**7. Transgenic methods in cardiotoxicity research**

Cardiotoxicity develops later as a result of stress, chronic diseases, cancer therapies, and immunotherapies in general, but it can occur *in vivo* and *in vitro* if the necessary genetic facilities are available. Mimicking proteomic and genetic disorders, in particular, can cause cardiomyopathies, cardiac transmission problems, and a variety of heart diseases. These models are created using a variety of transgenic methods. The method to be used in a study also varies depending on the pathophysiological mechanisms being studied.

The main methods for creating transgenic models are TALEN (transcription activation-like effector nucleases), CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9), sleeping beauty, piggyBac (PB), pronuclear microinjection (PMI), viral transgenesis, RNAi (RNA interference), and hiPSC (human induced pluripotent stem cells). Numerous studies are currently being conducted in which these methods are being used not only to create pathological models, but also to develop new treatment methods.

Following are a few examples of *in vivo* models of *transgenic methods* based on the literature;

Genetically modified cell lines are frequently used due to their ease of maintenance and manipulation. Unfortunately, cell culture results do not always accurately reflect human physiology.

hPSCs (human pluripotent stem cells) can be genetically reprogrammed and converted into iPSCs (induced pluripotent stem cells), which can then be used for functional analysis in a variety of studies. Isogenic hPSC lines derived from ZFNs (zinc-finger nucleases), TALENs, or CRISPR/Cas9 add to our understanding of a variety of cardiovascular diseases, particularly cardiomyopathies and electrophysiological disorders [136].

Disease models can be created in a matter of weeks using current gene-editing methods, including knock-in, knock-out or mutation of certain genes in experimental animals, such as rats. CRISPR/Cas9 systems have recently been used to edit DNA more efficiently based on direct injection of genome editing machines into single-celled mouse embryos. Various cardiovascular diseases have been studied in mammalian models, including but not limited to rats, rabbits, and pigs, to date. Even in zebrafish models alone, various cardiac development, cardiac regeneration, vascular development, and hereditary cardiomyopathy were studied. In addition to single-cell embryo studies, it has been demonstrated that somatic *in vivo* genome

#### *Experimental Model of Cardiotoxicity DOI: http://dx.doi.org/10.5772/intechopen.101401*

editing studies in adult animals using CRISPR/Cas9 delivery via viral vectors and lipid nanoparticles are possible. Adenovirus and adeno-associated viruses (AAVs) are viral vectors that can be used to efficiently present genetic material in adult animals [136].

Several transgenic models of inherited arrhythmias have been described. Long-QT syndrome (LQTS-1/2/3/8/15), Atrial fibrillation (AF), Brugada Syndrome, and Catecholaminergic polymorphic ventricular tachycardia (CPVT) are just a few of the inherited arrhythmias that have been created using the hPSC [137].

LQTS: Ion channel genes KCNQ1 and KCNH2 with dominant-negative mutations that cause LQTS Type 1 and 2, respectively, were successfully integrated into the AAVS1 locus, which is considered a safe haven using ZFN technology and created an LQTS model in a study on iPSC-CMs (iPSC-Cardiomyocytes). The potential duration of action in regulated iPSC-CMs was significantly longer than in control cells that were not regulated as characteristic phenotypes of the long-QT syndrome, according to patch-clamp results [138].

Dermal fibroblasts from two people in a family with LQTS-1 and two healthy people were infected with retroviral vectors encoding the human transcription factors OCT3/4, SOX2, KLF4, and c-MYC and converted into hPSCs in another study [139]. Another research examined the therapeutic potential of new IKs activators in LQTS using dermal fibroblasts differentiated into hPSCs [140].

To create the LQT15 model, an electrophysiological study mimicked mutations in CALM2 from the CALM1, CALM2, and CALM3 genes that encode Calmodulin, a Ca2+ sensor on hPSC [141].

Transgenic models are also frequently used in studies on other types of Long QT Syndrome [142–147].

Brugada Syndrome: a large class of arrhythmias caused by a mutation in SCN5A (sodium voltage-gated channel alpha subunit 5), a cardiac sodium channel gene. Brugada Syndrome, Bradycardia, Atrioventricular (AV) Blocks, and Ventricular Fibrillation are few examples (VF).

A scn5a+/− (heterozygous knock-out) transgenic rat model was created for therapeutic evaluation, and the Brugada Syndrome phenotype was mimicked in a study [148]. The models created in a study targeting the SCN5A gene demonstrate cardiac conduction slowdowns and ventricular tachycardia (VT) [149].

In an *in vitro* study, dermal fibroblasts from two patients with Brugada Syndrome and two healthy individuals were differentiated to iPSC-CMs using Sendai virus (SeV) [150].

CPVT: The mutation in the RYR2 gene, which encodes the cardiac ryanodine receptor and is associated with CPVT cases, is modeled on hiPSCs differentiated from dermal fibroblasts via retrovirus [151].

The main cardiomyopathies investigated by developing transgenic models are dilate cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), and restrictive cardiomyopathy (RCM).

Dilate cardiomyopathy: A study focused on striated muscle alpha-tropomyosin (α-TM), an essential thin filament protein involved in the pathogenesis of both dilate and hypertrophic cardiomyopathy. The Glu54Lys mutation, one of two prominent mutations in this protein (Glu40Lys and Glu54Lys), was expressed in transgenic rats. In echocardiography examinations, the successful dilate cardiomyopathy phenotype was observed [152].

In an iPSC-based study, an iPSC-CMs model was created from dermal fibroblasts from patients in a DCM family with a spot mutation (R173W) on the gene encoding cardiac troponin T (cTnT), a sarcomric protein [153].

