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The myogenic response of cerebral resistance arterial smooth muscle to intraluminal pressure elevation is a key physiological mechanism regulating blood flow to the brain. Rho-associated kinase plays a critical role in the myogenic response by activating Ca2+ sensitization mechanisms: (i) Rho-associated kinase inhibits myosin light chain phosphatase by phosphorylating its targeting subunit myosin phosphatase targeting subunit 1 (at T855), augmenting 20 kDa myosin regulatory light chain (LC20) phosphorylation and force generation; and (ii) Rho-associated kinase stimulates cytoskeletal actin polymerization, enhancing force transmission to the cell membrane. Here, we tested the hypothesis that abnormal Rho-associated kinase-mediated myosin light chain phosphatase regulation underlies the dysfunctional cerebral myogenic response of the Goto-Kakizaki rat model of type 2 diabetes. Basal levels of myogenic tone, LC20, and MYPT1-T855 phosphorylation were elevated and G-actin content was reduced in arteries of pre-diabetic 8–10 weeks Goto-Kakizaki rats with normal serum insulin and glucose levels. Pressure-dependent myogenic constriction, LC20, and myosin phosphatase targeting subunit 1 phosphorylation and actin polymerization were suppressed in both pre-diabetic Goto-Kakizaki and diabetic (18–20 weeks) Goto-Kakizaki rats, whereas RhoA, ROK2, and MYPT1 expression were unaffected. We conclude that abnormal Rho-associated kinase-mediated Ca2+ sensitization contributes to the dysfunctional cerebral myogenic response in the Goto-Kakizaki model of type 2 diabetes.

Our understanding of the molecular events contributing to myogenic control of diameter in cerebral resistance arteries in response to changes in intravascular pressure, a fundamental mechanism regulating blood flow to the brain, is incomplete. Myosin light chain kinase and phosphatase activities are known to be increased and decreased, respectively, to augment phosphorylation of the 20-kDa regulatory light chain subunits (LC20) of myosin II, which permits cross-bridge cycling and force development. Here, we assessed the contribution of dynamic reorganization of the actin cytoskeleton and thin filament regulation to the myogenic response and serotonin-evoked constriction of pressurized rat middle cerebral arteries. Arterial diameter and the levels of phosphorylated LC(20), calponin, caldesmon, cofilin, and HSP27, as well as G-actin content, were determined. A decline in G-actin content was observed following pressurization from 10 mm Hg to between 40 and 120 mm Hg and in three conditions in which myogenic or agonist-evoked constriction occurred in the absence of a detectable change in LC20 phosphorylation. No changes in thin filament protein phosphorylation were evident. Pressurization reduced G-actin content and elevated the levels of cofilin and HSP27 phosphorylation. Inhibitors of Rho-associated kinase and PKC prevented the decline in G-actin; reduced cofilin and HSP27 phosphoprotein content, respectively; and blocked the myogenic response. Furthermore, phosphorylation modulators of HSP27 and cofilin induced significant changes in arterial diameter and G-actin content of myogenically active arteries. Taken together, our findings suggest that dynamic reorganization of the cytoskeleton involving increased actin polymerization in response to Rho-associated kinase and PKC signaling contributes significantly to force generation in myogenic constriction of cerebral resistance arteries.

Key points • Blood flow to our organs is maintained within a defined range to provide an adequate supply of nutrients and remove waste products by contraction and relaxation of smooth muscle cells of resistance arteries and arterioles. • The ability of these cells to contract in response to an increase in intravascular pressure, and to relax following a reduction in pressure (the ‘myogenic response’), is critical for appropriate control of blood flow, but our understanding of its mechanistic basis is incomplete. • Small arteries of skeletal muscles were used to test the hypothesis that myogenic constriction involves two enzymes, Rho-associated kinase and protein kinase C, which evoke vasoconstriction by activating the contractile protein, myosin, and by reorganizing the cytoskeleton. • Knowledge of the mechanisms involved in the myogenic response contributes to understanding of how blood flow is regulated and will help to identify the molecular basis of dysfunctional control of arterial diameter in disease. Abstract The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca2+ entry via voltage-gated Ca2+ channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca2+ sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca2+ sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca2+ sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC20 phosphorylation, and a ROK- and PKC-evoked increase in actin polymerization) contribute to force generation in the myogenic response of skeletal muscle arterioles.

