Adverse Impact of Cannabis on Human Health.

Cannabis, the most commonly used recreational drug, is illicit in many areas of the world. With increasing decriminalization and legalization, cannabis use is increasing in the United States and other countries. The adverse effects of cannabis are unclear because its status as a Schedule 1 drug in the United States restricts research. Despite a paucity of data, cannabis is commonly perceived as a benign or even beneficial drug. However, recent studies show that cannabis has adverse cardiovascular and pulmonary effects and is linked with malignancy. Moreover, case reports have shown an association between cannabis use and neuropsychiatric disorders. With growing availability, cannabis misuse by minors has led to increasing incidences of overdose and toxicity. Though difficult to detect, cannabis intoxication may be linked to impaired driving and motor vehicle accidents. Overall, cannabis use is on the rise, and adverse effects are becoming apparent in clinical data sets.

Cryo-electron tomography reveals the structural diversity of cardiac proteins in their cellular context.

Cardiovascular diseases are a leading cause of death worldwide, but our understanding of the underlying mechanisms is limited, in part because of the complexity of the cellular machinery that controls the heart muscle contraction cycle. Cryogenic electron tomography (cryo-ET) provides a way to visualize diverse cellular machinery while preserving contextual information like subcellular localization and transient complex formation, but this approach has not been widely applied to the study of heart muscle cells (cardiomyocytes). Here, we deploy a platform for studying cardiovascular disease by combining cryo-ET with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). After developing a cryo-ET workflow for visualizing macromolecules in hiPSC-CMs, we reconstructed sub-nanometer resolution structures of the human thin filament, a central component of the contractile machinery. We also visualized a previously unobserved organization of a regulatory complex that connects muscle contraction to calcium signaling (the troponin complex), highlighting the value of our approach for interrogating the structures of cardiac proteins in their cellular context.

Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation.

Epidemiological studies reveal that marijuana increases the risk of cardiovascular disease (CVD); however, little is known about the mechanism. Δ9-tetrahydrocannabinol (Δ9-THC), the psychoactive component of marijuana, binds to cannabinoid receptor 1 (CB1/CNR1) in the vasculature and is implicated in CVD. A UK Biobank analysis found that cannabis was an risk factor for CVD. We found that marijuana smoking activated inflammatory cytokines implicated in CVD. In silico virtual screening identified genistein, a soybean isoflavone, as a putative CB1 antagonist. Human-induced pluripotent stem cell-derived endothelial cells were used to model Δ9-THC-induced inflammation and oxidative stress via NF-κB signaling. Knockdown of the CB1 receptor with siRNA, CRISPR interference, and genistein attenuated the effects of Δ9-THC. In mice, genistein blocked Δ9-THC-induced endothelial dysfunction in wire myograph, reduced atherosclerotic plaque, and had minimal penetration of the central nervous system. Genistein is a CB1 antagonist that attenuates Δ9-THC-induced atherosclerosis.

The use of new CRISPR tools in cardiovascular research and medicine.

Many novel CRISPR-based genome-editing tools, with a wide variety of applications, have been developed in the past few years. The original CRISPR-Cas9 system was developed as a tool to alter genomic sequences in living organisms in a simple way. However, the functions of new CRISPR tools are not limited to conventional genome editing mediated by non-homologous end-joining or homology-directed repair but expand into gene-expression control, epigenome editing, single-nucleotide editing, RNA editing and live-cell imaging. Furthermore, genetic perturbation screening by multiplexing guide RNAs is gaining popularity as a method to identify causative genes and pathways in an unbiased manner. New CRISPR tools can also be applied to ex vivo or in vivo therapeutic genome editing for the treatment of conditions such as hyperlipidaemia. In this Review, we first provide an overview of the diverse new CRISPR tools that have been developed to date. Second, we summarize how these new CRISPR tools are being used to study biological processes and disease mechanisms in cardiovascular research and medicine. Finally, we discuss the prospect of therapeutic genome editing by CRISPR tools to cure genetic cardiovascular diseases.

Deciphering pathogenicity of variants of uncertain significance with CRISPR-edited iPSCs.

Genetic variants play an important role in conferring risk for cardiovascular diseases (CVDs). With the rapid development of next-generation sequencing (NGS), thousands of genetic variants associated with CVDs have been identified by genome-wide association studies (GWAS), but the function of more than 40% of genetic variants is still unknown. This gap of knowledge is a barrier to the clinical application of the genetic information. However, determining the pathogenicity of a variant of uncertain significance (VUS) is challenging due to the lack of suitable model systems and accessible technologies. By combining clustered regularly interspaced short palindromic repeats (CRISPR) and human induced pluripotent stem cells (iPSCs), unprecedented advances are now possible in determining the pathogenicity of VUS in CVDs. Here, we summarize recent progress and new strategies in deciphering pathogenic variants for CVDs using CRISPR-edited human iPSCs.

