L. Fernando Santana

Downregulation of the BK Channel β1 Subunit in Genetic Hypertension

Abstract— The molecular mechanisms underlying increased arterial tone during hypertension are unclear. In vascular smooth muscle, localized Ca2+ release events through ryanodine-sensitive channels located in the sarcoplasmic reticulum (Ca2+ sparks) activate large-conductance, Ca2+-sensitive K+ (BK) channels. Ca2+ sparks and BK channels provide a negative feedback mechanism that hyperpolarizes smooth muscle and thereby opposes vasoconstriction. In this study, we examined Ca2+ sparks and BK channel function in Wistar-Kyoto (WKY) rats with borderline hypertension and in spontaneously hypertensive rats (SHR), a widely used genetic model of severe hypertension. We found that the amplitude of spontaneous BK currents in WKY and SHR cells were smaller than in normotensive cells even though Ca2+ sparks were of similar magnitude. BK channels in WKY and SHR cells were less sensitive to physiological changes in intracellular Ca2+ than normotensive cells. Our data indicate that decreased expression of the BK channel &bgr;1 subunit underlies the lower Ca2+ sensitivity of BK channels in SHR and WKY myocytes. We conclude that the lower expression of the &bgr;1 subunit during genetic borderline and severe hypertension reduced BK channel activity by decreasing the sensitivity of these channels to physiological changes in Ca2+. These results support the view that changes in the molecular composition of BK channels may be a fundamental event contributing to the development of vascular dysfunction during hypertension.

Anchored phosphatases modulate glucose homeostasis

Endocrine release of insulin principally controls glucose homeostasis. Nutrient‐induced exocytosis of insulin granules from pancreatic β‐cells involves ion channels and mobilization of Ca2+ and cyclic AMP (cAMP) signalling pathways. Whole‐animal physiology, islet studies and live‐β‐cell imaging approaches reveal that ablation of the kinase/phosphatase anchoring protein AKAP150 impairs insulin secretion in mice. Loss of AKAP150 impacts L‐type Ca2+ currents, and attenuates cytoplasmic accumulation of Ca2+ and cAMP in β‐cells. Yet surprisingly AKAP150 null animals display improved glucose handling and heightened insulin sensitivity in skeletal muscle. More refined analyses of AKAP150 knock‐in mice unable to anchor protein kinase A or protein phosphatase 2B uncover an unexpected observation that tethering of phosphatases to a seven‐residue sequence of the anchoring protein is the predominant molecular event underlying these metabolic phenotypes. Thus anchored signalling events that facilitate insulin secretion and glucose homeostasis may be set by AKAP150 associated phosphatase activity.

Down‐regulation of CaV1.2 channels during hypertension: how fewer CaV1.2 channels allow more Ca2+ into hypertensive arterial smooth muscle

•  A hallmark of essential hypertension is an anomalous vascular tone due to the increase in intracellular Ca2+ ([Ca2+]i) and/or Ca2+ sensitivity in vascular smooth muscle. Down‐regulation of K+ channels together with increased CaV1.2 channel function has been proposed as one pathogenic mechanism. •  Using a mouse model of essential hypertension (BPH line), we found a decrease in the global smooth muscle Ca2+ influx due to fewer CaV1.2 channels. However, these CaV1.2 channels are hyperactive, allowing a larger local Ca2+ influx at rest that triggers an increased Ca2+ release from intracellular stores (sparks). •  Large conductance, Ca2+‐activated K+ (BK) channels of BPH myocytes show reduced Ca2+ sensitivity, so that its activation by the increased [Ca2+]i is impaired. •  Our results suggest that changes in the molecular composition of both CaV1.2 and BK channels could explain vascular dysfunction during hypertension in BPH mice.

