HEART MUSCLE (View Printable Version)

Fig. H1.  Scheme of the heart compartments and the direction of the blood flow during diastole.  Note the pulmonary artery on the top of the right ventricle and the aorta on the top of the left ventricle.  The valves separating the atria from the ventricles are not shown.

Alternating contractions and relaxations of the heart muscle, called myocardium, causes the pumping of the heart.  There is a pacemaker in the right atrium that generates electrical impulses causing the atria to contract and thereby forcing blood into the ventricles.  Following contraction (systole) the ventricles relax (diastole); the entire process is called the cardiac cycle.  Normal human heart beats about 70-times per minute at rest.  The rate of heart beat increases during exercise, emotional excitement and fever, and decreases during sleep.

Ultrastructure: Cardiac muscle is composed of interconnected mono-nucleated cells.  The cells are imbedded in a weave of collagen.  The ultrastructure of the heart contains a large number of myofibrils, striated like in skeletal muscle.  A large fraction of the cell volume is occupied by mitochondria, which synthesize ATP to supply energy for the constantly working heart muscle.  Myofibrils and mitochondria occupy about 85% of the heart cell volume, the rest contains the sarcolemma, T-tubules, sarcoplasmic reticulum, and specialized structures such as the intercalated disk, which connects adjacent heart cells (cardiomyocytes), and gap junction or nexus which makes contact between the plasma membranes of adjacent heart cells (Fig. H2).

Fig. H2. Electron micrograph of cardiac muscle (Courtesy of Dr. Helen Rarick).

Contractile Proteins

Myosin. Cardiac myosin is composed of two heavy chains (HCs) and four light chains (LCs).  The ventricles contain only two types of light chains (skeletal muscle myosin possess three) with an estimated molecular mass of 19,000 dalton (regulatory light chain, RLC) and 27,000 dalton (essential light chain, ELC). The mass of entire cardiac myosin is about 480,000 and its length is approximately 1,700 Å (Katz 1992), similar to these parameters in skeletal muscle myosin (For structural organization see Figure M1).

The multigene families of HCs and LCs generate different isoforms that occur at various stages of heart development.  Two HC isoforms are known as α- and β-isoforms.  First it was thought that the functional properties of heart muscle are determined by their LC composition, but it turned out that HCs determine the myosin ATPase activity in vitro and the shortening velocity in the intact heart (Katz, 1992). In experimental diabetes the HC isoform expression was greatly altered (Rundell et al., 2004). After 6 weeks in diabetes the α-myosin isoform decreased to 41% and after 12 weeks to 33% as compared to the 78% and 74% control values, respectively. Cross-bridge cycling rate was significantly decreased in diabetes and the tension cost decreased linearly with the decreased α-myosin content.

In the atria there are two myosin isoforms, A₁ and A₂, with high and low ATPase activity, respectively, whereas in the ventricles there are three myosin isoforms, V₁ (α - α), V₂ (α - β), and V₃ (β - β), with high, intermediate, and low ATPase, respectively.  A comparative study on the various ATPase activities of rabbit heart and skeletal muscle myosins showed that under all conditions the activities of the skeletal source are higher (Bárány et al., 1964).

The atria and the ventricles contain different LC isoforms.  The atrial LCs are also found in developing heart and fast skeletal muscle, and in adult slow skeletal muscle.  The 19,000-dalton heart LC is phosphorylated and possibly participates in the regulation of heart contraction (Kopp and Bárány, 1979).

Actin: Purification   Heart actin has not been studied in detail. Recently we reported a procedure for the preparation of pure rat heart actin (Bárány and de Tombe, 2004). The hearts from freshly killed rats (15-20) were dropped into 1000 volumes of 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4 (kept in ice). The ventricles were isolated and repeatedly washed with the NaCl-Tris solution to remove blood. The ventricles were minced with a Latapie mincer, then myosin was extracted with 3 volumes of a solution containing 0.3 M KCl, 0.15 M KP_i, 2.5 mM MgSO₄ and 2.5 mM ATP, pH 6.5, in ice with stirring for 10 min. After centrifugation at 10,000 rpm for 10 min, the residue was washed, throughout in ice, with 5 vol of 0.4% NaHCO3, for 10 min. and centrifuged at 5,000 rpm for 10 min. The residue was washed with 10 vol of dist. water for 5 min and centrifuged at 10,000 rpm for 10 min. The residue was treated with acetone three times, 5, 3, and 2 vols, each time for 10 min of acetone, and centrifuged each time at  5,000 rpm for 10 min. The final residue was air dried overnight.

