CTAB enabled microwave-hydrothermal assisted mesoporous Zn-doped hydroxyapatite nanorods synthesis using bio-waste Nodipecten nodosus scallop for biomedical implant applications.

In biomedical exploration, the predominant characteristic is synthesizing and fabricating multifunctional nanostructure with intensified biocompatibility and excellent antibacterial applications to avoid post-surgical implant failure. The objective of the current study is to examine ideal mesoporous zinc-doped hydroxyapatite (HAp) for future use in the field of biomedical research. In the present investigation, we synthesized mesoporous Zn-doped HAp nanorods with varied mole concentrations using a profound microwave hydrothermal method utilizing bio-waste Nodipecten nodosus scallop as a calcium source and CTAB as an organic modifier. Bio-waste Nodipecten nodosus scallop is a widely available cheap calcium precursor which is converted into pure and zinc-doped hydroxyapatite nanorods with the help of the microwave hydrothermal method. Different analytical techniques like spectroscopy and electron microscopy were employed to evaluate and precisely characterize the structural and morphological characteristics in synthesized pure and mesoporous Zn-doped HAp nanorods. CTAB and microwave hydrothermal methods successfully create mesoporous Zn-doped hydroxyapatite nanorods with different sizes and morphology. Mesoporous Zinc-doped HAp nanorods show excellent antibacterial activity against Klebsiella pneumoniae (MTCC 7407) and Bacillus subtilis (MTCC 1133), compared to other nanorods. ZnHAp-3 shows notable excellent results of antibacterial effect towards K. pneumoniae and B. subtilis, by exhibiting 12.36 ± 0.12 and 13.12 ± 0.16 mm zone of inhibition. Furthermore, ZnHAp-1 shows the lower zone of inhibition, while the ZnHAp-3 sample shows the highest zone of inhibition. A foremost study performed was toxicity assays to validate safe attributes of mesoporous zinc-doped HAp intensified with the proliferation function of the zebrafish model. The results reveal the non-toxic behavior of pure and mesoporous zinc-doped HAp samples. Thus, our studies provide evidence for the synthesis technique for the mesoporous zinc-doped HAp nanorods using a novel CTAB-enabled microwave hydrothermal method utilizing bio-waste Nodipecten nodosus scallop as a calcium source will be alternative affordable biocidal antibacterial materials for controlling post-surgical implant failures.

Biomimetic facile synthesis of zinc oxide and copper oxide nanoparticles from Elaeagnus indica for enhanced photocatalytic activity.

The present study focused on synthesizing ZnO nanoparticles (NPs) and CuO NPs using Elaeagnus indica leaf extract as reducing and stabilizing agents using Zn(O2CCH3)2 and Cu2SO4, respectively, for the first time. We have confirmed the formation of aggregated ZnO NPs and CuO NPs with phytochemicals by various spectral analyses and electron microscopy studies. The size of synthesized ZnO NPs and CuO NPs were in the range of 20-30 nm and 30-40 nm, respectively. The antimicrobial activity of ZnO NPs at 75 µg concentration is superior against Salmonella typhimurium, Klebsiella pneumonia, Bacillus subtilis, Staphylococcus epidermidis, and Aspergillus niger. While CuO nanoparticles with 75 µg concentration effectively inhibited S. typhimurium, B. subtilis, S. epidermidis, and A. niger. Phytochemicals and reactive oxygen species generated by the prepared NPs may account for the antimicrobial effects observed. The photodegradation of methylene blue by ZnO NPs and CuO NPs was 91% and 76%, respectively, for 6 h of sunlight exposure. CuO NPs and ZnO NPs have different intrinsic properties and phytochemical compositions; hence ZnO NPs photodegrade faster than CuO NPs even though ZnO has higher bandgap energy than CuO. Consequently, CuO and ZnO NPs produced from E. indica leaf extract might be utilized as antimicrobials and photocatalysts in the future.

Citric Acid-Mediated Microwave-Hydrothermal Synthesis of Mesoporous F-Doped HAp Nanorods from Bio-Waste for Biocidal Implant Applications.

