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USING CLASSIC PAPERS TO TEACH PHYSIOLOGY

Teaching calcium-induced calcium release in cardiomyocytes using a classic paper by Fabiato

School of Biological Sciences, Nanyang Technological University, Singapore

Address for reprint requests and other correspondence: W. Liang, School of Biological Sciences, Nanyang Technological Univ., Singapore 637551 (e-mail: willmann{at}ntu.edu.sg)

Abstract

This teaching paper utilizes the materials presented by Dr. Fabiato in his review article entitled "Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum." In the review, supporting evidence of calcium-induced calcium release (CICR) is presented. Data concerning potential objections to the CICR theory are discussed as well. In closing, technical issues associated with the skinned cell model are mentioned. Based on this review article, teaching and learning points are put forth in this article to highlight two concepts: 1) the regulatory mechanisms of CICR in cardiomyocytes and 2) the recognition of contradicting hypotheses and limitations in experimental design. The first concept is certainly an important one for physiology students. The second concept is universally applicable to researchers in all fields of science. It is thus the aim of this article to cultivate a rewarding teaching and learning experience for both instructors and students.

Key words: cardiomyocyte; sarcoplasmic reticulum

TIGHT REGULATION of cardiac contractility is essential to the survival and well-being of many animals, including humans. Excitation-contraction (E-C) coupling is the underlying mechanism directing rhythmic contractions of the heart, allowing adequate delivery of blood and nutrients to other tissues in the body. One of the most important cell types in the heart are contractile cardiomyocytes (CMs). In E-C coupling, it has long been established that electrical excitation (i.e., action potential) will trigger contractions of individual CMs in an organized manner. Elucidating the link(s) between excitation and contraction, though, is still an ongoing task of many researchers in the cardiac research field. It is, however, agreed that the calcium ion (Ca²⁺) serves a central role in the signaling pathways leading to contractions of CMs and the heart.

The source(s) of Ca²⁺ in cardiac E-C coupling was not trivial to researchers decades ago. Contractile machinery is activated when the cytosolic Ca²⁺ level increases. Does Ca²⁺ from the extracellular space rush into a CM and contribute entirely to its contraction? Or is there a reservoir of stored Ca²⁺ within the CM that is responsible for the cytosolic Ca²⁺ increase? We know now that Ca²⁺-induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) is the major Ca²⁺ contributor in CM contractions (3). In fact, our present understanding of this concept is indebted to Dr. Alexandre Fabiato, who performed a series of experiments to illustrate the importance of CICR in the heart.

In his review article (6), which has been cited 800 times as of November 2007, Dr. Fabiato went into great detail explaining the mechanisms of CICR and its regulatory interactions with other Ca²⁺-transporting entities in CM contractions. The significance of this review article in explaining the cardiac CICR concept has earned the designation as a "classic paper." Classic papers are a selection of important works that the American Physiological Society envisions will foster students' appreciation of physiological concepts. Specifically, this classic paper (6) is a valuable tool for teaching students of the regulation of myocardial contractility by CICR. In Dr. Fabiato's article, the discussion of experimental findings relevant to opposing theories to CICR is also an important demonstration to us that we must remain open minded when encountering contradicting hypotheses. Finally, Dr. Fabiato gave an overview of the limitations of the skinned CM preparation, which is what his hypothesis of CICR was based on. As Dr. David Harder pointed out in his essay describing the significance of Dr. Fabiato's 1983 review article (10), the use of cell models (in this case, a skinned cell preparation) is both necessary but imperfect. The fact that Dr. Fabiato recognized his studies' methodological confines reminds us that we ought to consider the "whole" when we attempt to extrapolate our findings from the "small parts."

In this article, I propose some teaching and learning points from Dr. Fabiato's review article in three parts: 1) principles and properties of CICR and supporting evidence, 2) an opposing hypothesis to CICR, and 3) issues pertaining to the skinned CM preparation.

Principles and Properties of CICR
The skinned CM preparation.
Before we discuss the data supporting Dr. Fabiato's hypothesis, we should note that the skinned CM preparation forms the basis of the supporting evidence of CICR. The skinning procedures are briefly described here (please refer to Refs. 5 and 8 for details). The myocardial tissue is cut into small pieces and homogenized with a blender, resulting in bundled and isolated cells with varying degrees of sarcolemmal damage. The sarcolemma is then microdissected away with a glass microtool. After removal of the sarcolemma, the myoplasm is exposed to the bathing medium. In the skinned CM, the absence of the sarcolemmal barrier allows investigations of SR function without hindrance. Cellular components (e.g., the SR, mitochondria, and myofibrils that are located superficially) are also removed after skinning (8). Structures that are further away from the sarcolemma, including the majority of myofibrils, are preserved. Thus, sarcomere shortening and CM contraction can still be recorded. However, it is noted that subsarcolemmal cisternae of the SR, which form dyadic junctions with sarcolemmal L-type Ca²⁺ channels, are usually damaged in skinned CMs (12). Therefore, Ca²⁺ release in a skinned cell precludes the contribution of subsarcolemmal SR Ca²⁺-release channels, which account for 25% of total Ca²⁺ release (12). When [Ca²⁺] or any other constituents in the bathing medium are altered, the myoplasm and cellular components, including the SR, will be directly influenced. A "putative" SR Ca²⁺ channel at the time, now known as the ryanodine receptor (RyR), was proposed to release Ca²⁺ upon channel activation. Regulation of channel opening and closing is dependent on both the [Ca²⁺] immediately outside of the SR ([Ca²⁺]_Out-SR) and the rate of this [Ca²⁺] change.

