Flow of wormlike micellar solutions around microfluidic cylinders with high aspect ratio and low blockage ratio.

We employ time-resolved flow velocimetry and birefringence imaging methods to study the flow of a well-characterized shear-banding wormlike micellar solution around a novel glass-fabricated microfluidic circular cylinder. In contrast with typical microfluidic cylinders, our geometry is characterized by a high aspect ratio α = H/W = 5 and a low blockage ratio ß = 2r/W = 0.1, where H and W are the channel height and width, and the cylinder radius r = 20 µm. The small cylinder radius allows access up to very high Weissenberg numbers 1.9 ≤ Wi = λMU/r ≤ 3750 (where λM is the Maxwell relaxation time) while inertial effects remain entirely negligible (Reynolds number, Re < 10-4). At low Wi values, the flow remains steady and symmetric and a birefringent region (indicating micellar alignment and tensile stress) develops downstream of the cylinder. Above a critical value Wic ≈ 60 the flow transitions to a steady asymmetric state, characterized as a supercritical pitchfork bifurcation, in which the fluid takes a preferential path around one side of the cylinder. At a second critical value Wic2 ≈ 130, the flow becomes time-dependent, with a characteristic frequency f0 ≈ 1/λM. This initial transition to time dependence has characteristics of a subcritical Hopf bifurcation. Power spectra of the measured fluctuations become complex as Wi is increased further, showing a gradual slowing down of the dynamics and emergence of harmonics. A final transition at very high Wic3 corresponds to the re-emergence of a single peak in the power spectrum but at much higher frequency. We discuss this in terms of possible flow-induced breakage of micelles into shorter species with a faster relaxation time.

Hypoxia-induced interaction of filamin with Drp1 causes mitochondrial hyperfission-associated myocardial senescence.

Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission-accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission-associated myocardial senescence in mice after MI. In peri-infarct regions characterized by mitochondrial hyperfission and associated with myocardial senescence, filamin A colocalized with Drp1 around mitochondria. Hypoxic stress induced the interaction of filamin A with the GTPase domain of Drp1 and increased Drp1 activity in an actin-binding-dependent manner in rat cardiomyocytes. Expression of the A1545T filamin mutant, which potentiates actin aggregation, promoted mitochondrial hyperfission under normoxia. Furthermore, pharmacological perturbation of the Drp1-filamin A interaction by cilnidipine suppressed mitochondrial hyperfission-associated myocardial senescence and heart failure after MI. Together, these data demonstrate that Drp1 association with filamin and the actin cytoskeleton contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine, as well as provides a starting point for innovative Drp1 inhibitor development.

TRPC6 counteracts TRPC3-Nox2 protein complex leading to attenuation of hyperglycemia-induced heart failure in mice.

Excess production of reactive oxygen species (ROS) caused by hyperglycemia is a major risk factor for heart failure. We previously reported that transient receptor potential canonical 3 (TRPC3) channel mediates pressure overload-induced maladaptive cardiac fibrosis by forming stably functional complex with NADPH oxidase 2 (Nox2). Although TRPC3 has been long suggested to form hetero-multimer channels with TRPC6 and function as diacylglycerol-activated cation channels coordinately, the role of TRPC6 in heart is still obscure. We here demonstrated that deletion of TRPC6 had no impact on pressure overload-induced heart failure despite inhibiting interstitial fibrosis in mice. TRPC6-deficient mouse hearts 1 week after transverse aortic constriction showed comparable increases in fibrotic gene expressions and ROS production but promoted inductions of inflammatory cytokines, compared to wild type hearts. Treatment of TRPC6-deficient mice with streptozotocin caused severe reduction of cardiac contractility with enhancing urinary and cardiac lipid peroxide levels, compared to wild type and TRPC3-deficient mice. Knockdown of TRPC6, but not TRPC3, enhanced basal expression levels of cytokines in rat cardiomyocytes. TRPC6 could interact with Nox2, but the abundance of TRPC6 was inversely correlated with that of Nox2. These results strongly suggest that Nox2 destabilization through disrupting TRPC3-Nox2 complex underlies attenuation of hyperglycemia-induced heart failure by TRPC6.

