Objective: Heart failure in cancer patients remains underrepresented and challenging due to non-specific treatments and an incomplete understanding of cancer or cancer cachexia–induced cardiomyopathy. This study explores the roles of sarcomere impairment, altered Ca 2+ handling, and metabolic reprogramming in contributing to left ventricular (LV) dysfunction. Methods: Male BALB/c mice were subcutaneously inoculated with mouse colon-26 adenocarcinoma (C26) or IL-6–silenced C26 (C26 shIL-6) cells, while controls received PBS. Twenty days post-injection, cardiac function was evaluated via electrocardiography, transthoracic echocardiography, and ex vivo working heart assays. Isolated ventricular cardiomyocytes were analyzed for intracellular Ca 2+ transients and force–calcium relationships. Cardiac inflammation, metabolism, and fibrosis were quantified alongside proteomic analysis. Results: Despite similar tumor sizes, C26 mice exhibited cachexia with loss of subcutaneous fat and skeletal muscle and elevated serum IL-6. Both tumor-bearing groups trended toward impaired LV systolic and diastolic function, with a significant reduction in maximum calcium-activated tension (T_max; p < 0.05) in skinned cardiomyocyte preparations. Cachectic mice showed increased intracellular Ca 2+ transients, suggesting upregulated SERCA2 activity. While macrophage and T-cell infiltration and interstitial fibrosis were unchanged, neutrophil infiltration was enhanced in the cachectic group. Additionally, β-myosin heavy chain expression was upregulated, and metabolic profiling revealed a shift toward glucose utilization with reduced fatty acid oxidation, corroborated in h9c2 cells exposed to conditioned media from C26 and C26 shIL-6 cells. Unbiased proteomic analysis demonstrated significant downregulation of Bag3, Hspa4, Pln, Serca2, Pfkm, and Shdh exclusively in cachectic hearts. Discussion: In conclusion, cancer-induced cachexia in C26 mice leads to LV dysfunction, characterized by altered calcium handling in cardiomyocytes, metabolic reprogramming, and sarcomere impairment. These findings highlight key mechanisms contributing to cardiomyopathy in cachexia and provide potential targets for therapeutic intervention in cancer-related cardio metabolic dysfunction.

Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a severe muscle disease caused by dystrophin deficiency. Using the mdx mouse model for human DMD, we previously showed that the lack of dystrophin induces a significant loss of peak sodium current (INa) in ventricular cardiomyocytes. This provided a mechanistic explanation for ventricular conduction defects and concomitant arrhythmias in the dystrophic heart. The extracellular matrix protein tenascin C (TN-C), a major remodeling factor in the diseased heart, is strongly upregulated in DMD. The consequences of TN-C upregulation in the dystrophic heart, however, are unknown. Here, we tested if TN-C induces electrical remodeling in the dystrophic heart, and if inhibition of TN-C rescues peak INa loss in dystrophin-deficient ventricular cardiomyocytes. We found that cardiomyocytes from TN-C knockout (KO) mice had increased peak INa. The abnormally reduced peak INa in mdx myocytes was rescued to wild-type levels by additional TN-C KO, which was accompanied by enhanced Nav1.5 channel expression. Further, peak INa in mdx myocytes was increased by treatment of mdx mice with TN-C siRNA. Twenty-four-hour incubation of wild-type myocytes with human recombinant TN-C reduced their peak INa, an effect which could be abolished by blocking antibodies specific for the α-7 integrin subunit. Our findings suggest that TN-C induces peak INa loss in the dystrophic heart, and that inhibition of TN-C expression rescues abnormally reduced peak INa in dystrophin-deficient ventricular cardiomyocytes. TN-C inhibition emerges as a strategy to counteract ventricular conduction impairments and arrhythmias in patients with DMD.NEW & NOTEWORTHY Dystrophin deficiency in cardiomyocytes leads to abnormally reduced Na currents. These can be rescued by inhibition of the expression of tenascin C.

