**6. CL Function in Calcium Homeostasis**

Calcium is an important regulator of sarcomere contraction in the heart. In systole, Ca2<sup>+</sup> influx via L-type Ca2<sup>+</sup> channels triggers the release of Ca2<sup>+</sup> from ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR). Ca2<sup>+</sup> binding to troponin C induces contraction. During diastole, Ca2<sup>+</sup> is transported back into the SR by the sarcoplasmic reticulum Ca2+-ATPase (SERCA) or exported across the cell membrane via the Na+/Ca2<sup>+</sup> exchanger [96] (Figure 4). By means of the active transport of Ca2<sup>+</sup> ions, the P-type ATPase SERCA is required for muscle relaxation and the proper regulation of muscle contraction. It also ensures a sufficient Ca2<sup>+</sup> load in the sarcoplasmic reticulum for systolic contraction. Oxidative stress has been associated with a deregulation of excitation–contraction coupling in the BTHS. Peroxynitrite formed by increased levels of superoxide and nitric oxide (NO) can build adducts with tyrosine residues, which may change the structure or catalytic activity of target proteins [97]. Tyrosine nitrosylation of SERCA in BTHS leads on to a decrease in SERCA activity and results in a decline of SR Ca2<sup>+</sup> levels. These abnormalities may promote left ventricular diastolic dysfunction. A decrease in SR Ca2<sup>+</sup> levels has been observed in many forms of heart failure, including dilated cardiomyopathy [98,99].

Calcium is an important regulator of mitochondrial metabolism. Accelerated cardiac workload causes an enhanced demand of ATP. The conversion of ATP to ADP in energy-consuming processes such as contraction increases ADP levels, accelerates respiration and results in an elevated oxidation of reducing equivalents. To compensate for the higher demand for reducing equivalents, Ca2<sup>+</sup> plays an important role in activating the mitochondrial Krebs cycle [100]. During excitation–contraction coupling, Ca2<sup>+</sup> is emitted from the sarcoplasmic reticulum and is transmitted into the mitochondria via the Mitochondrial Calcium Uniporter (MCU) (Figure 4). To allow for an efficient calcium transmission, ryanodine receptors (RyRs) in the sarcoplasmic reticulum are located in close proximity to mitochondria, especially next to the mitochondrial calcium uniporter (MCU) in the inner membrane [101]. Ca2<sup>+</sup> stimulates key dehydrogenases of the Krebs cycle, including pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, accelerating the regeneration of NADH and FADH2. However, the forced regeneration of reducing equivalents activates the respiratory chain. Therefore, Ca <sup>2</sup><sup>+</sup> plays a prominent role in adapting mitochondrial metabolism to increased energy demands during accelerated cardiac workload [102]. The pore-forming unit of the mitochondrial calcium uniporter is the protein MCU, which associates EMRE and regulatory subunits MICU1, MICU2, and MCUb into a complex integrated into the inner membrane [103–106] (Figure 4). The association of phospholipids in MCU has been revealed in a recent structural analysis [107]. Moreover, a specific requirement of the MCU for CL was found. Consequently, the ability of MCU to assemble into functional complexes was reduced in cardiac patient samples of BTHS [108].

— **Figure 4.** Mitochondrial Calcium Uniporter: Calcium transport from the sarcoplasmic reticulum (SR) is mediated by different proteins—Ryanodine receptors (RyRs) and inositoltriphosphate receptors (iP3Rs) release Ca <sup>2</sup><sup>+</sup> under systolic conditions, while the voltage-dependent anion channel (VDAC) and mitochondrial calcium uniporter (MCU) allow the Ca <sup>2</sup><sup>+</sup> uptake in mitochondria. MCU is embedded in the inner mitochondrial membrane and requires CL for optimal activity. The sarcoplasmic reticulum Ca <sup>2</sup>+-ATPase (SERCA) controls the Ca <sup>2</sup><sup>+</sup> uptake by the SR. IMS, intermembrane space; IM, inner mitochondrial membrane.
