrenergic stimulation with isoprenaline (ISO). Here, we studied whether there are sex differences relating to structural organization of the transverse tubular network and ryanodine receptors (RyRs). Surprisingly, we found that female cardiomyocytes exhibited a higher spark frequency in a range of spark magnitudes. While overall RyR expression and phosphorylation were the same, female cardiomyocytes had larger but fewer RyR clusters. The density of transverse t-tubules was the same, but male cardiomyocytes had more longitudinal t-tubules. The Ca 2+ transients were similar in male and female cardiomyocytes under control conditions and in the presence of ISO. The synchrony of the Ca 2+ transients was similar between sexes as well. Overall, our data suggest subtle sex differences in the Ca 2+ influx and efflux pathways and their response to ISO, but these differences are balanced, resulting in similar Ca 2+ transients in field-stimulated male and female cardiomyocytes. The higher spark frequency in female cardiomyocytes is related to the organization of RyRs into larger, but fewer clusters. KEY POINTS: During a heartbeat, the force of contraction depends on the amplitude of the calcium transient, which in turn depends on the amount of calcium released as calcium sparks through ryanodine receptors in the sarcoplasmic reticulum. Previous studies suggest that cardiomyocytes from male compared to female mice exhibit larger calcium sparks, larger sarcoplasmic reticulum calcium release and greater response to adrenergic stimulation triggering a fight-or-flight response. In contrast, we show that cardiomyocytes from female mice have a higher spark frequency during adrenergic stimulation and similar spark morphology. The higher spark frequency is related to the organization of ryanodine receptors into fewer, but larger clusters in female compared to male mouse cardiomyocytes. Despite subtle sex differences in cardiomyocyte structure and calcium fluxes, the differences are balanced, leading to similar calcium transients in cardiomyocytes from male and female mice., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

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 Ca 2+ 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 Ca 2+ 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., Competing Interests: Disclosures None.

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 Ca 2+ -handling proteins. Together this ultrastructural and ionic remodelling is associated with aberrant Ca 2+ cycling and electrophysiological instabilities that underlie arrhythmic activity. However, due to the concurrent changes in TATs and Ca 2+ -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 Ca 2+ diffusion and TATS to investigate the isolated and interactive effects of changes in expression and localization of key Ca 2+ -handling proteins and variable TATS density on Ca 2+ -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) Ca 2+ buffer calsequestrin have varying pro- and anti-arrhythmic effects depending on the balance of opposing influences on SR Ca 2+ leak-load and Ca 2+ -voltage relationships. Interestingly, the impact of protein remodelling on Ca 2+ -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 Ca 2+ -handling protein remodelling that underlies dysfunctional Ca 2+ cycling and electrophysiological instability in disease. KEY POINTS: In our companion paper we developed a 3D human atrial myocyte model, coupling electrophysiology and Ca 2+ 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 Ca 2+ -handling proteins known to be remodelled in disease on Ca 2+ 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 Ca 2+ handling protein remodelling are profound in intermediately tubulated cells. Our work shows the interaction between TATS and Ca 2+ -handling protein remodelling that underlies the Ca 2+ -driven proarrhythmic behaviour observed in atrial fibrillation and may help to predict the effects of antiarrhythmic strategies at varying stages of ultrastructural remodelling., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Intracellular calcium (Ca 2+ ) 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 Ca 2+ -handling proteins such as the L-type Ca 2+ channel and Na + -Ca 2+ exchanger (NCX) on the cell surface with ryanodine receptors (RyRs) on the intracellular Ca 2+ 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 Ca 2+ -handling and Ca 2+ -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 Ca 2+ -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 Ca 2+ release. We found that varying TATS density and thus the localization of key Ca 2+ -handling proteins has profound effects on Ca 2+ handling. Following TATS loss, there is reduced NCX that results in increased cleft Ca 2+ concentration through decreased Ca 2+ extrusion. This elevated Ca 2+ increases RyR open probability causing spontaneous Ca 2+ 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 Ca 2+ -driven proarrhythmic behaviour that probably reflects the arrhythmogenic state observed in disease. KEY POINTS: Transverse-axial tubule systems (TATS) modulate Ca 2+ handling and excitation-contraction coupling in atrial myocytes, with TATS remodelling in heart failure and atrial fibrillation being associated with altered Ca 2+ cycling and subsequent arrhythmogenesis. To investigate the poorly understood mechanisms linking TATS variation and spontaneous Ca 2+ release, we built, parameterized and validated a 3D human atrial myocyte model coupling electrophysiology and spatially-detailed subcellular Ca 2+ handling governed by the TATS. Simulated TATS loss causes diastolic Ca 2+ and voltage instabilities through reduced Na + -Ca 2+ exchanger-mediated Ca 2+ removal, cleft Ca 2+ accumulation and increased ryanodine receptor open probability, resulting in spontaneous Ca 2+ release and promotion of arrhythmogenic waves and delayed afterdepolarizations. At fast electrical rates typical of atrial tachycardia/fibrillation, spontaneous Ca 2+ 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 Ca 2+ -driven instabilities that may ultimately contribute to the arrhythmogenic state in disease., (© 2022 The Authors. The Journal of Physiology © 2022 The Physiological Society.)

