Evolution of flower allometry and pigmentation in Mammillaria haageana (Cactaceae).

BACKGROUND: A puzzle in evolution is the understanding of how the environment might drive subtle phenotypic variation, and whether this variation is adaptive. Under the neutral evolutionary theory, subtle phenotypes are almost neutral with little adaptive value. To test this idea, we studied the infraspecific variation in flower shape and color in Mammillaria haageana, a species with a wide geographical distribution and phenotypic variation, which populations are often recognized as infraspecific taxa. RESULTS: We collected samples from wild populations, kept them in the greenhouse for at least one reproductive year, and collected newly formed flowers. Our first objective was to characterize tepal natural variation in M. haageana through geometric morphometric and multivariate pigmentation analyses. We used landmark-based morphometrics to quantify the trends of shape variation and tepal color-patterns in 20 M. haageana accessions, belonging to five subspecies, plus 8 M. albilanata accessions for comparison as the sister species. We obtained eight geometric morphometric traits for tepal shape and color-patterns. We found broad variation in these traits between accessions belonging to the same subspecies, without taxonomic congruence with those infraspecific units. Also the phenetic cluster analysis showed different grouping patterns among accessions. When we correlated these phenotypes to the environment, we also found that solar radiation might explain the variation in tepal shape and color, suggesting that subtle variation in flower phenotypes might be adaptive. Finally we present anatomical sections in M. haageana subsp. san-angelensis to propose some of the underlying tepal structural features that may give rise to tepal variation. CONCLUSIONS: Our geometric morphometric approach of flower shape and color allowed us to identify the main trends of variation in each accession and putative subspecies, but also allowed us to correlate these variation to the environment, and propose anatomical mechanisms underlying this diversity of flower phenotypes.

Post-transplant hepatic fibrosis in pediatric liver-small bowel transplant recipients: A single-center, retrospective, observational study.

BACKGROUND: Little is known about the prevalence of hepatic graft fibrosis in combined LSBT children. We aimed to determine the prevalence of and identify potential predictors for hepatic graft fibrosis in LSBT children and to compare them with those in LT children. METHODS: We retrospectively included children younger than 19 years who had received a primary LT/LSBT between 2000 and 2018 and had a liver biopsy performed at least 6 months post-transplant. A Cox proportional hazards regression model was used to determine predictors associated with significant hepatic graft fibrosis (≥F2) in LSBT vs LT children. RESULTS: Ninety-six children (47 LSBT, 54 females) were included. The median post-transplant follow-up (years) was 12.8 in LT vs 10.5 in LSBT patients (P = .06). Hepatic graft fibrosis was found in 81.6% of LT vs 70.2% of LSBT children (P = .19), after a median time of 2.5 years and 2.6 years, respectively. On multivariate analyses, having post-transplant biliary complications was found to be associated with significant graft fibrosis in LT children, whereas AST/ALT ratio was found to predict significant hepatic graft fibrosis in LSBT children. The use of parenteral nutrition after transplant was not associated with significant hepatic graft fibrosis. CONCLUSIONS: The prevalence of hepatic graft fibrosis in LSBT children did not significantly differ from that in LT children, but the predictors may differ. Future studies should investigate the role of post-transplant autoimmune antibodies and donor-specific antibodies in the development and progression of hepatic graft fibrosis in LSBT children.

Modeling structural and functional deficiencies of RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 (RBM20), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20-dependent splice variants affected sarcomeric (TTN and LDB3) and calcium (Ca(2+)) handling (CAMK2D and CACNA1C) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length (RBM20: 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P < 0.0001) and decreased sarcomeric width (RBM20: 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P < 0.0001). Additionally, CMs showed defective Ca(2+) handling machinery with prolonged Ca(2+) levels in the cytoplasm as measured by greater area under the curve (RBM20: 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P < 0.05) and higher Ca(2+) spike amplitude (RBM20: 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P < 0.05). ß-adrenergic stress induced with 10 µm norepinephrine demonstrated increased susceptibility to sarcomeric disorganization (RBM20: 86 ± 10.5% versus control: 40 ± 7%; P < 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool to dissect disease-relevant defects in RBM20 splicing as a global regulator of heart function.

