Altered Calcium Research Articles

The Relationship Between Length and Active Force for Submaximal Skeletal Muscle Contractions: a Review.

The force-length relationship is usually obtained for isometric contractions with maximal activation, but less is known about how sarcomere length affects force during submaximal activation. During submaximal activation, length-dependent alterations in calcium sensitivity, owing to changes in cross-bridge kinetics (rate of attachment and/or detachment), result in an activation-dependent shift in optimal length to longer sarcomere lengths. It is known that sarcomere length, as well as temperature and phosphorylation of the regulatory light chains of myosin, can modify Ca2⁺ sensitivity by altering the probability of cross-bridge interaction. This altered calcium sensitivity is particularly important for submaximal force levels, as it can change the shape of the length dependence of force, with peak force occurring at sarcomere lengths longer than those associated with maximal filament overlap. In athletic contexts, contractions typically do not reach maximal intensity. Therefore, understanding that the ability to produce force under both maximal and submaximal conditions can differ, and that peak force can be generated at different lengths, could influence the development of targeted training regimens optimal for each sport.

Insulin mitigates acute ischemia induced atrial fibrillation and sinoatrial node dysfunction ex vivo.

Acute atrial ischemia is a well-known cause of postoperative atrial fibrillation (POAF). However, mechanisms through which ischemia contributes to the development of POAF are not well understood. In this study, ex vivo Langendorff perfusion was used to induce acute ischemia and reperfusion in the heart in order to mimic POAF. Inducibility of atrial fibrillation (AF) was evaluated using programmed electrical stimulation and confirmed with open-atrium optical mapping. Compared to the control group without ischemia, 25 minutes of ischemia substantially increased the incidence of AF. The right atrium was more susceptible to AF than the left atrium. Administering insulin for 30 minutes before ischemia and during reperfusion with 25 minutes of ischemia greatly reduced the vulnerability to AF. However, insulin treatment during reperfusion only did not show substantial benefits against AF. Optical mapping studies showed that insulin mitigates ischemia-induced abnormal electrophysiology, including shortened action potential duration and effective refractory period, slowed conduction velocity, increased conduction heterogeneity, and altered calcium transients. In conclusion, insulin reduced the risk of acute ischemia/reperfusion-induced AF via improving the electrophysiology and calcium handling of atrial cardiomyocytes, which provides a potential therapy for POAF.

Alterations in store-operated calcium entry in neurodegenerative pathologies: history, facts, perspectives

Neurodegenerative disorders, along with cardiovascular and oncological pathologies, are one of the most actual issues facing modern medicine. Therefore, the study of the molecular mechanisms of their pathogenesis and the search for new drug targets is highly demanded. Neuronal calcium signalling has attracted close attention, as altered calcium homeostasis has been demonstrated in the pathogenesis of various neurodegenerative diseases. In this review, we focus on one of the most ubiquitous and important pathways for calcium uptake: store-operated calcium entry. Here we describe studies demonstrating disturbances in store-operated calcium entry in various neurodegenerative pathologies, including Alzheimer’s, Parkinson’s and Huntington’s diseases. Also, we analyse the molecular determinants underlying these disturbances and propose ways for pharmacological correction of altered calcium signaling. The information summarized in the review will allow us to consider store-operated calcium channels as promising targets for the drug development in order to treat neurodegenerative pathologies and outline further promising directions for the investigation.

Space Travel: The Radiation and Microgravity Effects on the Cardiovascular System.

Space flight modulates the functions of the cardiovascular system. The exposure to space conditions can alter the cerebral blood flow, as well as the venous return. Anemia, cardiac output changes, and increased activity of the sympathetic nervous system can also be seen. Understanding cardiac changes prepares astronauts for both better in-flight adaptations and long-term protection against cardiovascular diseases. The heart could undergo radio-degenerative effects when exposed to space radiation, increasing the risk of cardiovascular diseases in the long run. A high frequency of arrhythmias, such as ventricular/atrial premature complexes, have been reported during the Gemini and Apollo missions. Additionally, microgravity can lead to progressive degeneration of the myocytes and muscle atrophy with altered gene expression and calcium handling, along with impaired contractility. This review summarizes the potential cardiovascular effects of spaceflight and prevention measures.

