Langendorff Perfusion Research Articles

Effects of chronic stress on rat heart function following regional ischemia: a sex-dependent investigation.

Chronic psychological stress is a recognized, yet understudied risk factor for heart disease, with potential sex-specific effects. We investigated whether chronic stress triggers sex-dependent cardiac dysfunction in isolated Wistar rat hearts subjected to ischemia-reperfusion injury. The experimental cohort underwent 1 h of daily restraint stress for 4 wk versus matched controls, followed by euthanasia (sodium pentobarbital) and heart excision for ex vivo perfusion. Blood analysis revealed sex-specific alterations in stress hormones and inflammatory markers. When compared with controls, chronic restraint stress (CRS) males displayed decreased plasma brain-derived neurotrophic factor (BDNF) levels (P < 0.05), whereas CRS females exhibited elevated plasma adrenocorticotropic hormone (ACTH) (P < 0.01) and reduced corticosterone (P < 0.001) alongside lower serum estradiol (P < 0.001) and estradiol/progesterone ratio (P < 0.01). Of note, CRS females showed increased serum cardiac troponin T (P < 0.05) and tumor necrosis factor-α (TNF-α) (P < 0.01) with suppressed interleukin (IL)-1α, IL-1β, IL-6, and IL-10 levels (P < 0.05) when compared with controls. Ex vivo Langendorff perfusions revealed that CRS female hearts displayed impaired postischemic functional recovery for baseline stroke volume (SV, P < 0.01), work performance (P < 0.05), aortic output (AO, P < 0.05), coronary flow (CF, P < 0.01), and overall cardiac output (CO, P < 0.01) when compared with matched controls and CRS males (P < 0.05). Our findings reveal intriguing sex-specific responses at both the systemic and functional levels in stressed hearts. Here, the dysregulation of stress hormones, proinflammatory state, and potential underlying cardiomyopathy in females following the stress protocol renders them more prone to damage following myocardial ischemia. This study emphasizes the importance of incorporating sex as a biological variable in cardiac research focusing on stress-related cardiomyopathy.NEW & NOTEWORTHY Although chronic psychological stress is a risk factor for cardiovascular diseases, the underlying mechanisms remain poorly understood. This study revealed that chronic restraint stress resulted in systemic changes (dysregulated stress hormones, proinflammatory state) and potential cardiomyopathy in females versus controls and their male counterparts. The stressed female hearts also displayed reduced functional recovery following ex vivo ischemia-reperfusion. This highlights the importance of incorporating sex as a biological variable in cardiac research.

Cardiac Loading using Passive Left Atrial Pressurization and Passive Afterload for Graft Assessment.

Ex vivo machine perfusion or normothermic machine perfusion is a preservation method that has gained great importance in the transplantation field. Despite the immense opportunity for assessment due to the beating state of the heart, current clinical practice depends on limited metabolic trends for graft evaluation. Hemodynamic measurements obtained from left ventricular loading have garnered significant attention within the field due to their potential as objective assessment parameters. In effect, this protocol provides an easy and effective manner of incorporating loading capabilities to established Langendorff perfusion systems through the simple addition of an extra reservoir. Furthermore, it demonstrates the feasibility of employing passive left atrial pressurization for loading, an approach that, to our knowledge, has not been previously demonstrated. This approach is complemented by a passive Windkessel base afterload, which acts as a compliance chamber to maximize myocardial perfusion during diastole. Lastly, it highlights the capability of capturing functional metrics during cardiac loading, including left ventricular pulse pressure, contractility, and relaxation, to uncover deficiencies in cardiac graft function after extended periods of preservation times (˃6 h).

Impulse initiation in engrafted pluripotent stem cell-derived cardiomyocytes can stimulate the recipient heart

Transplantation of pluripotent stem cell-derived cardiomyocytes is a novel promising cell-based therapeutic approach for patients with heart failure. However, engraftment arrhythmias are a predictable life-threatening complication and represent a major hurdle for clinical translation. Thus, we wanted to experimentally study whether impulse generation by transplanted cardiomyocytes can propagate to the host myocardium and overdrive the recipient rhythm. We transplanted human induced pluripotent stem cell-derived cardiomyocytes expressing the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) in a guinea pig injury model. Eight weeks after transplantation ex vivo, Langendorff perfusion was used to assess electrical coupling. Pulsed photostimulation was applied to specifically activate the engrafted cardiomyocytes. Photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart with higher pacing frequency (4/9 hearts). Our study demonstrates that transplanted cardiomyocytes can (1) electrically couple to the host myocardium and (2) stimulate the recipient heart. Thus, our results provide experimental evidence for an important aspect of engraftment-induced arrhythmia induction and thereby support the current hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.

