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

Abstract

The force-frequency relationship (FFR) is an important intrinsic regulatory mechanism of cardiac contractility. However, a decrease (negative FFR) or no effect (flat FFR) on contractile force in response to an elevation of heart rate is present in the normal rat or in human heart failure. Reactive oxygen species (ROS) can act as intracellular signaling molecules activating diverse kinases as calcium-calmodulin-dependent protein kinase II (CaMKII) and p-38 MAP kinase (p-38K). Our aim was to elucidate the intracellular molecules implicated in the FFR of isolated rat ventricular myocytes. The myocytes were field-stimulated via two-platinum electrodes. Sarcomere length was recorded with a video camera. Ca transients and intracellular pH were recorded by epifluorescence. Increasing frequency from 0.5 to 3 Hz decreased cell shortening without changes in pH. This negative FFR was changed to positive FFR when the myocytes were pre-incubated with the ROS scavenger MPG, the NADPH oxidase blocker apocynin, or by inhibiting mitochondrial ROS production with 5-HD. Similar results were obtained when the cells were pre-incubated with the CaMKII blocker, KN-93, or the p-38K inhibitor, SB-202190. 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 transient amplitudes were reduced in MPG- and SB-202190-treated myocytes. In conclusion, our results indicate that the activation of the intracellular pathway involving ROS-CaMKII-p-38K contributes to the negative FFR of rat cardiomyocytes, likely by desensitizing the response of contractile myofilaments to Ca.

p38 MAPK Pathway in the Heart: New Insights in Health and Disease

“…Different mechanisms have been proposed for the negative effect of p38 on cardiac contractility. For example, p38 has been proposed to mediate the anti-inotropic effects of angiotensin II and ROS, which are also increased during HF, desensitizing the response of myofilaments to Ca 2+ [ 102 , 105 , 107 ]. The mechanism underlying p38-mediated dampening of Ca 2+ responsiveness is unknown, but two main possibilities have been proposed: modification of intracellular pH or phosphorylation of contractile proteins.…”

“…The mechanism underlying p38-mediated dampening of Ca 2+ responsiveness is unknown, but two main possibilities have been proposed: modification of intracellular pH or phosphorylation of contractile proteins. However, studies have disproved the involvement of pH modification in myofilament sensitivity to Ca 2+ [ 105 , 107 ], leaving phosphorylation of contractile proteins as the more likely mechanism. Analysis by Liao et al did not detect increased p38-mediated troponin I phosphorylation, which is known to reduce myofilament responsiveness to Ca 2+ [ 102 ].…”

Activation of G Protein Coupled Estrogen Receptors (GPER) Negatively Modulates Cardiac Excitation-Contraction Coupling (ECC) through the PI3K/NOS/NO Pathway

“…Calcium transient recordings were performed as previously described [49]. Briefly, isolated myocytes were loaded with 3 µmol/L Fura-2 AM (Thermo Fisher Scientific) for 10 minutes.…”