Long-wavelength traveling waves of vasomotion modulate the perfusion of cortex.

Brain arterioles are active, multicellular complexes whose diameters oscillate at ∼ 0.1 Hz. We assess the physiological impact and spatiotemporal dynamics of vaso-oscillations in the awake mouse. First, vaso-oscillations in penetrating arterioles, which source blood from pial arterioles to the capillary bed, profoundly impact perfusion throughout neocortex. The modulation in flux during resting-state activity exceeds that of stimulus-induced activity. Second, the change in perfusion through arterioles relative to the change in their diameter is weak. This implies that the capillary bed dominates the hydrodynamic resistance of brain vasculature. Lastly, the phase of vaso-oscillations evolves slowly along arterioles, with a wavelength that exceeds the span of the cortical mantle and sufficient variability to establish functional cortical areas as parcels of uniform phase. The phase-gradient supports traveling waves in either direction along both pial and penetrating arterioles. This implies that waves along penetrating arterioles can mix, but not directionally transport, interstitial fluids.

Right ventricular cardiomyocyte expansion accompanies cardiac regeneration in newborn mice after large left ventricular infarcts.

Cauterization of the root of the left coronary artery (LCA) in the neonatal heart on postnatal day 1 (P1) resulted in large, reproducible lesions of the left ventricle (LV), and an attendant marked adaptive response in the right ventricle (RV). The response of both chambers to LV myocardial infarction involved enhanced cardiomyocyte (CM) division and binucleation, as well as LV revascularization, leading to restored heart function within 7 days post surgery (7 dps). By contrast, infarction of P3 mice resulted in cardiac scarring without a significant regenerative and adaptive response of the LV and the RV, leading to subsequent heart failure and death within 7 dps. The prominent RV myocyte expansion in P1 mice involved an acute increase in pulmonary arterial pressure and a unique gene regulatory response, leading to an increase in RV mass and preserved heart function. Thus, distinct adaptive mechanisms in the RV, such as CM proliferation and RV expansion, enable marked cardiac regeneration of the infarcted LV at P1 and full functional recovery.

Adenylyl cyclase isoform 1 contributes to sinoatrial node automaticity via functional microdomains.

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by ß-adrenergic receptor (ß-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the ß-AR pathway that catalyzes the production of cAMP. There are current gaps in our knowledge regarding the dominant AC isoforms and the specific roles of Ca2+-activated ACs in the SAN. The current study tests the hypothesis that distinct AC isoforms are preferentially expressed in the SAN and compartmentalize within microdomains to orchestrate heart rate regulation during ß-AR signaling. In contrast to atrial and ventricular myocytes, SAN cells express a diverse repertoire of ACs, with ACI as the predominant Ca2+-activated isoform. Although ACI-KO (ACI-/-) mice exhibit normal cardiac systolic or diastolic function, they experience SAN dysfunction. Similarly, SAN-specific CRISPR/Cas9-mediated gene silencing of ACI results in sinus node dysfunction. Mechanistically, hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channels form functional microdomains almost exclusively with ACI, while ryanodine receptor and L-type Ca2+ channels likely compartmentalize with ACI and other AC isoforms. In contrast, there were no significant differences in T-type Ca2+ and Na+ currents at baseline or after ß-AR stimulation between WT and ACI-/- SAN cells. Due to its central characteristic feature as a Ca2+-activated isoform, ACI plays a unique role in sustaining the rise of local cAMP and heart rates during ß-AR stimulation. The findings provide insights into the critical roles of the Ca2+-activated isoform of AC in sustaining SAN automaticity that is distinct from contractile cardiomyocytes.

Genetically engineered mice for combinatorial cardiovascular optobiology.

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

Local IP3 receptor-mediated Ca2+ signals compound to direct blood flow in brain capillaries.

Healthy brain function depends on the finely tuned spatial and temporal delivery of blood-borne nutrients to active neurons via the vast, dense capillary network. Here, using in vivo imaging in anesthetized mice, we reveal that brain capillary endothelial cells control blood flow through a hierarchy of IP3 receptor-mediated Ca2+ events, ranging from small, subsecond protoevents, reflecting Ca2+ release through a small number of channels, to high-amplitude, sustained (up to ~1 min) compound events mediated by large clusters of channels. These frequent (~5000 events/s per microliter of cortex) Ca2+ signals are driven by neuronal activity, which engages Gq protein-coupled receptor signaling, and are enhanced by Ca2+ entry through TRPV4 channels. The resulting Ca2+-dependent synthesis of nitric oxide increases local blood flow selectively through affected capillary branches, providing a mechanism for high-resolution control of blood flow to small clusters of neurons.

