Ellen Heeren

Myocardial infarction (MI) causes extensive cardiomyocyte loss and adverse remodeling, frequently progressing to heart failure. Although stem cell-based therapies are being investigated for cardiac repair, clinical outcomes remain modest. Cardiac atrial appendage stem cells (CASCs) harbor cardiomyogenic potential, making them promising candidates for cardiac regeneration. This proof-of-concept pilot study investigates the feasibility and preliminary therapeutic potential of delivering CASCs within an elastin-like recombinamer (ELR) hydrogel scaffold following MI. MI was induced in rats by permanent ligation of the left anterior descending coronary artery. Immediately after occlusion, CASCs (MI+C), ELR hydrogel (MI+H), or CASCs embedded in ELR hydrogel (MI+C+H) were injected intramyocardially into the peri-infarct region. MI and SHAM-operated animals served as controls. Four weeks post-MI, echocardiography showed a large, non-significant left ventricular ejection fraction improvement in MI+C+H versus MI, with a trend toward reduced end-diastolic volume. MI+C+H significantly improved anterior wall thickness versus MI+H, with favorable trends for MI+C. Wall motion score index showed a similar large, non-significant effect. Transcriptomics suggested the MI+C+H molecular profile reflected complementary contributions of both monotherapies rather than distinct synergistic mechanisms. These preliminary findings support ELR-mediated CASC delivery and suggest potential benefits for preserving cardiac function and limiting adverse remodeling, warranting confirmation in adequately powered studies.

Rodents models of myocardial infarction (MI) continue to be frequently used in preclinical cardiovascular research, despite alternative approaches being on the rise. The commonly used coronary artery permanent ligation (PL) approach is often hampered by substantial perioperative mortality and variable success rates. We optimized the rat PL protocol by relying exclusively on isoflurane inhalation anesthesia by introducing a standardized intubation setup, maintaining strict control of body temperature throughout surgery, and surgical technique refinements. The latter included gentle mobilization of the Pectoralis major and thymus, a medial thoracotomy through the third intercostal space, and the use of a reference ligature to facilitate reliable identification and ligation of the left anterior descending coronary artery (LAD). Cardiac rhythm was continuously monitored, and extubation was carefully timed to reduce complications. With this protocol, perioperative mortality was reduced to zero and successful ligation was obtained in 94% of animals (n = 172). Echocardiography and histology confirmed consistent induction of infarcts. By lowering invasiveness and improving survival and reproducibility, the refined PL method enhances both the reliability of preclinical research and compliance with the 3Rs, representing a meaningful step forward for studies in cardiac regeneration.

Echocardiography-guided intramyocardial injection (EGI) is a minimally invasive technique for delivering stem cell therapies in preclinical myocardial infarction (MI) models. Compared to traditional open-chest approaches, EGI offers improved clinical translatability, reduced invasiveness, and minimized physiological impact on the animal. While EGI is well established in murine models, its application in rats remains limited due to anatomical and technical challenges. In particular, thinning of the left ventricular anterior wall (LVAW) in infarcted and peri-infarct regions complicates safe and accurate myocardial delivery, as wall thickness can fall below the needle bevel size of commonly used 27 G or 28 G needles, increasing the risk of ventricular perforation or failed delivery. To address this limitation, we optimized a protocol for EGI in rat MI models using 29 G Spinocan needles. The smaller-diameter, longer needle enables precise targeting of thin myocardial tissue, minimizing damage and enhancing injection accuracy, independent of LVAW thickness. This technique is compatible with standard transthoracic echocardiography platforms and eliminates the need for thoracotomy, allowing longitudinal studies in the same animal. Our refined method enables robust, reproducible delivery of therapeutic agents into viable myocardium adjacent to the infarct zone, where regenerative therapies are most effective. By improving safety and targeting precision, this approach increases the translational relevance of preclinical cardiac research and supports the development of standardized protocols across laboratories.

