Impaired modulation of the cardiac L-type Ca2+ channel activity by ahnak-1 after myocardial infarction

Introduction: The L-type cardiac Ca2+ channel (Cav 1.2) is an important determinant of cardiac repolarization and the main source of activator Ca2+ during excitation-contraction coupling in cardiac cells. Its defective regulation is a major cause of arrhythmias and contractile dysfunction. We have recently shown that the cytoskeletal protein ahnak-1 modulates Ca2+ current through Cav 1.2 channels (ICaL) by interacting with the regulatory beta-subunit of the Cav 1.2 channel and that the genetic variant of ahnak-1 I5483T (previously Ile5236Thr), interferes with the beta-adrenergic stimulation of ICaL. Objective: To extend our study of the I5483T variant to ventricular cardiomyocytes dissociated from remodelled infarcted rat hearts (PMI). Methods: The patch-clamp technique was used to record ICaL from enzymatically dissociated ventricular cardiomyocytes from young (2-month-old) and six-month-old sham-operated and PMI rats. Results: Basal ICaL was increased from 11 ± 0.5 A/F in young cardiomyocytes to 14.6 ± 1.1 A/F and 15.7 ± 1 A/F in sham and PMI cardiomyocytes respectively, while isoprenaline (ISO, 1 µmol/L) further increased ICaL by 101 ± 6%, 109 ± 10% and 104 ± 12% respectively. When cells were intracellularly perfused with a peptide containing the mutated ahnak-1 sequence (10 µmol/L) basal ICaL was increased to 20 ± 1 A/F, 22 ± 2 A/F and 21 ± 2 A/F in young, sham and PMI cardiomyocytes respectively. In these cells ISO increased ICaL by 11 ± 4%, 33 ± 6% and 79 ± 12% respectively. Conclusion: Modulation of ICaL by ahnak-1is impaired by myocardial ischemia and remodelling. Since ahnak-1 and Cav 1.2 channels co-localize in the transverse T-tubule system, remodelling of T-tubules could affect the interaction of ahnak-1 with the regulatory beta subunit of these channels

Auto-inhibitory effects of an IQ motif on protein structure and function

The denuded IQ2 domain, i.e. myosin heavy chain not associated with regulatory light chains, exerts an inhibitory effect on myosin ATPase activity. In this study, we elaborated a structural explanation for this auto-inhibitory effect of IQ2 on myosin function. We employed analytical ultracentrifugation, circular dichroism, and surface plasmon resonance spectroscopy to investigate structural and functional properties of a myosin heavy chain (MYH) head-rod fragment aa664-915. MYH(664-915) was monomeric, adopted a closed shape, and bound essential myosin light chains (HIS-MLC-1) with low affinity to IQ1. Deletion of IQ2, however opened MYH(664-915). Four amino acids present in IQ2 could be identified to be responsible for this auto-inhibitory structural effect: alanine mutagenesis of I814, Q815, R819, and W827 stretched MYH(664-915) and increased 30fold the binding affinity of HIS-MLC-1 to IQ1. In this study we show, that denuded IQ2 favours a closed conformation of myosin with a low HIS-MLC-1 binding affinity. The collapsed structure of myosin with denuded IQ2 could explain the auto-inhibitory effects of IQ2 on enzymatic activity of myosin

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart

Сложность алгоритмов криптографической системы Эль–Гамаля и ихэффективность

Objective. - Electrical remodeling as well as atrial contractile dysfunction after the conversion of atrial fibrillation (AF) to sinus rhythm (SR) are mainly caused by a reduction of the inward L-type Ca2+ current (ICaL). We investigated whether the expression of L-type Ca2+-channel subunits was reduced in atrial myocardium of AF patients. Methods. - Right atrial appendages were obtained from patients undergoing coronary artery bypass graft surgery (CAD, n = 35) or mitral valve surgery (MVD, n = 37). Seventeen of the CAD patients and 18 of the MVD patients were in chronic (>3 months) AF, whereas the others were in SR. The protein expression of the L-type Ca2+-channel subunits {alpha}1C and {beta}2 was quantified by western blot analysis. Furthermore, we measured the density of dihydropyridine (DHP)-binding sites of the L-type Ca2+ channel using 3H-PN220-100 as radioligand. Results. - Surprisingly, the {alpha}1C and the {beta}2-subunit expression was not altered in atrial myocardium of AF patients. Also, the DHP-binding site density was unchanged. Conclusion. - The protein expression of the L-type Ca2+-channel subunits {alpha}1C or {beta}2 is not reduced in atrial myocardium of AF patients. Therefore, the reduced ICaL might be due to downregulation of other accessory subunits ({alpha}2{delta}), expression of aberrant subunits, changes in channel trafficking or alterations in channel function

