Comparison of lentiviral vector titration methods

BACKGROUND: Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression. RESULTS: We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes. CONCLUSION: The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others

Myocyte remodeling due to fibro-fatty infiltrations influences arrhythmogenicity

The onset of cardiac arrhythmias depends on the electrophysiological and structural properties of cardiac tissue. Electrophysiological remodeling of myocytes due to the presence of adipocytes constitutes a possibly important pathway in the pathogenesis of atrial fibrillation. In this paper we perform an in-silico study of the effect of such myocyte remodeling on the onset of atrial arrhythmias and study the dynamics of arrhythmia sources-spiral waves. We use the Courtemanche model for atrial myocytes and modify their electrophysiological properties based on published cellular electrophysiological measurements in myocytes co-cultered with adipocytes (a 69-87 % increase in APD90 and an increase of the RMP by 2.5-5.5 mV). In a generic 2D setup we show that adipose tissue remodeling substantially affects the spiral wave dynamics resulting in complex arrhythmia and such arrhythmia can be initiated under high frequency pacing if the size of the remodeled tissue is sufficiently large. These results are confirmed in simulations with an anatomically accurate model of the human atria

Arrhythmogenicity of fibro-fatty infiltrations

The onset of cardiac arrhythmias depends on electrophysiological and structural properties of cardiac tissue. One of the most important changes leading to arrhythmias is characterised by the presence of a large number of non-excitable cells in the heart, of which the most well-known example is fibrosis. Recently, adipose tissue was put forward as another similar factor contributing to cardiac arrhythmias. Adipocytes infiltrate into cardiac tissue and produce in-excitable obstacles that interfere with myocardial conduction. However, adipose infiltrates have a different spatial texture than fibrosis. Over the course of time, adipose tissue also remodels into fibrotic tissue. In this paper we investigate the arrhythmogenic mechanisms resulting from the presence of adipose tissue in the heart using computer modelling. We use the TP06 model for human ventricular cells and study how the size and percentage of adipose infiltrates affects basic properties of wave propagation and the onset of arrhythmias under high frequency pacing in a 2D model for cardiac tissue. We show that although presence of adipose infiltrates can result in the onset of cardiac arrhythmias, its impact is less than that of fibrosis. We quantify this process and discuss how the remodelling of adipose infiltrates affects arrhythmia onset

Autonomic dysfunction increases cardiovascular risk in the presence of sleep apnea

The high prevalence of sleep apnea syndrome (SAS) and its direct relationship with an augmented risk of cardiovascular disease (CVD) have raised SAS as a primary public health problem. For this reason, extensive research aiming to understand the interaction between both conditions has been conducted. The advances in non-invasive autonomic nervous system (ANS) monitoring through heart rate variability (HRV) analysis have revealed an increased sympathetic dominance in subjects suffering from SAS when compared with controls. Similarly, HRV analysis of subjects with CVD suggests altered autonomic activity. In this work, we investigated the altered autonomic control in subjects suffering from SAS and CVD simultaneously when compared with SAS patients, as well as the possibility that ANS assessment may be useful for the early stage identification of cardiovascular risk in subjects with SAS. The analysis was performed over 199 subjects from two independent datasets during night-time, and the effects of the physiological response following an apneic episode, sleep stages, and respiration on HRV were taken into account. Results, as measured by HRV, suggest a decreased sympathetic dominance in those subjects suffering from both conditions, as well as in subjects with SAS that will develop CVDs, which was reflected in a significantly reduced sympathovagal balance (p < 0.05). In this way, ANS monitoring could contribute to improve screening and diagnosis, and eventually aid in the phenotyping of patients, as an altered response might have direct implications on cardiovascular health

Semiautomatic Training Load Determination in Endurance Athletes

Background: Despite endurance athletes recording their training data electronically, researchers in sports cardiology rely on questionnaires to quantify training load. This is due to the complexity of quantifying large numbers of training files. We aimed to develop a semiautomatic postprocessing tool to quantify training load in clinical studies. Methods: Training data were collected from two prospective athlete’s heart studies (Master Athlete’s Heart study and Prospective Athlete Heart study). Using in-house developed software, maximal heart rate (MaxHR) and training load were calculated from heart rate monitored during cumulative training sessions. The MaxHR in the lab was compared with the MaxHR in the field. Lucia training impulse score, based on individually based exercise intensity zones, and Edwards training impulse, based on MaxHR in the field, were compared. A questionnaire was used to determine the number of training sessions and training hours per week. Results: Forty-three athletes recorded their training sessions using a chest-worn heart rate monitor and were selected for this analysis. MaxHR in the lab was significantly lower compared with MaxHR in the field (183 ± 12 bpm vs. 188 ± 13 bpm, p < .01), but correlated strongly (r = .81, p < .01) with acceptable limits of agreement (±15.4 bpm). An excellent correlation was found between Lucia training impulse score and Edwards training impulse (r = .92, p < .0001). The quantified number of training sessions and training hours did not correlate with the number of training sessions (r = .20) and training hours (r = −.12) reported by questionnaires. Conclusion: Semiautomatic measurement of training load is feasible in a wide age group. Standard exercise questionnaires are insufficiently accurate in comparison to objective training load quantification

