Phospholamban Research Articles

AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and RYR.

The sarcoplasmic reticulum (SR) Ca2+-ATPase 2 (SERCA2) mediates Ca2+ reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates Ca2+ release from SR and triggers contraction. Ca2+/CaMKII (CaM [calmodulin]-dependent protein kinase II) regulates activities of SERCA2 through phosphorylation of PLN (phospholamban) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local Ca2+ signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. A role for AKAP18δ (A-kinase anchoring protein 18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adenoassociated virus injection, structural modeling, surface plasmon resonance, and alpha screen technology. Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via 2 distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to the natural CaMKII inhibitor peptide and the Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent Ca2+ threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster Ca2+ reuptake by SERCA2 and Ca2+ release through RYR, AKAP18δ-N had opposite effects. We propose a model where the 2 unique AKAP18δ regions fine-tune Ca2+-frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge, this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP (A-kinase anchoring protein) reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Abstract 9052: Upregulation of Cardiac SERCA 2a is a Key Molecular Mechanism of the Protective Effect on Myocardial Aging in Beta3-Adrenergic Receptor Deficiency Mice

Background: We have shown previously that aging-induced cardiomyocyte dysfunction and impaired β-adrenergic regulation were prevented in β 3 -adrenergic receptor (AR) knockout (β 3 KO) aged mice. However, the molecular mechanism is unclear. We hypothesize that reversal of aging-induced down-regulation of SR Ca 2+ -ATPase (SERCA 2a) and β 1 -ARs by β 3 -AR deficiency may play a key role for the protective effect. Methods: We compared cardiac SERCA 2a, phospholamban (PLB), β 1 - and β 3 -AR protein expression and myocyte contractile and [Ca 2+ ] i transient ([Ca 2+ ] iT ) responses to isoproterenol (ISO, 10 -8 M) in left ventriclar (LV) myocytes obtained from 2 young (Y)(~4-6 mo) and 2 aged (A) (~18-24 mo) groups (7/group) of wild-type (WT) and β 3 KO mice, respectively. Results: Compared with YWT, AWT myocytes had significantly decreased SERCA 2a (66%, AWT: 0.21 vs YWT: 0.61), SERCA 2a/PLB ratio (0.76 vs 1.77) and β 1 -AR (33%, 0.39 vs 0.58), but increased β 3 -AR (93%, 0.27 vs 0.14). These changes were associated with reduced basal cell contraction (dL/dt max ) (AWT: 84.3 vs YWT: 124.8 μm/s), relaxation (dR/dt max ) (60.1 vs 89.8 μm/s) and [Ca 2+ ] iT (0.17 vs 0.22). This was accompanied by diminished ISO-stimulated positive inotropic effect; in AWT myocytes, ISO (10 -8 M) caused significantly less increases in dL/dt max (AWT: 33% vs YWT: 81%), dR/dt max (24% vs 58%), and [Ca 2+ ] iT (14% vs 34%). Compared with YWT, Yβ 3 KO did not alter basal contraction and ISO response of myocytes, but SERCA 2a (Yβ 3 KO: 1.2 vs YWT: 0.61) and SERCA 2a/PLB ratio were doubled with relatively unchanged β 1 -AR (0.62 vs 0.58); Aβ 3 KO mice had similar alterations. Importantly, in contrast to AWT, in Aβ 3 KO myocytes, the increased SERCA 2a (1.1) and β 1 -AR (0.61) correlated with normal basal cell contraction and relaxation with preserved ISO-stimulated positive inotropic response. ISO caused similar increases in dL/dt max (84% vs 82%) and [Ca 2+ ] iT (32% vs 34%) compared to Yβ 3 KO mice. Conclusions: Chronic β 3 -AR deficiency upregulates cardiac SERCA 2a, prevents aging-induced downregulation of β 1 -ARs, and leads to the preservation of myocyte function, [Ca 2+ ] iT , and β-adrenergic responsiveness in aged hearts. Thus, antagonizing β 3 -AR could be a new therapeutic strategy for age-related decline in myocardial function.