Background: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca 2+ 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. Ca 2+ 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 Ca 2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca 2+ 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., Competing Interests: Disclosures J. Laporte is a cofounder of Dynacure. The other authors report no conflicts.

Background: Increasing SERCA2 (sarco[endo]-plasmic reticulum Ca 2+ 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.

Aims: Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in-depth assessment of lumican in clinical and experimental HCM., Methods: Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n = 15), and controls from hearts deemed unsuitable for transplantation (n = 8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild-type littermates (n = 8-10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid-ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n = 3) and examined using confocal microscopy., Results: Lumican mRNA was increased threefold in HCM patients (P < 0.05) and correlated strongly with expression of collagen I (R 2  = 0.60, P < 0.01) and III (R 2  = 0.58, P < 0.01). Lumican protein was increased by 40% in patients with HCM (P < 0.01) and correlated with total (R 2  = 0.28, P = 0.05) and interstitial (R 2  = 0.30, P < 0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P < 0.001), and lumican protein increased 20-fold (P < 0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co-localization measured in patients and mice with HCM (patients: +19%, P < 0.01; mice: +13%, P < 0.01). dSTORM super-resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (-15 nm, P < 0.05), overlapped more frequently with (+7.3%, P < 0.05) and to a larger degree with (+5.6%, P < 0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8 nm, P < 0.001), longer (+345.9 nm, P < 0.001), and fewer (-8.9%, P < 0.001) collagen fibres., Conclusions: The ECM proteoglycan lumican is increased in HCM and co-localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Ca 2+ sparks constitute the fundamental units of Ca 2+ 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 Ca 2+ sparks, by high-speed imaging. Two populations of Ca 2+ 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 β-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 Ca 2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca 2+ release., Competing Interests: Competing Interests Statement The authors declare no competing interests.

Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca 2+ sensor protein STIM1, which forms the Ca 2+ release-activated Ca 2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca 2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca 2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu 296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu 296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.

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)β drives cardiac fibroblast (CFB) to myofibroblast differentiation causing excessive extracellular matrix production and cardiac remodelling. New strategies to target pathological TGFβ signalling in heart failure are needed. Here we show that the secreted glycoprotein ADAMTSL3 regulates TGFβ in the heart. We found that Adamtsl3 knock-out mice develop exacerbated cardiac dysfunction and dilatation with increased mortality, and hearts show increased TGFβ activity and CFB activation after pressure overload by aortic banding. Further, ADAMTSL3 overexpression in cultured CFBs inhibits TGFβ signalling, myofibroblast differentiation and collagen synthesis, suggesting a cardioprotective role for ADAMTSL3 by regulating TGFβ activity and CFB phenotype. These results warrant future investigation of the potential beneficial effects of ADAMTSL3 in heart failure., (© 2022. The Author(s).)

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 Ca 2+ 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'.