In this current research, mesoporous nano-hydroxyapatite (HAp) and F-doped hydroxyapatite (FHAp) were effectively obtained through a citric acid-enabled microwave hydrothermal approach. Citric acid was used as a chelating and modifying agent for tuning the structure and porosity of the HAp structure. This is the first report to use citric acid as a modifier for producing mesoporous nano HAp and F-doped FHAp. The obtained samples were characterized by different analyses. The XRD data revealed that F is incorporated well into the HAp crystal structure. The crystallinity of HAp samples was improved and the unit cell volume was lowered with fluorine incorporation. Transmission electron microscopy (TEM) images of the obtained samples revealed that a nano rod-like shape was obtained. The mesoporous structures of the produced HAp samples were confirmed by Brunauer-Emmett-Teller (BET) analysis. In vivo studies performed using zebrafish and C. elegans prove the non-toxic behavior of the synthesized F doped HAp samples. The obtained samples are also analyzed for antimicrobial activity using Gram-negative and Gram-positive bacteria, which are majorly involved in implant failure. The F doped samples revealed excellent bactericidal activity. Hence, this study confirms that the non-toxic and excellent antibacterial mesoporous F doped HAp can be a useful candidate for biocidal implant application.

Mesoporous Mn-doped hydroxyapatite nanorods obtained via pyridinium chloride enabled microwave-assisted synthesis by utilizing Donax variabilis seashells for implant applications.

Manganese-doped mesoporous hydroxyapatite (MnHAp) nanorods, a bio-apatite were synthesized via pyridinium chloride mediated microwave approach using bio-waste Donax variabilis seashells to treat orthopedic infections. This is the first report on using pyridinium chloride mediated mesoporous MnHAp nanorods synthesis. Pure and Mn doped HAp samples were examined using Raman spectroscopy, X-ray powder diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) studies to confirm the prepared HAp nanorods. Furthermore, the fabrication of manganese-doped HAp was successful with the formation of a hexagonal crystal lattice without disturbing the HAp phase. It is because, at the time of synthesis, PO43- ions form an electrostatic interaction with the Mn ions. Furthermore, Mn-doped HAp samples showed a reduction in their sizes of 15, 10-15, 5-10 nm width, and 80-100, 10-15, 20-30 nm length with varied pore diameters and surface area. The pure HAp, MnHAp-1, MnHAp-2, and MnHAp-3 nanorods disclose the surface area of 39.4, 18.0, 49.2, and 80.4 m2 g-1, with a pore volume of 0.0102, 0.0047, 0.0143, and 0.0447 cm3 g-1, the corresponding pore diameter was estimated to be 6, 7, 6, and 4 nm, respectively. Moreover, antibacterial activity reveals effective bactericidal action against infections causing pathogens whereas cytotoxicity examination (MTT assay), and zebrafish results reveal their non-toxic behavior. Therefore, it is evident from the study, that rapid fabrication of mesoporous and diverse structured MnHAp nanorods could be convenient with pyridinium chloride enabled microwave-assisted method as a bactericidal biomaterial for implant applications.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury.

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Ascorbic Acid-Assisted Microwave Synthesis of Mesoporous Ag-Doped Hydroxyapatite Nanorods from Biowaste Seashells for Implant Applications.

Post-surgery implant infection is one of the most challenging issues in orthopedics and it is mainly caused by infective micro-organisms. A potential approach to overcome this issue is developing biomaterials with efficient antibacterial activity. The main intention of this present research is devoted to ascorbic acid-assisted microwave synthesis of mesoporous (silver) Ag-doped hydroxyapatite (HAp) nanorods using biowaste seashells with antibacterial properties. XRD, FTIR, and Raman spectroscopy results revealed that the synthesized nanoparticles are hexagonal crystalline HAp. Further, the silver-doped HAp was also successfully produced without affecting the HAp crystalline phase by forming electrostatic interaction with PO43- ions during the synthesis. The morphological features confirm that the pure HAp is elongated mesoporous nanorods with 20 nm width and 300-500 nm length. However, silver doped HAp nanoparticles such as AgHA-1, AgHA-2, and AgHA-3 are found to be similar mesoporous rods but with different aspect ratios in sizes of 15, 10-15, and 5-10 nm width and 80-100, 10-15, and 20-30 nm length. The BET specific surface areas were obtained as 29 ± 3, 84 ± 2, 87 ± 2, and 128 ± 3 m2 g-1, and pore diameters were 4.68, 4.18, 9.30, and 3.77 nm, respectively, for pure HA, AgHA-1, AgHA-2, and AgHA-3. Therefore, HAp nanoparticles with different dimensions and mesoporous structures could be rapidly prepared using a microwave-assisted method and ascorbic acid as a supporting material. In addition, the synthesized HAp nanoparticles are analyzed for its antibacterial and cytotoxicity studies. The antibacterial and cytotoxicity study clearly reveals that the Ag-doped HAp nanorods are efficiently antibacterial and nontoxic in nature. Hence, it is clear that the ascorbic acid-enabled microwave-assisted method will be one of the best methods for the rapid production of HAp nanoparticles with different dimensions and mesoporous structures for its application as an implant material.

