Exercise-induced cardiac mitochondrial reorganization and enhancement in spontaneously hypertensive rats.

The myocardium is a highly oxidative tissue in which mitochondria are essential to supply the energy required to maintain pump function. When pathological hypertrophy develops, energy consumption augments and jeopardizes mitochondrial capacity. We explored the cardiac consequences of chronic swimming training, focusing on the mitochondrial network, in spontaneously hypertensive rats (SHR). Male adult SHR were randomized to sedentary or trained (T: 8-week swimming protocol). Blood pressure and echocardiograms were recorded, and hearts were removed at the end of the training period to perform molecular, imaging, or isolated mitochondria studies. Swimming improved cardiac midventricular shortening and decreased the pathological hypertrophic marker atrial natriuretic peptide. Oxidative stress was reduced, and even more interesting, mitochondrial spatial distribution, dynamics, function, and ATP were significantly improved in the myocardium of T rats. In the signaling pathway triggered by training, we detected an increase in the phosphorylation level of both AKT and glycogen synthase kinase-3 ß, key downstream targets of insulin-like growth factor 1 signaling that are crucially involved in mitochondria biogenesis and integrity. Aerobic exercise training emerges as an effective approach to improve pathological cardiac hypertrophy and bioenergetics in hypertension-induced cardiac hypertrophy.

Multi-phased Kinetics and Interaction of Protein Kinase Signaling in Glycoprotein VI-Induced Platelet αIIbß3 Integrin Activation and Degranulation.

BACKGROUND: Platelet glycoprotein VI (GPVI) stimulation activates the tyrosine kinases Syk and Btk, and the effector proteins phospholipase Cγ 2 (PLCγ2) and protein kinase C (PKC). Here, the activation sequence, crosstalk, and downstream effects of this Syk-Btk-PKC signalosome in human platelets were analyzed. METHODS AND RESULTS: Using immunoblotting, we quantified 14 regulated phospho-sites in platelets stimulated by convulxin with and without inhibition of Syk, Btk, or PKC. Convulxin induced fast, reversible tyrosine phosphorylation (pY) of Syk, Btk, LAT, and PLCγ2, followed by reversible serine/threonine phosphorylation (pS/T) of Syk, Btk, and downstream kinases MEK1/2, Erk1/2, p38, and Akt. Syk inhibition by PRT-060318 abolished all phosphorylations, except Syk pY352. Btk inhibition by acalabrutinib strongly decreased Btk pY223/pS180, Syk pS297, PLCγ2 pY759/Y1217, MEK1/2 pS217/221, Erk1/2 pT202/Y204, p38 pT180/Y182, and Akt pT308/S473. PKC inhibition by GF109203X abolished most pS/T phosphorylations except p38 pT180/Y182 and Akt pT308, but enhanced most Y-phosphorylations. Acalabrutinib, but not GF109203X, suppressed convulxin-induced intracellular Ca2+ mobilization, whereas all three protein kinase inhibitors abolished degranulation and αIIbß3 integrin activation assessed by flow cytometry. Inhibition of autocrine ADP effects by AR-C669931 partly diminished convulxin-triggered degranulation. CONCLUSION: Kinetic analysis of GPVI-initiated multisite protein phosphorylation in human platelets demonstrates multiple phases and interactions of tyrosine and serine/threonine kinases with activation-altering feedforward and feedback loops partly involving PKC. The protein kinase inhibitor effects on multisite protein phosphorylation and functional readouts reveal that the signaling network of Syk, Btk, and PKC controls platelet granule exocytosis and αIIbß3 integrin activation.

Endothelium-mediated regulation of platelet activation: Involvement of multiple protein kinases.