TRPC3-GEF-H1 axis mediates pressure overload-induced cardiac fibrosis.

Structural cardiac remodeling, accompanying cytoskeletal reorganization of cardiac cells, is a major clinical outcome of diastolic heart failure. A highly local Ca2+ influx across the plasma membrane has been suggested to code signals to induce Rho GTPase-mediated fibrosis, but it is obscure how the heart specifically decodes the local Ca2+ influx as a cytoskeletal reorganizing signal under the conditions of the rhythmic Ca2+ handling required for pump function. We found that an inhibition of transient receptor potential canonical 3 (TRPC3) channel activity exhibited resistance to Rho-mediated maladaptive fibrosis in pressure-overloaded mouse hearts. Proteomic analysis revealed that microtubule-associated Rho guanine nucleotide exchange factor, GEF-H1, participates in TRPC3-mediated RhoA activation induced by mechanical stress in cardiomyocytes and transforming growth factor (TGF) ß stimulation in cardiac fibroblasts. We previously revealed that TRPC3 functionally interacts with microtubule-associated NADPH oxidase (Nox) 2, and inhibition of Nox2 attenuated mechanical stretch-induced GEF-H1 activation in cardiomyocytes. Finally, pharmacological TRPC3 inhibition significantly suppressed fibrotic responses in human cardiomyocytes and cardiac fibroblasts. These results strongly suggest that microtubule-localized TRPC3-GEF-H1 axis mediates fibrotic responses commonly in cardiac myocytes and fibroblasts induced by physico-chemical stimulation.

TRPC3 positively regulates reactive oxygen species driving maladaptive cardiac remodeling.

Reactive oxygen species (ROS) produced by NADPH oxidase 2 (Nox2) function as key mediators of mechanotransduction during both physiological adaptation to mechanical load and maladaptive remodeling of the heart. This is despite low levels of cardiac Nox2 expression. The mechanism underlying the transition from adaptation to maladaptation remains obscure, however. We demonstrate that transient receptor potential canonical 3 (TRPC3), a Ca2+-permeable channel, acts as a positive regulator of ROS (PRROS) in cardiomyocytes, and specifically regulates pressure overload-induced maladaptive cardiac remodeling in mice. TRPC3 physically interacts with Nox2 at specific C-terminal sites, thereby protecting Nox2 from proteasome-dependent degradation and amplifying Ca2+-dependent Nox2 activation through TRPC3-mediated background Ca2+ entry. Nox2 also stabilizes TRPC3 proteins to enhance TRPC3 channel activity. Expression of TRPC3 C-terminal polypeptide abolished TRPC3-regulated ROS production by disrupting TRPC3-Nox2 interaction, without affecting TRPC3-mediated Ca2+ influx. The novel TRPC3 function as a PRROS provides a mechanistic explanation for how diastolic Ca2+ influx specifically encodes signals to induce ROS-mediated maladaptive remodeling and offers new therapeutic possibilities.

[Function and role of transient receptor potential channels].

Transient receptor potential (TRP) proteins are components of Ca(2 +) -permeable non-selective cation channels activated by physical and chemical stimulus except for membrane depolarization. The pathophysiological role of TRP channels is documented in heart failure. Especially, canonical TRP subfamily C (TRPC) channels activated by neurohumoral factors have been implicated in the structural remodeling of the heart. TRPC proteins act not only as components of receptor-activated cation channels, but also as protein scaffold to form protein complex with intracellular signaling proteins, leading to amplification of receptor signaling. Recently, selective inhibitors of TRPC channels have been continuously identified, anticipating the possibility of drug discovery targeting TRPC channels for the treatment of heart failure.

Redox control of cardiovascular homeostasis by angiotensin II.

