Sarcoplasmic Reticulum In Cardiomyocytes Research Articles

Синдром недостаточности высвобождения кальция - редкий вариант катехоламинергической полиморфной желудочковой тахикардии

Authors present a difficult-to-diagnose clinical case of cotecholaminergic polymorphic ventricular tachycardia (CPVT), a rare hereditary disease, which is characterized by mutations most often in the RYR2 gene with increased function of the ryanodine receptor, which in its turn is the main protein of calcium channels in the sarcoplasmic reticulum of cardiomyocytes. The mechanism of development of the pathology is based on late post-depolarization, which is a trigger for the occurrence of severe ventricular arrhythmias associated with a high risk of sudden cardiac death. Ventricular arrhythmias in children with CPVT can causally be reproduced using an exercise test at a low heart rate threshold. However, there have been reports of sudden cardiac death in individuals who had the loss-of-function RyR2 mutations and who have nevertheless had negative stress test results. The main clinical manifestation is syncope, the genesis of which may remain unknown for a long time. The described clinical case demonstrates the complexity of diagnosis, for which the routine cardiological examination isn’t enough. Specialists therefore have difficulties in making the correct diagnosis and determining the patient management tactics.

Molecular Mechanism of a FRET Biosensor for the Cardiac Ryanodine Receptor Pathologically Leaky State.

Ca2+ leak from cardiomyocyte sarcoplasmic reticulum (SR) via hyperactive resting cardiac ryanodine receptor channels (RyR2) is pro-arrhythmic. An exogenous peptide (DPc10) binding promotes leaky RyR2 in cardiomyocytes and reports on that endogenous state. Conversely, calmodulin (CaM) binding inhibits RyR2 leak and low CaM affinity is diagnostic of leaky RyR2. These observations have led to designing a FRET biosensor for drug discovery targeting RyR2. We used FRET to clarify the molecular mechanism driving the DPc10-CaM interdependence when binding RyR2 in SR vesicles. We used donor-FKBP12.6 (D-FKBP) to resolve RyR2 binding of acceptor-CaM (A-CaM). In low nanomolar Ca2+, DPc10 decreased both FRETmax (under saturating [A-CaM]) and the CaM/RyR2 binding affinity. In micromolar Ca2+, DPc10 decreased FRETmax without affecting CaM/RyR2 binding affinity. This correlates with the analysis of fluorescence-lifetime-detected FRET, indicating that DPc10 lowers occupancy of the RyR2 CaM-binding sites in nanomolar (not micromolar) Ca2+ and lengthens D-FKBP/A-CaM distances independent of [Ca2+]. To observe DPc10/RyR2 binding, we used acceptor-DPc10 (A-DPc10). CaM weakens A-DPc10/RyR2 binding, with this effect being larger in micromolar versus nanomolar Ca2+. Moreover, A-DPc10/RyR2 binding is cooperative in a CaM- and FKBP-dependent manner, suggesting that both endogenous modulators promote concerted structural changes between RyR2 protomers for channel regulation. Aided by the analysis of cryo-EM structures, these insights inform further development of the DPc10-CaM paradigm for therapeutic discovery targeting RyR2.

PO-01-178 MAPPING OF PATHOGENIC VARIANT HOTSPOTS ON THE STRUCTURE OF RYANODINE RECEPTOR TYPE 2 DRIVES A RATIONAL DRUG DESIGN APPROACH FOR CATECHOLAMINERGIC POLYMORPHIC VENTRICULAR TACHYCARDIA

