Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.

Ca2+ signaling influences nearly every aspect of cellular life. Transient receptor potential (TRP) ion channels have emerged as cellular sensors for thermal, chemical and mechanical stimuli and are major contributors to Ca2+ signaling, playing an important role in diverse physiological and pathological processes. Notably, TRP ion channels are also one of the major downstream targets of Ca2+ signaling initiated either from TRP channels themselves or from various other sources, such as G-protein coupled receptors, giving rise to feedback regulation. TRP channels therefore function like integrators of Ca2+ signaling. A growing body of research has demonstrated different modes of Ca2+-dependent regulation of TRP ion channels and the underlying mechanisms. However, the precise actions of Ca2+ in the modulation of TRP ion channels remain elusive. Advances in Ca2+ regulation of TRP channels are critical to our understanding of the diversified functions of TRP channels and complex Ca2+ signaling.

PKD2 (polycystin-2, TRPP1), a TRP polycystin channel, is expressed in endothelial cells (ECs), but its physiological functions in this cell type are unclear. Here, we generated inducible, EC-specific Pkd2 knockout mice to examine vascular functions of PKD2. Data show that a broad range of intravascular flow rates stimulate EC PKD2 channels, producing vasodilation. Flow-mediated PKD2 channel activation leads to calcium influx that activates SK/IK channels and eNOS serine 1176 phosphorylation in ECs. These signaling mechanisms produce arterial hyperpolarization and vasodilation. In contrast, EC PKD2 channels do not contribute to acetylcholine-induced vasodilation, suggesting stimulus-specific function. EC-specific PKD2 knockout elevated blood pressure in mice without altering cardiac function or kidney anatomy. These data demonstrate that flow stimulates PKD2 channels in ECs, leading to SK/IK channel and eNOS activation, hyperpolarization, vasodilation and a reduction in systemic blood pressure. Thus, PKD2 channels are a major component of functional flow sensing in the vasculature.

The antidiabetic drug canagliflozin is reported to possess several cardioprotective effects. However, no studies have investigated protective effects of canagliflozin in isoprenaline (ISO)-induced cardiac oxidative damage-a model mimicking sympathetic nervous system (SNS) overstimulation-evoked cardiac injuries in humans. Therefore, we investigated protective effects of canagliflozin in ISOinduced cardiac oxidative stress, and their underlying molecular mechanisms in Long-Evans rat heart and in HL-1 cardiomyocyte cell line. Our data showed that ISO administration inflicts pro-oxidative changes in heart by stimulating production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In contrast, canagliflozin treatment in ISO rats not only preserves endogenous antioxidants but also reduces cardiac oxidative stress markers, fibrosis and apoptosis. Our Western blotting and messenger RNA expression data demonstrated that canagliflozin augments antioxidant and anti-inflammatory signaling involving AMP-activated protein kinase (AMPK), Akt, endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). In addition, canagliflozin treatment attenuates pro-oxidative, pro-inflammatory and proapoptotic signaling mediated by inducible nitric oxide synthase (iNOS), transforming growth factor beta (TGF-β), NADPH oxidase isoform 4 (Nox4), caspase-3 and Bax. Consistently, canagliflozin treatment improves heart function marker in ISO-treated rats. In summary, we demonstrated that canagliflozin produces cardioprotective actions by promoting multiple antioxidant and antiinflammatory signaling. Canagliflozin, a sodium-glucose cotransporter-2 (SGLT2) inhibitor, belongs to a new class of antidiabetic drugs prescribed for the management of type 2 diabetes mellitus (T2DM) 1. Accumulating evidence suggests that canagliflozin exhibits a range of cardiovascular effects that are independent of glucose lowering. According to recent preclinical and clinical data, canagliflozin treatment significantly reduced risk of cardiovascular death, myocardial infarction (MI), stroke and hospitalization due to heart failure in both diabetic and non-diabetic subjects 2-5. Proposed mechanisms for cardiovascular benefits of canagliflozin include improvement of cardiac metabolism and diastolic function, reduction of vascular stiffness, and an overall reduction of blood pressure 5-9. A growing body of evidence suggests that canagliflozin possess antioxidant and anti-inflammatory actions in various cellbased and animal models 10-14. Canagliflozin was shown to reduce vascular inflammation and atherosclerosis by

