Comprehensive meta-analysis of the effects of oral medroxyprogesterone acetate plus conjugated equine oestrogens on the lipid profile in women: Insights from randomized controlled trials.

BACKGROUND: Menopause is associated with elevated cardiovascular risk due to the loss of the cardioprotective effect of oestrogens. Postmenopausal women are often prescribed hormone replacement therapy (HRT) in order to control menopause symptoms and correct hormone imbalances; however, HRT can impact serum lipids' concentrations. At present, data on the effect of the administration of medroxyprogesterone acetate plus conjugated equine oestrogens (MPACEE) on the lipid profile in females are uncertain, as the investigations conducted so far have produced conflicting results. Thus, we aimed to clarify the impact of MPACEE prescription on the serum lipids' values in women by means of a systematic review and meta-analysis of randomized controlled trials (RCTs). METHODS: We employed a random-effects model based on the DerSimonian and Laird method to determine the combined estimates of the intervention's impact on the lipid profile. The computation of the weighted mean difference (WMD) and its corresponding 95% confidence interval (CI) relied on the mean and standard deviation values from both the MPACEE and control group, respectively. RESULTS: A total of 53 RCTs were included in the meta-analysis with 68 RCT arms on total cholesterol (TC), 70 RCT arms on low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG), and 69 RCT arms on high-density lipoprotein cholesterol (HDL-C). Administration of MPACEE resulted in a significant reduction of TC (WMD = -11.93 mg/dL; 95% CI: -13.42, -10.44; p < .001) and LDL-C (WMD = -16.61 mg/dL; 95% CI: -17.97, -15.26; p < .001) levels, and a notable increase in HDL-C (WMD = 3.40 mg/dL; 95% CI: 2.93, 3.86; p < .001) and TG (WMD = 10.28 mg/dL; 95% CI: 7.92, 12.64; p < .001) concentrations. Subgroup analysis revealed that changes in the lipid profile were influenced by several factors: body mass index (for TC, HDL-C, TG), MPACEE dosages (for TC, LDL-C, HDL-C, TG), age (for TC, LDL-C, HDL-C, TG), durations of the intervention (for TC, LDL-C, HDL-C, TG), continuous/sequential administration of MPACEE (continuous for TC; sequential for LDL-C, TG) administration of MPACEE and serum lipids' concentrations before enrolment in the RCT (for TC, LDL-C, HDL-C, TG). CONCLUSIONS: MPACEE administration can influence serum lipids' concentrations in females by raising HDL-C and TG levels and reducing LDL-C and TC values. Therefore, postmenopausal women who suffer from hypercholesterolaemia might benefit from this type of HRT.

The effect of tamoxifen on estradiol, SHBG, IGF-1, and CRP in women with breast cancer or at risk of developing breast cancer: a meta-analysis of randomized controlled trials.

BACKGROUND AND AIM: The effects of tamoxifen on the serum levels of hormones and acute phase reactants have been studied previously, but study results have been inconsistent, especially in women with breast cancer. Hence, we conducted this meta-analysis of randomized controlled trials (RCTs) to try to clarify the effects of tamoxifen on estradiol, insulin-like growth factor 1 (IGF-1), sex hormone binding globulin (SHBG), and C-reactive protein (CRP) serum levels in women with breast cancer or at risk of developing breast cancer. METHODS: Databases were systematically searched up to December 2023. The meta-analysis was generated through a random-effects model and is presented as the weighted mean difference (WMD) and 95 % confidence intervals (CI). RESULTS: Nine publications were included in the present meta-analysis. The comprehensive findings from the random-effects model revealed an elevation in estradiol (WMD: 13.04 pg/mL, 95 % CI: 0.79, 25.30, p = 0.037) and SHBG levels (WMD: 21.26 nmol/l, 95 % CI: 14.85, 27.68, p = 0.000), as well as a reduction in IGF-1 (WMD: -14.41 µg/L, 95 % CI: -24.23, -4.60, p = 0.004) and CRP concentrations (WMD: -1.17 mg/dL, 95 % CI: -2.29, -0.05, p = 0.039) following treatment with tamoxifen in women with breast cancer or at risk of developing breast cancer, with no impact on IGFBP-3 levels (WMD: 0.11 µg/mL, 95 % CI: -0.07, 0.30, p = 0.240). CONCLUSION: Tamoxifen administration seems to increase estradiol and SHBG levels and reduce CRP and IGF-1 levels in women with breast cancer or at risk of developing breast cancer. Further studies are needed to determine whether these changes have any clinical relevance.

