Special topics issue of microcirculation: ion channels and pulmonary vascular function

Unique features of the pulmonary circulation impact its function in health and disease, not the least of which is the existence of developmentally distinct, functionally heterogeneous extra-alveolar and septal capillary networks. The impact of ion channel expression and regulation in lung vascular smooth muscle or endothelium in these vascular compartments provides a focus for this special topics issue. Reviews and original contributions from experts in the field discuss two broad groups of ion channels, drawing on studies utilizing biophysical and molecular approaches in heterologous expression systems, in vitro approaches in pulmonary vascular smooth muscle and endothelial cells, and physiologic studies in animal models of chronic pulmonary hypertension. First, channels involved in membrane depolarization and related alterations in vascular tone, and shear sensing or exocytosis by endothelium are discussed: voltage-gated potassium channels, ATP-regulated potassium channels and L- and T- type voltage-gated calcium channels. The second series of reviews discusses the role of calcium influx pathways provided by transient receptor potential channels in regulation of pulmonary vascular tone or vascular remodeling, and endothelial barrier function. Understanding the role of ion channels in pulmonary vascular pathophysiology may be critical to development of new therapeutic strategies.     

New expression profiles of voltage-gated ion channels in arteries exposed to high blood pressure

The diameters of small arteries and arterioles are tightly regulated by the dynamic interaction between Ca(2+) and K(+) channels in the vascular smooth muscle cells. Calcium influx through voltage-gated Ca(2+) channels induces vasoconstriction, whereas the opening of K(+) channels mediates hyperpolarization, inactivation of voltage-gated Ca(2+) channels, and vasodilation. Three types of voltage-sensitive ion channels have been highly implicated in the regulation of resting vascular tone. These include the L-type Ca(2+) (Ca(L)) channels, voltage-gated K(+) (K(V)) channels, and high-conductance voltage- and Ca(2+)-sensitive K(+) (BK(Ca)) channels. Recently, abnormal expression profiles of these ion channels have been identified as part of the pathogenesis of arterial hypertension and other vasospastic diseases. An increasing number of studies suggest that high blood pressure may trigger cellular signaling cascades that dynamically alter the expression profile of arterial ion channels to further modify vascular tone. This article will briefly review the properties of Ca(L), K(V), and BK(Ca) channels, present evidence that their expression profile is altered during systemic hypertension, and suggest potential mechanisms by which the signal of elevated blood pressure may result in altered ion channel expression. A final section will discuss emerging concepts and opportunities for the development of new vasoactive drugs, which may rely on targeting disease-specific changes in ion channel expression as a mechanism to lower vascular tone during hypertensive diseases.     

Contribution of active membrane processes to conducted hyperpolarization in arterioles of hamster cheek pouch

OBJECTIVE: Conduction of vasodilation triggered by acetylcholine (ACh) in arteriolar networks reflects hyperpolarization and its spread from cell to cell along the vessel wall. The amplitude and distance of the vasomotor response appear greater than can be explained by simple passive decay of the electrical signal. The authors tested the hypothesis that the conduction of hyperpolarization involves active membrane processes as the signal travels along the arteriolar wall. METHODS: Intracellular recordings of membrane potential were made from either the smooth muscle or endothelial cell layer of arterioles of the hamster cheek pouch in vivo. Acetylcholine was delivered onto an arteriole using microiontophoresis at defined distances from the recording site, and transient hyperpolarizations were recorded. The area enclosed by the transients (voltage x time integral below baseline) was measured and compared to the area expected if the hyperpolarization was spreading passively. RESULTS: In 11 of 15 recordings from smooth muscle and 5 of 7 from endothelium, areas of the transients were larger than expected for purely passive spread of the electrical signal. CONCLUSIONS: Conduction of hyperpolarization is enhanced by active membrane processes as the signal travels along the arteriolar wall. Signal augmentation will promote blood flow to tissue regions from which hyperpolarization of arterioles originates.     

