Confocal Line-scan Imaging Research Articles

Oxidative Stress and Ca 2+ Sparks in Pulmonary Arterial Myocytes of High Altitude Acclimatized Sheep

High altitude (HA) can cause pulmonary hypertension and deregulate Ca2+ spark activity, which is due to local ryanodine receptor (RYR) activation and important to hypoxic-induced pulmonary vasoconstriction (HPV). HA also increases oxidative stress, which can impair Ca2+ spark activity. We tested the hypothesis that HA alters the impact of oxidative stress on Ca2+ spark activity in pulmonary arterial (PA) myocytes from low altitude (LA; 335m) and HA acclimatized adult sheep (~3,800m for <100 days). Ca2+ sparks were recorded using line-scan confocal imaging of the Ca2+ indicator Fluo-4. Oxidative stress was elevated with tert-butyl H2O2 (TbH2O2) or reduced with the antioxidant N-Acetyl-Cysteine (NAC). Ca2+ spark activity and spatial-temporal properties were analyzed with custom designed software (SparkLab). Oxidative stress decreased Ca2+ spark frequency in both LA and HA myocytes. NAC decreased frequency in HA but not LA myocytes. Spatial aspects of LA Ca2+ sparks were increased by both TbH2O2 and NAC, while HA decreased spatial aspects to TbH2O2. Temporal aspects in LA and HA were increased by NAC and TbH2O2. The alterations in Ca2+ spark activity in PA myocytes from LA and HA sheep support the premise that oxidative stress contributes to aberrant Ca2+ signaling seen following HA. Because RYRs are important to HPV, decreased Ca2+ spark activity following NAC in HA animals may indicate antioxidants can reduce pulmonary vascular pressures due to HA. This supports studies focused on resolving the mechanisms important to how HA mediated oxidative stress effects Ca2+ signaling (NIH and NSF support).

53Altered CaMKII and ROS microdomains favor sparks in orphaned RyR after myocardial infarction

Purpose: In ventricular myocytes of rodents, ryanodine receptors (RyRs) are typically organized at Z-lines where the sarcoplasmic reticulum forms dyads with T-tubules (TTs). In large mammals, TT density is lower and not all RyRs are coupled to the TTs. Recently, we have shown that non-coupled RyRs lack a CaMKII and ROS-dependent microdomain modulation during rate adaptation. Here we examine how this microdomain modulation may be altered in ischemic cardiomyopathy where the fraction of non-coupled RyRs is increased. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=7), we studied myocytes adjacent to the MI and compared these to myocytes from SHAM pigs (N=5) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator and confocal line scan imaging. Spontaneous Ca2+ sparks were recorded during a 15s period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyR using a specific algorithm. All data were obtained in at least 3 different animals. Results: In myocytes from SHAM, we confirmed the specific modulation of coupled, but not of non-coupled, RyR as seen in control hearts, i.e. an increase of spark frequency at 2 Hz stimulation that is dependent on CaMKII and NOX2-generated ROS (#sparks/100 μm/s in SHAM: 0.8 ± 0.1 at 0.5 Hz to 2.3 ± 0.2 at 2 Hz, vs. 1.5 ± 0.2 with AIP at 2 Hz, and 1.1 ± 0.2 with gp91 ds-tat peptide at 2 Hz, p-value < 0.05 vs. control, n=7-14 in each group). In MI myocytes this modulation of coupled RyR was absent (#sparks/100 μm/s at 2 Hz not different from at 0.5 Hz and no effect of AIP and gp-91 ds-tat peptide). The fraction of non-coupled RyR was larger in MI (orphaned RyR) and their spark frequency at 2 Hz was significantly higher compared to SHAM. In contrast to SHAM, the response of these orphaned RyR was sensitive to CaMKII inhibition (AIP) (at 2 Hz #sparks/100 μm/s was 5.3 ± 0.5 at baseline vs. 3.1 ± 0.6 with AIP, p-value < 0.05, n=8-10 in each group). A significant reduction in spark frequency was observed in these orphaned RyRs after global ROS scavenging (NAC) (2 Hz #sparks/100 μm/s was 5.2 ± 0.5 at baseline vs. 2.9 ± 0.6 with NAC, p-value < 0.05, n=9-10 in each group) and after mitochondrial ROS inhibition using mitoTEMPO (at 2 Hz #sparks/100 μm/s was 2.9 ± 0.4 at baseline vs. 1.1 ± 0.3 with mitoTEMPO, p-value < 0.001, n=8 in each group) while NOX2 inhibition had no effect (gp91 ds-tat peptide, n=10–14). Conclusion: After MI there is a novel RyR microdomain organization favoring sparks in orphaned RyR, possibly related to mitochondrial ROS production.

