Discovery of a pyrazolo[1,5-a]pyrimidine derivative (MT-3014) as a highly selective PDE10A inhibitor via core structure transformation from the stilbene moiety

We have developed a new class of PDE10A inhibitor, a pyrazolo[1,5-a]pyrimidine derivative MT-3014 (1). A previous compound introduced was deprioritized due to concerns for E/Z-isomerization and glutathione-adduct formation at the core stilbene structure. We discovered pyrazolo [1,5-a] pyrimidine as a new lead scaffold by structure-based drug design utilizing a co-crystal structure with PDE10A. The lead compound was optimized for in vitro activity, solubility, and selectivity against human ether-á-go-go related gene cardiac channel binding. We observed that MT-3014 shows excellent efficacy in rat conditioned avoidance response test and suitable pharmacokinetic properties in rats, especially high brain penetration.     

Method for isolation of intact titin (connectin) molecules from mammalian cardiac muscle

Cardiac titin was isolated from rabbit and ground squirrel ventricular muscles by a method that was used earlier to obtain myofibrils with intact minor proteins located in A-bands of sarcomeres (Podlubnaya, Z. A., et al. (1989) J. Mol. Biol., 210, 655–658). Small pieces of cardiac muscle were incubated for 2–3 weeks at 4°C in Ca²⁺-depleting solution before their homogenization to decrease activity of Ca²⁺-dependent proteases. Then the muscle was homogenized, and titin was isolated by the method of Soteriou, A., et al. (1993) J. Cell Sci., 14, 119–123. In control experiments, titin was isolated from cardiac muscle without its preincubation in Ca²⁺-depleting solution. Sometimes control titin preparations contained only T2-fragment, but generally they contained ～5–20% N2B-isoform of titin along with its T2-fragment. Preparations of titin obtained from rabbit cardiac muscle by our method contained ～30–50% of N2BA- and N2B-titin isoforms along with its T2-fragment. The content of α-structures in titin isolated by our method was increased. Actomyosin ATPase activity in vitro increased in the presence of titin preparations containing more intact molecules. This result confirms the significant role of titin in the regulation of actin-myosin interaction in muscles. The method used by us to preserve titin might be used for isolation of other proteins that are substrates of Ca²⁺-dependent proteases.     

Intracellular calcium mishandling leads to cardiac dysfunction and ventricular arrhythmias in a mouse model of propionic acidemia

Propionic acidemia (PA) is a rare metabolic disease associated with mutations in genes encoding the α and β subunits of the enzyme propionyl-CoA carboxylase. The accumulation of toxic metabolites results in mitochondrial dysfunction, increased reactive oxygen species production and oxidative damage, which have been associated with the disease pathophysiology. Clinical symptoms are heterogeneous and include cardiac complications, mainly cardiac dysfunction and arrhythmias, which are recognized as one of the major life-threatening manifestations in patients. We aimed to investigate the molecular mechanisms underlying the cardiac phenotype using a hypomorphic mouse model (Pcca^−/−(A138T)) that recapitulates some biochemical and clinical characteristics of PA. We demonstrate that Pcca^−/−(A138T) mice present with depressed cardiac function along with impaired cell contractility when compared to the wild-type mice. Cardiac dysfunction in Pcca^−/−(A138T) mice was associated with lower systolic Ca²⁺ release ([Ca²⁺]_i transients), impairment in the sarcoplasmic reticulum (SR) Ca²⁺ load and decreased Ca²⁺ re-uptake by SR-Ca²⁺ ATPase (SERCA2a). These functional changes correlated well with the depressed activity of SERCA2a, the elevated ROS levels and SERCA2a oxidation rate in cardiomyocytes isolated from Pcca^−/−(A138T) mice. In addition, decreased SR-Ca²⁺ load in Pcca^−/−(A138T) cardiomyocytes was associated with increased diastolic Ca²⁺ release. The increase in Ca²⁺ sparks, Ca²⁺ waves and spontaneous [Ca²⁺]_i transients in Pcca^−/−(A138T) cardiomyocytes could be responsible for the induction of ventricular arrhythmias detected in these mice. Overall, our results uncover the role of impaired Ca²⁺ handling in arrhythmias and cardiac dysfunction in PA, and identify new targets for the development of therapeutic approaches for this devastating metabolic disease.     