The dried powder was extracted with 30 vols of 0.5 mM ATP in ice for 1 hour; the supernatant was isolated by centrifugation at 15,000 rpm for 15 min. About half of the supernatant protein was actin that could be purified as described ((Bárány and de Tombe, 2004). To obtain the main bulk of actin in the heart powder, we extracted the residue of the first extract with 30 vol of a solution containing 0.25 M KI, 0.02 M Tris-HCl, pH 7.4 , and 1 mM ATP, in ice, for 1 hour. After centrifugation at 15,000 rpm for 30 min, the supernatant containing the actin was dialyzed against large volumes of a solution containing 0.1 M NaCl and 2 mM  MgCl₂ with frequent changes, at 4^oC for 20 h. To the viscous F-actin solution NaCl was added (from a concentrated solution) to 0.6 M , it was stirred in ice for 30 min, then centrifuged at 30,000 rpm, 5^oC,  for 3 h. The F-actin pellet was rinsed with dist water to remove salts, then dissolved by a Teflon-glass homogenizer in 0.5 mM ATP, pH 7.4, to yield a non-viscous G-actin (5-6 mg/ml). Upon addition of salts to 0.1 M NaCl and 2 mM MgCl₂, this actin solution became very viscous.

Fig. H3.  Electrophoresis of purified rat heart actin on 10% Bis-Tris gels. Actin 1 and Actin 2 refer to two different actin preparations. The gels were loaded with 10 and 15 μg actin protein, respectively.

Exchange of the actin-bound nucleotide in perfused rat heart:  In order to study the functional role of the actin bound nucleotide (ATP or ADP) in intact muscle there is need for a method to separate the free nucleotides in the cytoplasm from the actin-bound nucleotides in the myofibril structure. Common organic solvents do this job at -15 to -20^oC. We have used  50% ethanol for arterial smooth muscle (Bárány et al., 2001, see in the Smooth Muscle Chapter) and 75% methanol for rat heart (Bárány and de Tombe, 2004). If the muscles are incubated in physiological saline containing ³²P-orthophospate (³²P_i), first the γ-P and then the β-P of ATP will be labeled to generate [γ³²P, β³²P]ATP in a short time. The exchange of actin bound nucleotide under various physiological conditions can be followed by comparing the specific radioactivity of the ³²P-phosphate in the actin bound nucleotides with that of the ³²P-phosphate in the cytoplasmic nucleotides. The exchange of the actin-bound nucleotides is complete when its specific radioactivity equals that of the cytoplasmic nucleotides. In rat hearts perfused with ³²P_i the actin-bound nucleotide rapidly exchanged with the cytoplasmic ATP. Furthermore, the rate of exchange of the bound-nucleotide was comparable to the rate of the heart beat (Bárány and de Tombe, 2004).

Our work has also revealed the presence of monomeric-actin in the cytoplasm of rat heart (about 30% of the total actin).  The mechanism of exchange of actin-bound nucleotide is visualized in Fig. H4.

Fig. H4   Mechanism of exchange of actin-bound nucleotides in perfused rat heart.

Part A of the Figure shows that the double labeled ATP^32,32 in the cytoplasm (black) exchanges with the bound-ATP of  free monomeric-actin (G-actin) in the cytoplasm to yield G- ATP^32,32-actin (red). The polymeric-actin (F-actin) filament in the upper part of Part A contains non-radioactive ADP (gray).

Part B of the Figure shows the attachment of G-ATP^32,32-actin molecules to the barbed end of the polymeric-actin double filaments, while the terminal ADP-actin monomers are leaving the filaments at the pointed end.

In Part C, we see the attachment of new G-ATP^32,32-actin molecules, while the previously attached G-ATP^32,32-actins are hydrolyzed to actin-ADP³² monomers (pink) in the filaments. At the same time new terminal ADP-actin monomers are released from the pointed ends.

This process is repeated in Parts D, E, and F; all nucleotides in the filaments are radioactive at the end.

The chemical interpretation of Figure H4 is shown by the following two equations:

The double labeled ATP in the cytoplasm exchanges with the ATP bound to G-actin (equation 1). Subsequently, the double labeled G-actin polymerizes to F-actin containing single labeled ADP while  ³²P_i  is liberated (equation 2).

The data presented in Figure H4 and equations 1 and 2 suggest that actin polymerization takes place in the perfused rat heart.

Regulatory Proteins

TN-C. The cardiac muscle TN-C (cTN-C) differs from fast skeletal muscle TN-C in that it contains only one Ca²⁺-binding site in the N-terminal domain of the protein.  The neighboring site at the N-terminal does not contain aspartic acid, a prerequisite for coordination of Ca²⁺. This site is called site 1, whereas the Ca²⁺-binding site is called site 2 of  cTN-C, or the regulatory N-terminal lobe of   cTN-C. The binding of  Ca²⁺-ions has been shown to open a hydrophobic cleft in the regulatory N-terminal lobe of skeletal  TN-C, in cTN-C  Ca²⁺ alone is not sufficient to stabilize the open conformation so that a target peptide is necessary. The orientation of the regulatory N-terminal lobe of cTN-C with respect to its structural C-terminal lobe varies, with the central linker that connects them adopting a partially disordered or fully α-helical conformation (Brown and Cohen, 2005).

