Calcium Homeostasis In Cardiomyocytes Research Articles

Methylglyoxal induces cardiac dysfunction through mechanisms involving altered intracellular calcium handling in the rat heart

Methylglyoxal (MGO) is an endogenous, highly reactive dicarbonyl metabolite generated under hyperglycaemic conditions. MGO plays a role in developing pathophysiological conditions, including diabetic cardiomyopathy. However, the mechanisms involved and the molecular targets of MGO in the heart have not been elucidated. In this work, we studied the exposure-related effects of MGO on cardiac function in an isolated perfused rat heart ex vivo model. The effect of MGO on calcium homeostasis in cardiomyocytes was studied in vitro by the fluorescence indicator of intracellular calcium Fluo-4. We demonstrated that MGO induced cardiac dysfunction, both in contractility and diastolic function. In rat heart, the effects of MGO treatment were significantly limited by aminoguanidine, a scavenger of MGO, ruthenium red, a general cation channel blocker, and verapamil, an L-type voltage-dependent calcium channel blocker, demonstrating that this dysfunction involved alteration of calcium regulation. MGO induced a significant concentration-dependent increase of intracellular calcium in neonatal rat cardiomyocytes, which was limited by aminoguanidine and verapamil. These results suggest that the functionality of various calcium channels is altered by MGO, particularly the L-type calcium channel, thus explaining its cardiac toxicity. Therefore, MGO could participate in the development of diabetic cardiomyopathy through its impact on calcium homeostasis in cardiac cells.

Studying the role of random translocation of GLUT4 in cardiomyocytes on calcium oscillations

Defective excitation-contraction coupling (ECC) is one of the causes of contractile dysfunction linked to diabetic cardiomyopathy (DCM). This defect in ECC is mainly caused by the dysregulation of the calcium homeostasis in normal cardiomyocytes due to prolonged elevated blood glucose levels. Different types of channels inside the cardiomyocytes help maintain calcium homeostasis in the cell. Free cytosolic calcium concentration changes are one of several variables that affect complicated physiological cardiac processes and significantly impact proper heart functioning. Glucose concentration in the cardiac cell also plays a vital role in altering the calcium homeostasis in the DCM condition. In the current study, we develop a mathematical model that explicitly represents many of the known signalling components mediating translocation of the insulin-responsive glucose transporter type 4 (GLUT4) to gain insight into the complexities of metabolic insulin signalling pathways in cardiac cells. We examined the effect of the random movement of GLUT4 responsible for the entry of glucose in the cardiomyocytes. In non-paced cardiac cells calcium remains in the steady state in the absence of external pacing and avoid oscillation for healthy cardiomyocyte functioning. Intracellular calcium transients were observed during action potential. We investigated the circumstances under which the system transits from a stable state to an oscillatory state. We studied the effect of random translocation of GLUT4 on calcium oscillation. By altering system parameters, we induced insulin resistance in cardiomyocytes to mimic diabetic conditions and proposed some potential restoration strategies to restore normal calcium dynamics. Early bifurcation was observed with the introduction of randomness in the system.

Regulation of mitochondrial calcium uniporter expression and calcium-dependent cell signalling by lncRNA Tug1 in cardiomyocytes.

Cardiomyocyte calcium homeostasis is a tightly regulated process. The mitochondrial calcium uniporter (MCU) complex can buffer elevated cytosolic Ca2+ levels and consists of pore-forming proteins including MCU, and various regulatory proteins such as mitochondrial calcium uptake proteins 1 and 2 (MICU1/2). The stoichiometry of these proteins influences the sensitivity to Ca2+ and activity of the complex. However, the factors that regulate their gene expression remain incompletely understood. Long non-coding RNAs (lncRNAs) regulate gene expression through various mechanisms, and we recently found that the lncRNA Tug1 increased the expression of Mcu and associated genes. To further explore this, we performed antisense LNA knockdown of Tug1 (Tug1 KD) in H9c2 rat cardiomyocytes. Tug1 KD increased MCU protein expression, yet pyruvate dehydrogenase dephosphorylation, which is indicative of mitochondrial Ca2+ uptake, was not enhanced. However, RNA-seq revealed that Tug1 KD increased Mcu along with differential expression of >1000 genes including many related to Ca2+ regulation pathways in the heart. To understand the effect of this on Ca2+ signalling, we measured phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and its downstream target cAMP Response Element-Binding protein (CREB), a transcription factor known to drive Mcu gene expression. In response a Ca2+ stimulus, the increase in CAMKII and CREB phosphorylation was attenuated by Tug1 KD. Inhibition of CaMKII, but not CREB, partially prevented the Tug1 KD-mediated increase in Mcu. Together, these data suggest that Tug1 modulates MCU expression via a mechanism involving CAMKII and regulates cardiomyocyte Ca2+ signalling which could have important implications for cardiac function.

