**4. Myocardial remodeling during VAD support**

The myocardium consists of cardiomyocytes, composing nearly 56% of the adult heart, fibroblasts (27%), endothelial cells (7%), smooth muscle cells (10%), and various immune cells that transiently reside in the ECM [63]. These cell types are important in preserving normal cardiac function and morphology. The cells interact with each other using reciprocally secreted auto and paracrine factors, secretion of which is regulated by numerous molecules-messengers involving integrins, ET-1, BMPs, PECAM-1, VE-cadherin, VEGF, and TGFβ [64, 65]. Engineered heart tissue (EHT), created *in vitro* by seeding decellularized porcine myocardial sections with primary cardiomyocytes and fibroblasts isolated from neonatal rat ventricular myocardium or with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC), is a novel platform to study cardiac remodeling [66]. Characterization of EHTs demonstrated gradual normalization of stress-free tissue length after mechanical unloading and suggested that actomyosin contraction in cardiomyocytes and activity of fibroblasts may play crucial roles in reverse remodeling after mechanical unloading.

## **4.1 Cardiac fibroblasts and fibrosis**

Cardiac fibrosis in the failing heart is a final product of a series of biomechanical, molecular, and cellular changes that causes an imbalanced increase in ECM production and decreased ECM degradation [67]. The resultant increase in ECM deposition is accompanied by inflammatory and fibrotic scar formation in the interstitial and perivascular areas of the myocardium, interfering with the normal array of cardiomyocytes along with the disturbing supply of oxygen and nutrients to the myocardium. Moreover, cardiac fibrosis triggers further pathological remodeling and functional decline of the heart [68]. According to Tseng *et al.*, an increase in inflammation and fibrosis in the failing heart was associated with an increase in sST2 levels [58]. Synthesis and degradation of collagens I and III are highly regulated processes in human cardiac ECM. Collagen I is a major collagen component establishing the myocyte-collagen matrix, while collagen III contributes to elasticity, and changes in content may influence LV stiffness and size [69]. In HF, predominantly increased accumulation of collagens I and III in ECM results in cardiomyocyte injury, cardiac fibrosis, and the release of collagen-derived peptides into circulation [70]. Bruckner *et al.* recorded a significant decrease in intracardiac TNF-α, collagen I (by 66%), and collagen III (62%) in post-VAD myocardial samples of 18 patients compared to their pre-VAD levels [71]. They also found a decrease in cardiomyocyte size by 26% at post-VAD, demonstrating favorable reverse remodeling in cardiac hypertrophy.

Insulin-like growth factor I (IGF-1), released preferentially from cardiac fibroblasts, functions to negatively regulate atrophy and apoptosis, and stimulate cardiac repair by interacting with stromal cell-derived factor (SDF) [19]. SDF induces IGF-1 expression in cardiac myocytes *in vitro*. Patients with VAD support combined with β2-AR agonist clenbuterol have shown elevated *IGF-1* mRNA at the time of VAD explantation relative to the time of LVAD implantation [72].

## **4.2 Extracellular matrix remodeling**

Matrix metalloproteinases (MMPs) degrade the ECM, while tissue inhibitors of MMPs (TIMPs) prevent the ECM degradation during repair process of damaged

*Myocardial Remodeling with Ventricular Assist Devices DOI: http://dx.doi.org/10.5772/intechopen.110814*

tissues and cells. There are four variants of TIMPs that selectively inhibit different types of MMPs [73]. Typically, TIMP1 and MMP1 are increased in patients with deteriorating HF [74]. The increased ratio of MMP-1 to TIMP-1 in DCM has been shown to be almost normalized after LVAD, favoring decreased collagen degradation [17]. Felkin *et al.* found that high myocardial MMP1 and MMP8 expression is associated with high collagen content and increased IL-6 and IL-1β expression in HF patients requiring VAD support [75]. After VAD support, expression of *MMP-2* mRNA and active MMP-2 protein has been shown to be significantly increased compared to pre-VAD (P < 0.01), which was associated with a reduction of collagen IV content in the cardiomyocyte basement membrane. Furthermore, this was associated with a decrease in the thickness of cardiomyocyte membrane as revealed by electron microscopy [76]. MCS support increases collagen cross-linking and the ratio of collagen I to III in the heart as a result of decreased tissue MMP-1-to-TIMP-1 ratio and increased myocardial Ang I and II levels that stimulate ECM synthesis [17]. Therapy with ACEI drugs decreased Ang II levels and myocardial collagen content, resulting in enhanced myocardial recovery during VAD support [40]. In elderly patients with end-stage HF, VAD therapy is associated with decreased collagen turnover and cross-linking and increased tissue Ang II, whereas combined VAD and ACEI therapy normalizes LV end-diastolic pressure-volume relationships [77].