Musclin is an activity-stimulated myokine that enhances physical endurance.

Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin-a peptide with high homology to natriuretic peptides (NP)-as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca(2+)-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

Restrictions in ATP diffusion within sarcomeres can provoke ATP-depleted zones impairing exercise capacity in chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by the inability of patients to sustain a high level of ventilation resulting in perceived exertional discomfort and limited exercise capacity of leg muscles at average intracellular ATP levels sufficient to support contractility. METHODS: Myosin ATPase activity in biopsy samples from healthy and COPD individuals was implemented as a local nucleotide sensor to determine ATP diffusion coefficients within myofibrils. Ergometric parameters clinically measured during maximal exercise tests in both groups were used to define the rates of myosin ATPase reaction and aerobic ATP re-synthesis. The obtained parameters in combination with AK- and CK-catalyzed reactions were implemented to compute the kinetic and steady-state spatial ATP distributions within control and COPD sarcomeres. RESULTS: The developed reaction-diffusion model of two-dimensional sarcomeric space identified similar, yet extremely low nucleotide diffusion in normal and COPD myofibrils. The corresponding spatio-temporal ATP distributions, constructed during imposed exercise, predicted in COPD sarcomeres a depletion of ATP in the zones of overlap between actin and myosin filaments along the center axis at average cytosolic ATP levels similar to healthy muscles. CONCLUSIONS: ATP-depleted zones can induce rigor tension foci impairing muscle contraction and increase a risk for sarcomere damages. Thus, intra-sarcomeric diffusion restrictions at limited aerobic ATP re-synthesis can be an additional risk factor contributing to the muscle contractile deficiency experienced by COPD patients. GENERAL SIGNIFICANCE: This study demonstrates how restricted substrate mobility within a cellular organelle can provoke an energy imbalance state paradoxically occurring at abounding average metabolic resources.

Novel Cardiac Intracrine Mechanisms Based on Ang-(1-12)/Chymase Axis Require a Revision of Therapeutic Approaches in Human Heart Disease.

PURPOSE OF THE REVIEW: Drugs targeting the renin-angiotensin system (RAS), namely angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers, are the most commonly prescribed drugs for patients with or at risk for cardiovascular events. However, new treatment strategies aimed at mitigating the rise of the heart failure pandemic are warranted because clinical trials show that RAS blockers have limited benefits in halting disease progression. The main goal of this review is to put forward the concept of an intracrine RAS signaling through the novel angiotensin-(1-12)/chymase axis as the main source of deleterious angiotensin II (Ang II) in cardiac maladaptive remodeling leading to heart failure (HF). RECENT FINDINGS: Expanding traditional knowledge, Ang II can be produced in tissues independently from the circulatory renin-angiotensin system. In the heart, angiotensin-(1-12) [Ang-(1-12)], a recently discovered derivative of angiotensinogen, is a precursor of Ang II, and chymase rather than ACE is the main enzyme contributing to the direct production of Ang II from Ang-(1-12). The Ang-(1-12)/chymase axis is an independent intracrine pathway accounting for the trophic, contractile, and pro-arrhythmic Ang II actions in the human heart. Ang-(1-12) expression and chymase activity have been found elevated in the left atrial appendage of heart disease subjects, suggesting a pivotal role of this axis in the progression of HF. Recent meta-analysis of large clinical trials on the use of ACE inhibitors and angiotensin receptor blockers in cardiovascular disease has demonstrated an imbalance between patients that significantly benefit from these therapeutic agents and those that remain at risk for heart disease progression. Looking to find an explanation, detailed investigation on the RAS has unveiled a previously unrecognized complexity of substrates and enzymes in tissues ultimately associated with the production of Ang II that may explain the shortcomings of ACE inhibition and angiotensin receptor blockade. Discovery of the Ang-(1-12)/chymase axis in human hearts, capable of producing Ang II independently from the circulatory RAS, has led to the notion that a tissue-delimited RAS signaling in an intracrine fashion may account for the deleterious effects of Ang II in the heart, contributing to the transition from maladaptive cardiac remodeling to heart failure. Targeting intracellular RAS signaling may improve current therapies aimed at reducing the burden of heart failure.

