Direct Evidence for Microdomain-Specific Localization and Remodeling of Functional L-Type Calcium Channels in Rat and Human Atrial Myocytes

Supplemental Digital Content is available in the text.

macromolecular signaling complexes and scaffolding proteins, which enables the modulation of Ca 2+ signaling. 11 Although the main population of LTCCs is localized to dyadic junctions, extradyadic channels are also associated with the surface membrane. 12,13 Caveolin-3 (Cav3)-rich signaling microdomains are found to harbor specific LTCCs that may play an important role in modulation of Ca 2+ signaling, particularly in cells lacking organized T-tubules such as atrial 5,[14][15][16] and neonatal 17 myocytes. However, until recently, it was difficult to test this hypothesis because of the lack of appropriate experimental approaches.
The spatial compartmentation of Ca 2+ -signaling complexes was first assessed by immunofluorescence microscopy, 18 but imaging does not provide information on the functionality of channel proteins within a subcellular domain. Recent methodological advances have made it possible to image the topography of a live cardiomyocyte and study the clustering of functional ion channels within specific microdomains. 2 Here, we used a super-resolution scanning patchclamp technique to study the distribution of functional LTCCs in different structural microdomains of the sarcolemma of rat and human atrial myocytes. We provide direct evidence for 2 distinct subpopulations of atrial LTCCs: one localized in the T-tubules and another linked to caveolae structures. Our findings demonstrate that LTCCs, which are located in caveolae, critically contribute to atrial Ca 2+ signaling, particularly in cardiomyocytes lacking an organized T-tubule network. These different LTCC subpopulations may underlie the regional heterogeneity of Ca 2+ signaling and susceptibility to spontaneous Ca 2+ release events in the atrial. Using a rat model of heart failure (HF), we highlight the relevance of the concept of microdomain-specific remodeling of LTCCs: a disruption in the delicate balance of dynamic interactions between dyadic LTCCs and their cellular microenvironment can alter Ca 2+ signaling and lead to pathological changes in cellular physiology.

Materials and Methods
Detailed methods are provided in the online-only Data Supplement Methods.
All studies complied with the United Kingdom Home Office regulation governing the care and use of laboratory animals and with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).

Myocyte Isolation, T-Tubule Characterization, and Animal Models
Single atrial myocytes were isolated separately from both left (LA) and right (RA) atrial of control and 16-week post-myocardial infarction rats (online-only Data Supplement Table I). The subcellular T-tubule system was visualized by confocal imaging of Di-8-ANEPPS-stained cells. Surface topography was characterized by scanning ion conductance microscopy which uses a glass nanopipette as a sensitive probe. 19

Super-resolution Scanning Patch-Clamp With Pipette Clipping Modification
After generating a topographical image of the cell surface by scanning ion conductance microscopy, the tip diameter of the pipette was widened by clipping 19 to increase the area of attachment. The pipette was then lowered to a specific location until it touched the membrane, and a high-resistance seal was established. Recordings were then performed in a cell-attached mode. Controlled widening of the scanning nanopipette tip is described in detail in the online-only Data Supplement Methods. Macroscopic calcium currents were recorded by using the whole-cell patch-clamp technique. 20

Optical Mapping and Data Analysis
Optical mapping of cells loaded with the Ca 2+ -sensitive fluorescent dye Fluo-4 AM via a complementary metal-oxide semiconductor camera ULTIMA-L (SciMedia, USA Ltd, Costa Mesa, CA) was used to monitor localized changes in [Ca 2+ ] i .

Statistical Analysis
All graphs and statistical analysis were performed by using either GraphPad prism 5 or Origin version 6.1. The average values were calculated throughout all cells studied within the groups and then compared between the groups. Normality was tested using the Kolmogorov-Smirnov test. In cases where data failed the normality test, the nonparametric Mann-Whitney test was used instead of the unpaired Student t test, and the nonparametric Kruskal-Wallis test was used instead of analysis of variance. Statistical differences were assessed with analysis of variance, Student t test, Mann-Whitney test, Kruskal-Wallis test, χ 2 , and Fisher exact test as appropriate. All data are expressed as mean±standard error of the mean. A value of P<0.05 was considered statistically significant.

