Ventricular arrhythmias, a major cause of sudden cardiac death, are driven by Ca 2+ imbalance in cardiac myocytes, often linked to the overactivation of CaMKIIδ (Ca 2+ /calmodulin-dependent protein kinase II delta). As such, inhibiting CaMKIIδ represents a promising therapeutic strategy. Based on our previous finding that native secretoneurin (SN) is a weak CaMKIIδ inhibitor, we aimed to develop a more potent derivative of SN to effectively counter aberrant Ca 2+ handling and arrhythmia risk. Various regions of SN were tested for CaMKII binding, identifying the core region as the sequence with the strongest binding capacity. This region was subsequently optimised with two phenylalanine substitutions, resulting in the SN derivative SN-db-short. Structural homology modeling and ELISA-based assays revealed that SN-db-short bound both the substrate-binding (S-site) region of CaMKIIδ, in addition to the ATP-binding region, with 8-fold stronger binding compared to SN. Surface plasmon resonance experiments confirmed that SN-db-short exhibited a higher association rate and affinity for CaMKIIδ compared to SN. Consistent with only a partial calmodulin binding motif, SN-db-short showed no calmodulin binding, indicating selective CaMKIIδ inhibition. In functional studies, SN-db-short inhibited CaMKIIδ-mediated phosphorylation of ryanodine receptor 2 and appeared more effective than SN in reducing the incidence of Ca 2+ sparks and Ca 2+ waves. SN-db-short also more markedly inhibited CaMKIIδ phosphorylation of phospholamban, slowed Ca 2+ reuptake, and reduced the magnitude of Ca 2+ transients during isoproterenol stimulation. SN-db-short effectively inhibits CaMKIIδ and significantly counters aberrant Ca 2+ handling in cardiomyocytes. Thus, this optimised peptide holds therapeutic potential for reducing the risk of ventricular arrhythmias.

BACKGROUND:Interleukin-6 receptor (IL-6R) inhibition by tocilizumab improves myocardial salvage index (MSI) in ST-elevation myocardial infarction (STEMI). However, the mechanisms for this effect remain unclear. METHODS:This pre-defined exploratory sub-study of the ASSAIL-MI trial enumerated circulating monocytes and examined their transcriptome profile in relation to the MSI and peak troponin T (TnT) in STEMI patients randomiseded to tocilizumab (n = 101) or placebo (n = 98). RNA sequencing was performed on peripheral monocytes in 14 patients. To elaborate the in vivo findings, in vitro chemotaxis and apoptosis assays were performed on THP-1 monocytes and cardiomyocyte (HL-1) cell lines, respectively. FINDINGS:STEMI patients had increased monocyte counts at 24 h and 3-7 days after hospitalisation/PCI and this increase was attenuated by tocilizumab. Lower monocyte levels at 24 h were associated with lower TnT levels and higher MSI. Monocyte gene expression suggested that tocilizumab modulated cytokine signalling pathways related to myocardial remodelling, apoptosis, and chemotaxis, potentially through a decrease in suppressor of cytokine signalling 3 (SOCS3). In vitro, tocilizumab limited apoptosis of cardiomyocytes exposed to ischemia/reperfusion and reduced chemotaxis in monocytes exposed to IL-6. INTERPRETATION:These findings suggest that IL-6R inhibition by tocilizumab during STEMI is associated with reduced monocyte counts and cardioprotective alterations in monocyte signalling potentially linked to the downregulation of SOCS3. FUNDING:This work was supported by the South-Eastern Norway Regional Health Authority (no. 2019067) and The Research Council of Norway (no. 282867) The ASSAIL-MI main study was supported by an independent grant from ROCHE who also provided drugs/placebo for infusion.