Echocardiography-guided intramyocardial injection (EGI) is a minimally invasive technique for delivering stem cell therapies in preclinical myocardial infarction (MI) models. Compared to traditional open-chest approaches, EGI offers improved clinical translatability, reduced invasiveness, and minimized physiological impact on the animal. While EGI is well established in murine models, its application in rats remains limited due to anatomical and technical challenges. In particular, thinning of the left ventricular anterior wall (LVAW) in infarcted and peri-infarct regions complicates safe and accurate myocardial delivery, as wall thickness can fall below the needle bevel size of commonly used 27 G or 28 G needles, increasing the risk of ventricular perforation or failed delivery. To address this limitation, we optimized a protocol for EGI in rat MI models using 29 G Spinocan needles. The smaller-diameter, longer needle enables precise targeting of thin myocardial tissue, minimizing damage and enhancing injection accuracy, independent of LVAW thickness. This technique is compatible with standard transthoracic echocardiography platforms and eliminates the need for thoracotomy, allowing longitudinal studies in the same animal. Our refined method enables robust, reproducible delivery of therapeutic agents into viable myocardium adjacent to the infarct zone, where regenerative therapies are most effective. By improving safety and targeting precision, this approach increases the translational relevance of preclinical cardiac research and supports the development of standardized protocols across laboratories.

Individuals with type 2 diabetes mellitus (T2DM) are at an increased risk for heart failure, yet preventive cardiac care is suboptimal in this population. Pyridoxamine (PM), a vitamin B₆ analog, has been shown to exert protective effects in metabolic and cardiovascular diseases. In this study, we aimed to investigate whether PM limits adverse cardiac remodeling and dysfunction in rats who develop T2DM. Male rats received a standard chow diet or Western diet (WD) for 18 weeks to induce prediabetes. One WD group received additional PM (1 g/L) via drinking water. Glucose tolerance was assessed with a 1 h oral glucose tolerance test. Cardiac function was evaluated using echocardiography and hemodynamic measurements. Histology on left ventricular (LV) tissue was performed. Treatment with PM prevented the increase in fasting plasma glucose levels compared to WD-fed rats (p < 0.05). LV cardiac dilation tended to be prevented using PM supplementation. In LV tissue, PM limited an increase in interstitial collagen deposition (p < 0.05) seen in WD-fed rats. PM tended to decrease 3-nitrotyrosine and significantly lowered 4-hydroxynonenal content compared to WD-fed rats. We conclude that PM alleviates interstitial fibrosis and oxidative stress in the hearts of WD-induced prediabetic rats.

Endurance exercise training is a promising cardioprotective strategy in type 2 diabetes mellitus (T2DM), but the impact of its intensity is not clear. We aimed to investigate whether and how isocaloric moderate-intensity exercise training (MIT) and high-intensity interval exercise training (HIIT) could prevent the adverse cardiac remodeling and dysfunction that develop T2DM in rats. Male rats received a Western diet (WD) to induce T2DM and underwent a sedentary lifestyle (n = 7), MIT (n = 7) or HIIT (n = 8). Insulin resistance was defined as the HOMA-IR value. Cardiac function was assessed with left ventricular (LV) echocardiography and invasive hemodynamics. A qPCR and histology of LV tissue unraveled underlying mechanisms. We found that MIT and HIIT halted T2DM development compared to in sedentary WD rats (p < 0.05). Both interventions prevented increases in LV end-systolic pressure, wall thickness and interstitial collagen content (p < 0.05). In LV tissue, HIIT tended to upregulate the gene expression of an ROS-generating enzyme (NOX4), while both modalities increased proinflammatory macrophage markers and cytokines (CD86, TNF-α, IL-1β; p < 0.05). HIIT promoted antioxidant and dicarbonyl defense systems (SOD2, glyoxalase 1; p < 0.05) whereas MIT elevated anti-inflammatory macrophage marker expression (CD206, CD163; p < 0.01). We conclude that both MIT and HIIT limit WD-induced T2DM with diastolic dysfunction and pathological LV hypertrophy, possibly using different adaptive mechanisms.