In Situ T-Tubule Imaging in Isolated Rat Atrial Preparations
To characterize the atrial T-tubular network, we performed in situ T-tubule imaging on intact rat atrial using whole-mount fluorescence labeling with glycophilic lectin wheat germ agglutinin. We found significant region-dependent heterogeneity in T-tubule structure throughout the atrial. Although the LA myocardium predominantly consisted of cardiomyocytes with T-tubules ( Figure 1A, Right), in the RA, we observed 3 groups of cardiomyocytes: cells (1) with organized T-tubules, (2) with disorganized T-tubules, and (3) with absent T-tubules ( Figure 1A, Left).

Subcellular T-Tubule System in Rat Atrial Cardiomyocytes
Confocal imaging of Di-8-ANEPPS-stained cardiomyocytes isolated separately from the LA and RA revealed that, although about one-third of cells do not have T-tubules (≈39% in RA; 34% in LA), other cardiomyocytes possess a T-tubular network of some sort. We also found cells with organized T-tubular networks similar to those found in ventricular myocytes, and those with disorganized T-tubules ( Figure 1B). The majority of atrial myocytes with organized T-tubules were isolated from the LA (≈40% versus 2% in LA versus RA). Conversely myocytes isolated from the RA were more likely to have disorganized T-tubule network (26% versus 59% in LA versus RA) (onlineonly Data Supplement Figure I). In ventricular rat myocytes, T-tubules are distributed regularly at ≈2-µm intervals, as demonstrated by Fourier transform of binarized images of T-tubules ( Figure 1C). In contrast, the atrial T-tubular network is less dense and less regular than in ventricular myocytes. Atrial myocytes with organized T-tubules showed a smaller, but still distinct peak in the Fourier transform plot (3.1±0.6 AU versus 11.3±0.9 AU in atrial (n=31 cells from 4 rats) versus ventricular myocytes (n=45  cells from 3  disorganized or absent T-tubules (both in LA and RA) did not show a dominant peak on the Fourier transform plot ( Figure 1C). Organization of the atrial T-tubular network correlates with cell width: cells showing organized T-tubular networks were larger than cells with disorganized or absent T-tubules ( Figure 1D and online-only Data Supplement Figure II).

Surface Structures in Rat Atrial Myocytes
Our previous scanning ion conductance microscopy imaging of rat ventricular myocytes 21 has clearly showed the surface topography to be structured with T-tubule openings arranged along Z-grooves and the domed crests located in between them. To characterize cardiomyocyte topography, we have introduced the Z-groove index 21 : a ratio of the observed Z-groove length to the total extrapolated Z-groove length (as if they were present throughout the entire surface). We applied this analysis to characterize topography of atrial myocytes. We found that the larger an atrial myocyte, the more regular its surface topography. This is consistent with the more organized T-tubule network noted in these larger myocytes.
Atrial myocytes with organized surface structures and apparent T-tubule openings, similar to those seen in ventricular myocytes, were wider than those with patchy nonstructured areas on their surface, which in turn were wider than atrial myocytes that entirely lacked surface structures ( Figure 1E). Similarly, the Z-groove index was significantly higher in LA versus RA myocytes (0.66±0.03 versus 0.50±0.03 in LA versus RA, respectively, P<0.001), confirming the presence of more structured myocytes in the LA.

Rat Atrial Myocyte Ca 2+ Signaling
Atrial myocytes differ strikingly from ventricular myocytes in shape, magnitude, and kinetics of subcellular Ca 2+ transients and in the dynamic of spontaneous Ca 2+ release events, as well. 4,6 These differences were hypothesized to be largely attributed to the distinct structure of atrial T-tubules and to altered distribution of LTCCs and their coupling to RyR, as well. 10 Optical mapping of spontaneous Ca 2+ activity revealed that atrial myocytes have different Ca 2+ cycling than ventricular myocytes. Among all spontaneous Ca 2+ release events, Ca 2+ waves propagating throughout the entire cell ( Figure 2A) were distinguished from nonpropagating Ca 2+ release events ( Figure 2B). Although we did not distinguish Ca 2+ sparks from Ca 2+ puffs, 22,23 we observed 2 groups of nonpropagating Ca 2+ events: one with a smaller amplitude (23±1% of the amplitude of the corresponding paced Ca 2+ transients, 60% of all nonpropagating Ca 2+ events) and another with a higher amplitude (60±4% of the paced Ca 2+ transient amplitude, 40% of all nonpropagating Ca 2+ events), which could be attributed to Ca 2+ puffs and Ca 2+ sparks, respectively. 22 In comparison with ventricular myocytes, atrial cells showed a significantly higher frequency of nonpropagating Ca 2+ release events following a period of high-frequency stimulation ( Figures 2C and 2D and onlineonly Data Supplement Figure III). We found that wider rat atrial myocytes exhibited a significantly higher number of spontaneous Ca 2+ release events ( Figure 2E). No spontaneous Ca 2+ release events were observed in cells thinner than 11.0±0.4 µm (P=0.002 versus cells with events, Figure 2E). In addition, nonpropagating Ca 2+ release events were observed in atrial myocytes 13.2±0.6 µm wide, whereas propagating Ca 2+ waves were found in cells ≥14.9±1.1 µm wide (data not shown). Cardiomyocytes isolated from the LA demonstrated a higher number of Ca 2+ release events than RA myocytes ( Figures 2F and 2G). In atrial myocytes that exhibited spontaneous Ca 2+ release events, the number of events was found to be proportional to cell width, as shown in Figure 2H.