TN-I: The cardiac TN-I (cTN-I) differs from the fast skeletal muscle TN-I by containing an N-terminal extension of 33 amino acid residues (Fig. H5).

Fig. H5.   Comparison of the overall structure of cardiac muscle TN-I with that of skeletal muscle TN-I.  The shaded areas correspond to homologous regions in the structures (From Perry 1996).

This extension has two adjacent serine residues, No. 23 and 24 (at the time when Fig. H5 was prepared these residues were believed No. 22 and 23, respectively) in the sequence (Kobayashi and Solaro, 2005).  Solaro et al. (1976) discovered that TN-I is phosphorylated in perfused rabbit heart stimulated by adrenaline.  It was shown later (Mittmann et al., 1992) that both serines at positions 23 and 24 of rabbit cardiac TN-I can be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Ser-24 is phosphorylated first and, subsequently, much slower phosphorylation occurs at Ser-23.   Since PKA is activated by adrenaline in the beating heart it appears that PKA is responsible for the diphosphorylation of cTN-I in the heart. Phosphorylation of Ser-23 and Ser-24 appears specialized for regulation of sensitivity of the myofilaments to Ca²⁺ and for enhancing crossbridge cycling rate (Kobayashi and Solaro, 2005).

Protein kinase C (PKC) phosphorylates Ser-42, Ser-44 and Thr-106 residues in cTN-I. This phosphorylation appears specialized for depressing crossbridge cycling rate (Kobayashi and Solaro, 2005).

NMR, fluorescence resonance energy transfer, and mutation studies indicate that the N-terminal part of cTN-I combines with cTN-C.  Upon phosphorylation of cTN-I, the site 1 of cTN-C undergoes a conformational exchange consistent with an equilibrium between closed and opened forms of cTN-C (Gaponenko et al., 1999).  In addition, cTN-I phosphorylation changes the binding of Ca²⁺ to cTN-C, the structure of cTN-I, and the cooperative binding of cTN-I to actin-TM (Solaro and Van Eyk, 1996).  Previously, phosphorylation has been shown to modulate cardiac function by reducing the Ca²⁺ affinity for the N-terminal regulatory site of cTN-C (Solaro, 1986).  Thus, cTN-I phosphorylation is a unique property of the myocardium that plays a key role in cardiac function.

Fluorescence resonance energy transfer was used (Dong et al., 2001) to investigate the global conformation of the inhibitory region of a full-length TN-I mutant from cardiac muscle in the unbound state and in reconstituted complexes with the other cardiac TN subunits.  The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as an energy acceptor.  The distance between the donor and acceptor sites was found to be 19.4 Å and it was insensitive to reconstitution of cTN-I with cTN-T, cTN-C, or cTN-C plus cTN-T, in the absence of bound regulatory Ca²⁺ in cTN-C.  A large increase in the Trp129-Cys152 distance was observed upon saturation of the Ca²⁺ regulatory site of cTN-C in the complexes.  This increase suggests an extended conformation of the inhibitory region in the interface between cTN-C and cTN-I in the holo cardiac troponin, which may pull away the inhibitory region of cTN-I from actin upon Ca²⁺activation in cardiac muscle.

The review of Layland et al., (2005) on the regulation of cardiac function by cTN-I phosphorylation highlights the physiological and pathophysiological aspects of this reaction.

Many mutations of cTN-I have been described that are related to cardiomyopathy. The cause of the myopathy is the mechanical defect in the cardiac muscle fibers. For instance, inserting mutated human cTN-I from inherited restrictive cardiomyopathy, with a small structural change, into skinned cardiac muscle fibers greatly increased the Ca²⁺ sensitizing effect on force generation (Yumoto et al., 2005). Furthermore, increased Ca²⁺ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant  cTN-I was reported (Kobayashi and Solaro, 2006). This finding may be an important factor in triggering arrhythmias in cardiomyopathy.

In general, analyses of the mechanism by which a single mutation selectively perturbs one component of cardiac physiology one may get an insight into the complex process of heart failure (Morita et al., 2005)

TN-T: As skeletal TN-T, cardiac TN-T (cTN-T) also has several isoforms (McAuliffe et al., 1990).  Two TN-T isoforms have been identified in adult beef heart, which show differences in their sequence and activation of the actinS1 ATPase.  Five isoforms were found in rabbit heart and two in rat heart.  During development of rabbit heart, there are shifts in the isotype population of cTN-T and these are related to the differences in the Ca²⁺ regulation between neonate and adult hearts.

Mutations between residues 92 and 110 of cTN-T impair its TM dependent function (Palm et al., 2001).   Recombinant human cardiac TN, with wild type TN-T and ΔLys-210 TN-T mutant, altered thin filament regulation by increasing Ca²⁺ sensitivity   (ΔpCa₅₀  =  + 0.2 pCa units), while virtually abolishing the cooperativity of Ca²⁺ activation (Robinson et al., 2002).