Diabetic cardiomyopathy (DCM) is a complex condition linked to diabetes, characterized by cardiac and vascular dysfunction, frequently concomitant with heart failure with preserved ejection fraction. The extracellular matrix glycoprotein Tenascin-C (TNC) has been found to be upregulated under diabetic conditions. However, the potential contributory role of TNC in the progression of DCM remains largely unclear. This study was designed to elucidate the role of TNC in the pathogenesis of DCM. Diabetes was induced in adult male wild-type (WT) and TNC knockout (TNC-KO) mice, through the administration of streptozotocin (50 mg/kg) for five consecutive days. At 18 weeks cardiac and aortic vascular function was evaluated using echocardiography and wire myography. Myocardium and plasma samples were collected for biochemical, histological, and molecular analyses. Cardiomyocytes and cardiac fibroblasts were used to investigate the impact of diabetes on TNC expression, inflammation, myocardial stiffness and function. Additionally, transcriptomic analysis of cardiac tissue by RNA-sequencing was conducted. Plasma TNC levels were assessed by enzyme-linked immunosorbent assay in cohorts of heart failure patients and type 2 diabetes mellitus. TNC-KO diabetic mice showed preserved left ventricular systolic and diastolic function, significantly reduced cardiac fibrosis and mitigated endothelial dysfunction compared to WT diabetic animals. Compared with cardiomyocytes of diabetic WT animals, cardiomyocytes of TNC-KO mice developed less stiffness (Fpassive). Additionally, exposing mouse cardiomyocytes and human cardiac fibroblasts to high glucose stress (30 mM) led to a significant increase in TNC expression. Conversely, recombinant human TNC promoted pro-inflammatory and oxidative stress markers in cardiomyocytes. The role of TNC in fibrosis and DCM was found to involve pathways related to p53 signaling and Serpin1k, Ccn1, Cpt1a, and Slc27a1, as identified by RNA sequencing analysis. Additionally, plasma TNC levels were significantly elevated in patients with heart failure, irrespective of diabetes status, compared to healthy individuals. Our findings indicate that in diabetes, TNC contributes to cardiac contractile dysfunction, myocardial fibrosis, oxidative stress, inflammation, and metabolic disturbances in diabetic mouse heart. These results implicate the potential of TNC inhibition as a novel therapeutic approach for treating DCM.

OBJECTIVES:Myocardial protection is crucial for recovery after cardiac surgery. Despite older patients with more comorbidities, most surgeons still use potassium-induced depolarized arrest with cardioplegic solutions from more than 50 years ago. However, this can cause calcium overload, mitochondrial injury and energy depletion. We tested a new polarizing cardioplegia (STH-pol) versus traditional depolarized arrest (STH-control) in an ischaemia/reperfusion model of hearts with reduced ejection fraction. METHODS:Myocardial infarction (MI) was induced by permanent ligation of the left anterior descending artery in male Sprague-Dawley rats. Six weeks post-MI, the hearts were isolated and perfused on an erythrocyte-perfused working heart system. Cold ischaemic arrest (4°C, 60 min) was induced by STH-pol (n = 10) or STH-control (n = 8), followed by reperfusion (37°C, 45 min). Haemodynamic variables were recorded throughout reperfusion, and tissue samples were taken at the end of reperfusion to analyse high-energy phosphates (HEP). RESULTS:Administration of STH-pol resulted in comparable haemodynamic recovery after 60 min ischaemia as STH-control induced cardioplegic arrest (left ventricular systolic pressure: 109.1 ± 3.4 vs 103.3 ± 3.6). Similarly, HEP levels did not differ between the two groups (ATP: 2.5 ± 0.4 nmol/mg protein and 2.2 ± 0.9 nmol/mg). CONCLUSIONS:Polarizing cardioplegic arrest did not lead to a statistically significant difference in myocardial protection as compared to a clinically relevant, standard cold blood cardioplegia in rat hearts with chronic MI.