Calreticulin secures calcium-dependent nuclear pore competency required for cardiogenesis.

Calreticulin deficiency causes myocardial developmental defects that culminate in an embryonic lethal phenotype. Recent studies have linked loss of this calcium binding chaperone to failure in myofibrillogenesis through an as yet undefined mechanism. The purpose of the present study was to identify cellular processes corrupted by calreticulin deficiency that precipitate dysregulation of cardiac myofibrillogenesis related to acquisition of cardiac phenotype. In an embryonic stem cell knockout model, calreticulin deficit (crt(-/-)) compromised nucleocytoplasmic transport of nuclear localization signal-dependent and independent pathways, disrupting nuclear import of the cardiac transcription factor MEF2C. The expression of nucleoporins and associated nuclear transport proteins in derived crt(-/-) cardiomyocytes revealed an abnormal nuclear pore complex (NPC) configuration. Altered protein content in crt(-/-) cells resulted in remodeled NPC architecture that caused decreased pore diameter and diminished probability of central channel occupancy versus wild type counterparts. Ionophore treatment of impaired calcium handling in crt(-/-) cells corrected nuclear pore microarchitecture and rescued nuclear import resulting in normalized myofibrillogenesis. Thus, calreticulin deficiency alters nuclear pore function and structure, impeding myofibrillogenesis in nascent cardiomyocytes through a calcium dependent mechanism. This essential role of calreticulin in nucleocytoplasmic communication competency ties its regulatory action with proficiency of cardiac myofibrillogenesis essential for proper cardiac development.

Sarcolemmal α2-adrenoceptors control protective cardiomyocyte-delimited sympathoadrenal response.

Sustained cardiac adrenergic stimulation has been implicated in the development of heart failure and ventricular dysrhythmia. Conventionally, α2 adrenoceptors (α2-AR) have been assigned to a sympathetic short-loop feedback aimed at attenuating catecholamine release. We have recently revealed the expression of α2-AR in the sarcolemma of cardiomyocytes and identified the ability of α2-AR signaling to suppress spontaneous Ca2+ transients through nitric oxide (NO) dependent pathways. Herein, patch-clamp measurements and serine/threonine phosphatase assay revealed that, in isolated rat cardiomyocytes, activation of α2-AR suppressed L-type Ca2+ current (ICaL) via stimulation of NO synthesis and protein kinase G- (PKG) dependent activation of phosphatase reactions, counteracting isoproterenol-induced ß-adrenergic activation. Under stimulation with norepinephrine (NE), an agonist of ß- and α-adrenoceptors, the α2-AR antagonist yohimbine substantially elevated ICaL at NE levels >10nM. Concomitantly, yohimbine potentiated triggered intracellular Ca2+ dynamics and contractility of cardiac papillary muscles. Therefore, in addition to the α2-AR-mediated feedback suppression of sympathetic and adrenal catecholamine release, α2-AR in cardiomyocytes can govern a previously unrecognized local cardiomyocyte-delimited stress-reactive signaling pathway. We suggest that such aberrant α2-AR signaling may contribute to the development of cardiomyopathy under sustained sympathetic drive. Indeed, in cardiomyocytes of spontaneously hypertensive rats (SHR), an established model of cardiac hypertrophy, α2-AR signaling was dramatically reduced despite increased α2-AR mRNA levels compared to normal cardiomyocytes. Thus, targeting α2-AR signaling mechanisms in cardiomyocytes may find implications in medical strategies against maladaptive cardiac remodeling associated with chronic sympathoadrenal stimulation.