Activation of PKC affects the ventricular restitution properties and arrhythmogenesis through L-type Ca+ current.

To investigate the role of protein kinase C (PKC) in action potential duration (APD) restitution and ventricular tachyarrhythmias (VAs). Rabbits hearts were isolated and prepared for Langendorff perfusion technique. The stimuli-extra-stimulus (S1-S2) method and dynamic S1 pacing protocol were performed to construct APD restitution and to induce APD alternans or VA, respectively, at 10 sites throughout the ventricular chamber. Administration of phorbol-12-myristate-13-acetate (PMA) (100nM) (n=15) greatly steepened the restitution curves (Smax>1) (p<.01) at each site compared to the control group (n=15). Furthermore, treatment with PMA also induced larger spatial dispersions of Smax (p<.05) and decreased the thresholds of the VA and APD alternans (p<.01). However, perfused with the PKC inhibitor, bisindolylmaleimide (BIM) (500nM) (n=10), reversibly flattened the APD restitution curves at each site (Smax<1), decreased the spatial dispersions of Smax, and increased the thresholds of APD alternans and VA. According to the results of patch-clamp, peak amplitude of L-type Ca2+ current was significantly increased by addition of PMA compared with control (CTL) group (p<.05). Antagonize this current with verapamil (n=10) can fully inhibited the PMA induced increasing of Smax and inducibility of VA and alternans. PKC activation increased the dispersion of APD restitution and thus led to occurrence of VA, which possibly related to the increased Ca2+ influx.

Association between Aquaporin 7 Expression and Myocardial Protection with Nicorandil or Del Nido Cardioplegia: An Experimental Study

Background: Aquaporin 7 (AQP7), a member the aquaglyceroporin subgroup of the AQP family, is a water channel that controls transport of glycerol and water in heart tissues. It facilitates the uptake of glycerol, a substrate for cardiac energy production, in cardiomyocytes. St. Thomas' Hospital cardioplegic solution No. 2 has cardio-protective effect even in AQP7-deficient hearts. Here, we aimed to determine whether nicorandil or del Nido cardioplegia (DNC) solution can protect AQP7-deficient hearts. Methods: The hearts of male AQP7 knockout (KO) and wild-type (WT) C57/B6N mice (age &gt;15 weeks) were aerobically perfused using the Langendorff technique, and cardiac function was measured as left ventricular diastolic pressure (LVDP) throughout the study. Troponin T was measured as an indicator of myocardial damage after reperfusion for 60 min. We compared WT and KO controls subjected to 25 min of global ischemia as well as WT and KO groups infused with nicorandil (100 µM) for 10 min followed by 25 min of global ischemia. We also compared WT-DNC and KO-DNC hearts administered with DNC for 2 min followed by 23 min of global ischemia (Study 2). Results: The final recovery rates of LVDP were 20.8 ± 7.0%, 28.1 ± 7.6%, 40.0 ± 8.4%, and 38.7 ± 4.7% in the WT control, KO control, WT nicorandil, and KO nicorandil groups, respectively. The LVDP recovered faster in the hearts treated with DNC and reached a significantly higher plateau in the KO than in the WT hearts. Troponin T values were 2144 ± 493 and 1313 ± 717 in the WT and KO groups, respectively (p = 0.041). Conclusion: The Langendorff perfusion model revealed similar myocardial protective effects of nicorandil in AQP7-deficient mice as in WT mice. AQP7 deficiency did not impair the cardioprotective effects of DNC solution.

