New Insights in Cardiac Calcium Handling and Excitation-Contraction Coupling

Abstract

Excitation-contraction (EC) coupling denotes the conversion of electric stimulus in mechanic output in contractile cells. Several studies have demonstrated that calcium (Ca²⁺) plays a pivotal role in this process. Here we present a comprehensive and updated description of the main systems involved in cardiac Ca²⁺ handling that ensure a functional EC coupling and their pathological alterations, mainly related to heart failure.

1 Introduction

Each heartbeat is the result of calcium (Ca²⁺) release and reuptake. During this cycle, the presence of this cation is essential to convert electric stimulation (action potential) in mechanic output (contraction), in a process commonly termed excitation-contraction (EC) coupling (Santulli et al. 2017a; Fabiato and Fabiato 1975). The action potential is the electrical signal that depolarizes the plasma membrane of cardiac myocytes, allowing the entrance of a relatively low amount of extracellular Ca²⁺, which in turn induces high Ca²⁺ release from the sarcoplasmic reticulum (SR) (Lenzi and Caniggia 1953). Cytosolic Ca²⁺ binds to myofilaments, activating contractile machinery (Ebashi et al. 1967; Reddy and Honig 1972). This “Ca²⁺-induced Ca²⁺ release” (CICR), represents a positive feedback mechanism that allows the functional coupling between plasma membrane and SR (Katz 1967; Fabiato and Fabiato 1979; Fabiato 1983; Isenberg and Han 1994).

2 Microanatomy of EC Coupling

In cardiac cells, the structural units that mediate CICR are termed “diads” (Page et al. 1971; Soeller and Cannell 1997), consisting in specialized cellular micro-domains each composed by terminal cisternae of SR, also known as junctional SR, localized in close proximity to a tubular invagination of the plasma membrane, the transverse tubule (T-tubules) (Soeller and Cannell 1997; Shacklock et al. 1995; Clark et al. 2001). On the side of plasma membrane, along T-tubules, there are voltage-gated Ca²⁺ channels (T-Type and L-type, also known as dihydropyridine receptors, DHPRs), whereas on the side of SR cisternae there are intracellular Ca²⁺ release channels (ryanodine receptors, RyRs) (Pinali et al. 2017; Crocini et al. 2016; Li et al. 2015; Fameli et al. 2014).

Ca²⁺ channels on T-tubules are activated by depolarization and through them Ca²⁺ from the extracellular compartment goes in the cytosol, and activates RyR channels located on the SR of the same diad (Keizer and Levine 1996; Nakai et al. 1997). It has been estimated that for each Ca²⁺ channel opened on T-tubules, about 4–6 RyR channels are activated on SR, which in turn can activate neighboring RyR channels, resulting in massive release of Ca²⁺ from SR (Wang et al. 2001; Paolini et al. 2004).

3 Troponin, Tropomyosin, and Ca²⁺: How Force Is Produced

The troponin complex consists of three subunits: tropomyosin-binding (T), inhibitory (I), and Ca²⁺ sensor (C). Troponin-T anchors the other two subunits on tropomyosin. Troponin I, under resting intracellular levels of Ca²⁺, competes with tropomyosin for a common binding site on actin myofilaments, forcing tropomyosin into a position where it sterically blocks the binding of myosin heads (Lehman et al. 2013; Rao et al. 2014). The binding of cytosolic Ca²⁺ to Troponin-C induces conformational changes leading to the so-called activated state, in which myosin-binding sites on actin are exposed (Wang et al. 1999; Lehman et al. 2009). Then, the formation of cross-bridges between actin and myosin produces the sliding of myofilaments one over the other, eventually resulting in muscle contraction (Brunello et al. 2014; Kawai et al. 2006; Piazzesi and Lombardi 1996).