Cardiac involvement in patient-specific induced pluripotent stem cells of myotonic dystrophy type 1: unveiling the impact of voltage-gated sodium channels.

Myotonic dystrophy type 1 (DM1) is a genetic disorder that causes muscle weakness and myotonia. In DM1 patients, cardiac electrical manifestations include conduction defects and atrial fibrillation. DM1 results in the expansion of a CTG transcribed into CUG-containing transcripts that accumulate in the nucleus as RNA foci and alter the activity of several splicing regulators. The underlying pathological mechanism involves two key RNA-binding proteins (MBNL and CELF) with expanded CUG repeats that sequester MBNL and alter the activity of CELF resulting in spliceopathy and abnormal electrical activity. In the present study, we identified two DM1 patients with heart conduction abnormalities and characterized their hiPSC lines. Two differentiation protocols were used to investigate both the ventricular and the atrial electrophysiological aspects of DM1 and unveil the impact of the mutation on voltage-gated ion channels, electrical activity, and calcium homeostasis in DM1 cardiomyocytes derived from hiPSCs. Our analysis revealed the presence of molecular hallmarks of DM1, including the accumulation of RNA foci and sequestration of MBNL1 in DM1 hiPSC-CMs. We also observed mis-splicing of SCN5A and haploinsufficiency of DMPK. Furthermore, we conducted separate characterizations of atrial and ventricular electrical activity, conduction properties, and calcium homeostasis. Both DM1 cell lines exhibited reduced density of sodium and calcium currents, prolonged action potential duration, slower conduction velocity, and impaired calcium transient propagation in both ventricular and atrial cardiomyocytes. Notably, arrhythmogenic events were recorded, including both ventricular and atrial arrhythmias were observed in the two DM1 cell lines. These findings enhance our comprehension of the molecular mechanisms underlying DM1 and provide valuable insights into the pathophysiology of ventricular and atrial involvement.

Qi-Po-Sheng-Mai granule ameliorates Ach-CaCl2 -induced atrial fibrillation by regulating calcium homeostasis in cardiomyocytes

BackgroundAtrial fibrillation (AF) is one of the most common arrhythmias encountered in clinical settings. Currently, the pathophysiology of AF remains unclear, which severely limits the effectiveness and safety of medical therapies. The Chinese herbal formula Qi-Po-Sheng-Mai Granule (QPSM) has been widely used in China to treat AF. However, its pharmacological and molecular mechanisms remain unknown. PurposeThe purpose of this study was to investigate the molecular mechanisms and potential targets of QPSM for AF. Study design and methodsThe AF model was induced by Ach (66 μg/ml) and CaCl2 (10 mg/kg), and the dose of 0.1 ml/100 g was injected into the tail vein for 5 weeks. QPSM was administered daily at doses of 4.42 and 8.84 g/kg, and amiodarone (0.18 g/kg) was used as the positive control. The effect of QPSM on AF was assessed by electrocardiogram, echocardiography, and histopathological analysis. Then, we employed network pharmacology with single nucleus RNA sequencing (snRNA-Seq) to investigate the molecular mechanisms and potential targets of QPSM for AF. Furthermore, high performance liquid chromatography (HPLC) method was used for component analysis of QPSM, and molecular docking was used to verify the potential targets. Using the IonOptix single cell contraction and ion synchronization test equipment, single myocyte length and calcium ion variations were observed in real time. The expression levels of calcium Transporter-related proteins were detected by western blot and immunohistochemistry. ResultsBased on an Ach-CaCl2-induced AF model, we found that QPSM treatment significantly reduced atrial electrical remodeling-related markers, such as AF inducibility and duration, and attenuated atrial dilation and fibrosis. Network pharmacology identified 52 active ingredients and 119 potential targets for QPSM in the treatment of AF, and 45 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched, among which calcium pathway had the greatest impact. Using single nucleus sequencing (snRNA-seq), we identified cardiomyocytes as the most differentially expressed in response to drug treatment, with nine differentially expressed genes enriched in calcium signaling pathways. High performance liquid chromatography and molecular docking confirmed that the core components of QPSM strongly bind to the key factors in the calcium signaling pathway. Additional experiments have shown that QPSM increases calcium transients (CaT) and contractility in the individual cardiomyocyte. This was accomplished by increasing the expression of CACNA1C and SERCA2a and decreasing the expression of CAMK2B and NCX1. ConclusionThe present study has systematically elucidated the role of QPSM in maintaining calcium homeostasis in cardiomyocytes through the regulation of calcium transporters, which could lead to new drug development ideas for AF.

