Evaluation of laser induced sarcomere micro-damage: Role of damage extent and location in cardiomyocytes.

Whereas it is evident that a well aligned and regular sarcomeric structure in cardiomyocytes is vital for heart function, considerably less is known about the contribution of individual elements to the mechanics of the entire cell. For instance, it is unclear whether altered Z-disc elements are the reason or the outcome of related cardiomyopathies. Therefore, it is crucial to gain more insight into this cellular organization. This study utilizes femtosecond laser-based nanosurgery to better understand sarcomeres and their repair upon damage. We investigated the influence of the extent and the location of the Z-disc damage. A single, three, five or ten Z-disc ablations were performed in neonatal rat cardiomyocytes. We employed image-based analysis using a self-written software together with different already published algorithms. We observed that cardiomyocyte survival associated with the damage extent, but not with the cell area or the total number of Z-discs per cell. The cell survival is independent of the damage position and can be compensated. However, the sarcomere alignment/orientation is changing over time after ablation. The contraction time is also independent of the extent of damage for the tested parameters. Additionally, we observed shortening rates between 6-7% of the initial sarcomere length in laser treated cardiomyocytes. This rate is an important indicator for force generation in myocytes. In conclusion, femtosecond laser-based nanosurgery together with image-based sarcomere tracking is a powerful tool to better understand the Z-disc complex and its force propagation function and role in cellular mechanisms.

New Cardiac Abnormalities After Radiotherapy in Breast Cancer Patients Treated With Trastuzumab.

PURPOSE: To evaluate cardiac imaging abnormalities after modern radiotherapy and trastuzumab in breast cancer patients. PATIENTS AND METHODS: All patients treated with trastuzumab and radiotherapy for breast cancer between 2006 and 2014 with available cardiac imaging (echocardiogram or multigated acquisition scan) were retrospectively analyzed. Cardiac abnormalities included myocardial abnormalities (atrial or ventricular dilation, hypertrophy, hypokinesis, and impaired relaxation), decreased ejection fraction > 10%, and valvular abnormalities (thickening or stenosis of the valve leaflets). Breast laterality (left vs. right) and heart radiation dose volume parameters were analyzed for association with cardiac imaging abnormalities. RESULTS: A total of 110 patients with 57 left- and 53 right-sided breast cancers were evaluated. Overall, 37 patients (33.6%) developed a new cardiac abnormality. Left-sided radiotherapy was associated with an increase in new cardiac abnormalities (relative risk [RR] = 2.51; 95% confidence interval [CI], 1.34-4.67; P = .002). Both myocardial and valvular abnormalities were associated with left-sided radiotherapy (myocardial: RR = 2.21; 95% CI, 1.06-4.60; P = .029; valvular: RR = 3.30; 95% CI, 0.98-10.9; P = .044). There was no significant difference in decreased ejection fraction between left- and right-sided radiotherapy (9.6% vs. 2.1%; P = .207). A mean heart dose > 2 Gy as well as volume of the heart receiving 20 Gy (V20), V30, and V40 correlated with cardiac abnormalities (mean heart dose > 2 Gy: RR = 2.00; P = .040). CONCLUSION: New cardiac abnormalities, including myocardial and valvular dysfunction, are common after trastuzumab and radiotherapy. The incidence of new abnormalities correlates with tumor laterality and cardiac radiation dose exposure. Long-term follow-up is needed to understand the clinical significance of these early imaging abnormalities.

The impact of right ventricular function on prognosis in patients with stage III non-small cell lung cancer after concurrent chemoradiotherapy.

