CPVT cells demonstrated rightward and leftward shifts in the voltage-dependent activation and inactivation of <i>I_Ca</i>, respectively.

<p><i>A</i>. Activation kinetics of <i>I_Ca</i> over the whole voltage range were not significantly different between WT (open symbols) and CPVT (filled symbols) cells. <i>B</i>. The time course of inactivation of <i>I_Ca</i>, which encompass slow and fast components, were similar in WT and CPVT cells at all voltages studied. <i>C.</i> Superimposed voltage-dependence of <i>I_Ca</i> activation and inactivation. <i>I_Ca</i> activationis shifted to more positive values in CPVT <i>vs</i> WT cells, whereas inactivation of <i>I_Ca</i> is shifted to more hyperpolarized potential in CPVT cells compared with WT cells. <i>D</i>. Voltage dependence of <i>I_Ca</i> window current () display a bell-shaped voltage-dependence, however, the peak of is reduced in CPVT cells (continuous line) compared to WT cells (dashed line). * P<0.05 and ** P<0.005.</p

CICR gain is increased in CPVT mice due to reduced <i>I_Ca</i> at low voltages.

<p><i>A</i>. Representative examples of Ca²⁺ entry and release fluxes simultaneously recorded in WT (black traces) and CPVT (red traces) myocytes. Beneath the images is the corresponding profile of fluorescence, expressed as F/F₀, where F is fluorescence and F₀ is diastolic fluorescence, after background correction. <i>B</i>. Voltage-dependent Ca²⁺ induced-Ca²⁺ release gain (CICR-gain) decreased monotonically, giving rise to an L-shaped in CPVT (filled symbols, n = 14) and WT (open symbols, n = 16) cells. <i>C & D</i>. Voltage dependence of peak [Ca²⁺]_i transients (C) and peak of <i>I_Ca</i> density (D) displayed bell-shaped, graded function with the membrane potential. * P<0.05 and ** P<0.005.</p

Parameters of Boltzmann fittings of activation (d∞) and inactivation (f∞) curves (in mV).

<p>*p<0.05 vs WT;</p>#<p>p<0.05 vs control WT;</p>†<p>p<0.05 vs control CPVT.</p

Calmodulin inhibition reduced in WT cells.

<p><i>A & B</i>. CaM antagonists, W7 (<i>A</i>) or CALP2 (<i>B</i>) reduced in WT cells (dashed line) without affecting it in CPVT cells (continuous line). <i>C</i>. Comparison of the Ca²⁺ sparks occurrence at rest in myocytes from WT (open bars) and CPVT (closed bars) cells in control conditions and after incubation with W7 (left hatched bars) or CALP2 (right hatched bars). * P<0.05 vs WT. <i>D & E</i>. Representative immunoblots and quantification of CaM protein levels in cardiac heart lysates (<i>D</i>, normalized to the corresponding actin level and normalized to respective controls) and detected in the membrane fraction (<i>E</i>, normalized to the corresponding Ca_v1.2 level and normalized to respective controls) from WT (open bars, n = 4) and CPVT (closed bars, n = 4) mice.</p

Increased Ca²⁺ spark occurrence limits <i>I_Ca</i> window current ().

<p><i>A</i>. Analyze of the patch clamped-Ca²⁺ sparks revealed that the average of Ca²⁺ sparks frequencies in CPVT cells (filled circles, n = 10) were significantly higher compared to WT cells (open circles, n = 3). * P<0.05. <i>Right insets</i>. Representative examples of line-scan images of Ca²⁺ sparks elicited by depolarizing step to −48 mV. <i>B & C</i>. BAPTA dialysates (B) and thapsigargin-treatment (C) eliminates the difference in between CPVT and WT cells. <i>D</i>. In presence of Ryanodol in the perfusion solution, is reduced in WT cells compared to control condition, shown as light gray line.</p

MOESM1 of Urinary alpha-1 antitrypsin and CD59 glycoprotein predict albuminuria development in hypertensive patients under chronic renin-angiotensin system suppression