Residues Thr-194, Ser-198, Thr-203, and Thr-284 are phosphorylated by PKC in cTN-T. Among these four sites, Thr-203 is the most significant: it significantly inhibits tension and Ca²⁺ sensitivity of skinned heart fiber bundles (Kobayashi and Solaro, 2005).

Troponin T modulates sarcomere length-dependent recruitment of crossbridges in cardiac muscle (Chandra et al., 2006)

The troponin complex: A major advance in the understanding of the active and inhibited state of cardiac thin filaments came from the determination of the crystal structure of a ternary cardiac troponin complex (Takeda et al, 2003). These authors described that the core domain of human cardiac TN in the Ca²⁺ saturated form is divided into structurally distinct subdomains that are connected by flexible linkers, making the entire molecule highly flexible. The α-helical coiled-coil formed between TN-T and TN-I  is integrated in a rigid and asymmetric structure (about 80 Ǻ long), the IT arm, which bridges putative tropomyosin-anchoring regions. The structures of the TN ternary complex imply that Ca²⁺ binding to the regulatory site of TN-C  removes the carboxy-terminal portion of TN-I from actin, thereby allowing tropomyosin to move on the actin filament.

Structural model of  phosphorylation sites in cardiac troponin.  Most of the phosphorylation sites of cTN-I and cTN-T were not resolved in the crystal structure. Some information may be obtained about the surrounding areas of Ser-42 and Ser-44 in cTN-I and Thr-203 of cTN-T which were visible in the crystal structure (Fig. H6).

Ser-42 forms a side chain backbone hydrogen-bond network with Arg-45, which represents the most common pattern of α-helix capping (Part A of Fig.H6). Phosphorylation of Ser-42 may affect the capping and thus alter cTN-I local structure. Ser-44 of cTN-I interacts with Glu-10 of the N-lobe of cTN-C, indicating that phosphorylation of Ser-42/Ser44 might affect the interaction between cTN-I and cTN-C and thus Ca²⁺ activation. Another potential phosphorylation site visible in the crystal structure is Thr-203 of cTNT (Part B of Fig. H6). This residue also acts as an N-cap of a helix. Modeling of the structural change induced by phosphorylation of Thr-203 predicts an extension of the helix. Compared with controls, skinned fiber bundles containing cTN-T-Thr-203-Glu demonstrate a significantly reduced tension and ATPase rate, as well a reduced Ca²⁺ sensitivity (Kobayashi and Solaro, 2005).

Fig. H6.  Structural model of phosphorylation sites in cTN.  (A)  cTN-I (yellow)/ Ser-44 (red) and nearby residues. Ser-42 forms a capping box wit Arg-45 (magenta). Whereas Ser-44 interacts with Glu-10 (blue) from the N-terminal domain of cTN-C in the crystal structure, pseudophosphorylation (Asp substitution) of Ser-42/ Ser-44  perturbs  the N-terminal structure of the G-helix (also shown in blue) from the C-terminal domain of cTN-C.  (B)  cTN-T  Thr-203 (red) and nearby residues.  Thr-203 forms a capping box with Glu-206 (pale magenta) of cTN-T. (From Tomoyoshi Kobayashi and R. John Solaro, CALCIUM, THIN FILAMENTS, AND THE INTEGRATIVE  BIOLOGY  OF  CARDIAC  CONTRACTILITY. Annu. Rev. Physiol. 2005, 67: 39-67. Reproduced with permission. Copyright 2005 by Annual Reviews.

Structural and functional differences between cardiac and skeletal isoforms of troponin: Comparison of the structures and functions of cardiac and skeletal troponins shows several differences (Vinogradova et al., 2005). The Ca²⁺ binding site 1 in cTN-C is inactive because amino acid replacements. The N-terminal lobe of  cTN-C does not open as much as in skeletal TN-C in the presence of Ca²⁺. At the N-terminal cTN-I has an extension of 33 amino acid residues. Ca²⁺ has less effect on cTN than on skeletal TN.

On the other hand, the overall organization of the subunits in cTN and TN is similar.  However, the following features do not match: The structure of the TN-I inhibitory segment is ordered and visible only in the skeletal muscle isoform. The TN-C Ca²⁺ regulatory domain which is positioned in space by the central helix of TN-C is rigid in skeletal muscle TN-C, but it is melted in cardiac TN-C

TM: In skeletal muscle two isoforms of TM, α and β (each under different genetic control) are expressed.  In contrast, in the heart only the α-form of TM is expressed.  However, using transgenic approaches mice could be produced with overexpressed β-TM in the heart.  Novel functions of this TM isoform were detected (Palmiter et al., 1996).  Thus, the cardiac myofilaments, containing β-TM demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca²⁺ sensitivity of steady state force, and a decrease in the rightward shift of the Ca²⁺-force relation induced by cAMP-dependent phosphorylation.  These data indicate that switching of TM isoform has a major effect on heart myofilament activity.