Automated Detection and Analysis of Ca2+ Sparks in x-y Image Stacks Using a Thresholding Algorithm Implemented within the Open-Source Image Analysis Platform ImageJ

Previous studies have used analysis of Ca2+ sparks extensively to investigate both normal and pathological Ca2+ regulation in cardiac myocytes. The great majority of these studies used line-scan confocal imaging. In part, this is because the development of open-source software for automatic detection of Ca2+ sparks in line-scan images has greatly simplified data analysis. A disadvantage of line-scan imaging is that data are collected from a single row of pixels, representing only a small fraction of the cell, and in many instances x-y confocal imaging is preferable. However, the limited availability of software for Ca2+ spark analysis in two-dimensional x-y image stacks presents an obstacle to its wider application. This study describes the development and characterization of software to enable automatic detection and analysis of Ca2+ sparks within x-y image stacks, implemented as a plugin within the open-source image analysis platform ImageJ. The program includes methods to enable precise identification of cells within confocal fluorescence images, compensation for changes in background fluorescence, and options that allow exclusion of events based on spatial characteristics.

Wnt-11 Signaling in Cardiomyocytes

The Wnt/frizzled signalling pathways play a key role in cardiogenesis and in adult hearts during cardiac remodelling. We investigated whether the expression of Wnt-11 and its anticipated downstream signalling components is altered in human failing hearts and whether Wnt-11 acutely affects Ca2+ transients in adult murine cardiomyocytes.Left ventricles (LV) homogenates of human non-failing hearts (NF, n=4) and end-stage failing hearts with ischemic (ICM, n=5) and dilated (DCM, n=5) cardiomyopathy were used for western blotting with antibodies against Wnt-11, Fzd-4 and Fzd-7 (Wnt receptors) and Dvl-1 (downstream cytoplasmic regulator). Expression of proteins was normalized to GAPDH and is given as a fraction of average protein expression in NF. Ca2+ transients were measured during confocal line scan imaging with Fluo-4 in isolated murine adult cardiomyocytes after 5 (ncells=20) and 25 minutes (ncells=18) incubation with recombinant Wnt-11 (2 µg/ml). Untreated cells served as controls (ncells=27).Wnt-11 expression in LV in ICM (0.85 ± 0.05) and DCM (0.91 ± 0.34) was not significantly changed when compared to NF. However, the expression of Wnt-11 receptors was highly increased: Fzd-4 in ICM (6.25 ± 1.23; p<0.005) and in DCM (4.81 ± 0.82; p<0.005) as well Fzd-7 in ICM (4.48 ± 0.63; p<0.001) and in DCM (4.45 ± 0.76; p<0.001). The expression of Dvl-1 tended to be decreased in ICM (0.63 ± 0.15; p=0.15) and DCM (0.64 ± 0.29; p=0.17). The amplitude of Ca2+ transients (F/F0), time to peak, and time to 50 % decay (RT50) were not altered during 5 and 25 minutes incubation with Wnt-11 when compared to untreated control cells.The expression of the Wnt-receptors, Fzd-4 and Fzd-7 is significantly increased in human end-stage failing hearts, suggesting their regulation in cardiac remodelling and/or dysfunction. Wnt-11 did not acutely alter cardiomyocyte Ca2+ transients in non-diseased cardiomyocytes.

Altered CamkII and Ros Microdomains Favor Sparks in Orphaned RyR After Myocardial Infarction

