Physiological Biomimetic Culture System for Pig and Human Heart Slices

RATIONALE: Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. OBJECTIVE: To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. METHODS AND RESULTS: Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. CONCLUSIONS: We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3D heart model

mRNA Profiling for Myocardial Injury In Experimental Cardiac Arrest Models

Introduction: Cardiac arrest has a very poor prognosis and survival rate after restoration of spontaneous circulation (ROSC). Resuscitation techniques are one of the most significant contributors to the clinical outcome after cardiac arrest. Resuscitation aims for avoiding and/or alleviating the brain damage, myocardial function decline, and multi-organ failure due to ischemia/hypoxia during the event of cardiac arrest. Despite clinical research efforts to answer questions related to resuscitation techniques and the value of different resuscitation strategies, the 30-day mortality after an event of cardiac arrest is still high. Some of the great advancement of resuscitation techniques are Extracorporeal Life Support (ECLS) and Extracorporeal Membrane Oxygenator (ECMO) which provide means for restoring tissue perfusion, oxygenation, drug delivery and temperature control until cardiac function is restored. Several protocols that used ECLS and ECMO for neuroprotection and cardioprotection have been tested, including hypothermia and supplementation of different drugs. However, the results of such studies are not conclusive which can be attributed to lack of understanding of the molecular events associated with these protocols. Therefore, in this study we will investigate the genetic and molecular remolding associated with two models of post cardiac arrest resuscitation. Scope of the study: This study was conducted to assess the genetic and molecular changes associated with two different models of resuscitation in the sitting of cardiac arrest. First model is hypothermic arrest followed by different rate of rewarming. Second model is a cardiac arrest followed by cardiopulmonary bypass (CPB) resuscitation with NO supplementation using ECMO. This study is conducted to fulfill the requirements for doctoral degree in cardiovascular sciences at the university of Verona. Materials and methods: Male Sprague Dawley rats weighing around 400±50 g were used. Two main models of resuscitation in the sitting of cardiac arrest were used; the model of hypothermic cardiac arrest followed by different rates of rewarming using ECLS, and the model of experimental cardiac arrest and resuscitation using NO supplied through the ECMO. Cardiac tissues were collected after the experiment for molecular analysis. RNA sequencing was used to detect transcriptomic changes. The apoptosis was assessed using Tunnel assay Results: First our results highlight the effect of different rates of rewarming post cardiac arrest. Slow rewarming showed a significant lower level of myocardial apoptosis compared to rapid rewarming. Rapid rewarming was associated transcriptomic derangement involving pathways related to lipid metabolism, inflammatory and apoptotic pathways, and calcium handling. Second our results showed that resuscitation using nitric oxide (NO) has a deleterious effect of on the myocardium as indicated by a significant increase in the apoptosis. The known beneficial effects of NO include inhibition of the sympathetic signaling, and smooth muscle relaxation couldn’t overcome the complex metabolic derangement, and profound activation of inflammatory and apoptotic pathways. Conclusions: Our study explained the beneficial effect of the slow rewarming over the fast rewarming after hypothermic arrest at the molecular and genetic level. We also challenged the idea of using NO as a protective agent during resuscitation. NO has a deteriorating effect on the myocardium. This study highlights the genetic remodeling associated with resuscitation in the sitting of cardiac arrest which would guide clinical decisions. In addition, this study spots the light on the possible molecular pathways that could be targeted to improve resuscitation process post-cardiac arrest and would results in improving the survival rate post-cardiac arrest

Transient Cell Cycle Induction in Cardiomyocytes to Treat Subacute Ischemic Heart Failure

BackgroundThe regenerative capacity of the heart after myocardial infarction is limited. Our previous study showed that ectopic introduction of 4 cell cycle factors (4F; CDK1 [cyclin-dependent kinase 1], CDK4 [cyclin-dependent kinase 4], CCNB [cyclin B1], and CCND [cyclin D1]) promotes cardiomyocyte proliferation in 15% to 20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after myocardial infarction in mice.MethodsUsing temporal single-cell RNA sequencing, we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Using rat and pig models of ischemic heart failure, we aimed to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure.ResultsTemporal bulk and single-cell RNA sequencing and further biochemical validations of mature human induced pluripotent stem cell-derived cardiomyocytes treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 hours after infection with 4F, which was associated mainly with sarcomere disassembly and metabolic reprogramming (n=3/time point/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic nonintegrating lentivirus (NIL) encoding 4F; each is driven by a TNNT2 (cardiac troponin T isoform 2) promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially 1 week after myocardial infarction in rats (n=10/group) or pigs (n=6-7/group). Four weeks after injection, TNNT2-4Fpolycistronic-NIL-treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus-treated animals. At 4 months after treatment, rats that received TNNT2-4Fpolycistronic-NIL still showed a sustained improvement in cardiac function and no obvious development of cardiac arrhythmias or systemic tumorigenesis (n=10/group).ConclusionsThis study provides mechanistic insights into the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors owing to the use of a novel transient and cardiomyocyte-specific viral construct