The endothelial regulation of platelet activity is incompletely understood. Here we describe novel approaches to find molecular pathways implicated on the platelet-endothelium interaction. Using high-shear whole-blood microfluidics, employing coagulant or non-coagulant conditions at physiological temperature, we observed that the presence of human umbilical vein endothelial cells (HUVEC) strongly suppressed platelet adhesion and activation, via the collagen receptor glycoprotein VI (GPVI) and the PAR receptors for thrombin. Real-time monitoring of the cytosolic Ca2+ rises in the platelets indicated no major improvement of inhibition by prostacyclin or nitric oxide. Similarly under stasis, exposure of isolated platelets to HUVEC reduced the Ca2+ responses by collagen-related peptide (CRP-XL, GPVI agonist) and thrombin (PAR agonist). We then analyzed the label-free phosphoproteome of platelets (three donors), exposed to HUVEC, CRP-XL, and/or thrombin. High-resolution mass spectrometry gave 5463 phosphopeptides, corresponding to 1472 proteins, with good correlation between biological and technical replicates (R > .86). Stringent filtering steps revealed 26 regulatory pathways (Reactome) and 143 regulated kinase substrates (PhosphoSitePlus), giving a set of protein phosphorylation sites that was differentially (44) or similarly (110) regulated by HUVEC or agonist exposure. The differential regulation was confirmed by stable-isotope analysis of platelets from two additional donors. Substrate analysis indicated major roles of poorly studied protein kinase classes (MAPK, CDK, DYRK, STK, PKC members). Collectively, these results reveal a resetting of the protein phosphorylation profile in platelets exposed to endothelium or to conventional agonists and to endothelium-promoted activity of a multi-kinase network, beyond classical prostacyclin and nitric oxide actors, that may contribute to platelet inhibition.

Longitudinal changes of the femoral bone mineral density from first to third trimester of pregnancy: bone health assessment by means of non-ionizing REMS technology.

BACKGROUND: Throughout the pregnancy, there is a substantial transfer of calcium from the maternal skeleton to the fetus, which leads to a transient net reduction of the maternal bone mineral density. AIMS: To assess longitudinally the changes in the bone mineral density at the femoral neck between the first and third trimester of pregnancy in a cohort of healthy participants using Radiofrequency Echographic Multi Spectrometry (REMS) technology. METHODS: Prospective, cohort study conducted at the University hospital of Parma, Italy between July 2022 and February 2023. We recruited healthy participants with an uncomplicated singleton pregnancy before 14 completed weeks of gestation. All included participants were submitted to a sonographic examination of the femoral neck to assess the bone mineral density (and the corresponding Z-score values) using REMS at 11-13 and 36-38 weeks of pregnancy. The primary outcome was the change in the bone mineral density values at the maternal femoral neck between the first and third trimester of pregnancy. RESULTS: Over a period of 7 months, a total of 65 participants underwent bone mineral density measurement at the femoral neck at first and third trimester of the pregnancy using REMS. A significant reduction of the bone mineral density at the femoral neck (0.723 ± 0.069 vs 0.709 ± 0.069 g/cm2; p < 0.001) was noted with a mean bone mineral density change of - 1.9 ± 0.6% between the first and third trimester of pregnancy. At multivariable linear regression analysis, none of the demographic or clinical variables of the study population proved to be independently associated with the maternal bone mineral density changes at the femoral neck. CONCLUSIONS: Our study conducted on a cohort of healthy participants with uncomplicated pregnancy demonstrates that there is a significant reduction of bone mineral density at femoral neck from early to late gestation.

Differential Regulation of GPVI-Induced Btk and Syk Activation by PKC, PKA and PP2A in Human Platelets.