Purpose: In ventricular myocytes of rodents, ryanodine receptors (RyRs) are typically organized at Z-lines where the sarcoplasmic reticulum forms dyads with T-tubules (TTs). In large mammals, TT density is lower and not all RyRs are coupled to the TTs. Recently, we have shown that non-coupled RyRs lack a CaMKII and ROS-dependent microdomain modulation during rate adaptation. Here we examine how this microdomain modulation may be altered in ischemic cardiomyopathy where the fraction of non-coupled RyRs is increased. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=7), we studied myocytes adjacent to the MI and compared these to myocytes from SHAM pigs (N=5) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator and confocal line scan imaging. Spontaneous Ca2+ sparks were recorded during a 15s period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyR using a specific algorithm. All data were obtained in at least 3 different animals. Results: In myocytes from SHAM, we confirmed the specific modulation of coupled, but not of non-coupled, RyR as seen in control hearts, i.e. an increase of spark frequency at 2 Hz stimulation that is dependent on CaMKII and NOX2-generated ROS (#sparks/100 μm/s in SHAM: 0.8 ± 0.1 at 0.5 Hz to 2.3 ± 0.2 at 2 Hz, vs. 1.5 ± 0.2 with AIP at 2 Hz, and 1.1 ± 0.2 with gp91 ds-tat peptide at 2 Hz, p-value < 0.05 vs. control, n=7-14 in each group). In MI myocytes this modulation of coupled RyR was absent (#sparks/100 μm/s at 2 Hz not different from at 0.5 Hz and no effect of AIP and gp-91 ds-tat peptide). The fraction of non-coupled RyR was larger in MI (orphaned RyR) and their spark frequency at 2 Hz was significantly higher compared to SHAM. In contrast to SHAM, the response of these orphaned RyR was sensitive to CaMKII inhibition (AIP) (at 2 Hz #sparks/100 μm/s was 5.3 ± 0.5 at baseline vs. 3.1 ± 0.6 with AIP, p-value < 0.05, n=8-10 in each group). A significant reduction in spark frequency was observed in these orphaned RyRs after global ROS scavenging (NAC) (2 Hz #sparks/100 μm/s was 5.2 ± 0.5 at baseline vs. 2.9 ± 0.6 with NAC, p-value < 0.05, n=9-10 in each group) and after mitochondrial ROS inhibition using mitoTEMPO (at 2 Hz #sparks/100 μm/s was 2.9 ± 0.4 at baseline vs. 1.1 ± 0.3 with mitoTEMPO, p-value < 0.001, n=8 in each group) while NOX2 inhibition had no effect (gp91 ds-tat peptide, n=10–14). Conclusion: After MI there is a novel RyR microdomain organization favoring sparks in orphaned RyR, possibly related to mitochondrial ROS production.

CaMKII-Dependent Phosphorylation Modulates Ca2+ Cycling in Sinoatrial Node Cells to Regulate Cardiac Pacemaker Function

Spontaneous beating of rabbit sinoatrial node cells (SANC) is linked to rhythmic, submembrane, sarcoplasmic reticulum (SR) generated local Ca2+releases (LCR), which activate inward Na+/Ca2+exchange current to impart exponential increase of the late diastolic depolarization rate and fire an action potential (AP). Rabbit SANC have high basal PKA-dependent phosphorylation, but basal CaMKII-dependent phosphorylation has never been ascertained. Here we show that SANC maintain high basal PLB phosphorylation at Thr17 site (western blotting), employed as a marker of CaMKII-dependent protein phosphorylation, that is markedly suppressed by CaMKII inhibitor, KN-93, but not its inactive analog KN-92. Considering that CaMKII activity is critically dependent on intracellular [Ca2+]i we employed BAPTA-AM to chelate [Ca2+]i. Both KN-93 and BAPTA-AM markedly decrease the LCR size (confocal line-scan imaging), LCR number per each spontaneous cycle and prolong the LCR period (time between AP-induced Ca2+ transient and subsequent LCR). The increase in the LCR period predicts concurrent increase in the spontaneous cycle length. To delineate mechanisms of CaMKII-dependent regulation of Ca2+ cycling, avoiding interference of L-type Ca2+channels, which are also regulated by CaMKII, we permeabilized SANC with saponin. Elevation of cytosolic [Ca2+] from 50 to 150 nmol/L in permeabilized SANC significantly increases the LCR size and amplitude due, in part, to a marked increase of PLB phosphorylation at Thr17site and resultant increase in the SR Ca2+ content and, in part, to RyR phosphorylation at PKA- and CaMKII-dependent Ser2809 site (immunostaining). CaMKII-inhibitor AIP suppresses LCR characteristics in permeabilized SANC, decreases phosphorylation of RyR, PLB and reduces the SR Ca2+ load. Thus, normal automaticity of SANC is regulated by basal CaMKII-dependent phosphorylation, partly via modulation of intrinsic SR Ca2+ cycling, i.e. SR Ca2+ pumping and release attained through phosphorylation of PLB and RyR.