Right ventricular (RV) impairment after cancer therapy-related cardiotoxicity and its prognostic implications in lung cancer have not been examined. The present research sought to evaluate RV structure, function, and mechanics in stage III non-small-cell lung cancer (NSCLC) before and after concurrent chemoradiotherapy (CCRT), and to explore the associations between RV impairments, radiation dose, and all-cause mortality. This prospective investigation included 128 inoperable NSCLC patients who were scheduled to receive CCRT. Echocardiographic examination and strain evaluation was performed at baseline and 6 months post-CCRT in all participants. Conventional RV dimensions revealed no significant changes post-CCRT. However, a reduction in RV free wall strain (RV-fwLS) was observed at 6 months post-CCRT (- 28.3 ± 4.6% vs. - 25.5 ± 4.8%, P < 0.001). The same was revealed for global RV longitudinal strain (RV-GLS) (- 23.4 ± 2.9% vs. - 20.2 ± 3.4%, P < 0.001). Pearson correlation showed that RV radiation mean dose was related with the percentage change in RV-fwLS (r = 0.303, P = 0.001) and RV-GLS (r = 0.284, P = 0.002). In multivariable analysis, the percentage change in RV-fwLS was an independent predictor of all-cause mortality (HR 1.296, 95% CI 1.202-1.428, P < 0.001). RV longitudinal strain is deteriorated at 6 months post-CCRT. The RV mechanics deterioration was associated with radiation dose and affected the long-term outcome of stage III NSCLC patients treated with CCRT.

Effect of densely ionizing radiation on cardiomyocyte differentiation from human-induced pluripotent stem cells.

The process of human cardiac development can be faithfully recapitulated in a culture dish with human pluripotent stem cells, where the impact of environmental stressors can be evaluated. The consequences of ionizing radiation exposure on human cardiac differentiation are largely unknown. In this study, human-induced pluripotent stem cell cultures (hiPSCs) were subjected to an external beam of 3.7 MeV α-particles at low mean absorbed doses of 0.5, 3, and 10 cGy. Subsequently, the hiPSCs were differentiated into beating cardiac myocytes (hiPSC-CMs). Pluripotent and cardiac markers and morphology did not reveal differences between the irradiated and nonirradiated groups. While cell number was not affected during CM differentiation, cell number of differentiated CMs was severely reduced by ionizing radiation in a dose-responsive manner. ß-adrenergic stimulation causes calcium (Ca2+) overload and oxidative stress. Although no significant increase in Ca2+ transient amplitude was observed in any group after treatment with 1 µmol/L isoproterenol, the incidence of spontaneous Ca2+ waves/releases was more frequent in hiPSC-CMs of the irradiated groups, indicating arrhythmogenic activities at the single cell level. Increased transcript expression of mitochondrial biomarkers (LONP1, TFAM) and mtDNA-encoded genes (MT-CYB, MT-RNR1) was detected upon differentiation of hiPSC-CMs suggesting increased organelle biogenesis. Exposure of hiPSC-CM cultures to 10 cGy significantly upregulated MT-CYB and MT-RNR1 expression, which may reflect an adaptive response to ionizing radiation. Our results indicate that important aspects of differentiation of hiPSCs into cardiac myocytes may be affected by low fluences of densely ionizing radiations such as α-particles.

Radiotherapy-induced global and regional differences in early-stage left-sided versus right-sided breast cancer patients: speckle tracking echocardiography study.

Radiotherapy (RT) to the thoracic region increases late cardiovascular morbidity and mortality. The impact of breast cancer laterality on cardiac function is largely unknown. The aim of this prospective study was to compare RT-induced changes in left-sided and right-sided breast cancer patients using speckle tracking echocardiography (STE). Sixty eligible patients with left-sided breast cancer and 20 with right-sided breast cancer without chemotherapy were evaluated prospectively before and early after RT. A comprehensive echocardiographic examination included three dimensional measurements and STE of the left ventricle (LV). The global longitudinal strain (GLS) was reduced from -18.3 ± 3.1 to -17.2 ± 3.3% (p = 0.003) after RT in patients with left-sided breast cancer. Similarly, regional analysis showed a reduction in the apical strain from -18.7 ± 5.3 to -16.7 ± 4.9% (p = 0.002) and an increase in basal values from -21.6 ± 5.0 to -23.3 ± 4.9% (p = 0.024). Patients with right-sided breast cancer showed deterioration in basal anterior strain segments from -26.3 ± 7.6 to -18.8 ± 8.9% (p < 0.001) and in pulsed tissue Doppler by 0.825 [0.365, 1.710] cm/s (p < 0.001). In multivariable analysis, the use of aromatase inhibitor (ß = -2.002, p = 0.001) and decreased LV diastolic volume (ß = -0.070, p = 0.025) were independently associated with the decrease in GLS. RT caused no changes in conventional LV systolic measurements. RT induced regional changes corresponded to the RT fields. Patients with left-sided breast cancer experienced apical impact and global decline, whereas patients with right-sided breast cancer showed basal changes. The regional differences in cardiac impact warrant different methods in screening and in the follow-up of patients with left-sided versus right-sided breast cancer.

