MYBPC3 D389V Variant Induces Hypercontractility in Cardiac Organoids.

BACKGROUND: MYBPC3 , encoding cardiac myosin binding protein-C (cMyBP-C), is the most mutated gene known to cause hypertrophic cardiomyopathy (HCM). However, since little is known about the underlying etiology, additional in vitro studies are crucial to defining the underlying molecular mechanisms. Accordingly, this study aimed to investigate the molecular mechanisms underlying the pathogenesis of HCM associated with a polymorphic variant (D389V) in MYBPC3 by using human-induced pluripotent stem cell (hiPSC)-derived cardiac organoids (hCOs). METHODS: The hiPSC-derived cardiomyocytes (hiPSC-CMs) and hCOs were generated from human subjects to define the molecular, cellular, and functional changes caused by the MYBPC3 D389V variant. This variant is associated with increased fractional shortening and is highly prevalent in South Asian descendants. Recombinant C0-C2, N'-region of cMyBP-C (wildtype and D389V), and myosin S2 proteins were also utilized to perform binding and motility assays in vitro . RESULTS: Confocal and electron microscopic analyses of hCOs generated from noncarriers (NC) and carriers of the MYBPC3 D389V variant revealed the presence of highly organized sarcomeres. Furthermore, functional experiments showed hypercontractility with increased contraction velocity, faster calcium cycling, and faster contractile kinetics in hCOs expressing MYBPC3 D389V than NC hCOs. Interestingly, significantly increased cMyBP-C phosphorylation in MYBPC3 D389V hCOs was observed, but without changes in total protein levels, in addition to higher oxidative stress and lower mitochondrial membrane potential (ΔΨm). Next, spatial mapping revealed the presence of endothelial cells, fibroblasts, macrophages, immune cells, and cardiomyocytes in the hCOs. The hypercontractile function was significantly improved after treatment with the myosin inhibitor mavacamten (CAMZYOS®) in MYBPC3 D389V hCOs. Lastly, various in vitro binding assays revealed a significant loss of affinity in the presence of MYBPC3 D389V with myosin S2 region as a likely mechanism for hypercontraction. CONCLUSIONS: Conceptually, we showed the feasibility of assessing the functional and molecular mechanisms of HCM using highly translatable hCOs through pragmatic experiments that led to determining the MYBPC3 D389V hypercontractile phenotype, which was rescued by administration of a myosin inhibitor. Novelty and Significance: What Is Known?: MYBPC3 mutations have been implicated in hypertrophic cardiomyopathy. D389V is a polymorphic variant of MYBPC3 predicted to be present in 53000 US South Asians owing to the founder effect. D389V carriers have shown evidence of hyperdynamic heart, and human-induced pluripotent stem cells (hiPSC)-derived cardiomyocytes with D389V show cellular hypertrophy and irregular calcium transients. The molecular mechanism by which the D389V variant develops pathological cardiac dysfunction remains to be conclusively determined.What New Information Does This Article Contribute ?: The authors leveraged a highly translational cardiac organoid model to explore the role of altered cardiac calcium handling and cardiac contractility as a common pathway leading to pathophysiological phenotypes in patients with early HCM. The MYBPC3 D389V -mediated pathological pathway is first studied here by comparing functional properties using three-dimensional cardiac organoids differentiated from hiPSC and determining the presence of hypercontraction. Our data demonstrate that faster sarcomere kinetics resulting from lower binding affinity between D389V-mutated cMyBP-C protein and myosin S2, as evidenced by in vitro studies, could cause hypercontractility which was rescued by administration of mavacamten (CAMZYOS®), a myosin inhibitor. In addition, hypercontractility causes secondary mitochondrial defects such as higher oxidative stress and lower mitochondrial membrane potential (ΔΨm), highlighting a possible early adaptive response to primary sarcomeric changes. Early treatment of MYBPC3 D389V carriers with mavacamten may prevent or reduce early HCM-related pathology. GRAPHICAL ABSTRACT: A graphical abstract is available for this article.

Impairment of Endothelial Function by Aerosol From Marijuana Leaf Vaporizers.