A Luminal Calcium Sensing Mutation of the Cardiac Ryanodine Receptor Diminishes Calcium Waves and Stress -Induced Ventricular Tachycardias in Calsequestrin Null Mice

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease. Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) and calsequestrin (CASQ2) have been linked to CPVT. The sarcoplasmic reticulum calcium content and the sensitivity of RyR2 to luminal calcium are two important factors determining the propensity for store overload induced spontaneous calcium release, also known as store-overload induced calcium release (SOICR), and SOICR-evoked VTs. We recently found that residue E4872 of RyR2 is critical for luminal calcium sensing. In the present study, we determined whether the RyR2-E4872Q mutation is capable of rescuing the VT phenotype of the CASQ2 null mice. We bred E4872Q+/- mice with CASQ2 null mice to produce CASQ2-/- or CASQ2+/- mice with or without the RyR2-E4872Q mutation. CASQ2-/- and CASQ2+/- mice without the RyR2-E4872Q mutation showed long lasting stress-induced VTs, whereas CASQ2-/- / E4872Q+/- and CASQ2+/- / E4872Q +/- displayed little or no VTs. Thus, the RyR2-E4872Q mutation nearly completely protected the CASQ2 null mice from CPVT. We next studied the impact of the RyR2-E4872Q mutation on calcium handling in intact hearts using linescan confocal calcium imaging. The properties of calcium transients did not differ significantly between CASQ2-/- hearts with or without the RyR2-E4872Q mutation at resting conditions. However, upon elevation of external calcium from 2mM to 7mM or adding 100nM isoproterenol, frequent spontaneous calcium waves occurred in CASQ2-/- hearts without the RyR2-E4872Q mutation, but not in CASQ2-/- / E4872Q+/- hearts. These observations indicate that targeting the luminal calcium sensor of RyR2 represents a new therapeutic strategy for suppressing CPVT and other calcium mediated cardiac arrhythmias. (Support by NIH R01HL75210)

Scanned light sheet microscopy with confocal slit detection

In light sheet fluorescence microscopy optical sectioning is achieved by illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet fluorescence excitation and confocal slit detection. A high frame rate was achieved by using the "rolling shutter" of a scientific CMOS camera as a slit detector. Synchronizing the "rolling shutter" with the scanned illumination beam results in confocal line detection. Acquiring image data with selective plane illumination minimizes photo-damage while simultaneously enhancing contrast, optical sectioning and signal-to-noise ratio. Thus the imaging principle presented here merges the benefits of scanned light sheet microscopy and line-scanning confocal imaging.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Ranolazine Alters the Properties of Localized Ca2+ Sparks and Inhibits Global Ca2+ Waves in Rat Myocytes

The effect of ranolazine (10 μM) on local Ca2+ regulation was studied in isolated rat ventricular myocytes. Cells were loaded with fluo-4 AM and Ca2+ was detected using line-scan confocal imaging. Ranolazine increased Ca2+ spark frequency by 57.8 ± 17% (p<0.05, n=29). This was accompanied by significant (p<0.05) decreases in Ca2+ spark amplitude (44.5 ± 0.1%), duration (16.5 ± 1.9%) and width (5.6 ± 1.7%) resulting in a decrease in the calculated ‘spark mass’ of 41.3 ± 5.4% (data from 15-29 cells). In cells initially paced at 0.2 Hz in the presence of forskolin (10 μM), cessation of stimulation was followed by spontaneous diastolic Ca2+ waves. After introduction of ranolazine (but not in its absence), the frequency of spontaneous Ca2+ waves decreased markedly over 10-15 minutes and waves were completely abolished in 5 of 11 cells. Inhibition of Ca2+ waves also occurred in permeabilized cells, suggesting an intracellular site of action. We propose that the ranolazine-induced decrease in Ca2+ spark mass may contribute to inhibition of pro-arrhythmic Ca2+ waves by reducing the probability of saltatory propagation between Ca2+ release sites.

Lineage Reprogramming from Cardiomyocytes to Pacemaker Cells via a Single Transcription Factor