The inodilator levosimendan is a Ca²⁺ sensitizer that acts via binding the Ca²⁺-saturated Troponin-C (Gustafsson et al. 2017). In particular, hydrogen-bond donor and acceptor groups on the pyridazinone ring and on the mesoxalonitrile–hydrazone moieties of levosimendan bind to a hydrophobic pocket of the Ca²⁺-saturated amminoterminal domain of Troponin-C (Robertson et al. 2008). The main consequence of levosimendan binding is that the Ca²⁺-saturated Troponin-C is stabilized in the presence of the drug, thereby promoting contractile force without an increase in the amplitude of intracellular Ca²⁺ transient. Levosimendan also acts via activation of ATP-sensitive K⁺ channels (Grossini et al. 2009). In clinical trials levosimendan exhibited non-consistent results, especially in terms of mortality (Mebazaa et al. 2007; Polzl et al. 2017; Landoni et al. 2017).

Ca²⁺ handling within cardiac myocytes is in a delicate and dynamic equilibrium, which relies on several physiological modulators and subcellular systems, structurally and functionally related. Importantly, the amount of Ca²⁺ extruded by the cell during relaxation must be the same as the amount entering during the contraction (Karlstad et al. 2012). After the contraction phase, several mechanisms contribute to relaxation. Notably, Ca²⁺ is a fundamental determinant also during relaxation, since the correct functioning of channels responsible for cytosolic Ca²⁺ removal strictly depends on Ca²⁺ concentration (Allen et al. 1988; Plummer et al. 2011; Subramani et al. 2005).

4 Ca²⁺ Handling in the Pathogenesis of Heart Failure: New Insights

An accurate cardiac Ca²⁺ handling warrants the appropriate contraction that allows the heart to pump sufficient blood to meet the metabolic demand of the body. Impairment in cardiac pump function characterizes the pathological condition of heart failure (HF) (Karlstad et al. 2012; Kitsis and Narula 2008). Malfunction of the processes involved in Ca²⁺ handling plays an important role in the pathogenesis of HF, and this kind of alteration in myocytes is a primary defect causing contractile dysfunction in HF (Yano et al. 2005). Modifications in the expression and/or activity of Ca²⁺ channels, alongside with alterations of cardiomyocyte architecture (T-tubules), seem to have a determinant role in failing myocytes (Balke and Shorofsky 1998). The involvement of different Ca²⁺ transporters has been related to the stage and etiology of HF; indeed, studies in human failing hearts suggest that patients with ischemic cardiomyopathy are characterized by impaired Ca²⁺ uptake, while patients with idiopathic dilated cardiomyopathy exhibits mainly modifications in Ca²⁺ release (Sen et al. 2000).

Cardiac contraction is the result of isometric force (or ventricular pressure) and rapid shortening to allow ejection of the blood (Caremani et al. 2016; Colomo et al. 1994). Both of them rely on Ca²⁺, specifically on amplitude/duration of Ca²⁺ transients, and on sensitivity of myofilaments to Ca²⁺. To allow the generation of Ca²⁺ transients appropriate in terms of duration and amplitude, cardiac myocytes need a high – and at the same time flexible – capacity of Ca²⁺ buffering. Normally the amount of Ca²⁺ bound is in a 100:1 ratio compared to Ca²⁺ free in the cytosol, and during contraction such ratio immediately increases (about tenfold) in favor to free Ca²⁺ (MacGowan et al. 2006). Ca²⁺ buffering should ensure an optimal removal of free Ca²⁺ from cytosol in order to have an appropriate amount of Ca²⁺ available to the contraction in the next beat.

Each cycle of EC coupling needs a dynamic interplay between Ca²⁺ transient and myofilaments. The sensitivity of myofilaments to Ca²⁺ is high when the myofilaments are stretched, i.e. in the phase of cardiac blood filling (diastole) (Zhao et al. 2016). Therefore, right after relaxation the myofilaments are more responsive to Ca²⁺ binding. Such autoregulation is a key mechanism underlying the coordination in EC coupling. Hence, when a perfect overlap of high Ca²⁺ transient and high myofilaments sensitivity to Ca²⁺ is obtained, an appropriate force is generated, leading to an optimal contraction.

Although the exact mechanisms involved in cardiac Ca²⁺ handling are not entirely understood, it is known (Benitah et al. 2003) that EC coupling and cardiac performance are mainly based on two mechanisms: (1) Synchronized Ca²⁺ release during contraction; (2) Effective Ca²⁺ reuptake that ensure a good and robust termination of Ca²⁺ dependent contraction.