The effect of vitamin D2 supplementation on vitamin D levels in humans: A time and dose-response meta-analysis of randomized controlled trials.

BACKGROUND: Inconsistencies exist regarding the influence of vitamin D2 (ergocalciferol) supplementation on serum vitamin D levels. These inconsistencies could be attributed to numerous factors, such as dosage, baseline vitamin D levels, and duration of intervention. Hence, this dose-response meta-analysis of randomized controlled trials was conducted to assess the efficacy of vitamin D2 supplementation on vitamin D levels. METHODS: Relevant studies were searched in PubMed/Medline, Web of Science, Embase, and Scopus, from their inception to 3 January 2023. Variable alterations were considered to calculate the pooled weighted mean difference (WMD) with 95% confidence interval (CI) using the random effects model. RESULTS: Pooled results from 33 study arms demonstrated that Vitamin D2 treatment significantly increases total vitamin D concentrations (WMD: 11.47 ng/mL, 95 %CI: 9.29 to 13.64, p < 0.001), 25(OH)D2 concentrations (WMD: 11.40 ng/mL, 95 %CI: 4.72 to 18.09, p = 0.001), and 1,25(OH)D concentrations (WMD: 5.61 ng/mL, 95 %CI: 0.74 to 10.48, p = 0.024), but decreases 25(OH)D3 concentrations (WMD: -4.63 ng/mL, 95 %CI: -6.46 to -2.81, p < 0.001). In subgroup analyses, increase in total vitamin D concentrations was more significant in vitamin D2 doses >2000 IU/day (WMD: 13.82 ng/mL), studies with duration ≤12 weeks (WMD: 12.53 ng/mL), participants aged ≥60 years (WMD: 14.40 ng/mL), and trials with basal 25(OH)D concentrations <20 ng/mL (WMD: 11.47 ng/mL). CONCLUSIONS: This meta-analysis indicates that the supplementation of vitamin D2 significantly increases the serum concentrations of total vitamin D, 25(OH)D2, and 1,25(OH)D, but decreases 25(OH)D3 concentrations. Careful consideration of patient characteristics, dosage, and treatment duration is recommended for vitamin D2 supplementation.

The effect of subcutaneous Lixisenatide on weight loss in patients with type 2 Diabetes Mellitus: Systematic review and Meta-Analysis of randomized controlled trials.

BACKGROUND: The impacts of subcutaneous Lixisenatide on body weight in patients with type 2 DM, remain inadequately understood; consequently, this systematic review and meta-regression analysis of randomized controlled trials (RCTs) was conducted to evaluate the influence of subcutaneous Lixisenatide administration on BW and BMI values in individuals with Type 2 diabetes. METHODS: A comprehensive literature search was conducted across four databases, spanning from their inception to February 2023. We computed effect sizes employing the random-effects model and reported results in terms of weighted mean differences (WMD) along with their corresponding 95% confidence intervals (CI). RESULTS: 23 articles with 26 RCT arms were included in the meta-analysis. The combined findings from a random-effects model demonstrated a significant reduction in body weight (WMD: -0.97 kg, 95 % CI: -1.10, -0.83, p < 0.001) and BMI (WMD: -0.48 kg/m2, 95 % CI: -0.67, -0.29, P < 0.001) after subcutaneous administration of Lixisenatide. Furthermore, a more pronounced reduction in body weight was discovered in RCTs lasting less than 24 weeks (WMD: -1.56 kg, 95 % CI: -2.91, -0.20, p < 0.001), employing a daily dosage of subcutaneous Lixisenatide lower than 19 Âµg per day (WMD: -1.94 kg, 95 % CI: -2.54, -1.34, p < 0.001) and with a mean participant age of 60 years or more (WMD: -1.86 kg, 95 % CI: -3.16, -0.57, p = 0.005). CONCLUSIONS: Lixisenatide was found to significantly decrease BW and BMI in patients with type 2 DM and could be considered as a therapeutic option for those suffering from weight gain caused by other anti-diabetic agents. However, while prescribing Lixisenatide, careful consideration of patient-specific factors is recommended.

