A Low-Dose β1-Blocker in Combination with Milrinone Improves Intracellular Ca2+ Handling in Failing Cardiomyocytes by Inhibition of Milrinone-Induced Diastolic Ca2+ Leakage from the Sarcoplasmic Reticulum

Objectives

The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.

Background

The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca²⁺ handling in heart failure remains unclear.

Methods

We investigated the effect of milrinone plus landiolol on intracellular Ca²⁺ transient (CaT), cell shortening (CS), the frequency of diastolic Ca²⁺ sparks (CaSF), and sarcoplasmic reticulum Ca²⁺ concentration ({Ca²⁺}_SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).

Results

In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca²⁺}_SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca²⁺}_SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.

Conclusion

A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca²⁺ leak.     

Cytochrome oxidase of an acidophilic iron-oxidizing bacterium, Thiobacillus ferrooxidans, functions at pH 3.5

Cytochrome oxidase of Thiobacillus ferrooxidans was partially purified. The oxidase preparation had haems a and c, and oxidized ferrocytochrome c-552 of the bacterium. The optimal pH of the reaction was 3.5. The enzyme also oxidized the reduced form of rusticyanin, a copper protein of the bacterium. Our results indicate that the reduction of molecular oxygen by this enzyme may occur in the periplasm.     

ATP hydrolysis coupled to microtubule sliding in sea-urchin sperm flagella

Using sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1,000 microM) at 0-20 degrees C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes.     

Analysis of responses to endothelins in isolated porcine blood vessels by using a novel endothelin antagonist, BQ-153.

We examined the effects of a novel ETA-selective endothelin (ET) antagonist, BQ-153, on vascular responses to ET-1 and ET-3 in isolated porcine coronary and pulmonary blood vessels, to clarify the roles of ET receptor subtypes in the regulation of vascular smooth muscle tension. With endothelium-denuded vascular tissues, the concentration-contraction curve (CCC) for ET-1 appeared as a single sigmoidal shape for all types of tissue. The CCC for ET-1 was antagonized by BQ-153 (2 and 10 microM) in all tissues, but part of the contraction was resistant. The CCC for ET-3 usually consisted of two different phases with higher (first phase) and lower (second phase) sensitivities to the peptide. Only the second phase of CCC for ET-3 was completely inhibited by BQ-153 (2 microM) in all tissues, while the first phase was resistant. The BQ-153-resistant contractile phases of ET-1 and ET-3-induced vasoconstriction appeared to have similar sensitivity in all tissues, and the contractile activity varied with each type of tissue. With endothelium-intact materials, the potencies of ET-1 and ET-3 for endothelium-dependent vasorelaxation in pulmonary artery were almost equivalent. BQ-153 (10 microM) did not inhibit ET-induced vasorelaxation. These results indicate that ET-induced vasoconstriction is mediated not only through ETA but also through ETnonA (probably ETB), and that the relative proportions of the ET-receptor subtypes mediating contractions vary in different vascular areas. In addition, results showed that ET-induced endothelium-dependent vasorelaxation is mediated through ETB.     

Antimicrobial activity and secondary structure of a novel peptide derived from ovalbumin

A novel antimicrobial peptide derived from ovalbumin has been discovered. First, the peptide fragment RKIKVYLPRMK (TK9.1) was identified based on computerized predictions of the secondary structure of peptides in a protein data bank. Using HeliQuest, the sequence was developed into RKIKRYLRRMI (TK9.1.3), which was synthesized using Fmoc‐solid phase peptide synthesis, and found to have strongly antimicrobial activity against Gram‐positive and Gram‐negative bacteria, and fungi but not cytotoxic to HeLa cells and hemolysis in mouse red blood cells. Although ovalbumin itself does not have an antibacterial activity, our results suggest that it may supply the organisms that consume it with antimicrobial peptides, in support of their immunodefence.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.