Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing

Background: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. Methods: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. Results: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm−1s−1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [−0.1; 0.03], p = 0.387). Conclusions: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB

Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy

The multi-C2 domain protein dysferlin localizes to the plasma membrane and the T-tubule system in skeletal muscle; however, its physiological mode of action is unknown. Mutations in the DYSF gene lead to autosomal recessive limb-girdle muscular dystrophy type 2B and Miyoshi myopathy. Here, we show that dysferlin has membrane tubulating capacity and that it shapes the T-tubule system. Dysferlin tubulates liposomes, generates a T-tubule-like membrane system in non-muscle cells, and links the recruitment of phosphatidylinositol 4,5-bisphosphate to the biogenesis of the T-tubule system. Pathogenic mutant forms interfere with all of these functions, indicating that muscular wasting and dystrophy are caused by the dysferlin mutants' inability to form a functional T-tubule membrane system

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes

Background: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. Objective: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. Methods and Results: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. Conclusion: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF

SR Ca 2+ -leak and disordered excitation-contraction coupling as the basis for arrhythmogenic and negative inotropic effects of acute ethanol exposure

Aims: Ethanol has acute negative inotropic and arrhythmogenic effects. The underlying mechanisms, however, are largely unknown. Sarcoplasmic reticulum Ca2+-leak is an important mechanism for reduced contractility and arrhythmias. Ca2+-leak can be induced by oxidative stress and Ca2+/Calmodulin-dependent protein kinase II (CaMKII). Therefore, we investigated the influence of acute ethanol exposure on excitation-contraction coupling in atrial and ventricular cardiomyocytes. Methods and results: Isolated human atrial and murine atrial or ventricular cardiomyocytes were preincubated for 30 min and then superfused with control solution or solution containing ethanol. Ethanol had acute negative inotropic and positive lusitropic effects in human atrial muscle strips and murine ventricular cardiomyocytes. Accordingly, Ca2+-imaging indicated lower Ca2+-transient amplitudes and increased SERCA2a activity, while myofilament Ca2+-sensitivity was reduced. SR Ca2+-leak was assessed by measuring Ca2+-sparks. Ethanol induced severe SR Ca2+-leak in human atrial cardiomyocytes (calculated leak: 4.60 +/- 0.45 mF/F0 vs 1.86 +/- 0.26 in control, n = 80). This effect was dose-dependent, while spontaneous arrhythmogenic 2+-waves increased similar to 5-fold, as investigated in murine cardiomyocytes. Delayed afterdepolarizations, which can result from increased SR Ca2+-leak, were significantly increased by ethanol. Measurements using the reactive oxygen species (ROS) sensor CM-H2DCFDA showed increased ROS-stress in ethanol treated cells. ROS-scavenging with N-acetylcysteine prevented negative inotropic and positive lusitropic effects in human muscle strips. Ethanol-induced Ca2+-leak was abolished in mice with knockout of NOX2 (the main source for ROS in cardiomyocytes). Importantly, mice with oxidation-resistant CaMKII (Met281/282Val mutation) were protected from ethanol-induced Ca2+-leak. Conclusion: We show for the first time that ethanol acutely induces strong SR Ca2+-leak, also altering excitation-contraction coupling. Acute negative inotropic effects of ethanol can be explained by reduced systolic Ca2+-release. Mechanistically, ROS-production via NOX2 and oxidative activation of CaMKII appear to play central roles. This provides a mechanism for the arrhythmogenic and negative inotropic effects of ethanol and suggests a druggable target (CaMKII)

Generation of Highly Purified Human Cardiomyocytes from Peripheral Blood Mononuclear Cell-Derived Induced Pluripotent Stem Cells

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine

Postoperative complications after elective coronary artery bypass grafting surgery in patients with sleep-disordered breathing