Aging-induced adaptations of microvascular reactivity

Control of blood flow to skeletal muscle depends on the vasomotor tone present in the resistance vasculature. Although muscle blood flow has been shown to decline with advancing age, our knowledge of how alterations of reactivity of the resistance vasculature contribute to reduced delivery or altered distribution of blood in the aged is limited. Recent work has demonstrated that age alters the reactivity of resistance arteries and arterioles from skeletal muscle, and that impairment of both vasodilator and vasoconstrictor responses occurs with advancing age. The alterations in cellular mechanisms that contribute to age-related impairment of vasoreactive responses encompass both the vascular endothelium and smooth muscle, and differ in muscles of varying function and fiber type. Current research suggests that some degree of age-induced endothelial dysfunction occurs in resistance arteries and arterioles from most skeletal muscle; however, the severity of endothelial impairment appears greater in resistance arteries and arterioles from highly oxidative locomotory muscles. Age-related impairment of vasoconstrictor responses to metabolites and endogenous constrictor agents has also been documented. These age-related reductions in vasoreactivity that occur in the skeletal muscle resistance vasculature may contribute to inadequate delivery or distribution of blood flow during exercise and ultimately be a factor in loss of exercise capacity that occurs with advancing age.     

Hypoxia inhibits contraction but not calcium channel currents or changes in intracellular calcium in arteriolar muscle cells

OBJECTIVE: We tested the hypothesis that hypoxia inhibits currents through L-type Ca(2+) channels and inhibits norepinephrine-induced rises in intracellular Ca(2+) in cremasteric arteriolar muscle cells, thus accounting for the inhibitory effect of hypoxia on norepinephrine-induced contraction of these cells. METHODS: Single smooth muscle cells were enzymatically isolated from second-order and third-order arterioles from hamster cremaster muscles. The effects of hypoxia (partial pressure of oxygen: 10-15 mm Hg) were examined on Ba(2+) (10 mM) currents through L-type Ca(2+) channels by use of the perforated patch clamp technique. Also, the effect of hypoxia on norepinephrine-induced calcium changes was studied using Fura 2 microfluorimetry. RESULTS: Hypoxia inhibited the norepinephrine-induced (10 microM) contraction of single arteriolar muscle cells by 32.9 +/- 5.6% (mean +/- SE, n = 4). However, hypoxia had no significant effect on whole-cell currents through L-type Ca(2+) channels: the peak current densities measured at +20 mV were -3.83 +/- 0.40 pA/pF before hypoxia and -3.97 +/- 0.36 pA/pF during hypoxia (n = 15; p > 0.05). In addition, hypoxia did not inhibit Ca(2+) transients in arteriolar muscle cells elicited by 10 microM norepinephrine. Instead, hypoxia increased basal Ca(2+) (13.8 +/- 3.2%) and augmented peak Ca(2+) levels (29.4 +/- 7.3%) and steady-state Ca(2+) levels (15.2 +/- 5.4%) elicited by 10 microM norepinephrine (n = 21; p < 0.05). CONCLUSIONS: These data indicate that hypoxia inhibits norepinephrine-induced contraction of single cremasteric arteriolar muscle cells by a mechanism that involves neither L-type Ca(2+) channels nor norepinephrine-induced Ca(2+) mobilization. Instead, our findings suggest that hypoxia must inhibit norepinephrine-induced contraction by affecting a component of the signaling pathway that lies downstream from the increases in Ca(2+) produced by this neurotransmitter.     

The microcirculation of skeletal muscle in aging

Microvascular structure and function are key aspects of tissue and organ health. At approximately 40% of total body mass, skeletal muscle contains more microvessels than any other organ system in the body. Moreover, skeletal muscle is the most dynamic tissue in the body with the capacity to increase blood flow and metabolic rate 30- to 50- fold. Aging is associated with decrements in microvascular function and exercise tolerance that are poorly understood. Here, experts in their respective fields of microvascular structure and function are brought together to review the current state of knowledge regarding microvascular adaptations to aging. Reviews are drawn from human and animal studies and focus on age-related changes in sympathetic nervous system control of microvessels, capillary hemodynamics and oxygen pressure, microvascular network structure and functional integration, microvascular reactivity, whole muscle perfusion, and cellular contacts and inflammation.     

5-HT7 receptors mediate dilation of rat cremaster muscle arterioles in vivo

Objective

Serotonin (5-HT) infusion in vivo causes hypotension and a fall in total peripheral resistance. However, the vascular segment and the receptors that mediate this response remain in question. We hypothesized that 5-HT₇ receptors mediate arteriolar dilation to 5-HT in skeletal muscle microcirculation.

