Abstract
Sarcomeric reticulum (SR) Ca
2+
leak contributes to an impaired Ca
2+
cycling and contractile dysfunction of the heart. Leak is thought to be regulated by post-translational modifications of RyR2 associated with nitroso-redox (NO/redox) imbalance. Ca
2+
cycling is affected by both NO signaling and temperature.
We hypothesized that NO/redox balance modulates SR Ca
2+
leak in a temperature–dependent manner.
We used tetracaine to investigate SR Ca
2+
leak in cardiomyocytes (CMs) using a model of NO/redox imbalance (NOS1
−/−
mice) or WT mice. Reactive oxygen species (ROS) were measured by H
2
DCF. The expression of NOS isoforms was assessed by real time PCR. Experiments were carried out at 23°C - 37°C.
The SR Ca
2+
leak increased as the temperature decreased from 34°C to 23°C in WT CMs whereas, in NOS1
−/−
CMs, the leak suddenly increased when the temperature surpassed 30°C (Fig. A). SR Ca
2+
content in NOS1
−/−
CMs was reduced at 37°C compared to 23°C (42.7 mol/L vs. 115.6 mol/L, respectively;
p
=0.001), whereas it was unaffected in WT CMs. Differences in Ca
2+
leak between WT and NOS1
−/−
were magnified according the SR Ca
2+
load increased. Contrarily to WT, there was a trend to rise in ROS production at temperatures over 30°C in NOS1
−/−
CMs (Fig. B). There was a linear association between ROS and leak in both strains. The NOS isoforms were unaffected by temperature in WT.
Our observations show that Ca
2+
leak increases when the temperature drops below 34°C; however, in the absence of NOS1 activity, the leak rises at temperatures greater than 30°C, and is associated with more ROS production. These results suggest that Ca
2+
leak from the SR is regulated by a temperature-dependent balance between NO and ROS.