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Transcriptome analysis reveals potential mechanisms underlying differential heart development in fast- and slow-growing broilers under heat stress

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The greater reduction of growth rate in commercial broilers than the slow-growing broilers under heat stress reported in the present study agree well with the previous reports [47, 48], further indicating higher susceptibility of modern broilers than low BW broilers to heat stress. The undermined growth performance under heat stress is certainly related to various changes in different tissues, such as reported intestinal injury and decreased relative weight of bursa, thymus and spleen [49]. In the current study, we also observed reduced relative weight of heart in commercial broilers under heat stress and identified some correlate DE genes and altered pathways. To the best of our knowledge, this is the first study investigating cardiac response to heat stress in broilers through transcriptome analysis. With 21-day cyclic heat stress treatment, we have been able to see how differently the fast-growing broilers (Ross) and slow-growing broilers (Illinois) adapt to thermal stress conditions. In Illinois broilers, heat stress only induced three significant DE genes compared to thermoneutral group, so these birds may have already adapted to the heat stress and they no longer need to alter gene expression to adjust their physiological function. Among the three DE gene, WDR830S is a novel gene with unknown function. TNC is an essential promoter of cardiac angiogenesis [50] and its expression can be induced by mechanical overload [51]. Expression of PTGDS can be induced in vascular endothelial cells by laminar fluid shear stress and thus is associated with progression of atherosclerosis [52]. Because these genes only showed significant change in Illinois broilers but mild change in Ross broilers in the heat stress vs. thermoneutral comparison (Additional file 3: Table S3), the upregulation of TNC and downregulation of PTGDS in Illinois broilers may be important for their better adaption to heat stress than Ross broilers.

In the fast-growing broilers, more than 300 DE genes were identified, with most genes related to cell cycle regulation. According to prediction of IPA, several pathways in cell cycle promotion, such as “Mitotic Roles of Polo-like Kinase”, were downregulated, and several pathways in cell cycle arrest, such as “p53 signaling” in cell cycle, were upregulated. Therefore, it is highly possible that the slowed cardiac growth in Ross broilers is due to inhibited cell proliferation and increased cell apoptosis. Hyperthermia-induced apoptosis has been long-recognized in various cell lines and wildly utilized in cancer treatment [5355]. Previous studies suggested that hyperthermia-induced apoptosis is characterized by the occurrence of internucleosomal DNA damage [56]. It occurred during G1 phase of cell cycle in rat MFH-2NR cells and mouse FM3A cells [57, 58], and both G1 phase and S phase in human HL-60 cells [59, 60]. As the heat load increased with time or temperature, the apoptosis apparently transitions to necrosis, leading to pathological cell damage and an inflammatory response in the tissue [6062]. However, different cell types have varied resistance to hyperthermia-induced apoptosis [56]. For example, in normal rats treated with whole-body hyperthermia at 41.5 °C for 2 h, the highest apoptosis occurred in thymus, but only negligible apoptosis occurred in heart, lung and liver [63]. The different resistance to hyperthermia-induced apoptosis in different tissues may be related to different regulation mechanisms.