Creatine kinase (CK) is considered a crucial energy transfer system in cardiac muscle. Some studies have suggested that reduced CK energy transfer in the heart causes energy starvation, limits cardiac performance, and ultimately leads to heart failure. In agreement with this hypothesis, the hearts from creatine-deficient mice lacking arginine:glycine amidinotransferase (AGAT KO) have been shown, in some experiments, to resemble failing hearts. However, it is unclear if AGAT KO induces changes in cardiomyocyte substructure and Ca2+ cycling that resemble heart failure, including impairment of sarcoplasmic reticulum (SR) Ca2+ release and reuptake. To investigate this, we assessed functional and structural aspects of Ca2+ handling in cardiomyocytes from KO and WT littermates. We found minor, sex-dependent differences in the organization of transverse tubules and ryanodine receptors (RyRs), no differences in the expression and relative phosphorylation of RyR and PLB, but higher S100A1 expression levels. AGAT KO cardiomyocytes exhibited larger and longer Ca2+ transients with the same decay rate as WT. Ca2+ spark frequency and SR Ca2+ content were also increased in KO, while sodium-calcium exchanger activity was unchanged. Thus, our results strongly suggest that SR Ca2+ cycling is augmented in AGAT KO hearts. Although AGAT KO hearts also exhibited increased AMPK activation, suggesting higher levels of AMP/ADP, this did not detectably impair sarcoendoplasmic reticulum Ca2+-ATPase activity. In conclusion, the changes in AGAT KO cardiomyocytes are opposite to those in failing cardiomyocytes, showing that lifelong absence of CK energy transfer does not lead to heart failure.NEW & NOTEWORTHY Previous studies have suggested that reduced creatine kinase (CK) activity may lead to heart failure. Here, we studied calcium handling in the hearts of creatine-deficient arginine-glycine amidino-transferase knockout (AGAT KO) mice with lifelong inhibition of CK. In contrast to failing cardiomyocytes, AGAT KO cardiomyocytes exhibited larger calcium transients due to more readily firing RyR clusters releasing more calcium from the SR. Thus, lifelong creatine deficiency does not lead to the phenotype observed in heart failure.

AIMS:The heartbeat is triggered by the coordinated release of Ca2+ from the ryanodine receptor type-2 (RyR) in cardiomyocytes. Phosphorylation of RyR by Ca2+/calmodulin-dependent kinase IIδ (CaMKIIδ) fine-tunes this process in health, while hyperphosphorylation causes excessive, pathological Ca2+ release. We investigated how CaMKIIδ is spatially recruited and anchored to RyRs to achieve this functional regulation. METHODS AND RESULTS:We employed confocal and dSTORM microscopy to investigate the macro- and nanoscale distribution of CaMKIIδ across cardiomyocytes, respectively. We linked positional rearrangement of the kinase during β-adrenergic stimulation (isoproterenol, Iso) to alterations in RyR phosphorylation and function (Ca2+ sparks), and the requirement of the CaMKIIδ anchoring protein AKAP18δ by knockdown/knockout. Confocal microscopy revealed that macroscale CaMKIIδ localization was not markedly altered during Iso-treatment, although a narrowing of its distribution around the Z-lines occurred, where the RyR reside. Higher resolution dSTORM imaging confirmed that local mobilization of CaMKIIδ by Iso decreased the distance from Z-lines and RyRs to the nearest CaMKIIδ by 28 and 12%, respectively. Functionally, kinase translocation into the RyR nanodomain was accompanied by increased channel phosphorylation and Ca2+ spark frequency. These actions were dependent on CaMKIIδ activity, since kinase translocation, RyR phosphorylation, and activation were all mimicked by the upstream activator of CaMKIIδ (8-CPT) and prevented by direct CaMKIIδ inhibitors (AIP, N1 peptide). A critical role of AKAP18δ in this mechanism was supported by immunoprecipitation experiments, which showed greater kinase binding to AKAP18δ during Iso-stimulation. Furthermore, loss of AKAP18δ by viral-mediated AKAP18δ knockdown or knockout prevented CaMKIIδ translocation to Z-lines. Microtubular disruption also blocked CaMKIIδ translocation. CONCLUSION:Collectively, our results indicate that nanoscale movement of CaMKIIδ is closely associated with RyR activation following β-adrenergic stimulation. This translocation depends on an intact microtubular network and kinase binding to AKAP18δ.

Engineered heart tissues (EHTs), composed of human stem cell-derived cardiomyocytes and fibroblasts, have emerged as promising tools for disease modeling and drug discovery due to their ability to replicate aspects of the function and structure of the heart muscle. While bioimpedance and electrophysiology measurements are powerful tools for monitoring EHTs, these techniques can be challenging to perform due to the high conductivity of the surrounding cell medium. In this study, we explored the possibility of measuring bioimpedance and electrophysiology outside of the cell medium. To this end, we developed a platform that lifts EHTs from the culture medium during the measurements, thereby allowing the electrical current to flow directly through the tissues. The platform monitors contractility by fixed frequency impedance measurements, electrophysiology by field potential measurements, and structural changes through electrical impedance spectroscopy. Over two weeks of measurements, we observed increasing trends in contractility and field potential amplitudes, along with structural changes. No adverse effect on the EHTs were detected, ensuring their stability and viability throughout the monitoring process. Moreover, the impedance amplitude measured on the platform correlates with the contractile degree during drug experiments, serving as a reliable indicator of EHT functionality. Electrical stimulation with limited charge injection was also demonstrated. In conclusion, our platform offers a comprehensive and efficient method for monitoring EHTs and holds potential as a tool for cardiovascular disease modeling and drug screening.