Preparation, characterization, stability and replenishing calcium ability of Moringa oleifera leaf peptide-calcium chelates

Calcium deficiency has garnered significant attention as a global public health issue. A new generation of calcium supplements, peptide-calcium chelates, is expected to increase in market value. In this study, we produced MORP (MW < 1kDa) from Moringa oleifera leaf protein via enzymatic hydrolysis for chelation with Ca2+ to produce MORP-Ca. SEM, EDS, FTIR and FS characterized the structure of MORP-Ca. The results indicate alterations in both the appearance and internal structure of MORP following calcium chelation. The functional groups of N–H, C–H, C–N, −C = O, –COO–, C–O, and –OH in MORP are involved in chelating Ca2+ to form MORP-Ca. In addition, MORP-Ca exhibits poor stability in the stomach; however, it demonstrates high stability in the intestine and under various temperature conditions. The results of the cellular experiments demonstrated that MORP-Ca is an effective promoter of calcium transport and absorption. MORP-Ca effectively increased bone mineral density and improved bone formation in animal studies. In addition, MORP-Ca supplementation improved the gut microbiota imbalance in rats fed a calcium-deficient diet, resulting in an increase in Firmicutes and a decrease in Actinobacteria. Thus, there is a connection between altered gastrointestinal flora and calcium absorption. LC-MS/MS and molecular docking analyses identified ARNEGRDL, RELIIGDR, YTPDYETK, YYTPDYETK, and IKFEFPAVDTL as key peptide sequences for the calcium-supplementing role of MORP (MW < 1 kDa). These results establish a theoretical foundation for the use of MORP-Ca as a calcium supplement or functional food.

Early pathological mechanisms in a mouse model of heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) constitutes more than half of all HF cases, yet evidence-based therapies remain lacking due to limited understanding of its underlying pathological mechanisms. Our study aimed to uncover early pathological mechanisms in HFpEF by exposing mice to dietary conditions resembling a Western diet-rich in fats, salt, and low in fiber-alongside excess mineralocorticoids to replicate significant aspects of human HFpEF. Echocardiography was performed at both 3-wk and 6-wk intervals postchallenge, revealing cardiac alterations as early as 3 wk. While ejection fraction remained preserved, mice exhibited signs of diastolic dysfunction, reduced stroke volume, and left atrial enlargement. In addition, changes in pulmonary flow velocities were noted by the 3-wk mark, suggesting elevated pulmonary pressure. Extracardiac comorbidities included organ congestion, increased adiposity, impaired glucose tolerance, and hypercholesterolemia. Molecular analyses unveiled evidence of low-grade inflammation, oxidative stress, and impaired NO-cGMP-PKG signaling, contributing to the observed decrease in titin phosphorylation, thereby impacting myocardial stiffness. In addition, impaired nitric oxide (NO) signaling might have influenced the alterations observed in coronary flow reserve. Moreover, dysregulation of calcium signaling in cardiomyocytes and reduced sarcoplasmic reticulum (SR) load were observed. Interestingly, elevated phosphorylation of cMyBP-C was linked to preserved ejection fraction despite reduced SR load. We also observed intestinal atrophy, possibly due to a high-fat diet, low dietary fiber intake, and diminished gut perfusion, potentially contributing to systemic low-grade inflammation. These findings reveal how excess mineralocorticoid salt-induced hypertension and dietary factors, like high-fat and low-fiber intake, contribute to cardiac dysfunction and metabolic disturbances, offering insights into early HFpEF pathology in this model.NEW & NOTEWORTHY Our study demonstrates that feeding mice a Western diet rich in fat and salt and low in fiber alongside excess mineralocorticoids replicates aspects of human HFpEF. Cardiac alterations including diastolic dysfunction and decreased stroke volume with preserved ejection fraction were observed. Extracardiac effects included organ congestion, adiposity, glucose intolerance, and intestinal atrophy. Molecular analysis revealed inflammation, oxidative stress, impaired NO-cGMP-PKG signaling pathways, and altered calcium signaling in cardiomyocytes, shedding light on early pathological changes in HFpEF.

Skeletal muscle metabolic characteristics and fresh meat quality defects associated with wooden breast.