Background: In cardiac muscle, the ubiquitously expressed proteoglycan syndecan-4 is involved in the hypertrophic response to pressure overload. Protein kinase Akt signaling, which is known to regulate hypertrophy, has been found to be reduced in the cardiac muscle of exercised male syndecan-4 -/- mice. In contrast, we have recently found that pSer473-Akt signaling is elevated in the skeletal muscle ( tibialis anterior, TA) of female syndecan-4 -/- mice. To determine if the differences seen in Akt signaling are sex specific, we have presently investigated Akt signaling in the cardiac muscle of sedentary and exercised female syndecan-4 -/- mice. To get deeper insight into the female syndecan-4 -/- heart, alterations in cardiomyocyte size, a wide variety of different extracellular matrix components, well-known syndecan-4 binding partners and associated signaling pathways have also been investigated. Methods: Left ventricles (LVs) from sedentary and exercise trained female syndecan-4 -/- and WT mice were analyzed by immunoblotting and real-time PCR. Cardiomyocyte size and phosphorylated Ser473-Akt were analyzed in isolated adult cardiomyocytes from female syndecan-4 -/- and WT mice by confocal imaging. LV and skeletal muscle (TA) from sedentary male syndecan-4 -/- and WT mice were immunoblotted with Akt antibodies for comparison. Glucose levels were measured by a glucometer, and fasting blood serum insulin and C-peptide levels were measured by ELISA. Results: Compared to female WT hearts, sedentary female syndecan-4 -/- LV cardiomyocytes were smaller and hearts had higher levels of pSer473-Akt and its downstream target pSer9-GSK-3β. The pSer473-Akt inhibitory phosphatase PHLPP1/SCOP was lowered, which may be in response to the elevated serum insulin levels found in the female syndecan-4 -/- mice. We also observed lowered levels of pThr308-Akt/Akt and GLUT4 in the female syndecan-4 -/- heart and an increased LRP6 level after exercise. Otherwise, few alterations were found. The pThr308-Akt and pSer473-Akt levels were unaltered in the cardiac and skeletal muscles of sedentary male syndecan-4 -/- mice. Conclusion: Our data indicate smaller cardiomyocytes, an elevated insulin/pSer473-Akt/pSer9-GSK-3β signaling pathway, and lowered SCOP, pThr308-Akt/Akt and GLUT4 levels in the female syndecan-4 -/- heart. In contrast, cardiomyocyte size, and Akt signaling were unaltered in both cardiac and skeletal muscles from male syndecan-4 -/- mice, suggesting important sex differences., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor MEP declared a past collaboration with one of the authors CC., (Copyright © 2022 Støle, Lunde, Shen, Martinsen, Lunde, Li, Lockwood, Sjaastad, Louch, Aronsen, Christensen and Carlson.)

Ryanodine receptors (RyRs) exhibit dynamic arrangements in cardiomyocytes, and we previously showed that 'dispersion' of RyR clusters disrupts Ca 2+ homeostasis during heart failure (HF) (Kolstad et al., eLife, 2018). Here, we investigated whether prolonged β-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 Ca 2+ /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 Ca 2+ spark fidelity and magnitude, and slowed release kinetics from Ca 2+ propagation between more numerous RyR clusters. In healthy cells, this served to dampen the stimulatory actions of β-adrenergic stimulation over the longer term and protect against pro-arrhythmic Ca 2+ waves. However, during HF, RyR dispersion was linked to impaired Ca 2+ 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., Competing Interests: XS, Jv, AB, TK, EN, YH, ML, YA, AQ, EE, IS, XW, AE, CS, WL No competing interests declared, (© 2022, Shen et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