Usefulness of Released Cardiac Myosin Binding Protein-C as a Predictor of Cardiovascular Events.

Cardiac myosin binding protein-C (cMyBP-C) is a heart muscle-specific thick filament protein. Elevated level of serum cMyBP-C is an indicator of early myocardial infarction (MI), but its value as a predictor of future cardiovascular disease is unknown. Based on the presence of significant amount of cMyBP-C in the serum of previous study subjects independent of MI, we hypothesized that circulating cMyBP-C is a sensitive indicator of ongoing cardiovascular stress and disease. To test this hypothesis, 75 men and 83 women of similar ages were recruited for a prospective study. They underwent exercise stress echocardiography to provide pre- and poststress blood samples for subsequent determination of serum cMyBP-C levels. The subjects were followed for 1 to 1.5 years. Exercise stress increased serum cMyBP-C in all subjects. Twenty-seven primary events (such as death, MI, revascularization, invasive cardiovascular procedure, or cardiovascular-related hospitalization) and 7 critical events (CE; such as death, MI, stroke, or pulmonary embolism) occurred. After adjusting for sex and cardiovascular risk factors with multivariate Cox regression, a 96% sensitive prestress cMyBP-C threshold carried a hazard ratio of 8.1 with p = 0.041 for primary events. Most subjects (6 of 7) who had CE showed normal ejection fraction on echocardiography. Prestress cMyBP-C demonstrated area under receiver operating curve of 0.91 and multivariate Cox regression hazard ratio of 13.8 (p = 0.000472) for CE. Thus, basal cMyBP-C levels reflected susceptibility for a variety of cardiovascular diseases. Together with its high sensitivity, cMyBP-C holds potential as a screening biomarker for the existence of severe cardiovascular diseases.

Alterations in Multi-Scale Cardiac Architecture in Association With Phosphorylation of Myosin Binding Protein-C.

BACKGROUND: The geometric organization of myocytes in the ventricular wall comprises the structural underpinnings of cardiac mechanical function. Cardiac myosin binding protein-C (MYBPC3) is a sarcomeric protein, for which phosphorylation modulates myofilament binding, sarcomere morphology, and myocyte alignment in the ventricular wall. To elucidate the mechanisms by which MYBPC3 phospho-regulation affects cardiac tissue organization, we studied ventricular myoarchitecture using generalized Q-space imaging (GQI). GQI assessed geometric phenotype in excised hearts that had undergone transgenic (TG) modification of phospho-regulatory serine sites to nonphosphorylatable alanines (MYBPC3(AllP-/(t/t))) or phospho-mimetic aspartic acids (MYBPC3(AllP+/(t/t))). METHODS AND RESULTS: Myoarchitecture in the wild-type (MYBPC3(WT)) left-ventricle (LV) varied with transmural position, with helix angles ranging from -90/+90 degrees and contiguous circular orientation from the LV mid-myocardium to the right ventricle (RV). Whereas MYBPC3(AllP+/(t/t)) hearts were not architecturally distinct from MYBPC3(WT), MYBPC3(AllP-/(t/t)) hearts demonstrated a significant reduction in LV transmural helicity. Null MYBPC3((t/t)) hearts, as constituted by a truncated MYBPC3 protein, demonstrated global architectural disarray and loss in helicity. Electron microscopy was performed to correlate the observed macroscopic architectural changes with sarcomere ultrastructure and demonstrated that impaired phosphorylation of MYBPC3 resulted in modifications of the sarcomere aspect ratio and shear angle. The mechanical effect of helicity loss was assessed through a geometric model relating cardiac work to ejection fraction, confirming the mechanical impairments observed with echocardiography. CONCLUSIONS: We conclude that phosphorylation of MYBPC3 contributes to the genesis of ventricular wall geometry, linking myofilament biology with multiscale cardiac mechanics and myoarchitecture.