Previous study suggested that commercial broilers exposed to daily 7-h cycles of 35 °C showed physiological response to reduce hyperthermia-induced apoptosis in liver with four genes showing anti-apoptosis regulation among 40 DE genes [64]. But the Ross broilers exposed to daily 8-h cycles of 35–37 °C in this study remarkably showed apoptosis in heart with upregulation of PERP and sphingomyelin phosphodiesterase 3 (SMPD3), and downregulation of CDC7, CDC45, thymidylate synthase (TYMS), STMN1, ribonucleotide reductase M2 (RRM2), urokinase-plasminogen activator (PLAU), TRAF interacting protein (TRAIP), snail family zinc finger 2 (SNAI2) and NDC80 kinetochore complex component SPC25 (SPC25) (Additional file 3: Table S3). PERP is an important mediator of P53-dependent apoptotic pathway and it is directly activated by P53 and highly expressed in cells undergoing apoptosis [65]. SMPD3 catalyzes hydrolysis from sphingomyelin to ceramide [66] which accumulates in response to stress stimuli such as heat stress and promotes cell cycle arrest and apoptosis [67]. CDC7 is an essential cell cycle regulator, and its inactivation can lead to S-phase arrest and P53-dependent apoptosis in mouse embryonic stem cell culture [68]. Being rescued by an ectopically expressed transgene, CDC7 (−/−) mice could survive but showed decreased cell proliferation, impaired organ development and reduced body size [69]. CDC45 is a key replication helicase cofactor required for DNA replication, and its depletion was suggested to slow S-G2 progression in HCT116 cells [70]. TYMS and RRM2 are both indispensable enzymes for DNA synthesis. TYMS catalyzes the production of dTMP from dUMP [71], while RRM2 is the regulatory subunit of ribonucleotide reductase which catalyzes the conversion from ribonucleotides to deoxyribonucleotides [72]. Because both dTMP and deoxyribonucleotides are necessary for DNA replication and repair, inhibition of TYMS and RRM2 has been suggested to induce apoptosis in cancer cells [73, 74]. STMN1 is a key regulator in interphase and late mitosis to prevent assembly and promote disassembly of microtubule. Its upregulation has been observed in various cancers and its inhibition has been proposed as a therapeutic way for cancer treatment [7577]. PLAU regulates cell growth and apoptosis through regulation of several growth factors and control of cell-matrix contacts. Depletion of PLAU reduces cell proliferation and increases cell death in cell culture and during tissue regeneration [78]. TRAIP can inhibit cell death through inhibition of mediation of nuclear factor kappa-B by tumor necrosis factor receptor associated factor 2 [79]. TRAIP-deficient mice exhibited impaired embryonic development due to proliferative defects and excessive apoptosis [80]. SNAI2 functions as a survival factor that can prevent apoptosis of damaged cells. Disruption of SNAI2 can sensitize the tumor cells to apoptosis signaling and delay the development of mammary gland [81, 82]. As a component of NDC80 kinetochore complex, SPC25 is required not only to establish and maintain kinetochore-microtubule attachment in mitotic spindle and metaphase alignment of chromosomes, but also to move chromosomes to spindle poles in anaphase [83]. Depletion of SPC25 can cause aberrant spindles, aberrant mitosis and increased apoptosis in human HeLa cells [84]. Most of these genes are mainly involved in cell cycle progression from S phase to M phase and regulated by P53 signaling. Together with the highest ranks of “Mitotic Roles of Polo-like Kinase” and “G2/M DNA Damage Checkpoint” pathways, we speculate that the hyperthermia-induced apoptosis in the heart of Ross broilers may be P53-dependent and occur primarily in S-M phase. To confirm this speculation, in vivo cellular studies are still necessary in the future.

Due to the relatively smaller heart caused by cell cycle arrest and apoptosis, there also seems to be regulation of adaptive hypertrophy to cope with hypertension in the adverse situation of slow cardiac growth. The large increase of AGTR1 expression in Ross broilers under heat stress is an indicator of this change (Table 1), because overexpression of AGTR1 in cardiomyocytes has been reported to induce cardiac hypertrophy and remodeling [85]. In addition, downregulation of vav guanine nucleotide exchange factor 3 (VAV3) may also indicate cardiac dysfunction in Ross broilers under heat stress, because VAV3-deficient mice exhibited left ventricular hypertrophy, systemic arterial hypertension and tachycaridia [86]. BMP10, a cardiac cytokine expressed restrictively in the developing and postnatal heart, is essential for regulation of cardiac growth and chamber maturation [29]. Previous study suggested that BMP10 provides a positive growth signal for cardiomyocytes antagonizing cell cycle inhibitors, and regulates several key cardiogenic factors to maintain adequate cardiac function [29]. In the current study, BMP10 showed the most significant upregulation (more than 2,000 times higher) in the heart of Ross broilers under heat stress compared to the thermoneutral group. Upregulation of BMP10 has been reported in mice with myocardial hypertrophy and excessive trabeculation [87]. Therefore, BMP10 in Ross broilers may provide compensatory regulation through counteracting upregulated cell cycle inhibitors to promote cardiac hypertrophy under heat stress. Another indicator of possible cardiac remodeling is expression change of genes encoding different myosin heavy chain isoforms (MHCs). MYH7, which encodes β-MHC in slow fibers of cardiac ventricles [88], was upregulated more than 300 times in heart of Ross broilers under heat stress. Whereas MYH1E, which is an ortholog of human MYH4 and encodes MHC-IIb in fast fibers in skeletal muscle, was downregulated about 25 times by heat stress in Ross broilers. In human suffering from chronic heart failure, decrease of MHC-I encoded by MYH7 but increase of MHC-IIb was detected in costal diaphragm [89]. Therefore, the upregulation of MYH7 in Ross broilers’ hearts under heat stress in this study may also indicate a propensity to heart failure in these birds, because decrease in the ratio of fast MHC and slow MHC in cardiac muscle can reduce myofibrillar Ca2 + −activated ATPase activity and systolic function of heart [90]. The increase of slow MHC and decrease of fast MHC under continuous mild heat stress has also been reported in myoblast cell culture of human, mouse [91] and quail [92]. Therefore, the pro-slow shifting of MHC isoforms may be common response to heat stress in muscle cells both in vitro and in vivo.

