Inheritable Phenotypes Associated With Altered Intracellular Calcium Regulation

Calcium Cycling in Cardiac Excitation - Contraction Coupling

The series of events by which membrane depolarization in the form of an action potential is converted into mechanical contraction is called excitation-contraction (E-C) coupling. E-C coupling is discussed in detail elsewhere in this book ( Chapter 16 ), but a brief overview is necessary to place Ca ²⁺ homeostasis in the appropriate context ( Fig. 51.1A, D ). Every action potential (AP) in the heart is accompanied by a transient elevation of the free [Ca ²⁺ ] _i , from around 100 nM during diastole to nearly 1 μM during systole. This intracellular Ca ²⁺ transient activates the myofilaments and produces contraction. In adult cardiomyocytes, the amount of extracellular Ca ²⁺ entering the cell during an AP is insufficient to elicit full contractions. Instead, most of the Ca ²⁺ required for E-C coupling (approximately 70% in humans and 90% in mice) flows out of the sarcoplasmic reticulum (SR), ^, an intracellular Ca ²⁺ store. This is achieved via an amplification process termed Ca ²⁺ -induced Ca ²⁺ release (CICR). In this process, the relatively small inward Ca ²⁺ current (I _CaL ) from sarcolemmal L-type Ca ²⁺ channels (LTCCs) that occurs during AP activates cardiac ryanodine receptors (RyR2s), which release more Ca ²⁺ from the SR. During CICR, RyR2 acts as both a Ca ²⁺ sensor and a Ca ²⁺ channel, potentially turning CICR into a self-sustaining, regenerative event. CICR, however, reliably stops in healthy cardiomyocytes, and relaxation ensues once Ca ²⁺ is removed from the cytosol. Two main molecular players work in concert to achieve cytosolic Ca ²⁺ removal: the SR Ca ²⁺ -adenosine triphosphatase (ATPase; SERCA2a) refills the SR, whereas the Na ⁺ /Ca ²⁺ exchanger (NCX) extrudes Ca ²⁺ from the cell. To maintain equilibrium, the amount of Ca ²⁺ that enters the cell through I _CaL must be extruded by NCX, whereas the amount of Ca ²⁺ released by RyR2 channels must return to the SR. NCX is an electrogenic transporter that translocates three Na ⁺ and a single Ca ²⁺ in opposite directions across the cell membrane; hence, the extrusion of Ca ²⁺ creates a net inward depolarizing current.

CICR occurs at specialized regions of the cell where the sarcolemma is near the SR, mostly within the T-tubule (TT) network of the myocyte. In these tightly spaced microdomains, clusters of LTCCs provide the activating Ca ²⁺ signal for a group of RyR2s, forming a calcium release unit (CRU). A Ca ²⁺ release event that originates from a single CRU is referred to as a Ca ²⁺ spark. The global cytosolic Ca ²⁺ transient, on the other hand, results from the temporal and spatial summation of Ca ²⁺ sparks coordinated during E-C coupling by the AP, I _CaL , and CICR. Spontaneous sparks occur at relatively low frequency during diastole in healthy quiescent cardiomyocytes as a result of the finite open probability of RyR2 channels within a CRU. ^, Nevertheless, the propagation of spontaneous Ca ²⁺ waves or synchronization of spontaneous Ca ²⁺ release (SCR) events into discrete diastolic Ca ²⁺ transients is a sign of dysfunctional Ca ²⁺ handling.

Adrenergic Regulation of Calcium Homeostasis

The sympathetic branch of the autonomic nervous system increases the dynamic output of the heart in response to increased metabolic demand. This system provides an essential component of the cardiac fight-or-flight response through the activation of β ₁ -adrenergic receptors (β ₁ -ARs) by catecholamines (norepinephrine and epinephrine). Among other effects observed during β ₁ -AR activation, heart rate increases because of a direct effect of catecholamines on the sinus node (positive chronotropic effect), whereas contractility and rate of relaxation increase (positive inotropic and lusitropic effect, respectively) because of a Ca ²⁺ signaling cascade triggered by catecholamines in ventricular myocytes. The role of Ca ²⁺ signaling in sinus node automaticity has recently emerged and continues to be explored, but its importance for modulating inotropy and lusitropy has long been established. Canonical β ₁ -AR signaling in the ventricular myocytes involves an increase in cyclic adenosine monophosphate (cAMP), downstream activation of protein kinase A (PKA), and phosphorylation of at least three key E-C coupling components: (1) LTCC, which increases peak I _CaL ; (2) phospholamban (PLB), which relieves its partial inhibition on SERCA2a and accelerates SR Ca ²⁺ refilling; and (3) troponin I, which decreases the Ca ²⁺ affinity for the myofilaments and helps accelerate the cross-bridge detachment. These effects result in larger and faster Ca ²⁺ transients, thereby increasing contractile force, allowing for faster relaxation and filling the SR with Ca ²⁺ . PKA also phosphorylates RyR2 in at least two sites (S2808 and S2031, human nomenclature). The physiologic role of these modifications remains unclear, but recent studies suggest that, although S2808 is not critical for an integral adrenergic response, ^, S2031 may be a prominent regulator of RyR2.