Effect of [Ca²⁺]_Out-SR.
Consider the effect of [Ca²⁺]_Out-SR first. Table 1 (adapted from Table 1 in the original review article) compares the amplitude of force generated from skinned CMs across different [Ca²⁺]_Out-SR. In Table 1, [Ca²⁺]_Out-SR is represented by "pCa trigger," which is equivalent to the negative logarithm of myoplasmic [Ca²⁺] or free [Ca²⁺]. [Ca²⁺]_Out-SR acts as a stimulus for CICR, and the amplitude of the generated force is indicative of the amount of Ca²⁺ release from the SR. The rate of [Ca²⁺] change is kept constant at 0.1 s, so only [Ca²⁺]_Out-SR is the dependent variable here. Increasing force is initially observed when pCa changes from 7.00 to 6.25. Maximal force is obtained when the stimulus is at pCa 6.25. The pCa values translate to an increase in myoplasmic [Ca²⁺] from 10^–7 to 5.62 x 10^–7 mol/l, which is a close approximation to the physiological [Ca²⁺] during intact CM contraction (4). As [Ca²⁺]_Out-SR increases (or pCa decreases) further, smaller force is generated as a result of smaller SR Ca²⁺ release. These results show that the SR Ca²⁺ channel is activated at an optimal [Ca²⁺]_Out-SR, beyond which renders the channel less active or inactivated. Dr. Fabiato suggested that channel inactivation results from Ca²⁺ binding to a low-affinity site on the channel itself. This binding can only occur when [Ca²⁺]_Out-SR is high enough.

1. This is a checklist of students' general standings prior to taking the physiology course. Table 1 helps students recall fundamental knowledge they have learned from introductory chemistry and cell biology classes. The following questions can be used as indicators of the students' levels in basic science. Students should be able to do the following:
   1. Describe the directions of blood flow into and out of the heart (excluding coronary circulation) and name the relevant structures.
      1. Deoxygenated blood enters the right atrium via the vena cava, goes into the right ventricle through the opening of the tricuspid valve, and exits the heart through the opening of the pulmonary valve into the pulmonary artery and en route to the pulmonary circulation.
         
      2. Oxygenated blood enters the left atrium via the pulmonary vein, goes into the left ventricle through the opening of the mitral valve, and exits the heart through the opening of the aortic valve into the aorta and en route to systemic circulation.
         
      
      
   2. Name the major components of any animal cell: plasma membrane, nucleus, endoplasmic (or sarcoplasmic, in muscle cells) reticulum, mitochondria, cytosol, Golgi apparatus, and cytoskeleton.
      
   3. Calculate free [Ca²⁺] from pCa values: pCa = 10^–Ca, e.g., pCa 7.00 = 10^–7 mol/l.
      
   
   
2. To supplement lectures on muscle physiology, the following questions can be used to probe students' knowledge further. These may require the students to conduct some research on their own.
   1. What is the meaning of a "skinned" cell? As Dr. Fabiato described in the review article, skinning is of a physical kind, by removing the sarcolemma. There is another type of skinning (chemical skinning), where intact cells and tissues are treated with either a chelator or detergent to disrupt the plasma membrane.
      
   2. It is indicated in Table 1 that the intracellular solution contains EGTA, Mg²⁺, Mg-ATP, and phosphocreatine. What are the purposes of these constituents? Specifically, why is EGTA preferable over another common chelating agent (EDTA)? Both Mg²⁺ and Mg-ATP are substrates of the SR Ca²⁺ pump. Phosphocreatine provides a source of phosphate for the conversion of ADP to ATP in an anaerobic state. EGTA is a Ca²⁺ chelator and acts as a Ca²⁺ buffer in skinned cells, allowing the accurate estimation of free [Ca²⁺] in the myoplasm. EGTA is chosen over EDTA because of the former's better Ca²⁺-buffering capacity at physiological free [Ca²⁺], i.e., between pCa 7 to 6. Besides, EGTA has a relatively low affinity for Mg²⁺, so that the Mg²⁺ supply will not be limited in the skinned cell preparation. Bers (2) has shown that given the Ca-EGTA association constant (2.49 x 10⁶ M^–1) and n = 0.961, one can calculate the amount of total calcium required to achieve different free [Ca²⁺] based on a fixed [EGTA]. For example, if the bathing solution contains 0.001 mol/l EGTA, to achieve free [Ca²⁺] of 1 x 10^–6 mol/L, the amount of total calcium required = (1 x 10^–6 + {0.961 x 0.001(1 x 10^–6)/[1/(2.49 x 10⁶) + 1 x 10^–6]}) = 6.86 x 10^–4 mol/l.
      
   3. Explain the term "E-C coupling" in muscle function. There is a direct cause-effect relationship between the electrical excitation of a muscle cell and its consequent contraction due to activation of myofilaments. The electrical excitation is usually an action potential, causing membrane depolarization. Depending on the type of muscle in question, the signaling events that follow depolarization vary. In cardiac muscle, depolarization opens voltage-gated Ca²⁺ channels, and Ca²⁺ enters the cell. The Ca²⁺ influx induces CICR from the SR, largely increasing myoplasmic free [Ca²⁺]. The elevated Ca²⁺ binds to troponin C, exposing the myosin-binding sites on actin. Cross-bridges form between actin and myosin, resulting in sarcomere shortening and muscle contraction.
      
