PMC

Computational modeling of Ca²⁺ cycling at low level of ROS (A), intermediate level of ROS (B), and high level of ROS (C). Top trace represents the Ca²⁺ transient when the cell is paced for 30 beats using a periodic voltage clamp. Bottom trace is a longitudinal linescan of our spatially distributed model of 60 × 20 × 20 calcium release units. The linescan location is at position n_y = 10 and n_z = 15. Scale bar: 45 μm.

(A and B) More heterogeneous distribution of CaMKII-p-Na_v1.5 (S571) (A) and Na_v1.5 (B) in HF PLA, compared with control PLA in random nine 40× confocal images. Each number (yellow) represented the ratio of labeling frequency of CaMKII-p-Na_v1.5 (S571) (A, green) and Na_v1.5 (B, green) against cadherin (red) at ID. Scale bar: 40 μm. (C) Comparison of spatial distribution of CaMKII-p-Na_v1.5 (S571) and Na_v1.5 in control and HF PLA. Note that CaMKII-p-Na_v1.5 (S571) was more heterogeneously distributed than Na_v1.5 in HF PLA. Data are represented as mean ± SEM. *P <0.05, **P < 0.01. Independent t tests. HF, heart failure; PLA, posterior left atrium; ID, intercalated disc.

(A) Representative immunoblot and densitometric measurements of Ox-CaMKII (normalized to native CaMKII) from control and HF in PLA (left) and LAA (right). (B) Quantification of immunofluorescence in all myocytes in random nine 10× images in control and HF PLA. Coefficient of variation of Ox-CaMKII immunofluorescence for control vs. HF PLA is compared in the graph on the right. Scale bar: 40 μm. Data are represented as mean ± SEM. *P < 0.05. Independent t tests. HF, heart failure; PLA, posterior left atrium; LAA, left atrial appendage. See complete unedited blots in the supplemental material.

(A and B) Conduction velocity was decreased at 400 ms and 300 ms cycle length in PLA after 3 weeks of ventricular tachypacing, but not in LAA. (C and D) Conduction inhomogeneity was increased at NSR, 400 ms, and 300 ms cycle length in PLA after 3 weeks of ventricular tachypacing, but not in LAA. (E and F) Representative examples of activation maps in the PLA and LAA, respectively. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Repeated measures 2-way ANOVA with Bonferroni adjusted within cycle comparisons. HF, heart failure; PLA, posterior left atrium; LAA, left atrial appendage.

Simulations were performed on a 200 × 200 grid equivalent to a 5 × 5 mm sheet of tissue. Dense fibrotic region was modeled as inactive and nonconducting areas (gNa = 0). Regions were assigned either normal (gNa = 12) or reduced (gNa = 6) sodium conductance. Arrhythmia induction was performed in the presence of fibrosis alone (A) or reduced gNa regions juxtaposed to regions of fibrosis, either in a concentrated (B) or scattered configuration (C). In each condition, 25 screen shots of activation movie were taken every 75 ms. Sustained reentry could be induced in the setting of condition B and C, but not condition A.

(A) Representative immunoblot of CaMKII-p-Na_v1.5 (S571), native Na_v1.5, and cadherin (top) and densitometric measurements for CaMKII-p-Na_v1.5 (S571) (normalized to native Na_v1.5, bottom left) and native Na_v1.5 (normalized to cadherin, bottom right) from control and HF in PLA. (B) While Na_v1.5 was localized at both ID (white arrows) and the lateral membrane (LM, yellow arrows) in both control and HF (top), CaMKII-p-Na_v1.5 (S571) was only localized at ID in control (middle) and HF (bottom). Scale bar: 40 μm. (C) Quantification of myocytes with LM labeling of Na_v1.5 and CaMKII-p-Na_v1.5 (S571) in control and HF myocytes. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A) Independent t tests. (C) Two-way ANOVA, Bonferroni adjusted within level comparisons. HF, heart failure; PLA, posterior left atrium; ID, intercalated disc. See complete unedited blots in the supplemental material.