ß-Adrenergic response is counteracted by extremely-low-frequency pulsed electromagnetic fields in beating cardiomyocytes.

Proper ß-adrenergic signaling is indispensable for modulating heart frequency. Studies on extremely-low-frequency pulsed electromagnetic field (ELF-PEMF) effects in the heart beat function are contradictory and no definitive conclusions were obtained so far. To investigate the interplay between ELF-PEMF exposure and ß-adrenergic signaling, cultures of primary murine neonatal cardiomyocytes and of sinoatrial node were exposed to ELF-PEMF and short and long-term effects were evaluated. The ELF-PEMF generated a variable magnetic induction field of 0-6mT at a frequency of 75Hz. Exposure to 3mT ELF-PEMF induced a decrease of contraction rate, Ca(2+) transients, contraction force, and energy consumption both under basal conditions and after ß-adrenergic stimulation in neonatal cardiomyocytes. ELF-PEMF exposure inhibited ß-adrenergic response in sinoatrial node (SAN) region. ELF-PEMF specifically modulated ß2 adrenergic receptor response and the exposure did not modify the increase of contraction rate after adenylate cyclase stimulation by forskolin. In HEK293T cells transfected with ß1 or ß2 adrenergic receptors, ELF-PEMF exposure induced a rapid and selective internalization of ß2 adrenergic receptor. The ß-adrenergic signaling, was reduced trough Gi protein by ELF-PEMF exposure since the phosphorylation level of phospholamban and the PI3K pathway were impaired after isoproterenol stimulation in neonatal cardiomyocytes. Long term effects of ELF-PEMF exposure were assessed in cultures of isolated cardiomyocytes. ELF-PEMF counteracts cell size increase, the generation of binucleated of cardiomyocytes and prevents the up-regulation of hypertrophic markers after ß-adrenergic stimulation, indicating an inhibition of cell growth and maturation. These data show that short and long term exposure to ELF-PEMF induces a reduction of cardiac ß-adrenergic response at molecular, functional and adaptative levels.

Infrared inhibition of embryonic hearts.

Infrared control is a new technique that uses pulsed infrared lasers to thermally alter electrical activity. Originally developed for nerves, we have applied this technology to embryonic hearts using a quail model, previously demonstrating infrared stimulation and, here, infrared inhibition. Infrared inhibition enables repeatable and reversible block, stopping cardiac contractions for several seconds. Normal beating resumes after the laser is turned off. The block can be spatially specific, affecting propagation on the ventricle or initiation on the atrium. Optical mapping showed that the block affects action potentials and not just calcium or contraction. Increased resting intracellular calcium was observed after a 30-s exposure to the inhibition laser, which likely resulted in reduced mechanical function. Further optimization of the laser illumination should reduce potential damage. Stopping cardiac contractions by disrupting electrical activity with infrared inhibition has the potential to be a powerful tool for studying the developing heart.

ECG de noviembre de 2015 / ECG, November 2015

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2.1 GHz electromagnetic field does not change contractility and intracellular Ca2+ transients but decreases ß-adrenergic responsiveness through nitric oxide signaling in rat ventricular myocytes.

PURPOSE: Due to the increasing use of wireless technology in developing countries, particularly mobile phones, the influence of electromagnetic fields (EMF) on biologic systems has become the subject of an intense debate. Therefore, in this study we investigated the effect of 2.1 GHz EMF on contractility and beta-adrenergic (ß-AR) responsiveness of ventricular myocytes. MATERIALS AND METHODS: Rats were randomized to the following groups: Sham rats (SHAM) and rats exposed to 2.1 GHz EMF for 2 h/day for 10 weeks (EM-10). Sarcomere shortening and Ca(2+) transients were recorded in isolated myocytes loaded with Fura2-AM and electrically stimulated at 1 Hz, while L-type Ca(2+) currents (I(CaL)) were measured using whole-cell patch clamping at 36 ± 1°C. Cardiac nitric oxide (NO) levels were measured in tissue samples using a colorimetric assay kit. RESULTS: Fractional shortening and amplitude of the matched Ca(2+) transients were not changed in EM-10 rats. Although the isoproterenol-induced (10(-6) M) I(CaL) response was reduced in rats exposed to EMF, basal I(CaL) density in myocytes was similar between the two groups (p < 0.01). Moreover, EMF exposure led to a significant increase in nitric oxide levels in rat heart (p < 0.02). CONCLUSIONS: Long-term exposure to 2.1 GHz EMF decreases ß-AR responsiveness of ventricular myocytes through NO signaling.