Enhanced Ca ²⁺ cycling during β ₁ -AR signaling also promotes activation of the Ca ²⁺ /calmodulin (CaM)-dependent kinase II (CaMKII). A noncanonical branch of this signaling cascade, however, also activates CaMKII via the exchange protein activated by cAMP 2 (Epac2). ^, CaMKII phosphorylates many of the same protein targets of PKA, including LTCC, PLB, and RyR2, but chronic activation of CaMKII is considered deleterious. A key difference is that CaMKII also increases diastolic SR Ca ²⁺ leak, as observed in heart failure (HF), perhaps by prominently phosphorylating RyR2-S2814. ^, The role of PKA in pathologic SR Ca ²⁺ leak, in contrast, is a matter of intense debate. Whatever the downstream signaling pathways and regardless of the specific controversies, it is sufficiently clear that adrenergic signaling in the heart enhances Ca ²⁺ cycling by directly affecting the sarcolemmal, SR, and myofilament components of the E-C coupling apparatus.

Catecholaminergic Polymorphic Ventricular Tachycardia

CPVT is a severe inherited syndrome characterized by polymorphic ventricular tachycardia (VT) in the absence of structural disease or remarkable changes in the resting electrocardiogram. CPVT was initially described in 1975 as a case of bidirectional tachycardia in an infant with unexplained syncopal episodes. Arrhythmia episodes are triggered by peaks of sympathetic activation during emotional or physical stress (hence the name catecholaminergic ). The prevalence of the disease in the general population is unknown but is estimated between 1 in 5000 and 1 in 10,000. CPVT primarily affects young patients ; however, some do not display symptoms until adulthood. Patients often develop bidirectional tachycardia, potentially leading to ventricular fibrillation (VF) and sudden cardiac death (SCD); however, idiopathic VF has also been observed. A common feature of a CPVT episode is syncope, which could be mistaken for epilepsy; hence, exercise stress tests, Holter monitoring, and drug challenges with epinephrine or isoproterenol are important tools to establish a correct differential diagnosis. If untreated, 60% to 70% of patients may develop severe tachyarrhythmias, and around 30% may undergo cardiac arrest or SCD. ^, This high mortality rate is likely to impact estimates of disease prevalence, because cases where the first manifestation is sudden death may be unaccounted as cases of CPVT.

The study of the genetics of CPVT started when the disease was mapped to chromosome 1q42-43 in 1999. Shortly thereafter, this region was narrowed to the gene that codes for RyR2 when four specific mutations were reported in the same number of probands with catecholaminergic VT. One of those mutations, RyR2-R4497C, was later introduced in mice, and heterozygous animals recapitulated the most relevant clinical signs of CPVT: polymorphic VT and VF in response to catecholamines and exercise. Multiple genes are now associated with CPVT in the literature. Only four are supported by strong evidence and will be considered in the following sections: RYR2 (CPVT1), calsequestrin 2 ( CASQ2, CPVT2), calmodulin ( CALM1, CPVT4; CALM3, CPVT6), and triadin ( TRDN, CPVT5). The numbers assigned to each instance of CPVT are not universally accepted and can bring about confusion. For example, CPVT3 is given to the gene TECLR (the trans-2,3-enoyl-CoA reductase-like enzyme) based on a single inconclusive report, whereas CPVT4 and CPVT6 are given to two different genes ( CALM1 and CALM3 ) that code for the same protein (calmodulin). Therefore this nomenclature is not considered further in this chapter. RYR2 accounts for the vast majority of CPVT cases (around 60%), followed by CASQ2 (around 5%). All other instances are rare, and up to 12% of cases are idiopathic.

CPVT is a disorder that may be entirely attributed to abnormal intracellular Ca ²⁺ cycling. Nearly all molecular mechanisms reported to date converge on cellular events that involve diastolic SCR via hyperactive RyR2 destabilized by mutations in the channel itself or in its partner proteins (see Fig. 51.1B, E ). NCX is the fastest pathway for Ca ²⁺ removal from the cytosol but is also an electrogenic transporter. Extrusion of diastolic SCR via NCX produces a depolarization of the cell membrane called delayed afterdepolarization (DAD). If SCR is synchronous and of sufficient mass, a DAD can drive the membrane potential to threshold and trigger an extemporaneous AP (triggered activity). Further repetition of this cycle and triggered activity in a large group of myocytes may lead to ventricular arrhythmias, which become bidirectional or polymorphic as sites of ectopic electrical activity appear in different regions of the heart. Evidence suggests that arrhythmogenic focal activity is more prominent in Purkinje cells of the cardiac conduction system, where the subcellular structure, limited intercellular connections, and low electrotonicity favor the generation and propagation of triggered activity. ^,