Movement of tropomyosin during the heart cycle:  Fig. H7 shows that in diastole TM is fixed in the groove of the actin double helix by TN-T and  TN-I.  The position of the TM is such that the myosin cross-bridges (MHC, ELC, RLC) can not react with actin.  In systole, Ca²⁺-TN-C interacts with TN-I and TN-T. This allows TM to move on the thin filament removing the steric hindrance of the actin-crossbridge combination and systole ensues.

Fig. H7.   Illustration of  the movement of tropomyosin in the heart cycle (Courtesy of Dr. Helen Rarick).  For details see the text.

In summary, cardiac contraction is a series of interactions between Ca²⁺, the regulatory proteins, and the actomyosin system.  In the resting muscle, at low intracellular free Ca²⁺concentration, the TN-TM complex inhibits the actin-myosin combination and with an increase in the myoplasmic Ca²⁺ the inhibition is released.  The Ca²⁺ signaling process starts with the binding of Ca²⁺ to a single regulatory site of TN-C and by a tight binding of TN-C to TN-I.  The signal is transmitted by TN and TM to actin in the thin filament.  The final step is the combination of actin with myosin.

Methods

Displacement of endogenous TN in skinned fibers with TN mutants: A very sensitive method for mapping the functional domains of TN components is the replacement of native TN in the fibers with mutant or modified TN and then assaying force development or Ca²⁺-sensitivity of the modified fibers.  In principle, the fibers are briefly treated with 1% Triton X-100 to remove the sarcolemma and subsequently washed to remove the Triton.  The fibers are exposed to 10-20-fold excess of exogenous TN, over the endogenous TN, in a medium containing 250 mM KCl. Finally, unbound TN is removed by washing the fibers. For a long time, the replacement studies were performed with skeletal muscle fibers, but a method for exchanging the TN subunits in cardiac fibers was developed (Chandra et al., 1999). The skinned rat cardiac fiber bundles remained relaxed through much of the extraction/reconstitution procedure. The fibers were treated with a mixture of  cTN-T-cTN-I  to displace the endogenous TN; 70-80% of the endogenous TN subunits were removed.  After reconstitution with cTN-C the Ca²⁺-dependence of the force development by the fibers was restored to the extent of 80-85%.

Reconstitution of a modified TN can also be carried out at the level of myofibrils and the Mg²⁺-ATPase activity of the modified fibrils can be measured as a function of pCa (Rarick et al., 1997).

Purification of cardiac myofibrils is important for studying the interaction of the troponin components with each other, with tropomyosin and actin.  Myofibrils purified by conventional methods are contaminated with mitochondrial, sarcolemmal and sarcoplasmic reticulum membranes.  Treatment of these myofibrils by 1% Triton X-100 removes the contaminants (Solaro et al., 1971).  The MgATPase activity of the purified myofibrils corresponds to that of cardiac actomyosin and they exhibit the normal high Ca²⁺-sensitivity.

Gel electrophoresis of the regulatory proteins: In our laboratory, 10% polyacrylamide gels containing 1% SDS, 0.1 M Na-phosphate, pH 7.0 and 8.0 M urea are used for separation of cTM, cTN-T and cTN-I (Fig. H8).  Under these conditions cTM (zone A, 48-kDa) migrates behind actin, cTN-T (zone B, 38-kDa) migrates in front of actin, cTN-I (zones C and D, 30- and 29 kDa) splits into two components.  Zones E and F, correspond to the 27- and 19-kDa cardiac myosin LCs.  cTN-C migrates in front of the 19-kDa LC,  it is not visible because it does not stain with Coomassie blue.

Fig. H8.   Separation  of rat heart myofibril proteins by gel electrophoresis (From Kopp and Bárány, 1979).  Gel 1, myofibrils; gel 2, beef heart myosin; gel 3, beef heart troponin; and gel 4, beef heart TM.  For other details see the text.

Literature:  Reviews on the interaction of the TN complex, TM, and actin in cardiac thin filaments are available (Tobacman, 1996; Brown and Cohen, 2005; Kobayashi and Solaro, 2005).

Regulation of Ca²⁺ Flow

Sarcoplasmic Reticulum

Ca²⁺ transport into the sarcoplasmic reticulum (SR) occurs via the action of the SR Ca²⁺ pump.  As in skeletal muscle, 1 molecule of ATP has to be hydrolyzed per 2 molecules of Ca²⁺ that are pumped against a large concentration gradient into the lumen of the SR.  The SR Ca²⁺-ATPase, called sarco(endo)plasmic reticulum Ca-ATPase (SERCA), is regulated by phospholamban (described below).  Ca²⁺ is released from SR through a  Ca²⁺ release channel.  The channel tightly binds ryanodine and, therefore, it is also referred to as the ryanodine receptor (RYR), or Ca²⁺ release units (CRUs). In cardiac muscle, CRUs come in three subtypes that differ in geometry, but have common molecular components (Franzini-Armstrong et al., 2005). Peripherial couplings are formed by a specialized junctional domain of the SR (jSR) with the plasmalemma. Dyads occur where the jSR is associated with transverse (T).-tubules. Corbular SR is a jSR domain that is located within the cells and bears RYRs but does not associate with either plasmalemma or T-tubules.

