Diabetic Ocular Surface Has Defects in Oxygen Uptake Revealed by Optic Fiber Microsensor.

Purpose: Diabetes mellitus causes diabetic keratopathy (DK). This and other ocular surface disorders are underdiagnosed and problematic for affected patients as well as recipients of diabetic donor corneas. Thus, it is important to find noninvasive means to facilitate determination of the potentially vision-threatening DK. It has been reported that diabetic corneas uptake significantly less oxygen (O2) than healthy controls. However, an integral assessment of the ocular surface is missing. Methods: Using an optic-fiber O2 micro-sensor (optrode) we demonstrated recently that the healthy ocular surface displays a unique spatiotemporal map of O2 consumption. We hypothesize that diabetes impairs the spatiotemporal profile of O2 uptake at the ocular surface. Results: Using streptozotocin (STZ)-induced diabetic mice, we found diminished O2 uptake and loss of the unique pattern across the ocular surface. A diabetic cornea consumes significantly less O2 at the bulbar conjunctiva and limbus, but not the central and peripheral cornea, compared to controls. Further, we show that, contrary to the healthy cornea, the diabetic cornea does not increase the O2 consumption at the limbus in the evening as the normal control. Conclusions: Altogether, our measurements reveal a previously unknown impairment in O2 uptake at the diabetic cornea, making it a potential tool to diagnose ocular surface abnormalities and suggesting a new etiology mechanism.

SparkMaster 2: A New Software for Automatic Analysis of Calcium Spark Data.

BACKGROUND: Calcium (Ca) sparks are elementary units of subcellular Ca release in cardiomyocytes and other cells. Accordingly, Ca spark imaging is an essential tool for understanding the physiology and pathophysiology of Ca handling and is used to identify new drugs targeting Ca-related cellular dysfunction (eg, cardiac arrhythmias). The large volumes of imaging data produced during such experiments require accurate and high-throughput analysis. METHODS: We developed a new software tool SparkMaster 2 (SM2) for the analysis of Ca sparks imaged by confocal line-scan microscopy, combining high accuracy, flexibility, and user-friendliness. SM2 is distributed as a stand-alone application requiring no installation. It can be controlled using a simple-to-use graphical user interface, or using Python scripting. RESULTS: SM2 is shown to have the following strengths: (1) high accuracy at identifying Ca release events, clearly outperforming previous highly successful software SparkMaster; (2) multiple types of Ca release events can be identified using SM2: Ca sparks, waves, miniwaves, and long sparks; (3) SM2 can accurately split and analyze individual sparks within spark clusters, a capability not handled adequately by prior tools. We demonstrate the practical utility of SM2 in two case studies, investigating how Ca levels affect spontaneous Ca release, and how large-scale release events may promote release refractoriness. SM2 is also useful in atrial and smooth muscle myocytes, across different imaging conditions. CONCLUSIONS: SparkMaster 2 is a new, much-improved user-friendly software for accurate high-throughput analysis of line-scan Ca spark imaging data. It is free, easy to use, and provides valuable built-in features to facilitate visualization, analysis, and interpretation of Ca spark data. It should enhance the quality and throughput of Ca spark and wave analysis across cell types, particularly in the study of arrhythmogenic Ca release events in cardiomyocytes.

α1-Adrenergic receptor-PKC-Pyk2-Src signaling boosts L-type Ca2+ channel CaV1.2 activity and long-term potentiation in rodents.

The cellular mechanisms mediating norepinephrine (NE) functions in brain to result in behaviors are unknown. We identified the L-type Ca2+ channel (LTCC) CaV1.2 as a principal target for Gq-coupled α1-adrenergic receptors (ARs). α1AR signaling increased LTCC activity in hippocampal neurons. This regulation required protein kinase C (PKC)-mediated activation of the tyrosine kinases Pyk2 and, downstream, Src. Pyk2 and Src were associated with CaV1.2. In model neuroendocrine PC12 cells, stimulation of PKC induced tyrosine phosphorylation of CaV1.2, a modification abrogated by inhibition of Pyk2 and Src. Upregulation of LTCC activity by α1AR and formation of a signaling complex with PKC, Pyk2, and Src suggests that CaV1.2 is a central conduit for signaling by NE. Indeed, a form of hippocampal long-term potentiation (LTP) in young mice requires both the LTCC and α1AR stimulation. Inhibition of Pyk2 and Src blocked this LTP, indicating that enhancement of CaV1.2 activity via α1AR-Pyk2-Src signaling regulates synaptic strength.

