Gene therapy encoding cell cycle factors to treat chronic ischemic heart failure in rats.

AIMS: Gene therapies to induce cardiomyocyte (CM) cell cycle re-entry have shown a potential to treat subacute ischaemic heart failure (IHF) but have not been tested in the more relevant setting of chronic IHF. Our group recently showed that polycistronic non-integrating lentivirus encoding Cdk1/CyclinB1 and Cdk4/CyclinD1 (TNNT2-4Fpolycistronic-NIL) is effective in inducing CM cell cycle re-entry and ameliorating subacute IHF models and preventing the subsequent IHF-induced congestions in the liver, kidneys, and lungs in rats and pigs. Here, we aim to test the long-term efficacy of TNNT2-4Fpolycistronic-NIL in a rat model of chronic IHF, a setting that differs pathophysiologically from subacute IHF and has greater clinical relevance. METHODS AND RESULTS: Rats were subjected to a 2-h coronary occlusion followed by reperfusion; 4 weeks later, rats were injected intramyocardially with either TNNT2-4Fpolycistronic-NIL or LacZ-NIL. Four months post-viral injection, TNNT2-4Fpolycistronic-NIL-treated rats showed a significant reduction in scar size and a significant improvement in left ventricular (LV) systolic cardiac function but not in the LV dilatation associated with chronic IHF. A mitosis reporter system developed in our lab showed significant induction of CM mitotic activity in TNNT2-4Fpolycistronic-NIL-treated rats. CONCLUSION: This study demonstrates, for the first time, that TNNT2-4Fpolycistronic-NIL gene therapy induces CM cell cycle re-entry in chronic IHF and improves LV function, and that this salubrious effect is sustained for at least 4 months. Given the high prevalence of chronic IHF, these results have significant clinical implications for developing a novel treatment for this deadly disease.

Pharmacological or genetic inhibition of LTCC promotes cardiomyocyte proliferation through inhibition of calcineurin activity.

Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. Calcium (Ca2+) is an important signal transducer that regulates key cellular processes, but its role in regulating CM proliferation is incompletely understood. A drug screen targeting proteins involved in CM calcium cycling in human embryonic stem cell-derived cardiac organoids (hCOs) revealed that only the inhibition of L-Type Calcium Channel (LTCC), but not other Ca2+ regulatory proteins (SERCA or RYR), induced the CM cell cycle. Furthermore, overexpression of Ras-related associated with Diabetes (RRAD), an endogenous inhibitor of LTCC, induced CM cell cycle activity in vitro, in human cardiac slices, and in vivo. Mechanistically, LTCC inhibition by RRAD induces the cell cycle in CMs by modulating calcineurin activity and translocating Hoxb13 to the CM nucleus. Together, this represents a robust pathway for regenerative strategies.

A novel small molecule inhibitor of p38⍺ MAP kinase augments cardiomyocyte cell cycle entry in response to direct cell cycle stimulation.

BACKGROUND AND PURPOSE: Myocardial infarction (MI) is the leading cause of mortality globally due in part to the limited ability of cardiomyocytes (CMs) to regenerate. Recently, we demonstrated that overexpression of four-cell cycle factors, CDK1, CDK4, cyclin B1 and cyclin D1 (4F), induced cell division in ~20% of the post-mitotic CMs overexpressed 4F. The current study aims to identify a small molecule that augments 4F-induced CM cycle induction. EXPERIMENTAL APPROACH, KEY RESULTS: Screening of small molecules with a potential to augment 4F-induced cell-cycle induction in 60-day-old mature human induced pluripotent cardiomyocytes (hiPS-CMs) revealed N-(4,6-Dimethylpyridin-2-yl)-4-(pyridine-4-yl)piperazine-1-carbothioamide (NDPPC), which activates cell cycle progression in 4F-transduced hiPS-CMs. Autodock tool and Autodock vina computational methods showed that NDPPC has a potential interaction with the binding site at the human p38⍺ mitogen-activated protein kinase (p38⍺ MAP kinase), a critical negative regulator of the mammalian cell cycle. A p38 MAP kinase activity assay showed that NDPPC inhibits p38⍺ with 5-10 times lower IC50 compared to the other P38 isoforms in a dose-dependent manner. Overexpression of p38⍺ MAP kinase in CMs inhibited 4F cell cycle induction, and treatment with NDPPC reversed the cell cycle inhibitory effect. CONCLUSION AND IMPLICATIONS: NDPPC is a novel inhibitor for p38 MAP kinase and is a promising drug to augment CM cell cycle response to the 4F. NDPPC could become an adjunct treatment with other cell cycle activators for heart failure treatment.

Restored autophagy is protective against PAK3-induced cardiac dysfunction.