Ca²⁺ induced Ca²⁺ release (CICR) CICR is specific for the heart.  The small amount of Ca²⁺ that enters the cell through voltage dependent plasmalemmal Ca²⁺ channels (which open in response to the action potential) causes a much larger amount of Ca²⁺ to be released from within the SR.

Calsequestrin is a 44 kDa Ca²⁺ binding protein that is located within the lumen of SR and is primarily responsible for Ca²⁺ storage within the SR. Both cardiac and skeletal muscle calsequestrins have been crystallized and their structures and Ca²⁺ binding capacities compared (Park et al., 2004). The two crystal structures for cardiac and skeletal calsequestrins are nearly superimposable and both proteins can undergo dimerization, tetramerization and oligomerization  The Ca²⁺ binding capacities are; 60 Ca²⁺ ions per cardiac calsequestrin and 80 Ca²⁺ ions per skeletal calsequestrin, as compared with net charges for these molecules of -60 and -80, respectively. Authors suggest that Ca²⁺ binding to calsequestrin is coupled to the polymerization of the protein and the release of large number of Ca²⁺ ions during contraction occurs through surface diffusion from calsequestrin to the Ca²⁺ release channel.

Phospholamban (PLB): It is a pentamer made up of five identical subunits, each 6,000 dalton (52 amino acid residues).  PLB is found in cardiac, slow skeletal and smooth muscle but it is absent in fast skeletal muscle.  The protein is associated with the Ca²⁺-pump ATPase in the membrane of the SR.  The flexible N-terminus on which the phosphorylation sites are located extends out into the cytoplasm of the cardiac muscle cell.  The phosphorylation site at serine 16 is a substrate for PKA, whereas threonine 17 is phosphorylated by a Ca²⁺-calmodulin dependent protein kinase.  Phosphorylation of PLB by PKA increases the rate of Ca²⁺ transport and the Ca²⁺-sensitivity of the Ca²⁺-pump and thereby facilitates relaxation in heart exposed to β-adrenergic agonists (Katz, 1992; Perry, 1996).  Phosphorylation of PLB by a Ca²⁺-calmodulin dependent protein kinase II also stimulates Ca²⁺-uptake in vitro, and the physiological significance of this phosphorylation is extensively studied (Maier and Bers, 2002).

Research on the physiological significance of PLB reached an unexpected turn when a PLB deficient mouse was generated, the "PLB knockout (KO) mouse".  Surprisingly, there were no detrimental effects in the performance of such animals or in the function of their isolated heart.  For instance, the basal contractility and the Ca²⁺-transient of myocytes isolated from PLB deficient hearts were enhanced compared with cells isolated from wild type animals.  Furthermore, the contractility of PLB-deficient myocytes could be further enhanced by the β-adrenergic agonist, isoproterenol.  These results demonstrate that in the absence of PLB, there are mechanisms available in the heart to adjust its activity, and that phosphorylation of sites other than PLB may play an important role in regulation of contraction-relaxation dynamics of heart responding to β-adrenergic stimulation (Wolska et al., 1996).

Factors controlling the Ca²⁺ release from SR:

Ryanodine (a plant derived alkaloid) is a very effective drug that alters the SR Ca²⁺ release channel.

Phospholamban  phosphorylation by PKA.

Thapsigargin  and cyclopiazonic acid (CPA) that block the Ca²⁺ pump.

Digitalis, a drug that inhibits the Na⁺/K⁺ pump leading to a small increase in intracellular Na⁺.  This decreases the rate at which the Na⁺/Ca²⁺ exchanger extrudes Ca²⁺ from the cell and thus leads to enhanced loading of the SR.

Sarcolemma

Ca²⁺ enters the cardiac cell during the plateau phase of the action potential via the L-type Ca²⁺ channel (The channel tightly binds the Ca²⁺ channel blocking agents of the dihydropyridine family).  The sarcolemmal Na⁺/Ca²⁺ exchanger transports 3 Na⁺/Ca²⁺.  The energy for this transport is indirectly derived from ATP hydrolysis via the sodium gradient that is established by the Na⁺/K⁺ pump.  It is the major Ca²⁺ extrusion mechanism of the cardiac myocyte and may contribute significantly to myocardial relaxation.  The sarcolemma also houses the Na⁺/K⁺-ATPase that transports 3 Na⁺ out and 2 K⁺ into the cell per molecule of ATP, and thus moves out one net charge per cycle.  This is the key transporter that sets up the sarcolemmal ionic gradients for Na, K, and Ca, and consequently allows ion channels to function.  The Na⁺ and K⁺ channels in the sarcolemma are involved in action potential generation.  Fig. H12, illustrates the channels and the ion movements in the sarcolemma.

