AUDITORÍA DE CUENTAS ANUALES

A) HTR2A

The action of the neurotransmitter serotonin within the central nervous system is well-studied

(Berger, Gray, & Roth, 2009). In contrast, patho-physiological functions of serotonin on the

heart that involve regulation of heart rate, valve pathology, myocardial remodelling and during

cardiogenesis are less certain (Berger et al., 2009). Serotonin acts through a wide array of 5-

hydrotryptammine (5-HT) receptors; 5-HT1-7 that are targets of numerous neuropsychiatric

drugs used in management of psychiatric illnesses such as depression and psychosis (McCorvy

& Roth, 2015). The function of 5-HT2A receptor in cardiac hypertrophy is not well understood,

however a pro-hypertrophic role upon serotonin stimulation is suggested.

HTR2A was up-regulated in papillary muscle and cardiomyocytes isolated from the left

ventricle of rats subject to myocardial hypertrophy by transverse banding of the ascending

aorta (Brattelid et al., 2007). In addition, an increase in positive inotropic response on exposing

the papillary muscle to serotonin; 5-HT2A, which was directly proportional to the degree of

hypertrophy was reported (Brattelid et al., 2007). Activation of this receptor is therefore

speculated to improve cardiac function with reduced energy demand during pressure-overload

induced cardiac hypertrophy (Brattelid et al., 2007). Similar conclusions were reported in a rat

model of acute congestive heart failure (Qvigstad, 2005). A complementary study showed a

25-fold increase in expression of HTR2A in SHR compared to wild-type rats, suggesting a role

in cardiac remodelling associated with hypertension (Ayme-Dietrich et al., 2015). Additionally,

the role of HTR2A polymorphism to reveal the influence of serotonin on cardiovascular disease

has been investigated on a few occasions. For instance, T102C polymorphism in the 5-HT2A

receptor is potentially an independent risk factor for primary hypertension (Liolitsa et al.,

2001). Also, the severity of depression in men diagnosed with coronary heart disease was

shown to vary based on the presence of a specific 1438A/G HTR2A genotype (Golimbet et al.,

2014).

Our study is the first to show expression of the 5-HT2A receptor is up-regulated in the heart of

2-day old HHR that have cardiac hypertrophy. In addition, the study is the first to demonstrate

expression of HTR2A is down-regulated in H9C2-1 after transfection with miR-34a mimic. The

finding is indicative of the gene being regulated by the miR-34a. An exploratory research into

the biogenesis of microRNAs in cortical neural tissue in relation to schizophrenia reported a

probable miR-107, mir-195 and miR-15b mimic interaction with the gene (Beveridge et al.,

2010). Previous results from this study suggest that the expression of miR-34a is up-regulated

in hearts of 2-day old HHR. Therefore, the increase in expression of the microRNA is perhaps a

compensatory response to increased expression of HTR2A.

B) SGPP1

Sphingolipids are lipid mediators found in cardiomyocytes that respond to various extra-

cellular stimuli resulting in generation of sphingosine-1-phosphate (S1P) and ceramide

followed by up-regulation of key intra-cellular pathways such as RAS-MAPK and PI3K-AKT

(Levade et al., 2001). SGPP1 encodes for a phosphatase that is found primarily on intracellular

membranes of the endoplasmic reticulum that metabolises S1P into ceramide (Le Stunff et al.,

2002). Increased levels of ceramide are associated with apoptosis and arrest of the cell cycle,

whereas increased S1P activity induces cell proliferation and protects cells from apoptosis

(Borodzicz et al., 2015). The function of sphingolipids and related metabolites in cardio-

vascular disease such as myocardial infarctions, hypertension and stroke is poorly understood

while their role in cardiac hypertrophy is yet to be determined (Borodzicz et al., 2015)