Nueva causa de arrastre presistólico: extrasistolia-PR largo / New cause of presystolic drag: long PR extrasystoles

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Low Frequency Electromagnetic Field Conditioning Protects against I/R Injury and Contractile Dysfunction in the Isolated Rat Heart.

Low frequency electromagnetic field (LF-EMF) decreases the formation of reactive oxygen species, which are key mediators of ischemia/reperfusion (I/R) injury. Therefore, we hypothesized that the LF-EMF protects contractility of hearts subjected to I/R injury. Isolated rat hearts were subjected to 20 min of global no-flow ischemia, followed by 30 min reperfusion, in the presence or absence of LF-EMF. Coronary flow, heart rate, left ventricular developed pressure (LVDP), and rate pressure product (RPP) were determined for evaluation of heart mechanical function. The activity of cardiac matrix metalloproteinase-2 (MMP-2) and the contents of coronary effluent troponin I (TnI) and interleukin-6 (IL-6) were measured as markers of heart injury. LF-EMF prevented decreased RPP in I/R hearts, while having no effect on coronary flow. In addition, hearts subjected to I/R exhibited significantly increased LVDP when subjected to LF-EMF. Although TnI and IL-6 levels were increased in I/R hearts, their levels returned to baseline aerobic levels in I/R hearts subjected to LF-EMF. The reduced activity of MMP-2 in I/R hearts was reversed in hearts subjected to LF-EMF. The data presented here indicate that acute exposure to LF-EMF protects mechanical function of I/R hearts and reduces I/R injury.

Ultrasound-induced modulation of cardiac rhythm in neonatal rat ventricular cardiomyocytes.

Isolated neonatal rat ventricular cardiomyocytes were used to study the influence of ultrasound on the chronotropic response in a tissue culture model. The beat frequency of the cells, varying from 40 to 90 beats/min, was measured based upon the translocation of the nuclear membrane captured by a high-speed camera. Ultrasound pulses (frequency = 2.5 MHz) were delivered at 300-ms intervals [3.33 Hz pulse repetition frequency (PRF)], in turn corresponding to 200 pulses/min. The intensity of acoustic energy and pulse duration were made variable, 0.02-0.87 W/cm(2) and 1-5 ms, respectively. In 57 of 99 trials, there was a noted average increase in beat frequency of 25% with 8-s exposures to ultrasonic pulses. Applied ultrasound energy with a spatial peak time average acoustic intensity (Ispta) of 0.02 W/cm(2) and pulse duration of 1 ms effectively increased the contraction rate of cardiomyocytes (P < 0.05). Of the acoustic power tested, the lowest level of acoustic intensity and shortest pulse duration proved most effective at increasing the electrophysiological responsiveness and beat frequency of cardiomyocytes. Determining the optimal conditions for delivery of ultrasound will be essential to developing new models for understanding mechanoelectrical coupling (MEC) and understanding novel nonelectrical pacing modalities for clinical applications.

Long-term cardiac abnormalities after cranial radiotherapy in childhood cancer survivors.

