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. 2023 Apr 1;324(4):H430-H442.
doi: 10.1152/ajpheart.00562.2022. Epub 2023 Feb 3.

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury

Surawee Chuaiphichai  1 , Sandy M Chu  1 , Ricardo Carnicer  1 , Matthew Kelly  1 , Jenifer K Bendall  1 , Jillian N Simon  2 , Gillian Douglas  1 , Mark J Crabtree  3 , Barbara Casadei  1 , Keith M Channon  1
Affiliations

Endothelial cell-specific roles for tetrahydrobiopterin in myocardial function, cardiac hypertrophy, and response to myocardial ischemia-reperfusion injury

Surawee Chuaiphichai et al. Am J Physiol Heart Circ Physiol. .

Abstract

The cofactor tetrahydrobiopterin (BH4) is a critical regulator of nitric oxide synthase (NOS) function and redox signaling, with reduced BH4 implicated in multiple cardiovascular disease states. In the myocardium, augmentation of BH4 levels can impact on cardiomyocyte function, preventing hypertrophy and heart failure. However, the specific role of endothelial cell BH4 biosynthesis in the coronary circulation and its role in cardiac function and the response to ischemia has yet to be elucidated. Endothelial cell-specific Gch1 knockout mice were generated by crossing Gch1fl/fl with Tie2cre mice, generating Gch1fl/flTie2cre mice and littermate controls. GTP cyclohydrolase protein and BH4 levels were reduced in heart tissues from Gch1fl/flTie2cre mice, localized to endothelial cells, with normal cardiomyocyte BH4. Deficiency in coronary endothelial cell BH4 led to NOS uncoupling, decreased NO bioactivity, and increased superoxide and hydrogen peroxide productions in the hearts of Gch1fl/flTie2cre mice. Under physiological conditions, loss of endothelial cell-specific BH4 led to mild cardiac hypertrophy in Gch1fl/flTie2cre hearts. Endothelial cell BH4 loss was also associated with increased neuronal NOS protein, loss of endothelial NOS protein, and increased phospholamban phosphorylation at serine-17 in cardiomyocytes. Loss of cardiac endothelial cell BH4 led to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia-reperfusion injury. Taken together, these studies reveal a specific role for endothelial cell Gch1/BH4 biosynthesis in cardiac function and the response to cardiac ischemia-reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction and ischemia-reperfusion injury.NEW & NOTEWORTHY We demonstrate a critical role for endothelial cell Gch1/BH4 biosynthesis in coronary vascular function and cardiac function. Loss of cardiac endothelial cell BH4 leads to coronary vascular dysfunction, reduced functional recovery, and increased myocardial infarct size following ischemia/reperfusion injury. Targeting endothelial cell Gch1 and BH4 biosynthesis may provide a novel therapeutic target for the prevention and treatment of cardiac dysfunction, ischemia injury, and heart failure.

Keywords: cardiac function; hypertrophy; ischemia-reperfusion injury; nitric oxide synthase; tetrahydrobiopterin.

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Conflict of interest statement

No conflicts of interest, financial or otherwise, are declared by the authors.