Wooden breast (WB) is a myopathy that occurs in pectoralis major (PM) muscles, predominately affecting large, fast-growing broilers. Severe myodegeneration, increased hypoxia, reduced blood flow, and increased collagen deposition are hallmark characteristics of WB that culminate in unsatisfactory fresh meat quality attributes, such as poor water-holding capacity, tenderness, and processing characteristics. Therefore, WB meat is often downgraded resulting in economic losses for the United States poultry industry. Although WB has been well characterized, its etiology remains undefined. As the scientific community continues to resolve mechanisms responsible for WB onset, understanding biochemical changes associated with WB may facilitate solutions to negate its poor meat quality attributes. Given changes in metabolism of living muscle can alter biochemical processes during the conversion of muscle to meat, this review aims to summarize and discuss the current knowledge of WB muscle and meat biochemistry. For example, it appears metabolic pathways that support combating stress are upregulated in WB muscle at the expense of glycolytic flux, which presumably contributes to the high ultimate pH of WB meat. Further, perturbed function of WB mitochondria, such as altered calcium handling, impacts aspects of postmortem metabolism and proteolysis. Collectively, metabolic dysfunction of WB muscle alters the biochemical processes that occur during the conversion of muscle to meat, and thus contributes to the poor WB meat quality.

Phosphoproteomics of PLN R14del heart tissue reveals major myocyte degenerate phenotype, and is attenuated by PLN antisense treatment in vitro

Abstract Background/Purpose: Phospholamban (PLN) p.Arg14del (R14del, R14Δ/+) is a pathogenic variant that can cause cardiomyopathy, characterized by PLN protein aggregation, ultimately leading to heart failure (HF). The exact pathophysiology is unknown, we aim to uncover R14Δ/+ disease mechanisms and study potential treatment using PLN antisense oligonucleotides (PLN-ASOs). Methods Phosphoproteomics was performed on human heart tissue from end-stage R14Δ/+ patients (N=6) versus other etiologies of HF (N=10) as a control. CRISPR-Cas9 engineered R14Δ/+ and isogenic control (WT) induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were extensively characterized, including the phosphoproteome, calcium transients (FLUO 4-AM), mitochondrial respiration (Seahorse Mito Stress Test) and contractility in dynamic engineered heart tissues (dyn-EHTs) before and after PLN-ASO treatment, and the results were validated in R14Δ/+ iPSC-CMs derived from R14Δ/+ patients. Results Phosphoproteomics of heart tissue from R14Δ/+ vs. other etiologies of HF identified 138 differentially expressed proteins (DEPs) and 317 differentially expressed phosphoproteins (DEPPs), therewith establishing a disease-specific signature. Phosphoproteomics of WT vs. R14Δ/+ iPSC-CMs identified 451 DEPs and 1046 DEPPs. Gene ontology analysis of end-stage R14Δ/+ heart tissue and iPSC-CMs revealed a large overlap. Here, R14Δ/+ showed to have great impact on contraction (e.g. SYNPO2L, MYH10, TTN) and calcium (e.g. AHNAK, HRC, RYR2). In line with this, functional characterization revealed that R14Δ/+ iPSC-CMs have an increased Ca2+ systolic velocity and accelerated calcium transient (decreased T50 and T90), that associated with altered calcium-related DEPPs (e.g. RYR2). In dyn-EHTs, R14Δ/+ dyn-EHTs have a decreased contraction duration, time-to-peak and ability to pace, associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently altered PLN mRNA expression and protein levels in iPSC-CMs, and 62 DEPs and 372 DEPPs were identified. PLN-ASOs increased Ca2+ diastolic velocity, which associated with altered calcium-related DEPPs (e.g. PLN, AHNAK, HRC). PLN-ASOs increased the ability of dyn-EHTs to pace and therewith improved contractility, which associated with altered contractility-related DEPPs (e.g. SYNPO2L, MYH7, TTN). PLN-ASOs dose-dependently increased basal respiration and ATP production, which associated with altered mitophagy-related DEPs (GABARAPL2 and MAP1LC3). These functional characteristics were validated in R14Δ/+ patient iPSC-CMs. Discussion: R14Δ/+ cardiomyocytes have a degenerative phenotype characterized by altered calcium transients and reduced contractility and PLN-ASOs attenuate these characteristics and improve metabolism.

