Functional Analysis of Heat Shock Protein HSPA4

Abstract

The aims of this study were to determine the underlying causes of male infertility, growth retardation and cardiac hypertrophy in Hspa4-/- mice. In order to identify the spermatogenic stage, at which spermatogenesis is affected by Hspas4 deficiency in mice with hybrid C57BL/6J x 129/Sv genetic background, we performed histological and immunological analysis. Analysis of germ cell development during juvenile life of Hspa4-/- mice showed an arrest of first wave of spermatogenesis in late stage of prophase I. RNA analysis showed a marked reduction in expression of late meiotic and postmeiotic-specific marker genes, whereas expression of early meiotic-specific genes was unaffected in the Hspa4-/- testes. These results suggest that HSPA4 is required for the regulation of diverse chaperone processes and cell cycle during germ cell progression. The second aim of this study was concentrated to study the cause of growth retardation in Hspa4-/- mice with inbred 129/Sv genetic background. To investigate whether growth retardation is due to skeletal muscle myopathy, histological analyses of different skeletal muscles were carried out. Histological analyses revealed that Tibialis anterior, Vastus intermedius, Soleus and paraspinal muscles of Hspa4-/- adult mice displayed myopathic changes. Moreover, myopathic change was already present at an early postnatal stage of Hspa4 -/- mice development. This suggested that skeletal muscle myopathy is the cause of growth retardation. Furthermore, these results suggest that HSPA4 has protective effect that confers the protection of skeletal muscle against mechanical or metabolic stress, which induce skeletal muscle degradation. To determine whether skeletal muscle myopathy is also realized in heart as hypertrophic cardiomyopathy, histological molecular analyses and two-dimensional directed M-mode echocardiograms were performed. Histological analyses revealed the development of cardiac hypertrophy and fibrosis in heart of Hspa4-deficient mice. Ultrastructure analysis revealed myofibrillar disarray and disassembly in cardiomyocytes and increased collagen accumulation (fibrosis) in heart of Hspa4-deficient mice. Echocardiographic measurements further confirm the cardiac hypertrophy phenotype in Hspa4-null mice. To identify when the development of cardiac hypertrophy and fibrosis starts in Hspa4-/- hearts, we studied expression of hypertrophic and fibrotic marker genes in heart from different postnatal stages by Northern blot and real time PCR analyses. These results indicate that the development of cardiac hypertrophy and fibrosis in Hspa4-null mice starts in early postnatal life and let us to suggest that the early postnatal lethality in Hspa4-/- mice in an inbred 129/SV genetic background can be contributed to the progression of skeletal muscle myopathy and hypertrophic cardiomyopathy. In contrast to skeletal muscle and heart phenotype in Hspa4-/- mice with inbred background, histological analysis and expression analysis of hypertrophic genes showed that cardiac hypertrophy is not developed in Hspa4-/- mice with hybrid 129/Sv x C57BL genetic background. In order to identify the signal pathways that mediate cardiac hypertrophy in Hspa4-/- mice, we determined the expression of some genes and proteins, which are involved in Gp130/STAT3, MAPK and calcineurin-NFAT signalings, in heart of wild-type and Hspa4-/- mice. Results of these analyses suggest that STAT3 and MAPK signaling pathways are not responsible for the development of cardiac hypertrophy in Hspa4-null mice. On the contrary, increased activity of calcineurin/NFAT in Hspa4-/- heart seems to modulate the cardiac hypertrophy. Microarray analysis was used as a screening tool to identify altered gene expression in Hspa4-deficient heart at 25 days compared with that of wild-type mice. 98 genes were identified as differentially expressed. Several genes were selected and classified according to their function in the development of cardiac hypertrophy. To confirm the results of microarray analysis, quantitative real-time PCR was performed. We confirmed that genes involved in ion channel signaling (Kcne1, Kcnd2, Scn4a, Hcn1 and Irx4), in protection of cells against oxidative stress (Gnao1, Ptp4a1 and Mme1) and those inducing oxidative stress (Maob), are differentially expressed in heart of Hspa4-/- mice. The altered expression of these genes may be involved in the development of cardiac hypertrophy in Hspa4-/- mice.