Figures

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Graphical abstract
Figure 1.
Figure 1.
Myocardial endothelial cell targeted Gch1 deletion causes a tissue-specific decrease in Gch1 gene, GTPCH protein, and biopterin content. A: evaluation of Tie2cre-mediated excision of the loxP flanked DNA in heart tissues and primary heart endothelial cells derived from Gch1fl/flTie2cre and Gch1fl/fl [wild-type mice (WT)]. The predicted 1,030-bp product was detected in WT mice. In the presence of Tie2cre transgene a 1,392-bp knockout allele was detected, with efficient excision in primary endothelial cells from hearts. B and C: quantitative real-time PCR was used to quantify Gch1 gene expression in hearts and primary endothelial cells from hearts (*P < 0.05; n = 4 per group). D–F: representative immunoblot of GTPCH proteins in hearts, isolated primary endothelial cells, and isolated cardiomyocytes from WT and Gch1fl/flTie2cre hearts, respectively, with quantitative data, measured as percent band density in G–I: CD102 and β-tubulin were used as endothelial cell marker and loading control respectively. J: representative chromatograms of BH4 traces in hearts from WT and Gch1fl/flTie2cre mice. K and L: BH4 levels and BH4/BH2 + B ratio were reduced in hearts from Gch1fl/flTie2cre mice compared with wild-type littermates (*P < 0.05; n = 8 and 9 per group). M and N: BH4 levels were barely detectable in primary ECs from Gch1fl/flTie2cre compared with WT mice (*P < 0.05; n = 4–6 per group). O and P: BH4 levels were comparable between primary cardiomyocytes from Gch1fl/flTie2cre mice and wild-type littermates. Absolute BH2 levels in cardiomyocytes were significantly increased in Gch1fl/flTie2cre mice compared with wild-type mice, such that the BH4/BH2 and biopterin ratio was significantly reduced in cardiomyocytes in Gch1fl/flTie2cre mice (*P < 0.05; n = 4 per group). Each data point represents an individual adult male mouse.
Figure 2.
Figure 2.
Superoxide production is increased, and nitric oxide bioavailability is reduced in cardiac endothelial cell BH4-deficient mice. Quantification of superoxide production, as measured by 2-hydroxyethidium (2-HE), in whole heart homogenate from Gch1fl/flTie2cre and wild-type (WT) mice using dihydroethidine (DHE) high-performance liquid chromatograph (HPLC). A: superoxide production was markedly increased in hearts from Gch1fl/flTie2cre mice compared with wild-type controls (*P < 0.05, n = 5–6 per group). B: representative trances of 2-HE and ethidium peaks in hearts from WT and Gch1fl/flTie2cre mice detected by DHE HPLC. C and D: nonselective nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME; 100 µM)-inhibitable fraction and polyethylene glycol superoxide dismutase, PEG-SOD (100 U/ml)-inhibitable fraction were greatly increased in Gch1fl/flTie2cre hearts compared with wild-type controls (*P < 0.05, n = 5–6 per group), respectively. E: nitrite/nitrate production in whole heart homogenate. Nitrite/nitrate production in heart homogenate from Gch1fl/flTie2cre mice was significantly decreased when compared with that from WT controls (*P < 0.05; n = 4–6 animals per group). F: levels of hydrogen peroxide from wild-type and Gch1fl/flTie2cre hearts were determined using an amperometric hydrogen peroxide microsensor electrode. Level of hydrogen peroxide production was significantly increased in endothelial cell BH4-deficient hearts (Gch1fl/flTie2cre) compared with wild-type controls (*P < 0.05; n = 6 per group). G: representative immunoblots for antioxidant proteins: catalase, Es-SOD, Mn-SOD, and Cu/Zn-SOD in Gch1fl/flTie2cre and wild-type hearts, with quantitative data, measured as percent band density in H (n = 6 per group). Each data point represents an individual adult male mouse.
Figure 3.
Figure 3.
Loss of cardiac endothelial cell BH4 leads to cardiac dysfunction and hypertrophy. A: example of M-mode echocardiograms from Gch1fl/flTie2cre and wild-type littermates controls. B: ejection fraction. C: LV fractional shortening. D: peak systolic velocity, cardiac output (mL/min). E: systolic and diastolic LV end dimensions (mm). F and G: LV diastolic and systolic volume (μL). H: anterior and posterior LV end-diastolic thickness (mm). I: cardiac output (mL/min) in WT and Gch1fl/flTie2cre mice (*P < 0.05, n = 6 per group). J: systolic blood pressure in Gch1fl/flTie2cre and wild-type littermates were determined using tail-cuff plethysmography. K–M: gene expression of hypertrophic markers and fibrosis marker (N) in hearts from Gch1fl/flTie2cre and WT littermates (*P < 0.05; n = 7–8 per group). Each data point represents an individual adult male mouse.
Figure 4.
Figure 4.
Increased phosphorylated extracellular signal-regulated kinases 1/2 in hearts and cardiomyocytes from Gch1fl/flTie2cre mice. A: representative immunoblots for phosphorylated and total proteins for extracellular signal-regulated kinases 1/2 (ERK1/2) in hearts, isolated endothelial cells (ECs), and cardiomyocytes from Gch1fl/flTie2cre mice and wild-type littermate controls. B: summary data (*P < 0.05, n = 5–6 per group). Each data point represents an individual adult male mouse.
Figure 5.
Figure 5.
Deficiency in endothelial cell BH4 causes an increased nNOS expression and reduced eNOS expression in the hearts specifically in cardiomyocytes from Gch1fl/flTie2cre mice. A: quantitative real-time PCR was used to quantify endothelial cell nitric oxide synthase (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) gene expression in hearts from Gch1fl/flTie2cre mice and wild-type controls (*P < 0.05; n = 5–9 per group). B: representative immunoblots of eNOS, nNOS, and iNOS isoforms in hearts from Gch1fl/flTie2cre mice and wild-type controls. C: summary data (*P < 0.05; n = 6 per group). D: representative immunoblots of eNOS, nNOS, and iNOS isoforms in cardiomyocytes from Gch1fl/flTie2cre mice and wild-type controls. E: summary data (*P < 0.05; n = 6 per group). Each data point represents an individual adult male mouse.
Figure 6.
Figure 6.
Calcium-handling proteins in cardiomyocytes from wild-type and Gch1fl/flTie2cre mice. A and B: phosphorylation of phospholamban (PLB) at Thr (17) was significantly increased in cardiomyocytes from Gch1fl/flTie2cre mice (*P < 0.05; n = 6 per group), whereas the phosphorylation of phospholamban at Ser (16) was unchanged (n = 6 per group). C: representative immunoblots for sodium-calcium exchanger (NCX) and sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) in cardiomyocytes from Gch1fl/flTie2cre mice and wild-type littermate controls. D: summary data (n = 6 per group). Each data point represents an individual adult male mouse.
Figure 7.
Figure 7.
Loss of endothelial cell Gch1/BH4 biosynthesis leads to cardiac dysfunction and injury following cardiac ischemia-reperfusion. Cardiac function and infarct size were measured in isolated wild-type (WT) and Gch1fl/flTie2cre hearts perfused using Langendorff model. A: after baseline stabilization, wild-type and Gch1fl/flTie2cre hearts were subjected to 35 min of global ischemia and 60 min of reperfusion. B: LV developed pressure (LVDP). C: LV diastolic pressure. D: LV dP/dtmax. E: LV dP/dtmin. F: rate pressure product (RPP; mmHg). G: coronary perfusion pressure (CPP; mmHg). H: heart rate (beats/min). I: coronary flow was measured in hearts before ischemia and after periods of ischemia followed by 60 min of reperfusion (n = 7 per group). J and K: infarct size, defined by triphenyltetrazolium chloride staining and measurement as percentage of risk region, was significantly increased in hearts from Gch1fl/flTie2cre mice compared with wild-type littermate controls. Values are the means ± SE (*P < 0.05; n = 4–6 animals per group). Each data point represents an individual adult male mouse.
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