Bruton's tyrosine kinase (Btk) and spleen tyrosine kinase (Syk) are major signaling proteins in human platelets that are implicated in atherothrombosis and thrombo-inflammation, but the mechanisms controlling their activities are not well understood. Previously, we showed that Syk becomes phosphorylated at S297 in glycoprotein VI (GPVI)-stimulated human platelets, which limits Syk activation. Here, we tested the hypothesis that protein kinases C (PKC) and A (PKA) and protein phosphatase 2A (PP2A) jointly regulate GPVI-induced Btk activation in platelets. The GPVI agonist convulxin caused rapid, transient Btk phosphorylation at S180 (pS180↑), Y223 and Y551, while direct PKC activation strongly increased Btk pS180 and pY551. This increase in Btk pY551 was also Src family kinase (SFK)-dependent, but surprisingly Syk-independent, pointing to an alternative mechanism of Btk phosphorylation and activation. PKC inhibition abolished convulxin-stimulated Btk pS180 and Syk pS297, but markedly increased the tyrosine phosphorylation of Syk, Btk and effector phospholipase Cγ2 (PLCγ2). PKA activation increased convulxin-induced Btk activation at Y551 but strongly suppressed Btk pS180 and Syk pS297. PP2A inhibition by okadaic acid only increased Syk pS297. Both platelet aggregation and PLCγ2 phosphorylation with convulxin stimulation were Btk-dependent, as shown by the selective Btk inhibitor acalabrutinib. Together, these results revealed in GPVI-stimulated platelets a transient Syk, Btk and PLCγ2 phosphorylation at multiple sites, which are differentially regulated by PKC, PKA or PP2A. Our work thereby demonstrated the GPVI-Syk-Btk signalosome as a tightly controlled protein kinase network, in agreement with its role in atherothrombosis.

Multi-omics approaches to study platelet mechanisms.

Platelets are small anucleate cell fragments (2-4 µm in diameter) in the blood, which play an essential role in thrombosis and hemostasis. Genetic or acquired platelet dysfunctions are linked to bleeding, increased risk of thromboembolic events and cardiovascular diseases. Advanced proteomic approaches may pave the way to a better understanding of the roles of platelets in hemostasis, and pathophysiological processes such as inflammation, metastatic spread and thrombosis. Further insights into the molecular biology of platelets are crucial to aid drug development and identify diagnostic markers of platelet activation. Platelet activation is known to be an extremely rapid process and involves multiple post-translational mechanisms at sub second time scale, including proteolysis and phosphorylation. Multi-omics technologies and biochemical approaches can be exploited to precisely probe and define these posttranslational pathways. Notably, the absence of a nucleus in platelets significantly reduces the number of present proteins, simplifying mass spectrometry-based proteomics and metabolomics approaches.

Acid sphingomyelinase deactivation post-ischemia promotes brain angiogenesis and remodeling by small extracellular vesicles.

Antidepressants have been reported to enhance stroke recovery independent of the presence of depressive symptoms. They have recently been proposed to exert their mood-stabilizing actions by inhibition of acid sphingomyelinase (ASM), which catalyzes the hydrolysis of sphingomyelin to ceramide. Their restorative action post-ischemia/reperfusion (I/R) still had to be defined. Mice subjected to middle cerebral artery occlusion or cerebral microvascular endothelial cells exposed to oxygen-glucose deprivation were treated with vehicle or with the chemically and pharmacologically distinct antidepressants amitriptyline, fluoxetine or desipramine. Brain ASM activity significantly increased post-I/R, in line with elevated ceramide levels in microvessels. ASM inhibition by amitriptyline reduced ceramide levels, and increased microvascular length and branching point density in wildtype, but not sphingomyelinase phosphodiesterase-1 ([Smpd1]-/-) (i.e., ASM-deficient) mice, as assessed by 3D light sheet microscopy. In cell culture, amitriptyline, fluoxetine, and desipramine increased endothelial tube formation, migration, VEGFR2 abundance and VEGF release. This effect was abolished by Smpd1 knockdown. Mechanistically, the promotion of angiogenesis by ASM inhibitors was mediated by small extracellular vesicles (sEVs) released from endothelial cells, which exhibited enhanced uptake in target cells. Proteomic analysis of sEVs revealed that ASM deactivation differentially regulated proteins implicated in protein export, focal adhesion, and extracellular matrix interaction. In vivo, the increased angiogenesis was accompanied by a profound brain remodeling response with increased blood-brain barrier integrity, reduced leukocyte infiltrates and increased neuronal survival. Antidepressive drugs potently boost angiogenesis in an ASM-dependent way. The release of sEVs by ASM inhibitors disclosed an elegant target, via which brain remodeling post-I/R can be amplified.

Electronic cigarette liquids impair metabolic cooperation and alter proteomic profiles in V79 cells.