Covalent modification of sulfur-containing amino acids in proteins by reactive oxygen species (ROS) has been attracting attention as a major post-translational modification regulating intracellular signal transduction pathways. Angiotensin II Ang II, a major physiologically active substrate in renin-angiotensin (RAS) system, plays a central role in the pathophysiology of cardiovascular systems. Many evidences show that Ang II activates several signaling pathways via an oxidative modification of proteins by Ang II-induced ROS. Ang II induced ROS production is predominantly regulated by three enzymes: NADPH oxidase, mitochondrial respiratory complex, and nitric oxide synthase (NOS), and each enzyme-generating ROS are found to activate appropriate signaling pathways via selective oxidation of specific proteins. These reactions are negatively regulated by ROS-scavenging enzymes or disulfide bridge reducing enzymes, and functional disorders of these enzymes are found to cause cardiovascular dysfunctions. Thus, the spatial and temporal regulation of oxidative modification of signaling proteins by ROS is essential to maintain cardiovascular homeostasis by Ang II. This review brings in the new aspect in understanding ROS-mediated regulation of cardiovascular homeostasis by Ang II, and provides the possible mechanisms underlying metamorphosis of cardiovascular homeostasis by ROS.

Mammalian formin Fhod3 plays an essential role in cardiogenesis by organizing myofibrillogenesis.

Heart development requires organized integration of actin filaments into the sarcomere, the contractile unit of myofibrils, although it remains largely unknown how actin filaments are assembled during myofibrillogenesis. Here we show that Fhod3, a member of the formin family of proteins that play pivotal roles in actin filament assembly, is essential for myofibrillogenesis at an early stage of heart development. Fhod3(-/-) mice appear normal up to embryonic day (E) 8.5, when the developing heart, composed of premyofibrils, initiates spontaneous contraction. However, these premyofibrils fail to mature and myocardial development does not continue, leading to embryonic lethality by E11.5. Transgenic expression of wild-type Fhod3 in the heart restores myofibril maturation and cardiomyogenesis, which allow Fhod3(-/-) embryos to develop further. Moreover, cardiomyopathic changes with immature myofibrils are caused in mice overexpressing a mutant Fhod3, defective in binding to actin. These findings indicate that actin dynamics, regulated by Fhod3, participate in sarcomere organization during myofibrillogenesis and thus play a crucial role in heart development.

Hydrogen sulfide anion regulates redox signaling via electrophile sulfhydration.

An emerging aspect of redox signaling is the pathway mediated by electrophilic byproducts, such as nitrated cyclic nucleotide (for example, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP)) and nitro or keto derivatives of unsaturated fatty acids, generated via reactions of inflammation-related enzymes, reactive oxygen species, nitric oxide and secondary products. Here we report that enzymatically generated hydrogen sulfide anion (HS(-)) regulates the metabolism and signaling actions of various electrophiles. HS(-) reacts with electrophiles, best represented by 8-nitro-cGMP, via direct sulfhydration and modulates cellular redox signaling. The relevance of this reaction is reinforced by the significant 8-nitro-cGMP formation in mouse cardiac tissue after myocardial infarction that is modulated by alterations in HS(-) biosynthesis. Cardiac HS(-), in turn, suppresses electrophile-mediated H-Ras activation and cardiac cell senescence, contributing to the beneficial effects of HS(-) on myocardial infarction-associated heart failure. Thus, this study reveals HS(-)-induced electrophile sulfhydration as a unique mechanism for regulating electrophile-mediated redox signaling.

Recombinant mitochondrial transcription factor A protein inhibits nuclear factor of activated T cells signaling and attenuates pathological hypertrophy of cardiac myocytes.

The overexpression of mitochondrial transcription factor A (TFAM) attenuates the decrease in mtDNA copy number after myocardial infarction, ameliorates pathological hypertrophy, and markedly improves survival. However, non-transgenic strategy to increase mtDNA for the treatment of pathological hypertrophy remains unknown. We produced recombinant human TFAM protein (rhTFAM). rhTFAM rapidly entered into mitochondria of cultured cardiac myocytes. rhTFAM increased mtDNA and abolished the activation of nuclear factor of activated T cells (NFAT), which is well known to activate pathological hypertrophy. rhTFAM attenuated subsequent morphological hypertrophy of myocytes as well. rhTFAM would be an attractive molecule in attenuating cardiac pathological hypertrophy.