Calcium Signal Profiles in Vascular Endothelium from Cdh5-GCaMP8 and Cx40-GCaMP2 Mice.

INTRODUCTION: Studies in Cx40-GCaMP2 mice, which express calcium biosensor GCaMP2 in the endothelium under connexin 40 promoter, have identified the unique properties of endothelial calcium signals. However, Cx40-GCaMP2 mouse is associated with a narrow dynamic range and lack of signal in the venous endothelium. Recent studies have proposed many GCaMPs (GCaMP5/6/7/8) with improved properties although their performance in endothelium-specific calcium studies is not known. METHODS: We characterized a newly developed mouse line that constitutively expresses GCaMP8 in the endothelium under the VE-cadherin (Cdh5-GCaMP8) promoter. Calcium signals through endothelial IP3 receptors and TRP vanilloid 4 (TRPV4) ion channels were recorded in mesenteric arteries (MAs) and veins from Cdh5-GCaMP8 and Cx40-GCaMP2 mice. RESULTS: Cdh5-GCaMP8 mice showed lower baseline fluorescence intensity, higher dynamic range, and higher amplitudes of individual calcium signals than Cx40-GCaMP2 mice. Importantly, Cdh5-GCaMP8 mice enabled the first recordings of discrete calcium signals in the intact venous endothelium and revealed striking differences in IP3 receptor and TRPV4 channel calcium signals between MAs and mesenteric veins. CONCLUSION: Our findings suggest that Cdh5-GCaMP8 mice represent significant improvements in dynamic range, sensitivity for low-intensity signals, and the ability to record calcium signals in venous endothelium.

Contractile pericytes determine the direction of blood flow at capillary junctions.

The essential function of the circulatory system is to continuously and efficiently supply the O2 and nutrients necessary to meet the metabolic demands of every cell in the body, a function in which vast capillary networks play a key role. Capillary networks serve an additional important function in the central nervous system: acting as a sensory network, they detect neuronal activity in the form of elevated extracellular K+ and initiate a retrograde, propagating, hyperpolarizing signal that dilates upstream arterioles to rapidly increase local blood flow. Yet, little is known about how blood entering this network is distributed on a branch-to-branch basis to reach specific neurons in need. Here, we demonstrate that capillary-enwrapping projections of junctional, contractile pericytes within a postarteriole transitional region differentially constrict to structurally and dynamically determine the morphology of capillary junctions and thereby regulate branch-specific blood flow. We further found that these contractile pericytes are capable of receiving propagating K+-induced hyperpolarizing signals propagating through the capillary network and dynamically channeling red blood cells toward the initiating signal. By controlling blood flow at junctions, contractile pericytes within a functionally distinct postarteriole transitional region maintain the efficiency and effectiveness of the capillary network, enabling optimal perfusion of the brain.

Exposure to lipopolysaccharide (LPS) reduces contractile response of small airways from GSTCD-/- mice.

INTRODUCTION: Genome-Wide Association Studies suggest glutathione S transferase C terminal domain (GSTCD) may play a role in development of Chronic Obstructive Pulmonary Disease. We aimed to define the potential role of GSTCD in airway inflammation and contraction using precision cut lung slice (PCLS) from wild-type (GSTCD+/+) and GSTCD knockout mice (GSTCD-/-). METHODS: PCLS from age and gender matched GSTCD+/+ and GSTCD-/- mice were prepared using a microtome. Contraction was studied after applying either a single dose of Methacholine (Mch) (1 µM) or different doses of Mch (0.001 to 100 µM). Each slice was then treated with lipopolysaccharide (LPS) or vehicle (PBS) for 24 hours. PCLS contraction in the same airway was repeated before and after stimulation. Levels of TNFα production was also measured. RESULTS: There were no differences in contraction of PCLS from GSTCD+/+ and GSTCD-/- mice in response to Mch (EC50 of GSTCD+/+ vs GSTCD-/- animals: 100.0±20.7 vs 107.7±24.5 nM, p = 0.855, n = 6 animals/group). However, after LPS treatment, there was a 31.6% reduction in contraction in the GSTCD-/- group (p = 0.023, n = 6 animals). There was no significant difference between PBS and LPS treatment groups in GSTCD+/+ animals. We observed a significant increase in TNFα production induced by LPS in GSTCD-/- lung slices compared to the GSTCD+/+ LPS treated slices. CONCLUSION: GSTCD knockout mice showed an increased responsiveness to LPS (as determined by TNFα production) that was accompanied by a reduced contraction of small airways in PCLS. These data highlight an unrecognised potential function of GSTCD in mediating inflammatory signals that affect airway responses.

