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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.

Vascular complications are major causes of morbidity and mortality in diabetic patients. Recently, our group identified a novel purinergic signaling pathway involving the AKAP5/P2Y11/AC5/PKA/CaV1.2 protein network in arterial myocytes that is activated upon diabetic hyperglycemia leading to vasoconstriction. This purinergic complex is activated by the release of nucleotides (e.g. ATP) to the extracellular space in response to hyperglycemic conditions. Pannexin 1 (Panx1) is a channel that mediates ATP efflux, thus inducing purinergic signaling activation. In this study, we hypothesize that Panx1 is a critical regulator mediating ATP release and increasing cAMP synthesis, CaV1.2 potentiation, and vasoconstriction upon diabetic hyperglycemia. Consistent with this hypothesis, we found that elevated glucose (HG) induced an elevation in extracellular ATP concentrations, and this was reduced in the presence of the Panx1 inhibitor spironolactone (spiro). Panx1 was found in complex with AKAP5, P2Y11, AC5, PKA, and CaV1.2 in arterial myocytes. This protein complex was strengthened upon HG treatment, and spiro prevented this effect. Spiro and genetic ablation of smooth muscle Panx1 (iPanx1sm -/-) blocked cAMP production, CaV1.2 potentiation, sustained vasoconstriction, and in vivo elevations in cerebral artery myogenic tone and reduced blood flow in response to HG. In a mouse model of type 1 diabetes (e.g. STZ), the increased CaV1.2 potentiation and enhanced myogenic tone were prevented in arterial myocytes and arteries from iPanx1sm -/- mice. Altogether, these data suggest that Panx1 is part of the AKAP5/P2Y11/AC5/PKA/CaV1.2 signaling module in arterial myocytes. This Panx1-led complex modulates CaV1.2 activity and vascular reactivity in response to diabetic hyperglycemia. Thus, Panx1 could be a new therapeutic target to treat vascular complications during diabetes. This work was supported by NIH grants R01HL149127, R01HL121059, and R01HL161872 (to MFN). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

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.

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. (Figure presented.). in press

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 Ca

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 (50-100nM) 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.

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, Ca 2+ 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 Ca 2+ 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.

Sinoatrial node (SAN) cells are the heart's primary pacemaker. Their activity is tightly regulated by β-adrenergic receptor (β-AR) signaling. Adenylyl cyclase (AC) is a key enzyme in the β-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 β-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 β-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 β-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.

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 Ca

L-type CaA multiscale approach including patch-clamp electrophysiology, super-resolution nanoscopy, proximity ligation assay, calcium imaging' pressure myography, and Laser Speckle imaging was implemented to examine CaCaThese results suggest that PKA-dependent S1928 phosphorylation promotes the spatial reorganization of vascular α1

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 Ca 2+ 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 Ca 2+ 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.

Introduction: 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 entirely understood. Mitochondria are critical to cellular processes that determine life or death of the cell. Therefore, proper communication with the mitochondria is essential for normal cellular function. We tested the hypothesis that alterations in the mitochondria-sarcoplasmic reticulum (SR) connectomics underlie SAN dysfunction in HF. Methods: We took advantage of a mouse model of pressure-overload induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR/Cas9 mediated gene silencing of mitofusin-2 ( Mfn2 ). Results: 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 SR-mitochondria distance. The expression of the SR-mitochondria tether GTPase protein Mfn-2 was significantly down-regulated and showed reduced colocalization with ryanodine receptor (RyR2) in HF SAN cells. Indeed, SAN-specific gene silencing of Mfn-2 led to alterations in the mitochondrial-SR microdomains[MFN1] and SAN dysfunction. Finally, disruption in the mitochondrial-SR microdomains resulted in abnormal mitochondrial Ca 2+ handling, alterations in localized PKA activity, and impaired mitochondrial function in HF SAN cells. Conclusion: The study provides insight on the role of mitochondrial-SR microdomains in SAN automaticity and into possible therapeutic targets for SAN dysfunction in HF patients.

The L-type Ca

The cellular mechanisms mediating norepinephrine 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 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 norepinephrine. Indeed, a form of hippocampal 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.

