Validation of suitable house keeping genes for hypoxia-cultured human chondrocytes

<p>Abstract</p> <p>Background</p> <p>Hypoxic culturing of chondrocytes is gaining increasing interest in cartilage research. Culturing of chondrocytes under low oxygen tension has shown several advantages, among them increased synthesis of extracellular matrix and increased redifferentiation of dedifferentiated chondrocytes. Quantitative gene expression analyses such as quantitative real-time PCR (qRT-PCR) are powerful tools in the investigation of underlying mechanisms of cell behavior and are used routinely for differentiation and phenotype assays. However, the genes used for normalization in normoxic cell-cultures might not be suitable in the hypoxic environment. The objective of this study was to determine hypoxia-stable housekeeping genes (HKG) for quantitative real-time PCR (qRT-PCR) in human chondrocytes cultured in 21%, 5% and 1% oxygen by geNorm and NormFinder analyses.</p> <p>Results</p> <p>The chondrocytic response to the hypoxic challange was validated by a significant increase in expression of the hypoxia-inducible gene ankyrin repeat 37 as well as SOX9 in hypoxia. When cultured on the 3-dimentional (3D) scaffold TATA-binding protein (TBP) exhibited the highest expression stability with NormFinder while Ribosomal protein L13a (RPL13A) and beta2-microglobulin (B2M) were the most stable using geNorm analysis. In monolayer RPL13A were the most stable gene using NormFinder, while geNorm assessed RPL13A and human RNA polymerase II (RPII) as most stable. When examining the combination of (3D) culturing and monolayer RPL13A and B2M showed the highest expression stability from geNorm analysis while RPL13A also showed the highest expression stability using NormFinder. Often used HKG such as beta actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were the most unstable genes investigated in all comparisons. The pairwise variations for the two most stable HKG in each group were all below the cut-off value of 0.15, suggesting that the two most stable HKG from geNorm analysis would be sufficient for qRT-PCR.</p> <p>Conclusion</p> <p>All data combined we recommend RPL13A, B2M and RPII as the best choice for qRT-PCR analyses when comparing normoxic and hypoxic cultured human chondrocytes although other genes might also be suitable. However, the matching of HKG to target genes by means of a thorough investigation of the stability in each study would always be preferable.</p

Early Echocardiographic Deformation Analysis for the Prediction of Sudden Cardiac Death and Life-Threatening Arrhythmias After Myocardial Infarction

ObjectivesThis study sought to hypothesize that global longitudinal strain (GLS) as a measure of infarct size, and mechanical dispersion (MD) as a measure of myocardial deformation heterogeneity, would be of incremental importance for the prediction of sudden cardiac death (SCD) or malignant ventricular arrhythmias (VA) after acute myocardial infarction (MI).BackgroundSCD after acute MI is a rare but potentially preventable late complication predominantly caused by malignant VA. Novel echocardiographic parameters such as GLS and MD have previously been shown to identify patients with chronic ischemic heart failure at increased risk for arrhythmic events. Risk prediction during admission for acute MI is important because a majority of SCD events occur in the early period after hospital discharge.MethodsWe prospectively included patients with acute MI and performed echocardiography, with measurements of GLS and MD defined as the standard deviation of time to peak negative strain in all myocardial segments. The primary composite endpoint (SCD, admission with VA, or appropriate therapy from a primary prophylactic implantable cardioverter-defibrillator [ICD]) was analyzed with Cox models.ResultsA total of 988 patients (mean age: 62.6 ± 12.1 years; 72% male) were included, of whom 34 (3.4%) experienced the primary composite outcome (median follow-up: 29.7 months). GLS (hazard ratio [HR]: 1.38; 95% confidence interval [CI]: 1.25 to 1.53; p < 0.0001) and MD (HR/10 ms: 1.38; 95% CI: 1.24 to 1.55; p < 0.0001) were significantly related to the primary endpoint. GLS (HR: 1.24; 95% CI: 1.10 to 1.40; p = 0.0004) and MD (HR/10 ms: 1.15; 95% CI: 1.01 to 1.31; p = 0.0320) remained independently prognostic after multivariate adjustment. Integrated diagnostic improvement (IDI) and net reclassification index (NRI) were significant for the addition of GLS (IDI: 4.4% [p < 0.05]; NRI: 29.6% [p < 0.05]), whereas MD did not improve risk reclassification when GLS was known.ConclusionsBoth GLS and MD were significantly and independently related to SCD/VA in these patients with acute MI and, in particular, GLS improved risk stratification above and beyond existing risk factors

Baseline characteristics for patients in the intervention (n = 7) and control group (n = 7).