To satisfy the first point, there are systems that mediate Ca²⁺ flux towards the cytosol, from extracellular compartment or from intracellular Ca²⁺ stores, i.e. SR, during EC coupling (Fig. 1). For the second one, there are systems involved in retrograde Ca²⁺ flux, to terminate EC coupling (Fig. 2).

Fig. 1.

Role of Ca²⁺ in excitation-contraction coupling in cardiomyocyte. Ca²⁺: calcium; cal: calmodulin; CaMK: Ca²⁺/calmodulin-dependent protein kinase; CICR: Ca²⁺-induced Ca²⁺ release; Epac: exchange protein directly activated by cAMP; IP3R: inositol 1,4,5-trisphosphate receptor; MCU: mitochondrial Ca²⁺ uniporter; PKA: Protein Kinase A; RyR2: Type 2 ryanodine receptor Ca²⁺ release channel

Fig. 2.

Mechanistic role of Ca²⁺ in the relaxation phase. Ca²⁺: calcium; MCU: mitochondrial Ca²⁺ uniporter; Na⁺: sodium; RyR2: Type 2 ryanodine receptor Ca²⁺ release channel; SERCA: sarco/endoplasmic reticulum Ca²⁺-ATPase

5 Ca²⁺ Fluxes during Systole

Ca²⁺ flux towards the cytosol starts with the entrance of a small amount of Ca²⁺ from extracellular space, triggering EC coupling: the depolarization wave is “transformed” in high Ca²⁺ release from intracellular store for contraction (Nanasi et al. 2017). The individual events of local Ca²⁺ release from SR are named Ca²⁺ sparks and reflect the activation of a cluster of about 6–20 Ca²⁺ channels on SR (Xie et al. 2013). Single Ca²⁺ sparks occur also at rest, and a crucial difference from Ca²⁺ sparks events during EC coupling is the synchronization; indeed, the action potential is able to synchronize several thousand of Ca²⁺ sparks within the same cell (Louch et al. 2013). Thus, individual events of local Ca²⁺ release are overlapping in time and space during EC coupling.

The main cellular complexes mediating Ca²⁺ flux towards cytosol for EC coupling are: voltage sensitive Ca²⁺ channels (T- and L-type) on the plasma membrane and RyRs on the SR.

6 Voltage-Dependent Ca²⁺ Channels

In myocytes there are two main types of voltage-dependent Ca²⁺ channels: T-type and L-type Ca²⁺ channels (Gonzalez-Rodriguez et al. 2015; Shaw and Colecraft 2013). The T-type channels are not preferentially located in the junctional region inside the diads, and the Ca²⁺ current generated by this kind of channels is negligible, with a minimal contribute to EC coupling (Zhou and January 1998). The L-type channels, also known as dihydropyridine receptors (DHPRs), are the main voltage dependent Ca²⁺ channels involved in EC coupling; they mainly localize on the T-tubule, in close proximity of Ca²⁺ channels on SR terminal cisternae (Bodi et al. 2005). DHPRs are activated by depolarization, allowing the Ca²⁺ to enter inside the cytosol and trigger CICR. A strategic auto-regulation of the CICR mechanism occurs at this level: indeed, DHPRs are inhibited by Ca²⁺ itself at the cytosolic side, limiting the amount of Ca²⁺ entry after depolarization. This local inhibitory effect on DHPRs is mediated by calmodulin, which binds the C-terminal domain on the receptor (Pott et al. 2007). Ca²⁺ released from SR in the same sarcolemmal-SR junction has also a modulatory effect on L-type receptors: when SR Ca²⁺ release occurs, the Ca²⁺ flux through DHPRs is reduced (Shiferaw et al. 2003). These events confirm that the Ca²⁺ needed for the contraction primarily derives from SR, since Ca²⁺ channels on plasma membranes are quickly inhibited after the entrance of small amount of Ca²⁺.

As mentioned above, action potential is responsible for synchronization of Ca²⁺ release from SR. The specific organization of ion channels on the plasma membrane, and in particular on the specialized invaginations of the T-tubules, plays a pivotal role in EC coupling (Oyehaug et al. 2013; Galbiati et al. 2001). Indeed, the loss of these structures leads to aberrant Ca²⁺ handling with blunted contractility, and such alteration has been mechanistically associated to HF (Wei et al. 2010).