Duchenne muscular dystrophy (DMD), a severe muscle disease caused by mutations in the gene encoding for the intracellular protein dystrophin, is associated with impaired cardiac function and arrhythmias. A causative factor for complications in the dystrophic heart is abnormal calcium (Ca) handling in ventricular cardiomyocytes, and restoration of normal Ca homeostasis has emerged as therapeutic strategy. Here, we used a rodent model of DMD, the dystrophin-deficient DMDmdx rat, to test the following hypothesis: chronic administration of ivabradine (IVA), a drug clinically approved for the treatment of heart failure, improves Ca handling in dystrophic ventricular cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Intracellular Ca measurements revealed that 4-months administration of IVA to DMDmdx rats significantly improves Ca handling properties in dystrophic ventricular cardiomyocytes. In particular, IVA treatment increased electrically-evoked Ca transients and speeded their decay. This suggested enhanced sarcoplasmic reticulum Ca release and faster removal of Ca from the cytosol. Chronic IVA administration also enhanced the sarcoplasmic reticulum Ca load. Transthoracic echocardiography revealed a significant improvement of cardiac systolic function in IVA-treated DMDmdx rats. Thus, left ventricular ejection fraction and fractional shortening were enhanced, and end-systolic as well as end-diastolic diameters were diminished by the drug. Finally, chronic IVA administration neither significantly attenuated cardiac fibrosis and apoptosis, nor was vascular function improved by the drug. Collectively our findings suggest that long-term IVA administration enhances contractile function in the dystrophic heart by improvement of Ca handling in ventricular cardiomyocytes. Chronic IVA administration may be beneficial for DMD patients.

BACKGROUND:Venous thromboembolism is a major health problem. After thrombus formation, its resolution is essential to re-establish blood flow, which is crucially mediated by infiltrating neutrophils and monocytes in concert with activated platelets and endothelial cells. Thus, we aimed to modulate leukocyte function during thrombus resolution post-thrombus formation by blocking P-selectin/CD62P-mediated cell interactions. METHODS:Thrombosis was induced by inferior vena cava stenosis through ligation in mice. After 1 day, a P-selectin-blocking antibody or isotype control was administered and thrombus composition and resolution were analyzed. RESULTS:Localizing neutrophils and macrophages in thrombotic lesions of wild-type mice revealed that these cells enter the thrombus and vessel wall from the caudal end. Neutrophils were predominantly present 1 day and monocytes/macrophages 3 days after vessel ligation. Blocking P-selectin reduced circulating platelet-neutrophil and platelet-Ly6Chigh monocyte aggregates near the thrombus, and diminished neutrophils and Ly6Chigh macrophages in the cranial thrombus part compared with isotype-treated controls. Depletion of neutrophils 1 day after thrombus initiation did not phenocopy P-selectin inhibition but led to larger thrombi compared with untreated controls. In vitro, P-selectin enhanced human leukocyte function as P-selectin-coated beads increased reactive oxygen species production by neutrophils and tissue factor expression of classical monocytes. Accordingly, P-selectin inhibition reduced oxidative burst in the thrombus and tissue factor expression in the adjacent vessel wall. Moreover, blocking P-selectin reduced thrombus density determined by scanning electron microscopy and increased urokinase-type plasminogen activator levels in the thrombus, which accelerated caudal fibrin degradation from day 3 to day 14. This accelerated thrombus resolution as thrombus volume declined more rapidly after blocking P-selectin. CONCLUSIONS:Inhibition of P-selectin-dependent activation of monocytes and neutrophils accelerates venous thrombosis resolution due to reduced infiltration and activation of innate immune cells at the site of thrombus formation, which prevents early thrombus stabilization and facilitates fibrinolysis.

