2.2.1. Calcium Cycling in Healthy Cardiac Myocytes

During contraction in healthy cardiac myocytes, electrical stimulation of the muscle leads to an increase in intracellular calcium, first as a small amount which enters through L-type voltage-gated calcium channels (dihydropyridine receptors (DHPR)) in the sarcolemma. The initial calcium influx then triggers a larger calcium release from the sarcoplasmic reticulum (SR) through ryanodine receptor 2 (RyR2), located in the SR membrane. Together, this process is referred to as calcium-induced calcium release (CICR) [52]. Calcium rises in the cytoplasm and binds to troponin C (TnC), causing the protein to undergo a conformational change, which is facilitated by the binding of troponin I (TnI). As TnI switches binding from actin to TnC, tropomyosin (Tm) is then free to move, exposing myosin-binding sites on the actin filaments [29]. Troponin T (TnT)-Tm binding allows for the cooperative transmission of these conformational changes along the length of the thin filament in a complex series of protein-protein interactions, and cross-bridge binding of myosin to actin stabilizes Tm positioning [29]. Through these interactions the myofilament becomes activated and force can be generated [52] (Figure 2).

**Figure 2.** Normal excitation-contraction coupling in cardiac myocytes. Membrane depolarization leads to a small influx of calcium through the L-type calcium channel (LTCC/DHPR) (1), which triggers a larger release of calcium from the sarcoplasmic reticulum (SR) through ryanodine receptor 2 (RyR2) (2). Calcium then binds to the myofilaments, triggering myocyte contraction (3). During the relaxation phase, calcium reuptake occurs by pumping calcium out of the cytoplasm back into the SR via Serca2a or through the Na+/Ca2<sup>+</sup> exchanger (NCX) (4). Phospholamban negatively regulates Serca2a activity. β-adrenergic signaling leads to phospholamban (PLN) phosphorylation and dissociation from Serca2a, increasing the rate of calcium reuptake into the SR. Normal physiological stretch leads to NADPH oxidase 2 (NOX-2) production of reactive oxygen species (ROS), which increases calcium entry through stretch-activated channels (SACs). Dystrophin serves to stabilize the sarcolemma during the repeated stress of myocyte contraction and relaxation. Inset shows increased detail of the dystrophin glycoprotein complex (DCG) and myofilament proteins. NOS: nitric oxide synthase; MCU: mitochondrial calcium uniporter; NCLX: mitochondrial sodium calcium exchanger.

During relaxation, SR-associated proteins, notably sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and phospholamban (PLN), are essential to the removal of calcium from the cytoplasm back into the SR [53]. The Serca2a/PLN complex is the major regulator of calcium cycling in cardiac muscle [54]. Serca2a is an ATP-dependent calcium pump localized within the longitudinal membrane of the sarcoplasmic reticulum [55]. Serca2a has a key role in regulating both the rate of calcium reuptake and subsequent myocyte relaxation in diastole, as well as SR calcium load, which affects calcium transient peak height and contractility in systole [54,56]. PLN is a negative regulator of Serca2a function [54]. The monomeric, dephosphorylated form of PLN interacts with Serca2a and decreases its affinity for calcium, thus decreasing SR calcium reuptake velocity. Phosphorylation of PLN at Ser 16 or Thr 17 inhibits its interaction with Serca2a and increases oligomerization of PLN into its pentameric form, thereby relieving Serca2a inhibition [54,56]. The resulting increase in Serca2a function increases the rate of calcium reuptake in diastole and also increases SR calcium load, resulting in increased contraction amplitude and hastened relaxation (Figure 2).

The phosphorylation status of PLN is what determines its ability to alter Serca2a function. PLN phosphorylation is regulated in large part through β-adrenergic signaling. Stimulation of the sympathetic nervous system leads to increased activation and signaling through β-adrenergic receptors. The subsequent Protein Kinase A (PKA) activation leads to phosphorylation of PLN at Ser 16, relieving its inhibition on Serca2a and increasing SR calcium reuptake [54,56]. Phosphorylation at Thr 17 also relieves inhibition on Serca2a and is accomplished via Ca<sup>2</sup>+/CaM Kinase in response to physiological stressors including ischemic injury, pacing stress or increased calcium concentration [54,56]. Multiple accessory proteins, including HS-1 associated protein X-1 (HAX-1), Protein-Phosphatase 1 (PP1), endogenous inhibitors of PP1 (I-1 and I-2), Heat Shock Protein 20 (HSP20), and S100A1 proteins, are also associated with the Serca2a/PLN complex and serve to modulate PLN inhibition of Serca2a under various physiological conditions [54].