   4. Compare E-C coupling in cardiac muscle with that in skeletal and smooth muscles. When skeletal muscle is stimulated, the membrane depolarizes, but there is no Ca²⁺ entry. Membrane potential becomes more positive, activating dihydropyridine receptors that are coupled to RyRs, initiating Ca²⁺ release. This is very different from cardiac muscle, where Ca²⁺ entry is necessary to induce SR Ca²⁺ release. In smooth muscle, there are both CICR (from RyR activation by Ca²⁺ entry) and inositol 1,4,5-trisphosphate (IP₃)-induced Ca²⁺ release. Agonist binding to G protein-coupled receptors in smooth muscle activates phospholipase C, generating IP₃. Ca²⁺ release from the SR is induced by opening of IP₃ receptors. Contractile mechanisms in smooth muscle are also different from those in striated muscles. In smooth muscle, Ca²⁺ binds to calmodulin, activating myosin light chain (MLC) kinase (MLCK). This leads to phosphorylation of MLC, allowing myosin-actin interactions and cellular contraction. In cardiac muscle, MLCK is also present but only has a minor role in increasing myofilament Ca²⁺ sensitivity.
      
   
   
3. Students may be asked to plot a graph of tension versus pCa (or myoplasmic [Ca²⁺]). From this graph, the optimal pCa that generates the maximal force can be determined. This optimal value will indicate that CICR:
   1. is not necessarily larger when myoplasmic [Ca²⁺] is higher and that Ca²⁺ can act as both an activator or inactivator of CICR, a point illustrated by the traces shown in Fig. 2.
      
   2. is a graded rather than an all-or-none response, a point illustrated by the negative feedback system shown in Fig. 3.
      
   
   

View larger version (43K): [in this window] [in a new window]   Fig. 2. CICR and tension induced by free [Ca2+] in a skinned cardiomyocyte (CM). Tension traces are shown in A and D). Amplitudes of Ca2+ signals, represented by aequorin light intensities, are shown in B and E). Signals from solution exchanges are shown in C and F. [Adapted from Fig. 1 in the original review article.]

View larger version (16K): [in this window] [in a new window]   Fig. 3. Original Fig. 2 from the review article showing multiple events before and after CICR and the feedback mechanism that inhibits subsequent Ca2+ releases. Note that extra description and arrows have been added to this diagram compared with the original one. The original figure (shown as Fig. 4) is to be distributed to the students on the handout.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Relationship between free [Ca2+] trigger and the Ca2+-induced Ca2+ release (CICR)-induced tension based on the data shown in Table 1 of the original review article. Optimal free [Ca2+] where maximal tension is generated occurs at 5 x 10–7 mol/l.

1. Background knowledge.
   1. Describe the directions of blood flow into and out of the heart (excluding the coronary circulation) and name the relevant structures.
      
   2. Name the major components of any animal cell.
      
   3. Calculate [Ca²⁺] for each of the pCa values shown in Table 1.
      
   
   
2. Muscle physiology and experimental techniques.
   1. What are the features of a "skinned" cell? How is skinning done?
      
   2. What are the purposes of EGTA, Mg²⁺, Mg-ATP, and phosphocreatine in the intracellular or bathing solution of a skinned cell? Specifically, why is EGTA preferable over another common chelating agent (EDTA)?
      
   3. Explain the term "E-C coupling" in muscle function.
      
   4. Compare E-C coupling in cardiac muscle with that in skeletal and smooth muscles.
      
   
   
3. Plot a graph of tension versus free [Ca²⁺] and determine the optimal free [Ca²⁺] that gives the highest tension.
   

Importance of the rate of [Ca²⁺]_Out-SR change in CICR.
Figure 2 (Fig. 1 in the original review article) makes use of the data from Table 1 to highlight the importance of the rate of [Ca²⁺]_Out-SR change in CICR. Bathing media of three different pCa are used: pCa 7.40, 6.25, and 5.50. pCa 7.40 translates to 3.98 x 10^–8 mol/l, which is the basal or resting [Ca²⁺] in the cell normally. In the case of skinned cells, this resting [Ca²⁺] refers to [Ca²⁺]_Out-SR. From Table 1, maximal force is generated at pCa 6.25, so this [Ca²⁺]_Out-SR serves as the stimulus for CICR here. In another study (5), Dr. Fabiato calculated that 3.16 x 10^–6 mol/l Ca²⁺ (or pCa 5.50) is released during maximal contraction of a skinned CM. pCa 5.50 is used here to inactivate the SR Ca²⁺ channel, made possible by Ca²⁺ binding to the low-affinity site on RyRs. The high-affinity site on RyRs, when bound with low Ca²⁺ (e.g., pCa 6.25), activates SR Ca²⁺ release (4). Inactivation of SR Ca²⁺ release is also facilitated by calmodulin, which increases RyR close times (11). Phosphorylation of RyRs by Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) increases channel opening probability (4). It remains unclear how the interplay between Ca²⁺, calmodulin, and CaMKII regulates RyR activity and SR Ca²⁺ release in the intact CM.

When [Ca²⁺]_Out-SR is increased from pCa 7.40 to 6.25 (Fig. 2C), a rapid release of Ca²⁺ is observed, indicative of CICR (Fig. 2B). As peak [Ca²⁺]_Out-SR is reached from CICR in 0.6 s, the high [Ca²⁺] binds to the low-affinity site of the SR Ca²⁺ channel and inactivates it. Consequently, Ca²⁺ release stops, as illustrated by the descending phase of the Ca²⁺ response (Fig. 2B). The sequence of events happening here would simulate a normal CICR.

