Luis F. Santana

Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane.

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

Overexpression of Catalase Targeted to Mitochondria Attenuates Murine Cardiac Aging

Background— Age is a major risk for cardiovascular diseases. Although mitochondrial reactive oxygen species have been proposed as one of the causes of aging, their role in cardiac aging remains unclear. We have previously shown that overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17% to 21%. Methods and Results— We used echocardiography to study cardiac function in aging cohorts of wild-type and mCAT mice. Changes found in wild-type mice recapitulate human aging: age-dependent increases in left ventricular mass index and left atrial dimension, worsening of the myocardial performance index, and a decline in diastolic function. Cardiac aging in mice is accompanied by accumulation of mitochondrial protein oxidation, increased mitochondrial DNA mutations and deletions and mitochondrial biogenesis, increased ventricular fibrosis, enlarged myocardial fiber size, decreased cardiac SERCA2 protein, and activation of the calcineurin–nuclear factor of activated T-cell pathway. All of these age-related changes were significantly attenuated in mCAT mice. Analysis of survival of 130 mice demonstrated that echocardiographic cardiac aging risk scores were significant predictors of mortality. The estimated attributable risk to mortality for these 2 parameters was 55%. Conclusions— This study shows that cardiac aging in the mouse closely recapitulates human aging and demonstrates the critical role of mitochondrial reactive oxygen species in cardiac aging and the impact of cardiac aging on survival. These findings also support the potential application of mitochondrial antioxidants in reactive oxygen species–related cardiovascular diseases.

Mitochondrial Oxidative Stress Mediates Angiotensin II–Induced Cardiac Hypertrophy and Gαq Overexpression–Induced Heart Failure

Rationale: Mitochondrial dysfunction has been implicated in several cardiovascular diseases; however, the roles of mitochondrial oxidative stress and DNA damage in hypertensive cardiomyopathy are not well understood. Objective: We evaluated the contribution of mitochondrial reactive oxygen species (ROS) to cardiac hypertrophy and failure by using genetic mouse models overexpressing catalase targeted to mitochondria and to peroxisomes. Methods and Results: Angiotensin II increases mitochondrial ROS in cardiomyocytes, concomitant with increased mitochondrial protein carbonyls, mitochondrial DNA deletions, increased autophagy and signaling for mitochondrial biogenesis in hearts of angiotensin II–treated mice. The causal role of mitochondrial ROS in angiotensin II–induced cardiomyopathy is shown by the observation that mice that overexpress catalase targeted to mitochondria, but not mice that overexpress wild-type peroxisomal catalase, are resistant to cardiac hypertrophy, fibrosis and mitochondrial damage induced by angiotensin II, as well as heart failure induced by overexpression of G&agr;q. Furthermore, primary damage to mitochondrial DNA, induced by zidovudine administration or homozygous mutation of mitochondrial polymerase &ggr;, is also shown to contribute directly to the development of cardiac hypertrophy, fibrosis and failure. Conclusions: These data indicate the critical role of mitochondrial ROS in cardiac hypertrophy and failure and support the potential use of mitochondrial-targeted antioxidants for prevention and treatment of hypertensive cardiomyopathy.

Modulation of the molecular composition of large conductance, Ca 2+ activated K + channels in vascular smooth muscle during hypertension

Hypertension is a clinical syndrome characterized by increased vascular tone. However, the molecular mechanisms underlying vascular dysfunction during acquired hypertension remain unresolved. Localized intracellular Ca2+ release events through ryanodine receptors (Ca2+ sparks) in the sarcoplasmic reticulum are tightly coupled to the activation of large-conductance, Ca2+-activated K+ (BK) channels to provide a hyperpolarizing influence that opposes vasoconstriction. In this study we tested the hypothesis that a reduction in Ca2+ spark-BK channel coupling underlies vascular smooth muscle dysfunction during acquired hypertension. We found that in hypertension, expression of the beta1 subunit was decreased relative to the pore-forming alpha subunit of the BK channel. Consequently, the BK channels were functionally uncoupled from Ca2+ sparks. Consistent with this, the contribution of BK channels to vascular tone was reduced during hypertension. We conclude that downregulation of the beta1 subunit of the BK channel contributes to vascular dysfunction in hypertension. These results support the novel concept that changes in BK channel subunit composition regulate arterial smooth muscle function.