Diabetes is a leading cause of chronic kidney disease, and the high prevalence of sympathetic nervous system (SNS) hyperactivity in diabetic patients makes them further susceptible to SNS-mediated oxidative stress and accelerated kidney damage. Here, we investigated if canagliflozin can reverse isoprenaline (ISO)-induced renal oxidative damage in rats, a model that mimics SNS overstimulationinduced organ injuries in humans. We found that ISO administration elevates renal oxidative stress markers including malondialdehyde (MDA), advanced protein oxidation product (APOP), myeloperoxidase (MPO) and nitric oxide (NO), while depleting levels of endogenous antioxidants such as catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH). Strikingly, canagliflozin treatment of ISO-treated rats not only prevents elevation of oxidative stress markers but also rescues levels of depleted antioxidants. Our results also show that canagliflozin stimulates antioxidant/antiinflammatory signaling pathways involving AMP-activated protein kinase (AMPK), Akt and eNOS, and inhibits iNOS and NADPH oxidase isoform 4 (NOX4), all of which are associated with oxidative stress and inflammation. Further, canagliflozin prevents ISO-induced apoptosis of kidney cells by inhibiting Bax protein upregulation and caspase-3 activation. Histological examination of kidney sections reveal that canagliflozin attenuates ISO-mediated increases in inflammatory cell infiltration, collagen deposition and fibrosis. Finally, consistent with these findings, canagliflozin treatment improves kidney function in ISO-treated rats, suggesting that the antioxidant effects may be clinically translatable. Diabetic kidney disease is a major risk factor for the development of chronic kidney disease affecting approximately 40% of global diabetic population 1. Diabetic kidney disease is associated with vascular inflammation, loss of renal vascular integrity and hypertension, leading to a progressive loss of renovascular function and renal failure 1. Importantly, there is a high prevalence of sympathetic nervous system (SNS) hyperactivity in diabetic patients associated with autonomic neuropathy and concomitant vagal impairment, making diabetic patients twice as likely to develop hypertension 2. Diabetic patients are also highly susceptible to chronic kidney disease due to renal oxidative damage and inflammation 2. High SNS drive stimulates β1 adrenergic receptors (β1-AR) in juxtaglomerular cells, increasing renin secretion and subsequent activation of the renin-angiotensin-aldosterone system (RAAS). RAAS creates a feed-forward mechanism that accelerates renovascular dysfunction and kidney

PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specificPkd2knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca2+influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca2+influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2−mediated Na+currents (INa) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of INaactivation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.

Polycystin-1 (PC-1, PKD1), a receptor-like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms and physiological functions are poorly understood. Here, by studying tamoxifen-inducible, endothelial cell (EC)-specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC-1-mediated, Ca2+-dependent cation currents in ECs. EC-specific PC-1 knockout attenuates flow-mediated arterial hyperpolarization and vasodilation. PC-1-dependent vasodilation occurs over the entire functional shear stress range and via the activation of endothelial nitric oxide synthase (eNOS) and intermediate (IK)- and small (SK)-conductance Ca2+-activated K+ channels. EC-specific PC-1 knockout increases systemic blood pressure without altering kidney anatomy. PC-1 coimmunoprecipitates with polycystin-2 (PC-2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC-1 or PC-2 (Pkd2 ecKO mice) abolishes surface clusters of both PC-1 and PC-2 in ECs. Single knockout of PC-1 or PC-2 or double knockout of PC-1 and PC-2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow-mediated vasodilation. Flow stimulates non-selective cation currents in ECs that are similarly inhibited by either PC-1 or PC-2 knockout or by interference peptides corresponding to the C-terminus coiled-coil domains present in PC-1 or PC-2. In summary, we show that PC-1 regulates arterial contractility through the formation of an interdependent signaling complex with PC-2 in endothelial cells. Flow stimulates PC-1/PC-2 clusters in the EC plasma membrane, leading to eNOS, IK channel and SK channel activation, vasodilation and a reduction in blood pressure.

Membrane potential is a principal regulator of arterial contractility. Arterial smooth muscle cells express several different types of ion channel that control membrane potential, including K V channels. K V channel activation leads to membrane hyperpolariza- | INTRODUCTIONMembrane potential is a key regulator of arterial contractility. 1,2Arterial smooth muscle cells express several different types of ion channel that control membrane potential, including K V channels. | EXPRESSION AND DISTRIBUTION OF K V CHANNELS IN ARTERIAL SMOOTH MUSCLE CELLSK V channels are tetramers of four pore-forming α subunits that can associate with accessory β-subunits.