Central role of IP3R2-mediated Ca2+ oscillation in self-renewal of liver cancer stem cells elucidated by high-signal ER sensor.

Ca2+ oscillation is a system-level property of the cellular Ca2+-handling machinery and encodes diverse physiological and pathological signals. The present study tests the hypothesis that Ca2+ oscillations play a vital role in maintaining the stemness of liver cancer stem cells (CSCs), which are postulated to be responsible for cancer initiation and progression. We found that niche factor-stimulated Ca2+ oscillation is a signature feature of CSC-enriched Hep-12 cells and purified α2δ1+ CSC fractions from hepatocellular carcinoma cell lines. In Hep-12 cells, the Ca2+ oscillation frequency positively correlated with the self-renewal potential. Using a newly developed high signal, endoplasmic reticulum (ER) localized Ca2+ sensor GCaMP-ER2, we demonstrated CSC-distinctive oscillatory ER Ca2+ release controlled by the type 2 inositol 1,4,5-trisphosphate receptor (IP3R2). Knockdown of IP3R2 severely suppressed the self-renewal capacity of liver CSCs. We propose that targeting the IP3R2-mediated Ca2+ oscillation in CSCs might afford a novel, physiologically inspired anti-tumor strategy for liver cancer.

Extracellular histones induce calcium signals in the endothelium of resistance-sized mesenteric arteries and cause loss of endothelium-dependent dilation.

Histone proteins are elevated in the circulation after traumatic injury owing to cellular lysis and release from neutrophils. Elevated circulating histones in trauma contribute to coagulopathy and mortality through a mechanism suspected to involve endothelial cell (EC) dysfunction. However, the functional consequences of histone exposure on intact blood vessels are unknown. Here, we sought to understand the effects of clinically relevant concentrations of histones on the endothelium in intact, resistance-sized, mesenteric arteries (MAs). EC Ca2+ was measured with high spatial and temporal resolution in MAs from mice selectively expressing the EC-specific, genetically encoded ratiometric Ca2+ indicator, Cx40-GCaMP-GR, and vessel diameter was measured by edge detection. Application of purified histone protein directly to the endothelium of en face mouse and human MA preparations produced large Ca2+ signals that spread within and between ECs. Surprisingly, luminal application of histones had no effect on the diameter of pressurized arteries. Instead, after prolonged exposure (30 min), it reduced dilations to endothelium-dependent vasodilators and ultimately caused death of ~25% of ECs, as evidenced by markedly elevated cytosolic Ca2+ levels (793 ± 75 nM) and uptake of propidium iodide. Removal of extracellular Ca2+ but not depletion of intracellular Ca2+ stores prevented histone-induced Ca2+ signals. Histone-induced signals were not suppressed by transient receptor potential vanilloid 4 (TRPV4) channel inhibition (100 nM GSK2193874) or genetic ablation of TRPV4 channels or Toll-like receptor receptors. These data demonstrate that histones are robust activators of noncanonical EC Ca2+ signaling, which cause vascular dysfunction through loss of endothelium-dependent dilation in resistance-sized MAs. NEW & NOTEWORTHY We describe the first use of the endothelial cell (EC)-specific, ratiometric, genetically encoded Ca2+ indicator, Cx40-GCaMP-GR, to study the effect of histone proteins on EC Ca2+ signaling. We found that histones induce an influx of Ca2+ in ECs that does not cause vasodilation but instead causes Ca2+ overload, EC death, and vascular dysfunction in the form of lost endothelium-dependent dilation.

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45+ cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.