A mitotic kinase scaffold depleted in testicular seminomas impacts spindle orientation in germ line stem cells

Correct orientation of the mitotic spindle in stem cells underlies organogenesis. Spindle abnormalities correlate with cancer progression in germ line-derived tumors. We discover a macromolecular complex between the scaffolding protein Gravin/AKAP12 and the mitotic kinases, Aurora A and Plk1, that is down regulated in human seminoma. Depletion of Gravin correlates with an increased mitotic index and disorganization of seminiferous tubules. Biochemical, super-resolution imaging, and enzymology approaches establish that this Gravin scaffold accumulates at the mother spindle pole during metaphase. Manipulating elements of the Gravin-Aurora A-Plk1 axis prompts mitotic delay and prevents appropriate assembly of astral microtubules to promote spindle misorientation. These pathological responses are conserved in seminiferous tubules from Gravin−/− mice where an overabundance of Oct3/4 positive germ line stem cells displays randomized orientation of mitotic spindles. Thus, we propose that Gravin-mediated recruitment of Aurora A and Plk1 to the mother (oldest) spindle pole contributes to the fidelity of symmetric cell division. DOI: http://dx.doi.org/10.7554/eLife.09384.001

Oxidative stress decreases microtubule growth and stability in ventricular myocytes

Microtubules (MTs) have many roles in ventricular myocytes, including structural stability, morphological integrity, and protein trafficking. However, despite their functional importance, dynamic MTs had never been visualized in living adult myocytes. Using adeno-associated viral vectors expressing the MT-associated protein plus end binding protein 3 (EB3) tagged with EGFP, we were able to perform live imaging and thus capture and quantify MT dynamics in ventricular myocytes in real time under physiological conditions. Super-resolution nanoscopy revealed that EB1 associated in puncta along the length of MTs in ventricular myocytes. The vast (~80%) majority of MTs grew perpendicular to T-tubules at a rate of 0.06μm∗s(-1) and growth was preferentially (82%) confined to a single sarcomere. Microtubule catastrophe rate was lower near the Z-line than M-line. Hydrogen peroxide increased the rate of catastrophe of MTs ~7-fold, suggesting that oxidative stress destabilizes these structures in ventricular myocytes. We also quantified MT dynamics after myocardial infarction (MI), a pathological condition associated with increased production of reactive oxygen species (ROS). Our data indicate that the catastrophe rate of MTs increases following MI. This contributed to decreased transient outward K(+) currents by decreasing the surface expression of Kv4.2 and Kv4.3 channels after MI. On the basis of these data, we conclude that, under physiological conditions, MT growth is directionally biased and that increased ROS production during MI disrupts MT dynamics, decreasing K(+) channel trafficking.

Loss of AKAP150 promotes pathological remodelling and heart failure propensity by disrupting calcium cycling and contractile reserve

Aims Impaired Ca2 + cycling and myocyte contractility are a hallmark of heart failure triggered by pathological stress such as hemodynamic overload. The A-Kinase anchoring protein AKAP150 has been shown to coordinate key aspects of adrenergic regulation of Ca2+ cycling and excitation–contraction in cardiomyocytes. However, the role of the AKAP150 signalling complexes in the pathogenesis of heart failure has not been investigated. Methods and results Here we examined how AKAP150 signalling complexes impact Ca2+ cycling, myocyte contractility, and heart failure susceptibility following pathological stress. We detected a significant reduction of AKAP150 expression in the failing mouse heart induced by pressure overload. Importantly, cardiac-specific AKAP150 knockout mice were predisposed to develop dilated cardiomyopathy with severe cardiac dysfunction and fibrosis after pressure overload. Loss of AKAP150 also promoted pathological remodelling and heart failure progression following myocardial infarction. However, ablation of AKAP150 did not affect calcineurin-nuclear factor of activated T cells signalling in cardiomyocytes or pressure overload- or agonist-induced cardiac hypertrophy. Immunoprecipitation studies showed that AKAP150 was associated with SERCA2, phospholamban, and ryanodine receptor-2, providing a targeted control of sarcoplasmic reticulum Ca2+ regulatory proteins. Mechanistically, loss of AKAP150 led to impaired Ca2+ cycling and reduced myocyte contractility reserve following adrenergic stimulation or pressure overload. Conclusions These findings define a critical role for AKAP150 in regulating Ca2+ cycling and myocardial ionotropy following pathological stress, suggesting the AKAP150 signalling pathway may serve as a novel therapeutic target for heart failure.