In vitro RNA iodination with the aid of chloramine T

A new method for in vitro RNA radioiodination with the aid of chloramine T has been developed. The ¹²⁵I-labeled RNA preparations obtained by this method were uniformly labeled, nondegraded, and of high specific activity. The method is simple and the results are reproduced easily.     

IL-22 ameliorated cardiomyocyte apoptosis in cardiac ischemia/reperfusion injury by blocking mitochondrial membrane potential decrease,inhibiting ROS and cytochrome C

Irreversible cardiomyocyte death is one of the main reasons of heart failure following cardiac injury. Therefore, controlling cardiomyocyte death is an effective method to delay the progression of cardiac disease after injury. IL-22 plays critical roles in tissue homeostasis and repair, and has become an important bridge between the immune system and specific tissues or organs. However, whether IL-22 can prevent of cardiomyocyte apoptosis from cardiac injury remains unclear. Therefore, the present work would address the above question. Our results showed that, in vitro, IL-22 prevented cardiomyocyte apoptosis induced by Angiotensin II via enhancing the activity of SOD, blocking the decrease of mitochondrial membrane potential, inhibiting ROS production and release of cytochrome C. The similar results were also found in vivo and patients. Our results shed a light on the therapy of cardiac injury.     

藏羚羊与藏绵羊心肌、骨骼肌中肌红蛋白含量和乳酸脱氢酶活性的比较

为了探讨藏羚羊(Pantholops hodgsonii)对低氧环境的适应机制。以生活在同海拔(4 300 m)的藏绵羊(Tibetan Sheep)为对照,用分光光度法测定2种动物心肌、骨骼肌中肌红蛋白(myoglobin,Mb)含量、乳酸(lactic acid,LD)含量及乳酸脱氢酶(lactate dehydrogenase,LDH)活力。结果显示,藏羚羊心肌和骨骼肌中Mb含量明显高于藏绵羊(P0.05),但心肌和骨骼肌的Mb含量无差别(P0.05),而藏绵羊心肌Mb含量明显高于骨骼肌(P0.05);藏羚羊心肌和骨骼肌中LD含量及LDH活力明显低于藏绵羊(P0.05),且2种动物心肌中的LDH活力均明显低于其骨骼肌(P0.01)。结果表明,藏羚羊尽管生活在高寒缺氧地区,其心肌和骨骼肌细胞仍然能得到丰富的氧供应,并非处于缺氧状态,这可能是通过增加心肌和骨骼肌中Mb的含量,提高其在低氧环境获取和储存氧的能力,从而提高有氧获能水平。与之相反,藏绵羊尽管也生活在高寒缺氧地区,但其心肌和骨骼肌中Mb含量相对于藏羚羊较低,且LD含量和LDH活力较高,说明其心肌和骨骼肌细胞内氧供不如藏羚羊丰富,提示藏绵羊可能主要以糖酵解获能。我们推测这种差异可能与两种动物不同的运动习性密切相关,且认为藏羚羊较高的Mb含量可能是其适应高原缺氧条件的分子基础之一。     

Cardiac activity of freshwater crayfish at wakefulness, rest, and “animal hypnosis”

In laboratory experiments, simultaneous continuous recording of the circadian cycle of behavioral reactions and cardiac activity of freshwater crayfish Astacus astacus and Procambarus clarkii was carried out. A non-invasive fiber-optic method of recording of the heart rate (HR) was used. The obtained data were analyzed by the method of variational pulsometry (VP). It was revealed that certain values of HR and characteristics of VP corresponded to the physiological states of active wakefulness and rest. It was found that during long immobilization of crayfish it is possible to identify the states of operative rest and sleep by the animal behavioral reactions and parameters of cardiac activity. Crayfish were studied in the state of artificially evoked immobilization (“animal hypnosis”). During this state, a high HR level, an increase of indexes of tension and autonomic equilibrium, and changes of other VP characteristics were observed. It is suggested that the different level of cardiac activity in different physiological states of crayfish (active wakefulness, operative rest, sleep, and “animal hypnosis”) is regulated by nervous influences analogous to sympathetic and parasympathetic influences in vertebrates. It was concluded that freshwater crayfish as a representative of the highly organized invertebrates can serve an effective model for studying mechanisms of sleep-like states and “animal hypnosis” in animals.     