Nitric oxide synthase and reduced arterial tone contribute to arteriovenous malformation.

Mechanisms underlying arteriovenous malformations (AVMs) are poorly understood. Using mice with endothelial cell (EC) expression of constitutively active Notch4 (Notch4*EC), we show decreased arteriolar tone in vivo during brain AVM initiation. Reduced vascular tone is a primary effect of Notch4*EC, as isolated pial arteries from asymptomatic mice exhibited reduced pressure-induced arterial tone ex vivo. The nitric oxide (NO) synthase (NOS) inhibitor NG-nitro-l-arginine (L-NNA) corrected vascular tone defects in both assays. L-NNA treatment or endothelial NOS (eNOS) gene deletion, either globally or specifically in ECs, attenuated AVM initiation, assessed by decreased AVM diameter and delayed time to moribund. Administering nitroxide antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl also attenuated AVM initiation. Increased NOS-dependent production of hydrogen peroxide, but not NO, superoxide, or peroxynitrite was detected in isolated Notch4*EC brain vessels during AVM initiation. Our data suggest that eNOS is involved in Notch4*EC-mediated AVM formation by up-regulating hydrogen peroxide and reducing vascular tone, thereby permitting AVM initiation and progression.

Transfer of nuclear and ribosomal material from Sox10-lineage cells to neurons in the mouse brain.

Material transfer is an essential form of intercellular communication to exchange information and resources between cells. Material transfer between neurons and from glia to neurons has been demonstrated to support neuronal survival and activity. Understanding the extent of material transfer in the healthy nervous system is limited. Here we report that in the mouse central nervous system (CNS), neurons receive nuclear and ribosomal material of Sox10-lineage cell (SOL) origin. We show that transfer of SOL-derived material to neurons is region dependent, establishes during postnatal brain maturation, and dynamically responds to LPS-induced neuroinflammation in the adult mouse brain. We identified satellite oligodendrocyte-neuron pairs with loss of plasma membrane integrity between nuclei, suggesting direct material transfer. Together, our findings provide evidence of regionally coordinated transfer of SOL-derived nuclear and ribosomal material to neurons in the mouse CNS, with potential implications for the understanding and modulation of neuronal function and treatment of neurological disorders.

Diversity of cells and signals in the cardiovascular system.

This white paper is the outcome of the seventh UC Davis Cardiovascular Research Symposium on Systems Approach to Understanding Cardiovascular Disease and Arrhythmia. This biannual meeting aims to bring together leading experts in subfields of cardiovascular biomedicine to focus on topics of importance to the field. The theme of the 2022 Symposium was 'Cell Diversity in the Cardiovascular System, cell-autonomous and cell-cell signalling'. Experts in the field contributed their experimental and mathematical modelling perspectives and discussed emerging questions, controversies, and challenges in examining cell and signal diversity, co-ordination and interrelationships involved in cardiovascular function. This paper originates from the topics of formal presentations and informal discussions from the Symposium, which aimed to develop a holistic view of how the multiple cell types in the cardiovascular system integrate to influence cardiovascular function, disease progression and therapeutic strategies. The first section describes the major cell types (e.g. cardiomyocytes, vascular smooth muscle and endothelial cells, fibroblasts, neurons, immune cells, etc.) and the signals involved in cardiovascular function. The second section emphasizes the complexity at the subcellular, cellular and system levels in the context of cardiovascular development, ageing and disease. Finally, the third section surveys the technological innovations that allow the interrogation of this diversity and advancing our understanding of the integrated cardiovascular function and dysfunction.

Whole-heart multiparametric optical imaging reveals sex-dependent heterogeneity in cAMP signaling and repolarization kinetics.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological ß-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.

Aß efflux impairment and inflammation linked to cerebrovascular accumulation of amyloid-forming amylin secreted from pancreas.