Doxorubicin (DOX) is commonly used in cancer treatment but associated with cardiotoxicity. Pyridoxamine (PM), a vitamin B6 derivative, could be a cardioprotectant. This study investigated the effect of PM on DOX cardiotoxicity and DOX antitumor effectiveness. Sprague Dawley rats were treated intravenously with DOX (2 mg/kg/week) or saline over eight weeks. Two other groups received PM via oral intake (1 g/L in water bottles) next to DOX or saline. Echocardiography was performed after eight weeks. PM treatment significantly attenuated the DOX-induced reduction in left ventricular ejection fraction (72 ± 2% vs. 58 ± 3% in DOX; p < 0.001) and increase in left ventricular end-systolic volume (0.24 ± 0.02 µL/cm² vs. 0.38 ± 0.03 µL/cm² in DOX; p < 0.0001). Additionally, LA7 tumor cells were exposed to DOX, PM, or DOX and PM for 24 h, 48 h, and 72 h. Cell viability, proliferation, cytotoxicity, and apoptosis were assessed. DOX significantly reduced LA7 cell viability and proliferation (p < 0.0001) and increased cytotoxicity (p < 0.05) and cleaved caspase-3 (p < 0.001). Concomitant PM treatment did not alter the DOX effect on LA7 cells. In conclusion, PM attenuated DOX-induced cardiomyopathy in vivo without affecting the antitumor effect of DOX in vitro, highlighting PM as a promising cardioprotectant for DOX-induced cardiotoxicity.

Background: During myocardial infarction (MI), billions of cardiomyocytes are lost. The optimal therapy should effectively replace damaged cardiomyocytes, possibly with stem cells able to engraft and differentiate into adult functional cardiomyocytes. As such, cardiac atrial appendage stem cells (CASCs) are suitable candidates. However, the presence of elevated levels of advanced glycation end products (AGEs) in cardiac regions where CASCs are transplanted may affect their regenerative potential. In this study, we examine whether and how AGEs alter CASCs properties in vitro. Methods and Results: CASCs in culture were exposed to ranging AGEs concentrations (50 µg/mL to 400 µg/mL). CASCs survival, proliferation, and migration capacity were significantly decreased after 72 h of AGEs exposure. Apoptosis significantly increased with rising AGEs concentration. The harmful effects of these AGEs were partially blunted by pre-incubation with a receptor for AGEs (RAGE) inhibitor (25 µM FPS-ZM1), indicating the involvement of RAGE in the observed negative effects. Conclusion: AGEs have a time- and concentration-dependent negative effect on CASCs survival, proliferation, migration, and apoptosis in vitro, partially mediated through RAGE activation. Whether anti-AGEs therapies are an effective treatment in the setting of stem cell therapy after MI warrants further examination.

Background: The heart failure (HF)-syndrome is associated with neuro-hormonal activation, chronic kidney disease (CKD), inflammation and alterations in the phosphorus-metabolism, all of which are involved in regulation of mineral bone density. However, the role of HF as an independent factor associated with metabolic bone disease (MBD) remains unclear. Methods: HF-patients undergoing dual X-ray absorptiometry (DEXA) were matched in a 1:2 fashion against age and gender matched controls without HF, to determine the proportion of osteoporosis (T-score < −2.5). HF-status was tested against known predictors of MBD. Correlation analysis and Z-score analysis were used to assess the impact of HF on age-related bone demineralisation. Results: A total of 190 HF-patients (age = 80 ± 10 years, female = 61%) were age and gender matched to 380 controls. HF-patients had a higher proportion of osteoporosis (26 vs 17%; p = .007). HF patients had a lower averaged mineral bone density expressed in g/cm² (p = .030), T-scores (p = .001) and Z-scores (p < .001). After adjusting for the individual osteoporosis risk-factors of the FRAX-score, difference in baseline features, kidney function and phosphorus-metabolism alterations, heart failure remained independently associated with a lower averaged T-score (Adjusted β = –0.189; p = .017). Heart failure was associated with an accelerated age-related decline in mineral bone density (p = .0418). Therapies with ACE-I or ARBs and beta-blockers associated with ameliorated bone demineralisation (p = .023, respectively p = .029), while loop diuretic associated with worsened bone demineralization (p < .001). Conclusion: Heart failure independently associates with MBD and higher prevalence of osteoporosis. Heart failure aggravates the aged related loss in mineral bone density while treatment with neuro-hormonal blockers seemed to ameliorate this finding.