BackgroundSleep-disordered breathing (SDB) may increase the risk of postoperative complications in patients after cardiac surgery. This study evaluated the length of hospital stay as well as postoperative cardiac, respiratory, and renal complications after elective coronary artery bypass grafting (CABG) in patients without SDB, with central sleep apnea (CSA), or with obstructive sleep apnea (OSA).MethodsThe presence and type of SDB had been assessed with polygraphic recordings in 100 patients the night before elective CABG surgery. SDB was defined as an apnea-hypopnea index (AHI) of 15/h. Prolonged length of hospital stay (LOS) and postoperative hemodynamic instability due to any cause were retrospectively evaluated as primary endpoints and cardiac, respiratory, and renal complications as secondary endpoints.Results37% of patients had SDB, 14% CSA, and 23% OSA. LOS differed significantly between patients without SDB and those with CSA and OSA [median (25;75. percentile): 8.0days (7.5;11.0) vs. 9.5days (7.0;12.5) vs. 12.0days (9.0;17.0), Kruskal-Wallis test between three groups: p=0.023; OSA vs. no SDB: p=0.005]. AHI was significantly associated with prolonged LOS [>9days; odds ratio (OR) (95% confidence interval): 1.047 (1.001;1.095), p=0.044]. Prolonged need of vasopressors (48h) was observed in 36% of patients without SDB, in 64% with CSA, and in 62% with OSA (p=0.037). AHI was significantly associated with prolonged (48h) need of vasopressors [OR (95% CI): 1.052 (1.002;1.104), p=0.040], independent of any confounders.ConclusionsSDB, particularly OSA, is associated with prolonged LOS after CABG, independent of known confounders. Prolonged LOS in patients with SDB may be due to increased postoperative hemodynamic instability due to any cause

Cardiomyocyte yields after enrichment by MACS or purification with lactate metabolic selection.

<p>Cell counts and percentages shown are from single representative direct differentiations. Detailed information including data from several enrichments or purifications can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126596#pone.0126596.s008" target="_blank">S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126596#pone.0126596.s009" target="_blank">S3</a> Tables. Total cell counts and cTnT positive cell counts (cTnT+ cells) were calculated per T75 flask.</p><p>Cardiomyocyte yields after enrichment by MACS or purification with lactate metabolic selection.</p

AP characteristics of iPS cell-derived cardiomyocytes.

<p>Data are mean ± SEM. n indicates the cell number; C_m: membrane capacitance; SAF: spontaneous AP frequency; MDP: maximum diastolic potential; APA: AP amplitude; max dV/dt: maximum rate of rise of the AP upstroke; APD30/APD50/APD80: AP duration measured at 30%, 50%, 80% or 90% repolarization, respectively.</p><p>AP characteristics of iPS cell-derived cardiomyocytes.</p

Functional gap junctions in cardiac clusters.

<p><b>A:</b> Original recording of a iPS cell-derived cardiomyocyte cluster loaded with the gap junction permeable dye fluo-4 at baseline (left panel, scale bar 10 μm), immediate after photobleaching of a single cell (mid panel, arrow) and 10 minutes later (right panel). <b>B:</b> Analysis of fluorescence intensity as a function of time at two regions of interest: the bleached cell and an adjacent donor cell. The fluorescence recovery after photobleaching (FRAP) could be fit with a single exponential with a recovery rate constant k of 2.94 ms. The simultaneous drop in fluorescence in the adjacent donor cell that could also be fit with a single exponential (k = 0.51 ms).</p

Spontaneous intracellular Ca fluctuations in iPS cell-derived cardiomyocytes.

<p><b>A:</b> Exemplary traces of fluo-4 fluorescence in an iPS cell-derived cardiomyocyte at low-diastolic (left) and high-systolic [Ca]_I (right). Data was acquired in frame-scan mode (scale bar 10 μm). <b>B:</b> Original recording of a fast line-scan image (lower panel) and derived fluo-4 fluorescence intensity as a function of time (upper panel). A typical Ca transient is visible. In addition, spontaneous and localized diastolic Ca release, i.e. Ca sparks, are detectable. Lower right panel shows a surface plot of a Ca spark. <b>C:</b> Original recordings of spontaneous Ca transients (acquired in line-scan mode) at baseline and upon exposure to isoproterenol. Spontanous Ca transient frequency increased from 0.33 to 0.5 Hz.</p