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The Ca(2+) transport ATPase (SERCA) of sarcoplasmic reticulum (SR) plays an important role in muscle cytosolic signaling, as it stores Ca(2+) in intracellular membrane bound compartments, thereby lowering cytosolic Ca(2+) to induce relaxation. The stored Ca(2+) is in turn released upon membrane excitation to trigger muscle contraction. SERCA is activated by high affinity binding of cytosolic Ca(2+), whereupon ATP is utilized by formation of a phosphoenzyme intermediate, which undergoes protein conformational transitions yielding reduced affinity and vectorial translocation of bound Ca(2+). We review here biochemical and biophysical evidence demonstrating that release of bound Ca(2+) into the lumen of SR requires Ca(2+)/H(+) exchange at the low affinity Ca(2+) sites. Rise of lumenal Ca(2+) above its dissociation constant from low affinity sites, or reduction of the H(+) concentration by high pH, prevent Ca(2+)/H(+) exchange. Under these conditions Ca(2+) release into the lumen of SR is bypassed, and hydrolytic cleavage of phosphoenzyme may yield uncoupled ATPase cycles. We clarify how such Ca(2+)pump slippage does not occur within the time length of muscle twitches, but under special conditions and in special cells may contribute to thermogenesis.

The phospholamban (PLN) p.Arg14del mutation causes dilated cardiomyopathy, with the molecular disease mechanisms incompletely understood. Patient dermal fibroblasts were reprogrammed to hiPSC, isogenic controls were established by CRISPR/Cas9, and cardiomyocytes were differentiated. Mutant cardiomyocytes revealed significantly prolonged Ca 2+ transient decay time, Ca 2+ ‐load dependent irregular beating pattern, and lower force. Proteomic analysis revealed less endoplasmic reticulum (ER) and ribosomal and mitochondrial proteins. Electron microscopy showed dilation of the ER and large lipid droplets in close association with mitochondria. Follow‐up experiments confirmed impairment of the ER/mitochondria compartment. PLN p.Arg14del end‐stage heart failure samples revealed perinuclear aggregates positive for ER marker proteins and oxidative stress in comparison with ischemic heart failure and non‐failing donor heart samples. Transduction of PLN p.Arg14del EHTs with the Ca 2+ ‐binding proteins GCaMP6f or parvalbumin improved the disease phenotype. This study identified impairment of the ER/mitochondria compartment without SR dysfunction as a novel disease mechanism underlying PLN p.Arg14del cardiomyopathy. The pathology was improved by Ca 2+ ‐scavenging, suggesting impaired local Ca 2+ cycling as an important disease culprit. Synopsis The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism. Sarcoplasmic reticulum function remained unaltered in PLN p.Arg14del human cardiomyocytes. Impairment of the interface between endoplasmic reticulum and mitochondria was discovered as a novel disease phenotype. Cytoplasmic calcium‐scavenging improved the cardiomyopathy phenotype and revealed the role for cytoplasmic calcium in disease development. Graphical Abstract The disease mechanism linking the phospholamban (PLN) p.Arg14del mutation to dilated cardiomyopathy is incompletely understood. In this study, patient‐derived human induced pluripotent stem cell‐cardiomyocytes were used to elucidate this molecular mechanism.

PLN-R14del is a pathogenic Phospholamban (PLN) gene variant, characterized by ventricular arrhythmias and dilated cardiomyopathy in heterozygous carriers. Disease development is highly heterogeneous, indicating involvement of additional disease triggers, influencing both the onset and severity of the disease. A heterozygous PLN-R14del mouse model (R14 Δ/+ ) was used to investigate whether cardiac pressure induced by transverse aortic constriction (TAC), could accelerate disease onset. Wild-type littermates and sham operated animals were used as controls. surgery. At 6-weeks, both TAC groups exhibited increased in left ventricular wall thickness, ventricular and atrial weights, and reduced ejection fraction, with comparable hypertrophic and fibrotic responses. Furthermore, differential gene expression showed comparable activation of cardiac remodeling and stress pathways, and changes in metabolic genes expression. Importantly, TAC did not induce sarco-endoplasmic reticulum malformation in R14 Δ/+ mice, suggesting that general cardiac stress and remodeling pathways are insufficient to trigger PLN-R14del cardiomyopathy . In conclusion, TAC induced pressure overload provoked robust cardiac remodeling with activation of common stress pathways in young adult WT and R14 Δ/+ mice. It did not trigger PLN-R14del-specific sarco-endoplasmic malformation or accelerate disease progression. These findings imply that activation of common cardiac stress pathways alone may be insufficient to accelerate the onset of PLN-R14del cardiomyopathy in early adulthood.