The heartbeat originates in a small number of highly-specialized pacemaker cells found in the sinoatrial node (SAN). In an effort to create pacemaker cells from working cardiomyocytes, we expressed Tbx18, a transcription factor indispensible for embryonic development of the SAN. Within days, Tbx18-transduced neonatal rat cardiomyocytes (NRVMs) exhibited rhythmic spontaneous electrical firing. The mechanism of induced pacemaker activity resembled that of SAN cells, with enhanced voltage- and Ca2+-dependent “clock” pathways. The maximum diastolic potential was more positive in Tbx18-NRVMs in comparison to controls (−47±10 mV vs. −73±6 mV) with accompanying reduction in IK1. Line-scan confocal imaging of Tbx18-NRVMs loaded with Rhod-2 resolved localized Ca2+ release events (LCRs) that preceded each whole-cell Ca2+ transient resembling LCRs observed in native SAN cells. LCRs were not detected in control NRVMs. Spontaneous Ca2+-transients in Tbx18-NRVMs were suppressed on superfusion with ryanodine (10 μM) by 47±6% in comparison to 12±2% suppression in controls, indicative of increased relevance of [Ca2+]i stores. A 2.3-fold increase in [Ca2+]i stores was observed in Tbx18-NRVMs which can be attributed to increased sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity due to downregulation or phosphorylation of phospholamban (PLN), an inhibitor of SERCA. Tbx18-overexpression led to relative increase in the protein level of phosphorylated PLN (Ser16) similar to higher p-PLN level observed in the adult SAN relative to the left ventricle. In addition, Tbx18-NRVMs exhibited increased levels of cAMP and HCN4, shrank in size with disorganized myofibrils, and underwent epigenetic modifications consistent with durable reprogramming. Focal Tbx18 gene transfer in the guinea-pig ventricle consistently created ectopic pacemaker activity in vivo with a rhythm of 40±20 beats/min (n=5/7). Taken together, the data demonstrate faithful reprogramming of ventricular myocytes to pacemaker cells by a single transcription factor, Tbx18.

Abstract 17100: CaMKII Activity Modulates Basal Sarcoplasmic Reticulum Ca 2+ Cycling to Drive Normal Automaticity of Sinoatrial Node Cells

Spontaneous beating of rabbit sinoatrial node cells (SANC) is linked to rhythmic, sarcoplasmic reticulum (SR) generated, submembrane local Ca 2+ releases (LCR) during diastolic depolarization (DD). LCRs activate inward Na + /Ca 2+ exchange current which imparts an exponential increase to the later part of DD and brings the membrane potential to threshold to fire an action potential. Although the crucial dependence of normal SANC automaticity on CaMKII signaling has previously been linked to an effect to facilitate recovery from inactivation of L-type Ca 2+ channels, there may also be direct CaMKII-dependent regulation of LCRs in SANC. Here we show that in freshly isolated rabbit SANC CaMKII inhibitor, KN-93 (0.3 μmol/L), but not its inactive analog KN-92, markedly suppresses LCRs (confocal microscopy, Ca 2+ indicator Fluo-3) reducing LCR number per each spontaneous cycle by 64% (from 1.1±0.1 to 0.4±0.1); LCR size by 32% (from 7.0±0.6 to 4.8±0.2 μm); and by markedly prolonging the LCR period (the interval between AP-induced Ca 2+ transient and subsequent LCR). Prolongation of the LCR period predicted the concomitant increase in spontaneous cycle length, indicating that CaMKII is vitally important for spontaneous SANC firing. To further delineate mechanism of CaMKII-dependent regulation of Ca 2+ cycling, avoiding interference of L-type Ca 2+ channels, we permeabilized SANC with saponin. Confocal linescan imaging of permeabilized SANC showed that KN-93, but not KN-92, decreased the LCR number by 62% (from 16.8±3.7 to 6.4±2.0 per 1sec×100μm) and LCR size by 25% (from 6.5±1.1 to 4.9±0.8 μm). The suppression of LCR parameters was associated with a ~12% reduction of the SR Ca 2+ load, assessed by a rapid application of caffeine on permeabilized SANC. The SR Ca 2+ load is controlled by phospholamban (PLB) which could be phosphorylated by CaMKII at Thr 17 site. Basal PLB phosphorylation at Thr 17 site, assessed by western blotting, is ~3 times greater in SANC than in ventricular myocytes, and KN-93, but not KN-92, markedly decreases PLB phosphorylation by ~40%. Thus, normal automaticity of SANC is regulated by basal CaMKII-dependent phosphorylation, in part, through modulation of SR Ca 2+ cycling, i.e. SR Ca 2+ load controlled by CaMKII-dependent phosphorylation of PLB.