Effects of PMCA1 knockout on ventricular cardiomyocyte calcium homeostasis.

Plasma membrane calcium ATPase 1 (PMCA1), one of two PMCA isoforms expressed in the heart, is considered a minor Ca2+ regulatory transporter. However, this has been difficult to confirm due to lack of specific pharmacological blockers. To test the contribution of PMCA1 to Ca2+ regulation in ventricular tissue, we created a PMCA1 knockout (KO) model by breeding mice floxed at exon 10 of the PMCA1 gene with mice expressing Cre under the MLC2v promotor. The KO efficiency as determined by qPCR showed 60% reduction in PMCA1 transcript in ventricular cardiomyocytes when compared to PMCA1fx/fx mice (referred to as WT). Echocardiography and electrocardiography showed no significant differences in ejection fraction or heart rate in KO compared to WT. However, in field-stimulated isolated ventricular myocytes loaded with the Ca2+ indicator fura-2 AM, we found significant increases in systolic Ca2+ (KO: 1.9±0.5 vs WT: 1.5±0.3 F340/F380, p<0.0001) or Ca2+ transient amplitude (KO: 1.0±0.5 vs WT: 0.6±0.3 F340/F380, p<0.0001, n=30 from 3 mice). There was no change in diastolic Ca2+. We also found an increase in active sarcomere shortening in KO (7.2±3%) vs WT (5.9±3%, p=0.014, n=30), while the contraction and relaxation kinetics did not differ significantly. KO myocytes were less responsive than WT to the β-adrenergic agonist isoproterenol, with smaller proportional increases in systolic Ca2+, delta Ca2+ and myocyte shortening. In patch clamped myocytes, PMCA1 KO myocytes displayed prolonged action potential duration (APD) (KO: 227±100 vs WT: 72±30 ms, p=0.0002, n=10 from 5 mice) and more frequent early afterdepolarizations (EADs) than WT. 80% of KO cells had EADs, whereas there were none in WT. These results suggest that PMCA1 is critical for maintaining normal calcium homeostasis in ventricular cardiomyocytes and could be explored as a potential therapeutic target for Ca2+ regulation.

Эффекты кардиотоксинов кобры на папиллярной мышце и сердце крысы, перфузируемом по Лангендорфу, не связаны с выбросом адреналина

In a study of the effects of cobra cardiotoxins on myocardial tissue, both right ventricular (papillary muscle) and left ventricular contractility (isovolumic recording of left ventricular pulse pressure during Langendorff perfusion of the heart) were assessed. In papillary muscle, both toxins, at a concentration of 5 μg/mL, caused short-term increases in contractility to 200±25% and 171±15% for CTX-1 and CTX-2, respectively, at the point of maximum effect. At the same time, for CTX-1 and CTX-2, the time to peak tension (TPT) increased from 104±2 to 111±2 and from 96±2 to 104±5 ms, the relaxation time to 50%(TR50%) from 64±4 to 70±6 and from 64±6 to 69±7 ms, and the relaxation time to 95%(TR95%) from 163±10 to 190±22 and from 148±16 to 155±20 ms, respectively. This significantly differs from the positive inotropic effect of the β-adrenomimetic isoproterenol (170±31%), which causes acceleration of TPT from 106±5 to 89±4 ms, TR50% from 58±6 to 43±4 ms, and TR95% from 145±15 to 90±14 ms. When the whole heart was exposed to cardiotoxins, an increase in contractility was also observed, followed by its suppression and contracture, in contrast to isoproterenol, which caused a steady increase in contractility coupled with an increase in heart rate. Pretreatment of papillary muscles with the β-blocker propranolol &#x0D; (10 μM) did not prevent the development of cardiotoxin effects, but completely blocked the effects of isoproterenol. Our data indicate that the temporary increase in contractility under the action of cardiotoxins is not associated with the release of endogenous adrenaline, but rather is caused by changes in calcium homeostasis in cardiomyocytes.

