Ultrastructure of precapillary sphincters and the neurovascular unit.

Neurons communicate with vasculature to regulate blood flow in the brain, a process maintained by the neurovascular unit (NVU). This interaction, termed neurovascular coupling, is believed to involve astrocytes or molecules capable of traversing the astrocytic endfeet. The precise mechanism, however, remains elusive. Using large 3D electron microscopy datasets, we can now study the entire NVU in context of vascular hierarchy. This study presents evidence supporting the role of precapillary sphincters as a nexus for neurovascular coupling and endothelial transcytosis. It also highlights the role of fibroblast-synthesized collagen in fortifying first-order capillaries. Furthermore, I demonstrate how astrocytic endfeet establish a barrier for fluid flow and reveal that the cortex's microvasculature is semicircled by an unexpected arrangement of parenchymal neuronal processes around penetrating arterioles and arterial-end capillaries in both mouse and human brains. These discoveries offer insights into the NVU's structure and its operational mechanisms, potentially aiding researchers in devising new strategies for preserving cognitive function and promoting healthy aging.

Impaired dynamics of precapillary sphincters and pericytes at first-order capillaries predict reduced neurovascular function in the aging mouse brain.

The microvascular inflow tract, comprising the penetrating arterioles, precapillary sphincters and first-order capillaries, is the bottleneck for brain blood flow and energy supply. Exactly how aging alters the structure and function of the microvascular inflow tract remains unclear. By in vivo four-dimensional two-photon imaging, we reveal an age-dependent decrease in vaso-responsivity accompanied by a decrease in vessel density close to the arterioles and loss of vascular mural cell processes, although the number of mural cell somas and their alpha smooth muscle actin density were preserved. The age-related reduction in vascular reactivity was mostly pronounced at precapillary sphincters, highlighting their crucial role in capillary blood flow regulation. Mathematical modeling revealed impaired pressure and flow control in aged mice during vasoconstriction. Interventions that preserve dynamics of cerebral blood vessels may ameliorate age-related decreases in blood flow and prevent brain frailty.

Brain capillary pericytes and neurovascular coupling.

The neurovascular coupling ensures that cerebral activity is matched by the relevant blood flow. The control of the blood flow is mediated by capillaries and by the precapillary aterioles. It is the tone of the mural cells, which include pericytes, smooth muscle cells and cells with intermediate phenotypes between pericytes and smooth muscle cells, that determine the the diameter of the blood vessels and consequently the flow. Here we discuss the structure of these blood vessels and the excitationcontraction coupling of the mural cells.

Precapillary sphincters maintain perfusion in the cerebral cortex.

Active nerve cells release vasodilators that increase their energy supply by dilating local blood vessels, a mechanism termed neurovascular coupling and the basis of BOLD functional neuroimaging signals. Here, we reveal a mechanism for cerebral blood flow control, a precapillary sphincter at the transition between the penetrating arteriole and first order capillary, linking blood flow in capillaries to the arteriolar inflow. The sphincters are encircled by contractile mural cells, which are capable of bidirectional control of the length and width of the enclosed vessel segment. The hemodynamic consequence is that precapillary sphincters can generate the largest changes in the cerebrovascular flow resistance of all brain vessel segments, thereby controlling capillary flow while protecting the downstream capillary bed and brain tissue from adverse pressure fluctuations. Cortical spreading depolarization constricts sphincters and causes vascular trapping of blood cells. Thus, precapillary sphincters are bottlenecks for brain capillary blood flow.

Comparison of Cultured Human Cardiomyocyte Clusters Obtained from Embryos/Fetuses or Derived from Human Embryonic Stem Cells.

Cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs) are used to study cardiogenesis and mechanisms of heart disease, and are being used in methods for toxiological screening of drugs. The phenotype of stem-cell-derived CMs should ideally resemble native CMs. Here, we compare embryonic/fetal CMs with hESC-derived CMs according to function and morphology. CM clusters were obtained from human embryonic/fetal hearts from elective terminated pregnancies before gestational week 12, and separated into atrial and ventricular tissues. Specific markers for embryonic CMs and primary cilia were visualized using immunofluorescence microscopy analysis. Contracting human embryonic cardiomyocyte (hECM) clusters morphologically and phenotypically resemble CMs in the embryonic/fetal heart. In addition, the contracting hECM clusters expressed primary cilia similar to that of cells in the embryonic/fetal heart. The electrophysiological characteristics of atrial and ventricular CMs were established by recording action potentials (APs) using sharp electrodes. In contrast to ventricular APs, atrial APs displayed a marked early repolarization followed by a plateau phase. hESC-CMs displayed a continuum of AP shapes. In all embryonic/fetal clusters, both atrial and ventricular, AP duration was prolonged by exposure to the KV11.1 channel inhibitor dofetilide (50 nM); however, the prolongation was not significant, possibly due to the relatively small number of experiments. This study provides novel information on APs and functional characteristics of atrial and ventricular CMs in first trimester hearts, and demonstrates that Kv11.1 channels play a functional role already at these early stages. These results provide information needed to validate methods being developed on the basis of in vitro-derived CMs from either hESC or iPSC, and although there was a good correlation between the morphology of the two types of CMs, differences in electrophysiological characteristics exist.

Triggered intracellular calcium waves in dog and human left atrial myocytes from normal and failing hearts.

AIMS: Abnormal intracellular Ca2+ cycling contributes to triggered activity and arrhythmias in the heart. We investigated the properties and underlying mechanisms for systolic triggered Ca2+ waves in left atria from normal and failing dog hearts. METHODS AND RESULTS: Intracellular Ca2+ cycling was studied using confocal microscopy during rapid pacing of atrial myocytes (36 °C) isolated from normal and failing canine hearts (ventricular tachypacing model). In normal atrial myocytes (NAMs), Ca2+ waves developed during rapid pacing at rates ≥ 3.3 Hz and immediately disappeared upon cessation of pacing despite high sarcoplasmic reticulum (SR) load. In heart failure atrial myocytes (HFAMs), triggered Ca2+ waves (TCWs) developed at a higher incidence at slower rates. Because of their timing, TCW development relies upon action potential (AP)-evoked Ca2+ entry. The distribution of Ca2+ wave latencies indicated two populations of waves, with early events representing TCWs and late events representing conventional spontaneous Ca2+ waves. Latency analysis also demonstrated that TCWs arise after junctional Ca2+ release has occurred and spread to non-junctional (cell core) SR. TCWs also occurred in intact dog atrium and in myocytes from humans and pigs. ß-adrenergic stimulation increased Ca2+ release and abolished TCWs in NAMs but was ineffective in HFAMs making this a potentially effective adaptive mechanism in normals but potentially arrhythmogenic in HF. Block of Ca-calmodulin kinase II also abolished TCWs, suggesting a role in TCW formation. Pharmacological manoeuvres that increased Ca2+ release suppressed TCWs as did interventions that decreased Ca2+ release but these also severely reduced excitation-contraction coupling. CONCLUSION: TCWs develop during the atrial AP and thus could affect AP duration, producing repolarization gradients and creating a substrate for reentry, particularly in HF where they develop at slower rates and a higher incidence. TCWs may represent a mechanism for the initiation of atrial fibrillation particularly in HF.

Human Embryonic Stem Cell-Derived Cardiomyocytes Self-Arrange with Areas of Different Subtypes During Differentiation.

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of ß-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium.

The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K+ channel KV11.1 and its auxiliary ß subunit KCNE2. KV11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the KV11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death--conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine KV11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human KV11.1 and KV11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine KV11.1 currents were larger compared to human KV11.1, and the voltage dependence of activation was shifted to more negative values with V1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine KV11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine KV11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of KV11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.

Preservation of cardiac function by prolonged action potentials in mice deficient of KChIP2.

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVß2 protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice.

Loss of K+ currents in heart failure is accentuated in KChIP2 deficient mice.