BACKGROUND: Cardiac morbidity is an important late effect in long-term childhood cancer survivors (CCS) treated with cardiotoxic agents or radiotherapy (RT) on the chest. However, there is limited data on the long-term cardiac sequelae in CCS who only received cranial RT. We hypothesized that cranial RT might negatively influence cardiac structure and function. METHODS AND RESULTS: We studied 13 CCS [mean age 30.8 (18.1-39.3) years, 7 males] who received RT only on the head for a cranial tumor and 36 age- and sex-matched healthy sibling controls. Echocardiographic follow-up was performed at median 21.7 (12.6-30.8) years after diagnosis. CCS had lower indexed diastolic LV volumes [56.0 (31.4-68.3) vs. 60.5 (41.9-94.3) mL/m(2), p = 0.024]. CCS also had reduced LV systolic and diastolic function, reflected by lower systolic LV myocardial velocities (5.3 ± 0.9 vs. 7.1 ± 1.7 cm/s, p = 0.001) and longitudinal deformation (- 17.3 ± 3.1 vs. - 20.7 ± 2.0%, p < 0.001), as well as lower diastolic LV myocardial velocities (- 10.7 ± 1.7 vs. - 12.2 ± 1.5 cm/s, p = 0.006) and deformation speed (1.1 ± 0.3 vs. 1.5 ± 0.2 1/s, p = 0.005). Additionally, in CCS insulin-like growth factor levels [15.4 (9.2-34.6) vs. 24.4 (14.8-55.5) nmol/L, p = 0.007] were lower. CONCLUSION: Cranial RT in CCS is associated with smaller cardiac volumes and reduced systolic and diastolic LV function. This off target effect of RT might be related to lower insulin-like growth factor levels.

Extracorporeal acute cardiac pacing by high intensity focused ultrasound.

Ultrasound has been shown to produce Premature Ventricular Contractions (PVC's). Two clinical applications in which acute cardiac pacing by ultrasound may be valuable are: (1) preoperative patient screening in cardiac resynchronization therapy surgery; (2) Emergency life support, following an event of sudden death, caused by cardiac arrest. Yet, previously the demonstrated mean success rate of extra-systole induction by High Intensity Focused Ultrasound (HIFU) in rats is below 4.5% (Miller et al., 2011). This stands in contrast to previous work in rats using ultrasound (US) and ultrasound contrast agents (UCAs), where success rates of close to 100% were reported (Rota et al., 2006). Herein, bi-stage temporal sequences of accentuated negative pressure (rarefaction) and positive pressure HIFU transmission (insonation) patterns were applied to anaesthetized rats under real-time vital-signs monitoring and US imaging. This pattern of insonation first produces a gradual growth of dissolved gas cavities in tissue (cavitation) and then an ultrasonic impact. Results demonstrate sequences of successive successful HIFU pacing. Triggering insonation at different delays from the preceding ECG R-wave demonstrated successful HIFU pacing induction from mid ECG T-wave till the next ECG complex's PR interval. Spatially focusing the beam at different locations allows cumulative coverage of the whole left ventricle. Analysis of the acoustic wave patterns and temporal characteristics of paced PVCs is suggested to provide new insight into the mechanisms of HIFU cardiac pacing. Specifically, the observed HIFU pacing temporal success rate distribution suggests against sarcomere length modulation current being the dominant cellular level mechanism of HIFU cardiac pacing and may allow postulating that membrane deformation currents are dominant at the applied insonation conditions.

Myocardial effects of local shock wave therapy in a Langendorff model.

OBJECTIVE: Applying shock waves to the heart has been reported to stimulate the heart and alter cardiac function. We hypothesized that shock waves could be used to diagnose regional viability. METHOD: We used a Langendorff model to investigate the acute effects of shock waves at different energy levels and times related to systole, cycle duration and myocardial function. RESULTS: We found only a small time window to use shock waves. Myocardial fibrillation or extrasystolic beats will occur if the shock wave is placed more than 15 ms before or 30 ms after the onset of systole. Increased contractility and augmented relaxation were observed after the second beat, and these effects decreased after prolonging the shock wave delay from 15 ms before to 30 ms after the onset of systole. An energy dependency could be found only after short delays (-15 ms). The involved processes might include post-extrasystolic potentiation and simultaneous pacing. CONCLUSION: In summary, we found that low-energy shock waves can be a useful tool to stimulate the myocardium at a distance and influence function.

Acute cardiac impairment associated with concurrent chemoradiotherapy for esophageal cancer: magnetic resonance evaluation.

