Ca2+ spark latency and control of intrinsic Ca2+ release dyssynchrony in rat cardiac ventricular muscle cells.

Cardiac excitation-contraction coupling (ECC) depends on Ca2+ release from intracellular stores via ryanodine receptors (RyRs) triggered by L-type Ca2+ channels (LCCs). Uncertain numbers of RyRs and LCCs form 'couplons' whose activation produces Ca2+ sparks, which summate to form a cell-wide Ca2+ transient that switches on contraction. Voltage (Vm) changes during the action potential (AP) and stochasticity in channel gating should create variability in Ca2+ spark timing, but Ca2+ transient wavefronts have remarkable uniformity. To examine how this is achieved, we measured the Vm-dependence of evoked Ca2+ spark probability (Pspark) and latency over a wide voltage range in rat ventricular cells. With depolarising steps, Ca2+ spark latency showed a U-shaped Vm-dependence, while repolarising steps from 50 mV produced Ca2+ spark latencies that increased monotonically with Vm. A computer model based on reported channel gating and geometry reproduced our experimental data and revealed a likely RyR:LCC stoichiometry of âˆ¼ 5:1 for the Ca2+ spark initiating complex (IC). Using the experimental AP waveform, the model revealed a high coupling fidelity (Pcpl âˆ¼ 0.5) between each LCC opening and IC activation. The presence of âˆ¼ 4 ICs per couplon reduced Ca2+ spark latency and increased Pspark to match experimental data. Variability in AP release timing is less than that seen with voltage steps because the AP overshoot and later repolarization decrease Pspark due to effects on LCC flux and LCC deactivation respectively. This work provides a framework for explaining the Vm- and time-dependence of Pspark, and indicates how ion channel dispersion in disease can contribute to dyssynchrony in Ca2+ release.

Cardiac-specific overexpression of caveolin-3 preserves t-tubular ICa during heart failure in mice.

NEW FINDINGS: What is the central question of this study? What is the cellular basis of the protection conferred on the heart by overexpression of caveolin-3 (Cav-3 OE) against many of the features of heart failure normally observed in vivo? What is the main finding and its importance? Cav-3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t-tubular ICa , but not local t-tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav-3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release. ABSTRACT: Caveolin-3 (Cav-3) is an 18 kDa protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted and excitation-contraction coupling (ECC) is impaired. Previous work has suggested that Cav-3 overexpression (OE) is cardio-protective, but the effect of Cav-3 OE on these cellular changes is unknown. We therefore investigated whether Cav-3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks' transverse aortic constriction (TAC). Cav-3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t-tubule regularity and density, and impaired local Ca2+ release at the t-tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav-3 OE compared to WT mice, and t-tubular Ca2+ current (ICa ) density was maintained. These data suggest that Cav-3 OE helps prevent hypertrophy and loss of t-tubular ICa following TAC, but that other factors disrupt local Ca2+ release.

Caveolin-3 KO disrupts t-tubule structure and decreases t-tubular ICa density in mouse ventricular myocytes.

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current ( ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.

Solute movement in the t-tubule system of rabbit and mouse cardiomyocytes.

Cardiac transverse (t-) tubules carry both electrical excitation and solutes toward the cell center but their ability to transport small molecules is unclear. While fluorescence recovery after photobleaching (FRAP) can provide an approach to measure local solute movement, extraction of diffusion coefficients is confounded by cell and illumination beam geometries. In this study, we use measured cellular geometry and detailed computer modeling to derive the apparent diffusion coefficient of a 1-kDa solute inside the t-tubular system of rabbit and mouse ventricular cardiomyocytes. This approach shows that diffusion within individual t-tubules is more rapid than previously reported. T-tubule tortuosity, varicosities, and the presence of longitudinal elements combine to substantially reduce the apparent rate of solute movement. In steady state, large (>4 kDa) solutes did not freely fill the t-tubule lumen of both species and <50% of the t-tubule volume was available to solutes >70 kDa. Detailed model fitting of FRAP data suggests that solute diffusion is additionally restricted at the t-tubular entrance and this effect was larger in mouse than in rabbit. The possible structural basis of this effect was investigated using electron microscopy and tomography. Near the cell surface, mouse t-tubules are more tortuous and filled with an electron-dense ground substance, previously identified as glycocalyx and a polyanionic mesh. Solute movement in the t-tubule network of rabbit and mouse appears to be explained by their different geometric properties, which impacts the use of these species for understanding t-tubule function and the consequences of changes associated with t-tubule disease.

Caveolin 3-dependent loss of t-tubular ICa during hypertrophy and heart failure in mice.