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 myocyte damage in DCM hearts when compared to WT hearts. Intriguingly, DCM mouse heart homogenates had decreased glutathione (GSH/GSSG) ratio and increased protein carbonyl and lipid malondialdehyde content compared to WT heart homogenates, consistent with elevated oxidative stress. Importantly, a similar result was observed in human cardiomyopathy heart homogenate samples. These results were further supported by reduced signals for mitochondrial semiquinone radicals and Fe-S clusters in DCM mouse hearts measured using electron paramagnetic resonance spectroscopy. In conclusion, we demonstrate elevated oxidative stress in MYPBC3-mutated DCM mice, which may exacerbate the development of heart failure.

Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate.

BACKGROUND: Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGIα antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. METHODS AND RESULTS: We performed a screen for myocardial proteins interacting with the PKGIα leucine zipper (LZ)-binding domain to identify myocardial-specific PKGI antiremodeling substrates. Our screen identified cardiac myosin-binding protein-C (cMyBP-C), a cardiac myocyte-specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGIα-cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGIα phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGIα, but not in cells transfected with LZ mutant PKGIα, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. CONCLUSIONS: Taken together, these data support that PKGIα and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGIα kinase substrate. This study provides the first identification of a myocardial-specific PKGIα LZ-dependent antiremodeling substrate and supports further exploration of PKGIα myocardial LZ substrates as potential therapeutic targets for heart failure.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation.

ß-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 µm and SL = 2.3 µm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that ß-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

A hypertrophic cardiomyopathy-associated MYBPC3 mutation common in populations of South Asian descent causes contractile dysfunction.

Hypertrophic cardiomyopathy (HCM) results from mutations in genes encoding sarcomeric proteins, most often MYBPC3, which encodes cardiac myosin binding protein-C (cMyBP-C). A recently discovered HCM-associated 25-base pair deletion in MYBPC3 is inherited in millions worldwide. Although this mutation causes changes in the C10 domain of cMyBP-C (cMyBP-C(C10mut)), which binds to the light meromyosin (LMM) region of the myosin heavy chain, the underlying molecular mechanism causing HCM is unknown. In this study, adenoviral expression of cMyBP-C(C10mut) in cultured adult rat cardiomyocytes was used to investigate protein localization and evaluate contractile function and Ca(2+) transients, compared with wild-type cMyBP-C expression (cMyBP-C(WT)) and controls. Forty-eight hours after infection, 44% of cMyBP-C(WT) and 36% of cMyBP-C(C10mut) protein levels were determined in total lysates, confirming equal expression. Immunofluorescence experiments showed little or no localization of cMyBP-C(C10mut) to the C-zone, whereas cMyBP-C(WT) mostly showed C-zone staining, suggesting that cMyBP-C(C10mut) could not properly integrate in the C-zone of the sarcomere. Subcellular fractionation confirmed that most cMyBP-C(C10mut) resided in the soluble fraction, with reduced presence in the myofilament fraction. Also, cMyBP-C(C10mut) displayed significantly reduced fractional shortening, sarcomere shortening, and relaxation velocities, apparently caused by defects in sarcomere function, because Ca(2+) transients were unaffected. Co-sedimentation and protein cross-linking assays confirmed that C10(mut) causes the loss of C10 domain interaction with myosin LMM. Protein homology modeling studies showed significant structural perturbation in cMyBP-C(C10mut), providing a potential structural basis for the alteration in its mode of interaction with myosin LMM. Therefore, expression of cMyBP-C(C10mut) protein is sufficient to cause contractile dysfunction in vitro.

Myocardial infarction-induced N-terminal fragment of cardiac myosin-binding protein C (cMyBP-C) impairs myofilament function in human myocardium.

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca(2+) transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 µm) and long (2.3 µm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca(2+) sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca(2+) sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.

A sensitive and specific quantitation method for determination of serum cardiac myosin binding protein-C by electrochemiluminescence immunoassay.

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time. Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Cardiac myosin binding protein-C plays no regulatory role in skeletal muscle structure and function.