Spatial Localization of Functional LTCCs
In adult rat ventricular myocytes, functional LTCCs are predominantly localized in the T-tubules. 2 Here, we applied the same super-resolution scanning patch-clamp method 2 (Figures 3A through 3C) and found that, in contrast to ventricular myocytes, in rat atrial cells LTCCs were recorded with similar frequency from T-tubules (T-LTCC), crests (C-LTCC), and nonstructured areas ( Figure 3D Figure 3E). Like many other types of channels, LTCCs show multiple subconductance levels in addition to the largest and the main open state of the channel. 24,25 Examples of these substates are given in Figure 3F, Top, and online-only Data Supplement Figure IV. As summarized in Figure 3F, Bottom, C-LTCCs exhibited low-amplitude subconductance states more frequently than T-LTCCs: open probability of low-amplitude subconductance states was 0.027±0.005 and 0.005±0.002 for C-LTCCs (n=4) and T-LTCCs (n=5), respectively, P<0.01.
In addition, we explored microdomain-specific distribution of functional LTCCs in several human RA samples ( Figure 4A). On average, human atrial myocytes had a Z-groove index of 0.57±0.02 (n=27), which is similar to that measured in rat RA. Similar to rat, human LTCCs were recorded with similar frequency from T-tubules and crest ( Figure 4B). We did not observe any difference in the voltage-current characteristics between the areas ( Figure 4C). However, in contrast to rat, human T-LTCCs had significantly higher open probability than C-LTCCs: P(open) at -6.7mV: 0.03±0.002 for T-LTCCs versus 0.017±0.001 for C-LTCCs, P<0.001.

Caveolae as a Source of Extradyadic LTCCs
It has been demonstrated in ventricular myocytes that some LTCCs could be housed in caveolae. 12,13 We hypothesized that atrial extratubular LTCCs recorded from the crests and nonstructured areas might be localized to caveolae. To address this hypothesis, we used 2 different approaches to disrupt caveolae: treatment with methylβ-cyclodextrin (MβCD) and direct LTCC inhibition in Cav3-containig membranes using a specific peptide inhibitor Rem.

Ignition of Ca 2+ Sparks Through Caveolae
Along with changes in LTCC distribution, MβCD significantly suppressed the occurrence of spontaneous Ca 2+ release events in rat atrial myocytes (online-only Data Supplement Figure VIII). MβCD treatment significantly decreased the number of spontaneous Ca 2+ sparks, but not waves ( Figure 5E) and reduced the number of cells featuring spontaneous Ca 2+ release events ( Figure 5F). Moreover, removing caveolae via MβCD treatment also reduced the amplitude of Ca 2+ sparks in atrial myocytes ( Figure 5G), as has been previously demonstrated in neonatal ventricular myocytes and arterial smooth muscle cells. 17

Caveolae-Targeted LTCC Antagonist Eliminates Occurrence of Extradyadic LTCC Current
To confirm that extratubular LTCCs are localized to Cav3associated caveolae structures, rather than lipid rafts, we used a Cav3-targeted LTCC-blocking agent, Rem peptide. 12 The caveolae-targeted LTCC blocker (Rem 1-265 -Cav) was generated by molecular modification of Rem, a member of the RGK GTPase family that is known to potently inhibit LTCCs. 27 Makarewich