Subcellular Heterogeneity of Ryanodine Receptor Properties in Ventricular Myocytes with Low T-Tubule Density

RationaleIn ventricular myocytes of large mammals, not all ryanodine receptor (RyR) clusters are associated with T-tubules (TTs); this fraction increases with cellular remodeling after myocardial infarction (MI).ObjectiveTo characterize RyR functional properties in relation to TT proximity, at baseline and after MI.MethodsMyocytes were isolated from left ventricle of healthy pigs (CTRL) or from the area adjacent to a myocardial infarction (MI). Ca2+ transients were measured under whole-cell voltage clamp during confocal linescan imaging (fluo-3) and segmented according to proximity of TTs (sites of early Ca2+ release, F>F50 within 20 ms) or their absence (delayed areas). Spontaneous Ca2+ release events during diastole, Ca2+ sparks, reflecting RyR activity and properties, were subsequently assigned to either category.ResultsIn CTRL, spark frequency was higher in proximity of TTs, but spark duration was significantly shorter. Block of Na+/Ca2+ exchanger (NCX) prolonged spark duration selectively near TTs, while block of Ca2+ influx via Ca2+ channels did not affect sparks properties. In MI, total spark mass was increased in line with higher SR Ca2+ content. Extremely long sparks (>47.6 ms) occurred more frequently. The fraction of near-TT sparks was reduced; frequency increased mainly in delayed sites. Increased duration was seen in near-TT sparks only; Ca2+ removal by NCX at the membrane was significantly lower in MI.ConclusionTT proximity modulates RyR cluster properties resulting in intracellular heterogeneity of diastolic spark activity. Remodeling in the area adjacent to MI differentially affects these RyR subpopulations. Reduction of the number of sparks near TTs and reduced local NCX removal limit cellular Ca2+ loss and raise SR Ca2+ content, but may promote Ca2+ waves.

PGC-1α accelerates cytosolic Ca 2+ clearance without disturbing Ca 2+ homeostasis in cardiac myocytes

Energy metabolism and Ca 2+ handling serve critical roles in cardiac physiology and pathophysiology. Peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) is a multi-functional coactivator that is involved in the regulation of cardiac mitochondrial functional capacity and cellular energy metabolism. However, the regulation of PGC-1α in cardiac Ca 2+ signaling has not been fully elucidated. To address this issue, we combined confocal line-scan imaging with off-line imaging processing to characterize calcium signaling in cultured adult rat ventricular myocytes expressing PGC-1α via adenoviral transduction. Our data shows that overexpressing PGC-1α improved myocyte contractility without increasing the amplitude of Ca 2+ transients, suggesting that myofilament sensitivity to Ca 2+ increased. Interestingly, the decay kinetics of global Ca 2+ transients and Ca 2+ waves accelerated in PGC-1α-expressing cells, but the decay rate of caffeine-elicited Ca 2+ transients showed no significant change. This suggests that sarcoplasmic reticulum (SR) Ca 2+-ATPase (SERCA2a), but not Na +/Ca 2+ exchange (NCX) contribute to PGC-1α-induced cytosolic Ca 2+ clearance. Furthermore, PGC-1α induced the expression of SERCA2a in cultured cardiac myocytes. Importantly, overexpressing PGC-1α did not disturb cardiac Ca 2+ homeostasis, because SR Ca 2+ load and the propensity for Ca 2+ waves remained unchanged. These data suggest that PGC-1α can ameliorate cardiac Ca 2+ cycling and improve cardiac work output in response to physiological stress. Unraveling the PGC-1α-calcium handing pathway sheds new light on the role of PGC-1α in the therapy of cardiac diseases.

High-speed line-scan confocal imaging of stimulus-evoked intrinsic optical signals in the retina

A rapid line-scan confocal imager was developed for functional imaging of the retina. In this imager, an acousto-optic deflector was employed to produce mechanical vibration- and inertia-free light scanning, and a high-speed (68,000 Hz) linear CCD camera was used to achieve subcellular and submillisecond spatiotemporal resolution imaging. Two imaging modalities, i.e., frame-by-frame and line-by-line recording, were validated for the reflected light detection of intrinsic optical signals (IOSs) in visible light stimulus activated frog retinas. Experimental results indicated that fast IOSs were tightly correlated with retinal stimuli and could track visible light flicker stimulus frequency up to at least 2 Hz.