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. We identified 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 pre-associates with CaV1.2 to promote Po and CDI. We 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. 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 wildtype CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI identifying Ca2/CaM as an important mediator of Ca signaling.

The L-type Ca2+ channel CaV1.2 is essential for arterial myocyte excitability, gene expression and contraction. Elevations in extracellular glucose (hyperglycemia) potentiate vascular L-type Ca2+ channel via PKA, but the underlying mechanisms are unclear. Here, we find that cAMP synthesis in response to elevated glucose and the selective P2Y11 agonist NF546 is blocked by disruption of A-kinase anchoring protein 5 (AKAP5) function in arterial myocytes. Glucose and NF546-induced potentiation of L-type Ca2+ channels, vasoconstriction and decreased blood flow are prevented in AKAP5 null arterial myocytes/arteries. These responses are nucleated via the AKAP5-dependent clustering of P2Y11/ P2Y11-like receptors, AC5, PKA and CaV1.2 into nanocomplexes at the plasma membrane of human and mouse arterial myocytes. Hence, data reveal an AKAP5 signaling module that regulates L-type Ca2+ channel activity and vascular reactivity upon elevated glucose. This AKAP5-anchored nanocomplex may contribute to vascular complications during diabetic hyperglycemia., Molecular mechanisms by which glucose modulates L-type Ca2+ channel activity and vascular reactivity are unclear. Here the authors report a nanocomplex orchestrated by AKAP5 that facilitates local purinergic stimulation of L-type Ca2+ channels and vasoconstriction during diabetic hyperglycemia.

Diabetes affects millions of people worldwide. This devastating disease dramatically increases the risk of developing cardiovascular disorders. A hallmark metabolic abnormality in diabetes is hyperglycemia, which contributes to the pathogenesis of cardiovascular complications. These cardiovascular complications are, at least in part, related to hyperglycemia-induced molecular and cellular changes in the cells making up blood vessels. Whereas the mechanisms mediating endothelial dysfunction during hyperglycemia have been extensively examined, much less is known about how hyperglycemia impacts vascular smooth muscle function. Vascular smooth muscle function is exquisitely regulated by many ion channels, including several members of the potassium (K+) channel superfamily and voltage-gated L-type Ca2+channels. Modulation of vascular smooth muscle ion channels function by hyperglycemia is emerging as a key contributor to vascular dysfunction in diabetes. In this review, we summarize the current understanding of how diabetic hyperglycemia modulates the activity of these ion channels in vascular smooth muscle. We examine underlying mechanisms, general properties, and physiological relevance in the context of myogenic tone and vascular reactivity.

Formation of signaling complexes is crucial for the orchestration of fast, efficient, and specific signal transduction. Pharmacological disruption of defined signaling complexes has the potential for specific intervention in selected regulatory pathways without affecting organism-wide disruption of parallel pathways. Signaling by epinephrine and norepinephrine through α and β adrenergic receptors acts on many signaling pathways in many cell types. Here, we initially provide an overview of the signaling complexes formed between the paradigmatic β2 adrenergic receptor and two of its most important targets, the L-type Ca2+ channel CaV1.2 and the AMPA-type glutamate receptor. Importantly, both complexes contain the trimeric Gs protein, adenylyl cyclase, and the cAMP-dependent protein kinase, PKA. We then discuss the functional implications of the formation of these complexes, how those complexes can be specifically disrupted, and how such disruption could be utilized in the pharmacological treatment of disease.

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.