Dr. Fabiato pointed out a subtle difference between the CICR illustrated in Fig. 2 and that observed in other skinned CM preparations. He noted that in skinned CMs, repetitive cycles of CICR from a single stimulus are possible due to the slow process of returning myoplasmic [Ca²⁺] (and [Ca²⁺]_Out-SR) to baseline levels. Such cycles of CICR are not visible in Fig. 2, however. The reason for this lies in the rapid change of [Ca²⁺]_Out-SR from pCa 6.25 to 7.40 as soon as the Ca²⁺ response has completely descended from its peak.

We have seen a normal CICR in Fig. 2A. With experimental manipulation, CICR is not allowed to proceed fully (Fig. 2E). This is done by substituting the pCa 6.25 solution, shortly after it is added to stimulate CICR, with one containing pCa 5.50. Precisely, this solution change takes place within 0.1 s (Fig. 2F). In essence, this experiment introduces a positive change in the rate of [Ca²⁺]_Out-SR increase, by shortening the time involved from 0.6 to 0.1 s. The result is an early inactivation of the SR Ca²⁺ channel (RyR), as shown by the slower and smaller Ca²⁺ release (Fig. 2E). The early inactivation is due to premature exposure of the RyR to high free [Ca²⁺] (pCa 5.50) before completion of the Ca²⁺ release. Taken together, Ca²⁺ release is both activated and inactivated by Ca²⁺, depending on [Ca²⁺]_Out-SR and its rate of change.

TEACHING POINTS OF FIG. 2 (INSTRUCTOR'S REFERENCE).
The original Fig. 1 of the review article has been cropped, resized, and rearranged into Fig. 2 as part of the student handout below.

1. Summary of findings from Fig. 2.
   1. Greater tension as well as larger and more rapid CICR are seen when free [Ca²⁺] is elevated to trigger the responses (Fig. 2, A and B). Note that the Ca²⁺ response is allowed to proceed fully after the injection of pCa 6.25 (Fig. 2C). In Fig. 2F, CICR is again triggered by an injection of pCa 6.25. However, free [Ca²⁺] is increased further (injection of pCa 5.50; Fig. 2F) before CICR reaches completion. This premature free [Ca²⁺] increase results in a smaller and slower Ca²⁺ response and thus smaller tension (Fig. 2, D and E).
      
   
   
2. From the slower and smaller Ca²⁺ signal in Fig. 2E, it can be inferred that CICR can be inactivated when free [Ca²⁺] is too high. The injection of pCa 5.50 here causes an increase in free [Ca²⁺] to a level similar to when CICR has completed. Inactivation occurs when high [Ca²⁺] binds to a low-affinity site on the outer SR membrane. This site is generally agreed to be the RyR at present.
   
3. In Fig. 2B, only a portion of the full Ca²⁺ signal recordings are shown. What happened following the transient response is not visible in Fig. 2. Students should notice the reinjection of pCa 7.40 inhibits subsequent cycles of CICR. Students may be asked to predict what the Ca²⁺ signal will be if pCa 7.40 is not reinjected to replace pCa 6.25 after the completion of CICR. Slow cyclic Ca²⁺ signals indicating repetitive CICR may be seen. This is because the skinned cells lack sarcolemmal Ca²⁺-extruding entities to return myoplasmic or free [Ca²⁺] to the resting level efficiently, but the SR Ca²⁺ pump is still able to remove Ca²⁺ from the myoplasm. It may not be necessary for myoplasmic [Ca²⁺] to return to baseline before repetitive CICR can be generated. RyR opening can still occur in response to a subsequent, larger Ca²⁺ trigger (3). In a normal CM, however, repetitive CICR from a single Ca²⁺ trigger is rare. This ensures SR Ca²⁺ release and the resultant contraction are coupled to electrical stimulation and Ca²⁺ current (I_Ca).
   
4. If shortly (within 0.1 s) after the injection of pCa 6.25 (Fig. 2C), the bathing solution is substituted with pCa 7.40, what is expected of the CICR Ca²⁺ response then? The response will likely be a very short one with low amplitude. This manipulation is similar to an ultrashort trigger of CICR, where free [Ca²⁺] quickly returns to resting level just after CICR is initiated. Thus, there is incomplete Ca²⁺ release from the SR, as reflected by the small Ca²⁺ signal.
   
5. Depending on the level of students, the principle of using aequorin as a Ca²⁺-sensitive fluorescent indicator may be briefly discussed with students. Aequorin is a bioluminescent protein originally isolated from Aequorea forskalea. Active aequorin consists of apoaequorin, coelenterazine, and O₂. When bound with Ca²⁺, 465-nm light is emitted, together with apoaequorin and CO₂. In the experiment illustrated here, aequorin-emitted light intensity is used to represent the amount of SR Ca²⁺ release. Quick solution exchange is made possible by microprocessor-controlled microinjection and aspiration systems, with each of these connected to a micropipette. Micropipettes containing aequorin and different pCa solutions remain in the field of view of the photomultiplier tube. This allows a constant background or baseline luminescence to be recorded during the course of the experiment. It should be noted that before the injection of solutions from the micropipettes, Ca²⁺ in the SR is not bound to aequorin and does not contribute to the luminescence. The injection of pCa solution near the cell triggers Ca²⁺ release from the SR. The formerly unbound SR Ca²⁺ now binds to aequorin, emitting light as a result. Other Ca²⁺ fluxes (e.g., SR Ca²⁺ uptake) will not affect aequorin light intensity because these Ca²⁺ would have been bound to aequorin before they reach the cell.
   

