Cherrie H.T. Kong

Nanoscale organization of junctophilin-2 and ryanodine receptors within peripheral couplings of rat ventricular cardiomyocytes

The peripheral distributions of the cardiac ryanodine receptor (RyR) and a junctional protein, junctophilin-2 (JPH2), were examined using single fluorophore localization-based super-resolution microscopy in rat ventricular myocytes. JPH2 was strongly associated with RyR clusters. Estimates of the colocalizing fraction of JPH labeling with RyR was ~90% within 30 nm of RyR clusters. This is comparable to fractions estimated from confocal data (~87%). Similarly, most RyRs were associated with JPH2 labeling in super-resolution images (~81% within 30 nm of JPH2 clusters). The shape of associated RyR clusters and JPH2 clusters were very similar, but not identical, suggesting that JPH2 is dispersed throughout RyR clusters and that the packing of JPH2 into junctions and the assembly of RyR clusters are tightly linked.

Extraction of Sub-microscopic Ca Fluxes from Blurred and Noisy Fluorescent Indicator Images with a Detailed Model Fitting Approach

The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This ‘forward method’ allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

Altered distribution of ICa impairs Ca release at the t-tubules of ventricular myocytes from failing hearts

In mammalian cardiac ventricular myocytes, Ca influx and release occur predominantly at t-tubules, ensuring synchronous Ca release throughout the cell. Heart failure is associated with disrupted t-tubule structure, but its effect on t-tubule function is less clear. We therefore investigated Ca influx and release at the t-tubules of ventricular myocytes isolated from rat hearts ~ 18 weeks after coronary artery ligation (CAL) or corresponding Sham operation. L-type Ca current (ICa) was recorded using the whole-cell voltage-clamp technique in intact and detubulated myocytes; Ca release at t-tubules was monitored using confocal microscopy with voltage- and Ca-sensitive fluorophores. CAL was associated with cardiac and cellular hypertrophy, decreased ejection fraction, disruption of t-tubule structure and a smaller, slower Ca transient, but no change in ryanodine receptor distribution, L-type Ca channel expression, or ICa density. In Sham myocytes, ICa was located predominantly at the t-tubules, while in CAL myocytes, it was uniformly distributed between the t-tubule and surface membranes. Inhibition of protein kinase A with H-89 caused a greater decrease of t-tubular ICa in CAL than in Sham myocytes; in the presence of H-89, t-tubular ICa density was smaller in CAL than in Sham myocytes. The smaller t-tubular ICa in CAL myocytes was accompanied by increased latency and heterogeneity of SR Ca release at t-tubules, which could be mimicked by decreasing ICa using nifedipine. These data show that CAL decreases t-tubular ICa via a PKA-independent mechanism, thereby impairing Ca release at t-tubules and contributing to the altered excitation–contraction coupling observed in heart failure.

Stimulation of ICa by basal PKA activity is facilitated by caveolin-3 in cardiac ventricular myocytes

L-type Ca channels (LTCC), which play a key role in cardiac excitation–contraction coupling, are located predominantly at the transverse (t-) tubules in ventricular myocytes. Caveolae and the protein caveolin-3 (Cav-3) are also present at the t-tubules and have been implicated in localizing a number of signaling molecules, including protein kinase A (PKA) and β2-adrenoceptors. The present study investigated whether disruption of Cav-3 binding to its endogenous binding partners influenced LTCC activity. Ventricular myocytes were isolated from male Wistar rats and LTCC current (ICa) recorded using the whole-cell patch-clamp technique. Incubation of myocytes with a membrane-permeable peptide representing the scaffolding domain of Cav-3 (C3SD) reduced basal ICa amplitude in intact, but not detubulated, myocytes, and attenuated the stimulatory effects of the β2-adrenergic agonist zinterol on ICa. The PKA inhibitor H-89 also reduced basal ICa; however, the inhibitory effects of C3SD and H-89 on basal ICa amplitude were not summative. Under control conditions, myocytes stained with antibody against phosphorylated LTCC (pLTCC) displayed a striated pattern, presumably reflecting localization at the t-tubules. Both C3SD and H-89 reduced pLTCC staining at the z-lines but did not affect staining of total LTCC or Cav-3. These data are consistent with the idea that the effects of C3SD and H-89 share a common pathway, which involves PKA and is maximally inhibited by H-89, and suggest that Cav-3 plays an important role in mediating stimulation of ICa at the t-tubules via PKA-induced phosphorylation under basal conditions, and in response to β2-adrenoceptor stimulation.

Expansion and Characterization of Neonatal Cardiac Pericytes Provides a Novel Cellular Option for Tissue Engineering in Congenital Heart Disease

Background Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. Methods and Results CD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. Conclusions These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.

