Advances in the understanding of excitation-contraction coupling: the pulsing quest for drugs against heart failure and arrhythmias

Excitation-contraction coupling (ECC) converts electrical stimuli to mechanical responses. Herein we provide a concise summary of the most updated insights on, and nomenclature of, the main actors involved in cardiac ECC, posing the basis for pharmacologic interventions in heart failure (HF) and arrhythmias. Recently introduced therapeutic strategies targeting myocardial ECC are appraised as well, specifying their molecular mechanism of action.

Excitation-contraction coupling starts with the entry of Ca²⁺ into the cardiomyocyte through the L-type Ca²⁺ channels (LTCC, also known as dihydropyridine channel); this step triggers Ca²⁺-induced Ca²⁺-release from the sarcoplasmic reticulum (SR) by activating type 2 ryanodine receptor Ca²⁺ release channel (RyR2).¹ Ca²⁺-releasing units consisting of closely (∼15 nm) approximated LTCCs (on T-tubules, which are invaginations of the sarcolemma with a diameter of ∼200 nm that form a highly branched network) and RyRs (on the SR) are known as dyadic clefts or dyads; within each dyad, ∼25 LTCCs and ∼100 RyRs are closely associated (∼1:4 ratio). Junctophilin-2 is a membrane-binding protein that is responsible for the localization of LTCCs in close proximity to RyR2. Mutations in the joining region of Junctophilin-2 cause T-tubule remodeling and dyad loss with subsequent asynchronous Ca²⁺-release after β-adrenergic stimulation.²

LTCC contains the α₁ subunit, a tetramer of four six-transmembrane domains forming the pore, and auxiliary subunits (α₂δ, β, γ). The α₁ subunit, known as Ca²⁺-voltage-gated channel subunit α₁ (Ca_V1), has four isoforms: α₁S (Ca_V1.1), α₁C (Ca_V1.2), α₁D (Ca_V1.3), and α₁F (Ca_V1.3). Two groups of Ca²⁺-voltage-gated channels complete the family: Ca_V2 (P-type, N-type, R-type), and Ca_V3 (T-type).

RyR2, a tetrameric intracellular Ca²⁺-release channel located on the SR, can be modulated by post-translational modifications and by interactions with a number of other proteins, including calmodulin, striated muscle enriched protein kinase (SPEG), calstabin2, triadin/calsequestrin, calcineurin, and transmembrane protein 38A (TMEM-38A). Small molecules known as Rycals have been shown to stabilize RyRs, preventing the pathologic intracellular Ca²⁺ leak,¹ defined as inappropriate release of Ca²⁺ from the SR (e.g. during the diastolic phase).

The release of Ca²⁺ through RyRs increases its concentration in the dyad from ∼100 nMol to >∼100 µMol. How does this increase in Ca²⁺ concentration lead to contraction? Contraction of the cardiomyocyte relies on the synchronized movements of the main constituents of the sarcomere (the smallest functional unit of the striated muscle), with the mechanic translocation of the myosin thick filament respect to the thin filament, which is composed by a double-stranded actin polymer, two continuous tropomyosin polymers, and troponin (Tn) complex (Figure 1).

Figure 1.

Current pharmacologic strategies targeting the major players involved in cardiac excitation-contraction coupling. cGMP: 3′,5′-cyclic guanosine monophosphate (both the membrane-bound and the soluble forms are depicted); cMyBP-C: cardiac myosin binding protein-C; GTP: guanosine triphosphate; NO: nitric oxide; RyR2: ryanodine receptor; SERCA: sarco/endoplasmic-reticulum Ca²⁺-ATPase; SR: sarcoplasmic reticulum; Tn: troponin.

The Tn complex is formed by three distinct proteins (TnC, TnT, and TnI), encoded for by different genes that are expressed in cardiac and skeletal muscle (but not in smooth muscle). TnC is responsible for Ca²⁺ binding, which triggers conformational modifications of the Tn complex that cause tropomyosin to move deeper into the actin groove, exposing the myosin-binding sites that are covered in the relaxed state; while TnC in skeletal muscle has four Ca²⁺ binding sites, in cardiac muscle there are only three binding sites. The Ca²⁺ sensitizer levosimendan has been shown to interact with cardiac TnC.³ TnT interacts with tropomyosin and helps its positioning on actin; mutations in cardiac TnT are known to cause human dilated cardiomyopathy. TnI has an inhibitory role in tropomyosin/TnT interaction and alterations in its methylation at sites R74/R79 and R146/R148 have been linked to hypertrophic cardiomyopathy.

