Redox and Activation of Protein Kinase A Dysregulates Calcium Homeostasis in Pulmonary Vein Cardiomyocytes of Chronic Kidney Disease

Figure 1. Electrocardiography and spontaneous beating rates of pulmonary vein (PV) cardiomyocytes in control and chronic kidney disease (CKD) rabbits. A, Control rabbits had a regular beating rate, but CKD rabbits had repeated atrial premature complexes (↑, APCs). B, An example and average data show that CKD PV cardiomyocytes (n=8, from 4 hearts) had more rapid beating rates than control PV cardiomyocytes (n=10, from 4 hearts). *P<0.05 vs the control.

Figure 2. Intracellular calcium homeostasis, calcium regulatory protein, and sarcoplasmic reticulum (SR) Ca2+‐ATPase (SERCA2a) activity in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. A, Tracings and average data of SR calcium contents (left panels) from integrating caffeine‐induced sodium/calcium exchanger (NCX) currents in control (n=12, from 4 hearts) and CKD (n=10, from 4 hearts) PV cardiomyocytes. Tracings and average data of calcium transients from 2‐Hz field stimulation (right panels) in control (n=14, from 5 hearts) and CKD (n=12, from 6 hearts) PV cardiomyocytes. B, Representative immunoblot and average data of PKA, phosphorylated phospholamban (PLB) at PLB pThr17 (phosphorylated PLB at threonine 17), PLB pSer16 (phosphorylated PLB at serine 16), PLB, type 2 ryanodine receptor (RyR2) channels, RyR2 pS2814 (phosphorylated RyR2 at serine 2814), RyR2 pS2808 (phosphorylated RyR2 at serine 2808), from control (from 8 hearts) and CKD PV cardiomyocytes (from 8 hearts). C, The SERCA2a activity was higher in CKD PV cardiomyocytes (from 7 hearts) than control PV cardiomyocytes (from 6 hearts). *P<0.05, **P<0.01, ***P<0.005 vs the control.

Figure 3. The effect of calcium regulation signaling in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. Tracings and average data of calcium transients from 2‐Hz field stimulation in CKD PV cardiomyocytes before and after treatment with H89 (1 μmol/L, an inhibitor of PKA; n=8, from 4 hearts), KN93 (1 μmol/L, an inhibitor of CaMKII; n=8, from 3 hearts), and KN92 (1 μmol/L, an inactive analog of KN93; n=8, from 3 hearts). *P<0.05.

Figure 4. Calcium sparks in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. A, Examples of line scans to detect calcium sparks in control and CKD PV cardiomyocytes. Average data of the frequency, incidence of calcium sparks, duration, width, peak time, and decay time in control (n=33, from 7 hearts) and CKD PV cardiomyocytes (n=33, from 10 hearts). B, Examples of line scans to detect spontaneous calcium releases presented along with calcium spark in CKD PV cardiomyocytes. The spontaneous calcium releases (red asterisk) that precedes ectopic activity is presented along with a calcium spark (red arrow). The incidence of spontaneous calcium releases along the calcium spark in control (n=25, from 7 hearts) and CKD PV cardiomyocytes (n=29, from 10 hearts). *P<0.05, **P<0.01, ***P<0.005 vs the control.

Figure 5. Ionic currents in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. A, Tracings and current‐voltage relationship of the sodium/calcium exchanger (NCX) from control (n=9, from 4 hearts) and CKD PV cardiomyocytes (n=10, from 5 hearts). B, Tracings and I–V relationship of L‐type calcium currents (IC a‐L) from control (n=10, from 5 hearts) and CKD PV cardiomyocytes (n=10, from 5 hearts). C, Tracings and average data of late sodium currents (IN a‐Late) from control (n=11, from 6 hearts) and CKD PV cardiomyocytes (n=13, from 5 hearts). TTX indicates tetrodotoxin. *P<0.05, ***P<0.005 vs control.

Figure 6. Role of late sodium currents (IN a‐Late) in calcium transient in pulmonary vein (PV) cardiomyocytes. A, Tracings and average data of calcium transients from 2‐Hz field stimulation in control PV cardiomyocytes (n=10, from 3 hearts) before and after Anemonia sulcata toxin (ATXII, 100 nmol/L). B, Tracings and average data of calcium transients from 2‐Hz field stimulation in chronic kidney disease (CKD) PV cardiomyocytes (n=10, from 3 hearts) before and after ranolazine (10 μmol/L). *P<0.05, **P<0.01.

Figure 7. Reactive oxygen species (ROS) in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. A, An example and average data of mitochondrial ROS in control (n=10, from 3 hearts) and CKD (n=8, from 3 hearts) PV cardiomyocytes. B, Average data of plasma and cytosol malondialdehyde (MDA) in control and CKD rabbits and PV cardiomyocytes. *P<0.05, ***P<0.005 vs control.

Figure 8. The role of reactive oxygen species (ROS) in control and chronic kidney disease (CKD) pulmonary vein (PV) cardiomyocytes. A, Tracings and average data of calcium transients from 2‐Hz field stimulation in CKD PV cardiomyocytes before and after MPG (N‐[2‐mercaptopropionyl]glycine; 100 μmol/L, n=13, from 4 hearts). MPG‐treated CKD PV cardiomyocytes had smaller calcium transients than did CKD PV cardiomyocytes. B, Tracings and average data of calcium transients from 2‐Hz field stimulation in CKD PV cardiomyocytes treated with H89 (n=12, from 3 hearts) or KN93 (n=14, from 3 hearts) before and after MPG (100 μmol/L). **P<0.01.