A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A.

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in aging and metabolism regulation. Targeted sequencing of SIRT6 locus in a population of 450 Ashkenazi Jewish (AJ) centenarians and 550 AJ individuals without a family history of exceptional longevity identified enrichment of a SIRT6 allele containing two linked substitutions (N308K/A313S) in centenarians compared with AJ control individuals. Characterization of this SIRT6 allele (centSIRT6) demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double-strand break repair, and more robustly kill cancer cells compared with wild-type SIRT6. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which was correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

New N-aryl-N-alkyl-thiophene-2-carboxamide compound enhances intracellular Ca2+ dynamics by increasing SERCA2a Ca2+ pumping.

The type 2a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a central role in the intracellular Ca2+ homeostasis of cardiac myocytes, pumping Ca2+ from the cytoplasm into the sarcoplasmic reticulum (SR) lumen to maintain relaxation (diastole) and prepare for contraction (systole). Diminished SERCA2a function has been reported in several pathological conditions, including heart failure. Therefore, development of new drugs that improve SERCA2a Ca2+ transport is of great clinical significance. In this study, we characterized the effect of a recently identified N-aryl-N-alkyl-thiophene-2-carboxamide (or compound 1) on SERCA2a Ca2+-ATPase and Ca2+ transport activities in cardiac SR vesicles, and on Ca2+ regulation in a HEK293 cell expression system and in mouse ventricular myocytes. We found that compound 1 enhances SERCA2a Ca2+-ATPase and Ca2+ transport in SR vesicles. Fluorescence lifetime measurements of fluorescence resonance energy transfer between SERCA2a and phospholamban indicated that compound 1 interacts with the SERCA-phospholamban complex. Measurement of endoplasmic reticulum Ca2+ dynamics in HEK293 cells expressing human SERCA2a showed that compound 1 increases endoplasmic reticulum Ca2+ load by enhancing SERCA2a-mediated Ca2+ transport. Analysis of cytosolic Ca2+ dynamics in mouse ventricular myocytes revealed that compound 1 increases the action potential-induced Ca2+ transients and SR Ca2+ load, with negligible effects on L-type Ca2+ channels and Na+/Ca2+ exchanger. However, during adrenergic receptor activation, compound 1 did not further increase Ca2+ transients and SR Ca2+ load, but it decreased the propensity toward Ca2+ waves. Suggestive of concurrent desirable effects of compound 1 on RyR2, [3H]-ryanodine binding to cardiac SR vesicles shows a small decrease in nM Ca2+ and a small increase in µM Ca2+. Accordingly, compound 1 slightly decreased Ca2+ sparks in permeabilized myocytes. Thus, this novel compound shows promising characteristics to improve intracellular Ca2+ dynamics in cardiomyocytes that exhibit reduced SERCA2a Ca2+ uptake, as found in failing hearts.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

The transmembrane peptide DWORF activates SERCA2a via dual mechanisms.

The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF's precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a-binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA's enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

Differential phosphorylation of LZ+/LZ- MYPT1 isoforms regulates MLC phosphatase activity.

The vascular response to NO is due, in part, to a Ca(2+) independent activation of myosin light chain (MLC) phosphatase, a trimeric enzyme of 20kDa, 38kDa catalytic and 110-130kDa myosin targeting (MYPT1) subunits. Alternative mRNA splicing produces MYPT1 isoforms that differ by the presence or absence of a central insert (CI) and a leucine zipper (LZ), and the presence of a LZ+ MYPT1 isoform is important for protein kinase G (PKG) mediated activation of MLC phosphatase. This study was designed to determine the molecular basis for the differential sensitivity of the vasculature to NO. Our results demonstrate that the presence of the MYPT1 LZ domain is required for PKG to both phosphorylate MYPT1 at S668 and activate MLC phosphatase. Further for LZ+ MYPT1 isoforms, an S668A MYPT1 mutation prevents the PKG mediated, Ca(2+) independent activation of MLC phosphatase. These data demonstrate that differential PKG mediated S668 phosphorylation of LZ+/LZ- MYPT1 isoforms could be important for determining the diversity in the sensitivity of the vasculature to NO mediated vasodilatation. Thus, the relative expression of LZ+/LZ- MYPT1 isoforms, in part, defines the vascular response to NO and NO based vasodilators, and therefore, plays a role in the regulation of vascular tone in both health and disease.