Methods

Cremaster muscles of isoflurane-anesthetized male Sprague-Dawley rats were prepared for in vivo microscopy of third- and fourth-order arterioles and superfused with physiological salt solution at 34°C. Quantitative real-time PCR (RT-PCR) was applied to pooled samples of first- to third-order cremaster arterioles (2–4 rats/sample) to evaluate 5-HT₇ receptor expression.

Results

Topical 5-HT (1–10 nmols) or the 5-HT_1/7 receptor agonist, 5-carboxamidotryptamine (10–30 nM), dilated third- and fourth-order arterioles, responses that were abolished by 1 μM SB269970, a selective 5-HT₇ receptor antagonist. In contrast, dilation induced by the muscarinic agonist, methacholine (100 nmols) was not inhibited by SB269970. Serotonin (10 nmols) failed to dilate cremaster arterioles in 5-HT₇ receptor knockout rats whereas arterioles in wild-type litter mates dilated to 1 nmol 5-HT, a response blocked by 1 μM SB269970. Quantitative RT-PCR revealed that cremaster arterioles expressed mRNA for 5-HT₇ receptors.

Conclusions

5-HT₇ receptors mediate dilation of small arterioles in skeletal muscle and likely contribute to 5-HT-induced hypotension, in vivo.     

Responses of the skin microcirculation to acetylcholine and sodium nitroprusside in patients with NIDDM

Summary The mechanisms involved in the pathogenesis of microangiopathy occurring in non-insulin-dependent diabetes mellitus (NIDDM) are unclear. In the present study, blood flow responses to the vasodilators acetylcholine (which acts via the endothelium) and sodium nitroprusside (a smooth muscle relaxant) were evaluated in this patient group. In 14 male patients with NIDDM, treated with either diet alone (n=6) or diet plus insulin, (mean age 59 years) and 14 age-pair-matched control subjects, forearm skin perfusion following multiple doses of iontophoretically applied 1% acetylcholine and 0.01% sodium nitroprusside was recorded by laser Doppler perfusion imaging. Basal skin blood flow was not significantly different in the diabetic group compared with the control group. The following results are expressed as drug-minus-vehicle response. Acetylcholine significantly increased forearm skin perfusion (p<0.001, analysis of variance) in all subjects, but the vasodilatation was attenuated in the patient group compared with control subjects (0.86±0.09 vs 1.36±0.14 arbitrary units of volts (V) respectively, at the fifth measurement point, mean ± SEM, p<0.01). Skin perfusion significantly increased following sodium nitroprusside (p<0.001) but was lower in patients than control subjects (0.12±0.05 vs 0.45±0.11 V, respectively, at the fifth measurement point, p<0.01). These data suggest that endothelial and/or smooth muscle function may be impaired in the skin microcirculation of patients with NIDDM.Abbreviations IDDM Insulin-dependent diabetes mellitus - LDPI laser Doppler perfusion imager - mA milliamp - mC millicoulomb - NIDDM non-insulin-dependent diabetes mellitus - NO nitric oxide - ANOVA analysis of variance     

Time‐Course and Mechanisms of Restored Vascular Relaxation by Reduced Salt Intake and Angiotensin II Infusion in Rats Fed a High‐Salt Diet

Objective: This study determined the mechanisms and time‐course of recovery of vascular relaxation in middle cerebral arteries (MCAs) of salt‐fed Sprague‐Dawley rats returned to a low‐salt (LS) diet (0.4% NaCl) or infused with low‐dose angiotensin II (ANG II). Methods: Rats were fed a high‐salt (HS) diet (4% NaCl) for 3 days or 4 weeks before returning to an LS diet for various periods. Other rats fed a HS diet (HS+ANG II) received a chronic (3 days) intravenous (i.v.) infusion of a low dose of ANG II (5 ng kg^?1 min^?1) to prevent salt‐induced ANG II suppression. Results: The HS diet eliminated the increase in cerebral blood flow in response to acetylcholine (ACh) infusion and the relaxation of MCA in response to ACh, iloprost, cholera toxin, and reduced PO₂. Recovery of vascular relaxation was slow, requiring at least 2 weeks of the LS diet, regardless of the duration of exposure to a HS diet. Hypoxic dilation was mediated by cyclo‐oxygenase metabolites and ACh‐induced dilation was mediated via nitric oxide in LS rats and in HS rats returned to the LS diet or receiving ANG II infusion. Conclusions: Returning to a LS diet for 2 weeks or chronic 3‐day ANG II infusion restores the mechanisms that normally mediate cerebral vascular relaxation.     