Abstract 14952: Lysosomal Regulation of Calcium and Iron Homeostasis and Ferroptosis in Mouse Cardiomyocytes

Background: Lysosomes are specific organelles that contain various digestive enzymes and also store both iron (Fe) and calcium (Ca). However, it is not known whether cytosolic and mitochondrial Fe and Ca handling and ferroptosis are modulated by lysosomal dysfunction. We aimed to evaluate the interaction between lysosomal, mitochondrial and cytosolic Ca and Fe dynamics and its relevance to cardiomyocyte function and ferroptosis. Methods: Left ventricular myocytes were enzymatically isolated from mouse hearts. Cytosolic Ca i was imaged using fluo-4-AM. Cytosolic Fe was determined with Phen green while mitochondrial Fe was measured using rhodamine B-[(1,10-phenanthroline-5-yl)-aminocarbonyl]benzyl ester (RPA). Mitochondrial membrane potential (Δ Ψ m ) was monitored by TMRM. Ferroptosis was analyzed using the Live/Dead cell viability assay. Results: Treatment with nicotinic acid dinucleotide phosphate (NAADP-AM 50 nM), an agonist of the two pore channel in the lysosomal membrane, increased the aptitude of the Ca i transient (1.94 ± 0.09 to 2.41 ± 0.14, n = 12, p &lt; 0.05) and induced spontaneous Ca i waves. Diastolic Ca i became elevated (1.00 to 1.30 ± 0.10, n = 12, p &lt; 0.05) in intact myocytes. Elevation of basal Ca i level and an increase in spontaneous Ca i wave amplitude and frequency were confirmed in saponin-permeabilized myocytes. NAADP also increased the level of cytosolic Fe. These results suggest both Ca and Fe are released from lysosomes into the cytosol by NAADP. Our previous studies have shown that mitochondrial Fe uptake is essential for Fe overload-induced ferroptosis in cardiac myocytes. Therefore, we further assessed how lysosome function affects mitochondrial Fe loading and induction of ferroptosis. We found that NAAPD treatment resulted in an elevation of mitochondrial Fe levels (by 28.4 ± 3.5 %, n=23, p &lt;0.05), depolarization of Δ Ψ m as well as resulted in an increased sensitivity to Fe overload-induced ferroptosis. Similar results were observed using GPN that causes selective disruption of lysosomes. Conclusions: Lysosomal dysfunction may contribute to mishandling of Ca and Fe in myocytes and promote the occurrence of ferroptosis. A lysosomal storage disorder mouse model ( i.e. RagA/B KO) will be assessed in future studies.

Hypertensive vascular and cardiac remodeling protection by allicin in spontaneous hypertension rats via CaMK Ⅱ/NF-κB pathway

Allicin is the main active component of Traditional Chinese medicine, garlic. It is widely used to treat cardiovascular diseases. Our previous studies have confirmed that allicin significantly reduces blood pressure in Spontaneous Hypertension Rats (SHRs). However, the reports studying the effect of allicin on vascular and cardiac remodeling caused by hypertension are few, with their underlying mechanism not being studied in detail or fully elucidated. In this study, we treated 12-week-old SHRs with allicin for 4 weeks. After 4 weeks, allicin was shown to improve vascular and cardiac remodeling in SHRs, as evidenced by reduced cardiac left ventricular wall thickness, aortic vessel thickness, and reduced proliferating cell nuclear antigen (PCNA) and smooth muscle actin (α-SMA), and increased expression of and smooth muscle 22α (SM 22α). Additionally, allicin reduced serum IL-1β, IL-6, and TNF-α levels, improved calcium homeostasis in cardiomyocytes, downregulated calcium transportation-related CaMK II and inflammation-related NF-κB and NLRP3, which were observed in smooth muscle cells and cardiomyocytes. Thus, we inferred that allicin protected hypertensive vascular and cardiac remodeling in Spontaneous Hypertensive Rats by inhibiting the activation of the CaMK II/ NF-κB pathway. This study also provided new mechanistic insights into the anti-hypertensive vascular and cardiac remodeling effects of allicin, highlighting its therapeutic potential.

Generation of an induced pluripotent stem cell line from a Chinese Han child with catecholaminergic polymorphic ventricular tachycardia

TECRL, first reported in a Sudanese family with catecholaminergic polymorphic ventricular tachycardia (CPVT) in 2016. TECRL, is an endoplasmic reticulum (ER) protein preferentially expressed in the heart, playing a role in cardiomyocyte calcium homeostasis. Using Sendaivirus-mediated reprogramming, we generated an induced pluripotent stem cell (iPSC) line from the CPVT patient’s peripheral blood mononuclear cell. The iPSC exhibited stable amplification, expressed pluripotent markers, and differentiated spontaneously into three layers in vitro. Additionally, the iPSC line maintained a normal karyotype, retained the pathogenic TECRL mutation, and the cell resource facilitated a platform to explore the CPVT mechanisms related to TECRL mutations.

