|Year : 2019 | Volume
| Issue : 2 | Page : 63-69
Functional role of t-tubules on calcium transients in canine cardiac myocytes
Alicia A Barnes, Zachary E Williams, Samuel Olzcyk, Alida Cooke, Jonathan A Cordeiro, Tanya Zeina, Rueben Mathew, Jacqueline A Treat, Gary L Aistrup, Jonathan M Cordeiro
Department of Experimental Cardiology, Masonic Medical Research Institute, Utica, New York, USA
|Date of Submission||19-Sep-2019|
|Date of Acceptance||09-Oct-2019|
|Date of Web Publication||25-Nov-2019|
Dr. Jonathan M Cordeiro
Department of Experimental Cardiology, Masonic Medical Research Institute, 2150 Bleecker Street, Utica, New York 13501
Source of Support: None, Conflict of Interest: None
Background: A loss of t-tubules and alterations in ultrastructure occur in cultured ventricular myocytes. A similar alteration in t-tubules and ultrastructure is well documented under certain pathological conditions such as heart failure. We examined the ultrastructural changes in cultured canine cardiac cells and determined the functional impact these changes have on excitation-contraction coupling and Ca2+ transients. Materials, Methods and Results: Atrial, ventricle, and Purkinje myocytes were isolated by enzymatic dispersion. Atrial and ventricular myocytes were cultured for up to 48 h. Voltage clamp recordings were made with patch electrodes. Ca2+ transient was recorded as a laser scanning confocal microscope in myocytes loaded with Ca2+ fluorescent dyes. Membrane ultrastructure was imaged in myocytes stained with the membrane selective dye, di-8-ANEPP. Freshly isolated ventricular myocytes had a well-developed t-tubule system, while Purkinje cells had no t-tubules; some atrial cells exhibited a primitive t-tubule system. In atrial and Purkinje cells, the Ca2+ transient had a U-shaped profile with the fluorescence highest at the edge of the cell. In contrast, ventricular myocytes showed a homogeneous rise in Ca2+ at the edge and center of cells. Ventricular myocytes cultured for 2 days showed a nearly complete loss of t-tubules. The Ca2+ transients revealed a phenotypic switch from a homogeneous profile to a “U”-shaped profile. Interestingly, atrial cells in culture maintained their primitive t-tubule system. Conclusions: Our results show that atrial and ventricular myocytes respond differently to being placed in culture. Ventricular myocytes, but not atrial myocytes, quickly lose their t-tubules accompanied by a Ca2+ transient profile suggestive of a phenotypic switch in Ca2+ handling. The differential response also suggests that the various cardiac tissue types would respond differently to pathophysiological stresses.
Keywords: Atria, Ca2+ transients, cell culture, Purkinje, t-tubules, ventricle
|How to cite this article:|
Barnes AA, Williams ZE, Olzcyk S, Cooke A, Cordeiro JA, Zeina T, Mathew R, Treat JA, Aistrup GL, Cordeiro JM. Functional role of t-tubules on calcium transients in canine cardiac myocytes. Heart Mind 2019;3:63-9
|How to cite this URL:|
Barnes AA, Williams ZE, Olzcyk S, Cooke A, Cordeiro JA, Zeina T, Mathew R, Treat JA, Aistrup GL, Cordeiro JM. Functional role of t-tubules on calcium transients in canine cardiac myocytes. Heart Mind [serial online] 2019 [cited 2022 May 20];3:63-9. Available from: http://www.heartmindjournal.org/text.asp?2019/3/2/63/271535
| Introduction|| |
The functional role of transverse tubules (t-tubules) and its relation to the generation of Ca2+ transients in the ventricle is well established., T-tubules are discrete invaginations of the cell membrane which allows the spread of extracellular calcium influx via L-type Ca2+ channels to interior parts of the cell. This results in a nearly instantaneous rise of the Ca2+ transient in ventricular myocytes. T-tubules play a critical role in excitation-contraction (EC) coupling, and alterations in the t-tubule structure have been observed under pathological situations like heart failure (HF). In cell types that are devoid of t-tubules, such as neonate ventricular and Purkinje cells, a very different Ca2+ activation pattern has been observed.,, Confocal line scans in the transverse orientation showed a “U”-shaped profile suggesting that Ca2+ release starts at the cell periphery, and Ca2+ then diffuses toward the center of the cell.