Nitric Oxide-Dependent Feedback Loop Regulates Transient Receptor Potential Vanilloid 4 (TRPV4) Channel Cooperativity and Endothelial Function in Small Pulmonary Arteries.

BACKGROUND: Recent studies demonstrate that spatially restricted, local Ca2+ signals are key regulators of endothelium-dependent vasodilation in systemic circulation. There are drastic functional differences between pulmonary arteries (PAs) and systemic arteries, but the local Ca2+ signals that control endothelium-dependent vasodilation of PAs are not known. Localized, unitary Ca2+ influx events through transient receptor potential vanilloid 4 (TRPV4) channels, termed TRPV4 sparklets, regulate endothelium-dependent vasodilation in resistance-sized mesenteric arteries via activation of Ca2+-dependent K+ channels. The objective of this study was to determine the unique functional roles, signaling targets, and endogenous regulators of TRPV4 sparklets in resistance-sized PAs. METHODS AND RESULTS: Using confocal imaging, custom image analysis, and pressure myography in fourth-order PAs in conjunction with knockout mouse models, we report a novel Ca2+ signaling mechanism that regulates endothelium-dependent vasodilation in resistance-sized PAs. TRPV4 sparklets exhibit distinct spatial localization in PAs when compared with mesenteric arteries, and preferentially activate endothelial nitric oxide synthase (eNOS). Nitric oxide released by TRPV4-endothelial nitric oxide synthase signaling not only promotes vasodilation, but also initiates a guanylyl cyclase-protein kinase G-dependent negative feedback loop that inhibits cooperative openings of TRPV4 channels, thus limiting sparklet activity. Moreover, we discovered that adenosine triphosphate dilates PAs through a P2 purinergic receptor-dependent activation of TRPV4 sparklets. CONCLUSIONS: Our results reveal a spatially distinct TRPV4-endothelial nitric oxide synthase signaling mechanism and its novel endogenous regulators in resistance-sized PAs.

Cadherin-11 Overexpression Induces Extracellular Matrix Remodeling and Calcification in Mature Aortic Valves.

OBJECTIVE: Calcific aortic valve (AoV) disease is a significant clinical problem for which the regulatory mechanisms are poorly understood. Enhanced cell-cell adhesion is a common mechanism of cellular aggregation, but its role in calcific lesion formation is not known. Cadherin-11 (Cad-11) has been associated with lesion formation in vitro, but its function during adult valve homeostasis and pathogenesis is not known. This study aims to elucidate the specific functions of Cad-11 and its downstream targets, RhoA and Sox9, in extracellular matrix remodeling and AoV calcification. APPROACH AND RESULTS: We conditionally overexpressed Cad-11 in murine heart valves using a novel double-transgenic Nfatc1(Cre);R26-Cad11(TglTg) mouse model. These mice developed hemodynamically significant aortic stenosis with prominent calcific lesions in the AoV leaflets. Cad-11 overexpression upregulated downstream targets, RhoA and Sox9, in the valve interstitial cells, causing calcification and extensive pathogenic extracellular matrix remodeling. AoV interstitial cells overexpressing Cad-11 in an osteogenic environment in vitro rapidly form calcific nodules analogous to in vivo lesions. Molecular analyses revealed upregulation of osteoblastic and myofibroblastic markers. Treatment with a Rho-associated protein kinase inhibitor attenuated nodule formation, further supporting that Cad-11-driven calcification acts through the small GTPase RhoA/Rho-associated protein kinase signaling pathway. CONCLUSIONS: This study identifies one of the underlying molecular mechanisms of heart valve calcification and demonstrates that overexpression of Cad-11 upregulates RhoA and Sox9 to induce calcification and extracellular matrix remodeling in adult AoV pathogenesis. The findings provide a potential molecular target for clinical treatment.

Complex disease and phenotype mapping in the domestic dog.

The domestic dog is becoming an increasingly valuable model species in medical genetics, showing particular promise to advance our understanding of cancer and orthopaedic disease. Here we undertake the largest canine genome-wide association study to date, with a panel of over 4,200 dogs genotyped at 180,000 markers, to accelerate mapping efforts. For complex diseases, we identify loci significantly associated with hip dysplasia, elbow dysplasia, idiopathic epilepsy, lymphoma, mast cell tumour and granulomatous colitis; for morphological traits, we report three novel quantitative trait loci that influence body size and one that influences fur length and shedding. Using simulation studies, we show that modestly larger sample sizes and denser marker sets will be sufficient to identify most moderate- to large-effect complex disease loci. This proposed design will enable efficient mapping of canine complex diseases, most of which have human homologues, using far fewer samples than required in human studies.

