Abstract
Background
Insulin resistance and nonalcoholic fatty liver disease (NAFLD) both relate to cardiovascular mortality. Using a mouse model of chronic lipid overload and secondary-NAFLD-induced ...insulin resistance (SEC-NAFLD-IR), we recently deciphered that SEC-NAFLD-IR already at young age provoked myocardial lipotoxicity with reduced mitochondrial efficiency and increased vulnerability to cardiac ischemia. However, long-term consequences of SEC-NAFLD-IR remain elusive.
Purpose
Here we aimed to elucidate the impact of long-term SEC-NAFLD-IR on multiple mitochondrial quality control (mQC) mechanisms in the heart and its consequences for cardiac function.
Methods
We studied 36 SEC-NAFLD-IR mice (72-week-old). For mechanistic experiments, we applied palmitate-induced insulin resistant murine HL-1 cells. Cardiac mitochondrial dynamics were measured via quantification of mitochondrial morphology and expression of mitochondrial fusion and fission factors (Opa1, Drp1, Fis1, Mfn 1 & 2). Mitophagy level was evaluated via immunofluorescence and protein expression of key mitophagy-related genes (Parkin, NIX, LC3). Mitochondrial biogenesis and mass were examined via quantitation of PGC-1α expression, mtDNA and citrate synthase activity.
Results
72-week-old SEC-NAFLD-IR mice exhibited 21% (p=0.001) and 32% (p<0.001) higher body weight and heart weight compared with controls. Along with elevated oxidative stress, hepatic lipid accumulation and inflammation, 6h-fasted SEC-NAFLD-IR mice were characterized by increased plasma glucose, insulin and cholesterol. SEC-NAFLD-IR mice displayed a cardiac phenotype with 21% higher left ventricular mass (normalized to body weight, p<0.001) and 6% lower ejection fraction compared to controls (73.5% SEM 0.90 vs 69.4% SEM 1.65, p=0.04). We found several advantageous mQC mechanisms suppressed in aged SEC-NAFLD-IR mice including long form OPA1-mediated mitochondrial fusion, Parkin- and NIX-mediated mitophagy. Likewise, mitochondrial biogenesis was suppressed in the aged insulin-resistant heart, which was connected to a 65% downregulation of PGC-1α1 expression (p=0.01). Interestingly, downregulation of cardiac PGC-1α1 in aged SEC-NAFLD-IR mice coincided with upregulation of PARIS, indicating the crucial participation of the Parkin/PARIS pathway in mQC of the insulin-resistant heart. In addition, induction of insulin resistance in murine HL-1 cardiomyocytes also led to increased mitochondrial fragmentation and decreased PGC-1α1 expression.
Conclusion
This study demonstrated that regulation of mitochondrial network and turnover is hampered by SEC-NAFLD-IR in the hearts of aged mice, which may contribute to hypertrophy and cardiac dysfunction in insulin resistance.
Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)
Abstract
Background
Peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) acts as a transcriptional coactivator and regulates mitochondrial function. Various isoforms are ...generated by alternative splicing and differentially regulated promoters. In the heart, total PGC-1α deficiency knockout leads to dilatative cardiomyopathy, but knowledge on the complexity of cardiac isoform expression of PGC-1α remains sparse. Thus, this study aims to generate a reliable dataset on cardiac isoform expression pattern by long-read mRNA sequencing, followed by investigation of differential regulation of PGC-1α isoforms under metabolic and ischemic stress, using high-fat-high-sucrose-diet-induced obesity and a murine model of myocardial infarction.
Results
Murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery) was used for long-read mRNA sequencing, resulting in full-length transcriptomes including 58,000 mRNA isoforms with 99% sequence accuracy. Automatic bioinformatic analysis as well as manual similarity search against exonic sequences leads to identification of putative coding PGC-1α isoforms, validated by PCR and Sanger sequencing. Thereby, 12 novel transcripts generated by hitherto unknown splicing events were detected. In addition, we postulate a novel promoter with homologous and strongly conserved sequence in human heart. High-fat diet as well as ischemia/reperfusion (I/R) injury transiently reduced cardiac expression of PGC-1α isoforms, with the most pronounced effect in the infarcted area. Recovery of PGC-1α-isoform expression was even more decelerated when I/R was performed in diet-induced obese mice.
Conclusions
We deciphered for the first time a complete full-length transcriptome of the murine and human heart, identifying novel putative PGC-1α coding transcripts including a novel promoter. These transcripts are differentially regulated in I/R and obesity suggesting transcriptional regulation and alternative splicing that may modulate PGC-1α function in the injured and metabolically challenged heart.