#### **4.3 Endothelial and vasculature remodeling**

A gene ontology (GO) analysis implicated endothelial to mesenchymal transition (EndoMT) and *vice versa* (MEndoT) pathways in human end-stage HF based on dual expressed VE-Cadherin endothelial and FSP-1 mesenchymal markers [78]. Gene expression analysis of 19 paired pre-VAD and post-VAD human heart samples by Hall *et al.* revealed differential expression of neuropilin-1, *FGF9*, *Sprouty1*, SDF1, and endomucin, the genes involved in the regulation of vascular networks [79]. In addition, a significant downregulation of GATA-4 binding protein, a critical mediator of myocyte hypertrophy, was observed in these heart samples following mechanical unloading. Drakos *et al.* observed an increased density of endothelial cells by 33% and decreased microvascular lumen area (36%) in post-VAD *vs* pre-VAD myocardial samples of patients with chronic HF (n = 15). This was associated with the activation of endothelial cells evidenced by ultrastructural and immunohistochemical analysis [80]. In agreement with these findings, a significant increase in interstitial and total collagen content without structural changes in cardiomyocytes was suggestive of increased fibrosis accompanied by regression of cardiomyocyte hypertrophy.

#### **4.4 Reversal of cardiac hypertrophy**

The myocardium is typically subjected to three types of mechanical loading during every heartbeat, including cyclic stretch, static stretch, and shear stress, generated by blood flow and an increase in chamber volume and pressure. Cardiomyocytes are sensitive to mechanical stress, which is transduced to molecular transduction signaling by biomechanical sensors. Comparative analysis of cardiomyocyte size in pre- and post-VAD patients demonstrated a decrease of 26% (33.1 ± 1.32 to 24.4 ± 1.64 μm, P < 0.001) in all patients studied [71]. Long-term VAD support resulted in a 28% reduction in myocyte volume, 20% reduction in cell length, 20% reduction in cell width, and 32% reduction in cell length-to-thickness ratio [81]. Another study examined the effects of continuous-flow VAD on cardiomyocyte size and demonstrated

that cardiomyocyte cross-sectional area decreased after VAD, but not beyond that of normal donor hearts [82]. Electron microscopy, cardiac glycogen content, and echocardiographic assessment also did not suggest myocardial atrophy in post-VAD patients. Consistent with these findings, no upregulation of pro-atrophic genes and proteins of the ubiquitin-proteasome system (UPS) and no t-tubule pathologies have been demonstrated.

Myostatin (also called gdf-8) is a potent inhibitor of skeletal muscle growth from the TGF-β family and is secreted by cardiac muscle and adipocytes in response to pathological stress, such as myocardial infarction or obesity [83]. Myostatin has been shown to mediate the regression of cellular hypertrophy after unloading with LVAD support [84]. The nuclear factor (NF)-κB superfamily of transcription factors carries out broad functions by regulating immune cell maturation, cell survival, and inflammation in many cell types [85]. In the heart, NF-κB is shown to be cardioprotective during acute injury, however, prolonged activation of NF-κB enhances the release of TNF-α, IL-1, and IL-6 cytokines, triggering chronic inflammation, hypertrophy, and cell death [86, 87]. After VAD support, the NF-κB DNA-binding activity decreases in failing human hearts and this process has been associated with a decrease in cardiomyocyte diameter [88].

Several kinases such as mitogen-activated protein kinase (MAPK or MEK), ERK (extracellularly regulated kinase), AKT (protein kinase B, PKB), GSK-3b (glycogen synthase kinase-3 beta), JNK (c-Jun NH2-terminal kinase) and p38 are involved in the development of cardiac hypertrophy *via* kinase-mediated signal transduction pathways [89]. After VAD support, significantly decreased activities of ERKs and AKT were seen in failing hearts, while the activity of GSK-3β was increased [90]. These changes were associated with a decrease in TUNEL-positivity and myocyte diameter. The disparity in the regulation of MAPK activity with a concomitant decrease in ERK and JNK1/2 activities and an increase in p38 activity after VAD support has been also reported [91].