NEW FINDINGS: What is the central question of this study? Heart failure is associated with redistribution of L-type Ca2+ current (ICa ) away from the t-tubule membrane to the surface membrane of cardiac ventricular myocytes. However, the underlying mechanism and its dependence on severity of pathology (hypertrophy versus failure) are unclear. What is the main finding and its importance? Increasing severity of response to transverse aortic constriction, from hypertrophy to failure, was accompanied by graded loss of t-tubular ICa and loss of regulation of ICa by caveolin 3. Thus, the pathological loss of t-tubular ICa , which contributes to impaired excitation-contraction coupling and thereby cardiac function in vivo, appears to be attributable to loss of caveolin 3-dependent stimulation of t-tubular ICa . ABSTRACT: Previous work has shown redistribution of L-type Ca2+ current (ICa ) from the t-tubules to the surface membrane of rat ventricular myocytes after myocardial infarction. However, whether this occurs in all species and in response to other insults, the relationship of this redistribution to the severity of the pathology, and the underlying mechanism, are unknown. We have therefore investigated the response of mouse hearts and myocytes to pressure overload induced by transverse aortic constriction (TAC). Male C57BL/6 mice underwent TAC or equivalent sham operation 8 weeks before use. ICa and Ca2+ transients were measured in isolated myocytes, and expression of caveolin 3 (Cav3), junctophilin 2 (Jph2) and bridging integrator 1 (Bin1) was determined. C3SD peptide was used to disrupt Cav3 binding to its protein partners. Some animals showed cardiac hypertrophy in response to TAC with little evidence of heart failure, whereas others showed greater hypertrophy and pulmonary congestion. These graded changes were accompanied by graded cellular hypertrophy, t-tubule disruption, decreased expression of Jph2 and Cav3, and decreased t-tubular ICa density, with no change at the cell surface, and graded impairment of Ca2+ release at t-tubules. C3SD decreased ICa density in control but not in TAC myocytes. These data suggest that the graded changes in cardiac function and size that occur in response to TAC are paralleled by graded changes in cell structure and function, which will contribute to the impaired function observed in vivo. They also suggest that loss of t-tubular ICa is attributable to loss of Cav3-dependent stimulation of ICa .

Late Ca2+ Sparks and Ripples During the Systolic Ca2+ Transient in Heart Muscle Cells.

RATIONALE: The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. OBJECTIVE: To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. METHODS AND RESULTS: We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. CONCLUSIONS: These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.

Loss of caveolin-3-dependent regulation of ICa in rat ventricular myocytes in heart failure.

ß2-Adrenoceptors and L-type Ca2+ current ( ICa) redistribute from the t-tubules to the surface membrane of ventricular myocytes from failing hearts. The present study investigated the role of changes in caveolin-3 and PKA signaling, both of which have previously been implicated in this redistribution. ICa was recorded using the whole cell patch-clamp technique from ventricular myocytes isolated from the hearts of rats that had undergone either coronary artery ligation (CAL) or equivalent sham operation 18 wk earlier. ICa distribution between the surface and t-tubule membranes was determined using formamide-induced detubulation (DT). In sham myocytes, ß2-adrenoceptor stimulation increased ICa in intact but not DT myocytes; however, forskolin (to increase cAMP directly) and H-89 (to inhibit PKA) increased and decreased, respectively, ICa at both the surface and t-tubule membranes. C3SD peptide (which decreases binding to caveolin-3) inhibited ICa in intact but not DT myocytes but had no effect in the presence of H-89. In contrast, in CAL myocytes, ß2-adrenoceptor stimulation increased ICa in both intact and DT myocytes, but C3SD had no effect on ICa; forskolin and H-89 had similar effects as in sham myocytes. These data show the redistribution of ß2 -adrenoceptor activity and ICa in CAL myocytes and suggest constitutive stimulation of ICa by PKA in sham myocytes via concurrent caveolin-3-dependent (at the t-tubules) and caveolin-3-independent mechanisms, with the former being lost in CAL myocytes. NEW & NOTEWORTHY In ventricular myocytes from normal hearts, regulation of the L-type Ca2+ current by ß2-adrenoceptors and the constitutive regulation by caveolin-3 is localized to the t-tubules. In heart failure, the regulation of L-type Ca2+ current by ß2-adrenoceptors is redistributed to the surface membrane, and the constitutive regulation by caveolin-3 is lost.

The Effects of Aging on the Regulation of T-Tubular ICa by Caveolin in Mouse Ventricular Myocytes.

Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.

Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t-tubule membranes in mouse ventricular myocytes.

Cholesterol is a key component of the cell plasma membrane. It has been suggested that the t-tubule membrane of cardiac ventricular myocytes is enriched in cholesterol and that this plays a role in determining t-tubule structure and function. We have used methyl-ß-cyclodextrin (MßCD) to deplete cholesterol in intact and detubulated mouse ventricular myocytes to investigate the contribution of cholesterol to t-tubule structure, membrane capacitance, and the distribution of Ca flux pathways. Depletion of membrane cholesterol was confirmed using filipin; however, di-8-ANEPPS staining showed no differences in t-tubule structure following MßCD treatment. MßCD treatment had no significant effect on the capacitance:volume relationship of intact myocytes or on the decrease in capacitance:volume caused by detubulation. Similarly, Ca influx and efflux were not altered by MßCD treatment and were reduced by a similar amount following detubulation in untreated and MßCD-treated cells. These data show that cholesterol depletion has similar effects on the surface and t-tubule membranes and suggest that cholesterol plays no acute role in determining t-tubule structure and function.