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Austrian Science Fund (FWF) Objective Ivabradine (IVA) is indicated in symptomatic treatment of chronic stable angina, heart failure, and also in those who are unable to tolerate or have contraindications to the use of beta-blockers. It has been showed IVA may have cardiovascular benefits in Duchenne muscular dystrophy (DMD) patients. This study was aimed to investigate the sustained impact of chronic ivabradine (IVA) administration on cardiac function and potential underlying mechanism using high throutput proteomic analysis. Methods DMDmdx male and Sprague–Dawley wt (Sprague–Dawley) male rats were randomly allocated to vehicle (n = 6) or IVA (n=6; 10 mg/kg/day via drinking water for four months). Transthoracic echocardiography and unbiased proteomic analysis were performed to assess cardiac function and left ventricular (LV) tissue, respectively. Protein-protein interactions were graphically represented, and cluster analysis with subsequent enrichment analyses was conducted. Results Chronic IVA treatment significantly enhanced the LV ejection fraction (p<0.05 compared to vehicle-treated DMDmdx rats). Proteomic data identified possible transcription factors (SPI1, IRF1, PPARA), consistent with previous studies. The reduction in copper metabolic changes associated with DMD can be attributed to the upregulation of Atox1 following IVA treatment. Mitochondrial dysfunction was effectively mitigated by IVA, as indicated by the different abundance of mitochondrial proteins. In addition, intercellular adhesion molecule 1 showed a reduction in the number of animals treated with ivabradine, indicating a further positive effect of the treatment. Conclusion In summary, this study demonstrated the beneficial effects of IVA in DMDmdx rat hearts. Cluster analysis of proteomic data revealed notable changes in cardiac metabolism, inflammation, and mitochondrial function. Ivabradine has emerged as a potential therapeutic approach to address these shifts, while proteomic pathway analysis may uncover new drugable targets to alleviate cardiomyopathy progression in DMD.Interaction of IvabradinProtein-protein interaction network

Abstract Background Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease triggered by the deficiency of the structural protein dystrophin, which leads to muscular degeneration mainly affecting young males. Major contributors to early death in DMD patients are cardiac arrhythmias and dystrophic cardiomyopathy. One cause for arrhythmias is impaired ventricular impulse conduction, which leads to ventricular asynchrony and reentrant mechanisms. We recently showed that the disruption of dystrophin results in a significant reduction of Na current in ventricular cardiomyocytes (vCMs) of the dystrophin-deficient mdx mouse model for human DMD. Na current reduction provides a mechanistic explanation for the impaired ventricular conduction and accompanying arrhythmias in the dystrophic heart. The extracellular matrix protein tenascin-C (TN-C) is a significant remodeling factor in the injured and diseased heart and is strongly upregulated in dystrophic cardiomyopathy. To this date, it is unknown how the upregulation of TN-C in DMD patients affects dystrophic cardiomyopathy. Purpose In this study, we examined the effect of TN-C inhibition on diminished Na currents in dystrophin-deficient vCMs. Methods We compared four different mouse genotypes with each other, namely wild-type, dystrophin-deficient mdx, TN-C-deficient and dystrophin- plus TN-C-deficient mice. Furthermore, a cohort of mdx mice was injected with TN-C siRNA twice a week for 9 weeks to investigate the effect of TN-C knockdown. Hearts from adult male mice were enzymatically digested using a Langendorff system to isolate single vCMs. Na currents were then measured with the whole cell patch clamp technique. Results Na current densities were increased in TN-C deficient vCMs compared to wild-type vCMs. Accordingly, 24-hour incubation of wild-type vCMs with human recombinant TN-C resulted in a significant decrease in Na current. Na currents of vCMs from mdx mice were reduced, but restored to the wild-type level in vCMs from TN-C-deficient mdx mice. Moreover, vCMs of TN-C siRNA-treated mdx mice had significantly increased Na currents compared to control mdx vCMs. Conclusion Upregulation of TN-C in dystrophin-deficient vCMs reduces Na currents, whereas inhibition of TN-C prevents this reduction. Therefore, inhibition of TN-C in DMD patients may be considered as a potential new therapeutic strategy to improve ventricular conduction and reduce arrhythmia vulnerability.