Cardiomyocyte-specific estrogen receptor alpha increases angiogenesis, lymphangiogenesis and reduces fibrosis in the female mouse heart post-myocardial infarction

Experimental studies showed that 17{beta}-estradiol (E2) and activated Estrogen Receptors (ER) protect the heart from ischemic injury. However, the underlying molecular mechanisms are not well understood. To investigate the role of ER{alpha} in cardiomyocytes in the setting of myocardial ischemia, we generated transgenic mice with cardiomyocyte-specific overexpression of ER-{alpha} (ER{alpha}-OE) and subjected them to Myocardial Infarction (MI). At the basal level, female and male ER{alpha}-OE mice showed increased Left Ventricular (LV) mass, LV volume and cardiomyocyte length. Two weeks after MI, LV volume was significantly increased and LV wall thickness decreased in female and male WT-mice and male ER{alpha}-OE, but not in female ER{alpha}-OE mice. ER{alpha}-OE enhanced expression of angiogenesis and lymphangiogenesis markers (Vegf, Lyve-1), and neovascularization in the peri-infarct area in both sexes. However, attenuated level of fibrosis and higher phosphorylation of JNK signaling pathway could be detected only in female ER{alpha}-OE after MI. In conclusion, our study indicates that ER{alpha} protects female mouse cardiomyocytes from the sequelae of ischemia through induction of neovascularization in a paracrine fashion and impaired fibrosis, which together may contribute to the attenuation of cardiac remodelling

Detecting Different States of Ventilation with a Wearable Device through Minute Ventilation

Introduction: Detecting changes in respiration are essential to monitoring a patient’s vital signs. Few devices accomplish this in a non-invasive manner. We are developing a wearable Trachea Sound Sensor that measures respiratory rate (RR), tidal volume (TV), minute ventilation (MV = RR x TV). A prototypical Trachea Sound Sensor (TSS) was created and compared to a reference pneumotachometer. Both were used to record the sounds of breathing with research team members. Methods:The TSS recording device was tested on six research team members and breath sounds were recorded. Simultaneously, the member’s RR and MV was recorded using a calibrated pneumotachometer. The researchers were instructed to adjust their breathing rate and depth while intervals were recorded. Signal processing techniques were used to analyze and produce measurements of RR, TV, and characterize hyperventilatory or hypoventilatory states. Results: Based on the results, we found that it is possible to obtain accurate measures of RR and identify breathing patterns through the TSS. Signal processing and analysis calculated RR, states of hyperventilation and hypoventilation with 98% sensitivity and specificity. Results obtained for measuring TV were less accurate (±100 mL). Discussion: Our results suggest that it is viable to obtain accurate measures of RR and classify breathing sounds solely on measurements of breathing sounds from the TSS. The inaccuracy in TV measurements may be partly due to the systematic error from the pneumotachometer used. The prototypical TSS are suitable for upcoming NIH-funded clinical trials to test the TSS in volunteers and hospitalized patients

Smooth Muscle Myosin Inhibition: A Novel Therapeutic Approach for Pulmonary Hypertension

Pulmonary hypertension remains a major clinical problem despite current therapies. In this study, we examine for the first time a novel pharmacological target, smooth muscle myosin, and determine if the smooth muscle myosin inhibitor, CK-2019165 (CK-165) ameliorates pulmonary hypertension.Six domestic female pigs were surgically instrumented to measure pulmonary blood flow and systemic and pulmonary vascular dynamics. Pulmonary hypertension was induced by hypoxia, or infusion of the thromboxane analog (U-46619, 0.1 µg/kg/min, i.v.). In rats, chronic pulmonary hypertension was induced by monocrotaline.CK-165 (4 mg/kg, i.v.) reduced pulmonary vascular resistance by 22±3 and 28±6% from baseline in hypoxia and thromboxane pig models, respectively (p<0.01 and 0.01), while mean arterial pressure also fell and heart rate rose slightly. When CK-165 was delivered via inhalation in the hypoxia model, pulmonary vascular resistance fell by 17±6% (p<0.05) while mean arterial pressure and heart rate were unchanged. In the monocrotaline model of chronic pulmonary hypertension, inhaled CK-165 resulted in a similar (18.0±3.8%) reduction in right ventricular systolic pressure as compared with sildenafil (20.3±4.5%).Inhibition of smooth muscle myosin may be a novel therapeutic target for treatment of pulmonary hypertension