Summary 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 β-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., Graphical abstract, Highlights • Optimized culturing method of adult SAN cells from healthy and disease-state mice • Cultured adult SAN cells can be infected with diverse fluorescence biosensors • Examination of signaling networks, including discrete cAMP pools, in adult SAN cells • SAN cells cAMP and cGMP signals are impaired during heart failure, Cell biology; Functional aspects of cell biology; Biology experimental methods

Nuclear and ribosomal components define cell identity and function by regulating chromatin dynamics, gene expression, and protein turnover. Here we report that in the mouse central nervous system (CNS) under normal conditions, neurons accumulate nuclear and ribosomal material of oligodendrocyte (OL) origin. We show that neuronal accumulation of OL-derived nuclear and ribosomal material is brain area-specific, and in the cortex and hippocampal dentate gyrus gradually propagates during postnatal brain maturation. We further demonstrate that OL-to-neuron material transfer persists throughout adulthood and responds to neuroinflammation. We found that satellite OL of the gray matter form internuclear contacts with receiving neurons in the mouse brain. Similar close internuclear associations between satellite OL and neurons are present in the adult human cortex. Our findings provide the first evidence of wide-spread dynamic and selective OL-to-neuron nuclear and ribosomal material transfer in the mouse CNS and indicate that satellite OL serve as powerful mediators of neuronal function. Equivalent processes may occur in the human CNS and cause neurological disorders when dysregulated.One Sentence SummaryNeurons receive OL-derived nuclear and ribosomal material

Rationale: Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. The effective combination of these optical tools in mice requires designed genetic models that are optically compatible and efficiently assembled. We designed strains for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Objective: To address this lack of optogenetic resources by combining lineage-specific expression of optogenetic effectors and sensors in single mouse lines and demonstrating their utility. Methods and Results: Twenty-two lines of mice were created in which optogenetic effectors (11 lines) or Ca2+ sensors (11 lines) were expressed in a lineage-specific manner in cardiac pacemaker cells (SAN), cardiomyocytes, vascular endothelial and smooth muscle cells, as well as other cell types. We show functional responses associated with optical formation of the second messengers InsP3 (optoα1AR) and cAMP (optoβ2AR), or Ca2+-permeant membrane channels (CatCh2), in targeted cells in these mice. Green (GCaMP5, 8 and 8.1) and red (RCaMP 1.07) Ca2+ sensors were chosen to enable simultaneous fluorescent detection of cellular responses. Biallelic crosses were created with robust expression of sensor/effector pairs. We present examples of novel in vivo and ex vivo experiments in selected strains including cardiac pacing with stimulation of HCN4 conduction cells, intravital cardiac imaging of GCaMP8 with two-photon microscopy, optical vascular dilation/constriction by smooth muscle (Acta2) and vasodilation by light activation of endothelium (Cdh5). These experiments highlight the potential of these mice to uncover new insight into mechanisms regulating cardiovascular function. Conclusions: These new mouse lines efficiently express optical effectors and sensors in heart, blood vessels, and other tissues and can be efficiently combined enable novel in vivo and ex-vivo studies and can be combined or crossed with relevant cardiovascular disease models, constituting a new toolbox for cardiovascular biology.

> “The tragedies in life are largely arterial.” > > – Sir William Osler, 1908 (1) Hypertension is a debilitating cardiovascular disease characterized by the narrowing of arteries and arterioles which increases resistance to blood flow. This decrease in intraluminal diameter of arteries/arterioles is due to multiple factors, including arterial smooth muscle hypertrophy and hyperplasia as well as increased myogenic tone. Up-regulation of L-type Ca2+ channel activity is a major contributor to enhanced arterial smooth muscle contractility during hypertension (2, 3). Accordingly, current pharmacological guidelines for the treatment of hypertension include the use of L-type Ca2+ channel blockers such as nifedipine or amlodipine (4). In PNAS, Johnson et al. (5) report that chronic exposure to a broad range of these drugs promotes arterial smooth muscle to transition from a contractile to a proliferative phenotype by activating stromal-interacting molecule (STIM)/ORAI store-operated Ca2+ entry (SOCE) (Fig. 1). These findings have profound implications for our understanding of how L-type Ca2+ channel blockers affect vascular function during hypertension and may lead to changes on treatment of the pathology. Fig. 1. Response of contractile and synthetic smooth muscle to hypertension and L-type Ca2+ blockers. ( A ) During hypertension, contractile myocytes shorten in response to increased CaV1.2 channel activity, whereas synthetic myocytes experience a reduction in contractile proteins and increase in SOCE machinery. L-type Ca2+ blockers elicit a decrease in CaV1.2 influx, promoting relaxation and vasodilation. Synthetic myocytes see an increase in SOCE, proliferation, and migration, resulting in hyperplasia and increased incidence of heart failure. ( B ) L-type Ca2+ channel blockers stimulate the assembly of the STIM1/ORAI complexes via an unknown mechanism. The STIM1/ORAI complex promotes SOCE independent of S/ER Ca2+ content or CaV1.2 function. SOCE increases [Ca2+]i and promotes proliferation and migration of … [↵][1]1To whom correspondence may be addressed. Email: lfsantana{at}ucdavis.edu. [1]: #xref-corresp-1-1