Heritable mutations in RYR2 cause sudden-death predisposing arrhythmia syndromes, such as catecholaminergic polymorphic ventricular tachycardia (CPVT), through increased sarcoplasmic reticulum (SR) diastolic Ca2+ leak. RYR2-encoded ryanodine receptor 2 is an intracellular transmembrane Ca2+ release channel in the SR of cardiomyocytes. Even on optimal medical therapy, breakthrough life-threatening arrhythmias in patients with CPVT are common. We sought to correlate identified pathogenic hotspots with key functional domains on the 3D structure of RyR2 and possible mobile binding sites to determine small molecule targets for reducing SR Ca2+ leak. We performed “signal” to “noise” (S:N) analysis in RYR2 to establish pathogenic hotspots. The minor allele frequency (MAF) of literature-derived disease-associated genetic variants at a given amino acid locus was normalized to the MAF of rare population-based variants in Genome Aggregation Database (gnomAD). Using PyMOL, a heat map overlay was generated using S:N levels and possible molecular binding pockets were identified using software PyMOL and Maestro. The highest cumulative S:N intensity of pathogenic hotspots of each domain localized on the channel domain (28.2%), helical domain 1 (25.6%), central domain (17.7%), and N-terminal domain (16.5%) of RyR2. The SPRY1 (0.498%), P1 (0.147%), handle domain (2.32%), and central U motif (8.88%) had lower S:N. Cumulative S:N intensities were nonexistent for SPRY2, SPRY3, P2, and helical domain 2. Putative small molecule binding pockets were identified in all domains while the largest number localized to the channel domain. Once binding pockets were combined with the heat map structure of RyR2, activator binding pockets of low S:N threshold in the open state of RyR2 were shown to be located heavily on the P2 domain, where CaMKII and PKA binds. The highest S:N intensity was in the channel domain with high S:N levels in other proximal domains localizing near the SR transmembrane in the lumen. Mobile binding pockets between the open and closed state of RyR2 with low S:N intensity may be alternative areas for drug targeting, especially on domains that contain binding pockets for activators of RYR2.

ER stress and calcium-dependent arrhythmias.

The sarcoplasmic reticulum (SR) plays the key role in cardiac function as the major source of Ca2+ that activates cardiomyocyte contractile machinery. Disturbances in finely-tuned SR Ca2+ release by SR Ca2+ channel ryanodine receptor (RyR2) and SR Ca2+ reuptake by SR Ca2+-ATPase (SERCa2a) not only impair contraction, but also contribute to cardiac arrhythmia trigger and reentry. Besides being the main Ca2+ storage organelle, SR in cardiomyocytes performs all the functions of endoplasmic reticulum (ER) in other cell types including protein synthesis, folding and degradation. In recent years ER stress has become recognized as an important contributing factor in many cardiac pathologies, including deadly ventricular arrhythmias. This brief review will therefore focus on ER stress mechanisms in the heart and how these changes can lead to pro-arrhythmic defects in SR Ca2+ handling machinery.

Relationship between the Expression Level of Calcium Handling Proteins of the Sarcoplasmic Reticulum of Cardiomyocytes and the Structural and Functional State of the Patient Hearts with Permanent Atrial Fibrillation

Relationship between the Expression Level of Calcium Handling Proteins of the Sarcoplasmic Reticulum of Cardiomyocytes and the Structural and Functional State of the Patient Hearts with Permanent Atrial Fibrillation

Effects of different exercise modalities on cardiac dysfunction in heart failure with preserved ejection fraction.