Abstract
Objective
Optimal immunosuppression is an essential component of the best possible long-term survival after heart transplantation (HTX). Optimally patient-tailored immunosuppression is ...standard in Tx follow-up, balancing harmful side effects and preventing rejection episodes. Hereby, stable blood levels, as well as a stable and calculable absorption rate, is essential. Tacrolimus additionally has a narrow therapeutic range and is particularly difficult to dose stably. It has been shown that interindividual differences in absorption exist among various immunosuppressants, making adequate minimum levels and the avoidance of rejection events more difficult. For other solid organ transplantation procedures there are already studies on the proportion of patients (so-called "fast metabolizers") who experience different kinetics due to very rapid resorption. We here therefore aim to investigate the potential impact of the tacrolimus metabolizer status on outcome in a HTx cohort.
Methods
We defined fast metabolizers by a low concentration to dose ratio (C/D ratio) of <1.05. The C/D ratio was determined according to the following formula: C/D ratio (ng/ml x 1/mg) = Tacrolimus through level (ng/ml) / daily Tacrolimus dose (mg).
We included as a first step here 57 patients undergoing HTx in our center between 09/2010 and 10/2016 and calculated the C/D ratio retrospectivley from routine follow-ups. In both groups baseline characteristics, perioperative parameters and survival after 1 and 5 years after Tx were collected.
Results
We identified 24 patients (42%) as fast metabolizers and 33 patients as standard metabolizers (58%). The metabolizer status did not significantly correlate with reduced survival of the recipient up to 5 years in Kaplan-Meier-Analysis (Figure 1,A, Log Rank p=0.68). Additionally, regression analysis could not detect an association of 1-year-survival and C/D ratio or the fast metabolizer status definition (HR 1.1, 95% CI 0.17-7.2, p=0.92). Moreover, using ROC-analysis, no clear other cut-off for C/D ratio other than the used published definition (<1.05) could be derived (Figure 1,B, AUC 5-year survival 0.53, p = 0.72). Additionally, these parameters did not change significantly also when focusing on speficic substances with different pharmacodynamics such as Prograf (two doses per day) or Advagraf (delayed release, one dose per day). We could also not detect associations between C/D ratio or metabolizer status and unfavorable outcomes such as cellular or clinical relevant rejection episodes.
Conclusion
In this retrospective analysis, neither the metabolizer status or the raw tacrolimus concentration to dose ratio was associated with altered survival or outcome after heart transplantation. As we present here a yet small number of investigated patients (pilot study) with a potential era bias by currently not including advances in medication optimization over the last years, future studies are needed to elucidate this phenomen further.
Abstract
Objective
Several Risk assessment tools have been developed to help predict mortality after heart transplantation. While validated within their regional transplant cohorts, it remains ...unclear whether they can also be applied to other cohorts. We therefore aimed to test and compare the predictive value of an US-Score validated in the UNOS Database by Joyce et al., a French Score by Jasseron et al., and the BO-Score within the Eurotransplant area developed by Schramm et al.
Methods
We applied the three scores to our cohort using published model coefficients and variables (see Figure 1 for distribution). The initial BO-Score as well as the French Score were hereby modified to produce positive integer values within our cohort (mod. French Score = initial French Score + 2.24x10, mod. BO Score = initial BO initial score+10x3). C-statistics using ROC-analysis (Figure 2) were used to compare the three scores regarding their ability to discriminate between 30-day, 1 year and 5-year survivors and non-survivors within our heart transplant cohort.
Results
The French Score performed best predicting the 1 survival with an AUC of 0.69 95% CI: 0.59-0.79, p<0.0001, followed by the BO-Score with 0.66 (95% CI: 0.56-0.77, p=0.003) and the US-Score with an AUC of 0.67, CI 0.57-0.76, p=0.02). Regarding 30-day-Survival, only the French Score with an AUC of 0.64 95% CI: 0.52-0.76, p=0.006 as well as the BO-Score with 0.71 (95% CI: 0.60-0.81, p=0.008) could predict mortality sufficiently, while the US-Score could not discriminate statistically significant between survivors and non-survivors (AUC 0.64, CI 0.52-0.76, p=0.06). All scores performed worst predicting 5-year survival, with only the BO-Score being statistically significant (AUC 0.61, 95% CI: 0.56-0.77, p=0.003).
