Background: Nuclear envelope proteins play an important role in the pathogenesis of hereditary ca... more Background: Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. Methods: We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. Results: Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circu...
• The homozygous c.38T&gt;G mutation in the LEMD2 gene causes arrhythmic cardiomyopathy with ... more • The homozygous c.38T&gt;G mutation in the LEMD2 gene causes arrhythmic cardiomyopathy with bilateral juvenile cataract in the Hutterite population.<br>• The cardiac phenotype is characterized by localized inferior and inferolateral fibrosis of the left ventricle and mild impairment of left ventricular systolic function but severe ventricular arrhythmias leading to sudden cardiac death.<br>• Affected heart tissue and fibroblasts exhibit abnormally shaped nuclei with condensed peripheral heterochromatin.<br>• Functional assays on affected fibroblasts show decreased proliferation capacity, cellular senescence, and a prolonged G1 phase, suggesting premature aging and cellular senescence in proliferating cells.
<b>(A)</b> Pedigrees of 2 multigenerational Hutterite families of L-leut (family 600)... more <b>(A)</b> Pedigrees of 2 multigenerational Hutterite families of L-leut (family 600) and S-leut (family 290) descendants. <b>Filled black squares</b> (male subjects) and <b>circles</b> (female subjects) refer to affected individuals with cataract and arrhythmic cardiomyopathy. <b>Filled upper half symbols</b> indicate individuals diagnosed with arrhythmic cardiomyopathy. <b>Filled lower half symbols</b> refer to individuals with juvenile cataract. <b>Diagonal lines</b> indicate deceased individuals. <b>Double lines</b> refer to known consanguinity. The genotype indicated by a "+" is the p.L13 (wild-type) allele and that indicated by a "-" is the mutant p.R13 allele. <b>(B)</b> Short-axis views of late gadolinium enhancement cardiac magnetic resonance imaging of family 290, II-20 (a), II-22 (b), III-20 (c), and III-21 (d) confirming nearly transmural delayed enhancement of the inferior/inferolateral walls as indicated by the arrows. <b>(C)</b> Rhythm strip of individual 600, II-18 recorded by the implantable cardioverter-defibrillator before delivering an appropriate shock <b>(upper panel)</b>. Representative 12-lead electrocardiogram of the same individual showing deep T-wave inversions inferior and lateral corresponding to areas of fibrosis in the cardiac magnetic resonance image <b>(lower panel)</b>. DNA = deoxyribonucleic acid; LEMD2 = LEM domain containing protein 2.
Clinical and experimental pharmacology & physiology, 2015
SIRT7 with coenzyme NAD catalyzes protein de-acetylation. In stress response, SIRT7 regulates pro... more SIRT7 with coenzyme NAD catalyzes protein de-acetylation. In stress response, SIRT7 regulates protein folding in mitochondria with unknown mechanisms. Decreases in SIRT7 entrain hematopoietic stem cell senescence, but increasing SIRT7 causes elevation of hematopoietic stem cell regenerative function. We discuss the recent findings on SIRT7 and its binding proteins, NRF1 and GABPβ1, in decision making between the choices of inducing cell aging and immortality.
Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting te... more Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3 + 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telom...
<b>(A)</b> Apoptosis assays were conducted by labeling the deoxyribonucleic acid with... more <b>(A)</b> Apoptosis assays were conducted by labeling the deoxyribonucleic acid with 5-bromo-2′-deoxyuridine 5′-triphosphate) in heart and liver tissue from the affected patient (family 600, II-16) and control heart samples. A positive control was created by adding 1 μg/μl deoxyribonucleic acids. Apoptotic cells were detected as brown dots. No signs of apoptosis were detected in all tested tissue. <b>(B)</b> Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of fibroblasts and flow cytometry from patient and Ctrl1 and Ctrl3. The histograms of the fluorescein signal collected by using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling method are shown. There is no difference in the apoptotic signals between the 2 control subjects and patient cells. <b>(C)</b> Western blot analysis of apoptotic markers (annexin V, caspase 3 [2 subunits: 17 and 19 KD]) in patient and control fibroblasts on the left panel. GAPDH was used as loading control. Quantification of apoptotic markers by densitometry does not show a significant difference in expression levels (n = 3 experiments with each 2 to 3 replicates). Abbreviations as in Figures 2, 3, and 4.