Defining cardiac stability during Langendorff perfusion

The Langendorff heart preparation is a valuable ex vivo technique for studying cardiac electrophysiology. For the past century it has been used in toxicology studies, drug testing, and disease modeling and has contributed significantly to understanding cardiac physiology, development, and pathological states. This study aimed to investigate the stability and reproducibility of the modern Langendorff heart preparation under various experimental conditions and to establish cardiac metric standards with corresponding exclusion criteria. Isolated hearts were extracted from adult Sprague Dawley rats and retrograde perfused with a modified Krebs-Henseleit buffer. A cohort of isolated hearts were used to establish a control baseline of multiple physiological parameters measured over time (e.g., electrocardiogram, flow rate, electrophysiology, electrical and calcium dynamics). These control baseline values were then compared to hearts exposed to the most common media additives in Langendorff studies (e.g., blebbistatin(−/−), bovine serum albumin, and voltage and calcium indicator dye). Over a three-hour period, cardiac metrics were monitored in stable Langendorff-perfused hearts. The addition of blebbistatin, DMSO or dyes did not change EP or ECG measurements as compared to control baseline. The most significant drift in all the EP values was ventricular effective refractory period (VERP). With or without blebbistatin, VERP increased from 40 to 60ms over the course of three hours. In that time heart rate and flow also decreased slightly. Blebbistatin was associated with an increased action potential duration but did not alter calcium transient duration. It was also observed that 1% circulating bovine serum albumin quenched the rh237 voltage dye signal 80% as measured by signal to noise. Constant pressure Langendorff-perfusion with a crystalloid buffer provides a reliable and reproducible model for cardiac electrophysiology research with a few caveats. The total length of study should be carefully considered to account for adaptations in cardiac physiology during longer perfusion times. As an excitation-contraction uncoupler, blebbistatin reduces motion artifact but delays action potential duration. Although BSA is used to minimize cardiac edema, it interferes with membrane bound voltage dyes. This study sets physiological parameters as a useful guide for future cardiac studies that use the isolated heart as a research model. American Physiological Society, R01HD108839 to N.G.P. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Extending heart preservation to 24 h with normothermic perfusion.

Cold static storage (CSS) for up to 6 h is the gold standard in heart preservation. Although some hearts stored over 6 h have been transplanted, longer CSS times have increased posttransplant morbimortality. Transmedics® Organ Care System (OCS™) is the only FDA-approved commercial system that provides an alternative to CSS using normothermic ex situ heart perfusion (NEHP) in resting mode with aortic perfusion (Langendorff method). However, it is also limited to 6 h and lacks an objective assessment of cardiac function. Developing a system that can perfuse hearts under NEHP conditions for >24 h can facilitate organ rehabilitation, expansion of the donor pool, and objective functional evaluation. The Extracorporeal Life Support Laboratory at the University of Michigan has worked to prolong NEHP to >24 h with an objective assessment of heart viability during NEHP. An NEHP system was developed for aortic (Langendorff) perfusion using a blood-derived perfusate (leukocyte/thrombocyte-depleted blood). Porcine hearts (n = 42) of different sizes (6-55 kg) were divided into five groups and studied during 24 h NEHP with various interventions in three piglets (small-size) heart groups: (1) Control NEHP without interventions (n = 15); (2) NEHP + plasma exchange (n = 5); (3) NEHP + hemofiltration (n = 10) and two adult-size (juvenile pigs) heart groups (to demonstrate the support of larger hearts); (4) NEHP + hemofiltration (n = 5); and (5) NEHP with intermittent left atrial (iLA) perfusion (n = 7). All hearts with NEHP + interventions (n = 27) were successfully perfused for 24 h, whereas 14 (93.3%) control hearts failed between 10 and 21 h, and 1 control heart (6.6%) lasted 24 h. Hearts in the piglet hemofiltration and plasma exchange groups performed better than those in the control group. The larger hearts in the iLA perfusion group (n = 7) allowed for real-time heart functional assessment and remained stable throughout the 24 h of NEHP. These results demonstrate that heart preservation for 24 h is feasible with our NEHP perfusion technique. Increasing the preservation period beyond 24 h, infection control, and nutritional support all need optimization. This proves the concept that NEHP has the potential to increase the organ pool by (1) considering previously discarded hearts; (2) performing an objective assessment of heart function; (3) increasing the donor/recipient distance; and (4) developing heart-specific perfusion therapies.

ZBTB20 Regulates SERCA2a Activity and Myocardial Contractility Through Phospholamban.

