Bioenergetic and metabolic aberrations in induced pluripotent stem cell-derived cardiomyocytes generated from PRKAG2-mutated, WPW patient
Authors: Baskin P , Ben Jehuda R , Abramovich I , Agranovich B , Davidor M , Mekies L.N , Milman H , Shulman M , Arad M , Gottlieb E , Binah O 
Published: November 2020

Abstract

Introduction
Familial hypertrophic cardiomyopathy (HCM) is caused by over 400 mutations affecting mostly key sarcomere components, such as titin and myosin. However, HCM also results from mutations in non-structural genes such as PRKAG2 which is involved directly in a variety of bioenergetic and metabolic pathways. When the metabolic processes fail to work properly or effectively, structural and functional aberrations resulting in cardiac dysfunction can occur. Thus, mutations in the human PRKAG2 gene lead to HCM, autosomal dominant ventricular pre-excitation - Wolff-Parkinson-White syndrome (WPW), a progressive conduction system disease and vacuolar glycogen accumulation in cardiomyocytes.

Purpose
To investigate the hypothesis that intervening with the bioenergetic and metabolic consequences of the R302Q mutation in the PRKAG2 gene causing inherited cardiomyopathy, will attenuate the cardiac impairments.

Methods
We generated mutated and isogenic induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs) from a WPW patient carrying the R302Q mutation. As additional control, we used healthy volunteers' iPSC-CMs. Bioenergetic (Oxygen Consumption Rate, OCR) and metabolic status were measured using the Seahorse Flux Analyzer and LC-MS, respectively. To decipher the molecular basis underlying the bioenergetic and metabolic deficits, RNA-seq analysis was performed.

Results
The OCR results demonstrated in PRKAG2-mutated compared to isogenic and healthy iPSC-CMs, a 2-fold increase in maximal respiration rate and a 3.75-fold increase in spare respiratory capacity, while basal OCR parameters were similar in all groups. Importantly, when treated with the AMPK activator metformin (2.5 [mM]), all the abovementioned OCR parameters were similar in the three groups. RNA-seq analysis demonstrated that of the 553 differently expressed genes (DEGs), and of the 99 DEGs mutually differentially expressed, compared to isogenic and healthy cells, the most relevant altered pathways were glycolysis, carbon metabolism, biosynthesis of amino acids, HIF-1 signaling and fructose and mannose metabolism. These findings are consistent with the LC-MS results demonstrating in PRKAG2-mutated versus isogenic and healthy iPSC-CMs, at least a 3-fold decrease in metabolites linked to the abovementioned pathways: butyryl-carnitine, creatine, docosahexaenoic acid, GMP, IMP, myristoyl-carnitine, palmitoyl-carnitine, succinyl-Cys, UMP, UTP, UDP-GlcNAc.

Conclusion
PRKAG2-mutated iPSC-CMs exhibit bioenergetic and metabolic aberrations, which contribute to the cardiac pathological aspects of WPW syndrome. Importantly, treatment with the AMPK activator metformin eliminated the bioenergetic abnormalities in the mutated cells, while isogenic and healthy control cells remained unaffected. Based on these novel findings, a new therapeutic modality in WPW patients may be considered.

Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Israel Science Foundation (ISF)