BACKGROUND: Although still considered a safer alternative to classical cigarettes, growing body of work points to harmful effects of electronic cigarettes (e-cigarettes) affecting a range of cellular processes. The biological effect of e-cigarettes needs to be investigated in more detail considering their widespread use. METHODS: In this study, we treated V79 lung fibroblasts with sub-cytotoxic concentration of e-cigarette liquids, with and without nicotine. Mutagenicity was evaluated by HPRT assay, genotoxicity by comet assay and the effect on cellular communication by metabolic cooperation assay. Additionally, comprehensive proteome analysis was performed via high resolution, parallel accumulation serial fragmentation-PASEF mass spectrometry. RESULTS: E-cigarette liquid concentration used in this study showed no mutagenic or genotoxic effect, however it negatively impacted metabolic cooperation between V79 cells. Both e-cigarette liquids induced significant depletion in total number of proteins and impairment of mitochondrial function in treated cells. The focal adhesion proteins were upregulated, which is in accordance with the results of metabolic cooperation assay. Increased presence of posttranslational modifications (PTMs), including carbonylation and direct oxidative modifications, was observed. Data are available via ProteomeXchange with identifier PXD032071. CONCLUSIONS: Our study revealed impairment of metabolic cooperation as well as significant proteome and PTMs alterations in V79 cells treated with e-cigarette liquid warranting future studies on e-cigarettes health impact.

Toward Zero Variance in Proteomics Sample Preparation: Positive-Pressure FASP in 96-Well Format (PF96) Enables Highly Reproducible, Time- and Cost-Efficient Analysis of Sample Cohorts.

As novel liquid chromatography-mass spectrometry (LC-MS) technologies for proteomics offer a substantial increase in LC-MS runs per day, robust and reproducible sample preparation emerges as a new bottleneck for throughput. We introduce a novel strategy for positive-pressure 96-well filter-aided sample preparation (PF96) on a commercial positive-pressure solid-phase extraction device. PF96 allows for a five-fold increase in throughput in conjunction with extraordinary reproducibility with Pearson product-moment correlations on the protein level of r = 0.9993, as demonstrated for mouse heart tissue lysate in 40 technical replicates. The targeted quantification of 16 peptides in the presence of stable-isotope-labeled reference peptides confirms that PF96 variance is barely assessable against technical variation from nanoLC-MS instrumentation. We further demonstrate that protein loads of 36-60 µg result in optimal peptide recovery, but lower amounts ≥3 µg can also be processed reproducibly. In summary, the reproducibility, simplicity, and economy of time provide PF96 a promising future in biomedical and clinical research.

Profile of women choosing mixed-sex, women-only, and home-based cardiac rehabilitation models and impact on utilization.

This study compared characteristics and program utilization in women electing to participate in mixed-sex, women-only, or home-based cardiac rehabilitation (CR). In this retrospective cohort study, electronic records of CR participants in Toronto who were offered the choice of program model between January 2017-February 2020 were analyzed. There were 727 women (74.7% mixed, 22.0% women-only, 3.3% home-based) who initiated CR. There were significantly more women who were not working in women-only than mixed-sex (80.4% vs 64.1%; P = .009). Session adherence was significantly greater with mixed-sex (58.8 ± 28.9% sessions attended/25) than women-only (54.3 ± 26.3% sessions attended/25; P = .046); program completion was significantly lower with home-based (33.3%) than either supervised model (59.7%; P = .035). Participation in women-only CR may be less accessible. Further research is needed to investigate offering remote women-focused sessions or peer support.

Building Capacity Through ICCPR Cardiovascular Rehabilitation Foundations Certification (CRFC): EVALUATION OF REACH, BARRIERS, AND IMPACT.