Fluorescent-Based Methods for Gene Knockdown and Functional Cardiac Imaging in Zebrafish

A notable advantage of zebrafish as a model organism is the ease of gene knockdown using morpholino antisense oligonucleotide (MO). However, zebrafish morphants injected with MO for a target protein often show heterogeneous phenotypes, despite controlling the injection volume of the MO solution in all embryos. We developed a method for estimating the quantity of MO injected into each living morphant, based on the co-injection of a control MO labeled with the fluorophore lissamine. By applying this method for knockdown of cardiac troponin T (tnnt2a) in zebrafish, we could efficiently select the partial tnnt2a-depleted zebrafish with a decreased heart rate and impairment of cardiac contraction. To investigate cardiac impairment of the tnnt2a morphant, we performed fluorescent cardiac imaging using Bodipy-ceramide. Cardiac image analysis showed moderate reduction of tnnt2a impaired diastolic distensibility and decreased contraction and relaxation velocities. To the best of our knowledge, this is the first report to analyze the role of tnnt2a in cardiac function in tnnt2a-depleted living animals. Our combinatorial approach can be applied for analyzing the molecular function of any protein associated with human cardiac diseases.     

The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods

A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity.     

3,5-Dialkoxypyridine analogues of bedaquiline are potent antituberculosis agents with minimal inhibition of the hERG channel

Bedaquiline is a new drug of the diarylquinoline class that has proven to be clinically effective against drug-resistant tuberculosis, but has a cardiac liability (prolongation of the QT interval) due to its potent inhibition of the cardiac potassium channel protein hERG. Bedaquiline is highly lipophilic and has an extremely long terminal half-life, so has the potential for more-than-desired accumulation in tissues during the relatively long treatment durations required to cure TB. The present work is part of a program that seeks to identify a diarylquinoline that is as potent as bedaquiline against Mycobacterium tuberculosis, with lower lipophilicity, higher clearance, and lower risk for QT prolongation. Previous work led to the identification of compounds with greatly-reduced lipophilicity compounds that retain good anti-tubercular activity in vitro and in mouse models of TB, but has not addressed the hERG blockade. We now present compounds where the C-unit naphthalene is replaced by a 3,5-dialkoxy-4-pyridyl, demonstrate more potent in vitro and in vivo anti-tubercular activity, with greatly attenuated hERG blockade. Two examples of this series are in preclinical development.     

Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development

The heart is the first organ required to function during embryonic development and is absolutely necessary for embryo survival. Cardiac activity is dependent on both the sinoatrial node (SAN), which is the pacemaker of heart''s electrical activity, and the cardiac conduction system which transduces the electrical signal though the heart tissue, leading to heart muscle contractions. Defects in the development of cardiac electrical function may lead to severe heart disorders. The Erbb2 (Epidermal Growth Factor Receptor 2) gene encodes a member of the EGF receptor family of receptor tyrosine kinases. The Erbb2 receptor lacks ligand-binding activity but forms heterodimers with other EGF receptors, stabilising their ligand binding and enhancing kinase-mediated activation of downstream signalling pathways. Erbb2 is absolutely necessary in normal embryonic development and homozygous mouse knock-out Erbb2 embryos die at embryonic day (E)10.5 due to severe cardiac defects. We have isolated a mouse line, l11Jus8, from a random chemical mutagenesis screen, which carries a hypomorphic missense mutation in the Erbb2 gene. Homozygous mutant embryos exhibit embryonic lethality by E12.5-13. The l11Jus8 mutants display cardiac haemorrhage and a failure of atrial function due to defects in atrial electrical signal propagation, leading to an atrial-specific conduction block, which does not affect ventricular conduction. The l11Jus8 mutant phenotype is distinct from those reported for Erbb2 knockout mouse mutants. Thus, the l11Jus8 mouse reveals a novel function of Erbb2 during atrial conduction system development, which when disrupted causes death at mid-gestation.     