Rhynchophylline Regulates Calcium Homeostasis by Antagonizing Ryanodine Receptor 2 Phosphorylation to Improve Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a serious complication of diabetes that can lead to heart failure and death, for which there is no effective treatment. Rhynchophylline (Rhy) is the main effective component of the Chinese herbal medicine Uncaria rhynchophylla, which mainly acts on the cardiovascular and nervous systems. However, its role in protecting against DCM remains unexplored. The present study sought to reveal the mechanism of Rhy in improving type 2 diabetes mellitus (T2DM) myocardial lesions from the perspective of regulating calcium homeostasis in cardiomyocytes. We prepared a mouse model of T2DM using a high-fat diet combined with low doses of streptozotocin. The T2DM mice were given 40 mg/kg of Rhy for 8 weeks. The results showed that Rhy can attenuate cardiac pathological changes, slow down the heart rate, decrease serum cardiac enzyme levels, reduce cardiomyocyte apoptosis, enhance cardiomyocyte contractility, and raise the calcium transient amplitude in T2DM mice. Further, Rhy downregulated the phosphorylation level of ryanodine receptor 2, upregulated the phosphorylation level of phospholamban, protected mitochondrial structure and function, and increased adenosine triphosphate levels in the cardiac tissue of T2DM mice. Our results demonstrated that Rhy may protect against myocardial damage in T2DM mice and promote cardiomyocyte contraction, and its mechanism of action seems to be related to the regulation of intracellular calcium homeostasis.

Sex-specific aspects of phospholamban cardiomyopathy: The importance and prognostic value of low-voltage electrocardiograms.

A pathogenic variant in the gene encoding phospholamban (PLN), a protein that regulates calcium homeostasis of cardiomyocytes, causes PLN cardiomyopathy. It is characterized by a high arrhythmic burden and can progress to severe cardiomyopathy. Risk assessment guides implantable cardioverter-defibrillator therapy and benefits from personalization. Whether sex-specific differences in PLN cardiomyopathy exist is unknown. The purpose of this study was to improve the accuracy of PLN cardiomyopathy diagnosis and risk assessment by investigating sex-specific aspects. We analyzed a multicenter cohort of 933 patients (412 male, 521 female) with the PLN p.(Arg14del) pathogenic variant following up on a recently developed PLN risk model. Sex-specific differences in the incidence of risk model components were investigated: low-voltage electrocardiogram (ECG), premature ventricular contractions, negative T waves, and left ventricular ejection fraction. Sustained ventricular arrhythmias (VAs) occurred in 77 males (18.7%) and 61 females (11.7%) (P = .004). Of the 933 cohort members, 287 (31%) had ≥1 low-voltage ECG during follow-up (180 females [63%], 107 males [37%]; P = .006). Female sex, age, age at clinical presentation, and proband status predicted low-voltage ECG during follow-up (area under the curve: 0.78). Sustained VA-free survival was lowest in males with low-voltage ECG (P <.001). Low-voltage ECGs predict sustained VA and are a component of the PLN risk model. Low-voltage ECGs are more common in females, yet prognostic value is greater in males. Future studies should determine the impact of this difference on the risk prediction of PLN cardiomyopathy and possibly other cardiomyopathies.

Calcium dysregulation in heart diseases: Targeting calcium channels to achieve a correct calcium homeostasis

Intracellular calcium signaling is a universal language source shared by the most part of biological entities inside cells that, all together, give rise to physiological and functional anatomical units, the organ. Although preferentially recognized as signaling between cell life and death processes, in the heart it assumes additional relevance considered the importance of calcium cycling coupled to ATP consumption in excitation-contraction coupling. The concerted action of a plethora of exchangers, channels and pumps inward and outward calcium fluxes where needed, to convert energy and electric impulses in muscle contraction. All this without realizing it, thousands of times, every day. An improper function of those proteins (i.e., variation in expression, mutations onset, dysregulated channeling, differential protein-protein interactions) being part of this signaling network triggers a short circuit with severe acute and chronic pathological consequences reported as arrhythmias, cardiac remodeling, heart failure, reperfusion injury and cardiomyopathies. By acting with chemical, peptide-based and pharmacological modulators of these players, a correction of calcium homeostasis can be achieved accompanied by an amelioration of clinical symptoms.This review will focus on all those defects in calcium homeostasis which occur in the most common cardiac diseases, including myocardial infarction, arrhythmia, hypertrophy, heart failure and cardiomyopathies. This part will be introduced by the state of the art on the proteins involved in calcium homeostasis in cardiomyocytes and followed by the therapeutic treatments that to date, are able to target them and to revert the pathological phenotype.