Despite the development of clinical treatments, heart failure remains the leading cause of mortality. We observed that p21-activated kinase 3 (PAK3) was augmented in failing human and mouse hearts. Furthermore, mice with cardiac-specific PAK3 overexpression exhibited exacerbated pathological remodeling and deteriorated cardiac function. Myocardium with PAK3 overexpression displayed hypertrophic growth, excessive fibrosis, and aggravated apoptosis following isoprenaline stimulation as early as two days. Mechanistically, using cultured cardiomyocytes and human-relevant samples under distinct stimulations, we, for the first time, demonstrated that PAK3 acts as a suppressor of autophagy through hyper-activation of the mechanistic target of rapamycin complex 1 (mTORC1). Defective autophagy in the myocardium contributes to the progression of heart failure. More importantly, PAK3-provoked cardiac dysfunction was mitigated by administering an autophagic inducer. Our study illustrates a unique role of PAK3 in autophagy regulation and the therapeutic potential of targeting this axis for heart failure.

FGF21/FGFR1-ß-KL cascade in cardiomyocytes modulates angiogenesis and inflammation under metabolic stress.

Diabetes is a metabolic disorder with an increased risk of developing heart failure. Inflammation and damaged vasculature are the cardinal features of diabetes-induced cardiac damage. Moreover, systemic metabolic stress triggers discordant intercellular communication, thus culminating in cardiac dysfunction. Fibroblast growth factor 21 (FGF21) is a pleiotropic hormone transducing cellular signals via fibroblast growth factor receptor 1 (FGFR1) and its co-receptor beta-klotho (ß-KL). This study first demonstrated a decreased expression or activity of FGFR1 and ß-KL in both human and mouse diabetic hearts. Reinforcing cardiac FGFR1 and ß-KL expression can alleviate pro-inflammatory response and endothelial dysfunction upon diabetic stress. Using proteomics, novel cardiomyocyte-derived anti-inflammatory and proangiogenic factors regulated by FGFR1-ß-KL signaling were identified. Although not exhaustive, this study provides a unique insight into the protective topology of the cardiac FGFR1-ß-KL signaling-mediated intercellular reactions in the heart in response to metabolic stress.

Transient gene therapy using cell cycle factors reverses renin-angiotensin-aldosterone system activation in heart failure rat model.

The loss of cardiomyocytes after myocardial infarction (MI) leads to heart failure. Recently, we demonstrated that transient overexpression of 4 cell cycle factors (4F), using a polycistronic non-integrating lentivirus (TNNT2-4F-NIL) resulted in significant improvement in cardiac function in a rat model of MI. Yet, it is crucial to demonstrate the reversal of the heart failure-related pathophysiological manifestations, such as renin-angiotensin-aldosterone system activation (RAAS). To assess that, Fisher 344 rats were randomized to receive TNNT2-4F-NIL or control virus seven days after coronary occlusion for 2 h followed by reperfusion. 4 months after treatment, N-terminal pro-brain natriuretic peptide, plasma renin activity, and aldosterone levels returned to the normal levels in rats treated with TNNT2-4F-NIL but not in vehicle-treated rats. Furthermore, the TNNT2-4F-NIL-treated group showed significantly less liver and kidney congestion than vehicle-treated rats. Thus, we conclude that in rat models of MI, TNNT2-4F-NIL reverses RAAS activation and subsequent systemic congestion.

Biomimetic cardiac tissue culture model (CTCM) to emulate cardiac physiology and pathophysiology ex vivo.

There is need for a reliable in vitro system that can accurately replicate the cardiac physiological environment for drug testing. The limited availability of human heart tissue culture systems has led to inaccurate interpretations of cardiac-related drug effects. Here, we developed a cardiac tissue culture model (CTCM) that can electro-mechanically stimulate heart slices with physiological stretches in systole and diastole during the cardiac cycle. After 12 days in culture, this approach partially improved the viability of heart slices but did not completely maintain their structural integrity. Therefore, following small molecule screening, we found that the incorporation of 100 nM tri-iodothyronine (T3) and 1 µM dexamethasone (Dex) into our culture media preserved the microscopic structure of the slices for 12 days. When combined with T3/Dex treatment, the CTCM system maintained the transcriptional profile, viability, metabolic activity, and structural integrity for 12 days at the same levels as the fresh heart tissue. Furthermore, overstretching the cardiac tissue induced cardiac hypertrophic signaling in culture, which provides a proof of concept for the ability of the CTCM to emulate cardiac stretch-induced hypertrophic conditions. In conclusion, CTCM can emulate cardiac physiology and pathophysiology in culture for an extended time, thereby enabling reliable drug screening.

Paracrine signal emanating from stressed cardiomyocytes aggravates inflammatory microenvironment in diabetic cardiomyopathy.