CRISPRi/a Screening with Human iPSCs.

Identifying causative genes in a given phenotype or disease model is important for biological discovery and drug development. The recent development of the CRISPR/Cas9 system has enabled unbiased and large-scale genetic perturbation screens to identify causative genes by knocking out many genes in parallel and selecting cells with desired phenotype of interest. However, compared to cancer cell lines, human somatic cells including cardiomyocytes (CMs), neuron cells, and endothelial cells are not easy targets of CRISPR screens because CRISPR screens require a large number of isogenic cells to be cultured and thus primary cells from patients are not ideal. The combination of CRISPR screens with induced pluripotent stem cell (iPSC) technology would be a powerful tool to identify causative genes and pathways because iPSCs can be expanded easily and differentiated to any cell type in principle. Here we describe a robust protocol for CRISPR screening using human iPSCs. Because each screening is different and needs to be customized depending on the cell types and phenotypes of interest, we show an example of CRISPR knockdown screening using CRISPRi system to identify essential genes to differentiate iPSCs to CMs.

microRNA-33 maintains adaptive thermogenesis via enhanced sympathetic nerve activity.

Adaptive thermogenesis is essential for survival, and therefore is tightly regulated by a central neural circuit. Here, we show that microRNA (miR)-33 in the brain is indispensable for adaptive thermogenesis. Cold stress increases miR-33 levels in the hypothalamus and miR-33-/- mice are unable to maintain body temperature in cold environments due to reduced sympathetic nerve activity and impaired brown adipose tissue (BAT) thermogenesis. Analysis of miR-33f/f dopamine-ß-hydroxylase (DBH)-Cre mice indicates the importance of miR-33 in Dbh-positive cells. Mechanistically, miR-33 deficiency upregulates gamma-aminobutyric acid (GABA)A receptor subunit genes such as Gabrb2 and Gabra4. Knock-down of these genes in Dbh-positive neurons rescues the impaired cold-induced thermogenesis in miR-33f/f DBH-Cre mice. Conversely, increased gene dosage of miR-33 in mice enhances thermogenesis. Thus, miR-33 in the brain contributes to maintenance of BAT thermogenesis and whole-body metabolism via enhanced sympathetic nerve tone through suppressing GABAergic inhibitory neurotransmission. This miR-33-mediated neural mechanism may serve as a physiological adaptive defense mechanism for several stresses including cold stress.

Therapeutic genome editing in cardiovascular diseases.

During the past decade, developments in genome editing technology have fundamentally transformed biomedical research. In particular, the CRISPR/Cas9 system has been extensively applied because of its simplicity and ability to alter genomic sequences within living organisms, and an ever increasing number of CRISPR/Cas9-based molecular tools are being developed for a wide variety of applications. While genome editing tools have been used for many aspects of biological research, they also have enormous potential to be used for genome editing therapy to treat a broad range of diseases. For some hematopoietic diseases, clinical trials of therapeutic genome editing with CRISPR/Cas9 are already starting phase I. In the cardiovascular field, genome editing tools have been utilized to understand the mechanisms of diseases such as cardiomyopathy, arrythmia, and lipid metabolism, which now open the door to therapeutic genome editing. Currently, therapeutic genome editing in the cardiovascular field is centered on liver-targeting strategies to reduce cardiovascular risks. Targeting the heart is more challenging. In this review, we discuss the potential applications, recent advances, and current limitations of therapeutic genome editing in the cardiovascular field.

Lionheart LincRNA alleviates cardiac systolic dysfunction under pressure overload.

Recent high-throughput approaches have revealed a vast number of transcripts with unknown functions. Many of these transcripts are long noncoding RNAs (lncRNAs), and intergenic region-derived lncRNAs are classified as long intergenic noncoding RNAs (lincRNAs). Although Myosin heavy chain 6 (Myh6) encoding primary contractile protein is down-regulated in stressed hearts, the underlying mechanisms are not fully clarified especially in terms of lincRNAs. Here, we screen upregulated lincRNAs in pressure overloaded hearts and identify a muscle-abundant lincRNA termed Lionheart. Compared with controls, deletion of the Lionheart in mice leads to decreased systolic function and a reduction in MYH6 protein levels following pressure overload. We reveal decreased MYH6 results from an interaction between Lionheart and Purine-rich element-binding protein A after pressure overload. Furthermore, human LIONHEART levels in left ventricular biopsy specimens positively correlate with cardiac systolic function. Our results demonstrate Lionheart plays a pivotal role in cardiac remodeling via regulation of MYH6.