<b>(A)</b> Cell proliferation assay based on confluence from passage 2 (P2) in the &l... more <b>(A)</b> Cell proliferation assay based on confluence from passage 2 (P2) in the <b>left panel</b> and P15 in the <b>right panel</b> in patient (Pat) fibroblasts, Ctrl1, and an older age ("high-senescence") control (Ctrl3). There was a significant difference in the rate of proliferation detected between both control subjects and patient at P2 and P15. The cells were imaged every 2 h for 90 h. n = 8 wells, mean ± SD, ***p &lt; 0.001. <b>(B)</b> Images of β-galactosidase (β gal)-stained fibroblasts from patient and Ctrl1 as well as Ctrl3 at passage 6 (P6) and P15 in the <b>left panel</b>. Note that the amount of blue-stained cells is visibly higher in patient cells. <b>(Right)</b> Quantification of β gal–stained cells revealed increased cell senescence in patient cells compared with both control subjects at passages as indicated (n = 3; **p &lt; 0.01; ***p &lt; 0.001). <b>(C)</b> Fibroblasts stained with propidium iodide and measurements of the deoxyribonucleic acid content for each phase of the cell cycle by flow cytometry. Cells were taken from Ctrl1 at P6 and 9, from the patient (Pat) at passage 8 and passage 11, and from Ctrl3 at passage 12. Representative diagrams of each phase of the cell cycle are shown in the <b>left panel</b>. <b>(Right)</b> Quantification of the deoxyribonucleic acid content showed a potential arrest in the G1 phase in patient fibroblasts compared with Ctrl1 and similar to Ctrl3 at later passage. n = 3; ∗∗p &lt; 0.01; ∗p &lt; 0.05; ***p &lt; 0.001. <b>(D)</b> Western blot of Aurora B protein expression in patient and control cells on the left panel. GAPDH was used as loading control. <b>(Right)</b> Quantification by densitometry of Aurora B protein expression revealed a significant difference between both control subjects and patient fibroblasts. n = 3 experiments with each 2 to 3 replicates; ***p &lt; 0.001. Abbreviations as in Figures 2 and 3.
Background: Nuclear envelope proteins play an important role in the pathogenesis of hereditary ca... more Background: Nuclear envelope proteins play an important role in the pathogenesis of hereditary cardiomyopathies. Recently, a new form of arrhythmic cardiomyopathy caused by a homozygous mutation (p.L13R) in the inner nuclear membrane protein LEMD2 was discovered. The aim was to unravel the molecular mechanisms of mutant LEMD2 in the pathogenesis of cardiomyopathy. Methods: We generated a Lemd2 p.L13R knock-in mouse model and a corresponding cell model via CRISPR/Cas9 technology and investigated the cardiac phenotype as well as cellular and subcellular mechanisms of nuclear membrane rupture and repair. Results: Knock-in mice developed a cardiomyopathy with predominantly endocardial fibrosis, left ventricular dilatation, and systolic dysfunction. Electrocardiograms displayed pronounced ventricular arrhythmias and conduction disease. A key finding of knock-in cardiomyocytes on ultrastructural level was a significant increase in nuclear membrane invaginations and decreased nuclear circu...
• The homozygous c.38T&gt;G mutation in the LEMD2 gene causes arrhythmic cardiomyopathy with ... more • The homozygous c.38T&gt;G mutation in the LEMD2 gene causes arrhythmic cardiomyopathy with bilateral juvenile cataract in the Hutterite population.<br>• The cardiac phenotype is characterized by localized inferior and inferolateral fibrosis of the left ventricle and mild impairment of left ventricular systolic function but severe ventricular arrhythmias leading to sudden cardiac death.<br>• Affected heart tissue and fibroblasts exhibit abnormally shaped nuclei with condensed peripheral heterochromatin.<br>• Functional assays on affected fibroblasts show decreased proliferation capacity, cellular senescence, and a prolonged G1 phase, suggesting premature aging and cellular senescence in proliferating cells.
<b>(A)</b> Pedigrees of 2 multigenerational Hutterite families of L-leut (family 600)... more <b>(A)</b> Pedigrees of 2 multigenerational Hutterite families of L-leut (family 600) and S-leut (family 290) descendants. <b>Filled black squares</b> (male subjects) and <b>circles</b> (female subjects) refer to affected individuals with cataract and arrhythmic cardiomyopathy. <b>Filled upper half symbols</b> indicate individuals diagnosed with arrhythmic cardiomyopathy. <b>Filled lower half symbols</b> refer to individuals with juvenile cataract. <b>Diagonal lines</b> indicate deceased individuals. <b>Double lines</b> refer to known consanguinity. The genotype indicated by a "+" is the p.L13 (wild-type) allele and that indicated by a "-" is the mutant p.R13 allele. <b>(B)</b> Short-axis views of late gadolinium enhancement cardiac magnetic resonance imaging of family 290, II-20 (a), II-22 (b), III-20 (c), and III-21 (d) confirming nearly transmural delayed enhancement of the inferior/inferolateral walls as indicated by the arrows. <b>(C)</b> Rhythm strip of individual 600, II-18 recorded by the implantable cardioverter-defibrillator before delivering an appropriate shock <b>(upper panel)</b>. Representative 12-lead electrocardiogram of the same individual showing deep T-wave inversions inferior and lateral corresponding to areas of fibrosis in the cardiac magnetic resonance image <b>(lower panel)</b>. DNA = deoxyribonucleic acid; LEMD2 = LEM domain containing protein 2.
