Measurement of Subcellular CA2+ Redistribution in Cardiac Muscle In Situ: Time Resolved Rapid Freezing and Electron Probe Microanalysis

To directly assess the physiological roles of sarcoplasmic reticulum (SR) and miitochondria (MT), we have utilized energy dispersive electron probe microanalysis (EPMA) on ultrathin freeze-dried cryosections from isolated papillary muscles, rapidly frozen at precise time points of the contractile cycle. Using this approach, we can detect redistribution of subcellular Ca2+ during the cardiac contractile cycle. Changes in Ca2+ of less than 1.0 mmol/kg dry wt can be detected. By determining the variability of the Ca2+ measurements in preliminary experiments, we have also demonstrated that it is possible to optimize experimental design, i.e., to predict the number of animals per treatment group and the number of X-ray spectra per animal that are required in order to detect a specified Ca2+ difference. Quantitative EPMA of rapidly frozen contracting papillary muscle has also allowed us to correlate the Ca2+ content of SR and MT with the contractile state of the muscle. Our results show a decrease of 40% in the amount of Ca2+ stored in the junctional SR during a cardiac muscle twitch, thus providing direct evidence for a role of the SR as a primary site of Ca2+ release. In addition, we have demonstrated dissociation between MT Ca2+ uptake and activation of regulatory enzymes, such as pyruvate dehydrogenase, indicating that MT Ca2+ uptake is not required for activation of MT metabolism

Bayesian Integration of Genetics and Epigenetics Detects Causal Regulatory SNPs Underlying Expression Variability

The standard expression quantitative trait loci (eQTL) detects polymorphisms associated with gene expression without revealing causality. We introduce a coupled Bayesian regression approach—eQTeL, which leverages epigenetic data to estimate regulatory and gene interaction potential, and identifies combination of regulatory single-nucleotide polymorphisms (SNPs) that explain the gene expression variance. On human heart data, eQTeL not only explains a significantly greater proportion of expression variance but also predicts gene expression more accurately than other methods. Based on realistic simulated data, we demonstrate that eQTeL accurately detects causal regulatory SNPs, including those with small effect sizes. Using various functional data, we show that SNPs detected by eQTeL are enriched for allele-specific protein binding and histone modifications, which potentially disrupt binding of core cardiac transcription factors and are spatially proximal to their target. eQTeL SNPs capture a substantial proportion of genetic determinants of expression variance and we estimate that 58% of these SNPs are putatively causal

Pathologic gene network rewiring implicates PPP1R3A as a central regulator in pressure overload heart failure

Heart failure is a leading cause of mortality, yet our understanding of the genetic interactions underlying this disease remains incomplete. Here, we harvest 1352 healthy and failing human hearts directly from transplant center operating rooms, and obtain genome-wide genotyping and gene expression measurements for a subset of 313. We build failing and non-failing cardiac regulatory gene networks, revealing important regulators and cardiac expression quantitative trait loci (eQTLs). PPP1R3A emerges as a regulator whose network connectivity changes significantly between health and disease. RNA sequencing after PPP1R3A knockdown validates network-based predictions, and highlights metabolic pathway regulation associated with increased cardiomyocyte size and perturbed respiratory metabolism. Mice lacking PPP1R3A are protected against pressure-overload heart failure. We present a global gene interaction map of the human heart failure transition, identify previously unreported cardiac eQTLs, and demonstrate the discovery potential of disease-specific networks through the description of PPP1R3A as a central regulator in heart failure

Cardiomyocyte contractile impairment in heart failure results from reduced BAG3-mediated sarcomeric protein turnover

The association between reduced myofilament force-generating capacity (Fmax) and heart failure (HF) is clear, however the underlying molecular mechanisms are poorly understood. Here, we show impaired Fmax arises from reduced BAG3-mediated sarcomere turnover. Myofilament BAG3 expression decreases in human HF and positively correlates with Fmax. We confirm this relationship using BAG3 haploinsufficient mice, which display reduced Fmax and increased myofilament ubiquitination, suggesting impaired protein turnover. We show cardiac BAG3 operates via chaperone-assisted selective autophagy (CASA), conserved from skeletal muscle, and confirm sarcomeric CASA complex localization is BAG3/proteotoxic stress-dependent. Using mass spectrometry, we characterize the myofilament CASA interactome in the human heart and identify eight clients of BAG3-mediated turnover. To determine if increasing BAG3 expression in HF can restore sarcomere proteostasis/Fmax, HF mice were treated with rAAV9-BAG3. Gene therapy fully rescued Fmax and CASA protein turnover after four weeks. Our findings indicate BAG3-mediated sarcomere turnover is fundamental for myofilament functional maintenance