Syndecan-4 is a ubiquitously expressed transmembrane proteoglycan that links the extracellular matrix to intracellular protein networks. It is located at stress-sensing structures in cardiomyocytes, including costameres and Z-discs, and in male mice, it is involved in the hypertrophic response to cardiac pressure overload. We have recently found female syndecan-4 KO cardiomyocytes, without challenge, to be smaller in area. Smaller cardiomyocytes with elongation defects have been observed in animal models with β-parvin deficiency, where the loss of this mechano-sensor disrupts the guanine nucleotide exchange factor (GEF) β-PIX-GTPase Rac1 axis, which is essential for proper cell elongation. β-parvin, together with integrin-linked kinase (ILK) and particularly interesting new cysteine-histidine-rich protein (PINCH), constitutes the IPP complex (ILK-PINCH-parvin), which is part of the integrin consensus adhesome. Interestingly, in a previous large cardiac interactome study, we have identified β-parvin, as well as ILK, β-PIX, and Rac1 as potential syndecan-4 partners. To better understand the syndecan-4-β-parvin association, we mapped their interaction and investigated the effect of syndecan-4 ablation on the IPP complex, the β-parvin-β-PIX-Rac1 axis, and cardiomyocyte geometry in both females and males. Interestingly, genetic ablation of syndecan-4 resulted in shorter cardiomyocytes in females only. The syndecan-4-β-parvin interaction was mapped to accessible sequences within the N-terminal, linker, and CH2 domains of β-parvin and the unique variable C2 cytoplasmic region of syndecan-4. Syndecan-4 ablation resulted in lower levels of membrane-localized β-parvin in both sexes and sex-specific differences in its associated partners ILK and PINCH, suggesting that syndecan-4 is linked to integrin signaling through the IPP complex. Finally, Rac1, known for its involvement in cell size regulation, and some of its regulators, β-PIX, RhoGDIα, and the serine/threonine kinase PAK, showed sex-specific alterations following syndecan-4 ablation. Altogether, our data suggest that syndecan-4 binds directly to β-parvin and regulates cardiomyocyte length, the IPP complex, and the β-parvin-β-PIX-Rac1 in a sex-dependent manner. These findings highlight a sex-specific role for syndecan-4 in cardiomyocyte structure, offering new insight into the molecular basis for sex differences in cardiac biology.

IntroductionCD38, a regulator of intracellular calcium signalling, is highly expressed in immune cells. Mice lacking CD38 are very susceptible to acute bacterial infections, implicating CD38 in innate immune responses. The effects of CD38 inhibition on NLRP3 inflammasome activation in human primary monocytes and monocyte-derived macrophages have not been investigated. Apigenin is a naturally occurring flavonoid known to inhibit CD38. However, apigenin has also been proposed to inhibit the extracellular ATP receptor P2XR7, an upstream activator of NLRP3. In this study we aimed to investigate whether apigenin attenuates NLRP3 inflammasome activation in human monocytes and monocyte-derived macrophages through CD38 inhibition. MethodsLPS-primed human monocytes and monocyte-derived macrophages were treated with apigenin, the CD38 inhibitor 78c, antagonists of CD38 second messengers (8-br-ADPR and 8-br-cADPR) or the ATP hydrolase, apyrase, prior to NLRP3 activation with ATP, monosodium urate crystals (MSU) or nigericin. IL-1β and TNF secretion and mRNA expression, as well as N-terminal gasdermin-D formation were quantified. Ca2+ mobilization was determined by live confocal microscopy. NLRP3 activity was also compared in WT and CD38-/- mouse bone marrow-derived macrophages (BMDMs) with and without CD38 inhibitors.ResultsApigenin significantly inhibited IL-1β release from LPS-primed monocytes and macrophages activated with ATP, MSU, or nigericin. CD38 inhibition with 78c also attenuated NLRP3-dependent IL-1β release. Apigenin was a potent inhibitor of Ca2+ flux from the endoplasmic reticulum to the cytosol in human monocyte-derived macrophages. Apyrase attenuated IL-1β release induced by ATP or MSU, but not by nigericin. However, the NLRP3 inflammasome is not compromised in CD38-/- bone marrow-derived macrophages compared to corresponding WT cells, and apigenin moderated IL-1β release in both genotypes. DiscussionOur data support that apigenin attenuates NLRP3 activation independently of CD38. Our results also suggest that MSU crystals activate NLRP3 through autocrine or paracrine ATP signalling.

