Introduction: Intracellular sodium ([Na⁺]ᵢ) cycling is essential for cardiac electrophysiology and activation of excitation-contraction coupling (ECC) . First, we hypothesized that pathogenic NaV1.5 channel gain-of-function contributes to not only paroxysmal atrial fibrillation (pAF), but also affects mitochondrial metabolism and the ECC machinery. Second, we investigated if mild overexpression of Junctophilin2 (JP2) corrects the atrial phenotype.
Methods: Atrial tissue and isolated cardiomyocytes were obtained from FLAG-NaV1.5-F1759A double-transgenic (DTG) mice, expressing a cardiomyocyte-restricted gain-of-function mutation in the redox-competent C57BL/6N background. ECG, echocardiography, and immunoblotting characterized the DTG mouse model. Cardiac intracellular [Na⁺]ᵢ was measured using 23Na-NMR. Confocal microscopy visualised cardiomyocyte dimensions and changes of the transverse-axial tubule (TAT) network. Quantitative proteomics using tandem mass tag (TMT) labeling and untargeted metabolomics explored metabolic alterations. Triple-transgenic mice were generated expressing i) either a mitochondrial matrix redox biosensor or ii) JP2 in cardiomyocytes.
Results: C57BL/6N DTG mice reproduced paroxysmal AF episodes. Ex vivo, DTG hearts exhibited significantly elevated [Na⁺]ᵢ compared to WT control hearts under physiological conditions (WT: 4.26 ± 0.4 AU vs. DTG: 5.3 ± 0.4 AU, p<0.05). DTG mice showed an increased left atrial diameter and reduced fractional shortening. DTG atrial cardiomyocytes displayed hypertrophic remodeling with axialization of the TAT network, indicative of subcellular ECC disruption. JP2 and ryanodine receptor RyR2 protein expression was decreased. Proteomic analysis revealed major alterations in energetic substrate pathways in the mitochondria, further corroborated by metabolomic data. Specifically, we observed a significant reduction in high-energy metabolites such as NAD⁺, suggesting a shift from fatty acid oxidation to alternative substrates. TTG-mitochondrial redox biosensor atrial cardiomyocytes exhibited significantly decreased glutathione redox potential (EGSH) levels and increased mitochondrial matrix oxidation, indicating redox imbalance associated with elevated [Na⁺]ᵢ. In contrast, TTG-JP2OE mice showed improved atrial contractility, reduced BNP plasma levels, and restoration of TAT network architecture, rescuing the AF-associated atrial cardiomyoapthy.
Discussion: Our findings identify multi-factorial synergistic consequences of NaV1.5 gain-of-function in atrial cardiomyocytes. We demonstrate that elevated [Na⁺]ᵢ leads to severe metabolic dysfunction with mitochondrial redox imbalance and disruption of ECC. Near-physiological JP2 overexpression represents a promising strategy to restore atrial contractility and prevent episodic AF .