There has been much controversy regarding the presence of t-tubules in atrial tissue. Previous studies have shown the complete absence of t-tubules, the presence of a primitive transverse axial tubular system or a well-developed t-tubular system., T-tubules greatly increase the surface area of the cell resulting in unique localization patterns of ion channels. For example, immunocytochemical analysis has shown that L-type Ca2+ channels are localized predominantly within t-tubule structures in ventricular myocytes. This t-tubular localization results in a larger fraction of sarcoplasmic reticulum (SR) Ca2+ release (ryanodine receptor [RyR]) channels to be associated with L-type channels at junctions. Conversely, in cell types without t-tubules, only RyRs at the cell periphery form junctions with the surface membrane.
During HF, changes in EC coupling and Ca2+ transients are well established. In ventricular myocytes, alterations in the spatiotemporal profile of the Ca2+ transient has been well documented., In atrial myocytes, alterations in the Ca2+ transient have been reported, but it is unclear if these alterations were due to change in the cell ultrastructure or if other mechanisms were involved.
The culturing of cardiac myocytes has been used in many studies and applications.,, Associated with culturing of ventricular myocytes is a change in the electrophysiological properties. In addition, changes in the ultrastructure of the cell include a decline of t-tubules and loss of K+ currents resulting in prolongation of the ventricular action potential., Surprisingly, some of the alterations seen in HF can be found in cultured ventricular myocytes, such as loss of t-tubules and reduced repolarization reserve. There is little information in this regard for cultured atrial myocytes.
Using laser scanning confocal microscopy, we examined the Ca2+ transient profile in canine atria, ventricular, and Purkinje myocytes under identical recording conditions. Results of our study show that Purkinje cells have no t-tubules, ventricular myocytes have an extensive t-tubule network, while a percentage of atrial myocytes show a very sparse network system. Culturing of ventricular myocytes resulted in changes in t-tubule structure and alterations in the Ca2+ transient, whereas atrial myocytes placed in cultured exhibited little change in t-tubules and the Ca2+ transient.
| Matereials and Methods|| |
Atria and ventricular myocyte isolation
This study conforms to the Guide for Care and Use of Laboratory Animals published by the National Institutes of Health (The 8th Edition of the Guide for the Care and Use of Laboratory Animals [NRC 2011]). The Institutional Animal Care and Use Committee provided additional approval. Myocytes were prepared from canine ventricles or atria using established techniques., Right atrial or left ventricular preparations were removed and perfused with a Ca2+-free solution (mM): NaCl 129, NaHCO320, MgSO42.0, KCl 5.4, NaH2 PO40.9, glucose 5.5, and 0.1% bovine serum albumin (BSA). The solution was gassed with 95% O2/5% CO2 for approximately 5 min. The preparation was then perfused with Ca2+-free solution supplemented with 0.5 mg/ml collagenase (Type II, Worthington), 0.1 mg/ml protease (Type XIV, Sigma), and 1 mg/ml BSA for 8–12 min. For isolation of ventricular myocytes, slices of midmyocardial tissue were shaved from the wedge. For atrial myocytes, the pectinate muscle was isolated. The tissue was then placed in different beakers, chopped and incubated in fresh buffer containing 0.5 mg/ml collagenase, 1 mg/ml BSA and agitated. The cells in suspension were collected, filtered, and the myocytes were stored in 0.5 mM Ca2+ HEPES solution until.