Systolic Ca 2+ transients are shaped by the concerted summation of Ca 2+ sparks across cardiomyocytes. At high pacing rates, alterations of excitation-contraction coupling manifest as pro-arrhythmic Ca 2+ alternans that can be classified as concordant or discordant. Discordance is ascribed to out-of-phase alternation of local Ca 2+ 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 Ca 2+ transients. Index values were significantly increased by rapid pacing, and negatively correlated with Ca 2+ transient amplitude change, indicating that discordance is an important contributor to the negative Ca 2+ 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 Ca 2+ release impairment (DICRI). Diastolic Ca 2+ homeostasis was also observed to be disrupted by discordance, as late Ca 2+ release events elicited instability of resting Ca 2+ levels. Finally, the effects of two RyR2 inhibitors (VK-II-86 and dantrolene) were tested. While both compounds inhibited Ca 2+ wave generation, only VK-II-86 augmented subcellular discordance. Discordant Ca 2+ release is a quantifiable phenomenon, sensitive to pacing frequency, and impairs both systolic and diastolic Ca 2+ homeostasis. Interestingly, RyR2 inhibition can induce discordance, which should be considered when evaluating pharmacological RyR2 modulators for clinical use., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca 2+ 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., (© 2022. The Author(s).)

Complementary developments in microscopy and mathematical modeling have been critical to our understanding of cardiac excitation-contraction coupling. Historically, limitations imposed by the spatial or temporal resolution of imaging methods have been addressed through careful mathematical interrogation. Similarly, limitations imposed by computational power have been addressed by imaging macroscopic function in large subcellular domains or in whole myocytes. As both imaging resolution and computational tractability have improved, the two approaches have nearly merged in terms of the scales that they can each be used to interrogate. With this review we will provide an overview of these advances and their contribution to understanding ventricular myocyte function, including exciting developments over the last decade. We specifically focus on experimental methods that have pushed back limits of either spatial or temporal resolution of nanoscale imaging (e.g., DNA-PAINT), or have permitted high resolution imaging on large cellular volumes (e.g., serial scanning electron microscopy). We also review the progression of computational approaches used to integrate and interrogate these new experimental data sources, and comment on near-term advances that may unify understanding of the underlying biology. Finally, we comment on several outstanding questions in cardiac physiology that stand to benefit from a concerted and complementary application of these new experimental and computational methods., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Louch, Perdreau-Dahl and Edwards.)

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca 2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.

Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates Ca 2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca 2+ release from SR and triggers contraction. Ca 2+ /CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca 2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR., Methods: A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology., Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca 2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca 2+ reuptake by SERCA2 and Ca 2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca 2+ -frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR., Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

BIN1 (amphyphysin-II) is a structural protein involved in T-tubule (TT) formation and phosphatidylinositol-4,5-bisphosphate (PIP2) is responsible for localization of BIN1 to sarcolemma. The goal of this study was to determine if PIP2-mediated targeting of BIN1 to sarcolemma is compromised during the development of heart failure (HF) and is responsible for TT remodeling. Immunohistochemistry showed co-localization of BIN1, Cav1.2, PIP2, and phospholipase-Cβ1 (PLCβ1) in TTs in normal rat and human ventricular myocytes. PIP2 levels were reduced in spontaneously hypertensive rats during HF progression compared to age-matched controls. A PIP Strip assay of two native mouse cardiac-specific isoforms of BIN1 including the longest (cardiac BIN1 #4) and shortest (cardiac BIN1 #1) isoforms as well human skeletal BIN1 showed that all bound PIP2. In addition, overexpression of all three BIN1 isoforms caused tubule formation in HL-1 cells. A triple-lysine motif in a short loop segment between two helices was mutated and replaced by negative charges which abolished tubule formation, suggesting a possible location for PIP2 interaction aside from known consensus binding sites. Pharmacological PIP2 depletion in rat ventricular myocytes caused TT loss and was associated with changes in Ca 2+ release typically found in myocytes during HF, including a higher variability in release along the cell length and a slowing in rise time, time to peak, and decay time in treated myocytes. These results demonstrate that depletion of PIP2 can lead to TT disruption and suggest that PIP2 interaction with cardiac BIN1 is required for TT maintenance and function., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Zhou, Singh, Monnier, Marszalec, Gao, Jin, Frisk, Louch, Verma, Krishnamurthy, Nico, Mulla, Aistrup, Kishore and Wasserstrom.)