Arterial α2-Na+ pump expression influences blood pressure: lessons from novel, genetically engineered smooth muscle-specific α2 mice.

Arterial myocytes express α1-catalytic subunit isoform Na(+) pumps (75-80% of total), which are ouabain resistant in rodents, and high ouabain affinity α2-Na(+) pumps. Mice with globally reduced α2-pumps (but not α1-pumps), mice with mutant ouabain-resistant α2-pumps, and mice with a smooth muscle (SM)-specific α2-transgene (α2 (SM-Tg)) that induces overexpression all have altered blood pressure (BP) phenotypes. We generated α2 (SM-DN) mice with SM-specific α2 (not α1) reduction (>50%) using nonfunctional dominant negative (DN) α2. We compared α2 (SM-DN) and α2 (SM-Tg) mice to controls to determine how arterial SM α2-pumps affect vasoconstriction and BP. α2 (SM-DN) mice had elevated basal mean BP (mean BP by telemetry: 117 ± 4 vs. 106 ± 1 mmHg, n = 7/7, P < 0.01) and enhanced BP responses to chronic ANG II infusion (240 ng·kg(-1)·min(-1)) and high (6%) NaCl. Several arterial Ca(2+) transporters, including Na(+)/Ca(2+) exchanger 1 (NCX1) and sarcoplasmic reticulum and plasma membrane Ca(2+) pumps [sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 (SERCA2) and plasma membrane Ca(2+)-ATPase 1 (PMCA1)], were also reduced (>50%). α2 (SM-DN) mouse isolated small arteries had reduced myogenic reactivity, perhaps because of reduced Ca(2+) transporter expression. In contrast, α2 (SM-Tg) mouse aortas overexpressed α2 (>2-fold), NCX1, SERCA2, and PMCA1 (43). α2 (SM-Tg) mice had reduced basal mean BP (104 ± 1 vs. 109 ± 2 mmHg, n = 15/9, P < 0.02) and attenuated BP responses to chronic ANG II (300-400 ng·kg(-1)·min(-1)) with or without 2% NaCl but normal myogenic reactivity. NCX1 expression was inversely related to basal BP in SM-α2 engineered mice but was directly related in SM-NCX1 engineered mice. NCX1, which usually mediates arterial Ca(2+) entry, and α2-Na(+) pumps colocalize at plasma membrane-sarcoplasmic reticulum junctions and functionally couple via the local Na(+) gradient to help regulate cell Ca(2+). Altered Ca(2+) transporter expression in SM-α2 engineered mice apparently compensates to minimize Ca(2+) overload (α2 (SM-DN)) or depletion (α2 (SM-Tg)) and attenuate BP changes. In contrast, Ca(2+) transporter upregulation, observed in many rodent hypertension models, should enhance Ca(2+) entry and signaling and contribute significantly to BP elevation.

Optogenetic sensors and effectors: CHROMus-the Cornell Heart Lung Blood Institute Resource for Optogenetic Mouse Signaling.

Significant progress has been made in the last decade in the development of optogenetic effectors and sensors that can be deployed to understand complex biological signaling in mammals at a molecular level, without disrupting the distributed, lineage specific signaling circuits that comprise nuanced physiological responses. A major barrier to the widespread exploitation of these imaging tools, however, is the lack of readily available genetic reagents that can be easily combined to probe complex biological processes. Ideally, one could envision purpose-produced mouse lines expressing optically compatible sensors and effectors, sensor pairs in distinct lineages, or sensor pairs in discrete subcellular compartments, such that they could be crossed to enable in vivo imaging studies of unprecedented scientific power. Such lines could also be combined with mice to determine the alteration in signaling accompanying targeted gene deletion or addition. In order to address this lack, the National Heart Lung and Blood Institute has recently funded an optogenetic resource designed to create optically compatible, combinatorial mouse lines that will advance NHLBI research. Here we review recent advances in optogenetic sensor and effectors and describe the rationale and goals for the establishment of the Cornell/National Heart Lung Blood Resource for Optogenetic Mouse Signaling (CHROMus).