Impairment of vascular pathways of cerebral ß-amyloid (Aß) elimination contributes to Alzheimer disease (AD). Vascular damage is commonly associated with diabetes. Here we show in human tissues and AD-model rats that bloodborne islet amyloid polypeptide (amylin) secreted from the pancreas perturbs cerebral Aß clearance. Blood amylin concentrations are higher in AD than in cognitively unaffected persons. Amyloid-forming amylin accumulates in circulating monocytes and co-deposits with Aß within the brain microvasculature, possibly involving inflammation. In rats, pancreatic expression of amyloid-forming human amylin indeed induces cerebrovascular inflammation and amylin-Aß co-deposits. LRP1-mediated Aß transport across the blood-brain barrier and Aß clearance through interstitial fluid drainage along vascular walls are impaired, as indicated by Aß deposition in perivascular spaces. At the molecular level, cerebrovascular amylin deposits alter immune and hypoxia-related brain gene expression. These converging data from humans and laboratory animals suggest that altering bloodborne amylin could potentially reduce cerebrovascular amylin deposits and Aß pathology.

Mechanisms of Vascular CaV1.2 Channel Regulation During Diabetic Hyperglycemia.

Diabetes is a leading cause of disability and mortality worldwide. A major underlying factor in diabetes is the excessive glucose levels in the bloodstream (e.g., hyperglycemia). Vascular complications directly result from this metabolic abnormality, leading to disabling and life-threatening conditions. Dysfunction of vascular smooth muscle cells is a well-recognized factor mediating vascular complications during diabetic hyperglycemia. The function of vascular smooth muscle cells is exquisitely controlled by different ion channels. Among the ion channels, the L-type CaV1.2 channel plays a key role as it is the main Ca2+ entry pathway regulating vascular smooth muscle contractile state. The activity of CaV1.2 channels in vascular smooth muscle is altered by diabetic hyperglycemia, which may contribute to vascular complications. In this chapter, we summarize the current understanding of the regulation of CaV1.2 channels in vascular smooth muscle by different signaling pathways. We place special attention on the regulation of CaV1.2 channel activity in vascular smooth muscle by a newly uncovered AKAP5/P2Y11/AC5/PKA/CaV1.2 axis that is engaged during diabetic hyperglycemia. We further describe the pathophysiological implications of activation of this axis as it relates to myogenic tone and vascular reactivity and propose that this complex may be targeted for developing therapies to treat diabetic vascular complications.

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.

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2.

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100 nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

Diabetes and Excess Aldosterone Promote Heart Failure With Preserved Ejection Fraction.

Background The pathobiology of heart failure with preserved ejection fraction (HFpEF) is still poorly understood, and effective therapies remain limited. Diabetes and mineralocorticoid excess are common and important pathophysiological factors that may synergistically promote HFpEF. The authors aimed to develop a novel animal model of HFpEF that recapitulates key aspects of the complex human phenotype with multiorgan impairments. Methods and Results The authors created a novel HFpEF model combining leptin receptor-deficient db/db mice with a 4-week period of aldosterone infusion. The HFpEF phenotype was assessed using morphometry, echocardiography, Ca2+ handling, and electrophysiology. The sodium-glucose cotransporter-2 inhibitor empagliflozin was then tested for reversing the arrhythmogenic cardiomyocyte phenotype. Continuous aldosterone infusion for 4 weeks in db/db mice induced marked diastolic dysfunction with preserved ejection fraction, cardiac hypertrophy, high levels of B-type natriuretic peptide, and significant extracardiac comorbidities (including severe obesity, diabetes with marked hyperglycemia, pulmonary edema, and vascular dysfunction). Aldosterone or db/db alone induced only a mild diastolic dysfunction without congestion. At the cellular level, cardiomyocyte hypertrophy, prolonged Ca2+ transient decay, and arrhythmogenic action potential remodeling (prolongation, increased short-term variability, delayed afterdepolarizations), and enhanced late Na+ current were observed in aldosterone-treated db/db mice. All of these arrhythmogenic changes were reversed by empagliflozin pretreatment of HFpEF cardiomyocytes. Conclusions The authors conclude that the db/db+aldosterone model may represent a distinct clinical subgroup of HFpEF that has marked hyperglycemia, obesity, and increased arrhythmia risk. This novel HFpEF model can be useful in future therapeutic testing and should provide unique opportunities to better understand disease pathobiology.

