The inter-chamber differences in the contractile function between left and right atrial cardiomyocytes in atrial fibrillation in rats

Figure 1. Histological and morphological characteristics of left atrial (LA) and right atrial (RA) remodeling in rats with ACh-CaCl2-induced AF. (A) Example of an atrial fibrillation (AF) episode induced by an ACh-CaCl2 injection where C—is the control group. (B) Representative Sirius Red staining of LA tissue from AF and control rats for assessment of collagen content (40× magnification). (C) Collagen content in LA and RA tissues using Picrosirius red staining. (D) HE staining of LA (40× magnification). (E) The thickness of LA and RA walls in control and AF rats. (F) Relative tissue composition in LA and RA (*indicates a significant difference between AF and control groups). (G) Representative images of single isolated cardiomyocytes (CM) showing elongation of LA CM in AF (40× magnification). (H) Length of single LA and RA CM. Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents a median value from one animal. The number of N hearts in each group is shown below the boxplot. Samples are from the same animals in (C,E,F). Scheirer-Ray-Hare test with…

Figure 2. ROS and NO production in LA and RA CM in control rats and ACh-CaCl2-induced AF. (A) Representative confocal images of CM stained with the superoxide indicator dihydroethdium (DHE). (B) Intracellular ROS production in LA and RA CM. (C) Representative confocal images of CM from the control (C) and AF rats (AF) stained with diaminofluorescein-FM diacetate (DAF-FM). (D) Intracellular NO production in LA and RA CM. Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents an individual CM. The number of n CM from N hearts in each group is shown (5–12 CM from one rat). Statistical significance was determined by hierarchical clustering analysis with log-transformed data.

Figure 3. La vs. RA differences in sarcomere length (SL) dynamics in rats with ACh-CaCl2-induced AF. (A) Analyzed parameters derived from the SL change signal. (B) Representative recordings of the time-dependent SL changes in contracting LA and RA CM from the control rats (C) and AF rats (AF). (C) End-diastolic SL (EDSL). (D) Fractional sarcomere shortening amplitude (FS = sarcomere shortening amplitude/EDSL × 100%). (E) Maximum velocity of sarcomere shortening (vshort). (F) Maximum velocity of sarcomere relengthening (vrel). Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents an individual CM. The number of n CM from N hearts in each group is shown below the first boxplot (5–14 CM from one rat). Statistical significance was determined by hierarchical clustering analysis with log or square root-transformed data.

Figure 4. No LA vs. RA differences in Ca2+ handling in rats with ACh-CaCl2-induced AF. (A) Representative recordings of [Ca2+]i transients at a pacing frequency of 1 Hz followed by application of caffeine (10 mM) in LA and RA CM from the control rats (C) and AF rats (AF). F, fluorescence intensity; F0, fluorescence intensity at rest. (B) The amplitude of electrically evoked [Ca2+]i transients (CaT). (C) The sarcoplasmic reticulum (SR) Ca2+ load measured as the amplitude of [Ca2+]i transients evoked by caffeine application. Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents an individual CM. The number of n CM from N hearts in each group is shown below the boxplot. (4–12 CM (B) or 2–3 CM (C) from one rat). Statistical significance was determined by hierarchical clustering analysis with log-transformed data.

Figure 5. La vs. RA differences in auxotonic force characteristics in rats with ACh-CaCl2-induced AF. (A) Analyzed parameters derived from the auxotonic force signal. (B) Representative auxotonic force recordings in contracting LA and RA CM from the control and AF rats. (C) Normalized force amplitude. (D) Maximum velocity of force development (vFdev). (E) Maximum velocity of force relaxation (vFrel). Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents an individual CM. The number of n CM from N hearts in each group is shown below the first boxplot (1–3 CM from one rat). Statistical significance was determined by hierarchical clustering analysis with log-transformed data.

Figure 6. AF-induced changes in actin-myosin interaction and sarcomeric protein phosphorylation in LA and RA. (A) Sliding velocity of native thin filaments (NTF) and F-actin over myosin from LA and RA using the in vitro motility assay. (B) The example of gel electrophoresis of NTF and myosin extracted from the control (C) and in ACh-CaCl2-induced AF groups. MHC, myosin heavy-chain; cMyBP-C, cardiac myosin binding protein-C; ELC, myosin essential light chain; RLC, myosin regulatory light chain; TnT, troponin T; Tpm, tropomyosin; TnI, troponin I. Phosphorylation was assessed using Pro-Q Diamond and SYPRO Ruby (Invitrogen, Eugene, OR, USA). Precision Plus Protein™ unstained Standards (Bio-Rad, Hercules, CA, USA) was used as molecular weight markers for protein (M). (C) Phosphorylation levels of cMyBP-C and RLC. (D) Phosphorylation levels of TnT, Tpm, and TnI. Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers provide the 100% range of the values. Each dot represents a median value from one animal. The number of N hearts in each group is shown below the first boxplot. Scheirer-Ray-Hare test with Bonferroni…