Myosin binding protein-C (MyBP-C) exists in three major isoforms: slow skeletal, fast skeletal, and cardiac. While cardiac MyBP-C (cMyBP-C) expression is restricted to the heart in the adult, it is transiently expressed in neonatal stages of some skeletal muscles. However, it is unclear whether this expression is necessary for the proper development and function of skeletal muscle. Our aim was to determine whether the absence of cMyBP-C alters the structure, function, or MyBP-C isoform expression in adult skeletal muscle using a cMyBP-C null mouse model (cMyBP-C((t/t))). Slow MyBP-C was expressed in both slow and fast skeletal muscles, whereas fast MyBP-C was mostly restricted to fast skeletal muscles. Expression of these isoforms was unaffected in skeletal muscle from cMyBP-C((t/t)) mice. Slow and fast skeletal muscles in cMyBP-C((t/t)) mice showed no histological or ultrastructural changes in comparison to the wild-type control. In addition, slow muscle twitch, tetanus tension, and susceptibility to injury were all similar to the wild-type controls. Interestingly, fMyBP-C expression was significantly increased in the cMyBP-C((t/t)) hearts undergoing severe dilated cardiomyopathy, though this does not seem to prevent dysfunction. Additionally, expression of both slow and fast isoforms was increased in myopathic skeletal muscles. Our data demonstrate that i) MyBP-C isoforms are differentially regulated in both cardiac and skeletal muscles, ii) cMyBP-C is dispensable for the development of skeletal muscle with no functional or structural consequences in the adult myocyte, and iii) skeletal isoforms can transcomplement in the heart in the absence of cMyBP-C.

Increase in cardiac myosin binding protein-C plasma levels is a sensitive and cardiac-specific biomarker of myocardial infarction.

Earlier studies have shown that cardiac myosin binding protein-C (cMyBP-C) is easily releasable into the circulation following myocardial infarction (MI) in animal models and patients. However, since its release kinetics has not been clearly demonstrated, no parameters are available to judge its efficacy as a bona fide biomarker of MI in patients with MI. To make this assessment, plasma levels of cMyBP-C and six known biomarkers of MI were determined by sandwich enzyme-linked immunosorbent assay in patients with MI who had before and after Percutaneous Transcoronary Angioplasty (PTCA), as well as healthy controls. Compared to healthy controls (22.3 ± 2.4 ng/mL (n=54)), plasma levels of cMyBP-C were significantly increased in patients with MI (105.1 ± 8.8 ng/mL (n=65), P<0.001). Out of 65 patients, 24 had very high levels of plasma cMyBP-C (116.5 ± 13.3 ng/mL), indicating high probability of MI. Importantly, cMyBP-C levels were significantly decreased in patients (n=40) at 12 hours post-PTCA (41.2 ± 9.3 ng/mL, P<0.001), compared to the patients with MI. Receiver operating characteristic analysis revealed that a plasma cMyBP-C reading of 68.1 ng/mL provided a sensitivity of 66.2% and a specificity of 100%. Also, myoglobin, carbonic anhydrase and creatine kinase-MB levels were significantly increased in MI patients who also had higher cMyBP-C levels. In contrast, levels of cardiac troponin I, glycogen phosphorylase and heart-type fatty acid binding protein were not significantly changed in the samples, indicating the importance of evaluating the differences in release kinetics of these biomarkers in the context of accurate diagnosis. Our findings suggest that circulating cMyBP-C is a sensitive and cardiac-specific biomarker with potential utility for the accurate diagnosis of MI.

Pathogenic properties of the N-terminal region of cardiac myosin binding protein-C in vitro.

Cardiac myosin binding protein-C (cMyBP-C) plays a role in sarcomeric structure and stability, as well as modulating heart muscle contraction. The 150 kDa full-length (FL) cMyBP-C has been shown to undergo proteolytic cleavage during ischemia-reperfusion injury, producing an N-terminal 40 kDa fragment (mass 29 kDa) that is predominantly associated with post-ischemic contractile dysfunction. Thus far, the pathogenic properties of such truncated cMyBP-C proteins have not been elucidated. In the present study, we hypothesized that the presence of these 40 kDa fragments is toxic to cardiomyocytes, compared to the 110 kDa C-terminal fragment and FL cMyBP-C. To test this hypothesis, we infected neonatal rat ventricular cardiomyocytes and adult rabbit ventricular cardiomyocytes with adenoviruses expressing the FL, 110 and 40 kDa fragments of cMyBP-C, and measured cytotoxicity, Ca(2+) transients, contractility, and protein-protein interactions. Here we show that expression of 40 kDa fragments in neonatal rat ventricular cardiomyocytes significantly increases LDH release and caspase 3 activity, significantly reduces cell viability, and impairs Ca(2+) handling. Adult cardiomyocytes expressing 40 kDa fragments exhibited similar impairment of Ca(2+) handling along with a significant reduction of sarcomere length shortening, relaxation velocity, and contraction velocity. Pull-down assays using recombinant proteins showed that the 40 kDa fragment binds significantly to sarcomeric actin, comparable to C0-C2 domains. In addition, we discovered several acetylation sites within the 40 kDa fragment that could potentially affect actomyosin function. Altogether, our data demonstrate that the 40 kDa cleavage fragments of cMyBP-C are toxic to cardiomyocytes and significantly impair contractility and Ca(2+) handling via inhibition of actomyosin function. By elucidating the deleterious effects of endogenously expressed cMyBP-C N-terminal fragments on sarcomere function, these data contribute to the understanding of contractile dysfunction following myocardial injury.