Osteopontin (OPN) is a pleiotropic extracellular signal-regulated bone sialoprotein. Expression and activity of OPN are increased in myocardial tissues in accordance with the severity of HF [92]. Levels of *OPN* mRNA in heart biopsy specimens decreased significantly after VAD support, while OPN protein remained intact [93]. Moreover, VAD support induced a decrease of OPN levels in the plasma of some patients with VAD support, whereas OPN plasma levels were reduced significantly in all patients after a heart transplant.

#### **4.5 Cardiomyocyte apoptosis**

While MCS improves the survival of end-stage HF patients by reversing many biological processes activated during progression of HF, the reports on modulation of apoptotic cell death in response to VAD remain controversial. Prescimone *et al.* found a significant increase of Bax (pro-apoptotic), Bcl-2 (pro-apoptotic), and Hsp72 (antiapoptotic) molecules and a mild increase in cardiac caspase (Casp)-3 activity in post-VAD hearts compared to pre-VAD, suggesting involvement of mitochondria in apoptotic signaling [94]. The authors also found an increase in Casp-1 after VAD implant in HF patients and lack of apoptotic nuclei [95]. Conversely, Francis *et al.* found Bcl-2 being downregulated after VAD implant [96]. Another study found no significant differences in Bcl-2, while autophagy markers such as beclin-1, autophagyrelated gene 5 (Atg5), and microtubule-associated protein-1 light chain-3 (LC3) were all significantly decreased in response to unloading [97]. Moreover, Bedi *et al.*

*Myocardial Remodeling with Ventricular Assist Devices DOI: http://dx.doi.org/10.5772/intechopen.110814*

observed a highly variable expression of Fas among patients who had undergone MSC therapy [98]. Fas, also called Apo-1 or CD95, is a membrane receptor recognizing Fas ligand (Fas-L) and Fas/Fas-L coupling initiates an apoptotic cell death through the activation of caspase cascade in the heart [99]. Although apoptotic DNA fragmentation was attenuated in the myocardium, expression of antiapoptotic *Bcl-XL* and *FasExo6Del/Fas* genes was dependent on the duration of MCS [100]. Overall, no significant differences in number of TUNEL-positive cells between pre- and post-VAD samples have been reported by several groups [96, 97, 101, 102].

Abnormal Ca2+ cycling in HF triggers activation of UPS with an increase of binding immunoglobulin protein (BiP), eukaryotic initiation factor (eIF2α), and X-box binding protein 1 (XBP1) [103]. MCS support significantly decreases the levels of BiP and XBP1 and phosphorylation of eIF2α [104]. Moreover, a decrease in apoptosis observed during short-term VAD support has been associated with a decrease in phosphorylation of SMAD2 (mothers against decapentaplegic homolog 2), however, a long-term VAD support increased apoptosis and fibrosis in the heart *via* enhanced SMAD2 signaling and increased phosphorylation of HDAC4 (histone deacetylase 4) [101].

#### **4.6 Cardiomyocyte regeneration**

Diploid cardiomyocytes that are abundant in animal heart have a substantial capacity for cardiac repair and regeneration [105]. In human failing heart, polyploidy of cardiomyocytes is often observed as a precondition of heart hypertrophy [106], suggesting that cardiomyocyte polyploidization in HF may be associated with regeneration [107]. A study by Wohlschlaeger *et al.* demonstrated a marked reduction in the size of cardiomyocyte nuclei and in ratios between number of nuclei and cardiac myocytes after implantation of VAD [108]. They also reported a significant decrease in DNA content and reduction of polyploid cardiomyocytes in 23 myocardial samples studied after VAD, suggesting a decline in protein synthesis. On the contrary, an increase in the number of diploid cardiomyocytes was seen by other groups in post-VAD samples [108]. The decrease in polyploidy and increase in diploidy in response to MCS suggested an abundance of diploid cardiomyocytes going through cell cycle progression with the completion of mitosis or increase in stem cells. Prolonged MCS unloading increased the number of cardiomyocytes positive for phosphorylated histone H3 and Aurora B and this was associated with a decrease in cardiomyocyte size and mitochondrial content [109].