A single-nuclei paired multiomic analysis of the human midbrain reveals age- and Parkinson's disease-associated glial changes.

Age is the primary risk factor for Parkinson's disease (PD), but how aging changes the expression and regulatory landscape of the brain remains unclear. Here we present a single-nuclei multiomic study profiling shared gene expression and chromatin accessibility of young, aged and PD postmortem midbrain samples. Combined multiomic analysis along a pseudopathogenesis trajectory reveals that all glial cell types are affected by age, but microglia and oligodendrocytes are further altered in PD. We present evidence for a disease-associated oligodendrocyte subtype and identify genes lost over the aging and disease process, including CARNS1, that may predispose healthy cells to develop a disease-associated phenotype. Surprisingly, we found that chromatin accessibility changed little over aging or PD within the same cell types. Peak-gene association patterns, however, are substantially altered during aging and PD, identifying cell-type-specific chromosomal loci that contain PD-associated single-nucleotide polymorphisms. Our study suggests a previously undescribed role for oligodendrocytes in aging and PD.

Indirubin Inhibits TRAIL-Induced Activation of Death Receptor 5 in Jurkat Cells.

Death receptor 5 (DR5) is an apoptosis-inducing membrane receptor that mediates cell death in several life-threatening conditions. There is a crucial need for the discovery of DR5 antagonists for the therapeutic intervention of conditions in which the overactivation of DR5 underlies the pathophysiology. DR5 activation mediates cell death in non-alcoholic fatty liver disease (NAFLD) and neurodegenerative processes including amyloid-beta (Aß) accumulation, spinal cord injury (SCI), and brain ischemia. In the current work, we used fluorescence resonance energy transfer (FRET) to monitor the conformational dynamics of DR5 that mediate death signaling. We used a time-resolved FRET screening platform to screen the Selleck library of 2863 U.S. Food and Drug Administration (FDA)-approved compounds. The high-throughput screen (HTS) identified 13 compounds that modulated the FRET between DR5 monomers beyond 5 median absolute deviations (MADs) from the DMSO controls. Of these 13 compounds, indirubin was identified to specifically inhibit tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced caspase-8 activity without modulating DR5 surface expression or TRAIL binding. Indirubin inhibited Fas-associated death domain (FADD) oligomerization and increased cellular FLICE-inhibitory protein (c-FLIP) expression; both are molecular mechanisms involved in inhibiting the DR5 signaling cascade. This study has elucidated previously unknown properties of indirubin that make it a promising candidate for therapeutic investigation of diseases in which overactivation of DR5 underlies pathology.

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.

There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC50 of 34.2 µM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.

FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

Fluorescence lifetime FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA's function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening two different small validation libraries using novel microplate readers that detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from FRET-HTS of 50,000 compounds using the same biosensor, with hit compounds functionally evaluated using assays for Ca2+-ATPase activity and Ca2+-transport. We focused on 18 hit compounds, from which we identified eight structurally unique scaffolds and four scaffold classes as SERCA modulators, approximately half of which are activators and half are inhibitors. Five of these compounds were identified as promising SERCA activators, one of which activates Ca2+-transport even more than Ca2+-ATPase activity thus improving SERCA efficiency. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

FRET assay for live-cell high-throughput screening of the cardiac SERCA pump yields multiple classes of small-molecule allosteric modulators.

