Cellular and functional consequences in the heart after pre- and postnatal amino acid restriction

7.2.3.1 Consequences in neonatal hearts

Amino acids play a fundamental role in protein synthesis and cell growth²¹. We analyzed cardiomyocyte size in neonatal hearts upon intrauterine SPD or LPD exposure. We found that the cardiomyocyte CSA was increased in both LPD groups compared to the corresponding vehicle groups, while the difference in CSA was only significant for cHccs +/-neonates (see Chapter 6.5.3.2, Figure 20D). Increased CSA but unchanged HW (see Chapter 6.5.3.1, Figure 19C) point toward cell size compensation for a reduced cell number.

Evidence that cardiomyocyte CSA and cell number might be inversely correlated and that increased cardiomyocyte size in respect of normal HW can be considered an indirect measure for reduced cell number has recently been published⁸². The authors used a murine model for oxidative damage and revealed unchanged HW/BW ratio but increased cardiomyocyte size at P3⁸². This was accompanied by severely reduced cardiomyocyte proliferation, indicating reduced cardiomyocyte number⁸². Even though cardiomyocyte CSA is increased especially in neonatal LPD cHccs^+/- hearts (see Chapter 6.5.3.2, Figure 20D), the HW/BW ratio of this group is reduced (see Chapter 6.5.3.1, Figure 19D). This indicates that the increase in cell size might not be sufficient to compensate for the proposed reduction in cardiomyocyte number and that cardiomyocyte number in neonatal LPD cHccs^+/- hearts might even be further reduced compared to neonatal LPD Hccs^+/+ controls. Importantly, pathological cardiac hypertrophy is primarily achieved by an increase in width resulting in increased CSA, while physiologic cardiac growth is mediated by a combination of increased cardiomyocyte width and length²⁸⁴. Thus, the prediction of cardiomyocyte size from only measuring CSA is limited and it might therefore prove helpful to analyze isolated neonatal cardiomyocytes regarding their width, length, surface area and volume to get a precise estimate of their real size. It might be that cardiomyocytes in neonatal LPD hearts have increased CSA but are shorter in length. Hence, the overall cardiomyocyte volume might be unchanged, possibly explaining the unchanged HW.

In the rat model, neonatal LPD hearts had significantly less cardiomyocytes than neonatal SPD hearts²³, which was accompanied by decreased rates of myocardial cell proliferation²⁴.

DISCUSSION

We also focused on cardiac proliferation rates in neonatal SPD and LPD hearts and found no significant differences (see Chapter 6.5.3.2, Figure 20C). These observations suggest that the proposed reduction in cell number might be established by a proliferation defect during prenatal development and that altered proliferation rates might no longer be detectable in neonatal hearts. This discrepancy to the rat LPD model might again be explained by interspecies differences between rats and mice as discussed above. Importantly, Aroutiounova et al.²⁴ applied the more specific p-HH3 marker to assess neonatal proliferation rates, while we used KI67 staining, which labels proliferating cells in all active phases of the cell cycle²¹⁷. Given the known issue of cell cycle activity versus cell division in the perinatal heart, as discussed in Chapter 7.1.7, a more specific cell cycle activity marker might help to elucidate this discrepancy. Moreover, precise determination of cardiomyocyte number in neonatal SPD and LPD hearts for instance by stereological approaches (as described by Corstius et al.²³ and Lim et al.²⁵) is certainly of major importance to exclusively address the question of altered cell number in our murine LPD model.

7.2.3.2 Consequences in 13.5 dpc embryonic hearts

We postulated that a possible reduction in cardiomyocyte number might be established by a proliferation defect during prenatal development when amino acids are restricted. Hence, we analyzed 13.5 dpc SPD and LPD hearts; but we neither revealed differences in apoptosis (see Chapter 6.5.4.2, Figure 22E) nor proliferation rates (see Chapter 6.5.4.2, Figure 22B). In detail, we found no significant differences between the diet groups regarding cardiac proliferation in general but also compensatory hyperproliferation of healthy cells in the cHccs^+/- myocardium was not affected by prenatal LPD exposure (see Chapter 6.5.4.2, Figure 22B). These findings indicate that the demand for increased proliferation of healthy cells in cHccs^+/- 13.5 dpc embryos does not depend on amino acid availability. Hence survival of cHccs^+/- mice is ensured (see Chapter 6.5.2, Figure 18B) even if amino acids are restricted. Furthermore, morphology of 13.5 dpc embryonic LPD hearts was inconspicuous (see Chapter 6.5.4.1, Figure 21E), suggesting resistance of murine embryonic hearts against LPD exposure. However, it is conceivable that LPD might affect proliferation in the last trimester of prenatal development, the fetal phase, rather than in the embryonic phase. Previous studies (such as analyzing low oxygen conditions during pregnancy) revealed that IUGR can bring about changes in both embryonic and fetal development. For instance, increased apoptosis rates and premature terminal cardiomyocyte differentiation in fetal rat hearts³⁴ and ventricular dilation and myocardial hypoplasia in mid-gestation mouse embryos³⁶ were observed upon hypoxia. Moreover, Louey et al. previously showed that IUGR due to placental insufficiency causes decreased cell cycle activity in cardiomyocytes of fetal sheep hearts²⁸⁵. In the study on hand, the effects of prenatal LPD exposure on fetal cardiac development were not analyzed. Though, differential expression of cell cycle

DISCUSSION

regulating genes and accumulating evidence for reduced cell number in murine neonatal LPD hearts supports the general concept that LPD affects cardiomyocyte proliferative capacity. In conclusion, while effects of amino acid restriction in the rat become obvious in offspring at birth, we assume that in mice a reduced amino acid availability results in reduced cardiomyocyte proliferation capacity in fetal stages; a hypothesis that strongly urges for experiments focusing on proliferation rates in fetal hearts.