Additional file 1. Figure S1. Representative image of 2D-DIGE gel. Figure S2. Principal component analysis (PCA) graph. Each dot represents a urine sample used in the discovery phase (DIGE analysis). Table S1. Baseline medication for those patients including in the discovery phase. Data are expressed as percentages (%). ACEi: angiotensin converting enzyme inhibitors; ARB: angiotensin receptor blockers. N: normoalbuminuria; dnHA: de novo high albuminuria; MHA: maintained high albuminuria. Table S2. Baseline medication for those patients including in the validation phase. Data are expressed as percentages (%). ACEi: angiotensin converting enzyme inhibitors; ARB: angiotensin receptor blockers. N: normoalbuminuria; dnHA: de novo high albuminuria; MHA: maintained high albuminuria. Table S3. Proteins identifiedper gel spot with significant alteration (one-way ANOVA). The table shows the number of unique peptides identified, % sequence coverage and trends observed for each protein between compared groups (increase or decrease in the group located in the upper part of the ratio). Two spots contain a mixture of two proteins each, thus changes in expression forthose spots cannot be attributed to any of the two proteins initially. When one protein was identified in several spots, observed variations between groups followed the same trend (e.g. CD59 and alpha-1-antitrypsin). Table S4. SRM-LCMS/MS analysis conditions for those proteins confirmed in the validation phase with statistical signification (ANOVA <0.0001). Details of protein transitions (precursor and fragments masses), collision energy and peptide sequences are included

Image_1_Deficiency of NOD1 Improves the β-Adrenergic Modulation of Ca2+ Handling in a Mouse Model of Heart Failure.pdf

<p>Heart failure (HF) is a complex syndrome characterized by cardiac dysfunction, Ca²⁺ mishandling, and chronic activation of the innate immune system. Reduced cardiac output in HF leads to compensatory mechanisms via activation of the adrenergic nervous system. In turn, chronic adrenergic overstimulation induces pro-arrhythmic events, increasing the rate of sudden death in failing patients. Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) is an innate immune modulator that plays a key role in HF progression. NOD1 deficiency in mice prevents Ca²⁺ mishandling in HF under basal conditions, but its role during β-adrenergic stimulation remains unknown. Here, we evaluated whether NOD1 regulates the β-adrenergic modulation of Ca²⁺ signaling in HF. Ca²⁺ dynamics were examined before and after isoproterenol perfusion in cardiomyocytes isolated from healthy and from post-myocardial infarction (PMI) wild-type (WT) and Nod1^-/- mice. Isoproterenol administration induced similar effects on intracellular [Ca²⁺]_i transients, cell contraction, and sarcoplasmic reticulum (SR)-Ca²⁺ load in healthy WT and Nod1^-/- cells. However, compared with WT-PMI cells, isoproterenol exposure induced a significant increase in the [Ca²⁺]_i transients and cell contraction parameters in Nod1^-/--PMI cells, which mainly due to an increase in SR-Ca²⁺ load. NOD1 deficiency also prevented the increase in diastolic Ca²⁺ leak (Ca²⁺ waves) induced by isoproterenol in PMI cells. mRNA levels of β1 and β2 adrenergic receptors were significantly higher in Nod1^-/--PMI hearts vs WT-PMI hearts. Healthy cardiomyocytes pre-treated with the selective agonist of NOD1, iE-DAP, and perfused with isoproterenol showed diminished [Ca²⁺]_i transients amplitude, cell contraction, and SR-Ca²⁺ load compared with vehicle-treated cells. iE-DAP-treated cells also presented increased diastolic Ca²⁺ leak under β-adrenergic stimulation. The selectivity of iE-DAP on Ca²⁺ handling was validated by pre-treatment with the inactive analog of NOD1, iE-Lys. Overall, our data establish that NOD1 deficiency improves the β-adrenergic modulation of Ca²⁺ handling in failing hearts.</p