Rbm20-deficient cardiogenesis reveals early disruption of RNA processing and sarcomere remodeling establishing a developmental etiology for dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) due to mutations in RBM20, a gene encoding an RNA-binding protein, is associated with high familial penetrance, risk of progressive heart failure and sudden death. Although genetic investigations and physiological models have established the linkage of RBM20 with early-onset DCM, the underlying basis of cellular and molecular dysfunction is undetermined. Modeling human genetics using a high-throughput pluripotent stem cell platform was herein designed to pinpoint the initial transcriptome dysfunction and mechanistic corruption in disease pathogenesis. Tnnt2-pGreenZeo pluripotent stem cells were engineered to knockdown Rbm20 (shRbm20) to determine the cardiac-pathogenic phenotype during cardiac differentiation. Intracellular Ca(2+) transients revealed Rbm20-dependent alteration in Ca(2+) handling, coinciding with known pathological splice variants of Titin and Camk2d genes by Day 24 of cardiogenesis. Ultrastructural analysis demonstrated elongated and thinner sarcomeres in the absence of Rbm20 that is consistent with human cardiac biopsy samples. Furthermore, Rbm20-depleted transcriptional profiling at Day 12 identified Rbm20-dependent dysregulation with 76% of differentially expressed genes linked to known cardiac pathology ranging from primordial Nkx2.5 to mature cardiac Tnnt2 as the initial molecular aberrations. Notably, downstream consequences of Rbm20-depletion at Day 24 of differentiation demonstrated significant dysregulation of extracellular matrix components such as the anomalous overexpression of the Vtn gene. By using the pluripotent stem cell platform to model human cardiac disease according to a stage-specific cardiogenic roadmap, we established a new paradigm of familial DCM pathogenesis as a developmental disorder that is patterned during early cardiogenesis and propagated with cellular mechanisms of pathological cardiac remodeling.

Cocaine Hydrolase Gene Transfer Demonstrates Cardiac Safety and Efficacy against Cocaine-Induced QT Prolongation in Mice.

Cocaine addiction is associated with devastating medical consequences, including cardiotoxicity and risk-conferring prolongation of the QT interval. Viral gene transfer of cocaine hydrolase engineered from butyrylcholinesterase offers therapeutic promise for treatment-seeking drug users. Although previous preclinical studies have demonstrated benefits of this strategy without signs of toxicity, the specific cardiac safety and efficacy of engineered butyrylcholinesterase viral delivery remains unknown. Here, telemetric recording of electrocardiograms from awake, unrestrained mice receiving a course of moderately large cocaine doses (30 mg/kg, twice daily for 3 weeks) revealed protection against a 2-fold prolongation of the QT interval conferred by pretreatment with cocaine hydrolase vector. By itself, this prophylactic treatment did not affect QT interval duration or cardiac structure, demonstrating that viral delivery of cocaine hydrolase has no intrinsic cardiac toxicity and, on the contrary, actively protects against cocaine-induced QT prolongation.

Reward and Toxicity of Cocaine Metabolites Generated by Cocaine Hydrolase.

Butyrylcholinesterase (BChE) gene therapy is emerging as a promising concept for treatment of cocaine addiction. BChE levels after gene transfer can rise 1000-fold above those in untreated mice, making this enzyme the second most abundant plasma protein. For months or years, gene transfer of a BChE mutated into a cocaine hydrolase (CocH) can maintain enzyme levels that destroy cocaine within seconds after appearance in the blood stream, allowing little to reach the brain. Rapid enzyme action causes a sharp rise in plasma levels of two cocaine metabolites, benzoic acid (BA) and ecgonine methyl ester (EME), a smooth muscle relaxant that is mildly hypotensive and, at best, only weakly rewarding. The present study, utilizing Balb/c mice, tested reward effects and cardiovascular effects of administering EME and BA together at molar levels equivalent to those generated by a given dose of cocaine. Reward was evaluated by conditioned place preference. In this paradigm, cocaine (20 mg/kg) induced a robust positive response but the equivalent combined dose of EME + BA failed to induce either place preference or aversion. Likewise, mice that had undergone gene transfer with mouse CocH (mCocH) showed no place preference or aversion after repeated treatments with a near-lethal 80 mg/kg cocaine dose. Furthermore, a single administration of that same high cocaine dose failed to affect blood pressure as measured using the noninvasive tail-cuff method. These observations confirm that the drug metabolites generated after CocH gene transfer therapy are safe even after a dose of cocaine that would ordinarily be lethal.