TRPC3-mediated Ca2+ influx contributes to Rac1-mediated production of reactive oxygen species in MLP-deficient mouse hearts.

Dilated cardiomyopathy (DCM) is a myocardial disorder that is characterized by dilation and dysfunction of the left ventricle (LV). Accumulating evidence has implicated aberrant Ca(2+) signaling and oxidative stress in the progression of DCM, but the molecular details are unknown. In the present study, we report that inhibition of the transient receptor potential canonical 3 (TRPC3) channels partially prevents LV dilation and dysfunction in muscle LIM protein-deficient (MLP (-/-)) mice, a murine model of DCM. The expression level of TRPC3 and the activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) were increased in MLP (-/-) mouse hearts. Acitivity of Rac1, a small GTP-binding protein that participates in NADPH oxidase (Nox) activation, and the production of reactive oxygen species (ROS) were also increased in MLP (-/-) mouse hearts. Treatment with pyrazole-3, a TRPC3 selective inhibitor, strongly suppressed the increased activities of CaMKII and Rac1, as well as ROS production. In contrast, activation of TRPC3 by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or by mechanical stretch, induced ROS production in rat neonatal cardiomyocytes. These results suggest that up-regulation of TRPC3 is responsible for the increase in CaMKII activity and the Nox-mediated ROS production in MLP (-/-) mouse cardiomyocytes, and that inhibition of TRPC3 is an effective therapeutic strategy to prevent the progression of DCM.

Heterologous down-regulation of angiotensin type 1 receptors by purinergic P2Y2 receptor stimulation through S-nitrosylation of NF-kappaB.

Cross-talk between G protein-coupled receptor (GPCR) signaling pathways serves to fine tune cellular responsiveness by neurohumoral factors. Accumulating evidence has implicated nitric oxide (NO)-based signaling downstream of GPCRs, but the molecular details are unknown. Here, we show that adenosine triphosphate (ATP) decreases angiotensin type 1 receptor (AT(1)R) density through NO-mediated S-nitrosylation of nuclear factor κB (NF-κB) in rat cardiac fibroblasts. Stimulation of purinergic P2Y(2) receptor by ATP increased expression of inducible NO synthase (iNOS) through activation of nuclear factor of activated T cells, NFATc1 and NFATc3. The ATP-induced iNOS interacted with p65 subunit of NF-κB in the cytosol through flavin-binding domain, which was indispensable for the locally generated NO-mediated S-nitrosylation of p65 at Cys38. ß-Arrestins anchored the formation of p65/IκBα/ß-arrestins/iNOS quaternary complex. The S-nitrosylated p65 resulted in decreases in NF-κB transcriptional activity and AT(1)R density. In pressure-overloaded mouse hearts, ATP released from cardiomyocytes led to decrease in AT(1)R density through iNOS-mediated S-nitrosylation of p65. These results show a unique regulatory mechanism of heterologous regulation of GPCRs in which cysteine modification of transcriptional factor rather than protein phosphorylation plays essential roles.

Regulation of Angiotensin II receptor signaling by cysteine modification of NF-κB.

Angiotensin II (Ang II) is a major vasoactive peptide of the renin-angiotensin system. Ang II is originally found as one of potent vasoconstrictors, but is now attracted attention as an essential mediator of many cardiovascular problems, including endothelial dysfunction, arrhythmia and structural remodeling of cardiovascular systems. Most of the known pathophysiological effects of Ang II are mediated through Ang type1 receptors (AT(1)Rs), and the up-regulation of AT(1)Rs is one of important causes by which Ang II can contribute to cardiovascular diseases. A growing body of evidence has suggested that reactive oxygen species (ROS) and reactive nitrogen species (RNS) play important roles in the regulation of AT(1)R signaling. In cardiac fibroblasts, stimulation with cytokines or bacterial toxins induces AT(1)R up-regulation through NADPH oxidase-dependent ROS production. In contrast, nitric oxide (NO) decreases AT(1)R density through cysteine modification (S-nitrosylation) of a transcriptional factor, nuclear factor κB (NF-κB). The difference between the effects of ROS and NO on AT(1)R expression may be caused by the difference between intracellular location of ROS signaling and that of NO signaling, as the agonist-induced S-nitrosylation of NF-κB requires a local interaction between NO synthase (NOS) and NF-κB in the perinuclear region. Thus, the spatial and temporal regulation of cysteine modification by ROS or RNS may underlie the resultant changes of AT(1)R signaling induced by agonist stimulation.