Abstract Exercise‐induced muscle damage (EIMD) is characterized by a severe and prolonged decline in force‐generating capacity. However, the precise cellular mechanisms underlying the observed long‐lasting decline in force‐generating capacity associated with EIMD are still unclear. We investigated in vivo force generation and ex vivo Ca2+‐activated force generation, Ca2+ sensitivity, and myofiber Ca2+ handling systems (SR and t‐tubules) in human biceps brachii before and 2, 48, and 96 h after eccentrically muscle‐damaging contractions and in non‐exercised control arm. The force‐generating capacity declined by 50 ± 13% 3 h after exercise and was still not recovered after 96 h. The force‐Ca relationship of skinned myofibers revealed an impaired maximal Ca2+‐activated force in MHC I‐fibers, but not MHC II‐fibers 48 h after exercise. Further, Ca2+ sensitivity was increased in MHC II‐fibers, which was reversed after incubation with a strong reductant. There was a biphasic increase in SERCA sulfonylation, and a parallel reduction in the SR Ca2+ uptake rate, with no effects on SR vesicle leak or SR vesicle Ca2+ release rate. T‐tubules showed a progressive increase in the density of longitudinal tubules by 96 h after exercise. In conclusion, MHC II‐fiber Ca2+ sensitivity was increased 48 h after exercise, attributed to changes in the REDOX status. 96 h after exercise SR vesicle Ca2+ uptake was impaired, and an increased number of longitudinal tubules were observed. These alterations may contribute to the impaired force generation evident at the late stage of recovery.

BACKGROUND:Cardiac troponins and natriuretic peptides are benchmark biomarkers for heart failure (HF) with reduced ejection fraction (HFrEF) but have limited prognostic performance for HF patients with preserved ejection fraction (HFpEF). Non-coding RNA-based biomarkers represent an innovative approach to risk-stratify patients and might address the unmet need for minimally invasive prognostic and predictive tools for HF development and HF-related outcomes. Our aim is to investigate the prognostic performance and risk stratification potential of circulating panels of microRNAs (miRNAs) in HFrEF and HFpEF. METHODS:A systematic search on PubMed, Web of Science, and Scopus databases was performed for studies reporting miRNAs as prognostic biomarkers in HF patients. A total of 22 studies pooling 5736 participants were included for quantitative analysis. KM-based individual patient data (IPD) analysis was performed in 12 studies (5064 participants). RESULTS:KM-based IPD analysis in HFrEF allowed the identification of a panel of four miRNAs (miR-27a-3p, miR-129-5p, miR-145-5p, and miR-590-3p) predicting the risk of all-cause death with hazard ratio (HR) 4.26 [2.68-6.76]. MiR-122-5p and miR-423-5p predicted cardiovascular death of HFrEF patients (HR 3.61 [2.67-4.87]). In HFpEF, miR-19a-3p predicted all-cause death of HFpEF patients with HR 2.23 [1.16-4.27]. Moreover, a panel of eight miRNAs (miR-17-5p, miR-20a-5p, miR-21, miR-23, miR-27, miR-106b-5p, miR-210, and miR-221) showed significant association with HF incidence (HR 2.14 [1.81-2.53]). CONCLUSIONS:A comprehensive meta-analysis of KM-based IPD enabled the identification of unique miRNA panels predicting the incidence and severity of HFrEF and HFpEF, supporting the clinical usefulness of miRNA profiling for tailored healthcare and risk stratification in HF patients. Nonetheless, more rigorously designed longitudinal studies are needed to validate the clinical application of miRNAs as prognostic and predictive biomarkers.

Calcium release through the cardiac RyR2 (type-2 ryanodine receptor) is essential for cardiac contraction. RyR2 dysfunction is associated with a spectrum of cardiac pathologies, most notably arrhythmias. While excessive RyR2 activity was historically seen as the driver of arrhythmia, it is now clear that inadequate calcium release is equally detrimental. This homeostatic balance of activity requires precise tuning, which is provided by a swathe of regulating factors spanning posttranslational modifications, protein-protein interactions, and the more recently identified positioning of individual RyR2 channels within the cell. This review summarizes how too much, or too little, calcium release can lead to arrhythmia and explores how the multitude of regulating factors work synergistically to set and modify RyR2 physiologically and become impaired in disease. Finally, we examine how RyR2-targeted pharmacological approaches can therapeutically rebalance calcium handling and inhibit arrhythmia.