PURPOSE: To evaluate acute cardiac effects of concurrent chemoradiotherapy (CCRT) for esophageal cancer. METHODS AND MATERIALS: This prospective study was approved by the institutional review board, and written informed consent was obtained from all participants. The left ventricular function (LVF) of 31 patients with esophageal cancer who received cisplatin and 5-fluorouracil-based CCRT was evaluated using cardiac cine magnetic resonance imaging. The patients were classified into two groups according to mean LV dose. The parameters related to LVF were compared between before and during (40 Gy) or between before and after CCRT using a Wilcoxon matched-pairs single rank test, and parameter ratios (during/before CCRT, after/before CCRT) were also compared between the groups with a t test. Data were expressed as mean ± SE. RESULTS: In the low LV-dose group (n = 10; mean LV dose <0.6 Gy), LV ejection fraction decreased significantly (before vs. during vs. after CCRT; 62.7% ± 2.98% vs. 59.8% ± 2.56% vs. 60.6% ± 3.89%; p < 0.05). In the high LV-dose group (n = 21; mean LV dose of 3.6-41.2 Gy), LV end-diastolic volume index (before vs. after CCRT; 69.1 ± 2.93 vs. 57.0 ± 3.23 mL/m(2)), LV stroke volume index (38.6 ± 1.56 vs. 29.9 ± 1.60 mL/m(2)), and LV ejection fraction (56.9% ± 1.79% vs. 52.8% ± 1.15%) decreased significantly (p < 0.05) after CCRT. Heart rate increased significantly (before vs. during vs. after CCRT; 66.8 ± 3.05 vs. 72.4 ± 4.04 vs. 85.4 ± 3.75 beats per minute, p < 0.01). Left ventricle wall motion decreased significantly (p < 0.05) in segments 8 (before vs. during vs. after CCRT; 6.64 ± 0.54 vs. 4.78 ± 0.43 vs. 4.79 ± 0.50 mm), 9 (6.88 ± 0.45 vs. 5.04 ± 0.38 vs. 5.27 ± 0.47 mm), and 10 (9.22 ± 0.48 vs. 8.08 ± 0.34 vs. 8.19 ± 0.56 mm). The parameter ratios of LV end-diastolic volume index, stroke volume index, wall motion in segment 9, and heart rate showed significant difference (p < 0.05) after CCRT between the groups. CONCLUSIONS: Concurrent chemoradiotherapy for esophageal cancer impairs LVF from an early treatment stage. This impairment is prominent in patients with high LV dose.

Effects of far infrared rays irradiated from ceramic material (BIOCERAMIC) on psychological stress-conditioned elevated heart rate, blood pressure, and oxidative stress-suppressed cardiac contractility.

The present study examined the effects of BIOCERAMIC on psychological stress-conditioned elevated heart rate, blood pressure and oxidative stress-suppressed cardiac contractility using in vivo and in vitro animal models. We investigated the effects of BIOCERAMIC on the in vivo cardiovascular hemodynamic parameters of rats by monitoring their heart rates, systolic blood pressure, mean blood pressure and diastolic blood pressure. Thereafter, we assayed its effects on the heart rate in an isolated frog heart with and without adrenaline stimulation, and on cardiac contractility under oxidative stress. BIOCERAMIC caused significant decreases in heart rates and systolic and mean blood pressure in the stress-conditioned heart rate rat models (P < 0.05), as well as in the experimental models of an isolated frog heart with and without adrenaline stimulation (P < 0.05), and normalized cardiac contractility under oxidative stress (P < 0.05). BIOCERAMIC may, therefore, normalize the effects of psychological stress and oxidative stress conditions.

Light induced stimulation and delay of cardiac activity.

This article shows the combination of light activatable ion channels and microelectrode array (MEA) technology for bidirectionally interfacing cells. HL-1 cultures, a mouse derived cardiomyocyte-like cell line, transfected with channelrhodopsin were stimulated with a microscope coupled 473 nm laser and recorded with custom built 64 electrode MEAs. Channelrhodopsin induced depolarization of the cell can evoke action potentials (APs) in single cells. Spreading of the AP over the cell layer can then be measured with good spatiotemporal resolution using MEA recordings. The possibility for light induced pacemaker switching in cultures was shown. Furthermore, the suppression of APs can also be achieved with the laser. Possible applications include cell analysis, e.g. pacemaker interference or induced pacemaker switching, and medical applications such as a combined cardiac pacemaker and defibrillator triggered by light. Since current prosthesis research focuses on bidirectionality, this system may be applied to any electrogenic cell, including neurons or muscles, to advance this field.