We investigated whether hypothermia and hyperthermia can alter the efficacy and potency of histamine at increasing the force of cardiac contractions in mice that overexpress the human H 2 receptor only in their cardiac myocytes (labelled H 2 -TG). Contractile studies were performed in an organ bath on isolated, electrically driven (1 Hz) left atrial preparations and spontaneously beating right atrial preparations from H 2 -TG mice and wild-type (WT) littermate control mice. The basal beating rate in the right atrial preparations from H 2 -TG mice was lowered by hypothermia (23 °C) and elevated by hyperthermia (42 °C). Furthermore, the efficacy of histamine (0.01–100 µM) at exerting positive inotropic effects was more severely attenuated in the left and right H 2 -TG mouse atria under hypothermia and hyperthermia than under normothermia (37 °C). Similarly, the inotropic response to histamine was attenuated under hypothermia and hyperthermia in isolated electrically stimulated (1 Hz) right atrial preparations obtained from humans undergoing cardiac surgery. The phosphorylation state of phospholamban at serine 16 at 23 °C was inferior to that at 37 °C in left atrial preparations from H 2 -TG mice in the presence of 10 µM histamine. In contrast, in human atrial preparations, the phosphorylation state of phospholamban at serine 16 in the presence of 100 µM histamine was lower at 42 °C than at 37 °C. Finally, under hyperthermia, we recorded more and longer lasting arrhythmias in right atrial preparations from H 2 -TG mice than in those from WT mice. We conclude that the inotropic effects of histamine in H 2 -TG mice and in human atrial preparations, as well as the chronotropic effects of histamine in H 2 -TG mice, are temperature dependent. Furthermore, we observed that, even without stimulation of the H 2 receptors by exogenous agonists, temperature elevation can increase arrhythmias in isolated right atrial preparations from H 2 -TG mice. We propose that H 2 receptors play a role in hyperthermia-induced supraventricular arrhythmias in human patients.

We have previously shown that histamine (2-(1 H -imidazol-4-yl)ethanamine) exerted concentration-dependent positive inotropic effects (PIE) or positive chronotropic effects (PCE) on isolated left and right atria, respectively, of transgenic (H 2 R-TG) mice that overexpress the human H 2 histamine receptor (H 2 R) in the heart; however, the effects were not seen in their wild-type (WT) littermates. Amitriptyline, which is still a highly prescribed antidepressant drug, was reported to act as antagonist on H 2 Rs. Here, we wanted to determine whether the histamine effects in H 2 R-TG were antagonized by amitriptyline. Contractile studies were performed on isolated left and right atrial preparations, isolated perfused hearts from H 2 R-TG and WT mice and human atrial preparations. Amitriptyline shifted the concentration-dependent PIE of histamine (1 nM–10 μM) to higher concentrations (rightward shift) in left atrial preparations from H 2 R-TG. Similarly, in isolated perfused hearts from H 2 R-TG and WT mice, histamine increased the contractile parameters and the phosphorylation state of phospholamban (PLB) at serine 16 in the H 2 R-TG mice, but not in the WT mice. However, the increases in contractility and PLB phosphorylation were attenuated by the addition of amitriptyline in perfused hearts from H 2 R-TG. In isolated electrically stimulated human atria, the PIE of histamine that was applied in increasing concentrations from 1 nM to 10 μM was reduced by 10-μM amitriptyline. In summary, we present functional evidence that amitriptyline also acts as an antagonist of contractility at H 2 Rs in H 2 R-TG mouse hearts and in the human heart which might in part explain the side effects of amitriptyline.