PURPOSE: The International Council of Cardiovascular Prevention and Rehabilitation (ICCPR) developed an online Cardiovascular Rehabilitation Foundations Certification (CRFC; https://globalcardiacrehab.com/Certification) in October 2017, to build cardiac rehabilitation (CR) delivery capacity in low-resource settings based on their guidelines. Herein we evaluate its reach globally, barriers to its completion, as well as satisfaction and impact of the course among those completing it. METHODS: The country of origin of all applicants was tallied. An online survey was developed for learners who completed the CRFC (completers), and for those who applied but did not yet complete the program (noncompleters), administered using Google Forms. RESULTS: With regard to reach, 236 applications were received from 23/203 (11%) countries in the world; 51 (22%) were from low- or middle-income countries. A total of 130 (55%) have completed the CRFC; mean scores on the final examination were 88.3 ± 7.1%, with no difference by country income classification (P= .052). Sixteen (22%) noncompleters and 37 (34%) completers responded to the survey. Barriers reported by noncompleters were time constraints, cost, and technical issues. Overall satisfaction (scale 1-5) with the CRFC was high (4.49 ± 0.51); most completers would highly recommend the CRFC to others (4.30 ± 0.66), and perceived that the information provided will contribute to their work and/or the care of their patients (4.38 ± 0.89); 29 (78%) had used the information from the CRFC in their practice. CONCLUSIONS: The reach of the CRFC still needs to be broadened, in particular in low-resource settings. Learners are highly satisfied with the certification, and its impacts on CR practice are encouraging. Input has been implemented to improve the CRFC.

Evaluation of an Online Course in 5 Languages for Inpatient Cardiac Care Providers on Promoting Cardiac Rehabilitation: REACH, EFFECTS, AND SATISFACTION.

PURPOSE: Evidence proves that health care providers should promote cardiac rehabilitation (CR) to patients face-to-face to increase CR enrollment. An online course was designed to promote this at the bedside; it is evaluated herein in terms of reach, effect on knowledge, attitudes, discussion self-efficacy and practices, and satisfaction. METHODS: Design was observational, one-group pre- and post-test. Some demographics were requested from learners taking all language versions of the 20-min course: English, Portuguese, French, Spanish, and simplified Chinese, available at: https://globalcardiacrehab.com/CR-Utilization. Investigator-generated items in the pre- and post-test and evaluation survey administered using Google Forms were based on Kirkpatrick's training evaluation model. RESULTS: The course was initiated by 522 learners from 33 of 203 (16%) countries; most commonly female (n = 341, 65%) nurses (n = 180, 34%) from high-income countries (n = 259, 57%) completing the English (n = 296, 57%) and Chinese (n = 108, 21%) versions. A total of 414 (79%) learners completed the post-test and 302 (58%) completed the evaluation. Median CR attitudes were 5 of 5 on the Likert scale at pre-test, suggesting some selection bias. Mean CR knowledge ([7.22 ± 2.14]/10), discussion self-efficacy ([3.86 ± 0.85]/5), and practice ([4.13 ± 1.11]/5) significantly improved after completion of the course (all P < .001). Satisfaction was high regardless of language version ([4.44 ± 0.64]/5; P = .593). CONCLUSIONS: This free, open-access course is effective in increasing CR knowledge, self-efficacy, and encouragement practices among participating inpatient cardiac providers, with high satisfaction. While testing impact on actual CR use is needed, it should be more broadly disseminated to increase reach, in an effort to increase patient enrollment in CR, to reduce morbidity and mortality.

Women's outcomes following mixed-sex, women-only, and home-based cardiac rehabilitation participation and comparison by sex.