AimsHeart failure with preserved ejection fraction (HFpEF) is an increasingly prevalent disease. Physical exercise has been shown to alter disease progression in HFpEF. We examined cardiomyocyte Ca2+ homeostasis and left ventricular function in a metabolic HFpEF model in sedentary and trained rats following 8 weeks of moderate‐intensity continuous training (MICT) or high‐intensity interval training (HIIT).Methods and resultsLeft ventricular in vivo function (echocardiography) and cardiomyocyte Ca2+ transients (CaTs) (Fluo‐4, confocal) were compared in ZSF‐1 obese (metabolic syndrome, HFpEF) and ZSF‐1 lean (control) 21‐ and 28‐week‐old rats. At 21 weeks, cardiomyocytes from HFpEF rats showed prolonged Ca2+ reuptake in cytosolic and nuclear CaTs and impaired Ca2+ release kinetics in nuclear CaTs. At 28 weeks, HFpEF cardiomyocytes had depressed CaT amplitudes, decreased sarcoplasmic reticulum (SR) Ca2+ content, increased SR Ca2+ leak, and elevated diastolic [Ca2+] following increased pacing rate (5 Hz). In trained HFpEF rats (HIIT or MICT), cardiomyocyte SR Ca2+ leak was significantly reduced. While HIIT had no effects on the CaTs (1–5 Hz), MICT accelerated early Ca2+ release, reduced the amplitude, and prolonged the CaT without increasing diastolic [Ca2+] or cytosolic Ca2+ load at basal or increased pacing rate (1–5 Hz). MICT lowered pro‐arrhythmogenic Ca2+ sparks and attenuated Ca2+‐wave propagation in cardiomyocytes. MICT was associated with increased stroke volume in HFpEF.ConclusionsIn this metabolic rat model of HFpEF at an advanced stage, Ca2+ release was impaired under baseline conditions. HIIT and MICT differentially affected Ca2+ homeostasis with positive effects of MICT on stroke volume, end‐diastolic volume, and cellular arrhythmogenicity.

Relationship between the expression level of genes of Ca(2+)-transporting proteins of the cardiomyocytes' sarcoplasmic reticulum with the severity of heart failure

Abstract Introduction The sarcoplasmic reticulum of cardiomyocytes is the main Ca(2+) ion depot, the main functional proteins of which are Ca(2+)-ATPase SERCA2a, which re-traps ions from myoplasma, calsequestrin CASQ2, which binds most of Ca(2+), and ryanodine receptors RyR2 releasing Ca(2+) from the intracellular depot. A change in their functional activity can determine the progression of contractile myocardial dysfunction. The effectiveness of the Ca(2+)-transporting system and, consequently, the risk of development and progression of heart failure, may depend on the expression of the corresponding genes. Purpose To evaluate the association between the level of relative expression of the ATP2A2, CASQ2, RYR2 genes in the myocardium and the clinical parameters of heart failure in patients with coronary heart disease. Methods The study was carried out on the material of 90 patients with chronic heart failure developed on the background of coronary heart disease. Myocardial samples (a fragment of the right atrium appendage) obtained by connecting the cardiopulmonary bypass during planned coronary bypass surgery. The level of expression of the Ca(2+)-ATPase ATP2A2 gene, the ryanodine receptor RYR2 gene, and the calsequestrin CASQ2 gene was estimated. Samples were homogenized, RNA was isolated and cDNA was synthesized, and real-time PCR was performed. As reference, the glyceraldehyde-3-phosphate dehydrogenase GAPDH gene, the beta-actin ACTB gene, and the 18S gene were used. The level of gene expression was calculated automatically using the software of the thermocycler. For the analysis of quantitative data, the Kruskel-Wallis test or the Mann-Whitney test was used. The analysis of the strength of the linear relationship was carried out using the Spearman rank correlation coefficient. Results ATP2A2 gene expression was reduced in the myocardium of patients with left ventricular (LV) hypertrophy (p=0.027 for ATP2A2/GAPDH, p=0.043 for ATP2A2/ACTB, p=0.039 for ATP2A2/18S). A correlation was observed between an increase in the functional class of heart failure (NYHA) and a decrease in the expression level of the RYR2/GAPDH gene (p=0.023). Among patients with heart failure, there was a weak negative linear relationship between the expression level of CASQ2/18S and the LV ejection fraction (r=−0.288, p=0.047). A significant (p=0.040) increase in CASQ2/ACTB gene expression was also found in patients with diastolic dysfunction compared to individuals without it, as well as a similar tendency for CASQ2/GAPDH (p=0.068) and CASQ2/18S (p=0.090). Conclusion In patients with heart failure due to coronary heart disease with LV hypertrophy, there was a decrease in the expression of the Ca (2+)-ATPase ATP2A2 gene. A decrease in the expression of the ryanodine receptor RYR2 gene was found as the heart failure class worsened. However, CASQ2 expression increased with diastolic dysfunction and a decrease in LV ejection fraction. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Russian Foundation for Basic Research

Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators

The type 2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report 4 cryo-electron microscopy (cryo-EM) structures of porcine RyR2 bound to distinct modulators that, together with our published structures, provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the central domain that facilitates the dilation of the S6 bundle but is insufficient to open the pore. The small-molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state even in the presence of Ca2+ and PCB95. Although the channel is open when PCB95 is replaced by caffeine and adenosine 5'-triphosphate (ATP), neither of the modulators alone can sufficiently counter the antagonistic effect to open the channel. Our study marks an important step toward mechanistic understanding of the sophisticated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

Genetically targeted fluorescent probes reveal dynamic calcium responses to adrenergic signaling in multiple cardiomyocyte compartments.

Calcium (Ca2+), an important second messenger, regulates many cellular activities and varies spatiotemporally within the cell. Conventional methods to monitor Ca2+ changes, such as synthetic Ca2+ indicators, are not targetable, while genetically encoded Ca2+ indicators (GECI) can be precisely directed to cellular compartments. GECIs are chimeric proteins composed of calmodulin (or other proteins that change conformation on Ca2+ binding) coupled with two fluorescent proteins that come closer together after an increase in [Ca2+], and enhance Förster resonance energy transfer (FRET) that allows for ratiometric [Ca2+] assessment. Here, adult rat ventricular myocytes were transfected with specifically targeted calmodulin-based GECIs and Ca2+ responses to a physiological stimulus, norepinephrine (NE, 10 μM), were observed in a) sarcoplasmic reticulum (SR), b) mitochondria, c) the space between the mitochondria and SR, termed the Mitochondria Associated Membrane space (MAM) and d) cytosol for 10 min after stimulation. In SR and mitochondria, NE increased the [Ca2+] ratio by 17% and by 8%, respectively. In the MAM the [Ca2+] ratio decreased by 16%, while in cytosol [Ca2+] remained unchanged. In conclusion, adrenergic stimulation causes distinct responses in the cardiomyocyte SR, mitochondria and MAM. Additionally, our work provides a toolkit-update for targeted [Ca2+] measurements in multiple cellular compartments.

SPEG Controls Calcium Reuptake Into the Sarcoplasmic Reticulum Through Regulating SERCA2a by Its Second Kinase-Domain.

SPEG (Striated muscle preferentially expressed protein kinase) has 2 kinase-domains and is critical for cardiac development and function. However, it is not clear how these 2 kinase-domains function to maintain cardiac performance. To determine the molecular functions of the 2 kinase-domains of SPEG. A proteomics approach identified SERCA2a (sarcoplasmic/endoplasmic reticulum calcium ATPase 2a) as a protein interacting with the second kinase-domain but not the first kinase-domain of SPEG. Furthermore, the second kinase-domain of SPEG could phosphorylate Thr484 on SERCA2a, promote its oligomerization and increase calcium reuptake into the sarcoplasmic/endoplasmic reticulum in culture cells and primary neonatal rat cardiomyocytes. Phosphorylation of SERCA2a by SPEG enhanced its calcium-transporting activity without affecting its ATPase activity. Depletion of Speg in neonatal rat cardiomyocytes inhibited SERCA2a-Thr484 phosphorylation and sarcoplasmic reticulum calcium reuptake. Moreover, overexpression of SERCA2aThr484Ala mutant protein also slowed sarcoplasmic reticulum calcium reuptake in neonatal rat cardiomyocytes. In contrast, domain mapping and phosphorylation analysis revealed that the first kinase-domain of SPEG interacted and phosphorylated its recently identified substrate JPH2 (junctophilin-2). An inducible heart-specific Speg knockout mouse model was generated to further study this SPEG-SERCA2a signal nexus in vivo. Inducible deletion of Speg decreased SERCA2a-Thr484 phosphorylation and its oligomerization in the heart. Importantly, inducible deletion of Speg inhibited SERCA2a calcium-transporting activity and impaired calcium reuptake into the sarcoplasmic reticulum in cardiomyocytes, which preceded morphological and functional alterations of the heart and eventually led to heart failure in adult mice. Our data demonstrate that the 2 kinase-domains of SPEG may play distinct roles to regulate cardiac function. The second kinase-domain of SPEG is a critical regulator for SERCA2a. Our findings suggest that SPEG may serve as a new target to modulate SERCA2a activation for treatment of heart diseases with impaired calcium homeostasis.

