Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy

Background: Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology. Methodology and Results: We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively. Conclusions: Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity. Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology

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Low Plasma Hydrogen Sulfide Is Associated with Impaired Renal Function and Cardiac Dysfunction

American Journal of Nephrology ◽

10.1159/000489606 ◽

2018 ◽

Vol 47 (5) ◽

pp. 361-371 ◽

Cited By ~ 7

Author(s):

Qing Kuang ◽

Ning Xue ◽

Jing Chen ◽

Ziyan Shen ◽

Xiaomeng Cui ◽

...

Keyword(s):

Renal Function ◽

Hydrogen Sulfide ◽

Cardiac Dysfunction ◽

Mononuclear Cells ◽

Troponin T ◽

Left Ventricular ◽

Left Ventricular Ejection ◽

Mrna Levels ◽

Ventricular Ejection Fraction

Background: Chronic kidney disease (CKD) has been proposed to associate with decreased hydrogen sulfide (H2S) level. Nevertheless, the role of H2S in the pathogenesis of CKD has not been fully investigated. Our study aimed to investigate the plasma level of endogenous H2S in patients with different stages of CKD, and to identify the role of H2S in the progression of CKD and its relationship with cardiovascular diseases. Methods: A total of 157 non-dialysis CKD patients were recruited in our study, with 37 age- and sex-matched healthy individuals as control. Plasma concentration of H2S was measured with spectrophotometry. Sulfhemoglobin, the integration of H2S and hemoglobin, was characterized and measured by dual wavelength spectrophotometry. Serum levels of homocysteine (Hcy), cardiac troponin T (cTnT), and N-terminal pro B type natriuretic peptide were measured using automated analyzers. Conventional transthoracic echocardiography was performed and left ventricular ejection fraction (LVEF) was analyzed as a sensitive parameter of cardiac dysfunction. Results: The plasma H2S level (μmol/L) in CKD patients was significantly lower than those in healthy controls (7.32 ± 4.02 vs. 14.11 ± 5.24 μmol/L, p < 0.01). Plasma H2S level was positively associated with estimated glomerular filtration rate (eGFR; ρ = 0.577, p < 0.01) and negatively associated with plasma indoxyl sulfate concentration (ρ = –0.554, p < 0.01). The mRNA levels of cystathionine β-synthase and cystathionine γ-lyase, 2 catalytic enzymes of H2S formation, were significantly lower in blood mononuclear cells of CKD patients with respect to controls; however, the mRNA level of 3-mercaptopyruvate sulfurtransferase, as another H2S-producing enzyme, was significantly higher in CKD patients. The serum concentration of Hcy, acting as the substrate of H2S synthetase, was higher in the CKD group (p < 0.01). Specifically, the content of serum Hcy in CKD stages 3–5 patients was significantly higher than that in CKD stages 1–2, indicating an increasing trend of serum Hcy with the decline of renal function. Examination of ultrasonic cardiogram revealed a negative correlation between plasma H2S level and LVEF (ρ = –0.204, p < 0.05) in CKD patients. The H2S level also correlated negatively with cTnT concentration (ρ = –0.249, p < 0.01). Conclusions: Plasma H2S level decreased with the decline of eGFR, which may contribute to the cardiac dysfunction in CKD patients.

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Circulating Leukocytes and Oxidative Stress in Cardiovascular Diseases: A State of the Art

Oxidative Medicine and Cellular Longevity ◽

10.1155/2019/2650429 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 3

Author(s):

Speranza Rubattu ◽

Maurizio Forte ◽

Salvatore Raffa

Keyword(s):