Mitochondrial Targeted Antioxidant Peptide Ameliorates Hypertensive Cardiomyopathy

OBJECTIVES We investigated the effect of reducing mitochondrial oxidative stress by the mitochondrial-targeted antioxidant peptide SS-31 in hypertensive cardiomyopathy. BACKGROUND Oxidative stress has been implicated in hypertensive cardiovascular diseases. Mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase have been proposed as primary sites of reactive oxygen species (ROS) generation. METHODS The mitochondrial targeted antioxidant peptide SS-31 was used to determine the role of mitochondrial oxidative stress in angiotensin II (Ang)-induced cardiomyopathy as well as in Gαq overexpressing mice with heart failure. RESULTS Ang induces mitochondrial ROS in neonatal cardiomyocytes, which is prevented by SS-31, but not the nontargeted antioxidant N-acetyl cysteine (NAC). Continuous administration of Ang for 4 weeks in mice significantly increased both systolic and diastolic blood pressure, and this was not affected by SS-31 treatment. Ang was associated with up-regulation of NADPH oxidase 4 (NOX4) expression and increased cardiac mitochondrial protein oxidative damage, and induced the signaling for mitochondrial biogenesis. Reducing mitochondrial ROS by SS-31 substantially attenuated Ang-induced NOX4 up-regulation, mitochondrial oxidative damage, up-regulation of mitochondrial biogenesis, and phosphorylation of p38 mitogen-activated protein kinase and prevented apoptosis, concomitant with amelioration of Ang-induced cardiac hypertrophy, diastolic dysfunction, and fibrosis, despite the absence of blood pressure-lowering effect. The NAC did not show any beneficial effect. The SS-31 administration for 4 weeks also partially rescued the heart failure phenotype of Gαq overexpressing mice. CONCLUSIONS Mitochondrial targeted peptide SS-31 ameliorates cardiomyopathy resulting from prolonged Ang stimulation as well as Gαq overexpression, suggesting its potential clinical application for target organ protection in hypertensive cardiovascular diseases.

Sympathetic Stimulation of Adult Cardiomyocytes Requires Association of AKAP5 With a Subpopulation of L-Type Calcium Channels

Rationale: Sympathetic stimulation of the heart increases the force of contraction and rate of ventricular relaxation by triggering protein kinase (PK)A-dependent phosphorylation of proteins that regulate intracellular calcium. We hypothesized that scaffolding of cAMP signaling complexes by AKAP5 is required for efficient sympathetic stimulation of calcium transients. Objective: We examined the function of AKAP5 in the &bgr;-adrenergic signaling cascade. Methods and Results: We used calcium imaging and electrophysiology to examine the sympathetic response of cardiomyocytes isolated from wild type and AKAP5 mutant animals. The &bgr;-adrenergic regulation of calcium transients and the phosphorylation of substrates involved in calcium handling were disrupted in AKAP5 knockout cardiomyocytes. The scaffolding protein, AKAP5 (also called AKAP150/79), targets adenylyl cyclase, PKA, and calcineurin to a caveolin 3–associated complex in ventricular myocytes that also binds a unique subpopulation of Cav1.2 L-type calcium channels. Only the caveolin 3–associated Cav1.2 channels are phosphorylated by PKA in response to sympathetic stimulation in wild-type heart. However, in the AKAP5 knockout heart, the organization of this signaling complex is disrupted, adenylyl cyclase 5/6 no longer associates with caveolin 3 in the T-tubules, and noncaveolin 3–associated calcium channels become phosphorylated after &bgr;-adrenergic stimulation, although this does not lead to an enhanced calcium transient. The signaling domain created by AKAP5 is also essential for the PKA-dependent phosphorylation of ryanodine receptors and phospholamban. Conclusions: These findings identify an AKAP5-organized signaling module that is associated with caveolin 3 and is essential for sympathetic stimulation of the calcium transient in adult heart cells.

AKAP150 Is Required for Stuttering Persistent Ca2+ Sparklets and Angiotensin II–Induced Hypertension

Hypertension is a perplexing multiorgan disease involving renal primary pathology and enhanced angiotensin II vascular reactivity. Here, we report that a novel form of a local Ca2+ signaling in arterial smooth muscle is linked to the development of angiotensin II–induced hypertension. Long openings and reopenings of L-type Ca2+ channels in arterial myocytes produce stuttering persistent Ca2+ sparklets that increase Ca2+ influx and vascular tone. These stuttering persistent Ca2+ sparklets arise from the molecular interactions between the L-type Ca2+ channel and protein kinase C&agr; at only a few subsarcolemmal regions in resistance arteries. We have identified AKAP150 as the key protein, which targets protein kinase C&agr; to the L-type Ca2+ channels and thereby enables its regulatory function. Accordingly, AKAP150 knockout mice (AKAP150−/−) were found to lack persistent Ca2+ sparklets and have lower arterial wall intracellular calcium ([Ca2+]i) and decreased myogenic tone. Furthermore, AKAP150−/− mice were hypotensive and did not develop angiotensin II–induced hypertension. We conclude that local control of L-type Ca2+ channel function is regulated by AKAP150-targeted protein kinase C&agr; signaling, which controls stuttering persistent Ca2+ influx, vascular tone, and blood pressure under physiological conditions and underlies angiotensin II–dependent hypertension.