Simultaneous Recording of Subcellular Ca2+ Signals from the Cytosol and Sarco/Endoplasmic Reticulum: Compartmentalized Dye Loading, Imaging, and Analysis.

An increase in the cytosolic Ca2+ concentration triggers the contraction in cardiomyocytes. In these cells sarcoplasmic reticulum (SR) is the major source of Ca2+, and the release from this store is mediated by the ryanodine receptors (RyRs). These receptors are regulated by cytosolic and intra-SR [Ca2+]. The cytosolic Ca2+ regulation is well established, but there are some limitations to determine indirectly the intra-SR Ca2+ concentration and understand its role in the RyRs regulation. Therefore, the interest to directly measure the free intra-SR Ca2+ concentration ([Ca2+]SR) has led to the application of a low-affinity Ca2+ indicator (Fluo-5NAM) to follow changes of [Ca2+]SR in cardiomyocytes. However the loading of this AM-ester dye into the SR has remained a challenge in freshly isolated mouse cardiomyocytes. Here, we describe an optimized protocol to measure changes of [Ca2+]SR in mouse cardiomyocytes using fluorescent Ca2+ indicators and confocal microscopy. The application of this protocol allows to evaluate directly intra-SR Ca2+ in real time in various mouse models of cardiac disease, including transgenic animals harboring mutants of RyRs or other Ca2+ signaling proteins.

Abstract 16920: Permanent Reduction of Ryr2 Improves Lv Ejection Fraction in Heart Failure

Introduction: Ryanodine receptor type 2 (RyR-2), the main Ca 2+ release channel from sarcoplasmic reticulum in cardiomyocytes, plays an important role in regulation of myocardial contractile function and cardiac hypertrophy. Hypothesis: RyR-2 is a target of gene therapy in heart failure. Methods and Results: Two CRISPR-Cas9 gRNA targeting RyR-2 were identified: one located on the gene N terminal and the other on the C terminal. Adeno-associated virus was used to express CRISPR-Cas9 to knockdown RyR-2 in two weeks transverse aortic constriction (TAC) model of mice. After one week of administration of the virus, compared with the control, cardiac mass, heart size and fibrosis decreased and the LV ejection fraction increased significantly in the model with CRISPR-Cas9 gRNA on the RyR-2 N terminal, and the other one had a similar result but not significantly. Conclusions: Our results hint genome editing with CRISPR-Cas9 disrupts the RyR-2 in vivo with partly restore the function of the heart.

Activation of CaMKIIδA promotes Ca2+ leak from the sarcoplasmic reticulum in cardiomyocytes of chronic heart failure rats.

