Protein carbonylation causes sarcoplasmic reticulum Ca2+ overload by increasing intracellular Na+ level in ventricular myocytes

Abstract

Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca²⁺ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca²⁺ regulation in mouse ventricular myocytes. Analysis of intracellular Ca²⁺ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca²⁺ transients and sarcoplasmic reticulum (SR) Ca²⁺ load, with a limited effect on L-type Ca²⁺ channel-mediated Ca²⁺ transients and SERCA-mediated Ca²⁺ uptake. At the same time, MGO significantly slowed down cytosolic Ca²⁺ extrusion by Na⁺/Ca²⁺ exchanger (NCX). MGO also increased the frequency of Ca²⁺ waves during rest and these Ca²⁺ release events were abolished by an external solution with zero [Na⁺] and [Ca²⁺]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca²⁺ transients and SR Ca²⁺ load, but it also triggered spontaneous Ca²⁺ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca²⁺ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na⁺] revealed that MGO increases cytosolic [Na⁺] by 57% from the maximal effect produced by the Na⁺-K⁺ ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na⁺] was a result of activation of a tetrodotoxin-sensitive Na⁺ influx, but not an inhibition of Na⁺-K⁺ ATPase. An increase in cytosolic [Na⁺] after treating cells with ouabain produced similar effects on Ca²⁺ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca²⁺ regulation by increasing cytosolic [Na⁺] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca²⁺ extrusion by NCX, causing SR Ca²⁺ overload and spontaneous Ca²⁺ waves.

Keywords: Ca2+ signaling; Na+ homeostasis; Na+ pump; Na+/Ca2+ exchanger; heart; methylglyoxal; sarcoplasmic reticulum.