Another concern in fast-growing broilers under heat stress is the possible inhibitory effect on immune system. Decreased leukocytes and antibody production in peripheral blood has been reported in 31-week-old laying chickens under heat stress at 35 °C for 5 weeks [93]. In Ross broilers, heat stress in the range of 35-37 °C from 35 to 42 days posthatch also decreased T-helper (CD4+), T-cytotoxic (CD8+) lymphocytes and antibody titer in peripheral blood [94]. Therefore, similar inhibition effects of heat stress on the immune system may also exist in cardiac tissue, which is indicated by the expression changes of several key regulators of immune cell development, such as downregulation of CD4, CD40LG, lymphocyte cytosolic protein 2 (LCP2), STAT4, thrombospondin 4 (TSP4) and G protein-coupled receptor 183 (GPR183) and upregulation of SOCS3. Regarding their functions, we found the downregulated genes are generally important for cardiac immune response, while SOCS3 is an inhibitor of inflammatory response. CD4 plays a major role in cardiac allograft rejection, so anti-CD4 therapy is usually needed for a successful heart allograft [95]. Anti-CD40LG antibody reduced myocardial inflammation response in mice with acute viral myocarditis [96]. Deficiency of LCP2 has been found to lead to malfunction of mast cells and mild tachycardia in mouse heart [97]. STAT4-deficient mice are resistant to induction of myocarditis by cardiac myosin immunization [98]. TSP4 plays an important role in regulation of vascular inflammation. Knockout of TSP4 led to a reduced number of macrophages in aortic root lesions and reduced inflammatory factors in vascular walls [99]. GPR183 is highly regulated during cardiac inflammation, and may play key roles in pathogenesis of cardiovascular disease, inflammation and autoimmune diseases [100]. Antagonists of GPR183 have been suggested to be tested in paradigms relevant for cardiovascular diseases [101]. On the other hand, SOCS3 attenuated proinflammatory signaling mediated by activator of STAT family proteins, playing a negative role in a variety of inflammatory and autoimmune processes [102]. Therefore, the expression changes of all these genes point to decreased inflammatory response induced by heat stress in the heart of Ross broilers as predicted by IPA (Table 3).

Another merit of this study is the inclusion of a heritage line – Illinois. Through comparison between two broiler lines, we can determine how diversity in genetics and growth trait can lead to differential response to heat stress. When the two broiler lines are under thermoneutral condition, Ross broilers showed faster growth of BW with similar normalized heart weight compared to Illinois broilers, so their cardiac development must be faster than Illinois broilers to keep pace with their fast body growth. When the two broiler lines were under heat stress, normalized heart weight showed little change in Illinois but significant reduction in Ross broilers, indicating the slowed cardiac development of Ross under heat stress. However, to compare the heat stress effect on the two broiler lines, the intrinsic difference between thermoneutral groups must be subtracted for normalization. Through this approach, we identified “Mitotic Roles of Polo-like Kinase” Pathway as the primary pathway that contribute to the differential growth rate between the two broiler lines pre and post heat stress treatment. Expression of polo-like kinase has been reported to be highly correlated with proliferative activity of cardiomyocytes in rat [103]. Therefore, the lower expression of PLK1 and PLK4 (Table 2) in Ross broilers under heat stress compared to those under thermoneutral condition may be also an indicator of decreased proliferation of cardiomyocytes. PLK1 plays an important role in recovery of cells from G2 DNA damage-induced arrest in mammals. Overexpression of PLK1 enables cells to override cell cycle checkpoint even the DNA damage is still present [104]. Therefore, PLK1 may be a key regulator of cellular adaption to heat-induced DNA damage, and the downregulated polo-like kinases in Ross broilers under heat stress may be a key mechanism of the increased cell-cycle arrest and cell apoptosis.

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