Chronic loss of inhibitor-1 diminishes cardiac RyR2 phosphorylation despite exaggerated CaMKII activity

Inhibitor-1 (I-1) modulates protein phosphatase 1 (PP1) activity and thereby counteracts the phosphorylation by kinases. I-1 is downregulated and deactivated in failing hearts, but whether its role is beneficial or detrimental remains controversial, and opposing therapeutic strategies have been proposed. Overactivity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) with hyperphosphorylation of ryanodine receptors (RyR2) at the CaMKII-site is recognized to be central for heart failure and arrhythmias. Using an I-1-deficient mouse line as well as transfected cell lines, we investigated the effects of acute and chronic modulation of I-1 on CaMKII activity and RyR2 phosphorylation. We demonstrate that I-1 acutely modulates CaMKII by regulating PP1 activity. However, while ablation of I-1 should thus limit CaMKII-activation, we unexpectedly found exaggerated CaMKII-activation under beta-adrenergic stress upon chronic loss of I-1 in knockout mice. We unraveled that this is due to chronic upregulation of the exchange protein activated by cAMP (EPAC) leading to augmented CaMKII activation, and using computational modeling validated that an increase in EPAC expression can indeed explain our experimental findings. Interestingly, at the level of RyR2, the increase in PP1 activity more than outweighed the increase in CaMKII activity, resulting in reduced RyR phosphorylation at Ser-2814. Exaggerated CaMKII activation due to counterregulatory mechanisms upon loss of I-1 is an important caveat with respect to suggested therapeutic I-1-inhibition, as CaMKII overactivity has been heavily implicated in several cardiac pathologies

CaM kinase II regulates cardiac hemoglobin expression through histone phosphorylation upon sympathetic activation

Sympathetic activation of beta-adrenoreceptors (beta-AR) represents a hallmark in the development of heart failure (HF). However, little is known about the underlying mechanisms of gene regulation. In human ventricular myocardium from patients with end-stage HF, we found high levels of phosphorylated histone 3 at serine-28 (H3S28p). H3S28p was increased by inhibition of the catecholamine-sensitive protein phosphatase 1 and decreased by beta-blocker pretreatment. By a series of in vitro and in vivo experiments, we show that the beta-AR downstream protein kinase CaM kinase II (CaMKII) directly binds and phosphorylates H3S28. Whereas, in CaMKII-deficient myocytes, acute catecholaminergic stimulation resulted in some degree of H3S28p, sustained catecholaminergic stimulation almost entirely failed to induce H3S28p. Genome-wide analysis of CaMKII-mediated H3S28p in response to chronic beta-AR stress by chromatin immunoprecipitation followed by massive genomic sequencing led to the identification of CaMKII-dependent H3S28p target genes. Forty percent of differentially H3S28p-enriched genomic regions were associated with differential, mostly increased expression of the nearest genes, pointing to CaMKII-dependent H3S28p as an activating histone mark. Remarkably, the adult hemoglobin genes showed an H3S28p enrichment close to their transcriptional start or end sites, which was associated with increased messenger RNA and protein expression. In summary, we demonstrate that chronic beta-AR activation leads to CaMKII-mediated H3S28p in cardiomyocytes. Thus, H3S28p-dependent changes may play an unexpected role for cardiac hemoglobin regulation in the context of sympathetic activation. These data also imply that CaMKII may be a yet unrecognized stress-responsive regulator of hematopoesis

The poorly membrane permeable antipsychotic drugs amisulpride and sulpiride are substrates of the organic cation transporters from the SLC22 family.

Variations in influx transport at the blood-brain barrier might affect the concentration of psychotropic drugs at their site of action and as a consequence might alter therapy response. Furthermore, influx transporters in organs such as the gut, liver and kidney may influence absorption, distribution, and elimination. Here, we analyzed 30 commonly used psychotropic drugs using a parallel artificial membrane permeability assay. Amisulpride and sulpiride showed the lowest membrane permeability (P e -6 cm/s) and will require influx transport to penetrate the blood-brain barrier and other physiological barriers. We then studied the uptake of amisulpride and sulpiride by the organic cation transporters of the SLC22 family OCT1, OCT2, OCT3, OCTN1, and OCTN2 Amisulpride was found to be transported by all five transporters studied. In contrast, sulpiride was only transported by OCT1 and OCT2. OCT1 showed the highest transport ability both for amisulpride (CLint = 1.9 ml/min/mg protein) and sulpiride (CLint = 4.2 ml/min/mg protein) and polymorphisms in OCT1 significantly reduced the uptake of both drugs. Furthermore, we observed carrier-mediated uptake that was inhibitable by known OCT inhibitors in the immortalized human brain microvascular endothelial cell line hCMEC/D3. In conclusion, this study demonstrates that amisulpride and sulpiride are substrates of organic cation transporters of the SLC22 family. SLC22 transporters may play an important role in the distribution of amisulpride and sulpiride, including their ability to penetrate the blood-brain barrier