SERCA2a gain of function in patient-derived R14Del hiPSC-CMs

Phospholamban (PLN) is the natural inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a). Heterozygous PLN-R14del mutation is associated with an arrhythmogenic dilated cardiomyopathy (DCM), whose pathogenesis has been attributed to SERCA2a "superinhibition." The aim of the project is to test in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) harvested from a PLN-R14del carrier whether (1) Ca2+ dynamics and protein localization were compatible with SERCA2a superinhibition and (2) functional abnormalities could be reverted by pharmacological SERCA2a activation with PST3093. Ca2+ transients (CaT) were recorded at 36°C in hiPSC-CMs clusters during field stimulation. SERCA2a and PLN were immunolabeled in single hiPSC-CMs. Mutant (MUT) preparations were compared with isogenic WT ones obtained by mutation reversal. WT and MUT differed for the following properties: (1) CaT time to peak (tpeak) and half-time of CaT decay were shorter in MUT, (2) several CaT profiles were identified in WT, whereas "hyperdynamic" ones largely prevailed in MUT, (3) whereas tpeak rate-dependently declined in WT, it was shorter and rate independent in MUT, and (4) diastolic Ca2+ rate-dependently accumulated in WT, but not in MUT. When applied to WT, PST3093 changed all of the above properties to resemble those of MUT; when applied to MUT, PST3093 had no effect. Preferential perinuclear SERCA2a-PLN localization was lost in MUT hiPSC-CMs. In conclusion, functional data converge to argue for PLN-R14del incompetence in inhibiting SERCA2a in the tested case, thus weakening the rationale for therapeutic SERCA2a activation. Mechanisms alternative to SERCA2a superinhibition should be considered in the pathogenesis of DCM, including dysregulation of Ca2+-dependent transcription.

Phosphoprotein Phosphatase 1 but Not 2A Activity Modulates Coupled-Clock Mechanisms to Impact on Intrinsic Automaticity of Sinoatrial Nodal Pacemaker Cells.

Spontaneous AP (action potential) firing of sinoatrial nodal cells (SANC) is critically dependent on protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent protein phosphorylation, which are required for the generation of spontaneous, diastolic local Ca2+ releases (LCRs). Although phosphoprotein phosphatases (PP) regulate protein phosphorylation, the expression level of PPs and phosphatase inhibitors in SANC and the impact of phosphatase inhibition on the spontaneous LCRs and other players of the oscillatory coupled-clock system is unknown. Here, we show that rabbit SANC express both PP1, PP2A, and endogenous PP inhibitors I-1 (PPI-1), dopamine and cyclic adenosine 3′,5′-monophosphate (cAMP)-regulated phosphoprotein (DARPP-32), kinase C-enhanced PP1 inhibitor (KEPI). Application of Calyculin A, (CyA), a PPs inhibitor, to intact, freshly isolated single SANC: (1) significantly increased phospholamban (PLB) phosphorylation (by 2–3-fold) at both CaMKII-dependent Thr17 and PKA-dependent Ser16 sites, in a time and concentration dependent manner; (2) increased ryanodine receptor (RyR) phosphorylation at the Ser2809 site; (3) substantially increased sarcoplasmic reticulum (SR) Ca2+ load; (4) augmented L-type Ca2+ current amplitude; (5) augmented LCR’s characteristics and decreased LCR period in intact and permeabilized SANC, and (6) increased the spontaneous basal AP firing rate. In contrast, the selective PP2A inhibitor okadaic acid (100 nmol/L) had no significant effect on spontaneous AP firing, LCR parameters, or PLB phosphorylation. Application of purified PP1 to permeabilized SANC suppressed LCR, whereas purified PP2A had no effect on LCR characteristics. Our numerical model simulations demonstrated that PP inhibition increases AP firing rate via a coupled-clock mechanism, including respective increases in the SR Ca2+ pumping rate, L-type Ca2+ current, and Na+/Ca2+-exchanger current. Thus, PP1 and its endogenous inhibitors modulate the basal spontaneous firing rate of cardiac pacemaker cells by suppressing SR Ca2+ cycling protein phosphorylation, the SR Ca2+ load and LCRs, and L-type Ca2+ current.

Mitochondria Isolated from Hearts Subjected to Ischemia/Reperfusion Benefit from Adenine Nucleotide Translocase 1 Overexpression.

