Hypercontractile cardiac phenotype in mice overexpressing the regulatory subunit PR72 of protein phosphatase 2A.

Background: The activity, localization, and substrate specificity of the protein phosphatase 2A (PP2A) heterotrimer are controlled by various regulatory B subunits. PR72 belongs to the B'' gene family and has been shown to be upregulated in human heart failure. However, little is known about the functions of PR72 in the myocardium. Methods: To address this issue, we generated a transgenic mouse model with heart-specific overexpression of PP2A-PR72. Biochemical and physiological methods were used to determine contractility, Ca2+ cycling parameters, and protein phosphorylation. Results: A 2.5-fold increase in PR72 expression resulted in moderate cardiac hypertrophy. Maximal ventricular pressure was increased in catheterized transgenic mice (TG) compared to wild-type (WT) littermates. This was accompanied by an increased shortening of sarcomere length and faster relaxation at the single-cell level in TG. In parallel with these findings, the peak amplitude of Ca2+ transients was increased, and the decay in intracellular Ca2+ levels was shortened in TG compared to WT. The changes in Ca2+ cycling in TG were also evident from an increase in the full duration and width at half maximum of Ca2+ sparks. Consistent with the contractile data, phosphorylation of phospholamban at threonine-17 was higher in TG hearts. The lower expression of the Na+/Ca2+ exchanger may also contribute to the hypercontractile state in transgenic myocardium. Conclusion: Our results suggest that PP2A-PR72 plays an important role in regulating cardiac contractile function and Ca2+ cycling, indicating that the upregulation of PR72 in heart failure is an attempt to compensate functionally.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

Pharmacokinetics of Eleven Kratom Alkaloids Following an Oral Dose of Either Traditional or Commercial Kratom Products in Rats.

Kratom, Mitragyna speciosa Korth., is being widely consumed in the United States for pain management and the reduction of opioid withdrawal symptoms. The central nervous system (CNS) active alkaloids of kratom, including mitragynine, 7-hydroxymitragynine, and numerous additional compounds, are believed to derive their effects through opioid receptor activity. There is no literature describing the systemic exposure of many of these alkaloids after the consumption of kratom. Therefore, we have developed and validated a bioanalytical method for the simultaneous quantitation of 11 kratom alkaloids (mitragynine, 7-hydroxymitragynine, corynantheidine, speciogynine, speciociliatine, paynantheine, corynoxine, corynoxine-B, mitraphylline, ajmalicine, and isospeciofoline) in rat plasma. The validated method was used to analyze oral pharmacokinetic study samples of lyophilized kratom tea (LKT) and a marketed product, OPMS liquid shot, in rats. Among the 11 alkaloids, only mitragynine, 7-hydroxymitragynine, speciociliatine, and corynantheidine showed systemic exposure 8 h postdose, and the dose-normalized systemic exposure of these four alkaloids was higher (1.6-2.4-fold) following the administration of the commercial OPMS liquid. Paynantheine and speciogynine levels were quantifiable up to 1 h postdose, whereas none of the other alkaloids were detected. In summary, the method was successfully applied to quantify the exposure of individual kratom alkaloids after an oral dose of traditional or commercial products. This information will contribute to understanding the role of each alkaloid in the overall pharmacology of kratom and elucidating the pharmacokinetic differences between traditional and commercial kratom products.