BACKGROUND: Marijuana leaf vaporizers, which heat plant material and sublimate Δ-9-tetrahydrocannabinol without combustion, are popular alternatives to smoking cannabis that are generally perceived to be less harmful. We have shown that smoke from tobacco and marijuana, as well as aerosol from e-cigarettes and heated tobacco products, impair vascular endothelial function in rats measured as arterial flow-mediated dilation (FMD). METHODS AND RESULTS: We exposed 8 rats per group to aerosol generated by 2 vaporizer systems (Volcano and handheld Yocan) using marijuana with varying Δ-9-tetrahydrocannabinol levels, in a single pulsatile exposure session of 2 s/min over 5 minutes, and measured changes in FMD. To model secondhand exposure, we exposed rats for 1 minute to diluted aerosol approximating release of uninhaled Volcano aerosol into typical residential rooms. Exposure to aerosol from marijuana with and without cannabinoids impaired FMD by ≈50%. FMD was similarly impaired by aerosols from Yocan (237 °C), and from Volcano at both its standard temperature (185 °C) and the minimum sublimation temperature of Δ-9-tetrahydrocannabinol (157 °C), although the low-temperature aerosol condition did not effectively deliver Δ-9-tetrahydrocannabinol to the circulation. Modeled secondhand exposure based on diluted Volcano aerosol also impaired FMD. FMD was not affected in rats exposed to clean air or water vapor passed through the Volcano system. CONCLUSIONS: Acute direct exposure and modeled secondhand exposure to marijuana leaf vaporizer aerosol, regardless of cannabinoid concentration or aerosol generation temperature, impair endothelial function in rats comparably to marijuana smoke. Our findings indicate that use of leaf vaporizers is unlikely to reduce the vascular risk burden of smoking marijuana.

Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

AIMS: Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. METHODS AND RESULTS: Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). CONCLUSION: Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Impairment of Endothelial Function by Cigarette Smoke Is Not Caused by a Specific Smoke Constituent, but by Vagal Input From the Airway.

BACKGROUND: Exposure to tobacco or marijuana smoke, or e-cigarette aerosols, causes vascular endothelial dysfunction in humans and rats. We aimed to determine what constituent, or class of constituents, of smoke is responsible for endothelial functional impairment. METHODS: We investigated several smoke constituents that we hypothesized to mediate this effect by exposing rats and measuring arterial flow-mediated dilation (FMD) pre- and post-exposure. We measured FMD before and after inhalation of sidestream smoke from research cigarettes containing normal and reduced nicotine level with and without menthol, as well as 2 of the main aldehyde gases found in both smoke and e-cigarette aerosol (acrolein and acetaldehyde), and inert carbon nanoparticles. RESULTS: FMD was reduced by all 4 kinds of research cigarettes, with extent of reduction ranging from 20% to 46% depending on the cigarette type. While nicotine was not required for the impairment, higher nicotine levels in smoke were associated with a greater percent reduction of FMD (41.1±4.5% reduction versus 19.2±9.5%; P=0.047). Lower menthol levels were also associated with a greater percent reduction of FMD (18.5±9.8% versus 40.5±4.8%; P=0.048). Inhalation of acrolein or acetaldehyde gases at smoke-relevant concentrations impaired FMD by roughly 50% (P=0.001). However, inhalation of inert carbon nanoparticles at smoke-relevant concentrations with no gas phase also impaired FMD by a comparable amount (P<0.001). Bilateral cervical vagotomy blocked the impairment of FMD by tobacco smoke. CONCLUSIONS: There is no single constituent or class of constituents responsible for acute impairment of endothelial function by smoke; rather, we propose that acute endothelial dysfunction by disparate inhaled products is caused by vagus nerve signaling initiated by airway irritation.

Genetic, clinical, molecular, and pathogenic aspects of the South Asian-specific polymorphic MYBPC3Δ25bp variant.