Distance constraints on activation of TRPV4 channels by AKAP150-bound PKCα in arterial myocytes

TRPV4 (transient receptor potential vanilloid 4) channels are Ca2+-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca2+ influx, detectable optically as “TRPV4 sparklets.” TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C &agr; (PKC&agr;) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca2+ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKC&agr;.

Down-regulation of CaV1.2 channels during hypertension

•  A hallmark of essential hypertension is an anomalous vascular tone due to the increase in intracellular Ca2+ ([Ca2+]i) and/or Ca2+ sensitivity in vascular smooth muscle. Down‐regulation of K+ channels together with increased CaV1.2 channel function has been proposed as one pathogenic mechanism. •  Using a mouse model of essential hypertension (BPH line), we found a decrease in the global smooth muscle Ca2+ influx due to fewer CaV1.2 channels. However, these CaV1.2 channels are hyperactive, allowing a larger local Ca2+ influx at rest that triggers an increased Ca2+ release from intracellular stores (sparks). •  Large conductance, Ca2+‐activated K+ (BK) channels of BPH myocytes show reduced Ca2+ sensitivity, so that its activation by the increased [Ca2+]i is impaired. •  Our results suggest that changes in the molecular composition of both CaV1.2 and BK channels could explain vascular dysfunction during hypertension in BPH mice.

BIN1 induces the formation of T-tubules and adult-like Ca2+ release units in developing cardiomyocytes: BIN1 promotes hESC-CMs with ventricular phenotype

Human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) are at the center of new cell‐based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC‐CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse‐tubules (T‐tubules) with adult‐like Ca2+ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC‐CMs promotes T‐tubules formation, facilitates CaV1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca2+]i imaging, and super resolution microscopy, we found that BIN1 expression induced T‐tubule development in hESC‐CMs, while increasing differentiation toward a more ventricular‐like phenotype. Voltage‐gated CaV1.2 channels clustered along the surface sarcolemma and T‐tubules of hESC‐CM. The length and width of the T‐tubules as well as the expression and size of CaV1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased CaV1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1‐expressing cells had more CaV1.2‐ryanodine receptor junctions than control cells. This was associated with larger [Ca2+]i transients during excitation–contraction coupling. Our data support the view that BIN1 is a key regulator of T‐tubule formation and CaV1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC‐CMs, we show that BIN1 is also important for CaV1.2 channel clustering, junctional SR organization, and the establishment of excitation–contraction coupling. Stem Cells 2019;37:54–64

Down-regulation of CaV1.2 channels during hypertension: how fewer CaV1.2 channels allow more Ca2+into hypertensive arterial smooth muscle: Cav1.2 channels in essential hypertension

•  A hallmark of essential hypertension is an anomalous vascular tone due to the increase in intracellular Ca2+ ([Ca2+]i) and/or Ca2+ sensitivity in vascular smooth muscle. Down‐regulation of K+ channels together with increased CaV1.2 channel function has been proposed as one pathogenic mechanism. •  Using a mouse model of essential hypertension (BPH line), we found a decrease in the global smooth muscle Ca2+ influx due to fewer CaV1.2 channels. However, these CaV1.2 channels are hyperactive, allowing a larger local Ca2+ influx at rest that triggers an increased Ca2+ release from intracellular stores (sparks). •  Large conductance, Ca2+‐activated K+ (BK) channels of BPH myocytes show reduced Ca2+ sensitivity, so that its activation by the increased [Ca2+]i is impaired. •  Our results suggest that changes in the molecular composition of both CaV1.2 and BK channels could explain vascular dysfunction during hypertension in BPH mice.