Ion channels are often found in dense clusters within the plasma membranes of excitable cells. Based on experimental measurements of a wide range of channels in various cell types, Sato et al. propose that channel clusters form stochastically and that their size is regulated by a common feedback mechanism., Ion channels are often found arranged into dense clusters in the plasma membranes of excitable cells, but the mechanisms underlying the formation and maintenance of these functional aggregates are unknown. Here, we tested the hypothesis that channel clustering is the consequence of a stochastic self-assembly process and propose a model by which channel clusters are formed and regulated in size. Our hypothesis is based on statistical analyses of the size distributions of the channel clusters we measured in neurons, ventricular myocytes, arterial smooth muscle, and heterologous cells, which in all cases were described by exponential functions, indicative of a Poisson process (i.e., clusters form in a continuous, independent, and memory-less fashion). We were able to reproduce the observed cluster distributions of five different types of channels in the membrane of excitable and tsA-201 cells in simulations using a computer model in which channels are “delivered” to the membrane at randomly assigned locations. The model’s three parameters represent channel cluster nucleation, growth, and removal probabilities, the values of which were estimated based on our experimental measurements. We also determined the time course of cluster formation and membrane dwell time for CaV1.2 and TRPV4 channels expressed in tsA-201 cells to constrain our model. In addition, we elaborated a more complex version of our model that incorporated a self-regulating feedback mechanism to shape channel cluster formation. The strong inference we make from our results is that CaV1.2, CaV1.3, BK, and TRPV4 proteins are all randomly inserted into the plasma membranes of excitable cells and that they form homogeneous clusters that increase in size until they reach a steady state. Further, it appears likely that cluster size for a diverse set of membrane-bound proteins and a wide range of cell types is regulated by a common feedback mechanism.

Key points Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type CaV1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood.CaV1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails.We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of CaV1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca2+ influx.On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized CaV1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling. Abstract Voltage‐dependent L‐type CaV1.2 channels play an indispensable role in cardiac excitation–contraction coupling. Activation of the β‐adrenergic receptor (βAR)/cAMP/protein kinase A (PKA) signalling pathway leads to enhanced CaV1.2 activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. CaV1.2 channels exhibit a clustered distribution along the T‐tubule sarcolemma of ventricular myocytes where nanometer proximity between channels permits Ca2+‐dependent co‐operative gating behaviour mediated by dynamic, physical, allosteric interactions between adjacent channel C‐terminal tails. This amplifies Ca2+ influx and augments myocyte Ca2+ transient and contraction amplitudes. We investigated whether βAR signalling could alter CaV1.2 channel clustering to facilitate co‐operative channel interactions and elevate Ca2+ influx in ventricular myocytes. Bimolecular fluorescence complementation experiments reveal that the βAR agonist, isoproterenol (ISO), promotes enhanced CaV1.2–CaV1.2 physical interactions. Super‐resolution nanoscopy and dynamic channel tracking indicate that these interactions are expedited by enhanced spatial proximity between channels, resulting in the appearance of CaV1.2 ‘super‐clusters’ along the z‐lines of ISO‐stimulated cardiomyocytes. The mechanism that leads to super‐cluster formation involves rapid, dynamic augmentation of sarcolemmal CaV1.2 channel abundance after ISO application. Optical and electrophysiological single channel recordings confirm that these newly inserted channels are functional and contribute to overt co‐operative gating behaviour of CaV1.2 channels in ISO stimulated myocytes. The results of the present study reveal a new facet of βAR‐mediated regulation of CaV1.2 channels in the heart and support the novel concept that a pre‐synthesized pool of sub‐sarcolemmal CaV1.2 channel‐containing vesicles/endosomes resides in cardiomyocytes and can be mobilized to the sarcolemma to tune excitation–contraction coupling to meet metabolic and/or haemodynamic demands., Key points Prevailing dogma holds that activation of the β‐adrenergic receptor/cAMP/protein kinase A signalling pathway leads to enhanced L‐type CaV1.2 channel activity, resulting in increased Ca2+ influx into ventricular myocytes and a positive inotropic response. However, the full mechanistic and molecular details underlying this phenomenon are incompletely understood.CaV1.2 channel clusters decorate T‐tubule sarcolemmas of ventricular myocytes. Within clusters, nanometer proximity between channels permits Ca2+‐dependent co‐operative gating behaviour mediated by physical interactions between adjacent channel C‐terminal tails.We report that stimulation of cardiomyocytes with isoproterenol, evokes dynamic, protein kinase A‐dependent augmentation of CaV1.2 channel abundance along cardiomyocyte T‐tubules, resulting in the appearance of channel ‘super‐clusters’, and enhanced channel co‐operativity that amplifies Ca2+ influx.On the basis of these data, we suggest a new model in which a sub‐sarcolemmal pool of pre‐synthesized CaV1.2 channels resides in cardiomyocytes and can be mobilized to the membrane in times of high haemodynamic or metabolic demand, to tune excitation–contraction coupling.