Efficacy of atypical antipsychotics in the treatment of fecal incontinence in children and adolescents: a randomized clinical trial.

OBJECTIVES: Functional retentive overflow incontinence (retentive FI) is the most common cause of fecal soiling in children. Based on the clinical experiences, the treatment of retentive FI in patients with comorbid psychiatric disorders was accelerated when Risperidone was used as treatment for their psychiatric comorbidities; therefore, this study was conducted to evaluate the effect of risperidone in the treatment of retentive FI in children and adolescents. METHODS: In this double-blind, randomized, placebo-controlled trial, 140 patients aged 4-16 years eligible for the study were randomized into two groups, receiving either 0.25-0.5 mg of Risperidone syrup (n = 70) or maltodextrin syrup (placebo group, n = 70) every 12 h daily for 12 weeks. Sociodemographic data, including age, sex, weight, height, BMI, BMI z-score, and socioeconomic status, was recorded, and the number of nocturnal FI, diurnal FI, and painful defecations was measured. RESULTS: 136 participants (69 on Risperidone and 67 on placebo) were included in the study. Mean age of participants in the intervention and placebo groups were 7.2 ± 2.4 years and 8.0 ± 3.1 years, respectively. The mean number of nocturnal FI (Ptrend=0.39) and diurnal FI (Ptrend=0.48) in patients without psychiatric comorbidities, and the number of painful defecations for participants with and without psychiatric comorbidities (P = 0.49, P = 0.47, respectively) were not significantly different between the groups, but a significant effect was observed in diurnal FI after Risperidone treatment in patients with psychiatric comorbidities (P < 0.001). CONCLUSION: Risperidone, when used along with other non-pharmacological interventions, may be helpful in treating FI in pediatric patients with psychiatric comorbidities.