Amiodarone Induces Overexpression of Similar to Versican b to Repress the EGFR/Gsk3b/Snail Signaling Axis during Cardiac Valve Formation of Zebrafish Embryos

Although Amiodarone, a class III antiarrhythmic drug, inhibits zebrafish cardiac valve formation, the detailed molecular pathway is still unclear. Here, we proved that Amiodarone acts as an upstream regulator, stimulating similar to versican b (s-vcanb) overexpression at zebrafish embryonic heart and promoting cdh-5 overexpression by inhibiting snail1b at atrioventricular canal (AVC), thus blocking invagination of endocardial cells and, as a result, preventing the formation of cardiac valves. A closer investigation showed that an intricate set of signaling events ultimately caused the up-regulation of cdh5. In particular, we investigated the role of EGFR signaling and the activity of Gsk3b. It was found that knockdown of EGFR signaling resulted in phenotypes similar to those of Amiodarone-treated embryos. Since the reduced phosphorylation of EGFR was rescued by knockdown of s-vcanb, it was concluded that the inhibition of EGFR activity by Amiodarone is s-vcanb-dependent. Moreover, the activity of Gsk3b, a downstream effector of EGFR, was greatly increased in both Amiodarone-treated embryos and EGFR-inhibited embryos. Therefore, it was concluded that reduced EGFR signaling induced by Amiodarone treatment results in the inhibition of Snail functions through increased Gsk3b activity, which, in turn, reduces snail1b expression, leading to the up-regulation the cdh5 at the AVC, finally resulting in defective formation of valves. This signaling cascade implicates the EGFR/Gsk3b/Snail axis as the molecular basis for the inhibition of cardiac valve formation by Amiodarone.     

Response to Mechanical Stress Is Mediated by the TRPA Channel Painless in the Drosophila Heart

Mechanotransduction modulates cellular functions as diverse as migration, proliferation, differentiation, and apoptosis. It is crucial for organ development and homeostasis and leads to pathologies when defective. However, despite considerable efforts made in the past, the molecular basis of mechanotransduction remains poorly understood. Here, we have investigated the genetic basis of mechanotransduction in Drosophila. We show that the fly heart senses and responds to mechanical forces by regulating cardiac activity. In particular, pauses in heart activity are observed under acute mechanical constraints in vivo. We further confirm by a variety of in situ tests that these cardiac arrests constitute the biological force-induced response. In order to identify molecular components of the mechanotransduction pathway, we carried out a genetic screen based on the dependence of cardiac activity upon mechanical constraints and identified Painless, a TRPA channel. We observe a clear absence of in vivo cardiac arrest following inactivation of painless and further demonstrate that painless is autonomously required in the heart to mediate the response to mechanical stress. Furthermore, direct activation of Painless is sufficient to produce pauses in heartbeat, mimicking the pressure-induced response. Painless thus constitutes part of a mechanosensitive pathway that adjusts cardiac muscle activity to mechanical constraints. This constitutes the first in vivo demonstration that a TRPA channel can mediate cardiac mechanotransduction. Furthermore, by establishing a high-throughput system to identify the molecular players involved in mechanotransduction in the cardiovascular system, our study paves the way for understanding the mechanisms underlying a mechanotransduction pathway.     

Linear and non-linear analysis of cardiac health in diabetic subjects

Diabetes is a chronic disease characterized by hyperglycaemia, which leads to specific long-term complications: retinopathy, neuropathy, nephropathy and cardiomyopathy. Analysis of cardiac health using heart rate variation (HRV) has become a popular method to assess the activities of the autonomic nervous system (ANS). It is beneficial in the assessment of cardiac abnormalities, because of its ability to capture fast fluctuations that may be an indication of sympathetic and vagal activity.This paper documents work on the analysis of both normal and diabetic heart rate signals using time domain, frequency domain and nonlinear techniques. The study is based on data from 15 patients with diabetes and 15 healthy volunteers. Our results show that non-linear analysis of HRV is superior compared to time and frequency methods. Non-linear parameters namely,correlation dimension (CD), approximate entropy (ApEn), sample entropy (SampEn) and recurrence plot properties (REC and DET), are clinically significant.