Sarcomeric contraction in cardiomyocytes serves as the basis for the heart's pump functions. It has generally been considered that in cardiac muscle as well as in skeletal muscle, sarcomeres equally contribute to myofibrillar dynamics in myocytes at varying loads by producing similar levels of active and passive force. In the present study, we expressed α-actinin-AcGFP in Z-disks to analyze dynamic behaviors of sequentially connected individual sarcomeres along a myofibril in a left ventricular (LV) myocyte of the in vivo beating mouse heart. To quantify the magnitude of the contribution of individual sarcomeres to myofibrillar dynamics, we introduced the novel parameter "contribution index" (CI) to measure the synchrony in movements between a sarcomere and a myofibril (from -1 [complete asynchrony] to 1 [complete synchrony]). First, CI varied markedly between sarcomeres, with an average value of ∼0.3 during normal systole. Second, when the movements between adjacent sarcomeres were asynchronous (CI < 0), a sarcomere and the ones next to the adjacent sarcomeres and farther away moved in synchrony (CI > 0) along a myofibril. Third, when difference in LV pressure in diastole and systole (ΔLVP) was lowered to <10 mm Hg, diastolic sarcomere length increased. Under depressed conditions, the movements between adjacent sarcomeres were in marked asynchrony (CI, -0.3 to -0.4), and, as a result, average CI was linearly decreased in association with a decrease in ΔLVP. These findings suggest that in the left ventricle of the in vivo beating mouse heart, (1) sarcomeres heterogeneously contribute to myofibrillar dynamics due to an imbalance of active and passive force between neighboring sarcomeres, (2) the force imbalance is pronounced under depressed conditions coupled with a marked increase in passive force and the ensuing tug-of-war between sarcomeres, and (3) sarcomere synchrony via the distal intersarcomere interaction regulates the heart's pump function in coordination with myofibrillar contractility., (© 2021 Kobirumaki-Shimozawa et al.)

Dysfunctional sarcoplasmic reticulum Ca 2+ handling is commonly observed in heart failure, and thought to contribute to arrhythmogenesis through several mechanisms. Some time ago we developed a cardiomyocyte-specific inducible SERCA2 knockout mouse, which is remarkable in the degree to which major adaptations to sarcolemmal Ca 2+ entry and efflux overcome the deficit in SR reuptake to permit relatively normal contractile function. Conventionally, those adaptations would also be expected to dramatically increase arrhythmia susceptibility. However, that susceptibility has never been tested, and it is possible that the very rapid repolarization of the murine action potential (AP) allows for large changes in sarcolemmal Ca 2+ transport without substantially disrupting electrophysiologic stability. We investigated this hypothesis through telemetric ECG recording in the SERCA2-KO mouse, and patch-clamp electrophysiology, Ca 2+ imaging, and mathematical modeling of isolated SERCA2-KO myocytes. While the SERCA2-KO animals exhibit major (and unique) electrophysiologic adaptations at both the organ and cell levels, they remain resistant to arrhythmia. A marked increase in peak L-type calcium ( I CaL ) current and slowed I CaL decay elicited pronounced prolongation of initial repolarization, but faster late repolarization normalizes overall AP duration. Early afterdepolarizations were seldom observed in KO animals, and those that were observed exhibited a mechanism intermediate between murine and large mammal dynamical properties. As expected, spontaneous SR Ca 2+ sparks and waves were virtually absent. Together these findings suggest that intact SR Ca 2+ handling is an absolute requirement for triggered arrhythmia in the mouse, and that in its absence, dramatic changes to the major inward currents can be resisted by the substantial K + current reserve, even at end-stage disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Edwards, Mørk, Stokke, Lipsett, Sjaastad, Richard, Sejersted and Louch.)