INTRODUCTION: KV 4 together with KV Channel-Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (I(to,f)) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of I(to,f). We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K(+) currents in HF. METHODS AND RESULTS: HF was induced by aortic banding in wild-type (WT) and KChIP2 deficient (KChIP2(-/-)) mice, evaluated by echocardiography. Action potentials were measured by floating microelectrodes in intact hearts. Ventricular cardiomyocytes were isolated and whole-cell currents were recorded by patch clamp. Left ventricular action potentials in KChIP2(-/-) mice were prolonged in a rate dependent manner, consistent with patch-clamp data showing loss of a fast recovering outward K(+) current and upregulation of the slow recovering I(to,s) and I(Kur). HF decreased all outward K(+) currents in WT mice and did not change the relative contribution of I(to,f) in WT mice. Compared to WT HF, KChIP2(-/-) HF had a larger reduction of K(+) -current density. However, the relative APD prolongation caused by HF was shorter for KChIP2(-/-) compared with WT, and the APs of the 2 HF mouse types were indistinguishable. CONCLUSION: I(to,f) is just one of many K(+) currents being downregulated in murine HF. The downregulation of repolarizing currents in HF is accentuated in KChIP2(-/-) mice. However, the prolongation of APs associated with HF is less in KChIP2(-/-) compared to WT, suggesting other compensatory mechanism(s) in the KChIP2(-/-) mouse.

Development of heart failure is independent of K+ channel-interacting protein 2 expression.

Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K(+) channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K(+) current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2(-/-) mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2(-/-) mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2(-/-) control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham-operated mice of either genotype. Pacing-induced ventricular tachycardia (VT) was observed in 5/10 sham-operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham-operated KChIP2(-/-) mice were resistant to pacing-induced VT resulting in only 1/10 inducible mice. KChIP2(-/-) with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K(+) current.

TMEM16F (Anoctamin 6), an anion channel of delayed Ca(2+) activation.

Members of the TMEM16 (Anoctamin) family of membrane proteins have been shown to be essential constituents of the Ca(2+)-activated Cl(-) channel (CaCC) in many cell types. In this study, we have investigated the electrophysiological properties of mouse TMEM16F. Heterologous expression of TMEM16F in HEK293 cells resulted in plasma membrane localization and an outwardly rectifying ICl,Ca that was activated with a delay of several minutes. Furthermore, a significant Na(+) current was activated, and the two permeabilities were correlated according to PNa = 0.3 PCl. The current showed an EC50 of 100 µM intracellular free Ca(2+) concentration and an Eisenman type 1 anion selectivity sequence of PSCN > PI > PBr > PCl > PAsp. The mTMEM16F-associated ICl,Ca was abolished in one mutant of the putative pore region (R592E) but retained in two other mutants (K616E and R636E). The mutant K616E had a lower relative permeability to iodide, and the mutant R636E had an altered anion selectivity sequence (PSCN = PI = PBr = PCl > PAsp). Our data provide evidence that TMEM16F constitutes a Ca(2+)-activated anion channel or a pore-forming subunit of an anion channel with properties distinct from TMEM16A.

A novel KCND3 gain-of-function mutation associated with early-onset of persistent lone atrial fibrillation.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current (Ito) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K(+) Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. METHODS AND RESULTS: Two hundred and nine unrelated early-onset lone AF patients (<40 years) were recruited. The entire coding sequence of KCND3 and KCNIP2 was bidirectionally sequenced. One novel non-synonymous mutation A545P was found in KCND3 and was neither present in the control group (n = 432 alleles) nor in any publicly available database. The proband had onset of persistent AF at the age of 22, and no mutations in genes previously associated with AF were found. Electrophysiological analysis of KV4.3-A545P expressed in CHO-K1 cells, revealed that peak-current density was increased and the onset of inactivation was slower compared with WT, resulting in a significant gain-of-function both in the absence and the presence of KChIP2. CONCLUSION: Gain-of-function mutations in KV4.3 have previously been described in Brugada Syndrome, however, this is the first report of a KV4.3 gain-of-function mutation in early-onset lone AF. This association of KV4.3 gain-of-function and early-onset lone AF further supports the hypothesis that increased potassium current enhances AF susceptibility.

Characterization and mechanisms of action of novel NaV1.5 channel mutations associated with Brugada syndrome.