Intracellular Ca2+ cycling determines myocardial contraction and relaxation in response to physiological demands. SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a) is responsible for the sequestration of cytosolic Ca2+ into intracellular stores during cardiac relaxation, and its activity is reversibly inhibited by PLN (phospholamban). However, the regulatory hierarchy of SERCA2a activity remains unclear. Cardiomyocyte-specific ZBTB20 knockout mice were generated by crossing ZBTB20flox mice with Myh6-Cre mice. Echocardiography, blood pressure measurements, Langendorff perfusion, histological analysis and immunohistochemistry, quantitative reverse transcription-PCR, Western blot analysis, electrophysiological measurements, and chromatin immunoprecipitation assay were performed to clarify the phenotype and elucidate the molecular mechanisms. Specific ablation of ZBTB20 in cardiomyocyte led to a significant increase in basal myocardial contractile parameters both in vivo and in vitro, accompanied by an impairment in cardiac reserve and exercise capacity. Moreover, the cardiomyocytes lacking ZBTB20 showed an increase in sarcoplasmic reticular Ca2+ content and exhibited a remarkable enhancement in both SERCA2a activity and electrically stimulated contraction. Mechanistically, PLN expression was dramatically reduced in cardiomyocytes at the mRNA and protein levels by ZBTB20 deletion or silencing, and PLN overexpression could largely restore the basal contractility in ZBTB20-deficient cardiomyocytes. These data point to ZBTB20 as a fine-tuning modulator of PLN expression and SERCA2a activity, thereby offering new perspective on the regulation of basal contractility in the mammalian heart.

Early life stress exacerbates the obesogenic and anxiogenic effects of a Western diet without worsening cardiac ischaemic tolerance in male mice.

Early life stress (ELS) and a Western diet (WD) promote mood and cardiovascular disorders, however, how these risks interact in disease pathogenesis is unclear. We assessed effects of ELS with or without a subsequent WD on behaviour, cardiometabolic risk factors, and cardiac function/ischaemic tolerance in male mice. Fifty-six new-born male C57BL/6J mice were randomly allocated to a control group (CON) undisturbed before weaning, or to maternal separation (3h/day) and early (postnatal day 17) weaning (MSEW). Mice consumed standard rodent chow (CON, n = 14; MSEW, n = 15) or WD chow (WD, n = 19; MSEW + WD, n = 19) from week 8 to 24. Fasted blood was sampled and open field test and elevated plus maze (EPM) tests undertaken at 7, 15, and 23 weeks of age, with hearts excised at 24 weeks for Langendorff perfusion (evaluating pre- and post-ischaemic function). MSEW alone transiently increased open field activity at 7 weeks; body weight and serum triglycerides at 4 and 7 weeks, respectively; and final blood glucose levels and insulin resistance at 23 weeks. WD increased insulin resistance and body weight gain, the latter potentiated by MSEW. MSEW + WD was anxiogenic, reducing EPM open arm activity vs. WD alone. Although MSEW had modest metabolic effects and did not influence cardiac function or ischaemic tolerance in lean mice, it exacerbated weight gain and anxiogenesis, and improved ischaemic tolerance in WD fed animals. MSEW-induced increases in body weight (obesity) in WD fed animals in the absence of changes in insulin resistance may have protected the hearts of these mice.

DNA hypomethylation by fisetin preserves mitochondria functional genes and contributes to the protection of I/R rat heart.

Myocardial I/R can alter the expression of different sets of cardiac genes that negatively influence the I/R outcome via epigenetic modifications. Fisetin is known to be cardioprotective against I/R, but its underlying epigenetic mode of action is not known and is addressed in the present study. Male Wistar rats were subjected to I/R by using the Langendorff perfusion system. Fisetin (20mg/kg; i.p.) was administered before I/R induction, followed by the measurement of cardiac injury, hemodynamics, physiological indices, the differential expression of genes that regulate DNA methylation, and the function of mitochondria were performed. Fisetin administered I/R rat heart significantly reduced the global DNA hypermethylation and infarct size with an improved physiological recovery, measured via RPP (81%) and LVDP (82%) from the I/R control. Additionally, we noted decreased expression of the DNMT1 gene by 35% and increased expression of the TET1, TET2, and TET3 genes in fisetin-treated I/R rat hearts. Molecular docking analysis data reveals that the fisetin inhibits DNMT1 at the substrate binding site with minimum binding energy (- 8.2kcal/mol) compared to the DNMT1 inhibitor, 5-azacytidine. Moreover, fisetin-treated I/R heart reversed the expression of the I/R-linked declined expression of bioenergetics genes (MT-ND1, MT-ND2, MT-ND4, MT-Cyt B, MT-COX1, MT-COX2, MT-ATP6), mitochondrial fission gene (Fis1), replication control genes PGC-1α, POLG, and TFAM to near-normal level. Based on the above findings, we demonstrated that fisetin possesses the ability to modulate the expression of different mitochondrial genes via influencing the global DNA methylation in cardiac tissue, which contributes significantly to the improved contractile function and thereby renders cardioprotection against I/R.