In a study targeting Leiomodin proteins, which play a vital role in muscle thin filament length, the Lmod2 mutation was modeled in rats using the piggyBac transposon. It has been demonstrated that these rats with ventricular arrhythmias and increased postnatal mortality have a typical DCM phenotype [154].

In a recent study, a knock-in mouse model was created in which endogenous genes were altered to include the deletion of three base pairs encoded in cardiac troponin T for K210 in dilate cardiomyopathy patients [155].

In a study focusing on the titin protein, preserved blood samples from three DCM patients with the dominant TTN mutation were reprogrammed, and highquality iPSC clones were expanded, differentiated, and enriched by metabolic selection to create a culture with >% iPSC-CMs. To analyze the effects of titin mutation on sarcoma structure, the iPSC-CMs method was created [156].

Hypertrophic cardiomyopathy: A transgenic hypertrophic cardiomyopathy model based on the erasure of 468–527 amino acids, which is bridged by the addition of a point mutation (G1445A) and 9 nonmyosin amino acids (SerSerLeuProHisLeuLysLeu) resulting in Arg403Gln, was created in a study targeting two mutations in the myosin heavy chain gene. Transgenic sequences were shown to be extracted from prokaryotic vector sequences, purified on agarose gels, and injected into the pronuclei of fertilized rat eggs [157].

In a comparative study, two different mutations (R92Q and E163R) in the TNNT2 gene, which encodes cardiac troponin T, were used. Echocardiography showed left ventricular hypertrophy, increased contractility, and diastolic dysfunction in both models. These phenotypes, however, were found to be more pronounced in R92Q mice [158]. HCM rat models with these two mutations had previously been described [159, 160].

In a study on the actin protein, the molecular mechanisms of apical hypertrophic changes were tried to be clarified in rat models created by a mutation (E99K) in the cardiac actin gene (ACTC). The phenotypic investigation of the created models was carried out using echocardiography, electrocardiography, magnetic resonance imaging, and a conductance catheter [161].

In a study examining the central role of calcium-related disorders in the disease pathogenesis in HCM, the iPSC-CMs model was created using fibroblasts derived from HCM patients with the Arg663His mutation in the MYH7 gene, which encodes the heavy chain of myosin [162].

A recent study used transgenic mouse models with the cardiac troponin-I mutation (cTnIGly146) to try to demonstrate that the exosomally derived Y-RNA fragment could regress the HCM clinic [163].

Restrictive cardiomyopathy: Mogensen and colleagues first described six different TnI C-terminal mutations linked to restrictive cardiomyopathy (L144Q, R145W, A171T, K178E, D190G, and R192H) in 2003 [164, 165].

In a study in RCM examining troponin mutations, transgenic rat models were explained by focusing on cTnI193His (R193H) and R145W mutations [165].

Numerous transgenic methods from the past to the present have developed *in vivo* and *in vitro* models that are commonly utilized to describe the pathophysiology of the disease. Aside from these studies, which look at the phenotypic manifestations of disease molecular mechanisms, the fact that transgenic approaches give hope for the treatment of numerous diseases has sparked a lot of research. Transgenic methods have therapeutic potential, particularly for cardiotoxic conditions induced by proteomic and genetic disorders that cannot be treated with drugs.

Besides these, R14del mutation in the phospholamban gene (PLN) is another important cause of cardiomyopathy development. Correcting this mutation on induced cardiomyocytes (iCMs) using the TALEN vector method resulted in improved Ca2+ handling, hypertrophic phenotype regression, and homogeneous

#### *Experimental Model of Cardiotoxicity DOI: http://dx.doi.org/10.5772/intechopen.101401*

reticular distribution of phospholamban [166]. In another study using 3D human engineering heart tissue technology, the PLN R14del mutation disrupts cardiac contractility; however, the contractile function is restored in this model with TALEN-mediated genetic correction [167].

A study of the human embryo revealed that the heterozygous MYBPC3 mutation associated with HCM is corrected by an endogenous, germline-specific DNA repair response using the homology-directed repair (HDR) with CRISPR/Cas9, an up-todate genome editing method [168].

Long QT syndrome is caused by three major mutations: KCNQ1 (LQT1), KCNH2 (LQT2), and SCN5A (LQT3) [169]. These mutations disrupt potassium flow or prolong inward storage flows, prolonging the potential duration of cardiac action and reducing repolarization reserve. According to a study that modeled the E637K mutation in KCNH2, potassium currents were also corrected to relatively close levels after transfection with an optimized siRNA targeted against the mutant potassium channel [170].

A study of CASQ2 knockout rat models reported that exogenous CASQ2 expression, provided via intraperitoneal adeno-associated virus serotype 9 (AAV9) vector, improves CPVT phenotype by correcting arrhythmogenic phenotype and ultrastructural abnormalities [171].

In addition to these models, studies on the therapeutic potentials of transgenic studies for heart failure (HF) have been conducted. An earlier study revealed that overexpression of SERCA2a in cells isolated from HF patients improved myocyte contractile function [172]. Furthermore, genetic therapies for Duchenne muscular dystrophy (DMD), which causes HF with cardiomyopathy, were emphasized. Previous research tried to improve dystrophin mutations using transgenic methods such as ZFN, TALEN, and meganucleases [173–177]. Even more recently, the CRISPR/Cas9 method was used with AAV to treat mice with dystrophin deficiency induced by a spontaneous mutation in the dystrophin gene [178]. The aim was to remove exon 23 from the dystrophin gene, provide partial functional dystrophin expression in skeletal myofibers and heart muscle, and increase muscle strength [179–181].