Significance The L-type voltage-gated Ca2+ channel CaV1.2 is essential for excitation–contraction coupling in the heart. During the fight-or-flight response, CaV1.2 channel activity is augmented as a result of PKA-mediated phosphorylation, downstream of β-adrenergic receptor activation. We discovered that enhanced sarcolemmal abundance of CaV1.2 channels, driven by stimulated insertion/recycling of specific CaV1.2-containing endosomes, is essential for β-adrenergic receptor-mediated regulation of these channels in the heart. These data reveal a conceptual framework of this critical and robust pathway for on-demand tuning of cardiac excitation–contraction coupling during fight-or-flight., The number and activity of Cav1.2 channels in the cardiomyocyte sarcolemma tunes the magnitude of Ca2+-induced Ca2+ release and myocardial contraction. β-Adrenergic receptor (βAR) activation stimulates sarcolemmal insertion of CaV1.2. This supplements the preexisting sarcolemmal CaV1.2 population, forming large “superclusters” wherein neighboring channels undergo enhanced cooperative-gating behavior, amplifying Ca2+ influx and myocardial contractility. Here, we determine this stimulated insertion is fueled by an internal reserve of early and recycling endosome-localized, presynthesized CaV1.2 channels. βAR-activation decreased CaV1.2/endosome colocalization in ventricular myocytes, as it triggered “emptying” of endosomal CaV1.2 cargo into the t-tubule sarcolemma. We examined the rapid dynamics of this stimulated insertion process with live-myocyte imaging of channel trafficking, and discovered that CaV1.2 are often inserted into the sarcolemma as preformed, multichannel clusters. Similarly, entire clusters were removed from the sarcolemma during endocytosis, while in other cases, a more incremental process suggested removal of individual channels. The amplitude of the stimulated insertion response was doubled by coexpression of constitutively active Rab4a, halved by coexpression of dominant-negative Rab11a, and abolished by coexpression of dominant-negative mutant Rab4a. In ventricular myocytes, βAR-stimulated recycling of CaV1.2 was diminished by both nocodazole and latrunculin-A, suggesting an essential role of the cytoskeleton in this process. Functionally, cytoskeletal disruptors prevented βAR-activated Ca2+ current augmentation. Moreover, βAR-regulation of CaV1.2 was abolished when recycling was halted by coapplication of nocodazole and latrunculin-A. These findings reveal that βAR-stimulation triggers an on-demand boost in sarcolemmal CaV1.2 abundance via targeted Rab4a- and Rab11a-dependent insertion of channels that is essential for βAR-regulation of cardiac CaV1.2.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K+ channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K+ channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K+ current (IK1), but reduces the amplitude and inactivation recovery time of the transient outward K+ current (Ito) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, IK1 amplitude and Ito recovery effects (but not Ito amplitude) were prevented by the Ca2+/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. Ito reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both Ito and IK1 were reduced in line with the downregulated K+ channel expression. However, IK1 downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances IK1 and Ito recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces Ito amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K+ channel expression and function, which may further increase arrhythmia susceptibility.