Reperfusion is the only feasible therapy following myocardial infarction, but reperfusion has been shown to damage mitochondrial function and disrupt energy production in the heart. Adenine nucleotide translocase 1 (ANT1) facilitates the transfer of ADP/ATP across the inner mitochondrial membrane; therefore, we tested whether ANT1 exerts protective effects on mitochondrial function during ischemia/reperfusion (I/R). The hearts of wild-type (WT) and transgenic ANT1-overexpressing (ANT1-TG) rats were exposed to I/R injury using the standard Langendorff technique, after which mitochondrial function, hemodynamic parameters, infarct size, and components of the contractile apparatus were determined. ANT1-TG hearts expressed higher ANT protein levels, with reduced levels of oxidative 4-hydroxynonenal ANT modifications following I/R. ANT1-TG mitochondria isolated from I/R hearts displayed stable calcium retention capacity (CRC) and improved membrane potential stability compared with WT mitochondria. Mitochondria isolated from ANT1-TG hearts experienced less restricted oxygen consumption than WT mitochondria after I/R. Left ventricular diastolic pressure (Pdia) decreased in ANT1-TG hearts compared with WT hearts following I/R. Preserved diastolic function was accompanied by a decrease in the phospho-lamban (PLB)/sarcoplasmic reticulum calcium ATPase (SERCA2a) ratio in ANT1-TG hearts compared with that in WT hearts. In addition, the phosphorylated (P)-PLB/PLB ratio increased in ANT1-TG hearts after I/R but not in WT hearts, which indicated more effective calcium uptake into the sarcoplasmic reticulum in ANT1-TG hearts. In conclusion, ANT1-TG rat hearts coped more efficiently with I/R than WT rat hearts, which was reflected by preserved mitochondrial energy balance, diastolic function, and calcium dynamics after reperfusion.

Construction of a Stable Expression Cell Line of Human Phospholamban.

To construct a cell line that can stably express human phospholamban(PLN) and initially explore its application in the study of myocardial toxicity mechanism. FastCloning method was used to insert the open reading frame sequence of target gene PLN into eukaryotic expression vector pcDNA5/FRT/TO(hereinafter referred to as pDFT) to construct the pDFT-PLN-Flag plasmid. The Flp-InTM T-RExTM 293 cells were generated by cotransfection of the constructed plasmid and pOG44 plasmid to express the target gene. Successfully recombined monoclonal cell lines were screened by hygromycin B resistance. Western blot and indirect immunofluorescence (IFA) were used to examine the expression of the target protein in recombinant cells. After the cell line was exposed to aconitine, it was verified by Western blot to detect changes in PLN protein phosphorylation. After PCR amplification of the recombinant plasmid and DNA electrophoresis, the length of the amplified product is the same as the known PLN gene fragment, which is consistent with the open reading frame (ORF) sequence of the human PLN gene after sequencing. IFA and Western blot showed that the constructed proliferation cell line can stably express high levels of human PLN under induction and regulation. Preliminary results showed that the phosphorylation level of Thr17-PLN decreased after two hours of exposure to 1 μmol/L aconitine. This human cell line can stably express PLN and can be used to study the mechanism of action of aconitine on the cell at molecular level.

Cardiac contractility of the African sharptooth catfish, Clarias gariepinus: role of extracellular Ca2+, sarcoplasmic reticulum, and β-adrenergic stimulation.

This study investigated the dependence of contraction from extracellular Ca2+, the presence of a functional sarcoplasmic reticulum (SR), and the effects of β-adrenergic stimulation using isometric cardiac muscle preparations. Moreover, the expression of Ca2+-handling proteins such as SR-Ca2+-ATPase (SERCA), phospholamban (PLN), and Na+/Ca2+ exchanger (NCX) were also evaluated in the ventricular tissue of adult African sharptooth catfish, Clarias gariepinus, a facultative air-breathing fish. In summary, we observed that (1) contractility was strongly regulated by extracellular Ca2+; (2) inhibition of SR Ca2+-release by application of ryanodine reduced steady-state force production; (3) ventricular myocardium exhibited clear post-rest decay, even in the presence of ryanodine, indicating a decrease in SR Ca2+ content and NCX as the main pathway for Ca2+ extrusion; (4) a positive force-frequency relationship was observed above 60 bpm (1.0 Hz); (5) ventricular tissue was responsive to β-adrenergic stimulation, which caused significant increases in twitch force, kept a linear force-frequency relationship from 12 to 96 bpm (0.2 to Hz), and improved the cardiac pumping capacity (CPC); and (6) African catfish myocardium exhibited similar expression patterns of NCX, SERCA, and PLN, corroborating our findings that both mechanisms for Ca2+ transport across the SR and sarcolemma contribute to Ca2+ activator. In conclusion, this fish species displays great physiological plasticity of E-C coupling, able to improve the ability to maintain cardiac performance under physiological conditions to ecological and/or adverse environmental conditions, such as hypoxic air-breathing activity.