Integrins and regulation of the microcirculation: from arterioles to molecular studies using atomic force microscopy

Integrins are an important class of receptors for extracellular matrix proteins that can mediate both force transmission, by virtue of their connections with the cell matrix and cytoskeleton; and signal transduction, resulting from the assemblages of signaling proteins that associate with focal contacts. Consequently, integrins have been proposed to be the mechanosensor in vascular smooth muscle and endothelial cells and to play a central role in mechanotransduction. In this regard, mechanical force is an important stimulus for many vascular functions, including contractile and relaxation processes,proliferation, migration, attachment, and cell phenotype determination. Collectively, these functions define physiological properties of the vasculature such as control of blood flow, capillary pressure,permeability, and peripheral vascular resistance, and play a role in pathophysiological processes like hypertension, diabetes, and arteriosclerosis. Our knowledge concerning how integrins sense and transduce physical forces into cellular signals and which integrins are involved is incomplete. Compared to other cell surface receptors, integrins have a relatively low affinity for their binding sites on the extracellular matrix and their affinity can be regulated. These characteristics of integrin-ligand interaction may facilitate dynamic processes such as cell migration, cell remodeling, and contractile activation in response to external forces. Important questions remain concerning the nature and origin of integrin-mediated signaling in the vascular wall.     

ATP敏感性钾通道与低氧性肺动脉高压

研究证明肺血管上存在ATP敏感性钾通道（KATP通道），该通道参与了膜静息电位和血管紧张性的调节。在低氧性诱导的肺动脉高压中，KATP通道参与了血管收缩和重构的病理过程。了解KATP通道的结构和其生物学特性，对认识低氧性肺动脉高压的形成机制和发展新的临床治疗手段有重要意义。     

Signaling pathways of mechanotransduction in arteriolar endothelium and smooth muscle cells in hypertension

Hypertension, a disease with a high incidence in the population, affects all parts of the cardiovascular system. Studying the alteration of vasomotor responses of microvessels of hypertensive animals or responses of vessels following short-term increases in hemodynamic forces helps us to better understand the underlying cellular signaling events responsible for their functional adaptation. These adaptations are likely to precede the structural remodeling of arterioles, resulting in irreversible increases in peripheral vascular resistance in hypertension. Although malfunction of several mechanisms can lead to the development of hypertension, hemodynamic forces (such as pressure and shear stress) are increased in all forms of hypertension. Thus, local mechanisms that sense the level of these forces and transduce the signals into vasomotor responses must be affected in all forms of hypertension. The endothelium has a central role in the early functional adaptations. Pressure-induced myogenic constriction is enhanced due to the augmented release of endothelium-derived constrictor factors that modulate arteriolar smooth muscle sensitivity to Ca(2+). In contrast, flow/shear stress-induced dilation of arterioles is reduced in hypertension, due to the impaired mediation of the response by nitric oxide (NO). The magnitude of impairment is gender specific, primarily due to an estrogen-dependent enhancement of NO release in females. It is proposed that the elevated hemodynamic forces present in hypertension may themselves initiate these alterations, probably by enhancing the release of reactive oxygen species (ROS; produced by xanthine oxidase, NAD(P)H oxidoreductase, eNOS, etc.), which then interfere with the synthesis and/or action of endothelium-derived mediators. Interfering early on with these mechanisms may prevent the development of irreversible structural changes of the microcirculation observed in hypertension.     