Myocardial inflammation contributes to cardiomyopathy in diabetic patients through incompletely defined underlying mechanisms. In both human and time-course experimental samples, diabetic hearts exhibited abnormal ER, with a maladaptive shift over time in rodents. Furthermore, as a cardiac ER dysfunction model, mice with cardiac-specific p21-activated kinase 2 (PAK2) deletion exhibited heightened myocardial inflammatory response in diabetes. Mechanistically, maladaptive ER stress-induced CCAAT/enhancer-binding protein homologous protein (CHOP) is a novel transcriptional regulator of cardiac high-mobility group box-1 (HMGB1). Cardiac stress-induced release of HMGB1 facilitates M1 macrophage polarization, aggravating myocardial inflammation. Therapeutically, sequestering the extracellular HMGB1 using glycyrrhizin conferred cardioprotection through its anti-inflammatory action. Our findings also indicated that an intact cardiac ER function and protective effects of the antidiabetic drug interdependently attenuated the cardiac inflammation-induced dysfunction. Collectively, we introduce an ER stress-mediated cardiomyocyte-macrophage link, altering the macrophage response, thereby providing insight into therapeutic prospects for diabetes-associated cardiac dysfunction.

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

BACKGROUND: The 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. METHODS: Using 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. RESULTS: Temporal 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). 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 owing to the use of a novel transient and cardiomyocyte-specific viral construct.

Cell cycle induction in human cardiomyocytes is dependent on biosynthetic pathway activation.

AIMS: The coordinated gene and metabolic programs that facilitate cardiomyocyte entry and progression in the cell cycle are poorly understood. The purpose of this study was to identify the metabolic changes that influence myocyte proliferation. METHODS AND RESULTS: In adult mouse cardiomyocytes and human induced pluripotent stem cell cardiomyocytes (hiPS-CMs), cell cycle initiation by ectopic expression of Cyclin B1, Cyclin D1, CDK1, and CDK4 (termed 4F) downregulated oxidative phosphorylation genes and upregulated genes that regulate ancillary biosynthetic pathways of glucose metabolism. Results from metabolic analyses and stable isotope tracing experiments indicate that 4F-mediated cell cycle induction in hiPS-CMs decreases glucose oxidation and oxidative phosphorylation and augments NAD+, glycogen, hexosamine, phospholipid, and serine biosynthetic pathway activity. Interventions that diminish NAD+ synthesis, serine synthesis, or protein O-GlcNAcylation decreased 4F-mediated cell cycle entry. In a gain of function approach, we overexpressed phosphoenolpyruvate carboxykinase 2 (PCK2), which can drive carbon from the Krebs cycle to the glycolytic intermediate pool, and found that PCK2 augments 4F-mediated cell cycle entry. CONCLUSIONS: These findings suggest that a metabolic shift from catabolic to anabolic activity is a critical step for cardiomyocyte cell cycle entry and is required to facilitate proliferation.

Induced Cardiomyocyte Proliferation: A Promising Approach to Cure Heart Failure.

Unlike some lower vertebrates which can completely regenerate their heart, the human heart is a terminally differentiated organ. Cardiomyocytes lost during cardiac injury and heart failure cannot be replaced due to their limited proliferative capacity. Therefore, cardiac injury generally leads to progressive failure. Here, we summarize the latest progress in research on methods to induce cardiomyocyte cell cycle entry and heart repair through the alteration of cardiomyocyte plasticity, which is emerging as an effective strategy to compensate for the loss of functional cardiomyocytes and improve the impaired heart functions.

Heart slice culture system reliably demonstrates clinical drug-related cardiotoxicity.

The limited availability of human heart tissue and its complex cell composition are major limiting factors for the reliable testing of drug efficacy and toxicity. Recently, we developed functional human and pig heart slice biomimetic culture systems that preserve the viability and functionality of 300 µm heart slices for up to 6 days. Here, we tested the reliability of this culture system for testing the cardiotoxicity of anti-cancer drugs. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) with known different mechanisms of cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and gene expression. Slices incubated with any of these drugs for 48 h showed diminished in viability as well as loss of cardiomyocyte structure and function. Mechanistically, RNA sequencing of doxorubicin-treated tissues demonstrated a significant downregulation of cardiac genes and upregulation of oxidative stress responses. Trastuzumab treatment downregulated cardiac muscle contraction-related genes consistent with its clinically known effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes, in line with its mechanism of action. Similar to hiPS-derived-cardiomyocytes, heart slices recapitulated the expected toxicity of doxorubicin and trastuzumab, however, slices were superior in detecting sunitinib cardiotoxicity and mechanism in the clinically relevant concentration range of 0.1-1 µM. These results indicate that heart slice culture models have the potential to become a reliable platform for testing and elucidating mechanisms of drug cardiotoxicity.

Slicing and Culturing Pig Hearts under Physiological Conditions.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming 'first in man' trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

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 ß-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.