In cardiomyocytes, invaginations of the sarcolemmal membrane called t-tubules are critically important for triggering contraction by excitation-contraction (EC) coupling. These structures form functional junctions with the sarcoplasmic reticulum (SR), and thereby enable close contact between L-type Ca 2+ channels (LTCCs) and Ryanodine Receptors (RyRs). This arrangement in turn ensures efficient triggering of Ca 2+ release, and contraction. While new data indicate that t-tubules are capable of exhibiting compensatory remodeling, they are also widely reported to be structurally and functionally compromised during disease, resulting in disrupted Ca 2+ homeostasis, impaired systolic and/or diastolic function, and arrhythmogenesis. This review summarizes these findings, while highlighting an emerging appreciation of the distinct roles of t-tubules in the pathophysiology of heart failure with reduced and preserved ejection fraction (HFrEF and HFpEF). In this context, we review current understanding of the processes underlying t-tubule growth, maintenance, and degradation, underscoring the involvement of a variety of regulatory proteins, including junctophilin-2 (JPH2), amphiphysin-2 (BIN1), caveolin-3 (Cav3), and newer candidate proteins. Upstream regulation of t-tubule structure/function by cardiac workload and specifically ventricular wall stress is also discussed, alongside perspectives for novel strategies which may therapeutically target these mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Setterberg, Le, Frisk, Li and Louch.)

Background: The human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics., Methods: A panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing., Results: Our modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes., Conclusion: Our results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies., (© 2021. The Author(s).)

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca 2+ handling is a key feature of HF pathophysiology. Restoring the Ca 2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp -/- ), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF., (© 2021. The Author(s).)

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca 2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca 2+ alternans. We studied spontaneous Ca 2+ release events and Ca 2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca 2+ release events in RyR2-R2474S cardiomyocytes exposed to the β-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca 2+ alternans and increased Ca 2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca 2+ alternans was explained by prolongation of Ca 2+ release refractoriness. Importantly, the increased propensity for Ca 2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca 2+ release but promotes Ca 2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca 2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca 2+ alternans should include investigation of both phenomena. NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca 2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca 2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca 2+ release. Prolonged refractoriness of Ca 2+ release leads to arrhythmogenic Ca 2+ alternans. CaMKII inhibition promotes Ca 2+ alternans by prolonging Ca 2+ release refractoriness.

The goal of this work was to investigate the role of t-tubule (TT) remodeling in abnormal Ca 2+ cycling in ventricular myocytes of failing dog hearts. Heart failure (HF) was induced using rapid right ventricular pacing. Extensive changes in echocardiographic parameters, including left and right ventricular dilation and systolic dysfunction, diastolic dysfunction, elevated left ventricular filling pressures, and abnormal cardiac mechanics, indicated that severe HF developed. TT loss was extensive when measured as the density of total cell volume, derived from three-dimensional confocal image analysis, and significantly increased the distances in the cell interior to closest cell membrane. Changes in Ca 2+ transients indicated increases in heterogeneity of Ca 2+ release along the cell length. When critical properties of Ca 2+ release variability were plotted as a function of TT organization, there was a complex, nonlinear relationship between impaired calcium release and decreasing TT organization below a certain threshold of TT organization leading to increased sensitivity in Ca 2+ release below a TT density threshold of 1.5%. The loss of TTs was also associated with a greater incidence of triggered Ca 2+ waves during rapid pacing. Finally, virtually all of these observations were replicated by acute detubulation by formamide treatment, indicating an important role of TT remodeling in impaired Ca 2+ cycling. We conclude that TT remodeling itself is a major contributor to abnormal Ca 2+ cycling in HF, reducing myocardial performance. The loss of TTs is also responsible for a greater incidence of triggered Ca 2+ waves that may play a role in ventricular arrhythmias arising in HF. NEW & NOTEWORTHY Three-dimensional analysis of t-tubule density showed t-tubule disruption throughout the whole myocyte in failing dog ventricle. A double-linear relationship between Ca 2+ release and t-tubule density displays a steeper slope at t-tubule densities below a threshold value (∼1.5%) above which there is little effect on Ca 2+ release (T-tubule reserve). T-tubule loss increases incidence of triggered Ca 2+ waves. Chemically induced t-tubule disruption suggests that t-tubule loss alone is a critical component of abnormal Ca 2+ cycling in heart failure.