Massive expansion of human induced pluripotent stem cells resulting in efficient biobanking and functional 3D tissue analysis of genetic cardiomyopathies

Abstract Introduction Over the past decade, various protocols were established to ensure efficient differentiation of hiPSC into cardiomyocytes (CMs). A major limitation, however, remained the batch-to-batch variability of hiPSC-CM efficiency and cell number. Here, we suggest an approach in which concomitant GSK-3β inhibition and removal of cell-cell contact inhibition, resulted in a massive proliferative response of hiPSC-CMs1–3. This efficient method allows expansion and passaging of functional hiPSC-CMs, that routinely can be cryopreserved and subsequently used as a stable cell source for the downstream applications, such 3D in vitro models for the disease modelling of dilated cardiomyopathy (DCM). We focussed on the deletion of arginine 14 in the PLN gene (R14del), which is associated with severe heart failure in DCM patients, associated with arrhythmias, cardiac fibrosis and premature death. Methods Subsequent expansion of hiPSC-CM cultures is generally modest (<10 fold). Here, we describe a cost-effective strategy for massive expansion (up to 250-fold) of high-purity hiPSC-CMs relying on two aspects; 1) inhibition of cell-cell contact via low-density seeding and serial passaging in culture flask-format, 2)small molecular glycogen synthase kinase-3β inhibition with CHIR99021 (CHIR). Patient-specific hiPSC-CMs harbouring a PLNR14del mutation were generated and used for EHT formation and functional follow-up. Results We observed that proliferating hiPSC-CMs, especially within the first 2 passages, can routinely be cryopreserved and subsequently further expanded or utilized in downstream applications. Moreover, using this strategy, it is possible to produce ultimately >1 billion CMs within 3–5 weeks starting with one differentiation batch of day 11 hiPSC-CMs, without the need for cell sorting or selection. Expanded hiPSC-CMs retain their capacity to mature and allows fibrin-based engineered heart tissues (EHTs) formation. Previously expanded CMs from PLNR14del patient-specific hiPSC were used to generate EHT and displayed a reduced force phenotype (0.137±0.012 mN) vs healthy control (0.229±0.030 mN) and isogenic control (0.224±0.008 mN) in previously expanded CMs. Conclusion We provpresent a novel strategy for the massive expansion of functional hiPSC-CMs with concomitant GSK-3β inhibition and low cell density culture that ultimately generates up to a 250-fold increase in hiPSC-CM numbers. Expansion healthy control hiPSC-CMs does not limit the subsequent maturation process, and moreover cells remain fully functional such as required for downstream tissue engineering approaches. Therefore, CM expansion forms a well-controlled platform for upscaling hiPSC-CM production for functional 3-dimensionale PLN cardiac disease models, large drug screenings and multiple translational/regenerative applications. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): PLN Foundation

PDE1 Inhibition Modulates Cav1.2 Channel to Stimulate Cardiomyocyte Contraction.

[Figure: see text].

Optimal echocardiographic assessment of myocardial dysfunction for arrhythmic risk stratification in phospholamban mutation carriers.

Aims Phospholamban (PLN) p.Arg14del mutation carriers are at risk of developing malignant ventricular arrhythmias (VAs) and/or heart failure. Currently, left ventricular ejection fraction (LVEF) plays an important role in risk assessment for VA in these individuals. We aimed to study the incremental prognostic value of left ventricular mechanical dispersion (LVMD) by echocardiographic deformation imaging for prediction of sustained VA in PLN p.Arg14del mutation carriers.Methods and resultsWe included 243 PLN p.Arg14del mutation carriers, which were classified into three groups according to the ‘45/45’ rule: (i) normal left ventricular (LV) function, defined as preserved LVEF ≥45% with normal LVMD ≤45 ms (n = 139), (ii) mechanical LV dysfunction, defined as preserved LVEF ≥45% with abnormal LVMD >45 ms (n = 63), and (iii) overt LV dysfunction, defined as reduced LVEF <45% (n = 41). During a median follow-up of 3.3 (interquartile range 1.8–6.0) years, sustained VA occurred in 35 individuals. The negative predictive value of having normal LV function at baseline was 99% [95% confidence interval (CI): 92–100%] for developing sustained VA. The positive predictive value of mechanical LV dysfunction was 20% (95% CI: 15–27%). Mechanical LV dysfunction was an independent predictor of sustained VA in multivariable analysis [hazard ratio adjusted for VA history: 20.48 (95% CI: 2.57–162.84)].Conclusion LVMD has incremental prognostic value on top of LVEF in PLN p.Arg14del mutation carriers, particularly in those with preserved LVEF. The ‘45/45’ rule is a practical approach to echocardiographic risk stratification in this challenging group of patients. This approach may also have added value in other diseases where LVEF deterioration is a relative late marker of myocardial dysfunction.