BACKGROUND: Despite women's greater need for cardiac rehabilitation (CR), they are less likely to utilize it. Innovative CR models have been developed to better meet women's needs, yet there is little controlled, comparative data assessing the effects of these models for women. This study compared outcomes in women electing to participate in mixed-sex, women-only, or home-based CR, and a matched sample of men. METHODS: In this retrospective study, electronic records of CR participants in Toronto who were offered the choice of program model between January 2017 and July 2019 were analyzed; clinical outcomes comprised cardiorespiratory fitness, risk factors and psychosocial well-being. These were assessed at intake and post-6-month program and analyzed using general linear mixed models. RESULTS: There were 1181 patients (727 women [74.7% mixed, 22.0% women-only, 3.3% home-based]; 454 age and diagnosis-matched men) who initiated CR; Cardiorespiratory fitness among women was higher at initiation of mixed-sex than women-only (METs 5.1 ± 1.5 vs 4.6 ± 1.3; P = .007), but no other outcome differences were observed. 428 (58.9%) women completed the programs, with few women retained in the home-based model limiting comparisons. There were significant improvements in high-density lipoprotein cholesterol (P = .001) and quality of life (P = .001), and lower depressive symptoms (P = .030) as well as waist circumference (P = .001) with mixed-sex only. VO2peak was significantly higher at discharge in mixed-sex than women-only (estimate = 1.67, standard error = 0.63, 95% confidence interval = 0.43-2.91). CONCLUSION: Participation in non-gender-tailored women-only CR was not advantageous as expected. More research is needed, particularly including women participating in home-based programs.

Molecular Proteomics and Signalling of Human Platelets in Health and Disease.

Platelets are small anucleate blood cells that play vital roles in haemostasis and thrombosis, besides other physiological and pathophysiological processes. These roles are tightly regulated by a complex network of signalling pathways. Mass spectrometry-based proteomic techniques are contributing not only to the identification and quantification of new platelet proteins, but also reveal post-translational modifications of these molecules, such as acetylation, glycosylation and phosphorylation. Moreover, target proteomic analysis of platelets can provide molecular biomarkers for genetic aberrations with established or non-established links to platelet dysfunctions. In this report, we review 67 reports regarding platelet proteomic analysis and signalling on a molecular base. Collectively, these provide detailed insight into the: (i) technical developments and limitations of the assessment of platelet (sub)proteomes; (ii) molecular protein changes upon ageing of platelets; (iii) complexity of platelet signalling pathways and functions in response to collagen, rhodocytin, thrombin, thromboxane A2 and ADP; (iv) proteomic effects of endothelial-derived mediators such as prostacyclin and the anti-platelet drug aspirin; and (v) molecular protein changes in platelets from patients with congenital disorders or cardiovascular disease. However, sample sizes are still low and the roles of differentially expressed proteins are often unknown. Based on the practical and technical possibilities and limitations, we provide a perspective for further improvements of the platelet proteomic field.

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions.

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.

The effects of maintenance cardiac rehabilitation: A systematic review and Meta-analysis, with a focus on sex.

Phase III/IV cardiac rehabilitation (CR) is recommended to promote maintenance of benefits achieved during Phase II; there has been no meta-analysis to test this to date. This study determined the effects of maintenance CR on any outcome, with consideration of sex. Seven databases were searched from inception-January 2020. Randomized controlled trials on the effects of maintenance CR in cardiovascular disease patients who had graduated from CR were included. Level of evidence was evaluated with GRADEPro. 819 citations were identified, with 10 trials (21 papers) included (5238 participants; 859 [16.4%] female). Maintenance CR resulted in lower low-density lipoprotein (mean difference [MD]=-0.58; 95% confidence interval [CI]=-1.06--0.10, n = 392) and greater quality of life (MD = 0.28, 95% CI = 0.05-0.52, n = 118) when compared to usual care only. Outcomes for women and sex differences were mixed. In conclusion, maintenance programs appear to sustain patient's quality of life, but more focus on women's outcomes is needed.

Availability and delivery of cardiac rehabilitation in South-East Asia: How does it compare globally?