Remodeling in the microcirculation of rat skeletal muscle during chronic ischemia

OBJECTIVE: To establish the time course and extent of remodeling of terminal microcirculation in ischemic rat skeletal muscle during prolonged low flow that does not lead to inflammation. METHODS: One common iliac artery was ligated via laparotomy in adult Sprague-Dawley rats and extensor digitorum longus (EDL) muscles removed at intervals (1, 2, and 5 weeks) postsurgery. Serial frozen EDL sections were stained to show capillaries (alkaline phosphatase), cell proliferation (antibody to proliferating cell nuclear antigen [PCNA]), terminal microvessels (antibodies to alpha-smooth muscle actin (alpha-SMA) or endothelial nitric oxide synthase [eNOS]), and macrophages (antibodies to infiltrating and resident macrophages). Total muscle eNOS protein was quantified by standard Western blotting techniques. RESULTS: Capillary proliferation was very limited in ischemic EDLs, with a modest 12% increase in the capillary/fiber ratio after 5 weeks, preceded at 2 weeks by increased numbers of PCNA-positive nuclei at capillary sites. There was no muscle necrosis or evidence of inflammation, based on macrophage staining. The number of terminal microvessels that were positive for alpha-SMA and <10 microm in diameter was fewer in ischemic EDLs at all time points, whereas the number of larger positive vessels was unchanged. eNOS-positive vessels <10 microm in diameter were stained similarly throughout ischemic muscles as the controls, and showed a similar increase in vessel/fiber ratio as the capillaries. The total eNOS protein level was similar to that in controls in ischemic EDLs after 1 and 2 weeks, but was 28% lower after 5 weeks. CONCLUSIONS: Prolonged, moderate flow reduction to skeletal muscles does not necessarily lead to inflammation or extensive capillary growth. Based on eNOS staining, the terminal microcirculation remains intact, but the loss of alpha-SMA immunoreactivity may indicate remodeling involving the "deinvestment" of microvessels by smooth muscle.     

Transient receptor potential and other ion channels as pharmaceutical targets in airway smooth muscle cells

Regardless of the triggering stimulus in asthma, contraction of the airway smooth muscle (ASM) is considered to be an important pathway leading to the manifestation of asthmatic symptoms. Therefore, the various ion channels that modulate ASM contraction and relaxation are particularly attractive targets for therapy. Although voltage-operated Ca2+ channels (VOCC) are the most extensively characterised Ca(2+)-permeable channels in ASM cells and are obvious pharmacological targets, blockers of VOCC have not been successful in alleviating ASM contraction in asthma. Similarly, although the Cl- and K+ channels also modulate ASM contraction and relaxation by regulating plasma membrane potential, pharmacological interventions directed against these channels have failed to abrogate ASM contraction in asthma. A large body of evidence suggests that store-operated Ca2+ channels (SOCC) and Ca(2+)-permeable second messenger-activated non-selective cation channels (NSCC) predominantly mediate ASM contraction. However, development of pharmacological interventions involving these channels has been hampered by the paucity of information regarding their molecular identity. Members of the mammalian transient receptor potential (TRP) protein family, which form voltage-independent channels with variable Ca2+ selectivity that are activated by store depletion and/or by intracellular messengers, are potential molecular candidates for SOCC and NSCC in ASM cells. While the function of TRP channels in ASM cells remains to be elucidated and there are, at present, essentially no good TRP channel antagonists, this group of proteins is a potentially valuable pharmaceutical target for the treatment of asthma.     

Deteriorated function of cutaneous microcirculation in chronic congestive heart failure

Background Chronic congestive heart failure is a complex condition that leads to dysfunction in the peripheral microcirculation. We have previously shown that vascular reactivity is reduced with increasing age. In this study, we examined a group of very old patients with severe chronic heart failure to test the hypothesis that vascular function is further compromised by a combination of heart failure and aging. Methods Cutaneous forearm blood flow was measured by laser Doppler flowmetry and compared among three groups: Group 1 (n = 20, mean ± SE: 85.5 ± 4 years), heart failure patients with New York Heart Association class IV (NYHA IV) and with a NT-proBNP level ≥ 5000 ng/L; Group 2 (n = 15, mean ± SE: 76.5 ± 2 years), heart failure patients with NYHA II and NT-proBNP ≤ 2000 ng/L, and Group 3 (n = 10, mean ± SE: 67.6 ± 3.0 years), healthy controls with no clinical signs of heart failure. The vasodilator response to the iontophoretic administration of acetylcholine (ACh), acting via an endothelial mechanism, and sodium nitroprusside (SNP), acting via a smooth muscle cell mechanism, were studied. Results All patients with heart failure had significantly reduced vascular reactivity independent of the mode of stimulation (ACh, SNP or heat) when compared to healthy controls. However, the responses did not differ between the two groups of heart failure patients. Conclusions Cutaneous vascular reactivity is reduced in heart failure patients and does not correlate with the severity of the condition or age of patients.     

Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells

The endothelium plays a vital role in the control of vascular functions, including modulation of tone; permeability and barrier properties; platelet adhesion and aggregation; and secretion of paracrine factors. Critical signaling events in many of these functions involve an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)). This rise in [Ca(2+)](i) occurs via an interplay between several mechanisms, including release from intracellular stores, entry from the extracellular space through store depletion and second messenger-mediated processes, and the establishment of a favorable electrochemical gradient. The focus of this review centers on the role of potassium channels and membrane potential in the creation of a favorable electrochemical gradient for Ca(2+) entry. In addition, evidence is examined for the existence of various classes of potassium channels and the possible influence of regional variation in expression and experimental conditions.     

Faulty TRPM4 channels underlie age-dependent cerebral vascular dysfunction in Gould syndrome

Gould syndrome is a rare multisystem disorder resulting from autosomal dominant mutations in the collagen-encoding genes COL4A1 and COL4A2. Human patients and Col4a1 mutant mice display brain pathology that typifies cerebral small vessel diseases (cSVDs), including white matter hyperintensities, dilated perivascular spaces, lacunar infarcts, microbleeds, and spontaneous intracerebral hemorrhage. The underlying pathogenic mechanisms are unknown. Using the Col4a1^+/G394V mouse model, we found that vasoconstriction in response to internal pressure—the vascular myogenic response—is blunted in cerebral arteries from middle-aged (12 mo old) but not young adult (3 mo old) animals, revealing age-dependent cerebral vascular dysfunction. The defect in the myogenic response was associated with a significant decrease in depolarizing cation currents conducted by TRPM4 (transient receptor potential melastatin 4) channels in native cerebral artery smooth muscle cells (SMCs) isolated from mutant mice. The minor membrane phospholipid phosphatidylinositol 4,5 bisphosphate (PIP₂) is necessary for TRPM4 activity. Dialyzing SMCs with PIP₂ and selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that converts PIP₂ to phosphatidylinositol (3, 4, 5)-trisphosphate (PIP₃), restored TRPM4 currents. Acute inhibition of PI3K activity and blockade of transforming growth factor-beta (TGF-β) receptors also rescued the myogenic response, suggesting that hyperactivity of TGF-β signaling pathways stimulates PI3K to deplete PIP₂ and impair TRPM4 channels. We conclude that age-related cerebral vascular dysfunction in Col4a1^+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP₂ depletion, revealing an age-dependent mechanism of cSVD.