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 Ca2+-handling disturbances and Ca2+-driven arrhythmogenic events in male vs female atrial cardiomyocytes and establish their responses to Ca2+-targeted interventions. Methods We integrated reported sex differences and AF-associated changes (ie, expression and phosphorylation of Ca2+-handling proteins, cardiomyocyte ultrastructural characteristics, and dimensions) into our human atrial cardiomyocyte model that couples electrophysiology with spatially detailed Ca2+-handling processes. Sex-specific responses of atrial cardiomyocytes to arrhythmia-provoking protocols and Ca2+-targeted interventions were evaluated. Results Simulated quiescent cardiomyocytes showed 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. 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 Ca2+ current was protective against SCRs for male AF cardiomyocytes. Furthermore, simulated Ca2+-targeted interventions identified potential strategies (eg, t-tubule restoration, and inhibition of ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2⁺-ATPase) to attenuate Ca2+-driven arrhythmogenic events in women, and revealed enhanced efficacy when applied in combination. Conclusions Sex-specific modeling uncovers increased Ca2+-driven arrhythmogenic events in female vs male atria in AF, and suggests combined Ca2+-targeted interventions are promising therapeutic approaches in women.

Cardiac-related mortality is increasing in farmed salmon. Non-invasive tools for examining and screening for cardiac morphology and function are limited, and most common methodologies are lethal, time-consuming, and immobile. Echocardiography has previously been tested as a non-invasive, quick, and portable alternative, though its implementation is minimal. Improvements in echocardiographic techniques during the last decade have enabled more refined assessments of structure and function and hold potential for use in fish farms. Utilising a compact, transportable ultrasound system, we examined the applicability of echocardiography in Atlantic salmon (Salmo salar L.). Several protocols and projections were tested, and intra- and inter-variation for both operators (image acquisition) and observers (image analysis) were assessed. In addition, the accuracy of cardiac structure/function measurements was compared with standard methods. In general, high accuracy and reproducibility of cardiac dimensions and functional parameters were found within the same and between different observers analysing the same dataset (intra- and inter-observer). Measurements between recordings of the same operator (intra-operator) and between different operators (inter-operator) were less accurate and repeatable but comparable to observations in previous human and mammalian studies. Cardiac output was slightly higher when measured with echocardiography compared to transit time flow probe. Yet, a strong correlation exists between the two methods. Furthermore, morphology measured in excised hearts ex vivo was comparable to echocardiography measurements and strongly correlated. Thus, ultrasound presents a highly feasible, non-invasive, and swift alternative to current methods for detailed cardiac assessment of salmon hearts.

Intracellular calcium (Ca2+) is an essential mediator of normal cardiac function, and its dysregulation has been directly linked to atrial fibrillation (AF), the most common cardiac arrhythmia. Substantial sex differences exist in the prevalence, clinical representation, pathophysiology, therapy, and prognosis of AF, leading to less effective and sometimes harmful treatments for females. However, the associated mechanisms and causes remain largely unknown, partly due to female underrepresentation in both fundamental and clinical research. We recently revealed potential sex-specific mechanisms underlying the experimentally-observed increased female vs. male propensity for spontaneous Ca2+ release events (SCRs), and this has important implications for understanding sex-specific AF pathophysiology and treatment. Here, we propose a multi-scale mechanistic modeling approach to determine the impact of sex differences on human atrial tissue dynamics. We introduced reported sex differences in ionic and structural parameters into two-dimensional tissue-level constructs. These tissue models were built upon our new human atrial myocyte model, which incorporates spatially detailed Ca2+ handling and accounts for sex-specific functional and structural features in both AF and normal sinus rhythm. We reduced the complexity of the cell models via statistical functions describing the relationship between cell structure and SCR for efficient simulation of atrial tissue. Similar to findings in isolated myocytes, the female tissue model displayed greater and more frequent SCR-triggered delayed afterdepolarizations (DADs) compared to male. These disparities were further amplified in AF conditions and resulted from the interplay of heightened cellular SCR-DAD susceptibility and increased source-sink mismatch (worsened by fibrosis and cell-cell uncoupling), as revealed by a systematic parametric analysis. Our study demonstrates the interactive contributions of tissue-level sex differences to Ca2+-driven instability and provides a novel sex-specific multiscale simulation framework. The new mechanistic insight may inform novel sex-specific therapeutic strategies against AF.