In humans, HF secondary to dilated, hypertrophic, and ischemic cardiomyopathy, is related to aberrant alterations of T-tubule structure and the subsequent not-appropriate organization of voltage-dependent Ca²⁺ channels (Lyon et al. 2009). These anomalies could also be related to impaired function of junctophilins, proteins that are responsible for appropriate positioning of voltage-gated Ca²⁺ channels on T- tubules (Pinali et al. 2017; Schobesberger et al. 2017).

7 Key Role of RyR2 in Cardiac EC Coupling

RyRs are intracellular release channels, deputed to Ca²⁺ release from intracellular stores; the RyR2 isoform represents the most abundant isoform of this family in cardiomyocytes (Tunwell et al. 1996). This name was attributed based on the molecule with a high binding affinity to these receptors: the ryanodine, a natural product, known to induce a paralytic effect on striated muscle (Sutko et al. 1997; Rumberger and Ahrens 1972).

These channels have a tetrameric structure, similar to a mushroom, with the stalk across the SR membrane and the cap towards the cytosol. In this large structure, it is possible to identify at least two functional domains: C-terminus, which represents the ion pore across the membrane, and N-terminus that contains numerous modulatory sites (Gao et al. 1997). Indeed, RyR activity is highly regulated by several molecules – including calmodulin, FK-506 binding protein (FKBP), sorcin, junctin, and triadin (Balshaw et al. 2001; Anthony et al. 2007) – linked to the receptor, which thus represents the scaffold of a large macromolecular complex (Santulli et al. 2017b). Calmodulin and sorcin, two small proteins with high affinity for Ca²⁺, are able to inhibit RyR when Ca²⁺ levels in the cytosol are higher than a threshold value, preventing further Ca²⁺ release from SR. This phenomenon is involved in EC coupling termination.

RyR has also several sites modulated by Ca²⁺, Mg²⁺, phosphorylation, and oxidation, making this receptor also a sensor of intracellular redox state (Hain et al. 1995; Chugun et al. 2007; Xie et al. 2015). Therefore, RyR activity is the result of a multifaceted interactome specialized in Ca²⁺ handling.

Inositol-1,4,5, trisphosphate (IP3) receptors (IP3Rs) represent another example of intracellular Ca²⁺ release channel. Located on the SR as RyR, they are activated by a second messenger, IP3, involved in several pathways. These receptors are closely related to the RyR family, sharing a high structural homology (Santulli et al. 2017a, b; Maltsev et al. 2017). Although there are several stimuli that induce IP3 accumulation, the physiological depolarization of cardiac myocyte, does not seem to activate IP3Rs and their involvement seem to be limited to the regulation of transcription of important modulator of Ca²⁺ handling machinery (e.g. CamKII, calcineurin), indirectly contributing to regulate the capacity of the cells to maintain Ca²⁺ homeostasis, indispensable for appropriate EC coupling and contractility (Santulli et al. 2017a; Santulli and Marks 2015). Moreover, IP3R has a different distribution in atrial and ventricular myocytes, compared to RyR2; in particular, IP3Rs are principally located in the atrial myocyte and also in Purkinje fibers (Luo et al. 2008; Mignery et al. 1990). Purkinje fibers are a specialized conduction system, and IP3Rs can modulate the transfer of depolarization wave through ventricular mass.

8 Ca²⁺ Fluxes During Diastole

To ensure muscle relaxation, mechanisms of Ca²⁺ reuptake from the cytosol actively mediate a retrograde Ca²⁺ flux. The main “cytosolic Ca²⁺ scavengers”, Sarco-Endoplasmic reticulum Ca²⁺ ATPase (SerCa) and Na⁺/Ca²⁺ exchanger, achieve this function (Fig. 2). A large portion of Ca²⁺ is pumped by SerCa from the cytosol back to the SR, but the relative contribute of different Ca²⁺ removal systems can depend on the species.

