Unbalance Between Sarcoplasmic Reticulum Ca2 + Uptake and Release: A First Step Toward Ca2 + Triggered Arrhythmias and Cardiac Damage

Figures

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  FIGURE 1. Cardiac excitation-contraction coupling. When cardiomyocytes are reached by an AP, depolarization of the plasma membrane by Na+ entry (INa), induces the opening of L-type Ca2+ channels. Ca2+ entering through these channels (Ca2+ current, ICa), induces Ca2+ release by the RyR2s (Ca2+-induced- Ca2+-release), which subsequently activates myofilaments for muscle contraction. Relaxation occurs when SR -Ca2+ ATPase (SERCA2a) reuptakes Ca2+, lowering cytosolic Ca2+ concentration in combination with Ca2+ extrusion via the Na+- Ca2+ exchanger (NCX) working in reverse mode and the sarcolemma Ca2+-ATPase. Mitochondria also participate taking and extruding Ca2+ from the cytosol during Ca2+ cycle. Green arrows depict Ca2+ fluxes.

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  FIGURE 2. (A) Regulation of SR-Ca2+ uptake. SR- Ca2+ uptake takes place through the SR- Ca2+ ATPase (SERCA2a), being PLN the main regulatory protein of SERCA2a. PLN can be phosphorylated by PKA at Ser16 residue and by Ca2+-calmodulin-dependent protein kinase (CaMKII) at Thr17 site. Protein phosphatase 1 (PP1) dephosphorylates both sites. Either one or both phosphorylation sites relieve PLN inhibition on SERCA2a. Several other proteins regulate SERCA2 either directly, like S100A or Sumo1, or indirectly, through the regulation of PLN (like the hematopoietic lineage cell-specific protein-1 (HS-1) associated protein X-1 (Hax-1), the Heat-shot protein 20 (Hsp20), and the inhibitor 1 (I-1). As described in the text, PLN and SERCA2a are also regulated by redox processes and SERCA by acetylation and glycation (Not shown in the Figure for the sake of clarity). (B) Regulation of SR-Ca2+ release: SR-Ca2+ release occurs mainly through the RyR2 which are also highly regulated. CaMKII phosphorylates RyR2 at Ser2814 and S2808 sites and PKA at Ser2808 and 2030 sites. Ser2808 is also target of protein-kinase C (PKC) and protein-kinase G (PKG). PP1 and PP2 are the phosphatases that dephosphorylate RyR2.…

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  FIGURE 3. Afterdepolarizations. (A) Reactivation of L-type Ca2+ current (ICa) during prolongation of the AP, mainly during phase 2 or 3, increases the propensity for EADs. The Na+-Ca2+ current (NCX) also meets the criterion needed for the positive feedback required for an EAD to occur. Both currents can act synergistically facilitating EAD formation, and thus increasing the probability of an EAD-triggered AP. Red cross indicates inhibition of K+ channels. (B) Various conditions which increase SR-Ca2+ and/or sensitize RyR2 can induce spontaneous Ca2+ release from the SR. When this release achieves certain critical mass, Ca2+ can induce a saltatory propagation along cell sarcomeres, known as Ca2+ waves, which in turn activates the NCX that extrude 2 Ca2+ and enter 3 Na+, depolarizing the cell membrane and producing a delay after depolarization (DAD). If this depolarization attains the excitability threshold, a spontaneous AP occurs.

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  FIGURE 4. Threshold concept. (A) Cartoon that represents different conditions of RyR2 sensitivity and SR-Ca2+ load during Ca2+ release from SR. When the cell triggers a Ca2+ transient (1), SR Ca2+ load diminishes and RyR2s enter in a refractory period (2). SR Ca2+ uptake produces SR Ca2+ recovery (3). During this period there are no spontaneous releases, i.e., RyR2 remained refractory in spite of the fact that SR level has recovered completely (SR threshold). This period is followed by what has been named an “idle” period, by Belevych et al., 2012, (4) in which, after SR-Ca2+ load and RyR2s recovering, a stochastic activation of RyR2 may trigger a spontaneous release. (B) Schematic representation of time dependent changes of SR Ca2+ t (1 and 2), and refractoriness (2 and 3) after a stimulus-induced Ca2+ transient. (4) “Idle” period during which a spontaneous SR-Ca2+ release occurs. We called post-refilling refractory period to the period of time needed for refractoriness to recover after SR Ca2+ threshold was reached.

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  FIGURE 5. Confocal assessment of spontaneous Ca2+ release. (A) Representative confocal images showing that Ca2+ spark frequency is higher in isolated cardiomyocytes from SDKO mice in comparison to S2814D myocytes (unpublished confocal microscopy records representative of overall data shown in Mazzocchi et al., 2016). (B) Similar results were obtained in fluo-4 loaded intact isolated hearts from S2814D and SDKO mice under a confocal microscope, during reperfusion of the hearts submitted to a period of ischemia (unpublished confocal microscopy records representative of overall data shown in Valverde et al., 2019). In both examples, ablation of PLN increases Ca2+ spark frequency respect to hearts from S2814D mice.

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  FIGURE 6. Confocal assessment of spontaneous SR Ca2+ waves. (A) Representative confocal images showing Ca2+ waves in isolated cardiomyocytes from S2814D myocytes (left panel) when exposed to 6 mM extracellular Ca2+. When SDKO isolated myocytes are exposed to the same condition, they exhibit miniwaves instead of full propagating Ca2+ waves (Reproduced with permission from Mazzocchi et al., 2016). (B) Representative confocal images of epicardial Ca2+ waves in S2814D and SDKO intact isolated hearts during reperfusion after an ischemic period. SDKO hearts display fragmented Ca2+ waves or mini-waves. Red arrows indicate a series of mini-events conserving a Ca2+ wave pattern (reproduced with permission from Valverde et al., 2019).

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  FIGURE 7. Main factors developing arrhythmogenic Ca2+ waves. An increase in SR-Ca2+ content and/or a decrease in RyR2 refractoriness favor SR-Ca2+ leak and Ca2+ wave generation, meanwhile a decrease in propagation of Ca2+ by diffusion (e.g., like that produce by PLN ablation), will prevent Ca2+ wave propagation by conversion of Ca2+ waves into non-propagable Ca2+ miniwaves.

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  FIGURE 8. RyR2 dysregulation in DCM development. (A) Representative ECG recordings showing that fructose-rich diet (FRD) induces cardiac arrhythmias in WT but not in SR-AIP mice (CD, control diet). WT mice exhibit bradycardia (ii), bidirectional tachycardia (iii), ventricular ectopic beats (iv, arrow), and AV block (v, arrow) when treated with FRD. (B) FRD deteriorates mitochondria membrane potential, as shown by an increase in JC-1 green/red fluorescence ratio. (C) This alteration in FRD mitochondria is in accordance with a higher apoptotic ratio (Bax/Bcl-2 proteins), which can be prevented in mice expressing the CaMKII inhibitory peptide at the SR level (SR-AIP). (D) Histological samples (upper right) show a closer distance between mitochondria and SR in FRD vs. CD treated mice, which might be due to an alteration in structural proteins involved in SR-mitochondria communication. The cartoon in the left side of (D) represents this SR-mito interaction (reproduced with permission from Federico et al., 2017). *p < 0.05.