We have used FRET-based biosensors in live cells, in a robust high-throughput screening (HTS) platform, to identify small-molecules that alter the structure and activity of the cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA2a). Our primary aim is to discover drug-like small-molecule activators that improve SERCA’s function for the treatment of heart failure. We have previously demonstrated the use of an intramolecular FRET biosensor, based on human SERCA2a, by screening a small validation library using novel microplate readers that can detect the fluorescence lifetime or emission spectrum with high speed, precision, and resolution. Here we report results from a 50,000-compound screen using the same biosensor, with hit compounds functionally evaluated using Ca 2+ -ATPase and Ca 2+ -transport assays. We focused on 18 hit compounds, from which we identified eight structurally unique compounds and four compound classes as SERCA modulators, approximately half of which are activators and half are inhibitors. While both activators and inhibitors have therapeutic potential, the activators establish the basis for future testing in heart disease models and lead development, toward pharmaceutical therapy for heart failure.

MYPT1 protein isoforms are differentially phosphorylated by protein kinase G.

Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.

A Large-Scale High-Throughput Screen for Modulators of SERCA Activity.

The sarco/endoplasmic reticulum Ca-ATPase (SERCA) is a P-type ion pump that transports Ca2+ from the cytosol into the endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells. It is critically important in muscle, facilitating relaxation and enabling subsequent contraction. Increasing SERCA expression or specific activity can alleviate muscle dysfunction, most notably in the heart, and we seek to develop small-molecule drug candidates that activate SERCA. Therefore, we adapted an NADH-coupled assay, measuring Ca-dependent ATPase activity of SERCA, to high-throughput screening (HTS) format, and screened a 46,000-compound library of diverse chemical scaffolds. This HTS platform yielded numerous hits that reproducibly alter SERCA Ca-ATPase activity, with few false positives. The top 19 activating hits were further tested for effects on both Ca-ATPase and Ca2+ transport, in both cardiac and skeletal SR. Nearly all hits increased Ca2+ uptake in both cardiac and skeletal SR, with some showing isoform specificity. Furthermore, dual analysis of both activities identified compounds with a range of effects on Ca2+-uptake and ATPase, which fit into distinct classifications. Further study will be needed to identify which classifications are best suited for therapeutic use. These results reinforce the need for robust secondary assays and criteria for selection of lead compounds, before undergoing HTS on a larger scale.

Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules that Enhance Ca2+ Transport.

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators.

BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Nonmuscle myosin is regulated during smooth muscle contraction.

The participation of nonmuscle myosin in force maintenance is controversial. Furthermore, its regulation is difficult to examine in a cellular context, as the light chains of smooth muscle and nonmuscle myosin comigrate under native and denaturing electrophoresis techniques. Therefore, the regulatory light chains of smooth muscle myosin (SM-RLC) and nonmuscle myosin (NM-RLC) were purified, and these proteins were resolved by isoelectric focusing. Using this method, intact mouse aortic smooth muscle homogenates demonstrated four distinct RLC isoelectric variants. These spots were identified as phosphorylated NM-RLC (most acidic), nonphosphorylated NM-RLC, phosphorylated SM-RLC, and nonphosphorylated SM-RLC (most basic). During smooth muscle activation, NM-RLC phosphorylation increased. During depolarization, the increase in NM-RLC phosphorylation was unaffected by inhibition of either Rho kinase or PKC. However, inhibition of Rho kinase blocked the angiotensin II-induced increase in NM-RLC phosphorylation. Additionally, force for angiotensin II stimulation of aortic smooth muscle from heterozygous nonmuscle myosin IIB knockout mice was significantly less than that of wild-type littermates, suggesting that, in smooth muscle, activation of nonmuscle myosin is important for force maintenance. The data also demonstrate that, in smooth muscle, the activation of nonmuscle myosin is regulated by Ca(2+)-calmodulin-activated myosin light chain kinase during depolarization and a Rho kinase-dependent pathway during agonist stimulation.

Reduced force production during low blood flow to the heart correlates with altered troponin I phosphorylation.