COVID-19 and cardiovascular disease: from basic mechanisms to clinical perspectives.

Coronavirus disease 2019 (COVID-19), caused by a strain of coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic that has affected the lives of billions of individuals. Extensive studies have revealed that SARS-CoV-2 shares many biological features with SARS-CoV, the zoonotic virus that caused the 2002 outbreak of severe acute respiratory syndrome, including the system of cell entry, which is triggered by binding of the viral spike protein to angiotensin-converting enzyme 2. Clinical studies have also reported an association between COVID-19 and cardiovascular disease. Pre-existing cardiovascular disease seems to be linked with worse outcomes and increased risk of death in patients with COVID-19, whereas COVID-19 itself can also induce myocardial injury, arrhythmia, acute coronary syndrome and venous thromboembolism. Potential drug-disease interactions affecting patients with COVID-19 and comorbid cardiovascular diseases are also becoming a serious concern. In this Review, we summarize the current understanding of COVID-19 from basic mechanisms to clinical perspectives, focusing on the interaction between COVID-19 and the cardiovascular system. By combining our knowledge of the biological features of the virus with clinical findings, we can improve our understanding of the potential mechanisms underlying COVID-19, paving the way towards the development of preventative and therapeutic solutions.

Utility of collagen-derived peptides as markers of organ injury in patients with acute heart failure.

Objective: This study aims to investigate the time-dependent prognostic utility of two fibrosis markers representing organ fibrogenesis (N-terminal propeptide of procollagen III (PIIINP) and type IV collagen 7S (P4NP 7S)) in patients with acute heart failure (HF). Methods: 390 patients with acute HF were dichotomised based on the median value of fibrosis markers at discharge. The primary outcome measure was a composite of cardiac death and HF hospitalisation. Results: P4NP 7S significantly declined during hospitalisation, whereas PIIINP did not. The cumulative 90-day and 365-day incidence of the primary outcome measure was 16.6% vs 16.0% (p=0.42) and 33.3% vs 28.4% (p=0.34) in the patients with high versus low PIIINP; 19.9% vs 13.0% (p=0.04) and 32.3% vs 29.0% (p=0.34) in the patients with high and low P4NP 7S, respectively. After adjusting for confounders, high P4NP 7S correlated with significant excess risk relative to low P4NP 7S for both 90-day and 365-day primary outcome measure (adjusted HR, 1.50; 95% CI, 1.02 to 2.21; p=0.04 and adjusted HR, 1.89; 95% CI, 1.11 to 3.26; p=0.02, respectively), which was driven by significant association of high P4NP 7S with higher incidence of HF hospitalisation. Furthermore, P4NP 7S exhibited an additive value to conventional prognostic factors for predicting 90-day outcome (p=0.038 for net reclassification improvement; p=0.0068 for integrated discrimination improvement). High PIIINP did not correlate with significant excess risk for both 90-day and 365-day outcome. Conclusions: This study suggests a possible role of P4NP 7S in the risk stratification of patients with acute HF.

Cardioprotective Effects of VCP Modulator KUS121 in Murine and Porcine Models of Myocardial Infarction.

No effective treatment is yet available to reduce infarct size and improve clinical outcomes after acute myocardial infarction by enhancing early reperfusion therapy using primary percutaneous coronary intervention. The study showed that Kyoto University Substance 121 (KUS121) reduced endoplasmic reticulum stress, maintained adenosine triphosphate levels, and ameliorated the infarct size in a murine cardiac ischemia and reperfusion injury model. The study confirmed the cardioprotective effect of KUS121 in a porcine ischemia and reperfusion injury model. These findings confirmed that KUS121 is a promising novel therapeutic agent for myocardial infarction in conjunction with primary percutaneous coronary intervention.

Identification of Differential Roles of MicroRNA-33a and -33b During Atherosclerosis Progression With Genetically Modified Mice.