(E)-2-({2-[(E)-(Hy-droxy-imino)-meth-yl]phen-oxy}meth-yl)-3-phenyl-acrylonitrile.

In the title compound, C(17)H(14)N(2)O(2), the hy-droxy-ethanimine group adopts an anti-periplanar conformation. In the crystal, mol-ecules are linked by O-H⋯N hydrogen bonds, forming zigzag chains running along the c axis.

Cardiac myosin binding protein-C is a potential diagnostic biomarker for myocardial infarction.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament assembly protein that stabilizes sarcomeric structure and regulates cardiac function; however, the profile of cMyBP-C degradation after myocardial infarction (MI) is unknown. We hypothesized that cMyBP-C is sensitive to proteolysis and is specifically increased in the bloodstream post-MI in rats and humans. Under these circumstances, elevated levels of degraded cMyBP-C could be used as a diagnostic tool to confirm MI. To test this hypothesis, we first established that cMyBP-C dephosphorylation is directly associated with increased degradation of this myofilament protein, leading to its release in vitro. Using neonatal rat ventricular cardiomyocytes in vitro, we were able to correlate the induction of hypoxic stress with increased cMyBP-C dephosphorylation, degradation, and the specific release of N'-fragments. Next, to define the proteolytic pattern of cMyBP-C post-MI, the left anterior descending coronary artery was ligated in adult male rats. Degradation of cMyBP-C was confirmed by a reduction in total cMyBP-C and the presence of degradation products in the infarct tissue. Phosphorylation levels of cMyBP-C were greatly reduced in ischemic areas of the MI heart compared to non-ischemic regions and sham control hearts. Post-MI plasma samples from these rats, as well as humans, were assayed for cMyBP-C and its fragments by sandwich ELISA and immunoprecipitation analyses. Results showed significantly elevated levels of cMyBP-C in the plasma of all post-MI samples. Overall, this study suggests that cMyBP-C is an easily releasable myofilament protein that is dephosphorylated, degraded and released into the circulation post-MI. The presence of elevated levels of cMyBP-C in the blood provides a promising novel biomarker able to accurately rule in MI, thus aiding in the further assessment of ischemic heart disease.

Transcription regulation of ezrA and its effect on cell division of Bacillus subtilis.

The EzrA protein of Bacillus subtilis is a negative regulator for FtsZ (Z)-ring formation. It is able to modulate the frequency and position of Z-ring formation during cell division. The loss of this protein results in cells with multiple Z rings located at polar as well as medial sites; it also lowers the critical concentration of FtsZ required for ring formation (P. A. Levin, I. G. Kurster, and A. D. Grossman, Proc. Natl. Acad. Sci. USA 96:9642-9647, 1999). We have studied the regulation of ezrA expression during the growth of B. subtilis and its effects on the intracellular level of EzrA as well as the cell length of B. subtilis. With the aid of promoter probing, primer extension, in vitro transcription, and Western blotting analyses, two overlapping sigmaA-type promoters, P1 and P2, located about 100 bp upstream of the initiation codon of ezrA, have been identified. P1, supposed to be an extended -10 promoter, was responsible for most of the ezrA expression during the growth of B. subtilis. Disruption of this promoter reduced the intracellular level of EzrA very significantly compared with disruption of P2. Moreover, deletion of both promoters completely abolished EzrA in B. subtilis. More importantly, the cell length and percentage of filamentous cells of B. subtilis were significantly increased by disruption of the promoter(s). Thus, EzrA is required for efficient cell division during the growth of B. subtilis, despite serving as a negative regulator for Z-ring formation.