Apelin receptor inhibition in ischemia-reperfused mouse hearts protected by endogenous n-3 polyunsaturated fatty acids.

Background: While the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on cardiac ischemia-reperfusion (IR) injury have been previously reported, limited data are available regarding how these fatty acids affect membrane receptors and their downstream signaling following IR injury. We aimed to identify potential receptors activated by n-3 PUFAs in IR hearts to understand the regulatory mechanisms of these receptors. Methods: We used fat-1 mice, which naturally have elevated levels of n-3 PUFAs, and C57BL/6J mice as a control group to create a myocardial IR injury model through Langendorff perfusion. We assessed the impact of endogenous n-3 PUFAs on left ventricular function, myocardial infarct size, myocardial apoptosis, and ATP production. RNA sequencing (RNA-seq) and bioinformatics analysis were conducted to identify molecular targets affected by n-3 PUFAs. Based on these analyses we then treated IR hearts of WT and fat-1 mice with an antagonist (ML221) or an agonist (apelin-13) for the predicted receptor to assess cardiac contractile function and intracellular signaling pathways. An in vitro hypoxia-reoxygenation (HR) model was also used to confirm the effects of n-3 PUFAs on the examined intracellular signaling pathways. Results: Endogenous n-3 PUFAs protected cardiac structure and function in post-IR hearts, and modulated phosphorylation patterns in the PI3K-AKT-mTOR signaling pathways. RNA-seq analysis revealed that n-3 PUFAs affected multiple biological processes as well as levels of the apelin receptor (APLNR). Consistent with a role for the PLNNR, ML221 synchronized the activation of the PI3K-AKT-mTOR signaling axis, suppressed the expression of PKCδ and phosphorylated p38α, upregulated PKCε expression, upregulated or restored the phosphorylation of myofilaments, and prevented myocardial injury and contractile dysfunction in WT IR hearts. By contrast, apelin-13 disrupted the PI3K-AKT-mTOR signaling axis in post-IR fat-1 hearts. The phosphorylation signaling targeted by APLNR inhibition in post-IR fat-1 hearts was also observed after treating HR cells with eicosatetraenoic acid (EPA). Conclusion: Endogenous n-3 PUFAs protect against post-IR injury and preserve cardiac contractile function possibly through APLNR inhibition. This inhibition synchronizes the PI3K-AKT-mTOR axis, suppresses detrimental phosphorylation signaling, and restores or increases myofilament phosphorylation in post-IR hearts. The beneficial effects observed in fat-1 transgenic mouse hearts can be attributed, at least in part, to elevated EPA levels. This study is the first to demonstrate that n-3 PUFAs protect hearts against IR injury through APLNR inhibition.

The potential roles of stress-induced phosphoprotein 1 and connexin 43 in rats with reperfusion arrhythmia.

Connexin 43 (Cx43) is a critical gene for maintaining myocardial homeostasis. Interestingly, Cx43 and stress-induced phosphoprotein 1 (STIP1) were recorded to be lowly expressed in ischemia/reperfusion (I/R). However, their impacts on reperfusion arrhythmia (RA) remain to be explored. Our study aimed to find out the related underlying mechanisms. After the establishment of an isolated heart model through Langendorff perfusion, the heart rate, conduction activation time, conduction velocity, and conduction direction of the left ventricle were evaluated, along with the apoptotic rate detection in the collected myocardial tissues. After the construction of a hypoxia/reoxygenation (H/R)-induced cellular model, cell apoptosis, intercellular communication, cell viability, and the content of reactive oxygen species, superoxide dismutase, malondialdehyde, and lactic dehydrogenase were measured. The expression of Cx43 and STIP1 was determined in both rat heart and cell models. The bindings of STIP3 and Cx43 to heat shock protein 90 (HSP90) and heat shock protein 70 (HSP70) were verified. Relative to the corresponding controls, Cx43 and STIP1 were decreased in myocardial tissues of RA rats and H/R-stimulated H9C2 cells, where Cx43-binding HSP70 and HSP90 were respectively increased and decreased, and ubiquitination level of Cx43 was enhanced. STIP1 overexpression promoted protein expression of Cx43, intercellular communication, and cell viability, and reduced cell apoptosis and oxidative stress in H/R-stimulated H9C2 cells. STIP1 promoted Cx43 expression to improve intercellular communication and reduce oxidative stress in H/R-stimulated H9C2 cells.