Preliminary study on mechanism of electroacupuncture with the involvement of SERCA2a/PLB on the synergistic and attenuated effect of aconitine in treatment of heart failure

To investigate the mechanism of electroacupuncture (EA) with the involvement of sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a)/phospholamban (PLB) on the synergistic and attenuated effect of aconitine for heart failure. Thirty SPF-ranked SD rats were randomly divided into a control group, a model group, an EA group, an aconitine group and an EA plus aconitine group, with 6 rats in each group. The rat model of acute heart failure was established by infusion of high-dose propranolol hydrochloride solution into the right femoral vein. After stabilized for 10 min in the modeled rats, EA was exerted at "Neiguan" (PC 6), with disperse-dense wave, 2 Hz/15 Hz in frequency, 3 mA in intensity, for 30 min in the EA group and the EA plus aconitine group; aconitine solution (10 μg/kg) was injected from the left femoral veins in the rats in the aconitine group and the EA plus aconitine group. Hemodynamic indexes such as the left ventricular systolic pressure (LVSP) and the maximum rate of increase/decrease of left ventricular pressure (±dp/dtmax) were detected and arrhythmia types were observed and scored. SERCA2a protein and PLB protein expressions in left ventricular myocardial tissue of rats were detected by multiplex fluorescence Western blot. Compared with the control group, LVSP and ±dp/dtmax all were decreased after modeling and at each time point after intervention in the model group (P<0.01). Compared with the model group, ±dp/dtmax was increased in the aconitine group and the EA group at 1 min after intervention (P<0.01, P<0.05), +dp/dtmax was increased at 10 to 60 min after intervention in the aconitine group and at 20 to 60 min after intervention in the EA group (P<0.01, P<0.05), LVSP was increased at 1 min after intervention in the EA group (P<0.01), while LVSP and ±dp/dtmax were all increased at 1 to 60 min after intervention in the EA plus aconitine group (P<0.01, P<0.05). Compared with the aconitine group, LVSP and +dp/dtmax were increased at 1 min after intervention in the EA group (P<0.01, P<0.05), LVSP and ±dp/dtmax at 1 min after intervention while +dp/dtmax at 20 to 60 min after intervention were all increased in the EA plus aconitine group (P<0.01, P<0.05). Compared with the EA group, +dp/dtmax was higher at 10 to 60 min after intervention in the EA plus aconitine group (P<0.01). Compared with the model group, arrhythmia score was higher in the aconitine group (P<0.01). Compared with the aconitine group, arrhythmia score was lower in the EA group and the EA plus aconitine group (P<0.01). As compared with the control group, the expression of SERCA2a protein in the left ventricular cardiomyocytes was decreased (P<0.01), while the expression of PLB protein was increased in the model group (P<0.01). Compared with the model group, the expression of SERCA2a protein was increased in both the EA group and the EA plus aconitine group (P<0.05, P<0.01), and PLB protein expression was decreased in each intervention group respectively (P<0.01, P<0.05). As compared with the EA group and the aconitine group, the expression of SERCA2a protein was increased and the expression of PLB protein was decreased in the EA plus aconitine group separately (P<0.05, P<0.01). The intervention with electroacupuncture achieves the synergism/ attenuation effect of aconitine for the improvements in heart failure probably by up-regulating the expression of SERCA2a and down-regulating the expression of PLB in myocardial tissue.