Background: Geometrical alterations in the coronary resistance artery network and the potential involvement of Tenascin C (TNC) extracellular matrix protein were investigated in diabetic and control mice. Methods: Diabetes was induced by streptozotocin (STZ) injections (n = 7–11 animals in each group) in Tenascin C KO (TNC KO) mice and their Wild type (A/J) littermates. After 16–18 weeks the heart was removed and the whole subsurface network of the left coronary artery was prepared (down to branches of 40 μm outer diameter), in situ pressure-perfused and studied using video-microscopy. Outer and inner diameters, wall thicknesses and bifurcation angles were measured on whole network pictures reconstructed into collages at 1.7 μm pixel resolutions. Results: Diabetes induced abnormal morphological alterations including trifurcations, sharp bends of larger branches, and branches directed retrogradely (p < 0.001 by the χ2 test). Networks of TNC KO mice tended to form early divisions producing parallelly running larger branches (p < 0.001 by the χ2 probe). Networks of coronary resistance arteries were substantially more abundant in 100–180 μm components, appearing in 2–5 mm flow distance from orifice in diabetes. This was accompanied by thickening of the wall of larger arterioles (>220 μm) and thinning of the wall of smaller (100–140 μm) arterioles (p < 0.001). Blood flow should cover larger distances in diabetic networks, but interestingly STZ-induced diabetes did not generate further geometrical changes in TNC KO mice. Conclusions: Diabetes promotes hypertrophic and hypotrophic vascular remodeling and induces vasculogenesis at well defined, specific positions of the coronary vasculature. TNC plays a pivotal role in the formation of coronary network geometry, and TNC deletion causes parallel fragmentation preventing diabetes-induced abnormal vascular morphologies.

Transient receptor potential cation channel subfamily A member 1 (TRPA1), an ion channel primarily expressed on sensory neurons, can be activated by substances occurring during myocardial infarction. Aims were to investigate whether activation, inhibition, or absence of TRPA1 affects infarcts and to explore underlying mechanisms. In the context of myocardial infarction, rats received a TRPA1 agonist, an antagonist, or vehicle at different time points, and infarct size was assessed. Wild type and TRPA1 knockout mice were also compared in this regard. In vitro, sensory neurons were co-cultured with cardiomyocytes and subjected to a model of ischemia-reperfusion. Although there was a difference between TRPA1 activation or inhibition in vivo, no experimental group was different to control animals in infarct size, which also applies to animals lacking TRPA1. In vitro, survival probability of cardiomyocytes challenged by ischemia-reperfusion increased from 32.8% in absence to 45.1% in presence of sensory neurons, which depends, at least partly, on TRPA1. This study raises doubts about whether TRPA1 is a promising target to reduce myocardial damage within a 24 h period. The results are incompatible with relevant enlargements of infarcts by TRPA1 activation or inhibition, which argues against adverse effects when TRPA1 is targeted for other indications.

The muscular dystrophies caused by dystrophin deficiency, the so-called dystrophinopathies, are associated with impaired cardiac contractility and arrhythmias, which considerably contribute to disease morbidity and mortality. Impaired Ca handling in ventricular cardiomyocytes has been identified as a causative factor for complications in the dystrophic heart, and restoration of normal Ca handling in myocytes has emerged as a promising new therapeutic strategy. In the present study, we explored the hypothesis that ivabradine, a drug clinically approved for the treatment of heart failure and stable angina pectoris, improves Ca handling in dystrophic cardiomyocytes and thereby enhances contractile performance in the dystrophic heart. Therefore, ventricular cardiomyocytes were isolated from the hearts of adult dystrophin-deficient DMDmdx rats, and the effects of acutely applied ivabradine on intracellular Ca transients were tested. In addition, the drug's acute impact on cardiac function in DMDmdx rats was assessed by transthoracic echocardiography. We found that administration of ivabradine to DMDmdx rats significantly improved cardiac function. Moreover, the amplitude of electrically induced intracellular Ca transients in ventricular cardiomyocytes isolated from DMDmdx rats was increased by the drug. We conclude that ivabradine enhances Ca release from the sarcoplasmic reticulum in dystrophic cardiomyocytes and thereby improves contractile performance in the dystrophic heart.