Baseline characteristics for patients in the intervention (n = 7) and control group (n = 7).</p

Consort 2010 checklist.

ObjectiveHypokalemia is associated with increased risk of arrhythmias and it is recommended to monitor plasma potassium (p-K) regularly in at-risk patients with cardiovascular diseases. It is poorly understood if administration of potassium supplements and mineralocorticoid receptor antagonists (MRA) aimed at increasing p-K also increases intracellular potassium.MethodsAdults aged≥18 years with an implantable cardioverter defibrillator (ICD) were randomized (1:1) to a control group or to an intervention that included guidance on potassium rich diets, potassium supplements, and MRA to increase p-K to target levels of 4.5–5.0 mmol/l for six months. Total-body-potassium (TBK) was measured by a Whole-Body-Counter along with p-K at baseline, after six weeks, and after six months.ResultsFourteen patients (mean age: 59 years (standard deviation 14), 79% men) were included. Mean p-K was 3.8 mmol/l (0.2), and mean TBK was 1.50 g/kg (0.20) at baseline. After six-weeks, p-K had increased by 0.47 mmol/l (95%CI:0.14;0.81), p = 0.008 in the intervention group compared to controls, whereas no significant difference was found in TBK (44 mg/kg (-20;108), p = 0.17). After six-months, no significant difference was found in p-K as compared to baseline (0.16 mmol/l (-0.18;0.51), p = 0.36), but a significant increase in TBK of 82 mg/kg (16;148), p = 0.017 was found in the intervention group compared to controls.ConclusionsIncreased potassium intake and MRAs increased TBK gradually and a significant increase was seen after six months. The differentially regulated p-K and TBK challenges current knowledge on potassium homeostasis and the time required before the full potential of p-K increasing treatment can be anticipated.Trial registrationwww.clinicaltrials.gov (NCT03833089).</div

Supplementary appendix.

ObjectiveHypokalemia is associated with increased risk of arrhythmias and it is recommended to monitor plasma potassium (p-K) regularly in at-risk patients with cardiovascular diseases. It is poorly understood if administration of potassium supplements and mineralocorticoid receptor antagonists (MRA) aimed at increasing p-K also increases intracellular potassium.MethodsAdults aged≥18 years with an implantable cardioverter defibrillator (ICD) were randomized (1:1) to a control group or to an intervention that included guidance on potassium rich diets, potassium supplements, and MRA to increase p-K to target levels of 4.5–5.0 mmol/l for six months. Total-body-potassium (TBK) was measured by a Whole-Body-Counter along with p-K at baseline, after six weeks, and after six months.ResultsFourteen patients (mean age: 59 years (standard deviation 14), 79% men) were included. Mean p-K was 3.8 mmol/l (0.2), and mean TBK was 1.50 g/kg (0.20) at baseline. After six-weeks, p-K had increased by 0.47 mmol/l (95%CI:0.14;0.81), p = 0.008 in the intervention group compared to controls, whereas no significant difference was found in TBK (44 mg/kg (-20;108), p = 0.17). After six-months, no significant difference was found in p-K as compared to baseline (0.16 mmol/l (-0.18;0.51), p = 0.36), but a significant increase in TBK of 82 mg/kg (16;148), p = 0.017 was found in the intervention group compared to controls.ConclusionsIncreased potassium intake and MRAs increased TBK gradually and a significant increase was seen after six months. The differentially regulated p-K and TBK challenges current knowledge on potassium homeostasis and the time required before the full potential of p-K increasing treatment can be anticipated.Trial registrationwww.clinicaltrials.gov (NCT03833089).</div

Study protocol.