HF Model and Structural Changes in the Atrial
To study disease-associated atrial remodeling, we used the 16 weeks of post-myocardial infarction rat model of HF. 28 This model recapitulates many features of chronic HF in patients including adverse remodeling of the organ, characterized by left ventricle dilatation, reduced ejection fraction, and raised filling pressures (online-only Data Supplement Table I). Atrial cellular hypertrophy was noted with an increase in planar width ( Figure 7A). Similar to control, in HF we also observed 3 groups of cardiomyocytes, although cells with organized T-tubules were found exclusively in the LA ( Figure 7B). Despite cell hypertrophy, HF led to a profound degradation of the T-tubule system in all groups of cells, decreasing the proportion of structured myocytes and reducing the T-tubule density in both LA and RA ( Figure 7C). Similarly, HF resulted in the loss of surface structures and a reduction in Z-groove index (0.57±0.01 versus 0.45±0.02 in control versus HF, P<0.001).

Functional Atrial Remodeling in HF
Along with the degradation of the atrial T-tubular system, HF also caused altered Ca 2+ cycling. We found a ≈30% increase in spontaneous Ca 2+ spark frequency and a 3-fold increase in wave frequency ( Figure 8A and 8B). The average amplitude of nonpropagated Ca 2+ events was increased, perhaps suggesting a greater proportion of high-amplitude events (Ca 2+ events), as opposed to low-amplitude events (Ca 2+ puffs) (23/77 versus 60/40 for Ca 2+ puffs/sparks in HF versus control, respectively). In addition, failing atrial myocytes were associated with slower and wider Ca 2+ waves ( Figure 8D and online-only Data Supplement Figure IX), which may result from T-tubule degradation and increase time required for dyad-to-dyad Ca 2+ propagation.

Microdomain-Specific Remodeling of Atrial LTCCs in HF
When characterized by the super-resolution scanning patch clamp, LTCCs in HF cells demonstrate the same equal distribution between T-tubules and crests as observed in control cells (34.3% and 30.5% versus 28% and 30.1% for T-LTCCs and C-LTCCs in HF versus control, respectively, NS). This corresponds with the similar caveolae density in HF and control myocytes (3.7±0.5 caveolae/µm in control versus 4.6±0.4 caveolae/µm in HF, P=0.167). Although we did not observe any significant changes in open probability for T-LTCCs and C-LTCCs in HF, we found that T-LTCCs had ≈25% smaller amplitude in HF in comparison with control T-LTCCs ( Figure 8E). No changes in C-LTCC amplitude were revealed in HF. The decrease in T-LTCC amplitude in HF was associated with a change in the accessibility of the channel subconductance states ( Figure 8F). In HF, the occupancy of low-amplitude substates becomes more accessible than high-amplitude substates in comparison with control (0.018±0.006 and 0.006±0.002 versus 0.006±0.002 and 0.075±0.005 for low-and high-amplitude subconductance states in HF versus control, respectively).