Inherited cardiomyopathy caused by the p.(Arg14del) pathogenic variant of the phospholamban (PLN) gene is characterized by intracardiomyocyte PLN aggregation and can lead to severe dilated cardiomyopathy. We recently reported that pre-emptive depletion of PLN attenuated heart failure (HF) in several cardiomyopathy models. Here, we investigated if administration of a Pln-targeting antisense oligonucleotide (ASO) could halt or reverse disease progression in mice with advanced PLN-R14del cardiomyopathy. To this aim, homozygous PLN-R14del (PLN-R14 Δ/Δ) mice received PLN-ASO injections starting at 5 or 6 weeks of age, in the presence of moderate or severe HF, respectively. Mice were monitored for another 4 months with echocardiographic analyses at several timepoints, after which cardiac tissues were examined for pathological remodeling. We found that vehicle-treated PLN-R14 Δ/Δ mice continued to develop severe HF, and reached a humane endpoint at 8.1 ± 0.5 weeks of age. Both early and late PLN-ASO administration halted further cardiac remodeling and dysfunction shortly after treatment start, resulting in a life span extension to at least 22 weeks of age. Earlier treatment initiation halted disease development sooner, resulting in better heart function and less remodeling at the study endpoint. PLN-ASO treatment almost completely eliminated PLN aggregates, and normalized levels of autophagic proteins. In conclusion, these findings indicate that PLN-ASO therapy may have beneficial outcomes in PLN-R14del cardiomyopathy when administered after disease onset. Although existing tissue damage was not reversed, further cardiomyopathy progression was stopped, and PLN aggregates were resolved.

The features that stabilize the structures of membrane proteins remain poorly understood. Polar interactions contribute modestly, and the hydrophobic effect contributes little to the energetics of apolar side-chain packing in membranes. Disruption of steric packing can destabilize the native folds of membrane proteins, but is packing alone sufficient to drive folding in lipids? If so, then membrane proteins stabilized by this feature should be readily designed and structurally characterized—yet this has not been achieved. Through simulation of the natural protein phospholamban and redesign of variants, we define a steric packing code underlying its assembly. Synthetic membrane proteins designed using this code and stabilized entirely by apolar side chains conform to the intended fold. Although highly stable, the steric complementarity required for their folding is surprisingly stringent. Structural informatics shows that the designed packing motif recurs across the proteome, emphasizing a prominent role for precise apolar packing in membrane protein folding, stabilization, and evolution.

Heart failure with preserved ejection fraction (HFpEF) is a widespread syndrome with limited therapeutic options and poorly understood immune pathophysiology. Using a 2-hit preclinical model of cardiometabolic HFpEF that induces obesity and hypertension, we found that cardiac T cell infiltration and lymphoid expansion occurred concomitantly with cardiac pathology and that diastolic dysfunction, cardiomyocyte hypertrophy, and cardiac phospholamban phosphorylation were T cell dependent. Heart-infiltrating T cells were not restricted to cardiac antigens and were uniquely characterized by impaired activation of the inositol-requiring enzyme 1α/X-box-binding protein 1 (IRE1α/XBP1) arm of the unfolded protein response. Notably, selective ablation of XBP1 in T cells enhanced their persistence in the heart and lymphoid organs of mice with preclinical HFpEF. Furthermore, T cell IRE1α/XBP1 activation was restored after withdrawal of the 2 comorbidities inducing HFpEF, resulting in partial improvement of cardiac pathology. Our results demonstrated that diastolic dysfunction and cardiomyocyte hypertrophy in preclinical HFpEF were T cell dependent and that reversible dysregulation of the T cell IRE1α/XBP1 axis was a T cell signature of HFpEF.