BACKGROUND: Brugada syndrome is a heterogeneous heart rhythm disorder characterized by an atypical right bundle block pattern with ST-segment elevation and T-wave inversion in the right precordial leads. Loss-of-function mutations in SCN5A encoding the cardiac sodium channel Na(V)1.5 are associated with Brugada syndrome. We found novel mutations in SCN5A in 2 different families diagnosed with Brugada syndrome and investigated how those affected Na(V)1.5 channel function. METHODS AND RESULTS: We performed genetic testing of the probands' genomic DNA. After site-directed mutagenesis and transfection, whole-cell currents were recorded for Na(V)1.5 wild type and mutants heterologously expressed in Chinese hamster ovary-K1 cells. Proband 1 had two novel Na(V)1.5 mutations: Na(V)1.5-R811H and Na(V)1.5-R620H. The Na(V)1.5-R811H mutation showed a significant loss of function in peak Na(+) current density and alteration of biophysical kinetic parameters (inactivation and recovery from inactivation), whereas Na(V)1.5-R620H had no significant effect on the current. Proband 2 had a novel Na(V)1.5-S1218I mutation. Na(V)1.5-S1218I had complete loss of function, and 1:1 expression of Na(V)1.5-wild type and Na(V)1.5-S1218I mimicking the heterozygous state revealed a 50% reduction in current compared with wild type, suggesting a functional haploinsufficiency in the patient. CONCLUSIONS: Na(V)1.5-S1218I and R811H are novel loss-of-function mutations in the SCN5A gene causing Brugada syndrome.

Impact of KChIP2 on Cardiac Electrophysiology and the Progression of Heart Failure.

Electrophysiological remodeling of cardiac potassium ion channels is important in the progression of heart failure. A reduction of the transient outward potassium current (I(to)) in mammalian heart failure is consistent with a reduced expression of potassium channel interacting protein 2 (KChIP2, a K(V)4 subunit). Approaches have been made to investigate the role of KChIP2 in shaping cardiac I(to), including the use of transgenic KChIP2 deficient mice and viral overexpression of KChIP2. The interplay between I(to) and myocardial calcium handling is pivotal in the development of heart failure, and is further strengthened by the dual role of KChIP2 as a functional subunit on both K(V)4 and Ca(V)1.2. Moreover, the potential arrhythmogenic consequence of reduced I(to) may contribute to the high relative incidence of sudden death in the early phases of human heart failure. With this review, we offer an overview of the insights into the physiological and pathological roles of KChIP2 and we discuss the limitations of translating the molecular basis of electrophysiological remodeling from animal models of heart failure to the clinical setting.

Multiple arrhythmic syndromes in a newborn, owing to a novel mutation in SCN5A.

BACKGROUND: Mutations in the SCN5A gene have been linked to Brugada syndrome (BrS), conduction disease, Long QT syndrome (LQT3), atrial fibrillation (AF), and to pre- and neonatal ventricular arrhythmias. OBJECTIVE: The objective of this study is to characterize a novel mutation in Na(v)1.5 found in a newborn with fetal chaotic atrial tachycardia, post-partum intraventricular conduction delay, and QT interval prolongation. METHODS: Genomic DNA was isolated and all exons and intron borders of 15 ion-channel genes were sequenced, revealing a novel missense mutation (Q270K) in SCN5A. Na(v)1.5 wild type (WT) and Q270K were expressed in CHO-K1 with and without the Na(v)ß1 subunit. Results. Patch-clamp analysis showed ∼40% reduction in peak sodium channel current (I(Na)) density for Q270K compared with WT. Fast and slow decay of I(Na) were significantly slower in Q270K. Steady-state activation and inactivation of Q270K channels were shifted to positive potentials, and window current was increased. The tetrodotoxin-sensitive late I(Na) was increased almost 3-fold compared with WT channels. Ranolazine reduced late I(Na) in WT and Q270K channels, while exerting minimal effects on peak I(Na). CONCLUSION: The Q270K mutation in SCN5A reduces peak I(Na) while augmenting late I(Na), and may thus underlie the development of atrial tachycardia, intraventricular conduction delay, and QT interval prolongation in an infant.