Molecular mechanism regulating myosin and cardiac functions by ELC

The essential myosin light chain (ELC) is involved in modulation of force generation of myosin motors and cardiac contraction, while its mechanism of action remains elusive. We hypothesized that ELC could modulate myosin stiffness which subsequently determines its force production and cardiac contraction. We therefore generated heterologous transgenic mouse (TgM) strains with cardiomyocyte-specific expression of ELC with human ventricular ELC (hVLC-1; TgM(hVLC-1)) or E56G-mutated hVLC-1 (hVLC-1(E56G); TgM(E56G)). hVLC-1 or hVLC-1(E56G) expression in TgM was around 39% and 41%, respectively of total VLC-1. Laser trap and in vitro motility assays showed that stiffness and actin sliding velocity of myosin with hVLC-1 prepared from TgM(hVLC-1) (1.67pN/nm and 2.3{my}m/s, respectively) were significantly higher than myosin with hVLC-1(E56G) prepared from TgM(E56G) (1.25pN/nm and 1.7{my}m/s, respectively) or myosin with mouse VLC-1 (mVLC-1) prepared from C57/BL6 (1.41 pN/nm and 1.5+-0.03 {my}m/s, respectively). Maximal left ventricular pressure development of isolated perfused hearts in vitro prepared from TgM(hVLC-1) (80.0mmHg) were significantly higher than hearts from TgM(E56G) (66.2mmHg) or C57/BL6 (59.3+-3.9 mmHg). These findings show that ELCs decreased myosin stiffness, in vitro motility, and thereby cardiac functions in the order hVLC-1 > hVLC-1(E56G) ≈ mVLC-1. They also suggest a molecular pathomechanism of cardiomyopathies caused by hVLC-1 mutations

Pharmacogenomic evaluation of single nucleotide polymorphisms associated with oxaliplatin-induced peripheral neuropathy: a preliminary study

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Platinum-Fluoropyrimidine and Paclitaxel-Based Chemotherapy in the Treatment of Advanced Anal Cancer Patients.

Background Although treatment of localized anal cancer (AC) is well established, very little evidence is available to inform the management of advanced tumors, and the prognosis of these patients remains poor. We have analyzed treatment pathways and outcomes of a single-institution series of advanced AC patients in order to provide insight into the management of this rare condition.Materials and methods Inclusion criteria included epidermoid histology, inoperable locally recurrent or metastatic disease, and availability of full medical records. The primary objective was overall survival (OS). Prognostic factors were analyzed in univariate models.Results Sixty-four patients (1997-2014) were included: 16 (25.0%) with inoperable locally advanced and 48 (75.0%) with metastatic tumors. Fifty-one (79.7%) received at least one line of chemotherapy; of these, 37% underwent multimodality treatment. A combination of a platinum agent plus a fluoropyrimidine was the most common first-line regimen (74.5%), with an objective response rate (ORR) of 34.4% (95% confidence interval [CI], 18.6%-53.2%). Paclitaxel-based chemotherapy was used in 15 patients as front-line or salvage treatment, and the overall ORR was 53.3% (95% CI, 26.6%-78.7%). Median progression-free survival (PFS) after first- and second-line chemotherapy was 5.8 (interquartile range [IQR], 2.8-7.6) and 3.2 (IQR, 2.5-7.1) months, respectively. Five-year OS in the overall population was 15% (95% CI, 7.0%-25.0%). Age ≤65 years and liver metastases were predictive of better PFS (hazard ratio [HR], 0.39; 95% CI, 0.16-0.97; p = .04) and worse OS (HR, 2.25; 95% CI, 1.25-4.03; p = .01), respectively.Conclusion A platinum agent plus a fluoropyrimidine and paclitaxel-based chemotherapy are active regimens for advanced AC. Clinical trials are needed to standardize treatment pathways, investigate the potential of novel therapeutics, and improve the poor prognosis of this rare condition. The Oncologist 2017;22:402-408Implications for Practice: Because of the lack of randomized trials, the optimal management of advanced anal cancer is uncertain. Despite its retrospective analysis and relatively small sample size, this is the second largest study ever conducted in this setting, and, as such, it has the potential to serve as a valuable source of information for everyday clinical practice. These findings suggest that chemotherapy with a platinum agent plus a fluoropyrimidine or paclitaxel-containing regimens are reasonable treatment options for patients with inoperable locally recurrent or metastatic anal carcinoma