Alpha-2 adrenoceptors and imidazoline receptors in cardiomyocytes mediate counterbalancing effect of agmatine on NO synthesis and intracellular calcium handling.

Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α2 (α2-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine.

A study of complexation and biological fate of polyethyleneimine-siRNA polyplexes in vitro and in vivo by fluorescence correlation spectroscopy and positron emission tomography imaging.

A deeper knowledge on the formation and biological fate of polymer based gene vectors is needed for their translation into therapy. Here, polyplexes of polyethyleneimine (PEI) and silencing RNA (siRNA) are formed with theoretical N/P ratios of 2, 4 and 12. Fluorescence correlation spectroscopy (FCS) is used to study the formation of polyplexes from fluorescently labelled PEI and siRNA. FCS proves the presence of free PEI. From the analysis of the autocorrelation functions it was possible to determine the actual stoichiometry of polyplexes. FCS and fluorescence cross correlation spectroscopy (FCCS) are used to follow the fate of the polyplexes intracellularly. Polyplexes disassemble after 1 day inside cells. Positron emission tomography (PET) studies are conducted with radiolabelled polyplexes prepared with siRNA or PEI labelled with 2,3,5,6-tetrafluorophenyl 6-[18F]-fluoronicotinate ([18F]F-PyTFP). PET studies in healthy mice show that [18F]siRNA/PEI and siRNA/[18F]PEI polyplexes show similar biodistribution patterns with limited circulation in the bloodstream and accumulation in the liver. Higher activity for [18F]PEI in the kidney and bladder suggests the presence of free PEI.

Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution.

HYPOTHESIS: Renal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. EXPERIMENTS: We successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FINDINGS: CIT-PAH nanoparticles dissolves CaOx nanocrystals as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

Ranolazine decreases mechanosensitivity of the voltage-gated sodium ion channel Na(v)1.5: a novel mechanism of drug action.

BACKGROUND: Na(V)1.5 is a mechanosensitive voltage-gated sodium-selective ion channel responsible for the depolarizing current and maintenance of the action potential plateau in the heart. Ranolazine is a Na(V)1.5 antagonist with antianginal and antiarrhythmic properties. METHODS AND RESULTS: Mechanosensitivity of Na(V)1.5 was tested in voltage-clamped whole cells and cell-attached patches by bath flow and patch pressure, respectively. In whole cells, bath flow increased peak inward current in both murine ventricular cardiac myocytes (24±8%) and human embryonic kidney 293 cells heterologously expressing Na(V)1.5 (18±3%). The flow-induced increases in peak current were blocked by ranolazine. In cell-attached patches from cardiac myocytes and Na(V)1.5-expressing human embryonic kidney 293 cells, negative pressure increased Na(V) peak currents by 27±18% and 18±4% and hyperpolarized voltage dependence of activation by -11 mV and -10 mV, respectively. In human embryonic kidney 293 cells, negative pressure also increased the window current (250%) and increased late open channel events (250%). Ranolazine decreased pressure-induced shift in the voltage dependence (IC(50) 54 µmol/L) and eliminated the pressure-induced increases in window current and late current event numbers. Block of Na(V)1.5 mechanosensitivity by ranolazine was not due to the known binding site on DIVS6 (F1760). The effect of ranolazine on mechanosensitivity of Na(V)1.5 was approximated by lidocaine. However, ionized ranolazine and charged lidocaine analog (QX-314) failed to block mechanosensitivity. CONCLUSIONS: Ranolazine effectively inhibits mechanosensitivity of Na(V)1.5. The block of Na(V)1.5 mechanosensitivity by ranolazine does not utilize the established binding site and may require bilayer partitioning. Ranolazine block of Na(V)1.5 mechanosensitivity may be relevant in disorders of mechanoelectric dysfunction.

K(ATP) channels process nucleotide signals in muscle thermogenic response.