Effect of C reactive protein on the sodium-calcium exchanger 1 in cardiomyocytes.

Numerous previous studies have found that C-reactive protein (CRP) is associated with cardiac arrhythmia and cardiac remodeling. However, the underlying mechanisms of this association remain unclear. Sodium-calcium exchanger 1 (NCX1) serves an important role in the regulation of intracellular calcium concentration, which is closely related with cardiac arrhythmia and cardiac remodeling. The present study aimed to evaluate the effects of CRP on NCX1 and intracellular calcium concentration in cardiomyocytes. Primary neonatal mouse ventricular cardiomyocytes were cultured and treated with varying concentrations of CRP (0, 5, 10, 20 and 40 µg/ml). The cardiomyocytes were also treated with NF-κB-specific inhibitor PTDC and a specific inhibitor of the reverse NCX1 KB-R7943 before their intracellular calcium concentrations were measured. mRNA and protein expression levels of NCX1 were detected by reverse transcription-quantitative PCR and western blotting, respectively and intracellular calcium concentration was evaluated by flow cytometry. CRP treatment significantly increased mRNA and protein expression levels of NCX1 in myocytes (P=0.024), as well as intracellular calcium concentration (P=0.01). These results were significantly attenuated by the NF-κB-specific inhibitor PDTC and a specific inhibitor of the reverse NCX1, KB-R7943. CRP significantly upregulated NCX1 expression and increased intracellular calcium concentration in cardiomyocytes via the NF-κB pathway, suggesting that CRP may serve a pro-arrhythmia role via direct influence on the calcium homeostasis of cardiomyocytes.

PINK1 contained in huMSC-derived exosomes prevents cardiomyocyte mitochondrial calcium overload in sepsis via recovery of mitochondrial Ca2+ efflux

BackgroundSepsis is a systemic inflammatory response to a local severe infection that may lead to multiple organ failure and death. Previous studies have shown that 40–50% of patients with sepsis have diverse myocardial injuries and 70 to 90% mortality rates compared to 20% mortality in patients with sepsis without myocardial injury. Therefore, uncovering the mechanism of sepsis-induced myocardial injury and finding a target-based treatment are immensely important.ObjectiveThe present study elucidated the mechanism of sepsis-induced myocardial injury and examined the value of human umbilical cord mesenchymal stem cells (huMSCs) for protecting cardiac function in sepsis.MethodsWe used cecal ligation and puncture (CLP) to induce sepsis in mice and detect myocardial injury and cardiac function using serological markers and echocardiography. Cardiomyocyte apoptosis and heart tissue ultrastructure were detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) and transmission electron microscopy (TEM), respectively. Fura-2 AM was used to monitor Ca2+ uptake and efflux in mitochondria. FQ-PCR and Western blotting detected expression of mitochondrial Ca2+ distribution regulators and PTEN-induced putative kinase 1 (PINK1). JC-1 was used to detect the mitochondrial membrane potential (Δψm) of cardiomyocytes.ResultsWe found that expression of PINK1 decreased in mouse hearts during sepsis, which caused cardiomyocyte mitochondrial Ca2+ efflux disorder, mitochondrial calcium overload, and cardiomyocyte injury. In contrast, we found that exosomes isolated from huMSCs (huMSC-exo) carried Pink1 mRNA, which could be transferred to recipient cardiomyocytes to increase PINK1 expression. The reduction in cardiomyocyte mitochondrial calcium efflux was reversed, and cardiomyocytes recovered from injury. We confirmed the effect of the PINK1-PKA-NCLX axis on mitochondrial calcium homeostasis in cardiomyocytes during sepsis.ConclusionThe PINK1-PKA-NCLX axis plays an important role in mitochondrial calcium efflux in cardiomyocytes. Therefore, PINK1 may be a therapeutic target to protect cardiomyocyte mitochondria, and the application of huMSC-exo is a promising strategy against sepsis-induced heart dysfunction.