Dexmedetomidine abates myocardial ischemia reperfusion injury through inhibition of pyroptosis via regulation of miR-665/MEF2D/Nrf2 axis

The current study intended to delve into the mechanisms of dexmedetomidine (Dex) in regulating myocardial pyroptosis against myocardial ischemia/reperfusion injury (MIRI). The rat MIRI models were induced by ligation/release of the coronary artery in vivo and Langendorff perfusion ex vivo. Hemodynamic parameters, infarction sizes, and histopathological changes were assessed to understand the effects of Dex on MIRI. We explored the mechanisms through functional experiments on an H9c2 cell hypoxia/reoxygenation (H/R) model. Cell viability and apoptosis were evaluated using cell counting kit 8 (CCK-8) and AV/PI dual staining respectively. The expressions of miR-665 and MEF2D mRNA were detected by qRT-PCR. Western blot was employed to determine the expression levels of pyroptosis- and signaling pathway- related proteins. The interplays between miR-665 and MEF2D were validated by Dual-luciferase reporter assays. Our findings indicated that Dex preconditioning dramatically attenuated hemodynamic derangements, infarct size, and histopathological damage in rats undergoing MIRI. Dex markedly augmented cell viability, while suppressing cell apoptosis and expressions of NLRP3, cleaved-caspase-1, ASC, GSDMD, IL-1β, and IL-18 in H9c2 cells subjected to H/R injury. MiR-665 was significantly upregulated, MEF2D and Nrf2 downregulated following H/R, whereas Dex preconditioning reversed these changes. MEF2D was validated to be a target gene of miR-665. Overexpression of miR-665 decreased the expression of MEF2D and blunted the protective effects of Dex in H9c2 cells. Moreover, the functional rescue experiment further verified that Dex regulated MEF2D/Nrf2 pathway via miR-665. In conclusion, Dex mitigates MIRI through inhibiting pyroptosis via regulating miR-665/MEF2D/Nrf2 axis.

Sarcospan Deficiency Increases Oxidative Stress and Arrhythmias in Hearts after Acute Ischemia-Reperfusion Injury.

The protein sarcospan (SSPN) is an integral member of the dystrophin-glycoprotein complex (DGC) and has been shown to be important in the heart during the development and the response to acute stress. In this study, we investigated the role of SSPN in the cardiac response to acute ischemia-reperfusion (IR) injury in SSPN-deficient (SSPN-/-) mice. First, the hemodynamic response of SSPN-/- mice was tested and was similar to SSPN+/+ (wild-type) mice after isoproterenol injection. Using the in situ Langendorff perfusion method, SSPN-/- hearts were subjected to IR injury and found to have increased infarct size and arrhythmia susceptibility compared to SSPN+/+. Ca2+ handling was assessed in single cardiomyocytes and diastolic Ca2+ levels were increased after acute β-AR stimulation in SSPN+/+ but not SSPN-/-. It was also found that SSPN-/- cardiomyocytes had reduced Ca2+ SR content compared to SSPN+/+ but similar SR Ca2+ release. Next, we used qRT-PCR to examine gene expression of Ca2+ handling proteins after acute IR injury. SSPN-/- hearts showed a significant decrease in L-type Ca2+ channels and a significant increase in Ca2+ release channel (RyR2) expression. Interestingly, under oxidizing conditions reminiscent of IR, SSPN-/- cardiomyocytes, had increased H2O2-induced reactive oxygen species production compared to SSPN+/+. Examination of oxidative stress proteins indicated that NADPH oxidase 4 and oxidized CAMKII were increased in SSPN-/- hearts after acute IR injury. These results suggest that increased arrhythmia susceptibility in SSPN-/- hearts post-IR injury may arise from alterations in Ca2+ handling and a reduced capacity to regulate oxidative stress pathways.

Effect of SH2B1 on glucose metabolism during pressure overload-induced cardiac hypertrophy and cardiac dysfunction.