Cerebral small vessel diseases (cSVDs) are a group of familial and sporadic pathologies afflicting the blood vessels in the brain. cSVDs are a major cause of vascular contributions to cognitive impairment and dementia (VCID), second only to Alzheimer’s disease as the most common form of dementia in adults (1). VCID and cSVDs are more prevalent in the elderly and are expected to overburden health care systems globally as the world’s population ages (2). Idiopathic and familial forms of the disease have been described, but the pathogenesis remains poorly understood and specific treatment options are not available. Mutations in the genes encoding type IV collagen alpha 1 (COL4A1) and alpha 2 (COL4A2) cause Gould syndrome, an autosomal dominant multisystem disorder that encompasses all the hallmarks of cSVDs, including white matter hyperintensities, intracerebral hemorrhage (ICH), lacunes, and microinfarcts (3, 4). How COL4A1 and COL4A2 mutations cause cSVD and related brain defects are not known. Studies using a murine allelic series of Col4a1 and Col4a2 mutations show allelic heterogeneity with a position effect whereby mutations closer to the carboxyl terminus of the triple helical domain are associated with increased ICH severity (5, 6). These findings suggest that the mechanisms underlying cSVD associated with Gould syndrome are heterogeneous and complex. Here, we sought to elucidate the molecular links between a specific Col4a1 mutation and cerebrovascular dysfunction.COL4A1 and COL4A2 form a heterotrimer [α1α1α2(IV)] that is a fundamental component of all basement membranes. Collagen α1α1α2(IV) is secreted to the extracellular space, where it polymerizes into networks that are further cross-linked with other basement membrane components. COL4A1 and COL4A2 proteins are composed of a long triple helical domain flanked by 7S and NC1 domains at the amino and carboxyl termini, respectively (7). The triple helical regions consist of long stretches of G-X-Y repeats characteristic of all collagens. Glycine (G) is required at every third amino acid as the absence of a side chain allows it to fit into the core of the triple helix (8). The vast majority of pathogenic COL4A1 and COL4A2 mutations are missense mutations of one of these highly conserved glycine residues (9–11). Such mutations are thought to drive three potential adverse outcomes: diminishment of normal collagen α1α1α2(IV) in basement membranes; secretion and incorporation of mutant collagen α1α1α2(IV) that disrupts the functional integrity of the basement membrane; and intracellular retention of misfolded collagen α1α1α2(IV) molecules that cause ER/SR stress in collagen-producing cells (12). Col4a1 mutant mice display striking position-dependent heterogeneity in trafficking defects, with diminishing export and increased intracellular retention of collagen α1α1α2(IV) associated with mutations closer to the carboxyl terminus of the protein (13). For the current study, we used mice heterozygous for a point mutation in Col4a1 in which the G residue at position 394 (relatively near the amino terminus) had been replaced by valine (V). Col4a1^+/G394V mice traffic collagen α1α1α2(IV) out of cells relatively efficiently (13), suggesting that the pathology of these animals is independent of ER/SR stress and that structural and/or functional imperfections in the basement membrane is the primary defect.Autoregulation of blood flow in the brain is maintained in part by the vascular myogenic response, a process that sustains partial constriction of arteries and arterioles in response to intraluminal pressure (14). The myogenic response maintains near-constant blood flow within the microcirculation during beat-to-beat fluctuations in the force of perfusion, thereby preventing tissue ischemia during transient drops in pressure and protecting delicate capillary beds during temporary increases in pressure (14). Several types of cerebrovascular disease disrupt this process, including familial cSVD associated with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) (15). The effects of Col4a1 mutations on the myogenic tone of cerebral arteries are not known. Signaling pathways intrinsic to vascular smooth muscle cells (SMCs) forming the walls of arteries and arterioles generate the myogenic response (14). Increases in intraluminal pressure activate TRPM4 (transient receptor potential melastatin 4) channels, allowing an influx of Na⁺ ions that depolarize the SMC plasma membrane to initiate Ca²⁺ influx through voltage-gated Ca²⁺ channels and engage contractile pathways (16–18). The depolarizing effects of TRPM4 channels are balanced by hyperpolarizing K⁺ currents primarily conducted by voltage-dependent K⁺ (Kv) channels, large-conductance Ca²⁺-activated K⁺ (BK) channels, and inwardly rectifying K⁺ (K_IR) channels (19, 20). In the CADASIL mouse model, increased Kv channel current density in SMCs accounts for diminished myogenic tone (15). The electrophysiological properties of SMCs from Col4a1 mutant mice have not been reported.Here, we show that the myogenic response of cerebral pial arteries from Col4a1^+/G394V mice was dramatically impaired in an age-dependent manner. In contrast to CADASIL models, K⁺ currents in SMCs from Col4a1^+/G394V mice were not increased compared to controls. Instead, we found that the loss of myogenic tone was associated with decreased activity of inward Na⁺ currents conducted by TRPM4 channels. TRPM4 currents were restored by supplying exogenous phosphatidylinositol 4,5 bisphosphate (PIP₂), a minor membrane phospholipid that is a necessary co-factor for TRPM4 activity (21, 22), and by selective blockade of phosphoinositide 3-kinase (PI3K), an enzyme that depletes PIP₂ by converting it to phosphatidylinositol (3,4,5)-trisphosphate (PIP₃). Further, inhibition of PI3K activity and blockade of upstream transforming growth factor-beta (TGF-β) receptors rescued myogenic tone in arteries from 12-mo-old mutant mice. We conclude that age-dependent cerebral vascular dysfunction in Col4a1^+/G394V mice is caused by the loss of depolarizing TRPM4 currents due to PIP₂ depletion. Conversion of PIP₂ to PIP₃ by PI3K, acting downstream of TGF-β receptors, accounts for diminished PIP₂ levels in SMCs. Our findings reveal a mechanism of age-dependent cerebral vascular dysfunction and identify PI3K and TGF-β receptors as therapeutic targets for some forms of cSVDs.