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±18nm in rabbit and 169±15nm in mouse (p<0.001). Rabbit TT diameters were more variable than those of mouse (p<0.01) and the smallest TT detected was 41nm in mouse and the largest 695nm 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 ~175µm in rabbit and ~120µm 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.

Sarcolemmal distribution of ICa and INCX and Ca2+ autoregulation in mouse ventricular myocytes.

The balance of Ca2+ influx and efflux regulates the Ca2+ load of cardiac myocytes, a process known as autoregulation. Previous work has shown that Ca2+ influx, via L-type Ca2+ current (ICa), and efflux, via the Na+/Ca2+ exchanger (NCX), occur predominantly at t-tubules; however, the role of t-tubules in autoregulation is unknown. Therefore, we investigated the sarcolemmal distribution of ICa and NCX current (INCX), and autoregulation, in mouse ventricular myocytes using whole cell voltage-clamp and simultaneous Ca2+ measurements in intact and detubulated (DT) cells. In contrast to the rat, INCX was located predominantly at the surface membrane, and the hysteresis between INCX and Ca2+ observed in intact myocytes was preserved after detubulation. Immunostaining showed both NCX and ryanodine receptors (RyRs) at the t-tubules and surface membrane, consistent with colocalization of NCX and RyRs at both sites. Unlike INCX, ICa was found predominantly in the t-tubules. Recovery of the Ca2+ transient amplitude to steady state (autoregulation) after application of 200 µM or 10 mM caffeine was slower in DT cells than in intact cells. However, during application of 200 µM caffeine to increase sarcoplasmic reticulum (SR) Ca2+ release, DT and intact cells recovered at the same rate. It appears likely that this asymmetric response to changes in SR Ca2+ release is a consequence of the distribution of ICa, which is reduced in DT cells and is required to refill the SR after depletion, and NCX, which is little affected by detubulation, remaining available to remove Ca2+ when SR Ca2+ release is increased.NEW & NOTEWORTHY This study shows that in contrast to the rat, mouse ventricular Na+/Ca2+ exchange current density is lower in the t-tubules than in the surface sarcolemma and Ca2+ current is predominantly located in the t-tubules. As a consequence, the t-tubules play a role in recovery (autoregulation) from reduced, but not increased, sarcoplasmic reticulum Ca2+ release.

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 Ca(2+) 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 Ca(2+) spark frequency from about 7 to 32/100 µm(3)/s (n = 10), P = 0.05 with a reduction in the peak amplitude of the sparks. Simultaneous measurements of intracellular Ca(2+) and isometric force in multicellular trabeculae (n = 7, 1.5 mmol/L [Ca(2+)]o) revealed no effect of Epac activation on either the amplitude of Ca(2+) 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/mm(2) vs cpTOME 23 ± 5 mN/mm(2), P = 0.20). However, an effect of Epac in trabeculae was unmasked by lowering extracellular [Ca(2+)]o. In these depotentiated trabeculae, activation of the Epac pathway increased myofilament Ca(2+) sensitivity, an effect that was blocked by addition of KN-93, a Ca(2+)/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.

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 ~18weeks 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.

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: CD34(pos) 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, CD34(pos)/CD31(neg) 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-Kit(pos) 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.

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 ß2-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 ß2-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 ß2-adrenoceptor stimulation.

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.

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.

Local control in cardiac E-C coupling.

The development of local control theories in cardiac excitation-contraction coupling solved a major problem in the calcium-induced calcium release (CICR) hypothesis. Local control explained how regeneration, inherent in the CICR mechanism, might be limited spatially to enable graded Ca release (and force production). The key lies in the stochastic recruitment of individual calcium release units (couplons or CRUs) where adjacent CRUs are partially uncoupled by the distance between them. In the CRU, individual groups of sarcoplasmic reticulum calcium release channels (RyRs) are very close to the surface membrane where calcium influx, controlled by membrane depolarization, leads to high local Ca levels that enable a high speed response from RyRs that have a very low probability to opening at resting Ca levels. However, calcium diffusion from an activated CRU results in adjacent CRUs being exposed to much lower levels of Ca and probability of activation. This effectively uncouples the CRUs and limits overall regenerative gain to enable stability without compromising sensitivity. Nevertheless, it is still unclear how the CRU terminates its release of calcium on the physiological timescale, and possible mechanisms (and problems) are briefly reviewed. We suggest that modulation in RyR gating may serve to control average SR Ca levels to regulate other metabolic functions of the sarco(endo)plasmic reticulum beyond regulating contractility. This article is part of a special issue entitled "Local Signaling in Myocytes."