Alterations in Ca2+ Sparks and T-Tubules Promote Slowed, Dyssynchronous Ca2+ Release in Failing Cardiomyocytes

In heart failure, cardiomyocytes exhibit slowing of the rising phase of the Ca2+ transient which contributes to the impaired contractility in this condition. We investigated the underlying mechanisms in a murine model of congestive heart failure (CHF). Myocardial infarction (MI) was induced by left coronary artery ligation, and at 10 weeks post-MI, mice exhibited symptoms of CHF including reduced cardiac function and increased lung weight. Cardiomyocytes were isolated from viable regions of the septum, and septal myocytes from SHAM-operated mice served as controls. Confocal line-scan imaging revealed a slowed rate of rise of Ca2+ transients (fluo-4 AM, 1 Hz) in CHF cells, which largely resulted from spatially non-uniform Ca2+ release. Ca2+ sparks recorded in resting myocytes were also slower to peak in CHF than SHAM (11.5±0.6 ms vs 9.5±0.6 ms, P<0.05) and longer lasting (FWHM=24.5±0.7 ms vs 21.6±1.0 ms, P<0.05). The mean increase in these measurements resulted from a sub-population of sparks in CHF cells with very long rise times but small amplitudes. Local Ca2+ transients (width=2 μm) measured at the same coordinates as these sparks were also slow to rise, indicating that altered Ca2+ spark kinetics contributed to the dyssynchronous Ca2+ release pattern in CHF. As well, di-8-ANEPPS staining revealed disorganized T-tubular structure in failing myocytes, which is also known to promote Ca2+ release dyssynchrony. Specifically, we observed irregular gaps between adjacent tubules where Ca2+ release was markedly delayed, occurring only after Ca2+ diffusion from regions where tubules were present. Thus, slowed and dyssynchronous Ca2+ release in failing myocytes results from a combination of altered ryanodine receptor function and T-tubule disorganization. We suggest that the sub-population of slow, small amplitude Ca2+ sparks in CHF may represent ryanodine receptors which are functionally uncoupled from their neighbours.

Uniform Action Potential Repolarization within the Sarcolemma of In Situ Ventricular Cardiomyocytes

Previous studies have speculated, based on indirect evidence, that the action potential at the transverse (t)-tubules is longer than at the surface membrane in mammalian ventricular cardiomyocytes. To date, no technique has enabled recording of electrical activity selectively at the t-tubules to directly examine this hypothesis. We used confocal line-scan imaging in conjunction with the fast response voltage-sensitive dyes ANNINE-6 and ANNINE-6plus to resolve action potential-related changes in fractional dye fluorescence (Δ F/ F) at the t-tubule and surface membranes of in situ mouse ventricular cardiomyocytes. Peak Δ F/ F during action potential phase 0 depolarization averaged −21% for both dyes. The shape and time course of optical action potentials measured with the water-soluble ANNINE-6plus were indistinguishable from those of action potentials recorded with intracellular microelectrodes in the absence of the dye. In contrast, optical action potentials measured with the water-insoluble ANNINE-6 were significantly prolonged compared to the electrical recordings obtained from dye-free hearts, suggesting electrophysiological effects of ANNINE-6 and/or its solvents. With either dye, the kinetics of action potential-dependent changes in Δ F/ F during repolarization were found to be similar at the t-tubular and surface membranes. This study provides what to our knowledge are the first direct measurements of t-tubule electrical activity in ventricular cardiomyocytes, which support the concept that action potential duration is uniform throughout the sarcolemma of individual cells.

Voltage-activated elementary calcium release events in isolated mouse skeletal muscle fibers.

The elementary Ca(2+)-release events underlying voltage-activated myoplasmic Ca(2+) transients in mammalian muscle remain elusive. Here, we looked for such events in confocal line-scan (x,t) images of fluo-3 fluorescence taken from isolated adult mouse skeletal muscle fibers held under voltage-clamp conditions. In response to step depolarizations, spatially segregated fluorescence signals could be detected that were riding on a global increase in fluorescence. These discrete signals were separated using digital filtering in the spatial domain; mean values for their spatial half-width and amplitude were 1.99 +/- 0.09 microm and 0.16 +/- 0.005 DeltaF/F(0) (n = 151), respectively. Under control conditions, the duration of the events was limited by the pulse duration. In contrast, in the presence of maurocalcine, a scorpion toxin suspected to disrupt the process of repolarization-induced ryanodine receptor (RyR) closure, events uninterrupted by the end of the pulse were readily detected. Overall results establish these voltage-activated low-amplitude local Ca(2+) signals as inherent components of the physiological Ca(2+)-release process of mammalian muscle and suggest that they result from the opening of either one RyR or a coherently operating group of RyRs, under the control of the plasma membrane polarization.