Clinical and experimental pharmacology & physiology, 2015
SIRT7 with coenzyme NAD catalyzes protein de-acetylation. In stress response, SIRT7 regulates pro... more SIRT7 with coenzyme NAD catalyzes protein de-acetylation. In stress response, SIRT7 regulates protein folding in mitochondria with unknown mechanisms. Decreases in SIRT7 entrain hematopoietic stem cell senescence, but increasing SIRT7 causes elevation of hematopoietic stem cell regenerative function. We discuss the recent findings on SIRT7 and its binding proteins, NRF1 and GABPβ1, in decision making between the choices of inducing cell aging and immortality.
Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting te... more Telomere assumes intra-molecular G-quadruplex that is a significant drug target for inhibiting telomerase maintenance of telomeres in cancer. Metal cations have been recognized as playing important roles in stabilizing G-quadruplex, but their binding processes to human telomeric G-quadruplex remain uncharacterized. To investigate the detailed binding procedures, molecular dynamics simulations were conducted on the hybrid [3 + 1] form-one human telomeric intra-molecular G-quadruplex. We show here that the binding of a potassium ion to a G-tetrad core is mediated by two alternative pathways. Principal component analysis illustrated the dominant concerted motions of G-quadruplex occurred at the loop domains. MM-PBSA calculations revealed that binding was energetically favorable and driven by the electrostatic interactions. The lower binding site was found more constructive favorable for binding. Our data provide useful information on a potassium-mediated stable structure of human telom...
<b>(A)</b> Apoptosis assays were conducted by labeling the deoxyribonucleic acid with... more <b>(A)</b> Apoptosis assays were conducted by labeling the deoxyribonucleic acid with 5-bromo-2′-deoxyuridine 5′-triphosphate) in heart and liver tissue from the affected patient (family 600, II-16) and control heart samples. A positive control was created by adding 1 μg/μl deoxyribonucleic acids. Apoptotic cells were detected as brown dots. No signs of apoptosis were detected in all tested tissue. <b>(B)</b> Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining of fibroblasts and flow cytometry from patient and Ctrl1 and Ctrl3. The histograms of the fluorescein signal collected by using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling method are shown. There is no difference in the apoptotic signals between the 2 control subjects and patient cells. <b>(C)</b> Western blot analysis of apoptotic markers (annexin V, caspase 3 [2 subunits: 17 and 19 KD]) in patient and control fibroblasts on the left panel. GAPDH was used as loading control. Quantification of apoptotic markers by densitometry does not show a significant difference in expression levels (n = 3 experiments with each 2 to 3 replicates). Abbreviations as in Figures 2, 3, and 4.
<b>(A)</b> Cell proliferation assay based on confluence from passage 2 (P2) in the &l... more <b>(A)</b> Cell proliferation assay based on confluence from passage 2 (P2) in the <b>left panel</b> and P15 in the <b>right panel</b> in patient (Pat) fibroblasts, Ctrl1, and an older age ("high-senescence") control (Ctrl3). There was a significant difference in the rate of proliferation detected between both control subjects and patient at P2 and P15. The cells were imaged every 2 h for 90 h. n = 8 wells, mean ± SD, ***p &lt; 0.001. <b>(B)</b> Images of β-galactosidase (β gal)-stained fibroblasts from patient and Ctrl1 as well as Ctrl3 at passage 6 (P6) and P15 in the <b>left panel</b>. Note that the amount of blue-stained cells is visibly higher in patient cells. <b>(Right)</b> Quantification of β gal–stained cells revealed increased cell senescence in patient cells compared with both control subjects at passages as indicated (n = 3; **p &lt; 0.01; ***p &lt; 0.001). <b>(C)</b> Fibroblasts stained with propidium iodide and measurements of the deoxyribonucleic acid content for each phase of the cell cycle by flow cytometry. Cells were taken from Ctrl1 at P6 and 9, from the patient (Pat) at passage 8 and passage 11, and from Ctrl3 at passage 12. Representative diagrams of each phase of the cell cycle are shown in the <b>left panel</b>. <b>(Right)</b> Quantification of the deoxyribonucleic acid content showed a potential arrest in the G1 phase in patient fibroblasts compared with Ctrl1 and similar to Ctrl3 at later passage. n = 3; ∗∗p &lt; 0.01; ∗p &lt; 0.05; ***p &lt; 0.001. <b>(D)</b> Western blot of Aurora B protein expression in patient and control cells on the left panel. GAPDH was used as loading control. <b>(Right)</b> Quantification by densitometry of Aurora B protein expression revealed a significant difference between both control subjects and patient fibroblasts. n = 3 experiments with each 2 to 3 replicates; ***p &lt; 0.001. Abbreviations as in Figures 2 and 3.
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