The role of calcium homeostasis in modulating the immune response in cancer and infectious diseases

Calcium homeostasis is a pivotal determinant of physiological processes, particularly in cellular signaling and immune system regulation. This balance is crucial for modulating immune responses in the context of both infectious and oncological diseases. Disruptions in calcium homeostasis have been shown to influence disease progression, immune efficacy, and treatment outcomes. Our study investigates the critical role of calcium homeostasis in shaping the immune response to cancer and infectious diseases through a comprehensive literature review. The electronic databases Scopus, Google Scholar, and PubMed were utilized to gather relevant studies, identified using key terms such as "calcium homeostasis," "immune response," "cancer," and "infectious diseases." Emerging evidence underscores that calcium levels directly impact immune cell function, including the activation of immune effector cells, cytokine production, and the orchestration of the overall immune response. These processes are critical to the body's ability to combat infections and malignancies. Aberrant calcium signaling may lead to compromised immune defense mechanisms, negatively affecting the outcomes of therapeutic interventions. In cancer, for example, altered calcium homeostasis can suppress immune surveillance, while in infectious diseases, it can hinder the immune system's capacity to clear pathogens. Our review highlights that the pathophysiology of both cancer and infectious diseases is tightly interwoven with calcium regulation, suggesting a promising avenue for therapeutic targeting. Modulating calcium homeostasis may enhance immune functionality, thereby improving treatment efficacy. By restoring calcium balance, novel therapeutic approaches could be developed to optimize immune responses and bolster clinical outcomes across a range of diseases. Further research is warranted to elucidate the precise molecular mechanisms linking calcium homeostasis and immune regulation, with the goal of refining future therapeutic strategies. Maintaining calcium equilibrium is thus essential for the advancement of effective treatments in both cancer and infectious disease contexts.

Characterisation of infantile cardiomyopathy in Alström syndrome using ALMS1 knockout induced pluripotent stem cell derived cardiomyocyte model

Alström syndrome (AS) is an inherited rare ciliopathy characterised by multi-organ dysfunction and premature cardiovascular disease. This may manifest as an infantile-onset dilated cardiomyopathy with significant associated mortality. An adult-onset restrictive cardiomyopathy may also feature later in life. Loss of function pathogenic variants in ALMS1 have been identified in AS patients, leading to a lack of ALMS1 protein. The biological role of ALMS1 is unknown, particularly in a cardiovascular context. To understand the role of ALMS1 in infantile cardiomyopathy, the reduction of ALMS1 protein seen in AS patients was modelled using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), in which ALMS1 was knocked out. MuscleMotion analysis and calcium optical mapping experiments suggest that ALMS1 knockout (KO) cells have increased contractility, with altered calcium extrusion and impaired calcium handling dynamics compared to wildtype (WT) counterparts. Seahorse metabolic assays showed ALMS1 knockout iPSC-CMs had increased glycolytic and mitochondrial respiration rates, with ALMS1 knockout cells portraying increased energetic demand and respiratory capacity than WT counterparts. Using senescence associated β-galactosidase (SA-β gal) staining assay, we identified increased senescence of ALMS1 knockout iPSC-CMs. Overall, this study provides insights into the molecular mechanisms in AS, particularly the role of ALMS1 in infantile cardiomyopathy in AS, using iPSC-CMs as a ‘disease in a dish’ model to provide insights into multiple aspects of this complex disease.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms of myocardial injury in SLE remain poorly understood. In this study, we engineered human cardiac tissues and cultured them with IgG from patients with SLE, with and without myocardial involvement. IgG from patients with elevated myocardial inflammation exhibited increased binding to apoptotic cells within cardiac tissues subjected to stress, whereas IgG from patients with systolic dysfunction exhibited enhanced binding to the surface of live cardiomyocytes. Functional assays and RNA sequencing revealed that, in the absence of immune cells, IgG from patients with systolic dysfunction altered cellular composition, respiration and calcium handling. Phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. Coupling IgG profiling with cell surfaceome analysis identified four potential pathogenic autoantibodies that may directly affect the myocardium. Overall, these insights may improve patient risk stratification and inform the development of new therapeutic strategies.

Mitochondrial complex-1 as a therapeutic target for cardiac diseases.