An mRNA assay system demonstrates proteasomal-specific degradation contributes to cardiomyopathic phospholamban null mutation

BackgroundThe human L39X phospholamban (PLN) cardiomyopathic mutant has previously been reported as a null mutation but the detailed molecular pathways that lead to the complete lack of detectable protein remain to be clarified. Previous studies have shown the implication between an impaired cellular degradation homeostasis and cardiomyopathy development. Therefore, uncovering the underlying mechanism responsible for the lack of PLN protein has important implications in understanding the patient pathology, chronic human calcium dysregulation and aid the development of potential therapeutics.MethodsA panel of mutant and wild-type reporter tagged PLN modified mRNA (modRNA) constructs were transfected in human embryonic stem cell-derived cardiomyocytes. Lysosomal and proteasomal chemical inhibitors were used together with cell imaging and protein analysis tools in order to dissect degradation pathways associated with expressed PLN constructs. Transcriptional profiling of the cardiomyocytes transfected by wild-type or L39X mutant PLN modRNA was analysed with bulk RNA sequencing.ResultsOur modRNA assay system revealed that transfected L39X mRNA was stable and actively translated in vitro but with only trace amount of protein detectable. Proteasomal inhibition of cardiomyocytes transfected with L39X mutant PLN modRNA showed a fourfold increase in protein expression levels. Additionally, RNA sequencing analysis of protein degradational pathways showed a significant distinct transcriptomic signature between wild-type and L39X mutant PLN modRNA transfected cardiomyocytes.ConclusionOur results demonstrate that the cardiomyopathic PLN null mutant L39X is rapidly, actively and specifically degraded by proteasomal pathways. Herein, and to the best of our knowledge, we report for the first time the usage of modified mRNAs to screen for and illuminate alternative molecular pathways found in genes associated with inherited cardiomyopathies.

Abstract MP236: PI3Kgamma-PP2A Axis In Regulation Of SERCA Function

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ -/- ) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ -/- mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ -/- cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ -/- cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ -/- cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ -/- hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ -/- can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ inact ) in the global PI3Kγ -/- cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.

Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

Nothing Regular about the Regulins: Distinct Functional Properties of SERCA Transmembrane Peptide Regulatory Subunits.

The sarco-endoplasmic reticulum calcium ATPase (SERCA) is responsible for maintaining calcium homeostasis in all eukaryotic cells by actively transporting calcium from the cytosol into the sarco-endoplasmic reticulum (SR/ER) lumen. Calcium is an important signaling ion, and the activity of SERCA is critical for a variety of cellular processes such as muscle contraction, neuronal activity, and energy metabolism. SERCA is regulated by several small transmembrane peptide subunits that are collectively known as the “regulins”. Phospholamban (PLN) and sarcolipin (SLN) are the original and most extensively studied members of the regulin family. PLN and SLN inhibit the calcium transport properties of SERCA and they are required for the proper functioning of cardiac and skeletal muscles, respectively. Myoregulin (MLN), dwarf open reading frame (DWORF), endoregulin (ELN), and another-regulin (ALN) are newly discovered tissue-specific regulators of SERCA. Herein, we compare the functional properties of the regulin family of SERCA transmembrane peptide subunits and consider their regulatory mechanisms in the context of the physiological and pathophysiological roles of these peptides. We present new functional data for human MLN, ELN, and ALN, demonstrating that they are inhibitors of SERCA with distinct functional consequences. Molecular modeling and molecular dynamics simulations of SERCA in complex with the transmembrane domains of MLN and ALN provide insights into how differential binding to the so-called inhibitory groove of SERCA—formed by transmembrane helices M2, M6, and M9—can result in distinct functional outcomes.

Evolution of pectin synthesis relevant galacturonosyltransferase gene family and its expression during cotton fiber development

