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 3-dimensional heart model.

Background: The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI in mice. Methods: Here, using temporal single-cell RNAseq we aimed to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Also, 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. Results: Temporal bulk and single-cell RNAseq and further biochemical validations of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population at 48 h post-infection with 4F, which was mainly associated with sarcomere disassembly and metabolic reprogramming (n=3/timepoint/group). Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic non-integrating lentivirus (NIL) encoding the 4F; each is driven by a TNNT2 promoter (TNNT2-4Fpolycistronic-NIL). TNNT2-4Fpolycistronic-NIL or control virus was injected intramyocardially one week after MI in rats (n=10/group) or pigs (n=6-7/group). Four weeks post-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 four 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). Conclusions: This 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 thanks to the use of a novel transient and cardiomyocyte-specific viral construct.

The heart responds to pathological overload through myocyte hypertrophy. Here we show that this response is regulated by cardiac fibroblasts via a paracrine mechanism involving plasma membrane calcium ATPase 4 (PMCA4). Pmca4 deletion in mice, both systemically and specifically in fibroblasts, reduces the hypertrophic response to pressure overload; however, knocking out Pmca4 specifically in cardiomyocytes does not produce this effect. Mechanistically, cardiac fibroblasts lacking PMCA4 produce higher levels of secreted frizzled related protein 2 (sFRP2), which inhibits the hypertrophic response in neighbouring cardiomyocytes. Furthermore, we show that treatment with the PMCA4 inhibitor aurintricarboxylic acid (ATA) inhibits and reverses cardiac hypertrophy induced by pressure overload in mice. Our results reveal that PMCA4 regulates the development of cardiac hypertrophy and provide proof of principle for a therapeutic approach to treat this condition.

The adult mammalian heart is recalcitrant to regeneration after injury, in part due to the postmitotic nature of cardiomyocytes. Accumulating evidence suggests that cardiomyocyte proliferation in fetal or neonatal mammals and in regenerative non-mammalian models depends on a conducive metabolic state. Results from numerous studies in adult hearts indicate that conditions of relatively low fatty acid oxidation, low reactive oxygen species generation, and high glycolysis are required for induction of cardiomyocyte proliferation. Glycolysis appears particularly important because it provides branchpoint metabolites for several biosynthetic pathways that are essential for synthesis of nucleotides and nucleotide sugars, amino acids, and glycerophospholipids, all of which are required for daughter cell formation. In addition, the proliferative cardiomyocyte phenotype is supported in part by relatively low oxygen tensions and through the actions of critical transcription factors, coactivators, and signaling pathways that promote a more glycolytic and proliferative cardiomyocyte phenotype, such as hypoxia inducible factor 1α (Hif1α), Yes-associated protein (Yap), and ErbB2. Interventions that inhibit glycolysis or its integrated biosynthetic pathways almost universally impair cardiomyocyte proliferative capacity. Furthermore, metabolic enzymes that augment biosynthetic capacity such as phosphoenolpyruvate carboxykinase 2 and pyruvate kinase M2 appear to be amplifiers of cardiomyocyte proliferation. Collectively, these studies suggest that acquisition of a glycolytic and biosynthetic metabolic phenotype is a sine qua non of cardiomyocyte proliferation. Further knowledge of the regulatory mechanisms that control substrate partitioning to coordinate biosynthesis with energy provision could be leveraged to prompt or augment cardiomyocyte division and to promote cardiac repair.

Translational research in the cardiovascular field is hampered by the unavailability of cardiac models that can recapitulate organ-level physiology of the myocardium. Outside the body, cardiac tissue undergoes rapid dedifferentiation and maladaptation in culture. There is an ever-growing demand for preclinical platforms that allow for accurate, standardized, long-term, and rapid drug testing. Heart slices is an emerging technology that solves many of the problems with conventional myocardial culture systems. Heart slices are thin (<400 µm) slices of heart tissue from the adult ventricle. Several recent studies using heart slices have shown their ability to maintain the adult phenotype for prolonged periods in a multi cell-type environment. Here, we review the current status of cardiac culture systems and highlight the unique advantages offered by heart slices in the light of recent efforts in developing physiologically relevant heart slice culture systems.