BACKGROUND: Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid- like granulomas.METHODS AND RESULTS: The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c(+) cells (TSC2(fl/fl)CD11c-Cre; termed TSC2(KO)) and controls (TSC2(fl/fl)) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2(KO) animals were treated with either everolimus, an mTOR inhibitor, or Bay11- 7082, a nuclear factor- kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c(+) cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life-threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68(+) macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis.CONCLUSIONS: To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis.

Chronic kidney disease is a global health problem affecting 10% to 12% of the population. Uremic cardiomyopathy is often characterized by left ventricular hypertrophy, fibrosis, and diastolic dysfunction. Dysregulation of neuregulin-1β signaling in the heart is a known contributor to heart failure. The systemically administered recombinant human neuregulin-1β for 10 days in our 5/6 nephrectomy-induced model of chronic kidney disease alleviated the progression of uremic cardiomyopathy and kidney dysfunction in type 4 cardiorenal syndrome. The currently presented positive preclinical data warrant clinical studies to confirm the beneficial effects of recombinant human neuregulin-1β in patients with chronic kidney disease.

Duchenne muscular dystrophy (DMD) is characterized by wasting of muscles that leads to difficulty moving and premature death, mainly from heart failure. Glucocorticoids are applied in the management of the disease, supporting the hypothesis that inflammation may be driver as well as target. However, the inflammatory mechanisms during progression of cardiac and skeletal muscle dysfunction are still not well characterized. Our objective was to characterize the inflammasomes in myocardial and skeletal muscle in rodent models of DMD. Gastrocnemius and heart samples were collected from mdx mice and DMDmdx rats (3 and 9–10 months). Inflammasome sensors and effectors were assessed by immunoblotting. Histology was used to assess leukocyte infiltration and fibrosis. In gastrocnemius, a tendency towards elevation of gasdermin D irrespective of the age of the animal was observed. The adaptor protein was elevated in the mdx mouse skeletal muscle and heart. Increased cleavage of the cytokines was observed in the skeletal muscle of the DMDmdx rats. Sensor or cytokine expression was not changed in the tissue samples of the mdx mice. In conclusion, inflammatory responses are distinct between the skeletal muscle and heart in relevant models of DMD. Inflammation tends to decrease over time, supporting the clinical observations that the efficacy of anti-inflammatory therapies might be more prominent in the early stage.

