Na/K‐ATPase Alpha1 Isoform Signaling in Cardiac Metabolism

Abstract

Understanding cardiac metabolic signaling and its relation to pathological hypertrophy is critical to develop new therapeutic intervention in heart failure (HF). Decreased Na/K‐ATPase (NKA) activity and expression have long been associated with HF in experimental models and in patients. Classically known for its crucial role in transmembrane ion transport, electrical excitability, and contractility, NKA also supports non‐enzymatic receptor functions that differ among major cardiac isoforms (α1 and α2 in rodents). Studies from our group and others have suggested a role of these receptor functions in cardiac myocyte hypertrophy. Using epithelial (LLC‐PK1)‐derived cell lines, we have previously observed a NKA α1‐specific ability to modulate metabolic reserve and capacity. Mechanistically, this property is highly dependent upon NKA α1 ability to regulate the signaling partner Src kinase and is not supported by NKA α2, which lacks the Src binding domain. In this work, our goal was to assess the impact of NKA α1 ablation in the heart and demonstrate the importance of NKA α1 signaling function for proper cardiomyocyte metabolic function. We used a Myh6‐Cre/Lox approach to delete NKA α1 in the mouse heart (cardio α1‐/‐). These animals (3‐month‐old male mice) presented a normal cardiac structure, reduced NKA α1 expression (more than 100‐fold; n = 6‐7; p < 0.01) and increased NKA α2 expression (10‐fold; n = 6‐7; p < 0.01) by Western blot. RNA sequencing followed by gene set enrichment analysis revealed that cardio α1‐/‐ hearts have downregulated pathways related to mitochondrial metabolism, such as cytochrome complex, NADH dehydrogenase complex, respiratory chain, and mitochondrial fatty acid β‐oxidation (n = 3; FDR < 0.05). We next generated human induced pluripotent stem cells (hiPSCs) with a mutant form of the NKA α1 isoform that can pump ions but have blunted Src signaling (A420P mutant) using a CRISPR/Cas9‐mediated knock‐in approach. Genotyping and sequencing confirmed the substitution of alanine to proline in the NaKtide sequence to generate the desired mutation. These cells were differentiated into cardiomyocytes, as confirmed by overexpression of the cardiac markers myosin heavy chain 6 (MYH6) and cardiac muscle troponin T (TNNT2) (more than 10‐fold; n = 3; p < 0.05). Metabolic phenotyping using the Agilent Seahorse Extracellular Flux technology suggested that the A420P mutant hiPSC‐derived cardiomyocytes (A420P iCM) have decreased basal respiration, maximal respiration, spare respiratory capacity, and ATP production compared to the controls (WT iCM) based on changes in oxygen consumption rate (OCR) using the Agilent Seahorse XF Cell Mito Stress Test Kit. In addition, A420P iCM have reduced glycolytic capacity and reserve, based on changes in extracellular acidification rate (ECAR) using the Agilent Seahorse XF Glycolysis Stress Test Kit. Taken together, these results suggest that NKA α1 isoform signaling modulates the cardiomyocyte metabolism. Further studies may reveal a link between NKA/Src signaling, metabolic flexibility, and structural cardiac remodeling in the context of HF and cardiac hypertrophy.