Conclusion
In our heart transplant cohort, the available donor recipient risk assessment tools performed best predicting 1 year survival, with worse and heterogenous results for 30 day or 5 year survival. Further developments will have to incorporate risk factors not yet taken into account in order to make even more reliable predictions, especially for the Eurotransplant area.Figure 1 and 2
Abstract
Background
Alternative splicing is a process by which exons within a pre-mRNA are joined or skipped, resulting in isoforms being encoded by a single gene. Alternative Splicing affecting ...transcription factors may have substantial impact on cellular dynamics. The PPARG Coactivator 1 Alpha (PGC1-α), is a major modulator in energy metabolism. Data from murine skeletal muscle revealed distinctive isoform patterns giving rise to different phenotypes, i.e. mitogenesis and hypertrophy. Here, we aimed to establish a complete dataset of isoforms in murine and human heart applying single-molecule real-time (SMRT)-sequencing as novel approach to identify transcripts without need for assembly, resulting in true full-length sequences. Moreover, we aimed to unravel functional relevance of the various isoforms during experimental ischemia reperfusion (I/R).
Methods
RNA-Isolation was performed in murine (C57Bl/6J) or human heart tissue (obtained during LVAD-surgery), followed by library preparation and SMRT-Sequencing. Bioinformatic analysis was done using a modified IsoSeq3-Pipeline and OS-tools. Identification of PGC1-α isoforms was fulfilled by similarity search against exonic sequences within the full-length, non-concatemere (FLNC) reads. Isoforms with Open-Reading-Frame (ORF) were manually curated and validated by PCR and Sanger-Sequencing. I/R was induced by ligature of the LAD for 45 min in mice on standard chow as well as on high-fat-high-sucrose diet. Area At Risk (AAR) and remote tissue were collected three and 16 days after I/R or sham-surgery (n=4 per time point). Promotor patterns were analyzed by qPCR.
Results
Deciphering the full-length transcriptome of murine and human heart resulted in ∼60000 Isoforms with 99% accuracy on mRNA-sequence. Focusing on murine PGC1-α-isoforms we discovered and verified 15 novel transcripts generated by hitherto unknown splicing events. Additionally, we identified a novel Exon 1 originating between the known promoters followed by a valid ORF, suggesting the discovery of a novel promoter. Remarkably, we found a homologous novel Exon1 in human heart, suggesting conservation of the postulated promoter.
In I/R the AAR exhibited a significant lower expression of established and novel promoters compared to remote under standard chow 3d post I/R. 16d post I/R, the difference between AAR & Remote equalized in standard chow while remaining under High-Fat-Diet.
Conclusion
Applying SMRT-technique, we generated the first time a complete full-length-transcriptome of the murine and human heart, identifying 15 novel potentially coding transcripts of PGC1-α and a novel exon 1. These transcripts are differentially regulated in experimental I/R in AAR and remote myocardium, suggesting transcriptional regulation and alternative splicing modulating PGC1-α function in heart. Differences between standard chow and high fat diet suggest impact of impaired glucose metabolism on regulatory processes after myocardial infarction.
Funding Acknowledgement
Type of funding source: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)
Abstract
Objective
Donor hypernatremia has been associated with primary graft dysfunction in heart transplantation and is known to be associated with impaired outcome following liver and renal ...transplantation. However, controversial data exist regarding the impact of sodium deregulation in patient survival after heart transplantation (HTx). This study aims to investigate the impact of donor sodium levels on early morbidity and short- and midterm survival following HTx.
Methods
Between September 2010 and June 2021, a total of n=218 patients underwent HTx in our centre. From those, 214 could be included retrospectively in our study. For each donor, sodium levels were collected and different cut-off levels from 145 to 159 mmol/l were investigated by Kaplan-Meier-analysis. Then, recipients were divided in three groups regarding donor sodium: Normonatremia (133–145 mmol/l, n=73), mild hypernatremia (146–156 mmol/l, n=105) and severe hypernatremia (>156 mmol/l, n=35). Recipient and donor variables were reviewed and compared, including peri- and postoperative characteristics and recipient survival after up to 5 years after transplantation.
Results
All patients were comparable regarding baseline characteristics and perioperative parameters. Regarding early mortality, 90-day survival was significantly reduced only in patients with severe donor hypernatremia in comparison to normonatremia (see table 1 and fifgure 1, 90% vs. 71%, p=0.02), but not in mild normonatremia (89%, p=0.89). 1-year survival was comparable in all groups (p>0.28).
Conclusion
Donor hypernatremia was associated with reduced short-term survival, while correlation weakens >1 year after HTx. Future prospective studies are needed to confirm the possible cut-off value of 156 mmol/l for donor-acceptancy.
Funding Acknowledgement
Type of funding sources: None.
