Alternative Splicing of NOX4 in the Failing Human Heart

Abstract

Increased oxidative stress is a major contributor to the development and progression of heart failure, however, our knowledge on the role of the distinct NADPH oxidase (NOX) isoenzymes, especially on NOX4 is controversial. Therefore, we aimed to characterize NOX4 expression in human samples from healthy and failing hearts. Explanted human heart samples (left and right ventricular, and septal regions) were obtained from patients suffering from heart failure of ischemic or dilated origin. Control samples were obtained from donor hearts that were not used for transplantation. Deep RNA sequencing of the cardiac transcriptome indicated extensive alternative splicing of the NOX4 gene in heart failure as compared to samples from healthy donor hearts. Long distance PCR analysis with a universal 5′-3′ end primer pair, allowing amplification of different splice variants, confirmed the presence of the splice variants. To assess translation of the alternatively spliced transcripts we determined protein expression of NOX4 by using a specific antibody recognizing a conserved region in all variants. Western blot analysis showed up-regulation of the full-length NOX4 in ischemic cardiomyopathy samples and confirmed presence of shorter isoforms both in control and failing samples with disease-associated expression pattern. We describe here for the first time that NOX4 undergoes extensive alternative splicing in human hearts which gives rise to the expression of different enzyme isoforms. The full length NOX4 is significantly upregulated in ischemic cardiomyopathy suggesting a role for NOX4 in ROS production during heart failure.

NADPH Oxidases and Mitochondria in Vascular Senescence

“…Consistent with a role of Nox4 in senescence and oxidative stress in the cardiovascular system, increased expression of Nox4 and novel short isoforms of this oxidase were found in samples of failing hearts of human patients [ 111 ]. It is unknown, however, whether these novel isoforms are functional, and the type of ROS they can produce.…”

Mitochondrial dysfunction and oxidative stress in heart disease

“…While NOX2 causes cardiac hypertrophy through angiotensin II, NOX4, which partially localizes to the mitochondria, is associated with cardiac hypertrophy due to increased pressure overload from myocardial stresses in the failing human heart. While NOX4 is found to increase myocardial angiogenesis and protect against contractile dysfunction in HF 42 , recent evidence suggests that NOX4 in failing heart models undergoes alternative splicing, which may explain its detrimental involvement in HF 47 . Other studies have shown that NOX4 depletion decreased mitochondrial swelling and cytochrome c release and decreased mitochondrial DNA (mtDNA) damage.…”

Fibroblasts to Keratinocytes Redox Signaling: The Possible Role of ROS in Psoriatic Plaque Formation

“…The complex distribution of NOX4 in the human fibroblasts evidenced by the confocal microscopy and immunocytochemical staining confirms the role of NOX4 in redox signalling in physiological and pathological conditions. Indeed, other authors have shown on A549 and human heart tissue that the alternative splicing of NOX4 brings to the presence of several NOX4 isoforms that differ in intracellular localization [18,19]. The study of splicing isoforms in human skin fibroblasts and keratinocytes as well as the expression of other two members of NADPH oxidase family releasing H 2 O 2 as the main product, DUOX1 and DUOX2, may be the purpose of further studies.…”