Ca²⁺ Sensitivity

Ca²⁺ is the central factor in myocardial contraction and the potential of a cardiac system to be activated by Ca²⁺ is characterized by its Ca²⁺ sensitivity.  Originally, Ca²⁺ sensitivity was used to define the relationship between Ca²⁺ concentration and tension, but later the relationship was extended from tension to other parameters as well, e.g. myofibrillar ATPase activity or fluorescence intensity.  Conventionally, Ca²⁺ sensitivity is illustrated by plotting the pCa against the selected parameter, expressed as percentage of the control value (Fig. H9).

Fig. H9.   Graph for a pCa - activity relationship in a biological system.  "A" and "B" represent the variations within the same system, e.g. animals of different ages.

The pCa curve can shift to the left "increased Ca²⁺ sensitivity" or to the right "decreased Ca²⁺ sensitivity".  The extent of shift can be estimated from the difference of the pCa values at 50% activity.  The shift may be small, 0.05 pCa, or large >1.0 pCa.

There are several factors, which influence the Ca²⁺ sensitivity of the myocardium.  In case of the Force - pCa relationship the factors are: TN-I phosphorylation, β-tropomyosin, aging, acidosis, sarcomere length, temperature, ionic strength, caffeine, or other agents (Bers, 2001).  It should be mentioned that most Force - pCa relationships were measured on cardiac myofilaments with their sarcolemma removed by Triton treatment, "skinned fibres"; the Ca²⁺ sensitivity of the skinned preparations may differ from those of intact heart ventricles.

Some authors attribute the differences in Ca²⁺ sensitivity among hearts of various animals (frog, guinea pig, rat, rabbit, and cow) to differences in their TN-C content.  On the other hand, others feel that the Ca²⁺ sensitivity is not based on the single reaction between Ca²⁺ and TN-C but it rather reflects a series of reactions involved in the signal transduction initiated by Ca²⁺.

Excitation-Contraction Coupling

Ca²⁺ is an absolute requirement for excitation-contraction coupling (E-C) in the heart (Fig. H10). In the absence of Ca²⁺ the conformation of the troponin complex keeps tropomyosin in a blocking position along the actin filament. This effectively inhibits the myosin crossbridges to combine with actin and results in cardiac muscle relaxation. Upon release of Ca²⁺ , from the SR and through channels and transport system in the sarcolemma, the regulatory site in the N-terminal lobe of troponin-C binds Ca²⁺  thereby inducing a conformational change in the troponin complex. This causes tropomyosin to travel away from and expose binding sites on actin. Myosin crossbridges are now able to interact with actin, resulting in cardiac muscle contraction.

Fig.  H10.   (A)  Cardiac muscle relaxed in the absence of Ca2+.  (B)  Cardiac muscle activated in the presence of Ca2+ (Courtesy of Dr. Patti Engel).

There is a recurring theme that skeletal muscle contraction depends almost exclusively on Ca²⁺ released from SR with insignificant Ca²⁺ entry across the sarcolemma during a normal twitch.  Cardiac muscle contraction, on the other hand, depends on both Ca²⁺ entry across the sarcolemma and Ca²⁺ release from the SR.  There are notable differences in the ultrastructure: Skeletal muscle has an extensive and well organized SR network, abutting the narrow T-tubules.  In contrast, the SR of cardiac muscle is rather sparse and less organized, and surrounded with T-tubules of much larger diameter.  In addition, cardiac myocytes are only 0.02 nm thick, whereas the diameter of the skeletal muscle fibers goes up to 0.2 nm.  Thus, a substance from the extracellular space reaches the center of the heart cell much faster than the center of the skeletal muscle cell.  In general the structure of the cardiac cell is consistent with a larger role of the transsarcolemmal Ca²⁺ fluxes. Ca²⁺-calmodulin dependent protein kinase II regulates Ca²⁺-transporters in the heart (Maier and Bers, 2002).

Major events in cardiac E-C coupling (courtesy of Dr. Pieter de Tombe):

Excitation

Ca²⁺ induced Ca²⁺ release

Activation of contractile proteins

Ca²⁺ reuptake into the SR and Ca²⁺ extrusion leading to relaxation

Biochemistry of Starling’s Law

The Starling law describes an enhancement of contractile function of the heart resulting from the increased end-diastolic ventricular volume and consequently from an increase in muscle length.  The Starling law also applies to skeletal muscle but in cardiac muscle it is much more pronounced.  The simplest explanation of the phenomenon is based on the constant volume principle, i.e. the increased muscle length requires a decrease in the interfilament space in order to keep the volume of the muscle cell constant.  X-ray diffraction studies verified this assumption (Irving et al., 2000).  Accurate measurements of interfilament spacing, by synchrotron X-ray diffraction, as a function of sarcomere length, using skinned and intact rat trabecules demonstrated that the lattice spacing was decreased as sarcomere length increased.  Thus, the Starling law can be explained by the enhanced actin and myosin interaction as the result of moving the actin and myosin filaments closer to each other at the longer muscle length.