Mechanisms Underlying Heterogeneous Ca2+ Sparklet Activity in Arterial Smooth Muscle

In arterial smooth muscle, single or small clusters of Ca2+ channels operate in a high probability mode, creating sites of nearly continual Ca2+ influx (called “persistent Ca2+ sparklet” sites). Persistent Ca2+ sparklet activity varies regionally within any given cell. At present, the molecular identity of the Ca2+ channels underlying Ca2+ sparklets and the mechanisms that give rise to their spatial heterogeneity remain unclear. Here, we used total internal reflection fluorescence (TIRF) microscopy to directly investigate these issues. We found that tsA-201 cells expressing L-type Cavα1.2 channels recapitulated the general features of Ca2+ sparklets in cerebral arterial myocytes, including amplitude of quantal event, voltage dependencies, gating modalities, and pharmacology. Furthermore, PKCα activity was required for basal persistent Ca2+ sparklet activity in arterial myocytes and tsA-201 cells. In arterial myocytes, inhibition of protein phosphatase 2A (PP2A) and 2B (PP2B; calcineurin) increased Ca2+ influx by evoking new persistent Ca2+ sparklet sites and by increasing the activity of previously active sites. The actions of PP2A and PP2B inhibition on Ca2+ sparklets required PKC activity, indicating that these phosphatases opposed PKC-mediated phosphorylation. Together, these data unequivocally demonstrate that persistent Ca2+ sparklet activity is a fundamental property of L-type Ca2+ channels when associated with PKC. Our findings support a novel model in which the gating modality of L-type Ca2+ channels vary regionally within a cell depending on the relative activities of nearby PKCα, PP2A, and PP2B.

Activation of NFATc3 Down-regulates the β1 Subunit of Large Conductance, Calcium-activated K+ Channels in Arterial Smooth Muscle and Contributes to Hypertension

Large conductance, Ca2+-activated K+ (BK) channels modulate the excitability and contractile state of arterial smooth muscle. Recently, we demonstrated that during hypertension, expression of the accessory β1 subunit was decreased relative to the pore-forming α subunit of the BK channel. Reduced β1 subunit expression resulted in BK channels with impaired function due to lowered sensitivity to Ca2+. Here, we tested the hypothesis that activation of the calcineurin/NFATc3 signaling pathway down-regulates β1 expression during angiotensin II-induced hypertension. Consistent with this hypothesis, we found that in vivo administration of angiotensin II-activated calcineurin/NFATc3 signaling in arterial smooth muscle. During angiotensin II infusion, arterial smooth muscle BK channel function was decreased in wild type (WT) but not in NFATc3 null (NFATc3-/-) mice. Accordingly, β1 expression was decreased in WT but not in NFATc3-/- arteries. Angiotensin II-induced down-regulation of the β1 subunit required Ca2+ influx via L-type Ca2+ channels. However, in the absence of angiotensin II, moderate elevation of [Ca2+]i alone was not sufficient to activate NFAT transcriptional activity and, thus, decrease β1 subunit expression. Importantly, angiotensin II infusion increased systemic blood pressure to a lower extent in NFATc3-/- than in WT mice, indicating that this transcription factor is required for the development of severe hypertension during chronic angiotensin II signaling activation. We conclude that activation of calcineurin and NFATc3 during sustained angiotensin II signaling down-regulates the expression of the β1 subunit of the BK channel, which in turn contributes to arterial dysfunction and the development of hypertension.

Functional coupling of calcineurin and protein kinase A in mouse ventricular myocytes

We examined the role of the Ca2+‐regulated protein phosphatase calcineurin in controlling Ca2+ signalling in mouse ventricular myocytes. Membrane currents and voltage were measured in single myocytes using the patch‐clamp technique. Cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in cells loaded with the fluorescent Ca2+ indicators fluo‐4 or fura‐2 using a confocal or epifluorescence microscope. Inhibition of calcineurin with cyclosporin A (CsA, 100 nm) or the calcineurin auto‐inhibitory peptide (CiP, 100 μM), increased the amplitude and rate of decay of the evoked [Ca2+]i transient and also prolonged the action potential (AP) of ventricular myocytes to a similar extent. The effects of CsA (100 nm) and 100 μM CiP on the [Ca2+]i transient and AP were not additive. Calcineurin inhibition did not modify the K+ currents responsible for repolarisation of the mouse ventricle. Instead, inhibition of calcineurin increased the amplitude of the Ca2+ current (ICa) and the evoked calcium transient normalized to the ICa. Calcium sparks, which underlie the [Ca2+]i transient, had a higher frequency and amplitude, suggesting an elevation of SR calcium load. Inhibition of protein kinase A (PKA) prevented the effects of calcineurin inhibition, indicating that calcineurin opposes the actions of PKA. Finally, immunofluorescence images suggest that calcineurin and PKA co‐localize near the T‐tubules of ventricular myocytes. We propose that calcineurin and PKA are co‐localized to control Ca2+ influx through calcium channels and calcium release through ryanodine receptors.