9 Functional Role of Na⁺/Ca²⁺ Exchanger in Ca²⁺ Reuptake

Na⁺/Ca²⁺ exchanger mediates reversibly influx or efflux of Ca²⁺, depending on concentration of free Ca²⁺ inside the cytosol, but normally it works in Ca²⁺ efflux mode (Fujioka et al. 2000; Luongo et al. 2017). In cardiomyocytes this channel represents the major Ca²⁺ efflux system, with a critical role in the regulation of Ca²⁺ cellular content (Zhu et al. 2015; Acsai et al. 2011). Indeed, some alterations of ionic equilibrium inside the cell, that induce increase in Na⁺ intracellular levels, can trigger changes in the activity of the channel, which starts to work in reverse mode, mediating Na⁺ efflux, with influx of Ca²⁺ ions (Kang and Hilgemann 2004).

10 SerCa-ATPase

SerCa ATPase represents a fundamental pump system mediating Ca²⁺ reuptake in the SR/ER (Zima et al. 2014). In cardiomyocytes, SerCa removes the Ca²⁺ from cytosol during relaxation with an active process (ATP hydrolysis), in order to terminate the EC coupling cycle and to restore Ca²⁺ levels inside SR, necessary for the next contraction.

The correct activity of SerCa, and the indirect interplay with RyR2 that promotes Ca²⁺ flux in the opposite direction, are essential to have an EC coupling cycle with appropriate amplitude and synchronization in space and time. Ca²⁺ itself plays a pivotal role in mediating such interplay (Collins and Thomas 2001). SerCa, as well as RyR2, is highly regulated not only directly by Ca²⁺, but also by other molecules that are sensitive (CaMKII) or not (PKA) to Ca²⁺. These two kinases, as seen for RyR2, are able to regulate SerCa, but not via direct phosphorylation, but through phosphorylation of the most important regulator of SerCa, phospholamban. Phospholamban (PLB), is an endogenous inhibitor of SerCa that limits the affinity of the pump for Ca²⁺. PKA can phosphorylate PLB at Ser¹⁶, inhibiting PLB and therefore activating SerCa, increasing its pumping rate (Simmerman et al. 1986). CaMKII, whose activation relies also on the exchange protein directly activated by cAMP (Epac) pathway (de Rooij et al. 1998; Lymperopoulos et al. 2014; Parnell et al. 2015), is able to phosphorylate PLB at Thr¹⁷, leading also in this case to an acceleration of SR Ca²⁺ uptake (Simmerman et al. 1986). SerCa is also regulated by the metabolic state of the cell in terms of ATP/ADP ratio, and redox-status (Zima et al. 2014). Alterations in expression or activity of SerCa, strongly contribute to HF development and progression, through dysfunctional Ca²⁺ handling. In particular, several studies show that both mRNA and protein levels of SerCa pump are downregulated in HF (Currie and Smith 1999; Armoundas et al. 2007). The decrease in SerCa/PLB ratio results in impaired SR Ca²⁺ uptake (Linck et al. 2000). Moreover, the regulation of SerCa by both PKA (Schwinger et al. 1998) and CaMKII (Schwinger et al. 1999) is significantly impaired in failing hearts.

11 Intracellular Ca²⁺ Leak: Definition, Pathophysiology, and Interventions

RyR2 interactome ensures the appropriate amplitude and kinetics of Ca²⁺ cycling, indispensable for cardiac contractility. Alterations in RyR2 and/or in RyR2 interactome, are among the main identified aspects of HF (Ono et al. 2000; Kohno et al. 2003). All the events that are able to affect the opening frequency of RyR2, determining changes in amplitude or duration of Ca²⁺ release, can predispose to contractility dysfunction (Patel et al. 2000). In particular, the phenomenon of Ca²⁺ leak, defined as inappropriate release of Ca²⁺ from the SR (e.g. during the diastolic phase), is determinant in HF progression (Hofer et al. 1996). In pathological conditions such as HF, there is a high release of Ca²⁺ from SR during diastole, reducing the availability of SR Ca²⁺ for the subsequent contraction, thereby impairing contractility (Santulli et al. 2017b).