This study mainly explored the effect and mechanism of Src homology 2 (SH2) B adaptor protein 1 (SH2B1) on cardiac glucose metabolism during pressure overload-induced cardiac hypertrophy and dysfunction. A pressure-overloaded cardiac hypertrophy model was constructed, and SH2B1-siRNA was injected through the tail vein. Haematoxylin and eosin (H&E) staining was used to detect myocardial morphology. ANP, BNP, β-MHC and the diameter of myocardial fibres were quantitatively measured to evaluate the degree of cardiac hypertrophy, respectively. GLUT1, GLUT4, and IR were detected to assess cardiac glucose metabolism. Cardiac function was determined by echocardiography. Then, glucose oxidation and uptake, glycolysis and fatty acid metabolism were assessed in Langendorff perfusion of hearts. Finally, PI3K/AKT activator was used to further explore the relevant mechanism. The results showed that during cardiac pressure overload, with the aggravation of cardiac hypertrophy and dysfunction, cardiac glucose metabolism and glycolysis increased, and fatty acid metabolism decreased. After SH2B1-siRNA transfection, cardiac SH2B1 expression was knocked down, and the degree of cardiac hypertrophy and dysfunction was alleviated compared with the Control-siRNA transfected group. Simultaneously, cardiac glucose metabolism and glycolysis were reduced, and fatty acid metabolism was enhanced. The SH2B1 expression knockdown mitigated the cardiac hypertrophy and dysfunction by reducing cardiac glucose metabolism. After using PI3K/AKT activator, the effect of SH2B1 expression knockdown on cardiac glucose metabolism was reversed during cardiac hypertrophy and dysfunction. Collectively, SH2B1 regulated cardiac glucose metabolism by activating the PI3K/AKT pathway during pressure overload-induced cardiac hypertrophy and cardiac dysfunction.

Site-specific prolongation of repolarization prevents postmyocardial infarction tachycardia.

Site-specific prolongation of repolarization prevents postmyocardial infarction tachycardia.

Novel Molecular Vehicle-Based Approach for Cardiac Cell Transplantation Leads to Rapid Electromechanical Graft-Host Coupling.

Myocardial remodeling is an inevitable risk factor for cardiac arrhythmias and can potentially be corrected with cell therapy. Although the generation of cardiac cells ex vivo is possible, specific approaches to cell replacement therapy remain unclear. On the one hand, adhesive myocyte cells must be viable and conjugated with the electromechanical syncytium of the recipient tissue, which is unattainable without an external scaffold substrate. On the other hand, the outer scaffold may hinder cell delivery, for example, making intramyocardial injection difficult. To resolve this contradiction, we developed molecular vehicles that combine a wrapped (rather than outer) polymer scaffold that is enveloped by the cell and provides excitability restoration (lost when cells were harvested) before engraftment. It also provides a coating with human fibronectin, which initiates the process of graft adhesion into the recipient tissue and can carry fluorescent markers for the external control of the non-invasive cell position. In this work, we used a type of scaffold that allowed us to use the advantages of a scaffold-free cell suspension for cell delivery. Fragmented nanofibers (0.85 µm ± 0.18 µm in diameter) with fluorescent labels were used, with solitary cells seeded on them. Cell implantation experiments were performed in vivo. The proposed molecular vehicles made it possible to establish rapid (30 min) electromechanical contact between excitable grafts and the recipient heart. Excitable grafts were visualized with optical mapping on a rat heart with Langendorff perfusion at a 0.72 ± 0.32 Hz heart rate. Thus, the pre-restored grafts' excitability (with the help of a wrapped polymer scaffold) allowed rapid electromechanical coupling with the recipient tissue. This information could provide a basis for the reduction of engraftment arrhythmias in the first days after cell therapy.