Isolation of Purkinje cells
Purkinje cells from dogs were isolated using well-established methods., Adult mongrel dogs of either sex were anesthetized with sodium pentobarbital (35 mg/kg intravenous), their hearts were removed via a thoracotomy and put in Ca2+-free tyrode's solution to clear the blood. Purkinje fibers visualized with a dissecting scope, dissected out and put in a small beaker containing enzyme solution at 36°C. A small stir bar was used to assist with the dissociation of single Purkinje cells from the fibers. At 5 min intervals, enzyme solution containing Purkinje cells in suspension was removed and added to a storage solution. New enzyme solution was added to the undissociated Purkinje fibers. The isolation procedure of the Purkinje fibers into single cells usually lasted 45 min. Myocytes were kept in low Ca2+ storage solution at room temperature (20°C–22°C) and used the day of isolation.
The Ca2+-sensitive dye fluo-4/AM was utilized in this study. Dye was loaded into the myocytes by adding 5 μl of fluo-4/AM to 1 ml of myocytes in suspension. All experiments were performed with either a Zeiss or an Olympus Fluoview confocal microscope. Fluorescence signals were collected with ×40 water-immersion lens. Cells loaded with dye were placed in a perfusion chamber mounted on the stage of the confocal microscope. Excitation at 488 nm was provide by a krypton-argon laser, and the emitted fluorescence was detected through a 520 nm filter and photomultiplier tube. All confocal images were acquired with ZEN commercial acquisition software program from Zeiss and stored on a computer for analysis.
Staining with di-8-ANEPPS
Myocytes were stained with the membrane fluorescent dye di-8-ANEPPS (5 μM) for 5 min. After washout, XYZ scans were performed on cells and Z stage was changes in 1.5–2 μm increments through the entire volume of the cell. Excitation at 488 nm was provided by a krypton-argon laser, and the emitted fluorescence was detected through a 520 nm filter.
Culturing of adult myocytes
Freshly isolated myocytes from midmyocardial ventricular and left atrial regions were cultured. The cell debris was removed with a Percoll gradient, and the purified cells were resuspended in M199 media with the following additions (mM): carnitine 2, taurine 5, creatine 5, cytosine-D-arabinofuranoside 0.01, insulin-transferrin-selenium (1:100), and BSA 1.5%. Pen/strep (100 U/ml) was added to prevent bacterial growth. Myocytes were plated into 35 mm culture dishes and put in an incubator at 37°C. Recordings were made at 24–48 h time points.
Myocytes were perfused with a HEPES buffer of the following composition (mM): NaCl 126, HEPES 10, KCl 5.4, MgCl21.0, CaCl21.8, and glucose 11. pH = 7.4 with NaOH.
A MultiClamp 700A amplifier (Molecular Devices, Sunnyvale, CA) was used to perform both voltage and current-clamp recordings. Patch pipettes (1–3 MΩ) were made from glass capillaries (1.5 mm O.D., Fisher Scientific, Pittsburg, PA) and pulled using a gravity puller (Narishige Corporation, East Meadow, NY). Voltage clamp recordings of Ca2+ currents (ICa) from cells were done as described previously. Following a series of three prepulses, ICa was measured at potentials between −40 and +50 mV. Series resistance was reduced, 60%–70% by electronic compensation. Voltage clamp signals were acquired at 10–25 kHz, filtered at 4 kHz, digitized with a Digidata 1322 converter (Molecular Devices, Sunnyvale, CA), and recorded on a computer using pClamp9 software (Molecular Devices, Sunnyvale, CA). Myocyte experiments were performed at 36°C.
Di-8-ANEPPS and fluo-4/AM were purchased from Thermo Fisher Scientific (Eugene, Oregon).