Tropomyosins are integral components of actin filaments consisting of rod-shaped coiled-coil dimers (four genes are responsible for generating more than 40 isoforms) that lie along actin. Interaction occurs along the length of the actin filament, with dimers aligning in a head-to-tail fashion. In mammals, four genes (TPM1, TPM2, TPM3, and TPM4) are responsible for generating more than forty different tropomyosin isoforms. Mutations in TPM1 have been associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, and left ventricular non-compaction cardiomyopathy.

Myosin is a mechanochemical protein that converts chemical energy into mechanical force. The human genome contains >40 different myosin genes whose protein products share the basic properties of actin binding, ATP hydrolysis, and force transduction. By promoting the interaction between myosin and actin, omecamtiv mecarbil⁴ has been recently shown to significantly increase myocardial force production, although the exact underlying molecular mechanisms are not fully clear⁵; specifically, the GALACTIC-HF trial⁴ has revealed that in patients with HF and a reduced ejection fraction, those who received omecamtiv mecarbil, had a lower risk of a composite of HF or cardiovascular death than those who received placebo.⁴

After cytosolic Ca²⁺ has activated the contractile units, it is rapidly extruded from the cytosol in preparation for the following cycle; the main efflux mechanisms in cardiomyocytes are the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump and the Na⁺/Ca²⁺ exchanger (NCX).

Sarco/endoplasmic reticulum Ca²⁺ ATPase is responsible for the reuptake of Ca²⁺ from cytosol to SR. Three genes are present in vertebrates (ATP2A1-3), coding for three isoforms (SERCA-1–3); alternative splicing results in a total of ten different proteins: SERCA-1a/b, SERCA-2a/b, SERCA-3a/b/c/d/e/f. Sarco/endoplasmic reticulum Ca²⁺ ATPase is regulated by a number of different proteins, including phospholamban (ventricles and slow-twitch muscles), and sarcolipin (fast-twitch muscles and atria). Istaroxime is a Na⁺/K⁺ ATPase inhibitor with the distinctive property of increasing SERCA2a activity,⁶ most likely by relieving the inhibitory effect of phospholamban, thereby improving Ca²⁺ handling and diastolic dysfunction.

NCX, a.k.a. solute carrier family 8 (SLC8), is a low-affinity, high-capacitance antiporter that mediates the electrogenic exchange of three Na⁺ ions with one Ca²⁺ ion.

Other important components of the ECC machinery, for which therapeutic approaches are not (yet) available include cMyBP-C, titin, and nebulette.

cMyBP-C, a myosin-associated protein that binds at 43 nm intervals along the myosin thick filament backbone, influences contractile mechanics and myofilament orientation. Mutations of cMyBP-C have been linked to familial hypertrophic cardiomyopathy.

Titin is a giant protein (>1 µm) responsible for passive muscle elasticity, acting as a molecular spring, comprising 244 domains that unfold when the protein is stretched and refold once tension is removed. Titin mutations are among the most common causes of adult dilated cardiomyopathy.

The nebulin family includes five members: nebulin, nebulette, N-RAP, LASP-1, and LASP-2. Nebulin (∼800 kDa) extends along the thin filament of the skeletal muscle and consists of 185 copies of a 35 amino acid sequence (‘nebulin-repeat’) containing the actin-binding SDxxYK motif. Nebulette, a smaller protein (∼116 kDa, 23 nebulin repeats), replaces nebulin in cardiac muscle.⁷ Nebulin-related anchoring protein (NRAP) contains 46 nebulin repeats, while LASP-1 (not present in cardiomyocytes), and LASP-2, contain only two and three nebulin repeats, respectively.

We conclude this brief excursus presenting a molecular pathway that, although not being inherently involved in ECC, has been recently successfully targeted in HF: the generation of 3ʹ,5ʹ-cyclic guanosine monophosphate (cGMP), which has anti-inflammatory and anti-fibrotic properties. Sacubitril inhibits the degradation (mediated by neprilysin) of natriuretic peptides—which are known to activate the membrane-bound form of the enzyme responsible for cGMP production, guanylyl cyclase—and its association with valsartan has been shown to be effective in HF.^8,9 Vericiguat, instead, is a stimulator of the soluble form of guanylate cyclase, stabilizing its nitrosyl-heme interaction¹⁰. In patients with high-risk HF, the incidence of death from cardiovascular causes or hospitalization for HF was shown to be lower among those who received vericiguat than among those who received placebo.¹⁰

Conflict of interest: None declared.