Activation of the Ca2+/calmodulin-dependent protein kinase II isoform δA (CaMKIIδA) disturbs intracellular Ca2+ homeostasis in cardiomyocytes during chronic heart failure (CHF). We hypothesized that upregulation of CaMKIIδA in cardiomyocytes might enhance Ca2+ leak from the sarcoplasmic reticulum (SR) via activation of phosphorylated ryanodine receptor type 2 (P-RyR2) and decrease Ca2+ uptake by inhibition of SR calcium ATPase 2a (SERCA2a). In this study, CHF was induced in rats by ligation of the left anterior descending coronary artery. We found that CHF caused an increase in the expression of CaMKIIδA and P-RyR2 in the left ventricle (LV). The role of CaMKIIδA in regulation of P-RyR2 was elucidated in cardiomyocytes isolated from neonatal rats in vitro. Hypoxia induced upregulation of CaMKIIδA and activation of P-RyR2 in the cardiomyocytes, which both were attenuated by knockdown of CaMKIIδA. Furthermore, we showed that knockdown of CaMKIIδA significantly decreased the Ca2+ leak from the SR elicited by hypoxia in the cardiomyocytes. In addition, CHF also induced a downregulation of SERCA2a in the LV of CHF rats. Knockdown of CaMKIIδA normalized hypoxia-induced downregulation of SERCA2a in cardiomyocytes in vitro. The results demonstrate that the inhibition of CaMKIIδA may improve cardiac function by preventing SR Ca2+ leak through downregulation of P-RyR2 and upregulation of SERCA2a expression in cardiomyocytes in CHF.

S-Nitrosylation of STIM1 by Neuronal Nitric Oxide Synthase Inhibits Store-Operated Ca2 + Entry

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule-1 (STIM1) and Orai1 represents a major route of Ca2+ entry in mammalian cells and is initiated by STIM1 oligomerization in the endoplasmic or sarcoplasmic reticulum. However, the effects of nitric oxide (NO) on STIM1 function are unknown. Neuronal NO synthase is located in the sarcoplasmic reticulum of cardiomyocytes. Here, we show that STIM1 is susceptible to S-nitrosylation. Neuronal NO synthase deficiency or inhibition enhanced Ca2+ release-activated Ca2+ channel current (ICRAC) and SOCE in cardiomyocytes. Consistently, NO donor S-nitrosoglutathione inhibited STIM1 puncta formation and ICRAC in HEK293 cells, but this effect was absent in cells expressing the Cys49Ser/Cys56Ser STIM1 double mutant. Furthermore, NO donors caused Cys49- and Cys56-specific structural changes associated with reduced protein backbone mobility, increased thermal stability and suppressed Ca2+ depletion-dependent oligomerization of the luminal Ca2+-sensing region of STIM1. Collectively, our data show that S-nitrosylation of STIM1 suppresses oligomerization via enhanced luminal domain stability and rigidity and inhibits SOCE in cardiomyocytes.

Effect of sevoflurane preconditioning on function of sarcoplasmic reticulum in cardiomyocytes during ischemia-reperfusion in isolated rat hearts

Objective To evaluate the effect of sevoflurane preconditioning on the function of sarcoplasmic reticulum in cardiomyocytes during ischemia-reperfusion (I/R) in isolated rat hearts. Methods Healthy adult male Sprague-Dawley rats, weighing 270-300 g, were anesthetized with intraperitoneal pentobarbital.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃.Twenty-four isolated rat hearts successfully perfused in the Langendorff apparatus were divided into 3 groups (n=8 each) using a random number table: control group (group C), I/R group and sevoflurane preconditioning group (group SP). Myocardial ischemia was induced by interrupting perfusion for 30 min followed by 120 min reperfusion.In group SP, the hearts were perfused with 2.4% sevoflurane for 10 min starting from 10 min before ischemia.Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were recorded at 5, 10, 15, 30 and 60 min of reperfusion.Coronary effluent was collected at 10 min before reperfusion for measurement of the levels of lactate dehydrogenase (LDH) and cardiac troponin I (cTnI). Myocardial specimens were obtained at 120 min of reperfusion for examination of the pathological changes (using HE staining) and for determination of myocardial infarct size (by TTC staining), sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA2a) activity (with a spectrophotometer) and expression of Bcl-2, Bax and SERCA2a (by Western blot). Results Compared with group C, LVDP was significantly decreased and LVEDP was increased at each time point, myocardial infarct size was increased, the levels of LDH and cTnI in coronary effluent were increased, the expression of Bax was up-regulated, the expression of Bcl-2 and SERCA2a was down-regulated, and the activity of SERCA2a was decreased in group I/R (P<0.01). Compared with group I/R, LVDP was significantly increased and LVEDP was decreased at each time point, myocardial infarct size was decreased, the levels of LDH and cTnI in coronary effluent were decreased, the expression of Bax was down-regulated, the expression of Bcl-2 and SERCA2a was up-regulated, the activity of SERCA2a was increased (P<0.01), and the pathological changes were significantly attenuated in group SP. Conclusion The mechanism by which sevoflurane preconditioning reduces I/R injury in isolated rat hearts may be related to improving the function of sarcoplasmic reticulum in cardiomyocytes. Key words: Anesthetics, inhalation; Ischemic preconditioning; Myocardial reperfusion injury; Sarcoplasmic reticulum