Abstract 1522: Local Ca Releases And Spontaneous Beating Rate Of Rabbit Sinoatrial Node Cells Are Controlled By Interaction Of Basal Phosphodiesterase 3 And 4 Activity

A high basal phosphodiesterase (PDE) activity in sinoatrial node cells (SANC) regulates cAMP and modulates cAMP-mediated PKA-dependent phosphorylation of Ca 2+ cycling proteins that determines the characteristics of local subsarcolemmal Ca 2+ releases (LCR). LCRs occur during the terminal diastolic depolarization and activate the inward Na + -Ca 2+ exchange current, thus, regulating the SANC basal spontaneous beating rate. Though both PDE3 and PDE4 are present in rabbit SANC, a role of PDE4 or its interaction with PDE3 is not clear. To determine to what extent PDE4 controls LCRs and the SANC spontaneous beating rate, we used perforated patch to record spontaneous action potentials (APs) in conjunction with confocal linescan images or whole-cell patch clamp to measure L-type Ca 2+ current (I Ca,L ) at 35 o C. A specific PDE3 inhibitor, cilostamide (Cil, 0.3 μmol/L), increased the spontaneous beating rate by 28% (from 154 ± 21 to 191 ± 17 beat/min); in contrast a specific PDE4 inhibitor, rolipram (Rol, 2 μmol/L), had no effect on the firing rate. The substantial role of PDE4 in the control of basal spontaneous SANC firing was unmasked when PDE3 and PDE4 were simultaneously inhibited: the combination of Rol and Cil produced ~57% acceleration of the beating rate (from129 ± 10 to 200 ± 10 beat/min) equivalent to that produced by the broad-spectrum PDE inhibitor, IBMX (100μM), i.e. ~55% (from 145 ± 8 to 221 ± 6 beat/min). The combination of Cil and Rol increased LCR amplitude, size and decreased the LCR period similar to IBMX. Both IBMX and the combination of Cil and Rol produced a substantial increase in I Ca,L ~135% (from 10 ± 2 to 22 ± 3 pA/pF) and ~125% (from 6 ± 1 to 13 ± 3 pA/pF) respectively. The efficiency of PDE3 inhibition alone on spontaneous beating rate might be ascribed to its lower K m for cAMP, than that of PDE4. When PDE3 is suppressed level of cAMP is sufficiently elevated to activate PDE4. Therefore, in the presence of PDE3 inhibition, PDE4 inhibition contributes to the increase in the spontaneous beating rate. Thus, an interaction of basal PDE3 and PDE4 activities in SANC modulates cAMP level and I Ca,L which controls Ca 2+ influx, cell Ca 2+ load, determining LCR characteristics. When both PDE3 and PDE4 are inhibited the maximum spontaneous beating rate of SANC is achieved.

Paracrine effects of hypoxic fibroblast-derived factors on the MPT-ROS threshold and viability of adult rat cardiac myocytes.

Cardiac fibroblasts contribute to multiple aspects of myocardial function and pathophysiology. The pathogenetic relevance of cytokine production by these cells under hypoxia, however, remains unexplored. With the use of an in vitro cell culture model, this study evaluated cytokine production by hypoxic cardiac fibroblasts and examined two distinct effects of hypoxic fibroblast-conditioned medium (HFCM) on cardiac myocytes and fibroblasts. Hypoxia caused a marked increase in the production of tumor necrosis factor (TNF)-alpha by cardiac fibroblasts. HFCM significantly enhanced the susceptibility of cardiac myocytes to reactive oxygen species (ROS)-induced mitochondrial permeability transition (MPT), determined by high-precision confocal line-scan imaging following controlled, photoexcitation-induced ROS production within individual mitochondria. Furthermore, exposure of cardiac myocytes to HFCM for 5 h led to loss of viability, as evidenced by change in morphology and annexin staining. HFCM also decreased DNA synthesis in cardiac fibroblasts. Normoxic fibroblast-conditioned medium spiked with TNF-alpha at 200 pg/ml, a concentration comparable to that in HFCM, promoted loss of myocyte viability and decreased DNA synthesis in cardiac fibroblasts. These effects of HFCM are similar to the reported effects of hypoxia per se on these cell types, showing that hypoxic fibroblast-derived factors may amplify the distinct effects of hypoxia on cardiac cells. Importantly, because both hypoxia and oxidant stress prevail in a setting of ischemia and reperfusion, the effects of soluble factors from hypoxic fibroblasts on the MPT-ROS threshold and viability of myocytes may represent a novel paracrine mechanism that could exacerbate ischemia-reperfusion injury to cardiomyocytes.