Melatonin as a prospective metabolic regulator in pathologically altered cardiac energy homeostasis

A constant energy supply is indispensable for the relentlessly working heart. The unique metabolic flexibility of the cardiac tissue enables it to maintain its energy requirement under variable physiological conditions. However, some physiopathological statuses including aging, ischemia-reperfusion injury, diabetic cardiomyopathy, pathological cardiac hypertrophy, and heart failure frequently cause cardiac dysfunction and detrimental metabolic alteration. If the ATP supply fails to match the requirement of a working heart, the heart loses its functional capacity, resulting in slower recovery. A decrease in energy generation is often the ramifications of myocardial mitochondrial dysfunction and oxidative stress. Melatonin, a broad-spectrum antioxidant molecule has an appreciable role in the maintenance of metabolic homeostasis― from a single cell to an entire organism. Melatonin has the capacity to reduce ROS generation, preserve mitochondrial stability, and restore a robust mitochondrial function for unabated ATP production in cardiac tissues. Additionally, melatonin can promote carbohydrate and fat metabolism to further improve the ATP production in heart. In cardiac cells, melatonin upregulates GLUT4 expression either by impeding oxidative stress or by enhancing AMPK activation which accelerates fatty acid oxidation by upregulating PPAR-α and CPT-1α. Melatonin plays a pivotal role in the maintenance of calcium homeostasis in cardiomyocytes by obviating oxidative stress-mediated disruption of SERCA and NCX proteins. A possible role of melatonin to convert the Warburg effect to oxidative metabolism in pathological cardiac events has been recently contemplated. The current review will discuss the possible role of melatonin protecting against cardiac metabolic imbalances under pathological states.

Moderate Hydrogen Peroxide Postconditioning Ameliorates Ischemia/Reperfusion Injury in Cardiomyocytes via STAT3-Induced Calcium, ROS, and ATP Homeostasis

Introduction: Moderate hydrogen peroxide postconditioning (H₂O₂PoC) activates signal transducer and activator of transcription 3 (STAT3) to alleviate mitochondrial calcium overload during cardiac ischemia/reperfusion (I/R). However, the initial time window of STAT3-induced calcium hemostasis, the production of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in H₂O₂PoC, and its regulated mechanism remain unknown. This study aimed to investigate H₂O₂PoC-induced homeostasis of calcium, ROS and ATP, and the role of STAT3 in the regulation. Methods: Isolated rat cardiomyocytes were exposed to H₂O₂PoC and Janus kinase 2 (JAK2)/STAT3 inhibitor AG490 during I/R. Ca²⁺ transients, cell contraction, intracellular calcium concentration, ROS production, ATP contents, phosphorylation of STAT3, gene and protein expression of manganese superoxide dismutase (MnSOD), metallothionein 1 (MT1) and metallothionein 2 (MT2), as well as activities of mitochondrial complex I and complex II were detected. Results: Moderate H₂O₂PoC improved post-ischemic Ca²⁺ transients and cell contraction recovery as well as alleviated cytosolic and mitochondrial calcium overload, which were abrogated by AG490 in rat cardiomyocytes. Moderate H₂O₂PoC increased ROS production and rate of ROS production at early reperfusion in rat I/R cardiomyocytes, and this phenomenon was also abrogated by AG490. Notably, the expression of phosphorylated nuclear STAT3; gene and protein expression of MnSOD, MT1, and MT2; and activities of mitochondrial complex I and complex II were upregulated by moderate H₂O₂PoC but downregulated by AG490. Conclusion: These findings indicated that the cardioprotection of moderate H₂O₂PoC against cardiac I/R could be associated with activated STAT3 at early reperfusion to maintain calcium, ROS, and ATP homeostasis in rat cardiomyocytes.

VDAC2-mediated regulation of calcium homeostasis in cardiomyocytes (a review)

Background. Cardiovascular diseases, especially in association with arrhythmias, remain a prevailing cause of death worldwide. Arrhythmia related to imbalanced Ca2+ homeostasis is triggered by aberrant spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum through cardiac ryanodine receptor-Ca2+ release channel (RyR2). Voltage-dependent anion channel 2 (VDAC2) is the only mammalian specific isoform also carrying a specific cardiac function.Objectives. Description of VDAC2-mediated regulation of Ca2+ concentration in cardiomyocytes. Methods. Literature sources were mined in the MedLine/PubMed and eLibrary databases with keywords “heart AND calcium”, “heart AND VDAC2”, with a subsequent analysis.Results. From 36 English-language sources, 5 were included in the review. We summarise that potentiated VDAC2 promotes mitochondrial transport of Ca2+ ions, and suppression of the channel leads to Ca2+ imbalances. Efsevin renders the channel more cation-selective and downregulates Ca2+ concentration in diastole.Conclusion. VDAC2 comprises a potential drug target in therapy for severe arrhythmias. Efsevin is a promising agent for correcting abnormal Ca2+ transport in cardiomyocytes as an accelerator of mitochondrial Ca2+ uptake.