This study is the first to report up-regulation of SGPP1 in HHR neonates that exhibit cardiac

hypertrophy. It is implied increased apoptosis and reduced cell proliferation during foetal life

results in reduced number of cardiomyocytes, which is followed by compensatory cardiac

hypertrophy (Porrello et al., 2009). The cardiac changes observed in 2-day old HHR may

potentially be a consequence of increased SGPP1 expression (RT-PCR validation), which

warrant further investigation into the potential role of the gene in development of cardiac

hypertrophy. Furthermore, transfection of H9C2-1 with a mimic of miR-34a resulted in a 1.80-

fold decrease in expression of SGPP1 that was statistically insignificant potentially due to

increased variability seen in results from RT-PCR. Thus, the results are inconclusive, however

further investigation into the potential microRNA-mRNA relation is merited to verify the results

of the bioinformatics analysis.

C) GANC

GANC is proposed to be a member of glycosyl hydrolase gene family 31 and encodes for neutral

α-glucosidase C, an enzyme believed to be involved in glycogen metabolism (Hirschhorn, Huie,

& Kasper, 2002). The gene is currently understudied, while functional remarks in Rattus

norvegicus are primarily inferred from computational annotations that include alpha-1,4-

glucosidase activity, hydrolase activity and carbohydrate metabolic process (National Center

for Biotechnology Information, U.S. National Library of Medicine). To the best of the authors

knowledge, this study is the first to demonstrate down-regulation of GANC in hearts of 2-day

old HHR and is thus a significant discovery of biological function of the gene. Further, it is worth

mention that during cardiac hypertrophy there is a switch from fatty-acid oxidation to

glycolysis, a similar state of energy metabolism is seen in physiological cardiac growth during

foetal life (Bernardo et al., 2010). Hence, the function of neutral α-glucosidase C in altered

states of energy metabolism, which is characteristic of pathological cardiac hypertrophy

remains to be explored. In addition, the bioinformatics prediction analysis suggests GANC is

potentially a target gene for miR-351 and miR-490*. RT-PCR validation of GANC expression

following transfection of H9C2-1 with relevant mimics and inhibitors were inconclusive and

deserve further investigation.

D) ITGA7

ITGA7 encodes for integrin subunit ⍺7 that belongs to a vast family of cell adhesion alpha

receptors existing as heterodimers in conjugation with integrin subunit β1 (Takada, Ye, &

Simon, 2007). ⍺7β1 along with other heterodimeric integrins function as mechano-transducers

upon binding of an appropriate ligand to the receptor that detect and transmit changes in

mechanical forces occurring in the extra cellular matrix of myocytes (Takada et al., 2007). As a

consequence, several downstream intermediator molecules are activated including those

involved in PI3K and MAPK intra-cellular pathways, resulting in cellular changes concerned with

adhesion, apoptosis, proliferation, cell size and gene expression (Heineke & Molkentin, 2006;

Takada et al., 2007; Manso, Kang, & Ross, 2009). Furthermore, ⍺7 takes over from ⍺5 during

post-natal growth as the dominantly expressed alpha subunit (Israeli-Rosenberg et al., 2014).

Over-expression of ITGA7 is suggested to have a protective effect on cardiac and skeletal cells,

which was demonstrated by increased cellular regeneration, hypertrophy and reduced

cardiomyopathy in mice with skeletal muscular dystrophy, however, the mechanisms of the

indirect effect on the heart remain unclear (Israeli-Rosenberg et al., 2014). In another study,

overexpression of ⍺7β1 integrin in cardiomyocytes that were subjected to hypoxia was shown

to be protective by preserving mitochondrial membrane potential (Okada et al., 2013).

The role of integrin subunit ⍺7 in cardiac hypertrophy requires further analysis. Previously

reported protein expression of the subunit was significantly higher in cardiomyocytes isolated

from murine hearts with transverse aortic constriction (Babbitt et al., 2002). The results from

this study suggest expression of ITGA7 is down-regulated in hearts of 2-day old HHR. Hence,

further investigation into the role integrin subunit ⍺7 plays in cardiac hypertrophy is justified.

In addition, results from miR-351 transfection of H9C2-1 to determine microRNA-mRNA

interaction were inconclusive, therefore it is suggested the potential relation be considered in

future studies.