Introduction Increasing prevalence of ischemic heart disease and particularly myocardial infarction (MI) carries a high socioeconomic burden, and treatment strategies are limited. Emerging data from our group have indicated that reduced cardiac contractility in this condition is linked to degradation of subcellular structures called t-tubules. While the underlying mechanisms are unclear, our data suggest that the interplay between the scaffolding protein BIN1 and specific lipids called phospho-inositides (PIs) is critically involved. Objective The objective of this study is to establish that BIN1 and PI homeostasis precisely controls cardiac t-tubule growth, maturation, and maintenance. We additionally aim to target these pathways to reverse t-tubule remodeling during MI. Method We are making use of the HL-1 cardiomyocyte cell line, isolated mouse cardiomyocytes, and an in vivo mouse model of MI to thoroughly study the collaborative roles of BIN1 and PIs in controlling cardiac t-tubule growth. Transcript and protein levels of target proteins in heart tissue are assessed by RT-qPCR and Western-blotting. T-tubule organization is studied by immunofluorescence and immunohistochemistry followed by confocal microscopy. Results In a first part, we are working on the establishment of a cardiac “t-tubule interactome” in HL-1 cardiomyocytes, using affinity purification coupled to mass spectrometry to identify key pathways implicated in tubulogenesis and lipid homeostasis. Our initial confirmation by co-localization and Western-blotting, indicates that the BIN1-partners DNM2, MTM1, RIN2, RIN3, and SYNJ2 are the most propitious. We have also started to determine the lipid composition of t-tubules, using probes for membrane polarity and lipidomic.We are further investigating the promising target proteins in a cardiac disease setting. This work is carried out in a well-established mouse model of acute MI followed by 3h–28days of reperfusion. Our encouraging initial results suggest a downregulation of BIN1 and MTM1 as well as an upregulation of RIN3 at the protein level during MI. Conclusion Thus, this work will provide exciting new insight into both the pathophysiology and treatment of MI.

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.

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.

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a ∼560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision (∼3 nm) in all directions. This allows determining not only the location but also the 3D orientation of individual RyR2 channels in intact cells. In practice, this capability is currently limited by a relatively modest effective labeling efficiency (∼10 % subunit detection efficiency translating into ∼35% RyR2 labeling efficiency) which we measure in-situ using a novel procedure enabled by the true molecular resolution of MINFLUX microscopy. The new data suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data that may be at least partially explained by effects of labeling efficiency. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches may be applicable to other multi-subunit complexes in cardiac muscle and other cell types.### Competing Interest StatementThe authors declare that they have no competing interests in the production and presentation of results. I.J. works at Abberior Instruments that develops and manufactures superresolution fluorescence microscopes, including the 3D-MINFLUX system used here.

Creatine kinase (CK) is considered a crucial energy transfer system in cardiac muscle. Reduced CK energy transfer may exacerbate energy starvation in failing hearts, limiting cardiac performance. Furthermore, CK–/– mice eventually develop heart failure. However, CK is associated with many intracellular structures, raising the question of whether the CK–/– phenotype is due to its structural role. Here, we studied creatine-deficient mice lacking guanidinoacetate amidinotransferase (GAMT–/–) or arginine:glycine amidinotransferase (AGAT–/–). They have near-normal CK expression, but the absence of creatine inhibits energy transfer. Whereas the GAMT–/– phenotype is relatively mild, earlier studies have suggested that AGAT–/– hearts resemble failing hearts. To address this further, we studied how one of the major cardiac energy-consuming processes—Ca2+ handling—adapts to compromised CK energy transfer in cardiomyocytes from AGAT−/− and GAMT−/− mice. Here, we assessed Ca2+ sparks, the transverse tubular network, the ryanodine receptor (RyR) organization, and RyR and phospholamban expression and phosphorylation. We observed most changes in AGAT–/– cardiomyocytes, which exhibited larger and longer Ca2+ transients accompanied by higher Ca2+ spark frequency. We found no consistent differences in RyR expression or phosphorylation to explain this. However, taken together with the similar frequency of Ca2+ wave occurrences and changes in RyR arrangement, our results suggest a tighter dyadic space in AGAT–/– cardiomyocytes. Our results are opposite to failing cardiomyocytes, which have shallow Ca2+ transients suggesting that the absence of CK energy transfer does not lead to heart failure. Instead, in AGAT–/–, the heart adapts through changes in Ca2+ homeostasis and ultrastructural remodelling.