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Overexpression of the Na+/K+ ATPase ?2 but not ?1 isoform attenuates pathological cardiac hypertrophy and remodeling.

ABSTRACT: RATIONALE:The Na+ / K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+ / Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance. OBJECTIVE:To determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would enhance forward-mode Ca2+ clearance and prevent reverse-mode Ca2+ entry through NCX1 to protect the heart. METHODS AND RESULTS:Cardiac-specific transgenic mice overexpressing either NKA-?1 or NKA-?2 were generated and subjected to pressure overload hypertrophic stimulation. We found that although increased expression of NKA-?1 had no protective effect, overexpression of NKA-?2 significantly decreased cardiac hypertrophy after pressure overload in mice at 2, 10, and 16 weeks of stimulation. Remarkably, total NKA protein expression and activity were not altered in either of these 2 transgenic models because increased expression of one isoform led to a concomitant decrease in the other endogenous isoform. NKA-?2 overexpression but not NKA-?1 led to significantly faster removal of bulk Ca2+ from the cytosol in a manner requiring NCX1 activity. Mechanistically, overexpressed NKA-?2 showed greater affinity for Na+ compared with NKA-?1, leading to more efficient clearance of this ion. Furthermore, overexpression of NKA-?2 but not NKA-?1 was coupled to a decrease in phospholemman expression and phosphorylation, which would favor greater NKA activity, NCX1 activity, and Ca2+ removal. CONCLUSIONS:Our results suggest that the protective effect produced by increased expression of NKA-?2 on the heart after pressure overload is due to more efficient Ca2+ clearance because this isoform of NKA preferentially enhances NCX1 activity compared with NKA-?1.

SUBMITTER: Correll RN

PROVIDER: S-EPMC4001807 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Overexpression of the Na+/K+ ATPase α2 but not α1 isoform attenuates pathological cardiac hypertrophy and remodeling.

Correll Robert N RN Eder Petra P Burr Adam R AR Despa Sanda S Davis Jennifer J Bers Donald M DM Molkentin Jeffery D JD

Circulation research 20131111 2

<h4>Rationale</h4>The Na+ / K+ ATPase (NKA) directly regulates intracellular Na+ levels, which in turn indirectly regulates Ca2+ levels by proximally controlling flux through the Na+ / Ca2+ exchanger (NCX1). Elevated Na+ levels have been reported during heart failure, which permits some degree of reverse-mode Ca2+ entry through NCX1, as well as less efficient Ca2+ clearance.<h4>Objective</h4>To determine whether maintaining lower intracellular Na+ levels by NKA overexpression in the heart would ...[more]

PMID: 24218169

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Project description:Na⁺/K⁺-ATPase maintains electrochemical gradients of Na⁺ and K⁺ essential for a variety of cellular functions including neuronal activity. The α-subunit of the Na⁺/K⁺-ATPase exists in four different isoforms (α1-α4) encoded by different genes. With a view to future use of pig as an animal model in studies of human diseases caused by Na⁺/K⁺-ATPase mutations, we have determined the porcine coding sequences of the α1-α3 genes, ATP1A1, ATP1A2, and ATP1A3, their chromosomal localization, and expression patterns. Our ATP1A1 sequence accords with the sequences from several species at five positions where the amino acid residue of the previously published porcine ATP1A1 sequence differs. These corrections include replacement of glutamine 841 with arginine. Analysis of the functional consequences of substitution of the arginine revealed its importance for Na⁺ binding, which can be explained by interaction of the arginine with the C-terminus, stabilizing one of the Na⁺ sites. Quantitative real-time PCR expression analyses of porcine ATP1A1, ATP1A2, and ATP1A3 mRNA showed that all three transcripts are expressed in the embryonic brain as early as 60 days of gestation. Expression of α3 is confined to neuronal tissue. Generally, the expression patterns of ATP1A1, ATP1A2, and ATP1A3 transcripts were found similar to their human counterparts, except for lack of α3 expression in porcine heart. These expression patterns were confirmed at the protein level. We also report the sequence of the porcine ATP1A3 promoter, which was found to be closely homologous to its human counterpart. The function and specificity of the porcine ATP1A3 promoter was analyzed in transgenic zebrafish, demonstrating that it is active and drives expression in embryonic brain and spinal cord. The results of the present study provide a sound basis for employing the ATP1A3 promoter in attempts to generate transgenic porcine models of neurological diseases caused by ATP1A3 mutations.

Project description:Heart failure (HF) is a functional lack of myocardial performance due to a loss of molecular control over increases in calcium and ROS, resulting in proteolytic degradative advances and cardiac remodeling. Mitochondria are the molecular powerhouse of cells, shifting the sphere of cardiomyocyte stability and performance. Functional mitochondria rely on the molecular abilities of safety factors such as TFAM to maintain physiological parameters. Mitochondrial transcription factor A (TFAM) creates a mitochondrial nucleoid structure around mtDNA, protecting it from mutation, inhibiting NFAT (ROS activator/hypertrophic stimulator), and transcriptionally activates Serca2a to decrease calcium mishandling. Calpain1 and MMP9 are proteolytic degratory factors that play a major role in cardiomyocyte decline in HF. Current literature depicts major decreases in TFAM as HF progresses. We aim to assess TFAM function against Calpain1 and MMP9 proteolytic activity and its role in cardiac remodeling. To this date, no publication has surfaced describing the effects of aortic banding (AB) as a surgical HF model in TFAM-TG mice. HF models were created via AB in TFAM transgenic (TFAM-TG) and C57BLJ-6 (WT) mice. Eight weeks post AB, functional analysis revealed a successful banding procedure, resulting in cardiac hypertrophy as observed via echocardiography. Pulse wave and color doppler show increased aortic flow rates as well as turbulent flow at the banding site. Preliminary results of cardiac tissue immuno-histochemistry of HF-control mice show decreased TFAM and compensatory increases in Serca2a fluorescent expression, along with increased Calpain1 and MMP9 expression. Protein, RNA, and IHC analysis will further assess TFAM-TG results post-banding. Echocardiography shows more cardiac stability and functionality in HF-induced TFAM-TG mice than the control counterpart. These findings complement our published in vitro results. Overall, this suggests that TFAM has molecular therapeutic potential to reduce protease expression.

Dataset Information

Overexpression of the Na+/K+ ATPase ?2 but not ?1 isoform attenuates pathological cardiac hypertrophy and remodeling.

Publications

Overexpression of the Na+/K+ ATPase α2 but not α1 isoform attenuates pathological cardiac hypertrophy and remodeling.

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