Ryanodine receptor cluster fragmentation and redistribution in persistent atrial fibrillation enhance calcium release

In atrial fibrillation (AF), abnormalities in Ca(2+) release contribute to arrhythmia generation and contractile dysfunction. We explore whether RyR cluster ultrastructure is altered and is associated with functional abnormalities in AF.status: publishe

Altered adrenergic response in myocytes bordering a chronic myocardial infarction underlies <i>in vivo</i> triggered activity and repolarization instability

Ventricular arrhythmias are a major complication early after myocardial infarction (MI). The heterogeneous peri‐infarct zone forms a substrate for re‐entry while arrhythmia initiation is often associated with sympathetic activation. We studied the mechanisms triggering these post‐MI arrhythmias in vivo and their relation to regional myocyte remodelling. In pigs with chronic MI (6 weeks), in vivo monophasic action potentials were simultaneously recorded in the peri‐infarct and remote regions during adrenergic stimulation with isoproterenol (ISO). Sham animals served as controls. During infusion of ISO in vivo, the incidence of delayed afterdepolarizations (DADs) and beat‐to‐beat variability of repolarization (BVR) was higher in the peri‐infarct than in the remote region. Myocytes isolated from the peri‐infarct region, in comparison to myocytes from the remote region, had more DADs, associated with spontaneous Ca2+ release, and a higher incidence of spontaneous action potentials when exposed to ISO (9.99 ± 4.2 vs. 0.16 ± 0.05 APs/min, p = 0.004); these were suppressed by CaMKII inhibition. Peri‐infarct myocytes also had reduced repolarization reserve and increased BVR (26 ± 10 ms vs. 9 ± 7 ms, p 2+ handling at baseline and myocyte hypertrophy were present throughout the LV. Expression of some of the related genes was however different between the regions. In conclusion, altered myocyte adrenergic responses in the peri‐infarct, but not in the remote region, provide a source of triggered activity in vivo and of repolarization instability amplifying the substrate for re‐entry. These findings stimulate further exploration of region‐specific therapies targeting myocytes and autonomic modulation

QRS complex and T wave planarity for the efficacy prediction of automatic implantable defibrillators.

OBJECTIVE To test the hypothesis that in recipients of primary prophylactic implantable cardioverter-defibrillators (ICDs), the non-planarity of ECG vector loops predicts (a) deaths despite ICD protection and (b) appropriate ICD shocks. METHODS Digital pre-implant ECGs were collected in 1948 ICD recipients: 21.4% females, median age 65 years, 61.5% ischaemic heart disease (IHD). QRS and T wave three-dimensional loops were constructed using singular value decomposition that allowed to measure the vector loop planarity. The non-planarity, that is, the twist of the three-dimensional loops out of a single plane, was related to all-cause mortality (n=294; 15.3% females; 68.7% IHD) and appropriate ICD shocks (n=162; 10.5% females; 87.7% IHD) during 5-year follow-up after device implantation. Using multivariable Cox regression, the predictive power of QRS and T wave non-planarity was compared with that of age, heart rate, left ventricular ejection fraction, QRS duration, spatial QRS-T angle, QTc interval and T-peak to T-end interval. RESULTS QRS non-planarity was significantly (p<0.001) associated with follow-up deaths despite ICD protection with HR of 1.339 (95% CI 1.165 to 1.540) but was only univariably associated with appropriate ICD shocks. Non-planarity of the T wave loop was the only ECG-derived index significantly (p<0.001) associated with appropriate ICD shocks with multivariable Cox regression HR of 1.364 (1.180 to 1.576) but was not associated with follow-up mortality. CONCLUSIONS The analysed data suggest that QRS and T wave non-planarity might offer distinction between patients who are at greater risk of death despite ICD protection and those who are likely to use the defibrillator protection