DISCOVERY LEARNING OF FIG. 2 (STUDENT HANDOUT).
Figure 2 is included in the handout.

1. Summarize the tension and CICR findings in Fig. 2 with reference to each pCa treatment.
   
2. What can be inferred from the slower and smaller Ca²⁺ signal in Fig. 2E?
   
3. Notice that the Ca²⁺ signal recordings after reinjection of pCa 7.40 are not shown for an extended period in Fig. 2B. What do you expect the Ca²⁺ signals to look like after reinjection of pCa 7.40? Do you expect to see cyclic Ca²⁺ signals? If pCa 6.25 is not replaced with pCa 7.40 after the Ca²⁺ signal, will you expect to see cyclic Ca²⁺ signals?
   
4. In Fig. 2C, if pCa 7.40 is injected instead of pCa 5.50, what do you expect to see as the Ca²⁺ signal?
   

The homeostasis of CICR.
The dual purpose of Ca²⁺ in activating and inactivating CICR illustrates yet another key theme in physiology: homeostasis, or the maintenance of a dynamic steady state. Here, the homeostasis of CICR is achieved by a negative feedback system. Dr. Fabiato showed that CICR is graded rather than all or none. The graded CICR response is a result of the constant monitoring of [Ca²⁺]_Out-SR and its rate of changes based on a negative feedback system.

Figure 3 (Fig. 2 in the original review article) outlines the regulatory steps involved in CICR, Ca²⁺ refilling, and extrusion. The key to the CICR negative feedback lies in the interactions between myoplasmic [Ca²⁺] and the SR. Following the initial trigger of I_Ca on the sarcolemma, myoplasmic [Ca²⁺] increases as Ca²⁺ flows into the cell. This creates an increase in [Ca²⁺]_Out-SR, which activates CICR. Consequently, myoplasmic [Ca²⁺] increases to an even higher level, which, in turn, inactivates CICR. At this point, the fast I_Ca is partially inactivated, thus reducing the "triggering" effect to initiate CICR, and the SR retains its Ca²⁺. The smaller and slower tail of I_Ca, as well as inactivated CICR, leads to Ca²⁺ accumulation in the SR. SR Ca²⁺ accumulation is facilitated by Ca²⁺ uptake via the SR Ca²⁺ pump. The extent of SR Ca²⁺ accumulation also determines how easy it is to induce CICR. When the SR is fully loaded with Ca²⁺, a smaller trigger is able to induce CICR compared with when the SR is only half loaded. At the same time, the size of Ca²⁺ content in the SR will determine the amplitude of the graded CICR response.

As discussed earlier, a single response of CICR in skinned CMs is made possible by quickly returning [Ca²⁺] to baseline levels. In fact, CICR is also nonrepetitive in intact CMs. Sarcolemmal Ca²⁺ extruding entities [Na⁺/Ca²⁺ exchanger (NCX) and sarcolemmal (plasmalemmal) Ca²⁺ pump (PMCA)] present in intact but not skinned CMs are important in ensuing CICR is not induced in a cyclic manner without the proper trigger and stimuli, i.e., I_Ca and increased [Ca²⁺]_Out-SR. Soon after CICR, myoplasmic or free [Ca²⁺] is much increased, and large amounts of Ca²⁺ are extruded via the low-affinity NCX. As myoplasmic [Ca²⁺] decreases further, Ca²⁺ is extruded via the high-affinity PMCA. Both of these processes, together with SR Ca²⁺ uptake, inhibit the reinduction of CICR (Fig. 3).

For a more current understanding of CICR in CMs, readers are referred to recent reviews (1, 3). It is generally accepted that I_Ca is the predominant trigger for CICR. The physical apposition of L-type Ca²⁺ channels and RyRs allows few Ca²⁺ (from I_Ca) to concentrate within a narrow space. Thus, only small amounts of Ca²⁺ are required to generate a high local Ca²⁺ microdomain within the vicinity of RyRs, activating SR Ca²⁺ release. Without I_Ca as a trigger, spontaneous SR Ca²⁺ release is also possible when SR Ca²⁺ content is high (i.e., a fully loaded SR). As a result, the SR [Ca²⁺] can trigger CICR without membrane depolarization or I_Ca. Spontaneous Ca²⁺ releases, or Ca²⁺ sparks, usually occur at individual RyRs asynchronously, thus generating local Ca²⁺ increases only. Since the amount of Ca²⁺ release from each Ca²⁺ spark is very little, local Ca²⁺ increases hardly result in any contraction. The proximity of the SR and sarcolemma also creates an effective barrier to prevent small amounts of Ca²⁺ of a single Ca²⁺ spark from diffusing into the myoplasm. When multiple Ca²⁺ sparks occurs simultaneously, it is possible to generate global increases in myoplasmic [Ca²⁺]. This often leads to cardiac dysrhythmia since CMs contract without first receiving a proper electrical impulse from pacemaker cells. In contrast, I_Ca may trigger no or small CICR when SR Ca²⁺ content is low. Small CICR can facilitate SR Ca²⁺ refilling as I_Ca inactivation is slowed and reverse-mode NCX (i.e., Ca²⁺-influx mode) is favored.

TEACHING POINTS OF FIG. 3 (INSTRUCTOR'S REFERENCE).