Functional Anatomy of Atrial Myocardium
Atrial myocytes have long been perceived as having no or very few T-tubules. 3,10,29 However, recent experimental evidence demonstrates that atrial myocytes from certain species, such as sheep, 30 cows, horses, humans, 5 and even rodents 4,10,15 do possess an organized T-tubule network. In general, atrial T-tubules are sparse and less regular when compared with those in ventricular myocytes as assessed both in situ 31 and in vitro. 10 Our observations of T-tubules in rat atrial myocytes are consistent with those previously observed using electron microscopy 32,33 and fluorescent membrane labeling. 4,16 In the present study, we found significant heterogeneity of T-tubule organization between LA and RA, which might correlate with the arrangement of pectinate muscle bundles within the atrial appendages. It has been reported in rabbit atrial that myocytes isolated from the crista terminalis were significantly larger than those from the pectinate muscles, whereas the shape (the ratio of the length to the width) was found to be similar in the 2 types of cells. 34 Anatomic heterogeneity of the T-tubular system within the atrial has been proposed to underlie heterogeneous calcium current measured within the right atrium, in addition to different expression of LTCCs. In healthy dogs, Ca 2+ influx through LTCCs was found to be largest in crista terminalis cells, intermediate in cells from the appendage and pectinate muscles, and smallest in atrioventricular ring cells. 35 In contrast, in rabbit right atrial myocytes isolated from different areas, whole-cell clamp recordings showed no definite variation in the density of the voltage-dependent LTCCs. 34 Frisk and colleagues 16 used both isolated atrial cells and tissue to demonstrate that in pig and rat atrial there was a high variability in the distribution of T-tubules and I Ca,L among cells, with a steep dependence of I Ca,L on atrial myocyte capacitance and T-tubule density. The authors observed more T-tubules in the epicardium than in the endocardium, which may contribute to the synchronization of contraction across the atrial wall. Thus, anatomic heterogeneity of the T-tubule network and I Ca,L may explain complex features of atrial electric and mechanical activity including highly anisotropic physiological activation patterns, 36 action potential duration distribution, 37 and contractile function. In addition, disease-associated loss of T-tubules found in HF 30 and atrial fibrillation 38 may have an impact on calcium cycling and promotes the development of triggers of arrhythmia. One might hypothesize that distinct anatomic regions within the atrial will differentially respond on the stress and thus promote the propagation of arrhythmia triggers at specific locations aroused from superposition of areas of significant T-tubular degradation with those of profound molecular remodeling. 39 The mechanisms that result in the development of such trigger "hot spots" require special investigation and will be the focus of our follow-up studies.

Role of Caveolae Structures in Atrial Ca 2+ Signaling
It has been proposed that, because of the lack of a regular T-tubule system in atrial myocytes, differential spatial distribution of LTCCs with regard to their coupling to RyR2s may underlie a unique atrial myocyte Ca 2+ -signaling process. 9,40 In the present study, we uncovered for the first time the distinct distribution of functional atrial LTCCs in the sarcolemma where they appear at a similar frequency both in the T-tubules and the crests, in contrast to ventricular myocytes where LTCCs were primarily clustered in the T-tubules. 2 We demonstrated the importance of the extradyadic channels, which are predominantly located in caveolae, in the regulation of Ca 2+ signaling, particularly in cardiomyocytes lacking an organized T-tubule network. The function of LTCCs localized in caveolae remains open to question. It has been proposed that some LTCCs housed in Cav3-rich microdomains, could play an important role in the modulation of Ca 2+ signaling. Indeed, local spontaneous Ca 2+ release events are plentiful and, in contrast to ventricular myocytes, seem to be normally present in healthy atrial. 6,41 Walden and colleagues demonstrated important differences in Ca 2+ -handling mechanisms between ventricular and atrial myocytes: atrial myocytes were found to have a more robust and abundant Ca 2+ uptake mechanism and a higher SR Ca 2+ content than ventricular myocytes. 7 Higher SR Ca 2+ content in atrial myocytes has been proposed to lead to Ca 2+ overload and thus increase the sensitivity of RyR2s to cytosolic [Ca 2+ ] i . 9 This means that 1 Ca 2+ spark is more likely to trigger another Ca 2+ spark in atrial myocytes but remains a rare event in normal ventricular myocytes. Therefore, the benefit of the elevated SR Ca 2+ content is that it should improve the synchrony of the atrial [Ca 2+ ] i transients when T-tubules are disorganized or absent. The downside is that in atria the elevated SR Ca 2+ content and enhanced [Ca 2+ ] SR lead to the increased sensitivity of the Ca 2+ -induced Ca 2+ release process. For thin cells, or thick cells with organized T-tubules, the Ca 2+ signal propagation is likely to be relatively stable. However, larger myocytes with disorganized T-tubules may have an increased propensity toward subcellular Ca 2+ alternans and thus appear to be more prone to Ca 2+ sparks as demonstrated in our study (Figure 2). Uncontrolled [Ca 2+ ] i elevations, as occurs with Ca 2+ alternans, 42 will activate the Na/Ca exchanger and thereby generate Na/Ca exchanger current (I NCX ) between action potentials with subsequent induction of delayed afterdepolarizations. Thus, the high SR Ca 2+ content in atrial myocytes may be proarrhythmic. Such arrhythmogenic tendencies are unmasked in HF as cells undergo hypertrophy and the T-tubule system degrades (Figures 7 and 8). Slower and wider Ca 2+ waves observed in failing cells in the present study would lead to prolonged depolarization time required for Na/Ca exchanger activation and thus they are more likely to result in the formation of delayed afterdepolarizations observed in failing atrial. 43,44 Along with elevated diastolic [Ca 2+ ] i and SR Ca 2+ overload, observed in HF, it would result in the formation of foci of triggered ectopic activity located within areas of significant T-tubular degradation.