Background: Whereas heart failure with reduced ejection fraction (HFrEF) is associated with ventricular dilation and markedly reduced systolic function, heart failure with preserved ejection fraction (HFpEF) patients exhibit concentric hypertrophy and diastolic dysfunction. Impaired cardiomyocyte Ca 2+ homeostasis in HFrEF has been linked to disruption of membrane invaginations called t-tubules, but it is unknown if such changes occur in HFpEF., Objectives: This study examined whether distinct cardiomyocyte phenotypes underlie the heart failure entities of HFrEF and HFpEF., Methods: T-tubule structure was investigated in left ventricular biopsies obtained from HFrEF and HFpEF patients, whereas cardiomyocyte Ca 2+ homeostasis was studied in rat models of these conditions., Results: HFpEF patients exhibited increased t-tubule density in comparison with control subjects. Super-resolution imaging revealed that higher t-tubule density resulted from both tubule dilation and proliferation. In contrast, t-tubule density was reduced in patients with HFrEF. Augmented collagen deposition within t-tubules was observed in HFrEF but not HFpEF hearts. A causative link between mechanical stress and t-tubule disruption was supported by markedly elevated ventricular wall stress in HFrEF patients. In HFrEF rats, t-tubule loss was linked to impaired systolic Ca 2+ homeostasis, although diastolic Ca 2+ removal was also reduced. In contrast, Ca 2+ transient magnitude and release kinetics were largely maintained in HFpEF rats. However, diastolic Ca 2+ impairments, including reduced sarco/endoplasmic reticulum Ca 2+ -ATPase activity, were specifically observed in diabetic HFpEF but not in ischemic or hypertensive models., Conclusions: Although t-tubule disruption and impaired cardiomyocyte Ca 2+ release are hallmarks of HFrEF, such changes are not prominent in HFpEF. Impaired diastolic Ca 2+ homeostasis occurs in both conditions, but in HFpEF, this mechanism for diastolic dysfunction is etiology-dependent., Competing Interests: Author Disclosures This study was supported by the European Union’s Horizon 2020 Research and Innovation Programme (Consolidator grant to Dr. Louch) under grant agreement No. 647714. Additional support was provided by The South-Eastern Norway Regional Health Authority, Anders Jahre’s Fund for the Promotion of Science, the Research Council of Norway, the Norwegian Institute of Public Health, Oslo University Hospital, the University of Oslo, the K.G. Jebsen Center for Cardiac Research, Norway, the European Union Projects No. FP7-HEALTH-2010.2.4.2-4 (‘‘MEDIA-Metabolic Road to Diastolic Heart Failure’’), and the Marsden Fund administered by the Royal Society of New Zealand (UOO1501). The authors have reported that they have no relationships relevant to the contents of this paper to disclose., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aim: Dysfunction of the cardiac ryanodine receptor (RyR2) is an almost ubiquitous finding in animal models of heart failure (HF) and results in abnormal Ca 2+ release in cardiomyocytes that contributes to contractile impairment and arrhythmias. We tested whether exercise training (ET), as recommended by current guidelines, had the potential to stabilize RyR2-dependent Ca 2+ release in rats with post-myocardial infarction HF. Materials and Methods: We subjected male Wistar rats to left coronary artery ligation or sham operations. After 1 week, animals were characterized by echocardiography and randomized to high-intensity interval ET on treadmills or to sedentary behavior (SED). Running speed was adjusted based on a weekly VO 2max test. We repeated echocardiography after 5 weeks of ET and harvested left ventricular cardiomyocytes for analysis of RyR2-dependent systolic and spontaneous Ca 2+ release. Phosphoproteins were analyzed by Western blotting, and beta-adrenoceptor density was quantified by radioligand binding. Results: ET increased VO 2max in HF-ET rats to 127% of HF-SED ( P < 0.05). This coincided with attenuated spontaneous SR Ca 2+ release in left ventricular cardiomyocytes from HF-ET but also reduced Ca 2+ transient amplitude and slowed Ca 2+ reuptake during adrenoceptor activation. However, ventricular diameter and fractional shortening were unaffected by ET. Analysis of Ca 2+ homeostasis and major proteins involved in the regulation of SR Ca 2+ release and reuptake could not explain the attenuated spontaneous SR Ca 2+ release or reduced Ca 2+ transient amplitude. Importantly, measurements of beta-adrenoceptors showed a normalization of beta 1 -adrenoceptor density and beta 1 :beta 2 -adrenoceptor ratio in HF-ET. Conclusion: ET increased aerobic capacity in post-myocardial infarction HF rats and stabilized RyR2-dependent Ca 2+ release. Our data show that these effects of ET can be gained without major alterations in SR Ca 2+ regulatory proteins and indicate that future studies should include upstream parts of the sympathetic signaling pathway., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Danielsen, Sadredini, Manotheepan, Aronsen, Frisk, Hansen, Andressen, Hougen, Levy, Louch, Sejersted, Sjaastad and Stokke.)