The effect of subcutaneous dulaglutide on weight loss in patients with Type 2 diabetes mellitus: Systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Dulaglutide, a subcutaneously administered glucagon-like peptide 1 receptor agonist, has been hypothesized to lead to weight loss in patients with Type 2 diabetes mellitus (T2DM). However, the consequences of its prescription on body weight (BW) and other anthropometric indices, for example, body mass index (BMI) or waist circumference (WC), have not been completely clarified. Therefore, we aimed to assess the effects of subcutaneous dulaglutide administration on BW, BMI and WC values in T2DM subjects by means of a systematic review and meta-analysis of RCTs. METHODS: We computed a literature search in five databases (PubMed/Medline, Web of Science, EMBASE, Scopus and Google Scholar) from their inception to February 2023 to identify RCTs that examined the influence of subcutaneous dulaglutide on obesity indices. We calculated effect sizes using the random-effects model (using DerSimonian-Laird method). Results were derived across weighted mean differences (WMD) and 95% confidence intervals (CI). Subgroup analyses were applied to explore possible sources of heterogeneity among the RCTs. The current systematic review and meta-analysis was conducted in compliance with The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. RESULTS: In total, 18 studies with 33 RCT arms (BW = 33 RCT arms, 14,612 participants, 7869 cases and 6743 controls; BMI = 10 RCT arms, 14,612 subjects, 7869 cases and 6743 controls; WC = 10 RCT arms, 1632 participants, 945 cases and 687 cases) were included in the meta-analysis. BW (WMD: -0.86 kg, 95% CI: -1.22, -0.49, p < 0.001), BMI (WMD: -0.68 kg/m2 , 95% CI: -0.88, -0.49, p < 0.001) and WC (WMD: -1.23 cm, 95% CI: -1.82, -0.63, p < 0.001) values decreased notably following subcutaneous dulaglutide administration versus placebo. BW notably decreased in RCTs lasting >18 weeks (WMD: -1.42 kg, 95% CI: -1.90, -0.94, p < 0.001), whereas notable reductions in WC were seen in RCTs lasting ≤18 weeks (WMD: -1.78 cm, 95% CI: -2.59, -0.98, p < 0.001). Dulaglutide dosages >1 mg/day significantly decreased BW (WMD: -1.94 kg, 95% CI: -2.54, -1.34, p < 0.001), BMI (WMD: -0.80 kg/m2 , 95% CI: -1.07, -0.54, p < 0.001) and WC (WMD: -1.47 cm, 95% CI: -1.80, -1.13, p < 0.001). BW decreased particularly following dulaglutide prescription in individuals with obesity (WMD: -1.05 kg, 95% CI: -1.28, -0.82, p < 0.001) versus overweight. The dose-response meta-analysis revealed that BW decreased significantly when dulaglutide was prescribed in doses ≤3 mg/day versus >3 mg/day. CONCLUSIONS: Subcutaneous dulaglutide administration in T2DM reduces BW, BMI and WC. The decrease in BW and WC was influenced by the dose and the duration of dulaglutide administration. The reduction in BMI was only influenced by the dosage of dulaglutide. Moreover, T2DM patients who suffered from obesity experienced a notable decrease in BW versus T2DM subjects without obesity.

The effect of phytosterol supplementation on lipid profile: A critical umbrella review of interventional meta-analyses.

Despite multiple investigations assessing the impact of phytosterol supplementation on serum lipid levels, there is still a great deal of debate regarding the benefits of this intervention in the management of dyslipidemia. Therefore, we aimed at clarifying this dilemma by conducting the present umbrella review of interventional meta-analyses. Scopus, PubMed, Web of Science, and EMBASE were used to search for pertinent publications on the effect of phytosterol supplementation on the lipid profile in humans up to June 2023. To compute the overall effect size (ES) and confidence intervals (CI), the random-effects model was used. The I2 statistic and Cochrane's Q-test were applied to estimate the heterogeneity among the studies. Seventeen meta-analyses with 23 study arms were included in the umbrella meta-analysis. Data pooled from the 23 eligible arms revealed that phytosterol supplementation reduces low-density lipoprotein cholesterol (LDL-C) (ES = -11.47 mg/dL; 95% CI: -12.76, -10.17, p < 0.001), total cholesterol (TC) (ES = -13.02 mg/dL; 95% CI: -15.68, -10.37, p < 0.001), and triglyceride (TG) (ES = -3.77 mg/dL; 95% CI: -6.04, -1.51, p = 0.001). Subgroup analyses showed that phytosterol administration with dosage ≥2 g/day and duration over 8 weeks and in hypercholesterolemic subjects was more likely to decrease LDL-C, TC, and TG. Phytosterol administration did not significantly modify HDL-C (ES = 0.18 mg/dL; 95% CI: -0.13, -0.51, p = 258) levels when compared to controls. The present umbrella meta-analysis confirms that phytosterol administration significantly reduces LDL-C, TC, and TG, with a greater effect with doses of ≥2 g/day and treatment duration >8 weeks, suggesting its possible application as a complementary therapy for cardiovascular risk reduction. Further studies are needed to determine the efficacy of phytosterols in patients with specific health conditions, as well as to ascertain the adverse effects, the maximum tolerable dose, and the maximum recommended duration of phytosterol administration.

The impact of medroxyprogesterone acetate on lipid profiles in Women: A time and dose-response meta-analysis of randomized controlled trials.