1. Figure 3 can emphasize the importance of homeostasis in all levels of physiology. Students may be asked to first define homeostasis as a dynamic steady state maintained in the internal environment (i.e., the body) via a negative feedback system. Instructors may then ask students to give examples of other negative feedback mechanisms that they have learned in other physiology lectures. One such example, since myocardial contraction is the topic of interest here, is the regulation of blood pressure by the baroreceptor reflex. This reflex, when activated by changes in blood pressure, acts as a "sensor" to negatively feedback to inputs governing the maintenance of blood pressure. Consequently, cardiac output is adjusted, and blood pressure returns to normal.
   
2. Students may be asked to fill in the missing details of Fig. 4. First, there is no clear "feedback loop" depicted in Fig. 4. Students should be able to add in arrows to illustrate the feedback looping shown in Fig. 3. The loop includes negative input to CIRC from the following: "reinduction of Ca²⁺ release," "activation of nonselective cation channels," "repolarization total inactivation of I_Ca," and "NCX and sarcolemmal Ca²⁺ pump." Moreover, students should understand the effects of Ca²⁺ reaccumulation on the free [Ca²⁺] required to activate CICR. A fully loaded SR (i.e., higher Ca²⁺ reaccumulation) requires a smaller trigger (i.e., lower free [Ca²⁺]) to induce CICR.
   
3. Although high free [Ca²⁺] is the major inactivator of CICR, other factors also have a role in stopping Ca²⁺ release. Instructors may briefly mention about these inactivating factors: nonselective cation channel (NSCC) opening, increased K⁺ conductance, and sarcolemmal Ca²⁺-extruding entities. Opening of NSCCs increases both Na⁺ and K⁺ permeability. The electrochemical gradient of K⁺ is favored, increased K⁺ conductance leads to repolarization of the cell, and voltage-gated Ca²⁺ entry is inhibited. Sarcolemmal Ca²⁺ removal via NCX and PMCA lowers the free [Ca²⁺], effectively removing the trigger for further CICR.
   
4. In advanced physiology and pharmacology courses, it may be useful to ask students to describe the general mechanisms of common cardiac drugs. In the context of CICR, drugs that directly and indirectly alter free [Ca²⁺] may be discussed. Some examples are the cardiac glycosides and classes III and IV antidysrhythmics. Digoxin is a commonly used glycoside in congestive heart failure. Its main action is to inhibit Na⁺/K⁺-ATPase, resulting in NCX inhibition and increased free [Ca²⁺]. Subsequently, Ca²⁺ accumulation into the SR is increased, and fuller SR loading will result in greater Ca²⁺ release and greater myocardial contraction (positive inotropy). Another inotropic drug is amiodarone (class III antidysrhythmic), which blocks K⁺ channels. As shown in Fig. 3, increased K⁺ conductance (from K⁺ channel opening) repolarizes the cell. With K⁺ channel blockade, the cell remains depolarized for a longer time, prolonging the cardiac action potential plateau. There is stronger contraction as a result. Class IV antidysrhythmics, however, have opposite effects. Verapamil, as an example, blocks voltage-gated Ca²⁺ channels. The plateau phase of the action potential is shortened, resulting in weaker myocardial contraction.
   

View larger version (15K): [in this window] [in a new window]   Fig. 4. Original Fig. 2 from the review article showing the regulation of CICR.

1. Describe another example of negative feedback in the cardiovascular system.
   
2. Draw/fill in details in Fig. 4 to show the following:
   1. The feedback loop,
      
   2. Outcomes of "activation of nonselective cation channel," "total inactivation of I_Ca," and "NCX and the sarcolemmal Ca²⁺ pump."
      
   3. Differences between high and low Ca²⁺ reaccumulation.
      
   
   
3. Describe how the items in 2B may affect CICR.
   
4. Name and describe cardiac drugs that have direct or indirect effects in myocardial Ca²⁺ regulation.
   

Ca²⁺ movements in mammalian and frog CMs in generating contractions.
Figure 5 (Fig. 3 in the original review article) compares Ca²⁺ movements in mammalian and frog CMs in generating contractions. The importance of the SR in CICR is reinstated here. Frog CMs, unlike mammalian CMs, are devoid of CICR. This characteristic of frog CMs is because the SR is not involved in Ca²⁺ movements within the cell. As such, Ca²⁺ influx initiated by I_Ca simply diffuses in the myoplasm and activates myofilaments directly, a phenomenon not found in mammalian CMs. A possible reason, as stated by Dr. Fabiato, for the Ca²⁺ diffusion-induced myofilament activation in frog CMs is that these cells have a much smaller diameter than mammalian ones. Figure 5 may be distributed to students after the teaching and learning points of Fig. 3 are discussed. The key point of Fig. 5 is to highlight the SR's critical role in CICR in mammalian but not frog CMs. A minor point in Fig. 5, which serves as a good preview of the material in Table 2, is the inclusion of various Ca²⁺ buffers, e.g., calmodulin, phosphocreatine and troponin C. As we shall see later in Table 2, these Ca²⁺ buffers contribute to the great disparity between total [Ca²⁺] and myoplasmic or free [Ca²⁺].

View larger version (20K): [in this window] [in a new window]   Fig. 5. Ca2+ regulation in the myocardial contractility of mammals and frogs. SL, sarcolemma; SR, sarcoplasmic reticulum; TnC, troponin C.

1. Compare Ca²⁺ movements in mammalian and frog cardiomyocytes. In which one does CICR occur? Which cellular structure(s) is/are essential for CICR to take place?
   