Unique Atrial Myocyte Ca 2+ Signaling
It is possible that the mechanism behind the localized spontaneous Ca 2+ release events in atrial differs from that in ventricles. In rat ventricular myocytes, 85% of all Ca 2+ sparks evoked by electric stimulation occur within 0.5 µm of a T-tubule, 45 and formamide-induced detubulation significantly reduces Ca 2+ sparks in rat ventricular myocytes, 14 9 Nonjunctional Ca 2+ events might be attributed to inositol-1,4,5-trisphosphate (IP 3 ) dependent activation of nonjunctional RyRs. 22 Recently, Horn et al 47 have shown that IP 3 can effectively modulate RyR openings and Ca 2+ spark probability. Direct interaction between Cav3 and IP 3 -associated G q -protein-coupled receptor-signaling pathway has been demonstrated in canine ventricular myocytes 48 which can link IP 3 -dependent nonjunctional Ca 2+ events to caveolae and explain a significant decrease in occurrence of spontaneous Ca 2+ sparks observed in atrial myocytes treated with MβCD ( Figures 5E through 5G). Interestingly, a similar reduction in Ca 2+ sparks via direct inhibition of caveolae-housed LTCCs by Rem protein (Figure 6) highlights the importance of these channels in the initiation of Ca 2+ sparks. Alternatively, it is possible that MβCD-or Rem 1-265 -Cav-induced reduction in the whole-cell I Ca,L decreases the steady-state SR Ca 2+ load and thus suppresses the occurrence of spontaneous Ca 2+ transients observed in our experiments.

Microdomain-Specific Remodeling of Atrial LTCCs in HF
Decrease in atrial I Ca,L in HF has been shown in both animal models 49 and patients with congestive HF. 50 Taking into account the T-tubule degradation, one would expect a reduction of the number of functional channels to be mainly responsible  for the reduction in I Ca,L . However, as we demonstrated in the present study, an additional mechanism responsible for the reduction in I Ca,L in failing atrial might be represented by the reduction in the amplitude of T-LTCCs. Despite the decreased I Ca,L , an increase in SR Ca 2+ load (caffeine-induced [Ca 2+ ] i release) has been observed in failing atrial. 44,49 In addition to the increased SR Ca 2+ loading, a significant reduction in calsequestrin expression has been found in failing atrial, 44 and this has been linked to increase in SR Ca 2+ leak and atrial arrhythmogenesis, perhaps as a result of decreased SR Ca 2+ buffering. 39 Both SR Ca 2+ leak and elevated diastolic [Ca 2+ ] i may affect T-LTCC current, causing a reduction in amplitude, either through Ca 2+ -dependent inactivation or phosphorylation ( Figures 8E and 8F). Therefore, a disruption in the delicate balance of dynamic interactions between dyadic LTCCs and their microenvironment may alter Ca 2+ signaling and can lead to pathological changes in cellular physiology. This extends beyond the classical concept of electric remodeling, stressing that alterations of spatial compartmentation of ion channels and receptors are responsible for pathology, in addition to classically appreciated changes in protein expression and posttranslational modifications.

Limitations
In the present study, we used MβCD to disrupt caveolae structures that might have some potential side effects on cellular electrophysiology. However, it is the only available approach to physically destroy caveolae at the moment and it is widely used by many groups, without significant effects on T-tubule structure. 13,17,26 Accordingly, in our study, after 30 minutes treatment with 10 mM MβCD, no changes in cell topography and T-tubule density were found (online-only Data Supplement Figure V). In addition, MβCD treatment did not affect T-LTCC biophysical properties: neither T-LTCC occurrence nor their open probability and current-voltage characteristics were altered. Moreover, similar results on LTCC distribution and Ca 2+ activity were obtained with use of a specific Cav3-targeted LTCC-blocking agent, Rem peptide, 12 suggesting no direct effect of MβCD on LTCC. Therefore, the influence on the use of MβCD in this study should be limited, if there is any, and will not affect the result and the deducting conclusion regarding the LTCC distribution and functioning.