Data are illustrated as mean ± standard error of mean. Statistical comparisons were made using paired or unpaired Student's t-test (where appropriate) or one-way ANOVA. Post hoc ANOVA comparisons were performed using a Student Newman–Keuls test. P < 0.05 was considered statistically significant (*).
| Results|| |
Relationship between cell structure and Ca2+transients
Ventricular cells have an organized t-tubular system, whereas Purkinje cells appear to be devoid of any t-tubular network., Initially, we used the fluorescent dye di-8-ANEPPS to stain the cell membrane in canine ventricular and Purkinje myocytes [Figure 1], top panels]. Staining was present at the periphery of the Purkinje cell indicating this cell type lacks t-tubules. In ventricular cells, invaginations of the membrane were noted, indicating a well-developed t-tubular system. Interestingly, the staining pattern in atrial myocytes was highly variable; in a small percentage of cells, a t-tubular system was noted (~10%) but was not discernible in most cells [Figure 1], bottom panels].
|Figure 1: Confocal images of freshly isolated ventricular (top left), Purkinje (top right), and 2 different atrial myocytes (bottom panels). Most atrial cells were devoid of t-tubules, but some showed evidence of a primitive t-tubule system. Cells were stained with di-8-ANEPPS|
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Since the t-tubule system permits the influx and release the calcium to the interior of the cell, a cell type without a t-system should give a different Ca2+ transient pattern during excitation. Transverse line scan images from ventricular, atrial, and Purkinje cells revealed very different Ca2+ transient patterns associated with field stimulation [Figure 2]a. In ventricular myocytes, fluorescence increased synchronously across the cell, while in Purkinje cells, the fluorescence trace resembled a “U”-shaped profile. All atrial myocytes exhibited a “U”-shaped fluorescence profile that was less pronounced than that observed in Purkinje cells. The time course of fluorescence changes is shown in [Figure 2]b. The uniform rise in ventricular myocytes corresponds to the presence t-tubules which provides connections between the sarcolemma and the SR deep inside the cell, resulting in synchronous activation.
|Figure 2: (a) Representative transverse (X, T) line scans recorded from three cardiac cell types. (b) Ventricular myocytes exhibited a homogeneous rise in fluorescence across the cell. In contrast, Purkinje cells showed a “U”-shaped profile in fluorescence. Atrial cells exhibited a less pronounced “U”-shaped profile. Cells were paced at BCL = 2s. Ca2+ transient summary data for freshly isolated myocytes is shown in panel (c)|
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Cultured cardiomyocytes exhibit a differential loss of t-tubules
Previous investigations have shown that ventricular myocytes in culture show changes in the ultrastructure and certain ion channels such as Ito and IKr. In the next series of experiments, we put atrial and ventricular myocytes in culture and examined the alterations in t-tubular expression. [Figure 3]a shows representative confocal scans of 1-day-old cultured atrial and ventricular myocytes. Analysis of the ventricular myocytes showed that myocytes retained their rod-shaped appearance, but a loss of t-tubules was noted. In contrast, atrial myocytes did not show any change in t-tubules as cells maintained their primitive t-tubule structure.
|Figure 3: (a) Confocal images of a ventricular (left panel) and atrial myocytes (right panel) stained with di-8-ANNEPS. Ventricular cells kept in culture for 1 day showed a loss of t-tubules cells, whereas the pattern of staining in atrial cells was not changed. (b) Representative transverse line scan recorded from a ventricular and atrial cell that had been in culture for 1 day. Cells were paced at BCL = 2s. (c) Intensity plot showing fluorescence profile across the width of the cell. Ca2+ transient summary data for 1-day cultured myocyte s is shown in panel (d)|
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The changes in ultrastructure should result in an alteration in the Ca2+ transient profile in ventricular myocytes but no change in atrial myocytes when compared to freshly isolated cells. We next performed confocal XT line scans in the transverse orientation on 1-day-old cultured myocytes and recorded Ca2+ transients. Analysis of the transient in 1-day cultured atrial cells revealed little change in the Ca2+ transient profile or size compared to freshly isolated atrial cells. In contrast, line scan recordings from ventricular myocytes showed a fractionation of the Ca2+ transient as well as alterations in the fluorescence profile [Figure 3]b and [Figure 3]c. Ca2+ transient summary data for day 1 cultured cells is shown in [Figure 3]d.