Consideration of an Opposing Hypothesis to CICR
An important lesson for us all as researchers is the ability to acknowledge and evaluate hypotheses contradicting our own. Dr. Fabiato, in this review article, analyzed the probability of a direct activation of myofilaments consequent of transsarcolemmal Ca²⁺ influx, which is the main hypothesis in objection of CICR. There are other concurrent hypotheses regarding the activation of SR Ca²⁺ release, as outlined in Bers (3). However, it is generally agreed that these postulated mechanisms only have a minor role in modulating the predominant CICR in CMs.

Role of Ca²⁺ buffering.
As Dr. Fabiato pointed out, previous reports in support of direct Ca²⁺-influx activation of myofilaments only considered the [Ca²⁺] bound to myofilaments but failed to recognize the roles of other Ca²⁺ buffers in the cell. In an earlier section, we described the use of EGTA to mimic the effects of Ca²⁺ buffering. The physiological Ca²⁺ buffers in a CM are phosphocreatine, calmodulin, troponin C, an external binding site on the SR, and the high-affinity binding site on the sarcolemma. Using a computational program that Dr. Fabiato has described elsewhere (9), and taking into account the various Ca²⁺ buffers, values are obtained for total [Ca²⁺] and myoplasmic or free [Ca²⁺] needed to generate varying amplitudes of tension (Table 2; Table 4 in the original review article). The highest baseline myoplasmic [Ca²⁺] before CICR is induced is assumed to be pCa 7.10 (i.e., 7.94 x 10^–8 mol/l) based on experiments in skinned CMs. This baseline myoplasmic [Ca²⁺] is only achieved when total [Ca²⁺] is 7 x 10^–6 mol/l. That means over 98% of the Ca²⁺ is buffered or bound, leaving only 1–2% of free Ca²⁺ in the myoplasm, as reflected by myoplasmic [Ca²⁺]. As shown in Table 2, the ratio of free to bound Ca²⁺ increases slightly as total [Ca²⁺] gets higher. Nonetheless, even at the total [Ca²⁺] of 1.42 x 10^–4 mol/l, where near-maximal tension is obtained, 97% of the Ca²⁺ is still bound. These numbers reflect the importance of Ca²⁺ buffering in the consideration of a valid trigger to induce CM contraction.

Based on the values shown in Table 2, measurements of total [Ca²⁺] during I_Ca in other studies can serve as evidence to disprove the hypothesis of direct activation of myofilaments from Ca²⁺ influx. It has been found by Fabiato and Baumgarten (7) that the increase in total [Ca²⁺] during the first 20 ms of I_Ca is 1 x 10^–5 mol/l under physiological conditions. This amounts to a total [Ca²⁺] of 1.7 x 10^–5 mol/l when the baseline total [Ca²⁺] of 7 x 10^–6 mol/l (from Table 2) is taken into account. If over 98% of the total [Ca²⁺] is bound, as Table 2 suggests, the myoplasmic free [Ca²⁺] can only reach 1.92 x 10^–7 mol/l (pCa 6.72). Extrapolating from the values shown in Table 2, pCa 6.72 is likely to induce <5% of maximal tension only. However, the same pCa (6.72) is enough to induce CICR (7). In conclusion, Ca²⁺ influx from I_Ca under physiological conditions is able to induce CICR but not CM contraction directly.

TEACHING POINTS OF TABLE 2 (INSTRUCTOR'S REFERENCE).

1. Students may be asked to produce on the same graph a plot of free [Ca²⁺] and total [Ca²⁺] versus tension (Fig. 6). A dotted line is used to show the extrapolated values of free [Ca²⁺] and total [Ca²⁺] between 0% and 5% tension. It may be preferable to plot [Ca²⁺] in a logarithmic scale to accentuate the nearly 2 log step (i.e., 100-fold) difference between total and free [Ca²⁺]. Students should realize that almost 97-98% of all Ca²⁺ is buffered, meaning the actual Ca²⁺ trigger (from Ca²⁺ influx) for CICR as detected by the SR is of a very low concentration. Therefore, it is highly unlikely that Ca²⁺ entry, resulting in very small [Ca²⁺] increase, can activate myofilaments directly and cause contractions.
   
2. When Tables 1 and 2 are compared, we notice that the optimal free [Ca²⁺] generating 70% or near-maximal tension is very different. A likely explanation is that different Ca²⁺ buffers are included in the data from both tables. Only phosphocreatine is considered in Table 1, whereas Table 2 also accounts for other Ca²⁺ buffers. Thus, not all the free [Ca²⁺] in Table 1 is available to trigger CICR. However, the data in Table 1 should not be discounted completely, as we should acknowledge that the numbers in Table 2 are computed values but not actual experimental data.
   

View larger version (8K): [in this window] [in a new window]   Fig. 6. Relationships between total and free [Ca2+] in the generation of tension based on computed values shown in Table 2.

1. Plot on the same graph the relationship between tension and both total and free [Ca²⁺]. What is the ratio of total [Ca²⁺] to free [Ca²⁺] for any given tension in general?
   
2. Suggest reasons that may have contributed to the different free [Ca²⁺] values in generating 70% tension as shown in Tables 1 and 2.
   

Issues Pertaining to the Skinned CM Preparation
The final section of this teaching article aims to summarize the limitations of the skinned cell technique, as pointed out by Dr. Fabiato, although its extensive use throughout the years has largely enriched our understandings of physiology. Instructors may highlight the following teaching points during discussions of common experimental techniques employed in cellular, tissue, and systematic physiology. The objective here is to raise awareness among students regarding the pros and cons of any "cell model" systems, in this case, the skinned CM model.