The myogenic response of resistance arterioles and small arteries involving constriction in response to intraluminal pressure elevation and dilation on pressure reduction is fundamental to local blood flow regulation in the microcirculation. Integrins have garnered considerable attention in the context of initiating the myogenic response, but evidence indicative of mechanotransduction by integrin adhesions, for example established changes in tyrosine phosphorylation of key adhesion proteins, has not been obtained to substantiate this interpretation. Here, we evaluated the role of integrin adhesions and associated cellular signaling in the rat cerebral arterial myogenic response using function-blocking antibodies against α5ß1-integrins, pharmacological inhibitors of focal adhesion kinase (FAK) and Src family kinase (SFK), an ultra-high-sensitivity western blotting technique, site-specific phosphoprotein antibodies to quantify adhesion and contractile filament protein phosphorylation, and differential centrifugation to determine G-actin levels in rat cerebral arteries at varied intraluminal pressures. Pressure-dependent increases in the levels of phosphorylation of FAK (FAK-Y397, Y576/Y577), SFK (SFK-Y416; Y527 phosphorylation was reduced), vinculin-Y1065, paxillin-Y118 and phosphoinositide-specific phospholipase C-γ1 (PLCγ1)-Y783 were detected. Treatment with α5-integrin function-blocking antibodies, FAK inhibitor FI-14 or SFK inhibitor SU6656 suppressed the changes in adhesion protein phosphorylation, and prevented pressure-dependent phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) at T855 and 20kDa myosin regulatory light chains (LC20) at S19, as well as actin polymerization that are necessary for myogenic constriction. We conclude that mechanotransduction by integrin adhesions and subsequent cellular signaling play a fundamental role in the cerebral arterial myogenic response, This work was supported by grants from the Canadian Institutes of Health Research (CIHR MOP-97988 and MOP-13505) with no role in experimental design, manuscript writing or submission. OC, AMD and H-LZ were supported by Fellowship Awards from the Spanish Ministry of Education, Kertland Family Fellowship Fund and Andrew Family Professorship, and Alberta Innovates-Health Solutions, respectively. MTP G was supported by a senior scholarship of the Spanish Government (PR2011-0081) and by grant BFU2013-45867-R. MPW is an Alberta Heritage Foundation for Medical Research Scientist and recipient of a Canada Research Chair (Tier 1) in Vascular Smooth Muscle Research.

The myogenic response of resistance arteries to intravascular pressure elevation is a fundamental physiological mechanism of crucial importance for blood pressure regulation and organ-specific control of blood flow. The importance of Ca(2+) entry via voltage-gated Ca(2+) channels leading to phosphorylation of the 20 kDa myosin regulatory light chains (LC20) in the myogenic response is well established. Recent studies, however, have suggested a role for Ca(2+) sensitization via activation of the RhoA/Rho-associated kinase (ROK) pathway in the myogenic response. The possibility that enhanced actin polymerization is also involved in myogenic vasoconstriction has been suggested. Here, we have used pressurized resistance arteries from rat gracilis and cremaster skeletal muscles to assess the contribution to myogenic constriction of Ca(2+) sensitization due to: (1) phosphorylation of the myosin targeting subunit of myosin light chain phosphatase (MYPT1) by ROK; (2) phosphorylation of the 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) by PKC; and (3) dynamic reorganization of the actin cytoskeleton evoked by ROK and PKC. Arterial diameter, MYPT1, CPI-17 and LC20 phosphorylation, and G-actin content were determined at varied intraluminal pressures ± H1152, GF109203X or latrunculin B to suppress ROK, PKC and actin polymerization, respectively. The myogenic response was associated with an increase in MYPT1 and LC20 phosphorylation that was blocked by H1152. No change in phospho-CPI-17 content was detected although the PKC inhibitor, GF109203X, suppressed myogenic constriction. Basal LC20 phosphorylation at 10 mmHg was high at ∼40%, increased to a maximal level of ∼55% at 80 mmHg, and exhibited no additional change on further pressurization to 120 and 140 mmHg. Myogenic constriction at 80 mmHg was associated with a decline in G-actin content by ∼65% that was blocked by inhibition of ROK or PKC. Taken together, our findings indicate that two mechanisms of Ca(2+) sensitization (ROK-mediated phosphorylation of MYPT1-T855 with augmentation of LC20 phosphorylation, and a ROK- and PKC-evoked increase in actin polymerization) contribute to force generation in the myogenic response of skeletal muscle arterioles.

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K + current (Ito) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of Ito, even at low [Ca2+]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of Ito. Because channels underlying Ito are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvα-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca2+ was used in the pipette or when intracellular [Ca2+] ([Ca2+]i) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca2+- dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca2+ increase, and the association was inhibited by KN93. These results suggest that both channels underlying Ito are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca 2+]i increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca2+-dependent modulation by this enzyme of the associated Kv4.2 channels. Copyright © 2006 the American Physiological Society., This work was supported by Instituto de Salud Carlos III Grants G03/011 (Red Respira), G03/045 (Red Heracles), and PI041044 to J. R. López-López, Ministerio de Educación (MEC) Grant BFU2004-05551 to M. T. Pérez-García and MEC Grant SAF2005-00906 to O. Casis. O. Colinas is a fellow of the Spanish Ministerio de Ciencia y Tecnología, and M. Gallego and R. Setién are fellows of the University of Basque Country.