Oxidative Stress ◽

Cardiovascular Diseases ◽

Stress Level ◽

Mononuclear Cells ◽

Pathological Condition ◽

Clinical Manifestations ◽

Therapeutic Interventions ◽

Oxidative Stress Level ◽

The Impact

Increased oxidative stress from both mitochondrial and cytosolic sources contributes to the development and the progression of cardiovascular diseases (CVDs), and it is a target of therapeutic interventions. The numerous efforts made over the last decades in order to develop tools able to monitor the oxidative stress level in patients affected by CVDs rely on the need to gain information on the disease state. However, this goal has not been satisfactorily accomplished until now. Among others, the isolation of circulating leukocytes to measure their oxidant level offers a valid, noninvasive challenge that has been tested in few pathological contexts, including hypertension, atherosclerosis and its clinical manifestations, and heart failure. Since leukocytes circulate in the blood stream, it is expected that they might reflect quite closely both systemic and cardiovascular oxidative stress and provide useful information on the pathological condition. The results of the studies discussed in the present review article are promising. They highlight the importance of measuring oxidative stress level in circulating mononuclear cells in different CVDs with a consistent correlation between degree of oxidative stress and severity of CVD and of its complications. Importantly, they also point to a double role of leukocytes, both as a marker of disease condition and as a direct contributor to disease progression. Finally, they show that the oxidative stress level of leukocytes reflects the impact of therapeutic interventions. It is likely that the isolation of leukocytes and the measurement of oxidative stress, once adequately developed, may represent an eligible tool for both research and clinical purposes to monitor the role of oxidative stress on the promotion and progression of CVDs, as well as the impact of therapies.

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3213 Unraveling the role of Phospholamban (PLN) in humans via the characterization of Induced Pluripotent Stem Cell (iPSC) Cardiomyocytes (CM) derived from carriers of a lethal PLN mutation

Journal of Clinical and Translational Science ◽

10.1017/cts.2019.62 ◽

2019 ◽

Vol 3 (s1) ◽

pp. 26-26

Author(s):

Maria Giovanna Trivieri ◽

Francesca Stillitano ◽

Delaine Ceholski ◽

Irene Turnbull ◽

Kevin Costa ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

State Of The Art ◽

Induced Pluripotent Stem Cell ◽

Relevant Model ◽

Study Population ◽

Induced Pluripotent ◽

Ca Homeostasis

OBJECTIVES/SPECIFIC AIMS: To study the biology of Phosholamban (PLN) in a human relevant model. METHODS/STUDY POPULATION: State of the art stem-cell technologies using iPSC-CMs derived from carriers of a lethal PLN mutation. RESULTS/ANTICIPATED RESULTS: Our preliminary data demonstrate that this particular PLN mutation (L39) results in reduced expression and mis-localization of PLN as well as increased incidence of early after depolarization in isolated iPSC-CMs. DISCUSSION/SIGNIFICANCE OF IMPACT: Phospholamban (PLN) is a critical regulator of Ca++ homeostasis yet many uncertainties still remain regarding its role in humans. Our study will provide unique insights into the pathophysiology of this protein in HF.

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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

Circulation Research ◽

10.1161/res.129.suppl_1.p482 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Anichavezhi Devendran ◽

Rasheed Bailey ◽

Sumanta Kar ◽

Francesca Stillitano ◽

Irene Turnbull ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Model Systems ◽

Patient Specific ◽

Induced Pluripotent

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.

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Deficiency in MT5-MMP Supports Branching of Human iPSCs-Derived Neurons and Reduces Expression of GLAST/S100 in iPSCs-Derived Astrocytes

Cells ◽

10.3390/cells10071705 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1705

Author(s):

Nikita Arnst ◽

Pedro Belio-Mairal ◽

Laura García-González ◽

Laurie Arnaud ◽

Louise Greetham ◽

...

Keyword(s):

