Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.
Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts.
Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) β stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.
Summary Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1−/−). Orai1−/− mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1−/− mice were examined. Orai1-mediated SOCE was completely abolished in Orai1−/− osteoclast precursor cells and osteoclastogenesis in vitro from Orai1−/− mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1−/− osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1−/− mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1−/− osteoblast precursor cells and osteoblastogenesis in vitro from Orai1−/− stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.
Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission-associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding-dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1-filamin A interaction by cilnidipine suppressed mitochondrial hyperfission-associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development. , Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence.
The angiotensin (Ang) type 1 receptor (AT1R) promotes functional and structural integrity of the arterial wall to contribute to vascular homeostasis, but this receptor also promotes hypertension. In our investigation of how Ang II signals are converted by the AT1R from physiological to pathological outputs, we found that the purinergic P2Y6 receptor (P2Y6R), an inflammation-inducible G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR), promoted Ang II-induced hypertension in mice. In mice, deletion of P2Y6R attenuated Ang II-induced increase in blood pressure, vascular remodeling, oxidative stress, and endothelial dysfunction. AT1R and P2Y6R formed stable heterodimers, which enhanced G protein-dependent vascular hypertrophy but reduced β-arrestin-dependent AT1R internalization. Pharmacological disruption of AT1R-P2Y6R heterodimers by the P2Y6R antagonist MRS2578 suppressed Ang II-induced hypertension in mice. Furthermore, P2Y6R abundance increased with age in vascular smooth muscle cells. The increased abundance of P2Y6R converted AT1R-stimulated signaling in vascular smooth muscle cells from β-arrestin-dependent proliferation to G protein-dependent hypertrophy. These results suggest that increased formation of AT1R-P2Y6R heterodimers with age may increase the likelihood of hypertension induced by Ang II.
Purinergic signaling, mediated mainly by G protein-coupled P2Y receptors (P2YRs), is now attracting attention as a new therapeutic target for preventing or treating cardiovascular diseases. Observations using mice with genetically modified P2YRs and/or treated with a pharmacological P2YR inhibitor have helped us understand the physiological and pathological significance of P2YRs in the cardiovascular system. P2YR-mediated biological functions are predominantly activated by mononucleotides released from non-adrenergic, non-cholinergic nerve endings or non-secretory tissues in response to physical stress or cell injury, though recent studies have suggested the occurrence of ligand-independent P2YR function through receptor-receptor interactions (oligomerization) in several biological processes. In this review, we introduce the functions of P2YRs and possible dimerization with G protein-coupled receptors (GPCRs) in the cardiovascular system. We focus especially on the crosstalk between uridine nucleotide-responsive P2YR and angiotensin (Ang) II type1 receptor (AT1R) signaling, and introduce our recent finding that the P2YR antagonist MRS2578 interrupts heterodimerization between P2YR and AT1R, thereby reducing the risk of AT1R-stimulated hypertension in mice. These results strongly suggest that targeting P2YR oligomerization could be an effective new strategy to reduce the risk of cardiovascular diseases.
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