ObjectiveHypokalemia is associated with increased risk of arrhythmias and it is recommended to monitor plasma potassium (p-K) regularly in at-risk patients with cardiovascular diseases. It is poorly understood if administration of potassium supplements and mineralocorticoid receptor antagonists (MRA) aimed at increasing p-K also increases intracellular potassium.MethodsAdults aged≥18 years with an implantable cardioverter defibrillator (ICD) were randomized (1:1) to a control group or to an intervention that included guidance on potassium rich diets, potassium supplements, and MRA to increase p-K to target levels of 4.5–5.0 mmol/l for six months. Total-body-potassium (TBK) was measured by a Whole-Body-Counter along with p-K at baseline, after six weeks, and after six months.ResultsFourteen patients (mean age: 59 years (standard deviation 14), 79% men) were included. Mean p-K was 3.8 mmol/l (0.2), and mean TBK was 1.50 g/kg (0.20) at baseline. After six-weeks, p-K had increased by 0.47 mmol/l (95%CI:0.14;0.81), p = 0.008 in the intervention group compared to controls, whereas no significant difference was found in TBK (44 mg/kg (-20;108), p = 0.17). After six-months, no significant difference was found in p-K as compared to baseline (0.16 mmol/l (-0.18;0.51), p = 0.36), but a significant increase in TBK of 82 mg/kg (16;148), p = 0.017 was found in the intervention group compared to controls.ConclusionsIncreased potassium intake and MRAs increased TBK gradually and a significant increase was seen after six months. The differentially regulated p-K and TBK challenges current knowledge on potassium homeostasis and the time required before the full potential of p-K increasing treatment can be anticipated.Trial registrationwww.clinicaltrials.gov (NCT03833089).</div

POTCAST study protocol.

ObjectiveHypokalemia is associated with increased risk of arrhythmias and it is recommended to monitor plasma potassium (p-K) regularly in at-risk patients with cardiovascular diseases. It is poorly understood if administration of potassium supplements and mineralocorticoid receptor antagonists (MRA) aimed at increasing p-K also increases intracellular potassium.MethodsAdults aged≥18 years with an implantable cardioverter defibrillator (ICD) were randomized (1:1) to a control group or to an intervention that included guidance on potassium rich diets, potassium supplements, and MRA to increase p-K to target levels of 4.5–5.0 mmol/l for six months. Total-body-potassium (TBK) was measured by a Whole-Body-Counter along with p-K at baseline, after six weeks, and after six months.ResultsFourteen patients (mean age: 59 years (standard deviation 14), 79% men) were included. Mean p-K was 3.8 mmol/l (0.2), and mean TBK was 1.50 g/kg (0.20) at baseline. After six-weeks, p-K had increased by 0.47 mmol/l (95%CI:0.14;0.81), p = 0.008 in the intervention group compared to controls, whereas no significant difference was found in TBK (44 mg/kg (-20;108), p = 0.17). After six-months, no significant difference was found in p-K as compared to baseline (0.16 mmol/l (-0.18;0.51), p = 0.36), but a significant increase in TBK of 82 mg/kg (16;148), p = 0.017 was found in the intervention group compared to controls.ConclusionsIncreased potassium intake and MRAs increased TBK gradually and a significant increase was seen after six months. The differentially regulated p-K and TBK challenges current knowledge on potassium homeostasis and the time required before the full potential of p-K increasing treatment can be anticipated.Trial registrationwww.clinicaltrials.gov (NCT03833089).</div

Difference in changes between groups from baseline (reference) at six-weeks, and six-months with 95% intervals of confidence from a mixed linear random effects regression model.

Difference in changes between groups from baseline (reference) at six-weeks, and six-months with 95% intervals of confidence from a mixed linear random effects regression model.</p

CONSORT flow-chart of patient inclusion and follow-up.

CONSORT flow-chart of patient inclusion and follow-up.</p