Role of Ryanodine Receptor Subtypes in Initiation and Formation of Calcium Sparks in Arterial Smooth Muscle: Comparison with Striated Muscle

Calcium sparks represent local, rapid, and transient calcium release events from a cluster of ryanodine receptors (RyRs) in the sarcoplasmic reticulum. In arterial smooth muscle cells (SMCs), calcium sparks activate calcium-dependent potassium channels causing decrease in the global intracellular [Ca2+] and oppose vasoconstriction. This is in contrast to cardiac and skeletal muscle, where spatial and temporal summation of calcium sparks leads to global increases in intracellular [Ca2+] and myocyte contraction. We summarize the present data on local RyR calcium signaling in arterial SMCs in comparison to striated muscle and muscle-specific differences in coupling between L-type calcium channels and RyRs. Accordingly, arterial SMC Cav1.2 L-type channels regulate intracellular calcium stores content, which in turn modulates calcium efflux though RyRs. Downregulation of RyR2 up to a certain degree is compensated by increased SR calcium content to normalize calcium sparks. This indirect coupling between Cav1.2 and RyR in arterial SMCs is opposite to striated muscle, where triggering of calcium sparks is controlled by rapid and direct cross-talk between Cav1.1/Cav1.2 L-type channels and RyRs. We discuss the role of RyR isoforms in initiation and formation of calcium sparks in SMCs and their possible molecular binding partners and regulators, which differ compared to striated muscle

Indirect coupling between Cav1.2 channels and ryanodine receptors to generate Ca2+ sparks in murine arterial smooth muscle cells

In arterial vascular smooth muscle cells (VSMCs), Ca2+ sparks stimulate nearby Ca2+-activated K+ (BK) channels that hyperpolarize the membrane and close L-type Ca2+ channels. We tested the contribution of L-type Cav1.2 channels to Ca2+ spark regulation in tibial and cerebral artery VSMCs using VSMC-specific Cav1.2 channel gene disruption in (SMAKO) mice and an approach based on Poisson statistical analysis of activation frequency and first latency of elementary events. Cav1.2 channel gene inactivation reduced Ca2+ spark frequency and amplitude by ∼50% and ∼80%, respectively. These effects were associated with lower global cytosolic Ca2+ levels and reduced sarcoplasmic reticulum (SR) Ca2+ load. Elevating cytosolic Ca2+ levels reversed the effects completely. The activation frequency and first latency of elementary events in both wild-type and SMAKO VSMCs weakly reflected the voltage dependency of L-type channels. This study provides evidence that local and tight coupling between the Cav1.2 channels and ryanodine receptors (RyRs) is not required to initiate Ca2+ sparks. Instead, Cav1.2 channels contribute to global cytosolic [Ca2+], which in turn influences luminal SR calcium and thus Ca2+ sparks

Intrinsic Deregulation of Vascular Smooth Muscle and Myofibroblast Differentiation in Mesenchymal Stromal Cells from Patients with Systemic Sclerosis.

INTRODUCTION:Obliterative vasculopathy and fibrosis are hallmarks of systemic sclerosis (SSc), a severe systemic autoimmune disease. Bone marrow-derived mesenchymal stromal cells (MSCs) from SSc patients may harbor disease-specific abnormalities. We hypothesized disturbed vascular smooth muscle cell (VSMC) differentiation with increased propensity towards myofibroblast differentiation in response to SSc-microenvironment defining growth factors and determined responsible mechanisms. METHODS:We studied responses of multipotent MSCs from SSc-patients (SSc-MSCs) and healthy controls (H-MSCs) to long-term exposure to CTGF, b-FGF, PDGF-BB or TGF-β1. Differentiation towards VSMC and myofibroblast lineages was analyzed on phenotypic, biochemical, and functional levels. Intracellular signaling studies included analysis of TGF-β receptor regulation, SMAD, AKT, ERK1/2 and autocrine loops. RESULTS:VSMC differentiation towards both, contractile and synthetic VSMC phenotypes in response to CTGF and b-FGF was disturbed in SSc-MSCs. H-MSCs and SSc-MSCs responded equally to PDGF-BB with prototypic fibroblastic differentiation. TGF-β1 initiated myofibroblast differentiation in both cell types, yet with striking phenotypic and functional differences: In relation to H-MSC-derived myofibroblasts induced by TGF-β1, those obtained from SSc-MSCs expressed more contractile proteins, migrated towards TGF-β1, had low proliferative capacity, and secreted higher amounts of collagen paralleled by reduced MMP expression. Higher levels of TGF-β receptor 1 and enhanced canonical and noncanonical TGF-β signaling in SSc-MSCs accompanied aberrant differentiation response of SSc-MSCs in comparison to H-MSCs. CONCLUSIONS:Deregulated VSMC differentiation with a shift towards myofibroblast differentiation expands the concept of disturbed endogenous regenerative capacity of MSCs from SSc patients. Disease related intrinsic hyperresponsiveness to TGF-β1 with increased collagen production may represent one responsible mechanism. Better understanding of repair barriers and harnessing beneficial differentiation processes in MSCs could widen options of autologous MSC application in SSc patients