Background Micro RNA (miR)-33 targets cholesterol transporter ATP -binding cassette protein A1 and other antiatherogenic targets and contributes to atherogenic progression. Its inhibition or deletion is known to result in the amelioration of atherosclerosis in mice. However, mice lack the other member of the miR-33 family, miR-33b, which exists in humans and other large mammals. Thus, precise evaluation and comparison of the responsibilities of these 2 miRs during the progression of atherosclerosis has not been reported, although they are essential. Methods and Results In this study, we performed a comprehensive analysis of the difference between the function of miR-33a and miR-33b using genetically modified mice. We generated 4 strains with or without miR-33a and miR-33b. Comparison between mice with only miR-33a (wild-type mice) and mice with only miR-33b (miR-33a-/-/miR-33b+/+) revealed the dominant expression of miR-33b in the liver. To evaluate the whole body atherogenic potency of miR-33a and miR-33b, we developed apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice and apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice. With a high-fat and high-cholesterol diet, the apolipoprotein E-deficient/miR-33a-/-/miR-33b+/+ mice developed increased atherosclerotic plaque versus apolipoprotein E-deficient/miR-33a+/+/miR-33b-/- mice, in line with the predominant expression of miR-33b in the liver and worsened serum cholesterol profile. By contrast, a bone marrow transplantation study showed no significant difference, which was consistent with the relevant expression levels of miR-33a and miR-33b in bone marrow cells. Conclusions The miR-33 family exhibits differences in distribution and regulation and particularly in the progression of atherosclerosis; miR-33b would be more potent than miR-33a.

An in Vivo miRNA Delivery System for Restoring Infarcted Myocardium.

A major challenge in myocardial infarction (MI)-related heart failure treatment using microRNA is the efficient and sustainable delivery of miRNAs into myocardium to achieve functional improvement through stimulation of intrinsic myocardial restoration. In this study, we established an in vivo delivery system using polymeric nanoparticles to carry miRNA (miNPs) for localized delivery within a shear-thinning injectable hydrogel. The miNPs triggered proliferation of human embryonic stem cell-derived cardiomyocytes and endothelial cells (hESC-CMs and hESC-ECs) and promoted angiogenesis in hypoxic conditions, showing significantly lower cytotoxicity than Lipofectamine. Furthermore, one injected dose of hydrogel/miNP in MI rats demonstrated significantly improved cardiac functions: increased ejection fraction from 45% to 64%, reduced scar size from 20% to 10%, and doubled capillary density in the border zone compared to the control group at 4 weeks. As such, our results indicate that this injectable hydrogel/miNP composite can deliver miRNA to restore injured myocardium efficiently and safely.

MiR-33a is a therapeutic target in SPG4-related hereditary spastic paraplegia human neurons.

Recent reports, including ours, have indicated that microRNA (miR)-33 located within the intron of sterol regulatory element binding protein (SREBP) 2 controls cholesterol homeostasis and can be a potential therapeutic target for the treatment of atherosclerosis. Here, we show that SPAST, which encodes a microtubule-severing protein called SPASTIN, was a novel target gene of miR-33 in human. Actually, the miR-33 binding site in the SPAST 3'-UTR is conserved not in mice but in mid to large mammals, and it is impossible to clarify the role of miR-33 on SPAST in mice. We demonstrated that inhibition of miR-33a, a major form of miR-33 in human neurons, via locked nucleic acid (LNA)-anti-miR ameliorated the pathological phenotype in hereditary spastic paraplegia (HSP)-SPG4 patient induced pluripotent stem cell (iPSC)-derived cortical neurons. Thus, miR-33a can be a potential therapeutic target for the treatment of HSP-SPG4.

Hepatokine α1-Microglobulin Signaling Exacerbates Inflammation and Disturbs Fibrotic Repair in Mouse Myocardial Infarction.

Acute cardiac rupture and adverse left ventricular (LV) remodeling causing heart failure are serious complications of acute myocardial infarction (MI). While cardio-hepatic interactions have been recognized, their role in MI remains unknown. We treated cultured cardiomyocytes with conditioned media from various cell types and analyzed the media by mass spectrometry to identify α1-microglobulin (AM) as an Akt-activating hepatokine. In mouse MI model, AM protein transiently distributed in the infarct and border zones during the acute phase, reflecting infiltration of AM-bound macrophages. AM stimulation activated Akt, NFκB, and ERK signaling and enhanced inflammation as well as macrophage migration and polarization, while inhibited fibrogenesis-related mRNA expression in cultured macrophages and cardiac fibroblasts. Intramyocardial AM administration exacerbated macrophage infiltration, inflammation, and matrix metalloproteinase 9 mRNA expression in the infarct and border zones, whereas disturbed fibrotic repair, then provoked acute cardiac rupture in MI. Shotgun proteomics and lipid pull-down analysis found that AM partly binds to phosphatidic acid (PA) for its signaling and function. Furthermore, systemic delivery of a selective inhibitor of diacylglycerol kinase α-mediated PA synthesis notably reduced macrophage infiltration, inflammation, matrix metalloproteinase activity, and adverse LV remodeling in MI. Therefore, targeting AM signaling could be a novel pharmacological option to mitigate adverse LV remodeling in MI.