#### **4.7 Transcriptional changes during VAD therapy**

Accumulating evidence shows that the changes in transcriptome and metabolome profiles associated with HF persist in the reverse-remodeled myocardium despite apparent normalization on organ and cellular levels [110]. To identify transcriptional adaptations in failing and VAD-supported hearts, a comprehensive transcription analysis was performed in 199 human myocardial samples from nonfailing, failing, and VAD-supported human hearts. Although over 3088 transcripts exhibited alterations in HF samples, the number of differentially expressed genes (DEGs) with greater than or equal to a 2-fold difference was insignificant between HF and post-VAD samples, suggesting that many HF-associated transcriptional changes may have a limited role in regulating cardiac structure and function [111]. Significant elevation in myocardial arginine/glycine amidinotransferase (AGAT) expression is observed in

HF patients and myocardial *AGAT* is one of the DEGs that had a significant decrease during recovery [112]. In HF patients recovering after combination therapy, levels of *AGAT* mRNA decreased by 4.3-fold [P < 0.001] and 2.7-fold [P < 0.005] in VAD combined and VAD alone groups compared to donors, respectively, and *AGAT* levels returned to normal after recovery. These data highlighted the involvement of elevated local creatine synthesis specific to HF and its reversal during recovery. The genetic response of pediatric myocardium to MCS is distinct with approximately 40% of DEGs compared to adult hearts with VAD support, highlighting the importance of understanding features of reverse remodeling specific to pediatric myocardium to improve clinical strategies and LVAD management in children [113].

In long-term analysis of gene expression, data of patients studied for an average of 3.8 years post-explant revealed a significant association of integrin signaling and its downstream EPAC2 (exchange protein activated by cyclic-AMP2) during recovery of ventricular function by combined LVAD and clenbuterol therapy [20]. Downregulation of EPAC2 that regulates calcium involving cAMP pathway was associated with improvements in cardiac contractility and metabolism [114].

#### **4.8 miRNAs in response to LVAD therapy**

MicroRNAs (miRNAs) are small, endogenous noncoding RNAs that regulate posttranscriptional processes by repressing the translation of targeted protein-coding genes *via* binding to the 3' UTRs of mRNAs [115]. Therefore, cardiac miRNAs and circulating miRNAs (c-miRNAs) are promising biomarkers for HF diagnosis and prognosis [116]. Comprehensive microarray profiling of miRNAs and mRNAs, comparing myocardial specimens from adults with end stage HF with VAD and nonfailing hearts, showed upregulation of 28 miRNAs with almost normalization of miRNA profiles by VAD treatment [117]. Cardiac miRNAs have also been compared in 13 HF children at pre-VAD and at the moment of heart transplant (post-VAD) by next-generation sequencing [118]. The investigators found hsa-miR-199b-5p, hsamiR-19a-3p, and hsa-miR-1246 being differentially expressed at post-VAD compared to that at pre-VAD. The candidate targets of those differentially expressed miRNAs were sarcomeric troponins showing significantly higher post-VAD when compared with pre-VAD values, suggesting that miRNAs can be therapeutically targeted to improve heart function in pediatric HF. Levels of nine c-miRNAs were downregulated and four c-miRNAs were upregulated in the post-VAD samples *vs* pre-VAD levels [119]. In particular, the c-miR-409-3p has been shown to regulate coagulation factor 7 (F7) and F2, suggesting a role of c-miRNA-409-3p in thrombotic events during MCS.

#### **4.9 Beta-adrenergic receptor remodeling**

Myocardial beta-adrenergic receptor (β-AR) signaling is severely diminished in failing heart due to increase in phosphorylation of agonist-occupied β-ARs by GRK2 [120, 121]. In chronic HF, VAD support leads to the restoration of cardiac β-AR signaling *via* the reduction of myocardial GRK2 expression and activity [122]. Unloading with VAD normalizes the ability of cardiac muscle to respond to SNS stimulation, reversing the downregulation of β-ARs [123]. Both types of devices, continuous-flow and pulsatile, decreased the expression and activity of GRK2 and normalized neurohormonal homeostasis disturbed with HF [124]. In pediatric HF, VAD treatment also resulted in the recovery of total β-AR and β1-AR expressions and reversal of several pathologic processes in the heart [125].