Hypertrophic cardiomyopathy (HCM) is a cardiac genetic disease characterized by ventricular enlargement, diastolic dysfunction, and increased risk for sudden cardiac death. Sarcomeric genetic defects are the predominant known cause of HCM. In particular, mutations in the myosin-binding protein C gene (MYBPC3) are associated with ~ 40% of all HCM cases in which a genetic basis has been established. A decade ago, our group reported a 25-base pair deletion in intron 32 of MYBPC3 (MYBPC3Δ25bp) that is uniquely prevalent in South Asians and is associated with autosomal dominant cardiomyopathy. Although our studies suggest that this deletion results in left ventricular dysfunction, cardiomyopathies, and heart failure, the precise mechanism by which this variant predisposes to heart disease remains unclear. Increasingly appreciated, however, is the contribution of secondary risk factors, additional mutations, and lifestyle choices in augmenting or modifying the HCM phenotype in MYBPC3Δ25bp carriers. Therefore, the goal of this review article is to summarize the current research dedicated to understanding the molecular pathophysiology of HCM in South Asians with the MYBPC3Δ25bp variant. An emphasis is to review the latest techniques currently applied to explore the MYBPC3Δ25bp pathogenesis and to provide a foundation for developing new diagnostic strategies and advances in therapeutics.

Vascular endothelial function is impaired by aerosol from a single IQOS HeatStick to the same extent as by cigarette smoke.

BACKGROUND: Heated tobacco products (also called 'heat-not-burn' products) heat tobacco at temperatures below that of combustion, causing nicotine and other compounds to aerosolise. One such product, IQOS from Philip Morris International, is being marketed internationally with claims of harm reduction. We sought to determine whether exposure to IQOS aerosol impairs arterial flow-mediated dilation (FMD), a measure of vascular endothelial function that is impaired by tobacco smoke. METHODS: We exposed anaesthetised rats (n=8/group) via nose cone to IQOS aerosol from single HeatSticks, mainstream smoke from single Marlboro Red cigarettes or clean air for a series of consecutive 30 s cycles over 1.5-5 min. Each cycle consisted of 15 or 5 s of exposure followed by removal from the nose cone. We measured pre-exposure and postexposure FMD, and postexposure serum nicotine and cotinine. RESULTS: FMD was impaired comparably by ten 15 s exposures and ten 5 s exposures to IQOS aerosol and to cigarette smoke, but not by clean air. Serum nicotine levels were similar to plasma levels after humans have smoked one cigarette, confirming that exposure conditions had real-world relevance. Postexposure nicotine levels were ~4.5-fold higher in rats exposed to IQOS than to cigarettes, despite nicotine being measured in the IQOS aerosol at ~63% the amount measured in smoke. When IQOS exposure was briefer, leading to comparable serum nicotine levels to the cigarette group, FMD was still comparably impaired. CONCLUSIONS: Acute exposures to IQOS aerosol impairs FMD in rats. IQOS use does not necessarily avoid the adverse cardiovascular effects of smoking cigarettes.

One Minute of Marijuana Secondhand Smoke Exposure Substantially Impairs Vascular Endothelial Function.

BACKGROUND: Despite public awareness that tobacco secondhand smoke (SHS) is harmful, many people still assume that marijuana SHS is benign. Debates about whether smoke-free laws should include marijuana are becoming increasingly widespread as marijuana is legalized and the cannabis industry grows. Lack of evidence for marijuana SHS causing acute cardiovascular harm is frequently mistaken for evidence that it is harmless, despite chemical and physical similarity between marijuana and tobacco smoke. We investigated whether brief exposure to marijuana SHS causes acute vascular endothelial dysfunction. METHODS AND RESULTS: We measured endothelial function as femoral artery flow-mediated dilation (FMD) in rats before and after exposure to marijuana SHS at levels similar to real-world tobacco SHS conditions. One minute of exposure to marijuana SHS impaired FMD to a comparable extent as impairment from equal concentrations of tobacco SHS, but recovery was considerably slower for marijuana. Exposure to marijuana SHS directly caused cannabinoid-independent vasodilation that subsided within 25 minutes, whereas FMD remained impaired for at least 90 minutes. Impairment occurred even when marijuana lacked cannabinoids and rolling paper was omitted. Endothelium-independent vasodilation by nitroglycerin administration was not impaired. FMD was not impaired by exposure to chamber air. CONCLUSIONS: One minute of exposure to marijuana SHS substantially impairs endothelial function in rats for at least 90 minutes, considerably longer than comparable impairment by tobacco SHS. Impairment of FMD does not require cannabinoids, nicotine, or rolling paper smoke. Our findings in rats suggest that SHS can exert similar adverse cardiovascular effects regardless of whether it is from tobacco or marijuana.

A new method to make 24-hour urine collection more convenient: a validity study.