We further examine the functional alterations in 2-day cultured myocytes. In 2-day cultured ventricular myocytes, a nearly complete loss of t-tubules was observed, yet the myocytes still retained their rod-shaped appearance. Regarding 2-day cultured atrial myocytes, there was no change in the percentage of myocytes, in which t-tubules could be discerned, –i.e., ~10% of the cells retained primitive t-tubule structure [Figure 4]a.
|Figure 4: (a) Ventricular cells kept in culture for 2 days showed a loss of t-tubules (left panel). In contrast, the staining pattern of atrial cells in culture for 2 days was unchanged. Panel B: Representative transverse line scan recorded from a ventricular and atrial cell that had been in culture for 2 days. (c) Intensity plot showing fluorescence profile across the width of the cell. Ventricular cells exhibited a phenotypic switch from a homogenous to a “U”-shaped fluorescence profile when kept in culture. Ca2+ transient summary data for 2-day cultured myocyte is shown in panel (d)|
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The nearly complete loss of t-tubules in 2-day cultured ventricular myocytes should result in a complete alteration Ca2+ transient profile plot. Confocal XT line scans to measure Ca2+ transients on 2-day-old cultured ventricular myocytes showed a “U”-shaped profile plot, similar to the profile plots measured from freshly isolated atrial and Purkinje cells. In addition, the size and duration of the transient was altered compared to freshly isolated ventricular cells. No change in atrial myocyte Ca2+ transients was noted, consistent with no changes in t-tubules or ultrastructure in this cell type [Figure 4]b and [Figure 4]c. Ca2+ transient summary data for day 2 cultured cells is shown in [Figure 4]d.
Previous studies have demonstrated that alterations in t-tubules in ventricular cells result in a loss of repolarization reserved due to downregulation of the transient outward K+ current (Ito) and the rapid delayed rectifier K+ current (IKr). The loss of t-tubules as well as the changes in the Ca2+ transient parameters in ventricular myocytes suggests a reduction in Ca2+ influx and subsequent Ca2+ release. In the next series of experiments, we investigated the changes in ICa in ventricular myocyte at 0–2 days in culture. Following application of 3 prepulses (not shown), ICa was activated by applying 300-ms step depolarizations from −40 to +50 mV. The threshold for activation of ICa was −30 mV and the peak occurred at +10 mV [Figure 5]. The current–voltage relationship showed a reduction in ICa density after prolonged culturing, consistent with the loss of t-tubule.
|Figure 5: Representative whole-cell ICa current recordings from a freshly isolated ventricular cell (a) and a ventricular cultured for 2 days (b) using the voltage-clamp protocol located at the top of the figure. (c) Current-voltage (I-V) relation for day 0-, day 1-, and day 2-cultured ventricular myocytes|
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| Discussion|| |
Tissue-specific variations in Ca2+transients
The results of our study show that in field-stimulated myocytes, the Ca2+ transients in atrial, ventricular, and Purkinje cells showed very different profiles. Atrial and Purkinje cells showed a “U”-shaped transient [Figure 2], indicating that Ca2+ influx and release starts at the edge of the cell and diffuses to the cell center. In contrast, ventricular myocyte showed a linear profile suggesting influx and release is homogeneous throughout the cell. When we stained the cell with the membrane stain di-8-ANEPPS, all Purkinje cells exhibited a lack of t-tubules, some atrial myocytes had a primitive t-tubule system, whereas all ventricular myocytes had an extensive t-tubule system. When atrial and ventricular myocytes were placed in culture, ventricular myocytes showed rapid change in t-tubule expression, whereas atrial myocytes were unaffected. Line scan analysis of cultured myocytes showed that there was an alteration in the Ca2+ transient profile plot in ventricular myocytes; atrial myocytes appeared to be unaltered.