Uniquely gated by intracellular adenine nucleotides, sarcolemmal ATP-sensitive K(+) (K(ATP)) channels have been typically assigned to protective cellular responses under severe energy insults. More recently, K(ATP) channels have been instituted in the continuous control of muscle energy expenditure under non-stressed, physiological states. These advances raised the question of how K(ATP) channels can process trends in cellular energetics within a milieu where each metabolic system is set to buffer nucleotide pools. Unveiling the mechanistic basis of the K(ATP) channel-driven thermogenic response in muscles thus invites the concepts of intracellular compartmentalization of energy and proteins, along with nucleotide signaling over diffusion barriers. Furthermore, it requires gaining insight into the properties of reversibility of intrinsic ATPase activity associated with K(ATP) channel complexes. Notwithstanding the operational paradigm, the homeostatic role of sarcolemmal K(ATP) channels can be now broadened to a wider range of environmental cues affecting metabolic well-being. In this way, under conditions of energy deficit such as ischemic insult or adrenergic stress, the operation of K(ATP) channel complexes would result in protective energy saving, safeguarding muscle performance and integrity. Under energy surplus, downregulation of K(ATP) channel function may find potential implications in conditions of energy imbalance linked to obesity, cold intolerance and associated metabolic disorders.

Racial/Ethnic Differences in Non-Discretionary Risk Factors for COVID-19 Among Patients in an Early COVID-19 Hotspot.

BACKGROUND: Baseline disparities in non-discretionary risk factors, i.e., those not readily altered, like family size and work environment, appear to underlie the disproportionate COVID-19 infection rates seen among Hispanic persons and, at surge onsets, Black persons. No study has systematically compared such risk factors by race/ethnicity among infected individuals. METHODS: Using a cross-sectional survey, we compared household, job, and socioeconomic characteristics among 260 Hispanic, non-Hispanic Black, and non-Hispanic White adults with confirmed or probable COVID-19 in New York from March to May 2020. We used logistic regression to identify independent relationships. RESULTS: In bivariate analysis, we found significant differences by race/ethnicity in the following: (1) rates of household crowding (p < 0.001), which were highest for Hispanic patients (45.1%) and lowest for White patients (0.9%); (2) rates of non-healthcare frontline work (p < 0.001), which were highest for Hispanic patients (71.0% of those employed) and lowest for White patients (31.4%); (3) rates of working close to people (p < 0.001), which were highest for Black patients (69.4%) and lowest for Hispanic patients (32.3%); and (4) rates of frontline healthcare work (p = 0.004), which were higher for Black (44.9%) and White (44.3%) patients than Hispanic patients (19.4%). Adjusting for covariates eliminated most differences but not that for household crowding. CONCLUSIONS: Non-discretionary COVID-19 risk factors among patients in the initial surge differed substantially by race/ethnicity. Socioeconomic factors explained most differences, but household crowding was independently associated with Hispanic ethnicity. Our findings highlight the ongoing need for universal safeguards for US frontline workers, including mandated paid sick leave and expanded affordable housing options.

Compartmentation of membrane processes and nucleotide dynamics in diffusion-restricted cardiac cell microenvironment.

Orchestrated excitation-contraction coupling in heart muscle requires adequate spatial arrangement of systems responsible for ion movement and metabolite turnover. Co-localization of regulatory and transporting proteins into macromolecular complexes within an environment of microanatomical cell components raises intracellular diffusion barriers that hamper the mobility of metabolites and signaling molecules. Compared to substrate diffusion in the cytosol, diffusional restrictions underneath the sarcolemma are much larger and could impede ion and nucleotide movement by a factor of 10(3)-10(5). Diffusion barriers thus seclude metabolites within the submembrane space enabling rapid and vectorial effector targeting, yet hinder energy supply from the bulk cytosolic space implicating the necessity for a shunting transfer mechanism. Here, we address principles of membrane protein compartmentation, phosphotransfer enzyme-facilitated interdomain energy transfer, and nucleotide signal dynamics at the subsarcolemma-cytosol interface. This article is part of a Special Issue entitled "Local Signaling in Myocytes".