BackgroundPectin is a key substance involved in cell wall development, and the galacturonosyltransferases (GAUTs) gene family is a critical participant in the pectin synthesis pathway. Systematic and comprehensive research on GAUTs has not been performed in cotton. Analysis of the evolution and expression patterns of the GAUT gene family in different cotton species is needed to increase knowledge of the function of pectin in cotton fiber development.ResultsIn this study, we have identified 131 GAUT genes in the genomes of four Gossypium species (G. raimondii, G. barbadense, G. hirsutum, and G. arboreum), and classified them as GAUT-A, GAUT-B and GAUT-C, which coding probable galacturonosyltransferases. Among them, the GAUT genes encode proteins GAUT1 to GAUT15. All GAUT proteins except for GAUT7 contain a conserved glycosyl transferase family 8 domain (H-DN-A-SVV-S-V-H-T-F). The conserved sequence of GAUT7 is PLN (phospholamban) 02769 domain. According to cis-elemet analysis, GAUT genes transcript levels may be regulated by hormones such as JA, GA, SA, ABA, Me-JA, and IAA. The evolution and transcription patterns of the GAUT gene family in different cotton species and the transcript levels in upland cotton lines with different fiber strength were analyzed. Peak transcript level of GhGAUT genes have been observed before 15 DPA. In the six materials with high fiber strength, the transcription of GhGAUT genes were concentrated from 10 to 15 DPA; while the highest transcript levels in low fiber strength materials were detected between 5 and 10 DPA. These results lays the foundation for future research on gene function during cotton fiber development.ConclusionsThe GAUT gene family may affect cotton fiber development, including fiber elongation and fiber thickening. In the low strength fiber lines, GAUTs mainly participate in fiber elongation, whereas their major effect on cotton with high strength fiber is related to both elongation and thickening.

B-PO02-014 PLN VARIANTS IN PATIENTS WITH SUDDEN CARDIAC ARREST/DEATH WITHOUT DILATED OR ARRHYTHMOGENIC CARDIOMYOPATHY: A CASE SERIES

Phospholamban, encoded by the PLN gene, is a regulator of intracellular Ca2+. Pathogenic PLN variants cause cardiac remodeling and early death. It is plausible that PLN variants confer a pro-arrhythmic phenotype without overt structural remodeling. To date, most PLN variants associated with sudden cardiac arrest/death (SCA/D) are accompanied by structural cardiomyopathies. The prevalence of PLN variants in autopsy-negative SCA/D cases without overt cardiomyopathy is unclear. We present a case series of patients with PLN gene variants identified due to SCA/D without overt dilated or arrhythmogenic cardiomyopathies. We describe PLN gene variants presenting with primarily arrhythmic phenotypes. The PLN gene should be strongly considered in genetic testing strategies for SCA/D. Cases were ascertained through professional collaboration among cardiovascular genetic counselors. We prioritized cases with PLN variants presenting without recognized cardiomyopathy phenotypes and/or in autopsy-negative SCA/D. We describe five cases with pathogenic PLN variants. Two are previously reported (p.Arg14del, p.Leu39Ter), and one is novel (p.Gln22LeufsX19). There were 3 pediatric cases and 2 adult cases, and 2/3 pediatric cases presented with SCD and negative autopsies. The other pediatric case survived SCA, but follow-up cardiac investigations were normal. Four of five cases had uninformative cardiac phenotypes, though one case had some evidence of cardiac hypertrophy at autopsy. Interestingly, 4/5 cases had SCA/D occur during rest, and 1/5 cases occurred with some activity. Last, one adult with SCD had a history atrial fibrillation, though no other cases had known arrhythmias. Table 1 summarizes these findings for comparison and contrast. With a role in Ca2+ regulation, it is plausible that PLN variants confer a pro-arrhythmic substrate and increased of SCA/D in the absence of cardiomyopathy. We raise awareness for the possible role of PLN variants in phenotype- and autopsy-negative SCA/D and encourage further investigation. This may help guide genetic counseling for PLN variants found in patients at autopsy and/or in patients who have not yet developed cardiomyopathy.

Molecular noise filtering in the β-adrenergic signaling network by phospholamban pentamers.

SummaryPhospholamban (PLN) is an important regulator of cardiac calcium handling due to its ability to inhibit the calcium ATPase SERCA. β-Adrenergic stimulation reverses SERCA inhibition via PLN phosphorylation and facilitates fast calcium reuptake. PLN also forms pentamers whose physiological significance has remained elusive. Using mathematical modeling combined with biochemical and cell biological experiments, we show that pentamers regulate both the dynamics and steady-state levels of monomer phosphorylation. Substrate competition by pentamers and a feed-forward loop involving inhibitor-1 can delay monomer phosphorylation by protein kinase A (PKA), whereas cooperative pentamer dephosphorylation enables bistable PLN steady-state phosphorylation. Simulations show that phosphorylation delay and bistability act as complementary filters that reduce the effect of random fluctuations in PKA activity, thereby ensuring consistent monomer phosphorylation and SERCA activity despite noisy upstream signals. Preliminary analyses suggest that the PLN mutation R14del could impair noise filtering, offering a new perspective on how this mutation causes cardiac arrhythmias.