ZEB2 regulates a transcriptional network of calcium-handling genes in the injured heart

Abstract Intracellular calcium (Ca2+) overload is known to play a critical role in the development of cardiac dysfunction. Despite the remarkable progress in managing the progression of the disease, the development of effective therapies for heart failure (HF) remains challenging. Therefore, it is of great importance to understand the molecular mechanisms that maintain calcium level and contractility in homeostatic conditions. Here we identified a transcription factor ZEB2 that regulates the expression of numerous contractile and calcium-related genes. Zinc finger E-box-binding homeobox2 (ZEB2) is a transcription factor that plays a role during early fetal development and epithelial-to-mesenchymal transition (EMT); however, its function in the heart remains to be determined. Recently, we found that ZEB2 is upregulated in murine cardiomyocytes shortly after an ischemic event, but returns to baseline levels as the disease progresses. Gain- and loss-of-function genetic mouse models revealed the necessity and sufficiency of ZEB2 to maintain proper cardiac function after ischemic injury. We show that cardiomyocyte-specific ZEB2 overexpression (Zeb2 cTG) protected from ischemia-induced diastolic dysfunction and attenuated the structural remodeling of the heart. Moreover, RNA-sequencing of Zeb2 cTG hearts post-injury implicated ZEB2 in the regulation of numerous calcium-handling and contractile-related genes when compared to wildtype mice. Mechanistically, ZEB2 overexpression increased the phosphorylation of phospholamban (PLN) at both serine-16 and threonine-17, implying enhanced activity of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2A), thereby augmenting contractility. Improved cardiac function in ZEB2-overexpressing hearts correlated with higher expression of several sarcomeric proteins like myosin-binding protein C3 (MYBPC3), desmin (DES) and myosin regulatory light chain 2 (MYL2) further contributing to the observed protective phenotype. Furthermore, we observed a decrease in the activity of Ca2+-depended calcineurin/NFAT signaling, which is the main driver of pathological cardiac remodeling. Conversely to Zeb2 cTg mice, loss of ZEB2 from cardiomyocytes perturbed the expression of calcium- and contractile-related proteins and increased the activity of calcineurin/NFAT pathway, exacerbating cardiac dysfunction. Together, we show that ZEB2 is a central regulator of contractile and calcium-handling components, consequently mediating contractility in the mammalian heart. Further mechanistic understanding of the role of ZEB2 in the regulation of calcium homeostasis in cardiomyocytes is a critical step towards the development of improved therapies for various forms of heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): DR. E. Dekker from Dutch Heart Foundation

Klotho modulates electrical activity and calcium homeostasis in pulmonary vein cardiomyocytes via PI3K/Akt signalling.

Klotho, a potential antiageing protein has remarkable cardiovascular effects, which is lower in the patients with chronic kidney disease (CKD). Chronic kidney disease increases the risk of atrial fibrillation, majorly triggered by pulmonary vein (PV) arrhythmogenesis. This study investigated whether klotho protein can modulate PV electrical activity and the underlying potential mechanisms. A conventional microelectrode and whole-cell patch clamp were used to investigate the action potentials and ionic currents in isolated rabbit PV tissue preparations and single cardiomyocytes before and after klotho administration. Phosphoinositide 3-kinase (PI3K)/Akt signalling was studied using western blotting. Klotho significantly reduced PV spontaneous beating rates in PV tissue preparations at 1.0 and 3.0 ng/mL (but not at 0.1 and 0.3 ng/mL). In the presence of the Akt inhibitor (10 µM), klotho (1.0 and 3.0 ng/mL) did not change PV electrical activities. Klotho (1.0 ng/mL) significantly decreased the late sodium current (INa-Late) and L-type calcium current (ICa-L), similar to the Akt inhibitor (10 µM). Western blots demonstrated that klotho (1.0 ng/mL)-treated PV cardiomyocytes had less phosphorylation of Akt (Ser473) compared with klotho-untreated cardiomyocytes. Compared with control PVs, klotho at relatively lower concentrations (0.1 and 0.3 ng/mL) significantly reduced beating rates and decreased the amplitudes of delay afterdepolarizations in CKD PVs. Klotho modulated PV electrical activity by inhibiting PI3K/Akt signalling, which may provide a novel insight into CKD-induced arrhythmogenesis.