Functionally redundant control of cardiac hypertrophic signaling by inositol 1,4,5-trisphosphate receptors

Calcium plays an integral role to many cellular processes including contraction, energy metabolism, gene expression, and cell death. The inositol 1, 4, 5-trisphosphate receptor (IP3R) is a calcium channel expressed in cardiac tissue. There are three IP3R isoforms encoded by separate genes. In the heart, the IP3R-2 isoform is reported to being most predominant with regards to expression levels and functional significance. The functional roles of IP3R-1 and IP3R-3 in the heart are essentially unexplored despite measureable expression levels. Here we show that all three IP3Rs isoforms are expressed in both neonatal and adult rat ventricular cardiomyocytes, and in human heart tissue. The three IP3R proteins are expressed throughout the cardiomyocyte sarcoplasmic reticulum. Using isoform specific siRNA, we found that expression of all three IP3R isoforms are required for hypertrophic signaling downstream of endothelin-1 stimulation. Mechanistically, IP3Rs specifically contribute to activation of the hypertrophic program by mediating the positive inotropic effects of endothelin-1 and leading to downstream activation of nuclear factor of activated T-cells. Our findings highlight previously unidentified functions for IP3R isoforms in the heart with specific implications for hypertrophic signaling in animal models and in human disease.

Heart Failure Re-Distributes Phospholamban between Nuclear Membranes and Sarcoplasmic Reticulum in Cardiomyocytes

We recently documented that phospholamban (PLB), a sarcoplasmic reticulum (SR) resident protein regulator of cardiac SR Ca-ATPase (SERCA2a), is localized to the nuclear envelope (NE) of cardiomyocytes (CMs). It is well-known that heart failure (HF) remodeling involves altered protein expression of PLB and SERCA2a; however, with the demonstrated compartmentalization of PLB accumulation, we asked whether HF alters trafficking between ER/SR subcompartments affecting steady-state intracellular distribution of PLB and SERCA. In this study, we examined heart tissue from non-failing and pacing-induced HF dogs with the monoclonal PLB (2D12 or 1F1) and SERCA (2A7-A1) antibodies. Confocal immunofluorescence microscopy was used to compare subcellular concentration of PLB and SERCA. At least 3 regions from NE and SR in 10 CMs were analyzed for each dog, n = 4 dogs). Both PLB and SERCA antibodies stained SR and NE in these CMs. Fluorescence intensity ratios between NE and SR for PLB was 2.05±0.82 (mean ± SD) in control dog CMs, consistent with results just reported. In HF CMs we found a highly decreased ratio of PLB between NE and SR to 1.26 ± 0.34 (n = 8 dogs). In contrast to re-distribution of PLB, intensity ratios for SERCA in NE and SR remained unchanged (1.01±0.14 in Control, and 0.98±0.26 in HF). Furthermore, while there was only minor levels of phosphoPLB in Control CMs, we found that phosphoPLB (S16) were very significantly higher in NE of failed CMs. The decreased PLB relative to SERCA in the NE of CMs suggests that retention and accumulation of PLB in the NE is decreased in HF, leading to a smaller, possibly hyper-phosphorylated steady-state of PLB concentration in the NE.

Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1

Ryanodine receptor type 2 (RyR-2), the main Ca2+ release channel from sarcoplasmic reticulum in cardiomyocytes, plays a vital role in the regulation ofmyocardial contractile function and cardiac hypertrophy. However, the role of RyR-2 in cardiac fibrosis during the development of cardiac hypertrophy remains unclear.In this study, we examined whether RyR-2 regulates TGFβ1, which is secreted from cardiomyocytes and exerts on cardiac fibrosis using cultured cardiomyocytes and cardiac fibroblasts of neonatal rats. The expression of RyR-2 was found only in cardiomyocytesbut not in cardiac fibroblasts. Mechanical stretch induced upregulation of TGFβ1 in cardiomyocytes and RyR-2 knockdown significantly suppressed the upregulation of TGFβ1 expression. The transcript levels of collagen genes were also decreased in fibroblasts compare with wild type, although the expression of both two kinds was higher than those in stationary cardiomyocytes (non-stretch). With the inhibition of the TGFβ1-neutralizing antibody, the expression of collagen genes has no significant difference between the mechanically stretched cardiomyocytes and non-stretchedones. These results indicate that RyR-2 regulated TGFβ1 expression in mechanically stretched cardiomyocytes and TGFβ1 promoted collagen formation of cardiac fibroblasts by a paracrine mechanism.RyR-2 in mechanical stretch could promote the development of cardiac fibrosis involving TGFβ1-dependent paracrine mechanism. Our findings provided more insight into comprehensively understanding the molecular role of RyR-2 in regulating cardiac fibrosis.

Circulation Research “In This Issue” Anthology

Circulation Research “In This Issue” Anthology

Phospholamban Interaction with SR Ca-ATPase Investigated by Pre-Steady State Charge Measurements

Sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) promotes muscle relaxation by pumping Ca2+ ions from the cytoplasm into the SR lumen. The activity of SERCA is modulated by Phospholamban (PLN), a membrane protein present in the SR of cardiomyocytes (1). The molecular mechanism of PLN regulation is not yet fully understood.Proteoliposomes incorporating SERCA only or SERCA and PLN were adsorbed on a solid supported membrane (SSM) and then activated by an ATP concentration jump. The current transient observed following an ATP jump is related to Ca2+ translocation through the ATPase upon ATP phosphorylation during the first enzymatic cycle (2).We found that when PLN is co-reconstituted with SERCA into proteoliposomes, the ATP-dependent current signal is significantly reduced with respect to the control measurement (proteoliposomes containing SERCA only) at 1mM MgCl2. On the other hand, no effect of PLN on the ATP-induced current transient was observed at high Mg2+ (5mM).ATP jumps were performed on proteoliposomes incorporating SERCA and PLN in the presence of increasing Ca2+ concentrations yielding a K0.5 of 0.7 µM and a cooperativity coefficient of 1.37. Interestingly, we observed higher values of K0.5 (0.84 µM) and cooperativity coefficient (1.67) if simultaneous ATP and Ca2+ jumps were carried out on co-reconstituted SERCA/PLN proteoliposomes. The higher K0.5 and cooperativity values suggest that PLN may affect the SERCA conformational change that follows binding of the first calcium ion, as proposed in previous studies (3,4).Supported by the Ente Cassa di Risparmio di Firenze, POR CRO FSE 2007-2013 project and SIBPA.1) Kranias E. G., Hajjar R. J. 2012. Circ. Res.110, 1646–1660.2) Tadini-Buoninsegni F. et al. 2008. Arch. Biochem. Biophys. 476:75-86.3) Trieber C.A. et al. 2009. Biochemistry. 48(39):9287-96.4) Cantilina T. et al. 1993. J Biol Chem. 268(23):17018-25.