Mitochondrial dysfunction is critical for the development and progression of cardiovascular diseases (CVDs). Complex-1 (CI) is an essential component of the mitochondrial electron transport chain that participates in oxidative phosphorylation and energy production. CI is the largest multisubunit complex (~ 1 Mda) and comprises 45 protein subunits encoded by seven mt-DNA genes and 38 nuclear genes. These subunits function as the enzyme nicotinamide adenine dinucleotide hydrogen(NADH): ubiquinone oxidoreductase. CI dysregulation has been implicated in various CVDs, including heart failure, ischemic heart disease, pressure overload, hypertrophy, and cardiomyopathy. Several studies demonstrated that impaired CI function contributes to increased oxidative stress, altered calcium homeostasis, and mitochondrial DNA damage in cardiac cells, leading to cardiomyocyte dysfunction and apoptosis. CI dysfunction has been associated with endothelial dysfunction, inflammation, and vascular remodeling, critical processes in developing atherosclerosis and hypertension. Although CI is crucial in physiological and pathological conditions, no potential therapeutics targeting CI are available to treat CVDs. We believe that a lack of understanding of CI's precise mechanisms and contributions to CVDs limits the development of therapeutic strategies. In this review, we comprehensively analyze the role of CI in cardiovascular health and disease to shed light on its potential therapeutic target role in CVDs.

Loss of Calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy R1/2A (LGMD R1/2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wild-type (WT) mice, we determined whether loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD R1/2A pathology.

Therapeutic potential of transient receptor potential (TRP) channels in psychiatric disorders.

Psychiatric disorders such as Bipolar disorder, Anxiety, Major depressive disorder, Schizophrenia, Attention-deficit/hyperactivity disorder, as well as neurological disorders such as Migraine, are linked by the evidence of altered calcium homeostasis. The disturbance of intra-cellular calcium homeostasis disrupts the activity of numerous ion channels including transient receptor potential (TRP) channels. TRP channel families comprise non-selective calcium-permeable channels that have been implicated in variety of physiological processes in the brain, as well as in the pathogenesis of psychiatric disorders. Through a comprehensive review of current research and experimentation, this investigation elucidates the role of TRP channels in psychiatric disorders. Furthermore, this review discusses about the exploration of epigenetics and TRP channels in psychiatric disorders.

Transcriptomic meta-analysis and exploration of differentially expressed gene functions in wooden breast myopathy of broilers

Wooden breast (WB) myopathy is a common myopathy found in commercial broiler chickens worldwide. Although extensive research on WB has been conducted using transcriptomics, effectively screening and analyzing key target information remains a challenge. In this present study, 5 transcriptomic datasets obtained from the National Center for Biotechnology Information (NCBI) were used. A meta-analysis was conducted to identify meta-differentially expressed genes (meta-DEGs) involved in the response of broilers to WB myopathy. These meta-DEGs were further analyzed using Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA), supplemented by protein-protein interaction (PPI) network construction to pinpoint hub genes. These analyses help to reveal key genes, pathways, and biological processes associated with WB myopathy. The results showed that 645 up-regulated and 99 down-regulated significant meta-DEGs (|log2FC| ≥0.6, P-Meta < 0.05, and present in at least 4 datasets) were identified. GO analysis showed that multiple fibrosis-related pathways/biological processes, such as cell adhesion, connective tissue development, and collagen-rich extracellular matrix, as well as calcium ion binding were significantly upregulated. PPI analysis identified TGFB3, COL1A1, COL1A2, and COL3A1 as central hub genes involved in the fibrotic processes. KEGG analysis revealed significant upregulation of apoptosis and lysosomal pathways, with an enrichment of Ca2+-related signals and lysosomal cathepsins within the apoptosis pathway. Additionally, GSEA indicated a suppression of the tricarboxylic acid (TCA) cycle and the mitochondrial electron transport chain (ETC) in WB myopathy, with PPI analysis also identifying specific hub genes associated with these pathways. In conclusion, our comprehensive analysis of meta-DEGs elucidated key biological processes and pathways implicated in WB myopathy, including fibrosis, apoptosis, altered calcium signaling, and metabolic disruption. The identification of specific hub genes offers avenues for further investigation into the pathogenesis of this condition, potentially guiding targeted therapeutic strategies.

