Protection against endotoxemia‐induced contractile dysfunction in mice with cardiac‐specific expression of slow skeletal troponin I

Abstract

Gram negative endotoxemia is associated with an intrinsic impairment of cardiomyocyte contraction, in part due to a reduction in myofilament Ca2+ responsiveness. Endotoxemic rat hearts show increased cardiac troponin I (cTnI) phosphorylation at serines 23 and 24, residues required for the protein kinase A (PKA)-dependent reduction of myofilament Ca2+ sensitivity after beta-adrenoceptor stimulation. To investigate the functional significance of increased TnI phosphorylation in endotoxemia, we studied the contractile effects of systemic bacterial lipopolysaccharide (LPS) treatment in transgenic mice (TG) with cardiac-specific replacement of cTnI by slow skeletal TnI (ssTnI, which lacks the PKA phosphorylation sites) and matched nontransgenic littermates (NTG) on a CD1 background. In wild-type CD1 mice treated with LPS (6 mg/kg ip), after 16-18 h there was a significant reduction in the maximum rates of left ventricular pressure development and pressure decline in isolated Langendorff-perfused hearts compared with saline-treated controls and a decrease in isolated myocyte unloaded sarcomere shortening from 6.1 +/- 0.2 to 3.9 +/- 0.2% (1 Hz, 32 degrees C, P<0.05). Similarly, in NTG myocytes, endotoxemia reduced myocyte shortening by 42% from 6.7 +/- 0.2 to 3.9 +/- 0.1% (P<0.05) with no change in intracellular Ca2+ transients. However, in the TG group, LPS reduced myocyte shortening by only 13% from 7.5 +/- 0.2 to 6.5 +/- 0.2% (P<0.05). LPS treatment significantly reduced the positive inotropic effect of isoproterenol in NTG myocytes but not in TG myocytes, even though isoproterenol-induced increases in Ca2+ transient amplitude were similar in both groups. Only LPS-treated NTG hearts showed a significant increase in cTnI phosphorylation. Investigation of the sarcomere shortening-Ca2+ relationship in Triton-skinned cardiomyocytes revealed a significant reduction in myofilament Ca2+ sensitivity after LPS treatment in NTG myocytes, an effect that was substantially attenuated in TG myocytes. In conclusion, the replacement of cTnI with ssTnI in the heart provides significant protection against endotoxemia-induced cardiac contractile dysfunction, most probably by preserving myofilament Ca2+ responsiveness due to prevention of phosphorylation of TnI at PKA-sensitive sites.

Changes in myofilament proteins, but not Ca2+ regulation, are associated with a high-fat diet-induced improvement in contractile function in heart failure

“…LV tissue (n ϭ 6) was harvested, and myofilament proteins were purified using a previously described protocol (20). Ten micrograms of protein were loaded onto a 4 -12% XT Bis-Tris gel (Bio-Rad), followed by Pro-Q Diamond Phosphoprotein Gel staining (Invitrogen) per the manufacturer's protocol.…”

Aerobic interval training enhances cardiomyocyte contractility and Ca2+ cycling by phosphorylation of CaMKII and Thr-17 of phospholamban

“…The CaMKblockade maneuver in trained cardiomyocytes caused Ca 2+ sensitivity to revert toward levels closer to sedentary controls; hence, suggesting that CaMK partly modulates the traininginduced improvement in Ca 2+ sensitivity. A proposed mechanism for improved Ca 2+ sensitivity has been that troponins T [22] and I [23] undergo a shift in the isoforms expressed and that atrial myosin light chain 1 increases expression levels [24]. It has also been shown that CaMKII can act to mediate troponin I function [8] and the promoter-region of the atrial myosin light chain-1 gene [25].…”

p21-activated kinase improves cardiac contractility during ischemia-reperfusion concomitant with changes in troponin-T and myosin light chain 2 phosphorylation

“…ProQ Diamond (Invitrogen) stain was used to detect changes in phosphorylation states of myofilament proteins. Myofibrils were prepared from WT and Pak1-KO mouse hearts, and pellets were solubilized in a nonreducing 2ϫ Laemmli buffer (4% SDS, 20% glycerol, 0.004% bromphenol blue, and 0.125 M Tris·HCl, pH 6.8) (12). Twenty five millimolar N-ethylmaleimide (NEM) was added to the standard rigor buffer with Triton X-100, the standard rigor wash buffer, and the 2ϫ Laemmli buffer.…”

“…Myofibrils were prepared from WT and Pak1-KO mouse hearts, and pellets were solubilized in a nonreducing 2ϫ Laemmli buffer (12). Twenty five millimolar NEM was added to the standard rigor buffer with Triton X-100, the standard rigor wash buffer, and the 2ϫ Laemmli buffer.…”