Increasing Sensitivity of Ca2+ Spark Detection in Noisy Images by Application of a Matched-Filter Object Detection Algorithm

Microscopic calcium (Ca2+) events (such as Ca2+ sparks) are an important area for study, as they help clarify the mechanism(s) underlying intracellular signaling. In the heart, Ca2+ sparks occur as a result of Ca2+ release from the sarcoendoplasmic reticulum, via ryanodine receptor channels. Measurement of Ca2+ spark properties can provide valuable information about the control of ryanodine receptor channel gating in situ, but requires high spatiotemporal resolution imaging, which produces noisy datasets that are problematic for spark detection. Automated detection algorithms may overcome visual detection bias, but missed and false-positive events can distort the distribution of measured Ca2+ spark properties. We present a sensitive and reliable method for the automated detection of Ca2+ sparks in datasets obtained using confocal line-scanning or total internal reflection fluorescence microscopy. This matched-filter detection algorithm(MFDA) employs a user-defined object, chosen to mimic Ca2+ spark properties, and the experimental dataset is searched for instances of the object. Detection certainty is provided by nonparametric statistical testing. The supplied codes can also refine the search object on the basis of those detected to further increase detection sensitivity. In comparison to a commonly used, intensity-thresholding algorithm, the MFDA is more sensitive and reliable, particularly at low signal/noise ratios. The MFDA can also be easily adapted to other signal-detection problems in noisy datasets.

Sub-microscopic analysis of t-tubule geometry in living cardiac ventricular myocytes using a shape-based analysis method

Transverse-axial tubules (TTs) are key structures involved in cardiac excitation-contraction coupling and can become deranged in disease. Although optical measurement of TTs is frequently employed to assess TT abundance and regularity, TT dimensions are generally below the diffraction limit of optical microscopy so determination of tubule size is problematic. TT diameter was measured by labeling both local surface membrane area and volume with fluorescent probes (FM4-64 and calcein, respectively), correcting image asymmetry by image processing and using the relationship between surface area and volume for a geometric primitive. This method shows that TTs have a mean (± SEM) diameter of 356 ± 18 nm in rabbit and 169 ± 15 nm in mouse (p < 0.001). Rabbit TT diameters were more variable than those of mouse (p < 0.01) and the smallest TT detected was 41 nm in mouse and the largest 695 nm in rabbit. These estimates are consistent with TT diameters derived from the more limited sampling of high-pressure frozen samples by electron tomography (which examines only a small fraction of the cell volume). Other measures of TT abundance and geometry (such as volume, membrane fractions and direction) were also derived. On the physiological time scale of E-C coupling (milliseconds), the average TT electrical space constant is ~ 175 μm in rabbit and ~ 120 μm in mouse and is ~ 50% of the steady-state space constant. This is sufficient to ensure reasonable electrical uniformity across normal cells. The image processing strategy and shape-based 3D approach to feature quantification is also generally applicable to other problems in quantification of sub-cellular anatomy.

Heterogeneity of T-Tubules in Pig Hearts

Background T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue. Methods Cardiac tissue was obtained from young female Landrace White pigs (45–75 kg, 5–6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT). Results & Conclusion The spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 μm, n = 17) than in either ventricular cells (1.33±0.05 μm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 μm, n = 5, P<0.05). These data demonstrate considerable heterogeneity between pig cardiomyocytes in the extent of t-tubule network, which correlated with cell size.

Depotentiation of intact rat cardiac muscle unmasks an Epac-dependent increase in myofilament Ca2+ sensitivity

Recently, a family of guanine nucleotide exchange factors have been identified in many cell types as important effectors of cyclic adenosine 3′,5′‐monophospahte (cAMP) signalling that is independent of protein kinase A (PKA). In the heart, investigation of exchange protein directly activated by cAMP (Epac) has yielded conflicting results. Since cAMP is an important regulator of cardiac contractility, this study aimed to examine whether Epac activation modulates excitation–contraction coupling in ventricular preparations from rat hearts. The study used 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine‐3′, 5′‐cyclic monophosphate (cpTOME), an analogue of cAMP that activates Epac, but not PKA. In isolated myocytes, cpTOME increased Ca2+ spark frequency from about 7 to 32/100 μm3/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca2+ and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca2+]o) revealed no effect of Epac activation on either the amplitude of Ca2+ transients (Control 0.7 ± 0.1 vs cpTOME 0.7 ± 0.1; 340/380 fura‐2 ratio, P = 0.35) or on peak stress (Control 24 ± 5 mN/mm2 vs cpTOME 23 ± 5 mN/mm2, P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca2+]o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca2+ sensitivity, an effect that was blocked by addition of KN‐93, a Ca2+/calmodulin‐dependent protein kinase II (CaMK‐II) inhibitor. This study suggests that Epac activation may be a useful therapeutic target to increase the strength of contraction during low inotropic states.

Quenching the spark: Termination of CICR in the submicroscopic space of the dyad

Cannell and Kong discuss the different termination mechanisms proposed for CICR in cardiac myocytes.