Chronic hyperglycemia and diabetes lead to impaired cardiac repolarization, K(+) channel remodeling and increased arrhythmia risk. However, the exact signaling mechanism by which diabetic hyperglycemia regulates cardiac K(+) channels remains elusive. Here, we show that acute hyperglycemia increases inward rectifier K(+) current (I(K1)), but reduces the amplitude and inactivation recovery time of the transient outward K(+) current (I(to)) in mouse, rat, and rabbit myocytes. These changes were all critically dependent on intracellular O-GlcNAcylation. Additionally, I(K1) amplitude and I(to) recovery effects (but not I(to) amplitude) were prevented by the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor autocamtide-2-related inhibitory peptide, CaMKIIδ-knockout, and O-GlcNAc-resistant CaMKIIδ-S280A knock-in. I(to) reduction was prevented by inhibition of protein kinase C (PKC) and NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). In mouse models of chronic diabetes (streptozotocin, db/db, and high-fat diet), heart failure, and CaMKIIδ overexpression, both I(to) and I(K1) were reduced in line with the downregulated K(+) channel expression. However, I(K1) downregulation in diabetes was markedly attenuated in CaMKIIδ-S280A. We conclude that acute hyperglycemia enhances I(K1) and I(to) recovery via CaMKIIδ-S280 O-GlcNAcylation, but reduces I(to) amplitude via a NOX2-ROS-PKC pathway. Moreover, chronic hyperglycemia during diabetes and CaMKII activation downregulate K(+) channel expression and function, which may further increase arrhythmia susceptibility.

The L‐type Ca(2+) channel Ca(V)1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α‐actinin to the IQ motif of Ca(V)1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi‐functional mechanism that restricts Ca(V)1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646–1,664) bound to α‐actinin‐1 and to apo‐calmodulin (apoCaM). The Ca(V)1.2 K1647A and Y1649A mutations, which impair α‐actinin‐1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α‐actinin‐1 binding, decreased single‐channel open probability, gating charge movement, and its coupling to channel opening. Thus, α‐actinin recruits Ca(V)1.2 to defined surface regions and simultaneously boosts its open probability so that Ca(V)1.2 is mostly active when appropriately localized.

TRPML1 (transient receptor potential mucolipin 1) is a Ca(2+)-permeable, non-selective cation channel localized to the membranes of endosomes and lysosomes and is not present or functional on the plasma membrane. Ca(2+) released from endosomes and lysosomes into the cytosol through TRPML1 channels is vital for trafficking, acidification, and other basic functions of these organelles. Here, we investigated the function of TRPML1 channels in fully differentiated contractile vascular smooth muscle cells (SMCs). In live-cell confocal imaging studies, we found that most endosomes and lysosomes in freshly isolated SMCs from cerebral arteries were essentially immobile. Using nanoscale super-resolution microscopy, we found that TRPML1 channels present in late endosomes and lysosomes formed stable complexes with type 2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Spontaneous Ca(2+) signals resulting from the release of SR Ca(2+) through RyR2s (“Ca(2+) sparks”) and corresponding Ca(2+)-activated K(+) channel activity are critically important for balancing vasoconstriction. We found that these signals were essentially absent in SMCs from TRPML1-knockout (Mcoln1(−/−)) mice. Using ex vivo pressure myography, we found that loss of this critical signaling cascade exaggerated the vasoconstrictor responses of cerebral and mesenteric resistance arteries. In vivo radiotelemetry studies showed that Mcoln1(−/−) mice were spontaneously hypertensive. We conclude that TRPML1 is crucial for the initiation of Ca(2+) sparks in SMCs and the regulation of vascular contractility and blood pressure.