BACKGROUND: The effect of MPA on the lipid profile and CVD risk is still controversial; hence, this comprehensive dose-response meta-analysis of randomized controlled trials was conducted to assess the effect of MPA on lipid profiles in women. METHODS: A comprehensive search was conducted in the following databases: Web of Science, Scopus, PubMed/Medline, and Embase, up to October 20, 2023. A random-effects meta-analysis approach based on the DerSimonian and Laird method was used to compute the combined estimates of the intervention's impact on the lipid profile. RESULTS: 35 eligible studies with 58 arms were included in our meta-analyses analysis. Combined effect sizes suggested a significant effect of MPA on total cholesterol (TC) levels (WMD: -3.43 mg/dL, 95 % CI: -5.38 to -1.48, p < 0.001), HDL-C levels (WMD: -3.34 mg/dL, 95 % CI: -3.77 to -2.91, p < 0.001), and triglyceride (TG) levels (WMD: -9.13 mg/dL, 95 % CI: -10.92 to -7.33, p < 0.001). The subgroup meta-analysis revealed a more substantial reduction in TC in studies with dosages > 2.5 mg/day (WMD: -4.10 mg/dL), mean participant age lower than 60 years (WMD: -3.80 mg/dL), mean BMI lower than 25 kg/m2 (WMD: -5.61 mg/dL), duration of intervention of 12 months or more (WMD: -3.98 mg/dL), and when the baseline TC value was equal to or greater than 200 mg/dL (WMD: -4.13 mg/dL). CONCLUSIONS: The current meta-analysis showed a statistically significant decrease in TC, TG, and HDL-C levels and a non-significant increase in LDL-C levels after MPA administration in women.

Effects of Transdermal 17ß-Estradiol + Norethisterone Acetate on Cardiovascular Disease Risk Factors in Postmenopausal Women: A Meta-analysis of Data From Randomized, Controlled Trials.

PURPOSE: To date, no study has demonstrated the role of transdermal 17ß-estradiol + norethisterone acetate on all of the risk factors for cardiovascular disease in postmenopausal women. To overcome this knowledge gap, a systematic review and meta-analysis were conducted to determine the effects of this combination treatment on BMI, body weight, waist/hip ratio, fibrinogen, factor VII, lipoprotein(a), fasting blood sugar, insulin, HbA1c, TG, LDL-C, HDL-C, and TC in postmenopausal women. METHODS: PubMed/Medline, SCOPUS, Web of Science, Embase, and Google Scholar were searched for relevant articles published between the inception of each database and April 6, 2023. The sample size and mean (SD) were used to calculate overall effect size using a random-effects model. FINDINGS: A total of 10 articles with 14 arms were included in the meta-analysis. On pooled analysis of effect size, fibrinogen (weighted mean difference [WMD], -0.18 g/L; 95% CI, -0.25 to -0.10), factor VII (WMD, -9.58; 95% CI, -12.51 to -6.64), LDL-C (WMD, -13.09 mg/dL; 95% CI, -18.48 to -7.71), and TC (WMD, -12.61 mg/dL; 95% CI, -18.11 to -7.12) were significantly affected with the use of transdermal 17ß-estradiol + norethisterone acetate (all, P < 0.001), but effects on lipoprotein(a), TG, HDL-C, fasting blood sugar, insulin, HbA1c, BMI, body weight, and waist/hip ratio were not significant. IMPLICATIONS: Based on the findings from the present systematic review and meta-analysis, it was concluded that transdermal administration of 17ß-estradiol + norethisterone acetate had beneficial impacts on fibrinogen, factor VII, LDL-C, and TC, suggesting a possible application in the reduction of cardiovascular disease risk.

The effect of 17beta-estradiol plus norethisterone acetate on anthropometric indices: A systematic review and meta-analysis of randomized controlled trials.