Penerapan Metode Eksperimen untuk Meningkatkan Konsep Dasar Sains pada Anak Didik Kelompok A Tk Pkk Suruhwadang Kecamatan Kademangan Kabupaten Blitar

Tujuan penelitian ini adalah untuk memperoleh tentang kemampuan kognitif anak dalamhal konsep dasar sains dengan menggunakan metode eksperimen pada anak didik kelompokA TK PKK Suruhwadang sebelum dan sesudah dilakukan tindakan. Melakukan tindakanberupa penerapan metode eksperimen untuk meningkatkan kemampuan kognitif dalamkonsep dasar sains pada anak didik kelompok A TK PKK Suruhwadang. Mengetahui adatidaknya perbedaan kemampuan konsep dasar sains dengan menggunakan metodeeksperimen pada anak didik kelompok A TK PKK Suruhwadang antara sebelum dan setelahdilakukan tindakan. Rumusan masalah pada penitilian ini adalah apakah metode eksperimendapat meningkatkan kemampuan pemahaman konsep dasar sains pada anak didik kelompokA TK PKK Suruhwadang Kecamatan Kademangan Kabupaten Blitar. Untuk menjawabrumusan masalah digunakan jenis penelitian tindakan kelas (PTK) dengan model Kemmisdan Taggart melalui empat tahapan yaitu tahap perencanaan , pelaksanaan, observasi danrefleksiyang dilalui dengan dua siklus. Teknik pengumpulan data menggunakan teknikobservasi dan dokumentasi. Adapun instrumen yang digunakan adalah lembar observasikegiatan anak dan lembar observasi pembelajaran oleh guru.Hasil penelitian menunjukanbahwa kemampuan kognitif anak kelompok A pada konsep dasar sain pada pra penelitianmenunjukkan prosentase 56.25%. Setelah pelaksanaan siklus I tentang bidang kemampuankognitif pada konsep dasar sains menunjukkan 59% mengalami peningkatan .Setelahpelaksanaan siklus ke II naik menjadi 83%. Hal ini menunjukkan pelaksanaan siklus ke IItelah mencapai kriteria ketuntasan dan membuktikan bahwa dengan metode eksperimendapat meningkatkan kemampuan kognitif dalam konsep dasar sains

The Massive and Distant Clusters of WISE Survey VI: Stellar Mass Fractions of a Sample of High-Redshift Infrared-selected Clusters

We present measurements of the stellar mass fractions ($f_\star$) for a sample of high-redshift ($0.93 \le z \le 1.32$) infrared-selected galaxy clusters from the Massive and Distant Clusters of WISE Survey (MaDCoWS) and compare them to the stellar mass fractions of Sunyaev-Zel'dovich (SZ) effect-selected clusters in a similar mass and redshift range from the South Pole Telescope (SPT)-SZ Survey. We do not find a significant difference in mean $f_\star$ between the two selection methods, though we do find an unexpectedly large range in $f_\star$ for the SZ-selected clusters. In addition, we measure the luminosity function of the MaDCoWS clusters and find $m^*= 19.41\pm0.07$, similar to other studies of clusters at or near our redshift range. Finally, we present SZ detections and masses for seven MaDCoWS clusters and new spectroscopic redshifts for five MaDCoWS clusters. One of these new clusters, MOO J1521+0452 at $z=1.31$, is the most distant MaDCoWS cluster confirmed to date.Comment: Accepted to Ap

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD

Discovery of Genetic Variation on Chromosome 5q22 Associated with Mortality in Heart Failure

Failure of the human heart to maintain sufficient output of blood for the demands of the body, heart failure, is a common condition with high mortality even with modern therapeutic alternatives. To identify molecular determinant