(A) In HF PLA, there was a dropout of CaMKII-p-Na_v1.5 (S571) at certain ID (red arrow), where cadherin labeling was still intact at those ID (left). Quantification of myocytes with ID labeling of CaMKII-p-Na_v1.5 (S571) in control and HF PLA (right). (B) Representative immunoblot and densitometric measurements of AnkG (normalized to GAPDH and cadherin) in cytosolic and membrane fractions, respectively, from control and HF PLA. (C) ID localization of AnkG (red) was reduced in isolated HF myocytes, while labeling of cadherin (green) seemed similar in control and HF myocytes. (D) Colocalization of CaMKII-p-Na_v1.5 (S571) and AnkG at the ID (white arrows). Scale bar: 40 μm. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (A and B) Independent t test. (C) Generalized linear model, logit link comparing rates if ID localization of AnkG. (D) χ² test. HF, heart failure; PLA, posterior left atrium; ID, intercalated disc. See complete unedited blots in the supplemental material.

(A) Overall O₂^– generation in atria in control and HF. (B) Higher O₂^– generation in HF PLA compared with control PLA and HF LAA. (C and D) Contribution of various enzymatic sources of ROS generation in PLA and LAA. NADPH oxidase and mitochondrial ROS was found to be responsible for significant increase in ROS generation in HF PLA but not in HF LAA. (E) Representative immunoblot and densitometric measurements of gp91 (normalized to GAPDH) from control and HF atria. (F) General protein carbonylation levels in control and HF atria. (G) Comparison of protein carbonylation in HF PLA and HF LAA. (H) Comparison of protein carbonylation in HF LAA and HF RAA. Data are represented as mean ± SEM; *P < 0.05, **P < 0.01. (A, E, F, G, and H) Independent t test. (B) Two-way ANOVA test of main effects. (C and D) Two-way ANOVA with Bonferroni correction on comparison of HF vs. control within each enzyme. HF, heart failure; PLA, posterior left atrium; LAA, left atrial appendage; RAA, right atrial appendage. See complete unedited blots in the supplemental material.

Increase in ROS generation by NADPH oxidase (NOX2) and mitochondrial ROS results in enhanced oxidation of CaMKII in the HF PLA. Ox-CaMKII hyperphosphorylates downstream target molecules, including RyR2 and Na_v1.5. At the cellular level, CaMKII-dependent phosphorylation of RyR2 leads to emergence of triggered Ca²⁺ waves, which predispose to afterdepolarizations (triggered activity). At the tissue level, CaMKII-dependent phosphorylation of Na_v1.5 results in selective trafficking of the channel protein to the ID, with little or no trafficking to the lateral membrane (LM). An increase in the level of CaMKII-p-Na_v1.5 (S571) would therefore be expected to decrease peak I_Na and lead to conduction slowing. Furthermore, a decrease in AnkG levels in HF leads to defective forward trafficking of CaMKII-p-Na_v1.5 (S571) to the ID; this results in a patchy dropout of CaMKII-p-Na_v1.5 (S571) and, therefore, in increased heterogeneity of expression of CaMKII-p-Na_v1.5 (S571). The increased heterogeneity of expression of CaMKII-p-Na_v1.5 (S571) promotes inhomogeneous conduction in the HF PLA. The conduction slowing and increased inhomogeneity of conduction caused by altered expression patterns of CaMKII-p-Na_v1.5 (S571) leads to substrate for reentry. HF, heart failure; PLA, posterior left atrium; ID, intercalated disc.

(A) Representative immunoblot and densitometric measurements of CaMKII-p-RyR2 (S2814) (normalized to native RyR2) from control and HF in PLA (left) and LAA (right). (B) HF atrial myocytes showed higher incidence of triggered Ca²⁺ waves, compared with control. (C) Higher incidence of triggered Ca²⁺ waves in HF PLA myocytes, compared with HF LAA myocytes at 300 ms BCL. (D) Ca²⁺ imaging showed attenuation of incidence of triggered Ca²⁺ waves at 500 ms and 300 ms BCL in the presence of mito-TEMPO and apocynin in HF PLA myocytes, but not in HF LAA myocytes. Time bar: 1 second. The myocytes for these experiments were obtained from 4 HF dogs, and the number of myocytes for each experimental condition is given in each figure panel. Data are represented as mean ± SEM. *P < 0.05, ***P < 0.001. (A and B) Independent t test. (C) Paired t test. (D) Main effect of ANOVA (P = 0.017 at 300 ms, 0.053 at 500ms), post hoc t tests with each cycle with Bonferroni correction. HF, heart failure; PLA, posterior left atrium; LAA, left atrial appendage. See complete unedited blots in the supplemental material.