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

Subjects with functionally univentricular circulation who have completed staged single ventricle palliation, with the final stage culminating in the Fontan procedure, are often living into adulthood. However, high morbidity and mortality remain prevalent in these patients, as diastolic and systolic dysfunction of the single systemic ventricle are linked to Fontan circulatory failure. We presently investigated the effects of probenecid in post-Fontan patients. Used for decades for the treatment of gout, probenecid has been shown in recent years to positively influence cardiac function via effects on the Transient Receptor Potential Vanilloid 2 (TRPV2) channel in cardiomyocytes. Indeed, we observed that probenecid improved cardiac function and exercise performance in patients with a functionally univentricular circulation. This was consistent with our findings from a retrospective cohort of patients with single ventricle physiology where TRPV2 expression was increased. Experiments in isolated cardiomyocytes associated these positive actions to augmentation of diastolic calcium homeostasis.

Cardiac contraction is the result of integrated cellular, tissue and organ function. Biophysical in silico cardiac models offer a systematic approach for studying these multi-scale interactions. The computational cost of such models is high, due to their multi-parametric and nonlinear nature. This has so far made it difficult to perform model fitting and prevented global sensitivity analysis (GSA) studies. We propose a machine learning approach based on Gaussian process emulation of model simulations using probabilistic surrogate models, which enables model parameter inference via a Bayesian history matching (HM) technique and GSA on whole-organ mechanics. This framework is applied to model healthy and aortic-banded hypertensive rats, a commonly used animal model of heart failure disease. The obtained probabilistic surrogate models accurately predicted the left ventricular pump function ( R 2  = 0.92 for ejection fraction). The HM technique allowed us to fit both the control and diseased virtual bi-ventricular rat heart models to magnetic resonance imaging and literature data, with model outputs from the constrained parameter space falling within 2 SD of the respective experimental values. The GSA identified Troponin C and cross-bridge kinetics as key parameters in determining both systolic and diastolic ventricular function. This article is part of the theme issue 'Uncertainty quantification in cardiac and cardiovascular modelling and simulation'.

Cardiac hypertrophy and renal damage associated with hypertension are independent predictors of morbidity and mortality. In a model of hypertensive heart disease and renal damage, we tested the actions of continuous administration of Vastiras, a novel compound derived from the linear fragment of ANP (atrial natriuretic peptide), namely pro-ANP 31-67 , on blood pressure and associated renal and cardiac function and remodeling. Of note, this peptide, unlike the ring structured forms, does not bind to the classic natriuretic peptide receptors. Dahl/Salt-Sensitive rats fed a 4% NaCl diet for 6 weeks developed hypertension, cardiac hypertrophy, and renal damage. Four weeks of treatment with 50 to 100 ng/kg per day of Vastiras exhibited positive effects on renal function, independent of blood pressure regulation. Treated rats had increased urine excretion, natriuresis, and enhanced glomerular filtration rate. Importantly, these favorable renal effects were accompanied by improved cardiac structure and function, including attenuated cardiac hypertrophy, as indicated by decreased heart weight to body weight ratio, relative wall thickness, and left atrial diameter, as well as reduced fibrosis and normalized ratio of the diastolic mitral inflow E wave to A wave. A renal subtherapeutic dose of Vastiras (25 ng/kg per day) induced similar protective effects on the heart. At the cellular level, cardiomyocyte size and t-tubule density were preserved in Vastiras-treated compared with untreated animals. In conclusion, these data demonstrate the cardiorenal protective actions of chronic supplementation of a first-in-class compound, Vastiras, in a preclinical model of maladaptive cardiac hypertrophy and renal damage induced by hypertension.