The N terminus of myosin-binding protein C extends toward actin filaments in intact cardiac muscle

Myosin and actin filaments are highly organized within muscle sarcomeres. Myosin-binding protein C (MyBP-C) is a flexible, rod-like protein located within the C-zone of the sarcomere. The C-terminal domain of MyBP-C is tethered to the myosin filament backbone, and the N-terminal domains are postulated to interact with actin and/or the myosin head to modulate filament sliding. To define where the N-terminal domains of MyBP-C are localized in the sarcomere of active and relaxed mouse myocardium, the relative positions of the N terminus of MyBP-C and actin were imaged in fixed muscle samples using super-resolution fluorescence microscopy. The resolution of the imaging was enhanced by particle averaging. The images demonstrate that the position of the N terminus of MyBP-C is biased toward the actin filaments in both active and relaxed muscle preparations. Comparison of the experimental images with images generated in silico, accounting for known binding partner interactions, suggests that the N-terminal domains of MyBP-C may bind to actin and possibly the myosin head but only when the myosin head is in the proximity of an actin filament. These physiologically relevant images help define the molecular mechanism by which the N-terminal domains of MyBP-C may search for, and capture, molecular binding partners to tune cardiac contractility

Oxidative Stress in Dilated Cardiomyopathy Caused by MYBPC3 Mutation

Cardiomyopathies can result from mutations in genes encoding sarcomere proteins including MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). However, whether oxidative stress is augmented due to contractile dysfunction and cardiomyocyte damage in MYBPC3-mutated cardiomyopathies has not been elucidated. To determine whether oxidative stress markers were elevated in MYBPC3-mutated cardiomyopathies, a previously characterized 3-month-old mouse model of dilated cardiomyopathy (DCM) expressing a homozygous MYBPC3 mutation (cMyBP-C(t/t)) was used, compared to wild-type (WT) mice. Echocardiography confirmed decreased percentage of fractional shortening in DCM versus WT hearts. Histopathological analysis indicated a significant increase in myocardial disarray and fibrosis while the second harmonic generation imaging revealed disorganized sarcomeric structure and my

Targeted Genome-Wide Enrichment of Functional Regions

Only a small fraction of large genomes such as that of the human contains the functional regions such as the exons, promoters, and polyA sites. A platform technique for selective enrichment of functional genomic regions will enable several next-generation sequencing applications that include the discovery of causal mutations for disease and drug response. Here, we describe a powerful platform technique, termed “functional genomic fingerprinting” (FGF), for the multiplexed genomewide isolation and analysis of targeted regions such as the exome, promoterome, or exon splice enhancers. The technique employs a fixed part of a uniquely designed Fixed-Randomized primer, while the randomized part contains all the possible sequence permutations. The Fixed-Randomized primers bind with full sequence complementarity at multiple sites where the fixed sequence (such as the splice signals) occurs within the genome, and multiplex amplify many regions bounded by the fixed sequences (e.g., exons). Notably, validation of this technique using cardiac myosin binding protein-C (MYBPC3) gene as an example strongly supports the application and efficacy of this method. Further, assisted by genomewide computational analyses of such sequences, the FGF technique may provide a unique platform for high-throughput sample production and analysis of targeted genomic regions by the next-generation sequencing techniques, with powerful applications in discovering disease and drug response genes

Impaired right ventricular calcium cycling is an early risk factor in r14del-phospholamban arrhythmias

The inherited mutation (R14del) in the calcium regulatory protein phospholamban (PLN) is linked to malignant ventricular arrhythmia with poor prognosis starting at adolescence. However, the underlying early mechanisms that may serve as prognostic factors remain elusive. This study generated humanized mice in which the endogenous gene was replaced with either human wild type or R14del-PLN and addressed the early molecular and cellular pathogenic mechanisms. R14del-PLN mice exhibited stress-induced impairment of atrioventricular conduction, and prolongation of both ventricular activation and repolarization times in association with ventricular tachyarrhythmia, originating from the right ventricle (RV). Most of these distinct electrocardiographic features were remarkably similar to those in R14del-PLN patients. Studies in isolated cardiomyocytes revealed RV-specific calcium defects, including prolonged action potential duration, depressed calcium kinetics and contractile parameters, and elevated diastolic Ca-levels. Ca-sparks were also higher although SR Ca-load was reduced. Accordingly, stress conditions induced after contractions, and inclusion of the CaMKII inhibitor KN93 reversed this proarrhythmic parameter. Compensatory responses included altered expression of key genes associated with Ca-cycling. These data suggest that R14del-PLN cardiomyopathy originates with RV-specific impairment of Ca-cycling and point to the urgent need to improve risk stratification in asymptomatic carriers to prevent fatal arrhythmias and delay cardiomyopathy onset

Multiplexed amplification of exons by splice signal FR primers.

<p>The FR primers for the donor (5′ splice signal) and the acceptor (3′ splice signal) splice sequences bind the exons with complementary base pairing over the entire length of the primers, by virtue of the presence of all the possible sequences within the randomized sequence portion of the FR primer. Only the specific primer molecule from the FR primer population is expected to bind selectively with full complementarity at the fixed sequence target site at a high Tm condition. The FR primers amplify multiple exons since they are capable of binding many exons within the human genome with complete sequence complementarity, wherever the fixed sequence binds.</p

Multiplex genome-wide exon amplification based on MYBPC3 gene exons 7 and 8.

<p>The human genomic DNA was PCR-amplified under standard conditions at 58°C with primers designed from the donor (5′) splice signal sequence (from exon 7) and the acceptor (3′) splice signal sequence (from exon 8) of the MYBPC3 gene. It was also amplified by the same FR primer pairs with decreasing number of fixed bases and increasing number of random bases (Ns) as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011138#pone-0011138-t001" target="_blank">Table 1</a>. The expected fragment (438 bases for the combined exon 7, intron 7 and exon 8) is present in all the lanes, and the number of fragments amplified increased with increasing Ns in the FR primers. M1 & M2 are marker lanes. Lane CS shows the computer simulated exon fingerprint obtained with the same primers used for lane 5, with four bases removed from the 5′ end of the forward primer and three bases removed from the 5′ end of the reverse primer (see text).</p

FR Primers used in the amplification experiments shown in Figure 4.

a<p>Excess FR primers required theoretically for each specific primer sequence within the primer population to be the same as in the standard PCR reaction compared to the actual excess used in the experiment.</p

Design of an FR primer.

<p>To a core fixed sequence (ATCTG), a series of Ns (A, T, G and C) are added in equimolar concentrations at each step of the oligo-nucleotide synthesis. This process generates all possible sequences of length n, when n bases are randomized, such that each variable sequence is attached to the end of the fixed sequence.</p