The Starling law is also characterized by an increase in Ca²⁺ sensitivity of the cardiac myofilaments as the sarcomere length increases ( Solaro, 1999). At a longer sarcomere length the distance between thick and thin filaments decreases and hence the length of the diffusion pathway for Ca²⁺, in the interfilament space, to reach TN-C decreases.  One can assume that saturation of TN-C with Ca²⁺ and subsequent activation of the thin filaments is easier and faster at smaller interfilament spacing than at the larger spacing.  However, the explanation is more complex because tension development induced by Ca²⁺ is not a linear but a cooperative activation of the actin-myosin combination.

Fig. H11 illustrates a current  method to measure force-length relation in cardiac muscle.  A small strip of cardiac muscle is mounted in an experimental setup.  Sarcomere length is measured by laser diffraction techniques.  Force is measured by a sensitive force transducer.  Muscle length is controlled by a high speed device.

Fig. H11.  Force-length measurement with small heart strips (Courtesy of Dr. Pieter de Tombe).

Energetics

The energy that the heart uses, to pump the blood, is generated through the hydrolysis of ATP to ADP and P_i.  ATP is constantly generated by the mitochondria that are abundant in heart muscle cells.  Since the outer mitochondrial membrane is impermeable to adenine nucleotides there is need for "energy carriers" to transport the energy into the cytosol.  This is achieved by the phosphorylcreatine (PCr) shuttle (Bessman and Geiger, 1981), that is, excess ATP is transformed to PCr within the mitochondrial inner membrane through creatine kinase isoforms, located in the mitochondria.  The PCr formed diffuses into the cytoplasm to saturate the myofibrillar water.  When ATP is hydrolyzed by actomyosin, during heart beat, the ADP formed will be immediately regenerated by PCr with aid of specific creatine kinase isoforms.

In addition to the contractile apparatus, ATP is also used by the Ca²⁺-ATPase and Na⁺/K⁺-ATPase of the sarcolemma, by the Ca²⁺-ATPase of SR to store Ca²⁺, and by biosynthetic processes.

About 90% of the ATP is synthesized by oxidative phosphorylation in the mitochondria and about 10% by glycolysis, that take place in the cytosol.  Mitochondria are strictly dependent on O₂, they mainly oxidize fatty acids (note: of all the food we eat, fat has the highest calorie value) and pyruvate, arising from the glycolysis of glucose.

Since PCr plays an important role in cardiac energetics, creatine depletion and creatine supplementation studies were carried out in order to gain more insight into muscle energetics.  Feeding rats with the creatine analogue βguanidinopropionate (β-GP) reduced myocardial PCr and Cr by about 80%, the velocity of the creatine kinase reaction decreased by 90%, but the level of ATP remained unchanged (Neubauer et al., 1999).  The same biochemical alterations were found in isolated rat hearts perfused with β-GP; this was accompanied by reduced contractile performance in vitro.  However, in intact rats only a minimal functional impairment was observed.  Thus, in intact rat heart cardiac and/or humoral compensatory mechanisms are sufficient to maintain normal hemodynamics in spite of the greatly reduced PCr concentration.  The same conclusion could be drawn from studies on creatine kinase knock-out animals, which exhibited normal muscle activity suggesting that neither creatine kinase nor PCr are central to cellular energy metabolism.  However, both creatine kinase knock out and creatine analogue fed animals showed marked myofibrillar and mitochondrial remodeling, suggesting that energy transduction was altered.

Five days high dose creatine feeding enhanced creatine disposal and glycogen storage in rat skeletal muscles (Op't et al., 2001).  The creatine and glycogen response was markedly greater in oxidative than in glycolytic muscles.  This investigation contributes to the understanding of how the increased use of creatine by athletes, as a dietary supplement, improves their physical performance.

Summary: Fig. H12 shows the factors which determine contractility in the heart.

Fig. H12.  An overview of the chemical events taking place in the working heart (Courtesy of Dr. Pieter de Tombe).

The movements of Na⁺ and K⁺ determine the electrical properties of the heart membrane.  The Ca²⁺ homeostasis is established by the Ca²⁺ channel which lets the Ca²⁺ in and the Ca²⁺ pump and Na⁺/Ca²⁺ exchanger which remove the excess Ca²⁺ from the heart cell.  The intracellular Ca²⁺ is partially stored in the SR, but its main function is to activate the sarcomere to produce force and shortening.  The energy cost for the external mechanical work and Ca²⁺ storage is covered by ATP, produced by the mitochondrion.  The mitochondria burn  glucose, acetate and other fatty acids to CO₂ and H₂O, which leave the cell by diffusion.

Suggested readings: The following books (Solaro, 1986; Katz, 1992; Bers, 2001) and reviews (Solaro and Van Eyk, 1996; Tobacman, 1996;  Janssen, 1997; Solaro, 1999; Maier and Bers, 2002; Kobayashi and Solaro, 2005) can help to increase knowledge in the biochemistry of cardiac contractility

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