Background Phospholamban ( PLN ) p.Arg14del (R14del, R14 ∆/+ ) is the most commonly identified pathogenic variant that causes cardiomyopathy in the Netherlands. Many disease characteristics are still unclear, including the phenotypic triggers, disease progression and disease-specific biomarkers. We aim to gain a better understanding of the R14 ∆/+ pathophysiology by establishing a cohort across the R14 ∆/+ disease spectrum. Methods The Disease spECifIc PatHways and modifiERs in PhosphoLambaN r14del cardiomyopathy ( DECIPHER-PLN) cohort includes 101 participants, categorised as unaffected R14 ∆/+ ( n  = 21), early affected R14 ∆/+ ( n  = 42), end-stage R14 ∆/+ ( n  = 28) and heart failure (HF) of another aetiology ( n  = 10). R14 ∆/+ category was based on left ventricular ejection fraction, HF symptoms, electrocardiogram (ECG) and N‑terminal pro-brain natriuretic peptide concentrations. Of the 91 included R14 ∆/+ carriers, 46 (51%) were female, with a mean age of 55 years (standard deviation: 14). Low-voltage ECG older age, arrhythmias, and conduction and repolarisation abnormalities were common in (early) affected R14 ∆/+ carriers. Serum and plasma were collected from all participants. Induced pluripotent stem cells were generated from fibroblasts of end-stage R14 ∆/+ patients and unaffected R14 ∆/+ family members ( n  = 4) and differentiated into cardiomyocytes. Explanted heart tissue was obtained from R14 ∆/+ patients undergoing cardiac surgery and patients with other HF aetiologies as control. Abnormal PLN protein localisation was confirmed in R14 ∆/+ carriers. Conclusion DECIPHER-PLN comprises R14 ∆/+ carriers across the disease and non-disease spectrum and can be used to identify disease-specific biological pathways and modifiers that play a role in R14 ∆/+ cardiomyopathy. Using a multi-omics approach and in vitro disease modelling, we aim to identify novel biomarkers and improve our understanding of R14 ∆/+ pathophysiology. Material is available upon request.

Pre-existing Ca 2+ handling abnormalities constitute the arrhythmogenic substrate in patients developing postoperative atrial fibrillation (POAF), a common complication after cardiac surgery. Postoperative interleukin (IL)-6 levels are associated with atrial fibrosis in several animal models of POAF, contributing to atrial arrhythmias. Here, we hypothesize that IL-6-mediated-Ca 2+ handling abnormalities contribute to atrial fibrillation (AF) in sterile pericarditis (SP) rats, an animal model of POAF. SP was induced in rats by dusting atria with sterile talcum powder. Anti-rat-IL-6 antibody (16.7 μg/kg) was administered intraperitoneally at 30 min after the recovery of anesthesia. In vivo electrophysiology, ex vivo optical mapping, western blots, and immunohistochemistry were performed to elucidate mechanisms of AF susceptibility. IL-6 neutralization ameliorated atrial inflammation and fibrosis, as well as AF susceptibility in vivo and the frequency of atrial ectopy and AF with a reentrant pattern in SP rats ex vivo . IL-6 neutralization reversed the prolongation and regional heterogeneity of Ca 2+ transient duration, relieved alternans, reduced the incidence of discordant alternans, and prevented the reduction and regional heterogeneity of the recovery ratio of Ca 2+ transient. In agreement, western blots showed that IL-6 neutralization reversed the reduction in the expression of ryanodine receptor 2 (RyR2) and phosphorylated phospholamban. Acute IL-6 administration to isolated rat hearts recapitulated partial Ca 2+ handling phenotype in SP rats. In addition, intraperitoneal IL-6 administration to rats increased AF susceptibility, independent of fibrosis. Our results reveal that IL-6-mediated-Ca 2+ handling abnormalities in SP rats, especially RyR2-dysfunction, independent of IL-6-induced-fibrosis, early contribute to the development of POAF by increasing propensity for arrhythmogenic alternans.