Background: The aims of this study were to establish cardiac rehabilitation (CR) availability and density, as well as the nature of programs in South-East Asian Region (SEAR) countries, and to compare this with other regions globally. Methods: In 2016/2017, the International Council of Cardiovascular Prevention and Rehabilitation engaged cardiac associations to facilitate program identification globally. An online survey was administered to identify programs using REDCap, assessing capacity and characteristics. CR density was computed using Global Burden of Disease study annual ischemic heart disease (IHD) incidence estimates. The program audit was updated in 2020. Results: CR was available in 6/11 (54.5%) SEAR countries. Data were collected in 5 countries (83.3% country response); 32/69 (68.1% response rate from 2016/2017) programs completed the survey. These data were compared to 1082 (32.1%) programs in 93/111 (83.3%) countries with CR. Across SEAR countries, there was only one CR spot per 283 IHD patients (vs. 12 globally), with an unmet regional need of 4,258,968 spots annually. Most programs were in tertiary care centers (n = 25, 78.1%; vs. 46.1% globally, P < 0.001). Most were funded privately (n = 17, 56.7%; vs. 17.9%, P < 0.001), and 22 (73.3%) patients were paying out of pocket (vs. 36.2% globally; P < 0.001). The mean number of staff on the multidisciplinary teams was 5.5 ± 3.0 (vs. 5.9 ± 2.8 globally P = 0.268), offering 8.6 ± 1.7/11 core components (consistent with other countries) over 16.8 ± 12.6 h (vs. 36.2 ± 53.3 globally, P = 0.01). Conclusion: Funded CR capacity must be augmented in SEAR. Where available, services were consistent with guidelines, and other regions of the globe, despite programs being shorter than other regions.

Mild hyperlipidemia in mice aggravates platelet responsiveness in thrombus formation and exploration of platelet proteome and lipidome.

Hyperlipidemia is a well-established risk factor for cardiovascular diseases. Millions of people worldwide display mildly elevated levels of plasma lipids and cholesterol linked to diet and life-style. While the prothrombotic risk of severe hyperlipidemia has been established, the effects of moderate hyperlipidemia are less clear. Here, we studied platelet activation and arterial thrombus formation in Apoe-/- and Ldlr-/- mice fed a normal chow diet, resulting in mildly increased plasma cholesterol. In blood from both knockout mice, collagen-dependent thrombus and fibrin formation under flow were enhanced. These effects did not increase in severe hyperlipidemic blood from aged mice and upon feeding a high-fat diet (Apoe-/- mice). Bone marrow from wild-type or Ldlr-/- mice was transplanted into irradiated Ldlr-/- recipients. Markedly, thrombus formation was enhanced in blood from chimeric mice, suggesting that the hyperlipidemic environment altered the wild-type platelets, rather than the genetic modification. The platelet proteome revealed high similarity between the three genotypes, without clear indication for a common protein-based gain-of-function. The platelet lipidome revealed an altered lipid profile in mildly hyperlipidemic mice. In conclusion, in Apoe-/- and Ldlr-/- mice, modest elevation in plasma and platelet cholesterol increased platelet responsiveness in thrombus formation and ensuing fibrin formation, resulting in a prothrombotic phenotype.

Impaired iloprost-induced platelet inhibition and phosphoproteome changes in patients with confirmed pseudohypoparathyroidism type Ia, linked to genetic mutations in GNAS.

Patients diagnosed with pseudohypoparathyroidism type Ia (PHP Ia) suffer from hormonal resistance and abnormal postural features, in a condition classified as Albright hereditary osteodystrophy (AHO) syndrome. This syndrome is linked to a maternally inherited mutation in the GNAS complex locus, encoding for the GTPase subunit Gsα. Here, we investigated how platelet phenotype and omics analysis can assist in the often difficult diagnosis. By coupling to the IP receptor, Gsα induces platelet inhibition via adenylyl cyclase and cAMP-dependent protein kinase A (PKA). In platelets from seven patients with suspected AHO, one of the largest cohorts examined, we studied the PKA-induced phenotypic changes. Five patients with a confirmed GNAS mutation, displayed impairments in Gsα-dependent VASP phosphorylation, aggregation, and microfluidic thrombus formation. Analysis of the platelet phosphoproteome revealed 2,516 phosphorylation sites, of which 453 were regulated by Gsα-PKA. Common changes in the patients were: (1) a joint panel of upregulated and downregulated phosphopeptides; (2) overall PKA dependency of the upregulated phosphopeptides; (3) links to key platelet function pathways. In one patient with GNAS mutation, diagnosed as non-AHO, the changes in platelet phosphoproteome were reversed. This combined approach thus revealed multiple phenotypic and molecular biomarkers to assist in the diagnosis of suspected PHP Ia.