Ca2+ transients in freshly dissociated cells
The initiation of the Ca2+ in cardiac myocytes is mainly due to Ca2+ influx via L-type Ca2+ channels and SR Ca2+ release. Since L-type Ca2+ channels are located on the sarcolemma, it stands to reason that efficient Ca2+ release would occur where the RyRs and the sarcolemma form junctions. In freshly isolated ventricular myocytes, these RyR/Ca2+ channel junctions are found throughout the cells resulting in the homogeneous rise in the Ca2+ transient throughout the cell [Figure 2]. In Purkinje and atrial myocytes, these junctions could only occur at the cell periphery, since these cell types have little or no t-tubules. Previous studies have shown that Ca2+ transients in ventricular cells represent the summation of many Ca2+ sparks. These Ca2+ sparks form at junctions between the surface membrane and SR.,, Consistent with the structural data, the peripheral activation of calcium transients in atrial and Purkinje cells suggests a requirement for Ca2+ influx via Ca2+ channels to be in close proximity to RyRs to elicit a Ca2+ transient., In connection with this point, perhaps the proximity of RyRs to L-type Ca2+ channels would increase local Ca2+ levels to promote the activation of junctional RyRs. Our observations demonstrate that the microarchitecture of the myocyte is important in modulating the Ca2+ transient.
Alterations in Ca2+transients due to loss of t-tubules
Ventricular cells in culture still had a rod-shaped morphology but showed a profound loss of t-tubules in much of the cell interior. The loss of t-tubules resulted in an alteration in the profile plot of the Ca2+ transient. As the t-tubules progressively disappeared from ventricular cells, the Ca2+ transient went from a homogenous activation pattern to a dyssynchronous activation pattern. After 2 days in culture, a dramatic loss of t-tubules was observed, resulting in a Ca2+ transient with a “U”-shaped profile similar to the transient in atrial and Purkinje cells. These observations highlight the role of cell microarchitecture in modulating the Ca2+ transient.
Differential loss of t-tubules
Ventricular myocytes had an extensive t-tubular system, whereas a percentage of atrial myocytes showed a less well developed t-tubular system. Interestingly, atrial myocytes retained their primitive t-tubule system even after 2 days in culture. It is unclear why there is this differential effect, but these differences may be important under pathophysiological conditions. For example, HF is associated with a reduction of t-tubules and changes in the Ca2+ transient in the ventricle. Since many ion channels are localized in the t-tubules, the loss of t-tubules could be associated with a loss in ion channels. Indeed, previous studies showed cultured ventricular myocytes were associated with a loss of K+ currents, namely IKr and Ito. Our current observation that ICa is reduced would further support the t-tubular location of ion channels. Since atrial myocytes lack t-tubules or have primitive t-tubular network, the loss of this network would have little effect on ion channel density or EC coupling.
Functional consequence of t-tubule loss
Previous studies on Purkinje and atrial cells have shown that RyR2 receptors are distributed at regularly spaced intervals throughout the cell, likely adjacent to z-lines., These observations suggest that the RyR2 comprise a mix of junctional and nonjunction/corbular RyR2 channels. In atrial and Purkinje cells, the “U”-shaped profile indicates that Ca2+ transients are confined to the cell periphery suggesting that only peripheral junctional RyRs appear to be activated during a normal Ca2+ transient.
The loss of t-tubules from ventricular cells would result in a orphaned RyR2 receptor, resulting in a greater proportion of RyR2 receptors converting from junctional to corbular channels. The phenotypic switch to atrial or Purkinje-like myocytes may have implications on Ca2+ regulation and arrhythmias. In conditions of elevated intracellular Ca2+, uncontrolled Ca2+ release in the form of triggered Ca2+ waves and spontaneous Ca2+ release has been observed from the cell interior of atrial and Purkinje cells, presumably from corbular SR. In ventricular cells which have lost t-tubules, similar uncontrolled release of Ca2+ would occur. This uncontrolled Ca2+ release coupled with a reduction in repolarization reserve may increase the susceptibility of arrhythmias in ventricular cells.
We are grateful to Judy Hefferon and Bob Goodrow for excellent technical assistance.
Financial support and sponsorship
This study was supported by the Free and Accepted Masons of New York, Florida, Massachusetts, Connecticut, Maryland, Wisconsin, Washington, and Rhode Island (to JMC).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]