The major finding of this study is the identification of GIT1 as a novel regulator of cardiac biogenesis, which is essential for maintaining normal postnatal mitochondrial integrity and cardiac function. The GIT1 KO mice demonstrated four important cardiac phenotypes. First, GIT1 was highly expressed postnatally (P2–3 days) and GIT1 KO exhibited increased cardiomyocyte diameter and heart weight/body weight ratio. Second, mitochondria in GIT1 KO exhibited multiple abnormalities including cristae swelling, decreased volume density, decreased ATP production, and decreased membrane potential. Third, these mitochondrial defects were likely caused by a decrease in PGC-1α and PGC-1β, key regulators of mitochondrial biogenesis. Fourth, the GIT1 KO exhibited significant cardiac dysfunction characterized by dilation, impaired EF and increased cardiomyocyte apoptosis.
Previously we showed that GIT1 KO mice demonstrate disrupted postnatal lung vascular development [9
]. In contrast, the vasculature in heart, brain and kidney were normal, suggesting that the cardiac defects were not related to vascular abnormalities [22
].We do not believe that the lung phenotype caused cardiac dysfunction in GIT1 KO mice for the following reasons: 1) PGC-1α protein and mRNA expression was downregulated in GIT1 KO at P1. In contrast, VEGF mediated lung vascular development is just initiated at that point and doesn’t peak until P5 [23
]. 2) GIT1 KO mice showed about 35% increase in cardiomyocyte size in both left ventricle and right ventricle as early as P5, suggesting a biventricular mechanism. 3) Our recently published data showed that the pulmonary artery pressure and right ventricle pressure in GIT1 KO mice at 2–3 months of age were similar to the age-matched WT mice [9
]. Thus the cardiac hypertrophy in GIT1 KO mice is not consequent to pulmonary hypertension. We also don’t believe that increased blood pressure is a major contributor to GIT1 KO cardiac hypertrophy, since blood pressure as well as heart rate were similar in WT and GIT1 KO mice (Supplemental Table 1
The cardiac phenotype of the GIT1 KO was somewhat unexpected because Premont’s group found no expression of GIT1 in the heart. Specifically, this group used mice with β-galactosidase (β-Gal) reporters inserted into the two GIT genes to visualize GIT1 and GIT2 gene expression in mouse tissues [26
]. β-Gal staining showed that GIT2 was expressed inmost cells of the body, while GIT1 was highly restricted to brain, vessels (both EC and VSMC), lung bronchi and liver bile ducts. There was no expression of GIT1 in heart, skeletal, or smooth muscle cells (except vascular). However, the expression of GIT1 in neonatal heart was not investigated in the Premont study. We discovered that GIT1 is highly expressed in neonatal cardiomyocytes with lesser expression in adult cardiomyocytes. No compensation of GIT2 in hearts of GIT1 KO mice was observed (data not shown). These findings demonstrate a novel role for GIT1 in postnatal cardiac maturation.
After birth, there is a dramatic increase in mitochondrial biogenesis, and a transition of major mitochondrial metabolism substrates from glucose and lactate to fatty acids. The coactivator PGC-1α has been identified as the most important regulator of mitochondrial biogenesis and substrate transition in this period [5
]. PGC-1α is highly expressed in mitochondrial-enriched tissues such as heart. PGC-1α coactivates nuclear respiratory factor 1 (NRF-1) and NRF2, which are crucial for expression of Tfam. Tfam then binds to the promoter enhancer of the mitochondrial DNA (mtDNA) and drives the transcription and replication of mitochondrial DNA [16
]. Our data showed that Tfam was remarkably inhibited by deletion of GIT1,while NRF1 and NRF2 expression were not altered (data not shown). These results imply that GIT1 mediated PGC-1 signaling targets specific genes. Consistent with reduced expression of mitochondrial biogenesis-related genes, GIT1 KO mice displayed profound abnormalities in mitochondrial morphology in heart, including decreased mitochondrial DNA, mitochondrial volume density, reduced cristae density and increased vacuoles. Accordingly, adult cardiomyocytes from GIT1 KO mice produced less ATP and had significantly more dissipated mitochondrial potentials compared to WT, which is evidence of the disrupted mitochondrial function in the KO animals. Interestingly, the mitochondrial morphology in brain, kidney and lung was not affected by loss of GIT1 (data not shown) and PGC-1 expression in skeletal muscle was not altered in GIT1 KO mice. These data suggest the effects of GIT1 deletion on mitochondria are restricted to cardiomyocytes.
The key role of PGC-1 regulated genes in mitochondrial biogenesis in the postnatal heart is supported by the fact that the PGC-1αβ double KO mice die quickly after birth due to impaired mitochondrial function [1
]. The present study supports a key role for GIT1 in regulating PGC-1α gene expression. Specifically, we showed altered mitochondria ultra-structure, biogenesis, and reduced mitochondrial potential. Moreover, the severe lung phenotype obviously increases the oxidative stress and the GIT1 KO mice quickly develop cardiac dysfunction from 2 to 3 months old. The exact mechanisms by which GIT1 regulates PGC-1α expression remains under investigation, though evidence suggests that the ARF-GAP domain of GIT1 may play a critical role (unpublished data).
In summary, our study identifies GIT1 as a novel regulator of cardiac mitochondrial biogenesis and cell survival, as well as a new pathway for regulation of PGC-1α mediated gene expression.