Background and Objectives. This study proposes a novel urine collection device that can divide each urine collection into 20 parts and store and cool just one part. The aim of the current study is to compare measured biomarkers from the proposed urine collection device to those of conventional 24-hour sampling method. We also hypothesized that the new method would significantly increase patients' adherence to the timed urine collection. Methods. Two 24-hour urine samples with the conventional method and with the new automated urine collection device that uses just one-twentieth of each void were obtained from 40 healthy volunteers. Urine parameters including volume, creatinine, and protein levels were compared between the two methods and the agreement of two measurements for each subject was reported through Bland-Altman plots. Results. Our results confirmed that for all three variables, there is a positive correlation (P < 0.001) between the two measurements and high degree of agreement could be seen in Bland-Altman plots. Moreover, more subjects reported the new method as "more convenient" for 24-hour urine collection. Conclusions. Our results clearly indicate that a fixed proportion of each void may significantly reduce the urine volume in timed collections and this, in turn, may increase subjects' adherence to this difficult sampling.

Can procalcitonin reduce unnecessary voiding cystoureterography in children with first febrile urinary tract infection?

OBJECTIVE: Recently, new predictors of vesicoureteral reflux (VUR) in children with a first febrile UTI such as Procalcitonin (PCT) were introduced as selective approaches for cystography. This study wants to show the capability of PCT in predicting presence of VUR at the first febrile UTI in children. METHODS: Patients between 1 month and 15 years of age with febrile UTI were included in this prospective study. PCT values were measured through a semi-quantitative method in four grades comprising values less than 0.5, 0.5-2.0, 2.0-10.0 and above 10.0 ng/ml. The independence of PCT levels in predicting VUR were assessed after adjustment for all potential confounders using a logistic-regression model. FINDINGS: A total of 68 patients, 54 (79.4%) girls and 14 (20.6%) boys were evaluated. PCT level demonstrated a significant difference between patients with positive VUR and those with negative VUR (P=0.012). To calculate the independent factors that may predict the presence of VUR, all included variables were adjusted for age and sex. Results of logistic regression showed that a PCT level between 2.0 and 10.0 ng/mL could independently predict presence of VUR (Odds ratio=6.11, CI 95%= 1.22-30.77, P=0.03). CONCLUSION: Our finding in this study showed that readily available semi-quantitative measures for PCT are feasible for detecting patients with VUR. We suggest that in semi-quantitative measurements of PCT, levels between 2.0 and 10.0 ng/ml could be an independent predictor of positive VUR.

Intra-arterial injection of acrylic cement as a complication of percutaneous vertebroplasty.

Percutaneous vertebroplasty (PVP) with polymethylmethacrylate (PMMA) is a minimally invasive procedure that provides significant pain relief in a high percentage of patients with osteoporotic fractures. The complication rate of PVP is reported to be below 6%. This case illustrates, for the first time, an arterial PMMA embolus to the aorta and its branches as a complication of PVP.

A role for opioid system in the proconvulsant effects of sildenafil on the pentylenetetrazole-induced clonic seizure in mice.

There are several lines of evidence that opioidergic and nitrergic systems could modulate the seizure threshold. We previously have shown that sildenafil had proconvulsant effects in a model of clonic seizure induced by pentylenetetrazole (PTZ) or bicuculline. In the present study, we examined whether the opioid system participates in the action of sildenafil on the PTZ-induced clonic seizures in mice. Sildenafil (1, 5, 10 and 20 mg/kg, i.p.) significantly decreased the seizure threshold in a dose-dependent manner, whereas morphine had both anticonvulsant and proconvulsant effects at low (0.5, 1, and 3 mg/kg, s.c.) and high (60 mg/kg, s.c.) doses. A sub-effective dose of sildenafil (5 mg/kg) combined with a dose of morphine (7.5 mg/kg) which was sub-effective for its proconvulsant effects significantly decreased the seizure threshold. Although naltrexone at 0.5 and 1 mg/kg had no effect on the seizure threshold, it significantly prevented both the proconvulsant effects of sildenafil as well as the anticonvulsant and proconvulsant effects of morphine on the PTZ-induced seizure thresholds. Our data suggested a role for opioidergic system in the proconvulsant effects of sildenafil on the PTZ-induced clonic seizures in mice.