Physiological Role ◽

Neuronal Morphology ◽

App Processing ◽

Amyloidogenic Processing ◽

Model Of Alzheimer’S Disease ◽

Human Neurons ◽

Human Ipscs ◽

Induced Pluripotent ◽

The Impact

For some time, it has been accepted that the β-site APP cleaving enzyme 1 (BACE1) and the γ-secretase are two main players in the amyloidogenic processing of the β-amyloid precursor protein (APP). Recently, the membrane-type 5 matrix metalloproteinase (MT5-MMP/MMP-24), mainly expressed in the nervous system, has been highlighted as a new key player in APP-processing, able to stimulate amyloidogenesis and also to generate a neurotoxic APP derivative. In addition, the loss of MT5-MMP has been demonstrated to abrogate pathological hallmarks in a mouse model of Alzheimer’s disease (AD), thus shedding light on MT5-MMP as an attractive new therapeutic target. However, a more comprehensive analysis of the role of MT5-MMP is necessary to evaluate how its targeting affects neurons and glia in pathological and physiological situations. In this study, leveraging on CRISPR-Cas9 genome editing strategy, we established cultures of human-induced pluripotent stem cells (hiPSC)-derived neurons and astrocytes to investigate the impact of MT5-MMP deficiency on their phenotypes. We found that MT5-MMP-deficient neurons exhibited an increased number of primary and secondary neurites, as compared to isogenic hiPSC-derived neurons. Moreover, MT5-MMP-deficient astrocytes displayed higher surface area and volume compared to control astrocytes. The MT5-MMP-deficient astrocytes also exhibited decreased GLAST and S100β expression. These findings provide novel insights into the physiological role of MT5-MMP in human neurons and astrocytes, suggesting that therapeutic strategies targeting MT5-MMP should be controlled for potential side effects on astrocytic physiology and neuronal morphology.

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Transgenic incorporation of skeletal TnT into cardiac myofilaments blunts PKC-mediated depression of force

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2001.280.3.h1011 ◽

2001 ◽

Vol 280 (3) ◽

pp. H1011-H1018 ◽

Cited By ~ 35

Author(s):

David E. Montgomery ◽

Murali Chandra ◽

Qi-Quan Huang ◽

Jian-Ping Jin ◽

R. John Solaro

Keyword(s):

Troponin I ◽

Troponin T ◽

Tension Development ◽

Maximum Tension ◽

Kinase C ◽

Cardiac Myofilament ◽

Skinned Fiber Bundles ◽

The Impact ◽

Pkc Activation

Protein kinase C (PKC)-mediated phosphorylation of cardiac troponin I (cTnI) and troponin T (cTnT) has been shown to diminish maximum activation of myofilaments. The functional role of cTnI phosphorylation has been investigated. However, the impact of cTnT phosphorylation on myofilament force is not well studied. We tested the effect of endogenous PKC activation on steady-state tension development and Ca2+ sensitivity in skinned fiber bundles from transgenic (TG) mouse hearts expressing fast skeletal TnT (fsTnT), which naturally lacks the PKC sites present in cTnT. The 12- O-tetradecanoylphorbol 13-acetate (TPA) treatment induced a 29% (46.1 ± 2.5 vs. 33.4 ± 2.6 mN/mm2) reduction in maximum tension in the nontransgenic (NTG) preparations ( n = 7) and was inhibited with chelerythrine. However, TPA did not induce a change in the maximum tension in the TG preparations ( n = 11). TPA induced a small but significant ( P < 0.02) increase in Ca2+sensitivity (untreated pCa50 = 5.63 ± 0.01 vs. treated pCa50 = 5.72 ± 0.01) only in TG preparations. In TG preparations, 32P incorporation was not evident in TnT and was also significantly diminished in cTnI, compared with NTG. Our data indicate that incorporation of fsTnT into the cardiac myofilament lattice blunts PKC-mediated depression of maximum tension. These data also suggest that cTnT may play an important role in amplifying the myofilament depression induced by PKC-mediated phosphorylation of cTnI.

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Intergroup Threat and Outgroup Attitudes

Social Psychology ◽

10.1027/1864-9335/a000127 ◽

2013 ◽

Vol 44 (5) ◽

pp. 311-319 ◽

Cited By ~ 12

Author(s):

Marco Brambilla ◽

David A. Butz

Keyword(s):

Gay Men ◽

Social Group ◽

Welfare Recipients ◽

Intergroup Attitudes ◽

Social Policies ◽

Intergroup Threat ◽

Economic Threat ◽

The Impact ◽

General Climate

Two studies examined the impact of macrolevel symbolic threat on intergroup attitudes. In Study 1 (N = 71), participants exposed to a macrosymbolic threat (vs. nonsymbolic threat and neutral topic) reported less support toward social policies concerning gay men, an outgroup whose stereotypes implies a threat to values, but not toward welfare recipients, a social group whose stereotypes do not imply a threat to values. Study 2 (N = 78) showed that, whereas macrolevel symbolic threat led to less favorable attitudes toward gay men, macroeconomic threat led to less favorable attitudes toward Asians, an outgroup whose stereotypes imply an economic threat. These findings are discussed in terms of their implications for understanding the role of a general climate of threat in shaping intergroup attitudes.

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