Specific TRPC6 Channel Activation, a Novel Approach to Stimulate Keratinocyte Differentiation*S⃞

The protective epithelial barrier in our skin undergoes constant regulation, whereby the balance between differentiation and proliferation of keratinocytes plays a major role. Impaired keratinocyte differentiation and proliferation are key elements in the pathophysiology of several important dermatological diseases, including atopic dermatitis and psoriasis. Ca2+ influx plays an essential role in this process presumably mediated by different transient receptor potential (TRP) channels. However, investigating their individual role was hampered by the lack of specific stimulators or inhibitors. Because we have recently identified hyperforin as a specific TRPC6 activator, we investigated the contribution of TRPC6 to keratinocyte differentiation and proliferation. Like the endogenous differentiation stimulus high extracellular Ca2+ concentration ([Ca2+]o), hyperforin triggers differentiation in HaCaT cells and in primary cultures of human keratinocytes by inducing Ca2+ influx via TRPC6 channels and additional inhibition of proliferation. Knocking down TRPC6 channels prevents the induction of Ca2+- and hyperforin-induced differentiation. Importantly, TRPC6 activation is sufficient to induce keratinocyte differentiation similar to the physiological stimulus [Ca2+]o. Therefore, TRPC6 activation by hyperforin may represent a new innovative therapeutic strategy in skin disorders characterized by altered keratinocyte differentiation

Measurement of functional L-type Cav1.2 Ca2+ channels in response to growth factors.

<p>Functional L-type Cav1.2 Ca²⁺ channels measured as nimodipine sensitive Ca²⁺-influx upon depolarisation with 60 mmol/L KCl in response to treatment with systemic sclerosis microenvironment defining growth factors for 6 days. <b>A,</b> Bars represent the mean+SEM of nimodipine sensitive Ca²⁺-influx from 3 independent experiments. Control was set to 1. *P<0.05, **P<0.01, ***P<0.001. <b>B-F,</b> Representative recordings of Ca²⁺ dependent intracellular fluorescence intensities in response to depolarisation with KCl. Mean±SEM F/F0 of 30–40 cells per growth factor are plotted against time. Black lines represent tracings without the specific L-type calcium channel blocker nimodipine, grey lines those obtained after preincubation with 1 mmol/L nimodipine.</p

Cell functions of mesenchymal stromal cells upon treatment with systemic sclerosis microenvironment defining growth factors.

<p><b>A,</b> Chemotactic response towards a 5 ng/ml gradient of each growth factor. Bars represent the mean+SEM of 3 independent experiments, 3 replicates each. Control was set to 1. <b>B,</b> Proliferation measured by BrdU incorporation after stimulation with 10 ng/ml of each growth factor for 24 h. Bars represent the mean+SEM of 3 independent experiments, 5 replicates each. Control was set to 1. <b>C,</b> Total collagen content in cell culture supernatants after 6 days of treatment with 10 ng/ml of each growth factor. Bars represent the mean+SEM of 6 independent experiments, 2 replicates each. *P<0.05, **P<0.01, ***P<0.001. <b>D,</b> Illustration of the induction of specific cell types of vascular smooth muscle cell (VSMC) and fibroblast lineages by systemic sclerosis microenvironment defining growth factors.</p

Analysis of the Transforming growth factor-β1 signaling network.

<p><b>A,</b> Expression of TGF-β1 mRNA and <b>B,</b> secreted TGF-β1 protein for assessment of the autocrine TGF-β feedback loop. Bars represent the mean+SEM of 6 independent experiments, 3 replicates each. <b>C,</b> Expression of CTGF mRNA. Quantitative real-time PCR analysis of mRNA transcripts. Bars represent the mean+SEM of 6 independent experiments quantified with the ΔΔCt method in duplicates. <b>D,</b> Expression of TGF-β receptor 1 protein. <b>E-G,</b> Induction of canonic and non-canonic TGF-β signaling. <b>E,</b> Phosphorylation of SMAD3 at Ser423/425. <b>F,</b> Phosphorylation of AKT at Ser473. <b>G,</b> Phosphorylation of ERK1/2 at Thr202/Tyr204. Analysis of all experiments was performed after 6 days treatment with 10 ng/ml transformin growth factor -β1. Representative western blots of 6 independent experiments are shown. Bars represent the mean+SEM of densitometrically determined band intensities after normalization to GAPDH (TGFBR1, pSMAD3) or total kinase (pAKT, pERK). Control was set to 1. *P<0.05, **P<0.01, ***P<0.001.</p