7.2.3.3 Consequences in adult hearts

In line with the developmental programming concept, studies in the classical LPD rat model (diet changed to SPD after birth) revealed that under baseline conditions prenatal amino acid restriction causes significant changes in heart morphology and function later in life, including interstitial fibrosis²⁶, hypertrophy²⁷ and increased stiffening²⁸ of the LV in adulthood.

In these models, the IUGR insult is released at birth and the rats display postnatal catch-up growth. To investigate if cardiac growth and function in adulthood is also affected if postnatal conditions resemble that of in utero, we analyzed 11 week old mice after pre- and postnatal amino acid restriction. It is conceivable that maintaining similar conditions during postnatal life prevents CVD caused by developmental programming. Interestingly, we did not observe significant changes regarding BW, HW and HW/BW ratio in adult mice after pre- and postnatal LPD exposure (see Chapter 6.5.5.1, Figure 23G-I). LPD rats also normalized HW until weaning; however they were on SPD after birth²⁵. Strikingly, when a SPD was administered to the pups not immediately after birth but after 2 weeks postnatally, LPD rat offspring remained significantly smaller and demonstrated reduced HW, resulting in unchanged HW/BW ratio compared to SPD offspring at 18 weeks of life²⁸⁶. However, to the best of our knowledge, our study is the first approach that investigated long-term consequences of LPD treatment without changing the diet to SPD at any time; though that there are no studies we can directly compare our results with.

Given that postnatal growth of the myocardium primarily occurs by cardiomyocyte hypertrophy and deposition of extracellular matrix (ECM)²⁸⁷, we focused on cardiomyocyte CSA and ECM deposition. According to previous findings in the cardiac-specific Hccs KO model (see Chapter 3.4), cHccs^+/- hearts compensate the hypoplastic neonatal phenotype by cardiomyocyte hypertrophy in adulthood (unpublished data, see Chapter 3.4). Strikingly, while compensatory hypertrophy is preserved in cHccs^+/- females after pre- and postnatal LPD exposure (even though it misses statistical significance in the sample set used in this study), cardiomyocyte CSA in adult Hccs^+/+ as well as cHccs^+/- LPD hearts is significantly smaller than in adult SPD hearts (see Chapter 6.5.5.2, Figure 24C). These finding contradicts the adult HW, which was not reduced in LPD compared to SPD hearts in both genotypes (see Chapter 6.5.5.1, Figure 23H). We speculate that other cardiac cell types (e.g. fibroblasts or smooth muscle cells) might be present in a higher number in adult LPD hearts, thus compensating for the

DISCUSSION

decreased cardiomyocyte CSA resulting in normal HW. Evidence comes from studies showing that upon pathological remodeling following injury dramatic shifts in the various cardiac cell populations and tissue composition occur, such that fibroblasts contribute to a larger extend to the heart tissue²⁸⁸. It will be important to investigate this potential mechanism by FACS quantification of non-cardiomyocytes within the LV myocardium of our LPD hearts. Besides, it is tempting to speculate that even though cardiomyocyte CSA is reduced, overall cell volume might be unchanged due to increase in length, as discussed for neonatal hearts above.

Analyzing adult cardiomyocyte dimensions after fixation and dissociation of the LV myocardium might prove useful to investigate this alternative. Furthermore, ECM deposition, determined by measuring the percentage of fibrotic tissue, was not increased in adult LPD hearts (see Chapter 6.5.5.2, Figure 24D); excluding the possibility that aggregated ECM deposition is responsible for the normalization of the HW. This finding was also in line with normal expression of genes encoding for ECM components (see Chapter 6.5.5.2, Figure 24E).

Moreover, prenatal LPD in the rat was shown to be associated with cardiac dysfunction in the first 2 weeks of life, as obvious by a severe depression in the EF, but recovery of cardiac function in the LPD offspring up to 40 weeks of age²⁷. Investigation of heart function in our 11 week old LPD mice revealed normal cardiac contractility (see Chapter 6.5.5.3, Figure 25D and Supplementary Table S7). However, we did not analyze cardiac function in murine LPD offspring during the lactation period. Furthermore, since 11 weeks old mice are rather young adults, investigating heart function and morphology of LPD Hccs^+/+ and cHccs^+/- mice at older age (e.g. after 1 year) or if cardiac stress (such as TAC or angiotensin) is applied, might reveal differences compared to adult SPD animals. Importantly, our data are in line with recent data from LPD rats, which revealed that maintaining a poor nutritional environment after birth might not increase the risk for CVD²⁸⁶. In this study, LPD rat offspring remained on LPD and were switched to SPD not until 2 weeks postnatally²⁸⁶. At 18 weeks of life, heart rate and FS of the cardiac muscle was not different between the treatment groups²⁸⁶.

In conclusion, we did not find evidence that pre- and postnatal LPD alters postnatal cardiac growth and function in general or in the cHccs^+/- myocardium in particular. This is in line with the developmental programming concept stating that postnatal cardiac catch-up growth, which does not occur in our approach, contributes to increased susceptibility for adulthood disease rather than being beneficial^15,16. However, given that the IUGR insult is not released at birth, our postnatal LPD model might be limited when interpreting the results in regard of developmental programming.

7.2.4 Consequences of pre- and postnatal amino acid restriction on mTOR pathway