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Swiss National Foundation The development of novel adjuvant angiogenic therapies to restore the low-perfused microvascular network upon myocardial infarction (MI) is crucial to avoid a possible end-stage heart failure. Of the current adult cell-based therapies, human adipose tissue-derived stromal vascular fraction cell (SVF) has vast reparative potential, principally due to: 1) its heterogeneous composition rich in mesenchymal stem cells (MSC), endothelial cells (EC), pericytes and hematopoietic cells, among others. In vitro engineering of SVF-based patches under unidirectional flow, applied by the help of a perfusion-based bioreactor, was found to increase certain cellular SVF subgroups such as pericytes, compared to static culture. In this study, we aimed at studying the potential of SVF-based engineered tissues in a model of chronic MI in nude rats. Human SVF cells were isolated upon liposuction and cultured on 3D collagen sponges (8 mm diameter, 3 mm thickness) either under constant unidirectional perfusion or in static condition for 5 days. Patches were characterized in terms of cellular composition prior to implantation. MI was induced by permanent ligation of the left anterior descending (LAD) coronary artery in male nude rats. Cardiac MRI was performed 4 weeks after MI; prior to the suture of patches and before sacrifice (4 weeks after implantation). Left ventricular ejection fraction (EF) was the surrogate marker and primary end point for cardiac pump function. Controls included untreated MI animals. Following perfusion culture, SVF cells were composed with a statistically superior percentage of pericytes, identified as CD45- CD34- CD146+ compared to static culture (28.06±10.03 and 3.37±2.50, respectively, p<0.0007). The presence of other cell subpopulations was similar in the patches generated in perfusion or static culture. While the percentage of EF at the time of sacrifice resulted to be not statistically different between static and perfusion-based patches, statically generated constructs showed a general trend of decrease in the % EF before and after treatment (rat 1: 61.96 vs 52.90; rat 2: 55.39 vs 53.00; rat 3: 52.34 vs 50.62, respectively). Perfusion-cultured patches, instead, rather improved the cardiac function, measured as % EF (rat 1: 51.82 vs 58.72; rat 2: 51.66 vs 60.45; rat 3: 53.50 vs 52. 36, respectively for 4 weeks following MI and 4 weeks following treatment). When comparing the ratio of the % EF 8 weeks and 4 weeks between static or perfusion-based patches and the untreated controls, rats treated with patches generated under perfusion resulted to show higher levels of % EF, with an almost statistically difference (p=0.0556), compared to the control group. The observed results showed the great potential of human SVF-based patches in the improvement of the heart pump function.

Abstract Funding Acknowledgements Type of funding sources: None. Background Cancer patients undergoing heart-related complications result in high incidences of mortality. Nevertheless, it is still not fully understood whether localized tumors affect heart function prior to the onset of cachexia, hence, making the heart more vulnerable for functional abnormalities in later stages of the disease. In addition to analyse heart function, we focus on the expression BCL-2–associated athanogene 3 (BAG3), a co-chaperone protein and Hsp70, which are highly expressed in tumor but decrease in cardiomyocytes (CM) in heart failure (HF). Methods Colon-26 adenocarcinoma cells (C26; n=22) with/without shIL-6 (C26 shIL-6; n=22) were injected subcutaneously into the right flank of 10-11 weeks old BALB/c male mice. Control mice were injected with vehicle (PBS; n=8). Cardiac function was assessed by echocardiography and invasive hemodynamic measurements 10 (early) and 20 (late) days after the injection, respectively. In addition, the expression of BAG3 and Hsp70 were determined by Western blot as well as the extend of cardiac fibrosis was determined by Masson-Goldner's trichrome staining. Results The tumor size was comparable between the two injected groups. However, only C26 group showed a significant loss of subcutaneous fat and skeletal muscle (p<0.05, respectively), suggesting cachexia. Heart weight normalized to tibia length was not changed in the injected groups as compared to controls (day 20). However, left ventricular ejection fraction (LVEF) showed a tendency to decline in the early phase (p~0.08) in both injected group and it reached significance at late stage (p<0.05). Invasive hemodynamic assessment also confirmed the contractile dysfunction, resulting in a decrease in LV systolic pressure and increase of LV end-diastolic pressure (p<0.05, respectively). Importantly, these functional changes in the heart in tumor-bearing mice were associated with a marked reduction in both BAG3 and Hsp70 in the myocardium. Furthermore, there was no sign of cardiac fibrosis in the injected groups. Discussion Our study shows for the first time that tumor rather than cancer cachexia plays a significant maladaptive role in the progression of cardiac dysfunction in a mouse model of C26 injection-induced cachexia. The progression of cardiac contractile dysfunction was associated with a decline in BAG3 and Hsp70 in tumor-bearing mice, suggesting changes of BAG3/Hsp 70 signalling may be a critical component as well as target.