OBJECTIVE: Little evidence exists on the effect of 17beta-estradiol plus norethisterone acetate on all the anthropometric indices. Hence, this systematic review and meta-analysis of Randomized Controlled Trials was conducted to give an evidence-based report on the effect of 17beta-estradiol plus norethisterone acetate on anthropometric indices. METHODS: The literature search was executed in databases including PubMed/Medline, Web of Science, Scopus, Embase, and Google Scholar to recognize clinical trials that examined the influence of 17beta-estradiol plus norethisterone acetate on obesity indices from database inception to Jan 2023. RESULTS: Combined findings were generated from 20 eligible articles. The meta-analysis showed that body weight (Weighted Mean Difference (WMD): -0.47 kg, 95% CI: -1.32, 0.37, p = 0.274), body fat (WMD: 0.16 kg, 95% CI: -1.26, 1.59, p = 0.821), WHR (WMD: 0.001 kg, 95% CI: -0.006, 1.15, p = 0.872), and LBM (WMD: -0.02 kg, 95% CI: -1.19, 1.15, p = 0.970) were not modified in DHEA group compared to the control, but BMI levels were significantly reduced in 17beta-estradiol plus norethisterone acetate group (WMD: -0.15 kg/m2, 95% CI: -0.30, -0.008, p = 0.039). Moreover, based on intervention duration (months), a more significant reduction in BMI was found in trials that were performed on studies with ˃3 months duration (WMD: -0.176 kg/m2) than studies with ≤ 3 months (WMD: 0.05 kg/m2). CONCLUSION: Administration of 17beta-estradiol plus norethisterone acetate for more than 3 months results in a decrease in BMI, which helps to reduce cardiovascular disease risk.

Correction: Rehnelt et al. Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation. Int. J. Mol. Sci. 2017, 18, 2634.

The authors wish to make the following corrections to this paper [...].

Cell Force-Driven Basement Membrane Disruption Fuels EGF- and Stiffness-Induced Invasive Cell Dissemination from Benign Breast Gland Acini.

Local basement membrane (BM) disruption marks the initial step of breast cancer invasion. The activation mechanisms of force-driven BM-weakening remain elusive. We studied the mechanical response of MCF10A-derived human breast cell acini with BMs of tuneable maturation to physical and soluble tumour-like extracellular matrix (ECM) cues. Traction force microscopy (TFM) and elastic resonator interference stress microscopy (ERISM) were used to quantify pro-invasive BM stress and protrusive forces. Substrate stiffening and mechanically impaired BM scaffolds induced the invasive transition of benign acini synergistically. Robust BM scaffolds attenuated this invasive response. Additional oncogenic EGFR activation compromised the BMs' barrier function, fuelling invasion speed and incidence. Mechanistically, EGFR-PI3-Kinase downstream signalling modulated both MMP- and force-driven BM-weakening processes. We show that breast acini form non-proteolytic and BM-piercing filopodia for continuous matrix mechanosensation, which significantly push and pull on the BM and ECM under pro-invasive conditions. Invasion-triggered acini further shear and compress their BM by contractility-based stresses that were significantly increased (3.7-fold) compared to non-invasive conditions. Overall, the highest amplitudes of protrusive and contractile forces accompanied the highest invasiveness. This work provides a mechanistic concept for tumour ECM-induced mechanically misbalanced breast glands fuelling force-driven BM disruption. Finally, this could facilitate early cell dissemination from pre-invasive lesions to metastasize eventually.

Frequency-Dependent Multi-Well Cardiotoxicity Screening Enabled by Optogenetic Stimulation.

Side effects on cardiac ion channels causing lethal arrhythmias are one major reason for drug withdrawals from the market. Field potential (FP) recording from cardiomyocytes, is a well-suited tool to assess such cardiotoxic effects of drug candidates in preclinical drug development, but it is currently limited to the spontaneous beating of the cardiomyocytes and manual analysis. Herein, we present a novel optogenetic cardiotoxicity screening system suited for the parallel automated frequency-dependent analysis of drug effects on FP recorded from human-induced pluripotent stem cell-derived cardiomyocytes. For the expression of the light-sensitive cation channel Channelrhodopsin-2, we optimised protocols using virus transduction or transient mRNA transfection. Optical stimulation was performed with a new light-emitting diode lid for a 96-well FP recording system. This enabled reliable pacing at physiologically relevant heart rates and robust recording of FP. Thereby we detected rate-dependent effects of drugs on Na⁺, Ca2+ and K⁺ channel function indicated by FP prolongation, FP shortening and the slowing of the FP downstroke component, as well as generation of afterdepolarisations. Taken together, we present a scalable approach for preclinical frequency-dependent screening of drug effects on cardiac electrophysiology. Importantly, we show that the recording and analysis can be fully automated and the technology is readily available using commercial products.