FKBP12.6, also known as Calstabin2, plays an important pathophysiological role in the regulation of RyR2 function. Indeed, it stabilizes a conformation of the channel that prevents Ca²⁺ leak (Timerman et al. 1996; Lam et al. 1995; Xin et al. 1999; Yuan et al. 2014). There are numerous events, both acute and chronic, that trigger instability of RyR macromolecular complex. One typical example is represented by its phosphorylation, which induces the dissociation from the receptor of its stabilizing molecule, FKBP12.6 (Yuan et al. 2014). Chronic activation of beta-adrenergic system increases PKA activity (Santulli et al. 2013), with consequent phosphorylation of RyR (Takasago et al. 1991). Phosphorylation of RyR by CaMKII has also been reported in HF (Kushnir et al. 2010). Furthermore, ROS-linked oxidation of RyR affects the normal Ca²⁺ handling, compromising the correct contraction of myocytes during HF (Santulli et al. 2015).

Several benzothiazepine derivatives, including JTV519 (also known as K201, 4-[−3{1-(4-Benzyl) piperidinyl}propionyl]-7-methoxy-2,3,4,5-tetrahydro-1,4-benzothiazepine) (Kohno et al. 2003; Hachida et al. 1999a, b; Sacherer et al. 2012) and its substructure S107 (7-methoxy-4-methyl-2,3,4,5-tetrahydro-1,4-benzothiazepine) (Thevis et al. 2009; Matecki et al. 2016; Lukyanenko et al. 2017) have shown cardioprotective and antiarrhythmic properties by decreasing Ca²⁺ leak (Santulli et al. 2017a; Xie et al. 2013, 2015).

Henceforth, RyR2 represents the central target of many pathways dysregulated in cardiac pathological conditions, including adrenergic dysfunction, metabolic disorders, ROS production: all of these conditions are accompanied by alterations in Ca²⁺ handling and subsequent impairment in contractility.

12 Role of Mitochondrial Ca²⁺ in Metabolism-Contraction Coupling

Mitochondria play a strategic role in ensuring an adequate EC coupling (Miragoli and Cabassi 2017; Umanskaya et al. 2014; Gambardella et al. 2017; Torrealba et al. 2017). Indeed, cardiac myocytes are critically dependent on constant and appropriate energy supply, alongside with a finely tuned Ca²⁺ handling (Torrealba et al. 2017; Sorriento et al. 2017; Sheeran and Pepe 2017). Mitochondria and SR are functionally and structurally associated and, at the point of interaction (mitochondrial associated membranes) Ca²⁺ transits from SR to mitochondria (Min et al. 2012; Bononi et al. 2017). Ca²⁺ that enters in mitochondria is able to increase their bioenergetics, but at the same time, mitochondria, allowing Ca²⁺ entrance through the mitochondrial Ca²⁺ uniporter (De Stefani et al. 2011; Liu et al. 2017; Granatiero et al. 2017), can act as local Ca²⁺ buffers, actively participating in Ca²⁺ handling (Walsh et al. 2009; Gunter and Sheu 2009; Drago et al. 2012; Morciano et al. 2017). However, mitochondrial Ca²⁺ overload has been shown to be detrimental in various cell types (Liu et al. 2016; Kostic et al. 2015; Charles et al. 2017), and, specifically, it has been proven to play a mechanistic role in the pathogenesis of HF (Santulli et al. 2015). Therefore, our hypothesis is that whereas transient mitochondrial Ca²⁺ uptake promotes ATP production, prolonged or excessive Ca²⁺ uptake can be harmful.

The ability of mitochondria to respond to changes in Ca²⁺ levels, increasing metabolic outcome, and acting as Ca²⁺ scavenger, contributes to the adequate contractile response of cardiomyocytes. Indeed, alterations in these processes can lead to increase in cytosolic Ca²⁺ with potential activation of detrimental pathways that have been associated with HF, including CaMKII (Zhang and Brown 2004). Additionally, alterations in SR-mitochondria contacts have been found in cardiac dysfunction and HF (Lopez-Crisosto et al. 2017).

Whether mitochondrial Ca²⁺ transients exist during physiological EC coupling, in a beat-to-beat fashion, remains to be determined. Anyway, an important aspect of these phenomena is that Ca²⁺ proves once again its ability to act as a messenger, communicating to mitochondria the energetic demand of the cell, for adequate contraction.