The reduced myofilament responsiveness to calcium contributes to the negative force-frequency relationship in rat cardiomyocytes: role of reactive oxygen species and p-38 MAP kinase.

ESPEJO MS; AIELLO I; IBAÑEZ AM; SEPULVEDA M; MARTÍN G. VILA PETROFF; AIELLO EA; DE GIUSTI VC

Congreso; 38th Congress of the International Union of Physiological Sciences (IUPS); 2017

The force?frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) in contractile force in response to an elevation of heart rate is present in the normal rat heart and in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as CaMKII and p38 MAP Kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. Sarcomere length shortening (SLS) was recorded with a video-camera. The stimulation frequency was set to 0.5, 1, 2 or 3 Hz. Ca transients and intracellular pHi were recorded by epifluorescence. Increasing frequencyfrom 0.5 to 3 Hz decreased SLS (at 2 Hz vs. 0.5 Hz SLS decreased approximately 18%) without changes in pHi. This negative FFR was changed significantly to positive FFR when the myocytes were pre-incubated with th ROS scavenger N-(2-Mercapto-Propionyl) Glycine (MPG 2 mM, at 2 Hz SLS increased 27.9±4.6%* vs. 0.5 Hz; n=11); the NADPH oxidase blocker, Apocynin (Apo 300 µM, 16.5±3.2%* increase in SLS at 2 Hz vs. 0.5 Hz; n=6) or by inhibiting mitochondrial ROS production with 5-Hydroxydecanoic Acid (5-HD 500µM, 30.3±5.8%*increase in SLS at 2 Hz vs. 0.5 Hz; n=10). Same FFR results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93 (2.5 µM, 23.0±6.3%* increase in SLS at 2 Hz vs. 0.5 Hz; n=7) or the p-38K inhibitor, SB-202190 (10 µM, 37.1±7.6%* increase in SLS at 2 Hz vs. 0.5 Hz; n=7). Consistently, the levels of phosphorylation of p-38K and the oxidation of CaMKII were significantly higher at 2 Hz than at 0.5 Hz. Despite the presence of positive inotropic effect during stimulation frequency enhancement, Ca transients amplitude were reduced in MPG- and SB-202190-treated myocytes, without differences between treatments and control. Finally we studied the Ca -contractility loop to assess the involvement of the myofilament sensitivity to Ca in the FFR. We found a significantly increase in the slope of the relaxation phase of the loops in MPG- and SB202190-treated myocytes, indicating enhanced myofilament Ca sensitivity. In conclusion, our results indicate that the activation of the intracellular pathways involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca2+