Conditional HIF-1alpha expression produces a reversible cardiomyopathy

Abstract

Background: The response to hypoxia in tissues is regulated by the heterodimeric transcription factor Hypoxia Inducible Factor-1 (HIF-1).

Methodology/principal findings: We have created a strain of mice with inducible cardiomyocyte-specific expression of a mutated, oxygen-stable, form of HIF-1alpha. Cardiac function steadily decreased with transgene expression, but recovered after the transgene was turned off. Using long-oligo microarrays, we identified 162 transcripts more than 3-fold dysregulated in these hearts after transgene expression. Among the down-regulated genes the transcript for SERCA was reduced 46% and the protein 92%. This led us to an evaluation of calcium flux that showed diminished reuptake of cytoplasmic calcium in myocytes from these hearts, suggesting a mechanism for cardiac dysfunction.

Conclusions/significance: These results provide a deeper understanding of transcriptional activity of HIF in the heart, and show that enhanced HIF-1 activity is sufficient to cause contractile dysfunction in the adult heart. HIF is stabilized in the myocardium of patients with ischemic cardiomyopathy, and our results suggest that HIF could be contributing directly to the contractile dysfunction in this disease.

Figure 1. VEGF expression in SVEC and cardiomyocyte cell culture 72 hrs after infection with adenoviruses encoding HIF-1αPPN or GFP.

Figure 2. Western blot of anti-HA immunoprecipitated HIF-1α-PPN from mouse hearts induced by doxycycline withdrawal for (A) 14 days and (B) 3, 5, and 7 days.

Each lane contains cardiac lysate from a different animal.

Figure 3. Echocardiographic fractional shortening of tTA/HIF-1α-PPN vs. tTA mouse hearts at (A) 0, 3, 7, and 14 days after doxycycline removal and (B) after 3 days of transgene induction followed by 7 days of recovery.

Figure 4. Changes in heart weight and epicardial vessels after HIF expression.

(A) Heart weight to body weight ratio following doxycycline removal. (B) Macroscopic view of tTA heart compared to HIF/tTA seven days after doxycycline removal. Arrows point to epicardial blood vessels that are much more prominent after.

Figure 5. Representative histology of HIF-1α-PPN expressing hearts.

Top panels: hematoxylin and eosin; Lower panels: trichrome, Scale bar: 50 µm.

Figure 6. Western blot of HIF-1α-PPN and SERCA levels following doxycycline restoration.

Figure 7. Transcriptional regulation by cardiac HIF.

(A) Venn diagram of genes regulated >3-fold in the heart as determined by microarray. (B) RT-PCR Gene expression results for tTA/HIF-1α-PPN mice compared to tTA control mice.

Figure 8. Semi-quantitative real-time PCR analysis of transcript abundance in hearts after HIF induction.

Metabolism-related genes showing the expected up-regulation of glycolytic genes and down-regulation of genes involved in oxidative metabolism. Evaluation of select genes involved in calcium cycling shows down-regulation of SERCA, along with moderate down-regulation of RYR-2 and PLB.

Figure 9. Effect of HIF expression on cardiac ATP:ADP ratio.

Progressive increase in ATP:ADP ratio was observed after doxycycline was withheld in the hearts of HIF/tTA mice. (n =  three hearts per time point).

Figure 10. SERCA protein levels and calcium reuptake after HIF expression.

(A) SERCA western blot. SERCA is markedly reduced after even a single day of transgene induction; the western blot below the graph shows results from two animals at each timepoint. (B) Calcium reuptake in cardiomyocytes is slower after transgene induction.