Spatiotemporal Control of Vascular CaV1.2 by α1C S1928 Phosphorylation.

BACKGROUND: L-type CaV1.2 channels undergo cooperative gating to regulate cell function, although mechanisms are unclear. This study tests the hypothesis that phosphorylation of the CaV1.2 pore-forming subunit α1C at S1928 mediates vascular CaV1.2 cooperativity during diabetic hyperglycemia. METHODS: A multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaV1.2 cooperativity, α1C clustering, myogenic tone, and blood flow in human and mouse arterial myocytes/vessels. RESULTS: CaV1.2 activity and cooperative gating increase in arterial myocytes from patients with type 2 diabetes and type 1 diabetic mice, and in wild-type mouse arterial myocytes after elevating extracellular glucose. These changes were prevented in wild-type cells pre-exposed to a PKA inhibitor or cells from knock-in S1928A but not S1700A mice. In addition, α1C clustering at the surface membrane of wild-type, but not wild-type cells pre-exposed to PKA or P2Y11 inhibitors and S1928A arterial myocytes, was elevated upon hyperglycemia and diabetes. CaV1.2 spatial and gating remodeling correlated with enhanced arterial myocyte Ca2+ influx and contractility and in vivo reduction in arterial diameter and blood flow upon hyperglycemia and diabetes in wild-type but not S1928A cells/mice. CONCLUSIONS: These results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1C into "superclusters" upon hyperglycemia and diabetes. This triggers CaV1.2 activity and cooperativity, directly impacting vascular reactivity. The results may lay the foundation for developing therapeutics to correct CaV1.2 and arterial function during diabetic hyperglycemia.

Vascular CaV1.2 channels in diabetes.

Diabetic vasculopathy is a significant cause of morbidity and mortality in the diabetic population. Hyperglycemia, one of the central metabolic abnormalities in diabetes, has been associated with vascular dysfunction due to endothelial cell damage. However, studies also point toward vascular smooth muscle as a locus for hyperglycemia-induced vascular dysfunction. Emerging evidence implicates hyperglycemia-induced regulation of vascular L-type Ca2+ channels CaV1.2 as a potential mechanism for vascular dysfunction during diabetes. This chapter summarizes our current understanding of vascular CaV1.2 channels and their regulation during physiological and hyperglycemia/diabetes conditions. We will emphasize the role of CaV1.2 in vascular smooth muscle, the effects of elevated glucose on CaV1.2 function, and the mechanisms underlying its dysregulation in hyperglycemia and diabetes. We conclude by examining future directions and gaps in knowledge regarding CaV1.2 regulation in health and during diabetes.

Ion channel molecular complexes in vascular smooth muscle.

Ion channels that influence membrane potential and intracellular calcium concentration control vascular smooth muscle excitability. Voltage-gated calcium channels (VGCC), transient receptor potential (TRP) channels, voltage (KV), and Ca2+-activated K+ (BK) channels are key regulators of vascular smooth muscle excitability and contractility. These channels are regulated by various signaling cues, including protein kinases and phosphatases. The effects of these ubiquitous signaling molecules often depend on the formation of macromolecular complexes that provide a platform for targeting and compartmentalizing signaling events to specific substrates. This manuscript summarizes our current understanding of specific molecular complexes involving VGCC, TRP, and KV and BK channels and their contribution to regulating vascular physiology.

Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 (Mfn2), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Deciphering cellular signals in adult mouse sinoatrial node cells.

Sinoatrial node (SAN) cells are the pacemakers of the heart. This study describes a method for culturing and infection of adult mouse SAN cells with FRET-based biosensors that can be exploited to examine signaling events. SAN cells cultured in media with blebbistatin or (S)-nitro-blebbistatin retain their morphology, protein distribution, action potential (AP) waveform, and cAMP dynamics for at least 40 h. SAN cells expressing targeted cAMP sensors show distinct ß-adrenergic-mediated cAMP pools. Cyclic GMP, protein kinase A, Ca2+/CaM kinase II, and protein kinase D in SAN cells also show unique dynamics to different stimuli. Heart failure SAN cells show a decrease in cAMP and cGMP levels. In summary, a reliable method for maintaining adult mouse SAN cells in culture is presented, which facilitates studies of signaling networks and regulatory mechanisms during physiological and pathological conditions.

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.