SUMMARY Cardiac contractility is mediated by variable flux in intracellular calcium (Ca2+), thought to be integrated into mitochondria via the mitochondrial calcium uniporter (MCU) channel to match energetic demand. Here we examine a conditional, cardiomyocyte-specific, mutant mouse lacking Mcu, the pore-forming subunit of the MCU channel, in adulthood. Mcu−/− mice display no overt baseline phenotype and are protected against mCa2+-overload in an in vivo myocardial ischemia-reperfusion injury model by preventing the activation of the mitochondrial permeability transition pore, decreasing infarct size, and preserving cardiac function. In addition, we find that Mcu−/− mice lack contractile responsiveness to acute β-adrenergic receptor stimulation and in parallel are unable to activate mitochondrial dehydrogenases and display reduced bioenergetic reserve capacity. These results support the hypothesis that MCU may be dispensable for homeostatic cardiac function but required to modulate Ca2+-dependent metabolism during acute stress.

TRPM2 is a Ca 2؉ -permeable channel that is activated by oxidative stress and confers susceptibility to cell death. Here, an isoform of TRPM2 was identified in normal human bone marrow that consists of the TRPM2 N terminus and the first two predicted transmembrane domains. Because of alternative splicing, a stop codon (TAG) is located at the splice junction between exons 16 and 17, resulting in deletion of the four C-terminal transmembrane domains, the putative calcium-permeable pore region, and the entire C terminus. This splice variant was found in other hematopoietic cells including human burst forming unit-erythroid-derived erythroblasts and TF-1 erythroleukemia cells. Endogenous expression of both the short form of TRPM2 (TRPM2-S) and the full length (TRPM2-L) was determined by reverse transcriptase-PCR, and localization of endogenous TRPM2 to the plasma membrane was demonstrated by confocal microscopy.

G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. Herein we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine-Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

SUMMARY Mitochondrial Ca2+ Uniporter (MCU)-dependent mitochondrial Ca2+ uptake is the primary mechanism for increasing matrix Ca2+ in most cell types. However, a limited understanding of the MCU complex assembly impedes the comprehension of the precise mechanisms underlying MCU activity. Here we report that mouse cardiomyocytes and endothelial cells lacking MCU regulator 1, MCUR1, have severely impaired [Ca2+]m uptake and IMCU current. MCUR1 binds to MCU and EMRE and function as a scaffold factor. Our protein binding analyses identified the minimal, highly conserved regions of coiled-coil domain of both MCU and MCUR1 that are necessary for heterooligomeric complex formation. Loss of MCUR1 perturbed MCU heterooligomeric complex and functions as a scaffold factor for the assembly of MCU complex. Vascular endothelial deletion of MCU and MCUR1 impaired mitochondrial bioenergetics, cell proliferation and migration but elicited autophagy. These studies establish the existence of a MCU complex which assembles at the mitochondrial integral membrane and regulates Ca2+-dependent mitochondrial metabolism.

PKA-dependent, alpha 1-specific NKA activation may be mediated through phosphorylation of the accessory protein PLM, rather than direct alpha1 subunit phosphorylation.