A rat model of low myocardial blood flow was established to test the hypothesis that post-translational changes to proteins of the thin and thick muscle filaments correlate with decreased cardiac contractility. Following 3 days of low blood flow by constriction of the left anterior descending artery, rat hearts demonstrated a reduction in fractional shortening at rest and a relative decline in fractional shortening when challenged with high dose versus low dose dobutamine, reflecting reduced energy reserves. Permeabilized fibers from low blood flow hearts demonstrated a decline in maximum force per cross-section and Ca2+ sensitivity as compared to their sham operated counterparts. An examination of sarcomeric proteins by twodimensional gel electrophoresis, mass spectrometry, and phospho-specific antibodies provided evidence for Ser23/24 and Ser43/45 phosphorylation of troponin I (TnI). Total TnI phosphorylation was not different between the groups, but Ser23/24 phosphorylation declined with low blood flow, implying an accompanying increase in phosphorylation at other sites of TnI. Affinity chromatography demonstrated that TnI from low blood flow myocardium had reduced relative affinity to Ca2+ bound troponin C compared to TnI from sham operated hearts, providing a mechanism for reduced Ca2+ sensitivity of force production in low blood flow fibers. These findings suggest that altered TnI function, due to changes in the distribution of phosphorylated sites, is an early contributor to reduced contractility of the heart.

Red-Shifted FRET Biosensors for High-Throughput Fluorescence Lifetime Screening.

We have developed fluorescence resonance energy transfer (FRET) biosensors with red-shifted fluorescent proteins (FP), yielding improved characteristics for time-resolved (lifetime) fluorescence measurements. In comparison to biosensors with green and red FRET pairs (GFP/RFP), FPs that emit at longer wavelengths (orange and maroon, OFP/MFP) increased the FRET efficiency, dynamic range, and signal-to-background of high-throughput screening (HTS). OFP and MFP were fused to specific sites on the human cardiac calcium pump (SERCA2a) for detection of structural changes due to small-molecule effectors. When coupled with a recently improved HTS fluorescence lifetime microplate reader, this red-shifted FRET biosensor enabled high-precision nanosecond-resolved fluorescence decay measurements from microliter sample volumes at three minute read times per 1536-well-plate. Pilot screens with a library of small-molecules demonstrate that the OFP/MFP FRET sensor substantially improves HTS assay quality. These high-content FRET methods detect minute FRET changes with high precision, as needed to elucidate novel structural mechanisms from small-molecule or peptide regulators discovered through our ongoing HTS efforts. FRET sensors that emit at longer wavelengths are highly attractive to the FRET biosensor community for drug discovery and structural interrogation of new therapeutic targets.

Targeting protein-protein interactions for therapeutic discovery via FRET-based high-throughput screening in living cells.

We have developed a structure-based high-throughput screening (HTS) method, using time-resolved fluorescence resonance energy transfer (TR-FRET) that is sensitive to protein-protein interactions in living cells. The membrane protein complex between the cardiac sarcoplasmic reticulum Ca-ATPase (SERCA2a) and phospholamban (PLB), its Ca-dependent regulator, is a validated therapeutic target for reversing cardiac contractile dysfunction caused by aberrant calcium handling. However, efforts to develop compounds with SERCA2a-PLB specificity have yet to yield an effective drug. We co-expressed GFP-SERCA2a (donor) in the endoplasmic reticulum membrane of HEK293 cells with RFP-PLB (acceptor), and measured FRET using a fluorescence lifetime microplate reader. We screened a small-molecule library and identified 21 compounds (Hits) that changed FRET by >3SD. 10 of these Hits reproducibly alter SERCA2a-PLB structure and function. One compound increases SERCA2a calcium affinity in cardiac membranes but not in skeletal, suggesting that the compound is acting specifically on the SERCA2a-PLB complex, as needed for a drug to mitigate deficient calcium transport in heart failure. The excellent assay quality and correlation between structural and functional assays validate this method for large-scale HTS campaigns. This approach offers a powerful pathway to drug discovery for a wide range of protein-protein interaction targets that were previously considered "undruggable".