P85: Development Of The University of Pittsburgh Ex-vivo Cardiac Perfusion System: System Minimization Update

Background: Heart transplantation remains the definitive treatment for end-stage heart failure; however, the waitlist exceeds the donor organs available. It is difficult to obtain an assessment of organ viability for transplant in many circumstances. We have developed a system that can allow for assessment of organ viability and potentially expand the donor pool. The device will allow for ex vivo assessment of the heart both via Langendorff and physiologic perfusion modes. Initial experiments have been performed on the system with mixed success. This is in part due to the system design and components. The current focus is developing an updated prototype to mitigate some of these problems. Methods: A brief series of perfusion experiments were performed. Hearts were purchased donated by a local slaughterhouse and purchased from Animal Technologies (Tyler, TX) and were used for both recovery and static perfusion experiments to enhance system design. The hearts were connected to the circuit which consisted of an integrated perfusion chamber/reservoir, a PediMag (Abbott) pump, and a Eurosets (Abbott) oxygenator. Whole blood from the donor animal was the perfusate with physiological correction guided by blood chemistries. A sweep gas control unit was utilized to manage perfusate pH and PCO2 during organ perfusion. Slaughterhouse procured hearts were cannulated and rewarmed slowly to test the protocol. Results: Based on initial experiments, we chose to modify our existing design to minimize the overall footprint of our perfusion system. A new organ chamber has been designed with a scalloped heart platform (Figure 1). Circulatory support components will be housed in the rear of the organ chamber. Additionally, we have determined that a pediatric sized oxygenator will meet the VO2 requirements of the perfused organ, which also allows us to investigate the use of an integrated pump/oxygenator as a further system minimization.

Sevoflurane Improves Ventricular Conduction by Exosomes Derived from Rat Cardiac Fibroblasts After Hypothermic Global Ischemia-Reperfusion Injury.

This study investigated the effect of exosomes derived from sevoflurane-treated cardiac fibroblasts (Sev-CFs-Exo) on reperfusion arrhythmias (RA), ventricular conduction, and myocardial ischemia-reperfusion injury (MIRI). Primary cardiac fibroblasts (CFs) were isolated from the hearts of neonatal rats and identified by morphology and immunofluorescence. Exosomes were isolated from CFs at passages 2-3 after they had been treated with 2.5% sevoflurane for an hour and cultivated for 24-48 hours. The control group was CFs that did not receive any treatment. The hypothermic global ischemia-reperfusion injury model was established using the Langendorff perfusion technique following injection with exosomes through the caudal vein. Multi-electrode array (MEA) mapping was used to investigate the changes in RA and ventricular conduction in isolated hearts. Western blots and immunofluorescence were used to examine the relative expression and location of connexin 43 (Cx43). In addition, the MIRI was evaluated with triphenyl tetrazolium chloride and Hematoxylin-Eosin staining. The primary CFs had a variety of morphologies, no spontaneous pulsation, and were vimentin-positive, which confirmed their successful isolation. Sev-CFs-Exo increased the heart rate (HR) at reperfusion for 15 minutes (T2) and 30 minutes (T3) and lowered the score and duration of RA and the time for restoration of heartbeat in reperfusion. Meanwhile, Sev-CFs-Exo increased conduction velocity (CV), decreased absolute inhomogeneity (P5-95) and inhomogeneity index (P5-95/P50) at T2 and T3, as well as promoted the recovery of HR, CV, P5-95 and P5-95/P50 after hypothermic global ischemia-reperfusion injury. Furthermore, Sev-CFs-Exo raised expression and reduced lateralization of Cx43, and improved myocardial infarct sizes and cellular necrosis. However, while cardiac fibroblast-derived exosomes (CFs-Exo) showed similar cardioprotective effects, the outcomes were not as significant. Sevoflurane reduces the risk of RA and improves ventricular conduction and MIRI by CFs-Exo, and this may be driven by the expression and location of Cx43.

Isolation of porcine adult cardiomyocytes: Comparison between Langendorff perfusion and tissue slicing-assisted enzyme digestion.

Tissue slicing-assisted digestion (TSAD) of adult cardiomyocytes has shown significant improvements over conventional chunk methods. However, it remains unclear how this method compares to Langendorff perfusion, the current standard of adult cardiomyocyte isolation. Using adult Bama minipigs, we performed cardiomyocyte isolation via these two distinct methods, and compared the resulting cellular quality, including viability, cellular structure, gene expression, and electrophysiological properties, of cardiomyocytes from 3 distinct anatomical regions, namely the left ventricle, right ventricle, and left atrial appendage. Our results revealed largely indistinguishable cell quality in all of the measured parameters. These findings suggest that that TSAD can be reliably used to isolate adult mammalian cardiomyocytes as a reliable alternative to perfusion in cardiomyocyte isolation from larger mammals, particularly when Langendorff perfusion is not feasible.