Biotin-conjugated fusogenic liposomes for high-quality cell purification.

Purification of defined cell populations from mixed primary cell sources is essential for many biomedical and biotechnological applications but often very difficult to accomplish due to missing specific surface markers. In this study, we developed a new approach for efficient cell population separation based on the specific membrane fusion characteristics of distinct cell types upon treatment with fusogenic liposomes. When such liposomes are conjugated with biotin, specific cell populations can be efficiently surface functionalized by biotin after liposomal treatment while other populations remain unlabeled. Due to the high affinity of biotin for avidin-like proteins, biotin functionalized cells are ideal targets for conjugation of e.g. avidin tagged magnetic beads, fluorophores or antibodies with bioanalytical relevance. Here, based on the differential biotinylation of distinct cell populations high quality separation of cardiac fibroblasts from myocytes, and cerebromicrovascular endothelial cells from fibroblasts was successfully established.

Defined 2-D microtissues on soft elastomeric silicone rubber using lift-off epoxy-membranes for biomechanical analyses.

Surface patterning with complex molecules has become a valuable tool in cell biology and biotechnology, as it enables one to control cell shape and function in culture. However, this technique for micro-contact printing is normally performed on rigid substrates, e.g. Petri dishes or glass. Despite the fact that these substrates can easily be patterned they are artificially stiff environments for cells affecting their morphology and function. Those artifacts can be avoided on tissue elasticity resembling substrates, leading to a nature like cell morphology and behavior. However, reproducible patterning of very soft elastomeric substrates is challenging. Here, we describe a simple and highly accurate method through cavities of lift-off membranes for protein patterning of silicone rubber substrates in an elasticity range down to 1.5 kPa without altering their mechanical properties. Membranes are made of epoxy resin with feature sizes that can be chosen almost arbitrarily including widths down to 5 µm and aspect ratios of 100 and more. Different feature shapes were used to actively manipulate cell adhesion, cell morphology and the actin cytoskeleton on soft substrates. Manipulation of cytoskeletal organization furthermore allowed the comparison of myofibril alignment and cellular forces of cardiac myocytes. These data could show that cell forces are largely unaffected upon active disordering of overall myofibril alignment on a single cell level while aligned multicellular systems generate cell forces in an additive manner.

The constant beat: cardiomyocytes adapt their forces by equal contraction upon environmental stiffening.

Cardiomyocytes are responsible for the permanent blood flow by coordinated heart contractions. This vital function is accomplished over a long period of time with almost the same performance, although heart properties, as its elasticity, change drastically upon aging or as a result of diseases like myocardial infarction. In this paper we have analyzed late rat embryonic heart muscle cells' morphology, sarcomere/costamere formation and force generation patterns on substrates of various elasticities ranging from ∼1 to 500 kPa, which covers physiological and pathological heart stiffnesses. Furthermore, adhesion behaviour, as well as single myofibril/sarcomere contraction patterns, was characterized with high spatial resolution in the range of physiological stiffnesses (15 kPa to 90 kPa). Here, sarcomere units generate an almost stable contraction of ∼4%. On stiffened substrates the contraction amplitude remains stable, which in turn leads to increased force levels allowing cells to adapt almost instantaneously to changing environmental stiffness. Furthermore, our data strongly indicate specific adhesion to flat substrates via both costameric and focal adhesions. The general appearance of the contractile and adhesion apparatus remains almost unaffected by substrate stiffness.