Rationale and design of the PHOspholamban RElated CArdiomyopathy intervention STudy (i-PHORECAST).

BackgroundThe p.Arg14del (c.40_42delAGA) phospholamban (PLN) pathogenic variant is a founder mutation that causes dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM). Carriers are at increased risk of malignant ventricular arrhythmias and heart failure, which has been ascribed to cardiac fibrosis. Importantly, cardiac fibrosis appears to be an early feature of the disease, occurring in many presymptomatic carriers before the onset of overt disease. As with most monogenic cardiomyopathies, no evidence-based treatment is available for presymptomatic carriers.AimsThe PHOspholamban RElated CArdiomyopathy intervention STudy (iPHORECAST) is designed to demonstrate that pre-emptive treatment of presymptomatic PLN p.Arg14del carriers using eplerenone, a mineralocorticoid receptor antagonist with established antifibrotic effects, can reduce disease progression and postpone the onset of overt disease.MethodsiPHORECAST has a multicentre, prospective, randomised, open-label, blinded endpoint (PROBE) design. Presymptomatic PLN p.Arg14del carriers are randomised to receive either 50 mg eplerenone once daily or no treatment. The primary endpoint of the study is a multiparametric assessment of disease progression including cardiac magnetic resonance parameters (left and right ventricular volumes, systolic function and fibrosis), electrocardiographic parameters (QRS voltage, ventricular ectopy), signs and/or symptoms related to DCM and ACM, and cardiovascular death. The follow-up duration is set at 3 years.Baseline resultsA total of 84 presymptomatic PLN p.Arg14del carriers (n = 42 per group) were included. By design, at baseline, all participants were in New York Heart Association (NHYA) class I and had a left ventricular ejection fraction > 45% and < 2500 ventricular premature contractions during 24-hour Holter monitoring. There were no statistically significant differences between the two groups in any of the baseline characteristics. The study is currently well underway, with the last participants expected to finish in 2021.ConclusioniPHORECAST is a multicentre, prospective randomised controlled trial designed to address whether pre-emptive treatment of PLN p.Arg14del carriers with eplerenone can prevent or delay the onset of cardiomyopathy. iPHORECAST has been registered in the clinicaltrials.gov-register (number: NCT01857856).

Adaptive versus maladaptive cardiac remodelling in response to sustained β-adrenergic stimulation in a new 'ISO on/off model'.

On the one hand, sustained β-adrenergic stress is a hallmark of heart failure (HF) and exerts maladaptive cardiac remodelling. On the other hand, acute β-adrenergic stimulation maintains cardiac function under physiological stress. However, it is still incompletely understood to what extent the adaptive component of β-adrenergic signaling contributes to the maintenance of cardiac function during chronic β-adrenergic stress. We developed an experimental catecholamine-based protocol to distinguish adaptive from maladaptive effects. Mice were for 28 days infused with 30 mg/kg body weight/day isoproterenol (ISO) by subcutaneously implanted osmotic minipumps (‘ISO on’). In a second and third group, ISO infusion was stopped after 26 days and the mice were observed for additional two or seven days without further ISO infusion (‘ISO off short’, ‘ISO off long’). In this setup, ‘ISO on’ led to cardiac hypertrophy and slightly improved cardiac contractility. In stark contrast, ‘ISO off’ mice displayed progressive worsening of left ventricular ejection fraction that dropped down below 40%. While fetal and pathological gene expression (increase in Nppa, decrease in Myh6/Myh7 ratios, increase in Xirp2) was not induced in ‘ISO on’, it was activated in ‘ISO off’ mice. After ISO withdrawal, phosphorylation of phospholamban (PLN) at the protein kinase A (PKA) phosphorylation site Ser-16 dropped down to 20% as compared to only 50% at the Ca2+/Calmodulin-dependent kinase II (CaMKII) phosphorylation site Thr-17 in ‘ISO off’ mice. PKA-dependent cardioprotective production of the N-terminal proteolytic product of histone deacetylase 4 (HDAC4-NT) was reduced in ‘ISO off’ as compared to ‘ISO on’. Taken together, these data indicate that chronic ISO infusion induces besides maladaptive remodelling also adaptive PKA signalling to maintain cardiac function. The use of the ‘ISO on/off’ model will further enable the separation of the underlying adaptive from maladaptive components of β-adrenergic signalling and may help to better define and test therapeutic targets downstream of β-adrenergic receptors.