Calcium – a possible element behind hypertension

Background. Hypertension is the one of the important reasons of mortality and morbidity worldwide which affects around 6% to 32% of the general population. It has been documented that the serum calcium level has the impact on cardiovascular functions. Patients with essential hypertension display altered calcium metabolism, including elevated cytosolic calcium, reduced serum calcium levels and augmented urine excretion of calcium. Therefore, this study was undertaken in the direction for evaluate the link of calcium in newly diagnosed hypertensive patients. Materials and methods. This cross-sectional study was conducted in isolated newly diagnosed essential Hypertensive subjects who came to the hypertensive OPD, who were free from any other illness or under any medication. 100 cases and 50 controls were included in this study. Body mass index, Blood pressure and serum calcium levels were evaluated in the enrolled subjects. Student's t test and chi-square test were used for statistical analysis. Results. The mean serum calcium in newly diagnosed hypertensive group was 8.35 ± 1.04 and in control group it was 10.04 ± 0.66. The mean corrected calcium in newly diagnosed hypertensive group was 8.40 ± 1.01 and in control group it was 9.87 ± 4.76. These results were highly statistically significant p=0.000. Negative correlation was found between serum calcium and blood pressure among our subjects. Conclusion. Patients with essential hypertension had lower corrected serum calcium levels, and there was a substantial inverse association (negative correlation) between corrected serum calcium levels and blood pressures. So, while treating patients with hypertension, more attention to be given for the serum calcium levels.

Modulation of cream cheese physicochemical and functional properties with ultrafiltration and calcium reduction

The production of cream cheese from ultrafiltered (UF) milk can reduce acid whey generation but the effect of altered protein and calcium concentration on the physicochemical properties of cream cheese is not well understood. In this study, the effect of skim milk concentration by UF (2.5 and 5 fold) was assessed both with and without calcium reduction using 2% (w/v) cation resin treatment. UF concentration increased the concentration of peptides and free amino acids and led to a more heterogeneous and porous microstructure, resulting in a softer, less viscous and less thermally stable cream cheese. Calcium reduction decreased peptide generation, increased the size of corpuscular structures, decreased porosity and increased thermal stability but did not significantly decrease cheese hardness or viscosity. The study illustrates how protein or calcium concentration, can be used to alter functional properties.

MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids.

MYBPC3 , encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro . Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The MYBPC3 D389V -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.

FgF23 and soluble klotho effects on the myocardium: an ex vivo study

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research Background Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2). Purpose We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices. Methods We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR. Results Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling. Conclusions sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell function

Aims/hypothesisRegulatory factor X 6 (RFX6) is crucial for pancreatic endocrine development and differentiation. The RFX6 variant p.His293LeufsTer7 is significantly enriched in the Finnish population, with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 and gestational diabetes. However, the precise mechanism of this predisposition remains unknown.MethodsTo understand the role of this variant in beta cell development and function, we used CRISPR technology to generate allelic series of pluripotent stem cells. We created two isogenic stem cell models: a human embryonic stem cell model; and a patient-derived stem cell model. Both were differentiated into pancreatic islet lineages (stem-cell-derived islets, SC-islets), followed by implantation in immunocompromised NOD-SCID-Gamma mice.ResultsStem cell models of the homozygous variant RFX6−/− predictably failed to generate insulin-secreting pancreatic beta cells, mirroring the phenotype observed in Mitchell–Riley syndrome. Notably, at the pancreatic endocrine stage, there was an upregulation of precursor markers NEUROG3 and SOX9, accompanied by increased apoptosis. Intriguingly, heterozygous RFX6+/− SC-islets exhibited RFX6 haploinsufficiency (54.2% reduction in protein expression), associated with reduced beta cell maturation markers, altered calcium signalling and impaired insulin secretion (62% and 54% reduction in basal and high glucose conditions, respectively). However, RFX6 haploinsufficiency did not have an impact on beta cell number or insulin content. The reduced insulin secretion persisted after in vivo implantation in mice, aligning with the increased risk of variant carriers to develop diabetes.Conclusions/interpretationOur allelic series isogenic SC-islet models represent a powerful tool to elucidate specific aetiologies of diabetes in humans, enabling the sensitive detection of aberrations in both beta cell development and function. We highlight the critical role of RFX6 in augmenting and maintaining the pancreatic progenitor pool, with an endocrine roadblock and increased cell death upon its loss. We demonstrate that RFX6 haploinsufficiency does not affect beta cell number or insulin content but does impair function, predisposing heterozygous carriers of loss-of-function variants to diabetes.Data availabilityUltra-deep bulk RNA-seq data for pancreatic differentiation stages 3, 5 and 7 of H1 RFX6 genotypes are deposited in the Gene Expression Omnibus database with accession code GSE234289. Original western blot images are deposited at Mendeley (https://data.mendeley.com/datasets/g75drr3mgw/2).Graphical