[Regulation of cardiovascular functions by the phosphorylation of TRPC channels].

Calcium ions (Ca²(+)) play an essential role in homeostasis and the activity of cardiovascular cells. Ca²(+) influx across the plasma membrane induced by neurohumoral factors or mechanical stress elicits physiologically relevant timing and spatial patterns of Ca²(+) signaling, which leads to the activation of various cardiovascular functions, such as muscle contraction, gene expression, and hypertrophic growth of myocytes. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca²(+) signaling pathway required for pathological hypertrophy. We have recently found that the inhibition of cGMP-selective phosphodiesterase 5 (PDE5) suppresses agonist- and mechanical stretch-induced hypertrophy through inhibition of Ca²(+) influx in rat cardiomyocytes. The inhibition of PDE5 suppressed the increase in frequency of Ca²(+) spikes induced by receptor stimulation or mechanical stretch. Activation of protein kinase G by PDE5 inhibition phosphorylated TRPC6 proteins at Thr69 and prevented TRPC6-mediated Ca²(+) influx. Substitution of Ala for Thr69 in TRPC6 abolished the antihypertrophic effects of PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlies the prevention of cardiac hypertrophy by PDE5 inhibition. As TRPC6 proteins are also expressed in vascular smooth muscle cells and reportedly participate in vascular remodeling, TRPC6 blockade may be an effective therapeutic strategy for preventing pathologic cardiovascular remodeling.

Phosphorylation of TRPC6 channels at Thr69 is required for anti-hypertrophic effects of phosphodiesterase 5 inhibition.

Activation of Ca(2+) signaling induced by receptor stimulation and mechanical stress plays a critical role in the development of cardiac hypertrophy. A canonical transient receptor potential protein subfamily member, TRPC6, which is activated by diacylglycerol and mechanical stretch, works as an upstream regulator of the Ca(2+) signaling pathway. Although activation of protein kinase G (PKG) inhibits TRPC6 channel activity and cardiac hypertrophy, respectively, it is unclear whether PKG suppresses cardiac hypertrophy through inhibition of TRPC6. Here, we show that inhibition of cGMP-selective PDE5 (phosphodiesterase 5) suppresses endothelin-1-, diacylglycerol analog-, and mechanical stretch-induced hypertrophy through inhibition of Ca(2+) influx in rat neonatal cardiomyocytes. Inhibition of PDE5 suppressed the increase in frequency of Ca(2+) spikes induced by agonists or mechanical stretch. However, PDE5 inhibition did not suppress the hypertrophic responses induced by high KCl or the activation of protein kinase C, suggesting that PDE5 inhibition suppresses Ca(2+) influx itself or molecule(s) upstream of Ca(2+) influx. PKG activated by PDE5 inhibition phosphorylated TRPC6 proteins at Thr(69) and prevented TRPC6-mediated Ca(2+) influx. Substitution of Ala for Thr(69) in TRPC6 abolished the anti-hypertrophic effects of PDE5 inhibition. In addition, chronic PDE5 inhibition by oral sildenafil treatment actually induced TRPC6 phosphorylation in mouse hearts. Knockdown of RGS2 (regulator of G protein signaling 2) and RGS4, both of which are activated by PKG to reduce G alpha(q)-mediated signaling, did not affect the suppression of receptor-activated Ca(2+) influx by PDE5 inhibition. These results suggest that phosphorylation and functional suppression of TRPC6 underlie prevention of pathological hypertrophy by PDE5 inhibition.