TEACHING POINTS OF THE "SKINNED CM PREPARATION" (INSTRUCTOR'S REFERENCE).

1. Understanding the differences between various "skinning" preparations.
   1. In Dr. Fabiato's experiments, the sarcolemma was physically removed, thus the term "physical skinning." However, there is another way of "skinning" done by Ca²⁺-chelating agents (e.g., EGTA) or detergents (e.g., saponin). This latter method of "skinning" is commonly termed as "chemical skinning." Chemically skinned preparations are useful in studying whole tissues or multicellular preparations, thereby allowing a certain extent of intercellular interactions to be observed. The disadvantage of chemically skinned cells is the slow diffusion of substances from the extracellular space. The slower diffusion rate greatly affects the accurate measurements of the CICR trigger, which is dependent not only on [Ca²⁺]_Out-SR but also on its rate of change. Although [Ca²⁺]_Out-SR for single physically skinned CMs is determined as described earlier, this method of skinning rids the cell of subsarcolemmal SR, the equivalent of terminal cisternae in skeletal muscle. In cardiac muscle, CICR also occurs at the subsarcolemmal SR, and skinning eliminates the contribution of this SR to Ca²⁺ release.
      
   
   
2. Structural alteration of the SR associated with skinning.
   1. As mentioned earlier, the skinning procedure can cause structural damage to the SR and myofibrils as well. When the sarcolemma is removed, the skinned cell is exposed to a bathing solution that should mimic the intracellular fluid in an intact cell. Quite often, the ionic and chemical composition of the bathing solution is suboptimal in terms of osmolarity. Swelling of the SR is not uncommon but can be rectified by increasing the osmolarity of the bathing solution with the addition of sucrose.
      
   
   
3. Composition of a physiological bathing solution (intracellular milieu).
   1. Osmolarity is just one of several concerns in constituting the bathing solution of skinned cells. One other major problem is with EGTA. In skinned cell experiments, [EGTA] can alter the level of free [Ca²⁺] available for CICR induction. Moreover, it has been found that zinc is present in large quantity in striated (skeletal and cardiac) muscles. Zn²⁺, being a divalent ion, can be chelated by EGTA as well. Thus, the actual [EGTA] available for Ca²⁺ buffering will be much lower than when Zn²⁺ is not present. The ratio of Zn²⁺ and Ca²⁺ chelation by EGTA was not estimated by Dr. Fabiato, and he acknowledged that this could be a source of uncertainty in the skinned cell preparation. If [EGTA] is increased to accommodate Zn²⁺, too much EGTA has been shown to disrupt charge distribution on the SR. High [EGTA] in a skinned cell preparation also results in a constant myoplasmic or free [Ca²⁺] globally, a phenomenon not true in intact cells. Aside from these issues that are known, there may be many others that are yet to be discovered. Therefore, the challenges in determining an intracellular milieu that resembles the in vivo environment may undermine the conclusions drawn from skinned cell studies.
      
   
   

Summary
In summary, this article makes suggestions in the teaching of CICR using a skinned CM preparation based on Dr. Fabiato's review article (6). Not only are the mechanisms of CICR explained but also evidence to disprove an opposed hypothesis to CICR is presented in Dr. Fabiato's article. This sets a good example for us to always consider data and evidence that are in disagreement with our own hypotheses. Finally, Dr. Fabiato's discussion of problems associated with the skinned CM technique provides a reality check to the limitations of cell models we routinely use in experiments. A detailed reading of the original review article in combination with reference to the points discussed in this article should provide a valuable learning experience for all.

Received for publication September 24, 2007. Accepted for publication November 9, 2007.

REFERENCES

1. Berridge MJ. Calcium microdomains: organization and function. Cell Calcium 40: 405–412, 2006.[CrossRef][ISI][Medline]
2. Bers DM. A simple method for the accurate determination of free [Ca] in Ca-EGTA solutions. Am J Physiol Cell Physiol 242: C404–C408, 1982.[Abstract/Free Full Text]
3. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
4. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. Boston, MA: Kluwer Academic, 2003.
5. Fabiato A. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J Gen Physiol 78: 457–497, 1981.[Abstract/Free Full Text]
6. Fabiato A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am J Physiol Cell Physiol 245: C1–C14, 1983.[Abstract/Free Full Text]
7. Fabiato A, Baumgarten CM. Methods for detecting calcium release from the sarcoplasmic reticulum of skinned cardiac cells and the relationships between calculated transsarcolemmal calcium movements and calcium release. In: Physiology and Pathophysiology of the Heart, edited by Sperelakis N. Boston, MA: Martinus Nijhoff, 1984, p. 215–254.
8. Fabiato A, Fabiato F. Contractions induced by a calcium-triggered release of calcium from the sarcoplasmic reticulum of single skinned cardiac cells. J Physiol 249: 469–475, 1975.[Abstract/Free Full Text]
9. Fabiato A, Fabiato F. Calculator programs for computing the composition of solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol 75: 463–505, 1979.
10. Harder D. Ca-induced Ca release: lessons regarding cell models. Am J Physiol Cell Physiol 287: C1165–C1166, 2004.[Abstract/Free Full Text]
11. Meissner G. Molecular regulation of cardiac ryanodine receptor ion channel. Cell Calcium 35: 621–628, 2004.[CrossRef][ISI][Medline]
12. Page E, Surdyk-Droske M. Distribution, surface density, and membrane area of dyadic junctional contacts between plasma membrane and terminal cisterns in mammalian ventricle. Circ Res 45: 260–267, 1979.[Abstract]

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