The inhibition of pyruvate dehydrogenase kinase improves impaired cardiac function and electrical remodeling in two models of right ventricular hypertrophy: resuscitating the hibernating right ventricle

Experimental protocols

The University of Chicago Institutional Animal Care and Use Committee approved all protocols. In the monocrotaline model, adult male Sprague-Dawley rats weighing 260–280 g were randomized to the following groups: Control, Dichloroacetate, Monocrotaline, and Monocrotaline+Dichloroacetate. A single subcutaneous injection of monocrotaline (60 mg/kg, Sigma, St. Louis, MO) produced severe PAH and RVH at 1 month. The water for the Dichloroacetate and Monocrotaline+Dichloroacetate groups contained dichloroacetate 0.75 g/L, pH 7.0 (Sigma, St. Louis, MO) and rats consumed an estimated daily dose of 70 mg/kg. Dichloroacetate therapy started on day 10 post-monocrotaline, at which time PAH is established[18].

A pressure-overload RVH model was created by PAB. Penicillin G (600,000 U) was administered preoperatively. The rat was anesthetized with 3% isoflurane and then intubated. A median sternotomy was performed and the PA was dissected free from the aorta and left atrium. A silk suture was placed around the PA and a loose knot formed. A 16-gauge needle was inserted through the knot, parallel to PA. The suture was tied tightly and the needle was withdrawn, creating a stenosis equal to the needle’s diameter (~1.6 mm). After PAB, the thorax was closed. Pain relief was achieved using buprenorphine (15 µg/kg sc). The PAB protocol included four groups: Sham, Dichloroacetate, PAB, PAB+Dichloroacetate (0.75 g/L in the drinking water). Dichloroacetate therapy started on the same day as PAB surgery. The hearts were studied 7 weeks after PAB. Sham and Sham+DCA groups had gained 316±19 g and 318±17 g body weight (n=6–7, P>0.05) seven weeks after sham surgery. Although the increase of the body weight in PAB and PAB+DCA groups are less than the increase in Sham and Sham+DCA groups due to the surgery, PAB and PAB+Sham groups still had gained 268±16 g and 249±14 g body weight, respectively. There are no differences in weight gain between the two groups (n=6–7, P>0.05).

Echocardiography

Doppler, 2D and M-mode echo was applied to measure the PA acceleration time (PAAT), diastolic thickness and systolic thickness of the right ventricular free wall thickness (RVFW) (see methods in on-line Supplement).

Positron Emission Tomography (PET)

Glucose uptake was imaged in vivo in rats anesthetized using isoflurane gas anesthesia (2% in oxygen) using FDG as a radiotracer. PET Imaging was performed using a Triumph PET/SPECT/CT system (GE Healthcare, Northridge, CA). Rats fasted overnight and were given a sucrose load 30-minutes before FDG injection (20 × 45 mg sucrose pellets, Bilaney Consultants, NJ). Images were acquired 20 minutes after FDG injection. To provide anatomical references, the rat thorax was first imaged using micro-CT followed by a 60-minute PET data acquisition. Images are shown using a pseudo color map (red indicates high and green indicates low glucose uptake). Data were analyzed by manual segmentation of the LV and RV free walls of the 3D PET image dataset.

RV Langendorff perfusion and measurement of MAPD

Rats were anesthetized with isoflurane and hearts were excised, perfused with a saline solution for 1–2 min at 25 °C and then mounted on a Langendorff perfusion apparatus for retrograde aortic perfusion with oxygenated Krebs-Henseleit buffer (95% O₂+5% CO₂) at a constant pressure of 75–85 cm H₂O (37_C). The PO₂ in the solution exceeded 560 mm Hg in all experiments. The AV node was ablated and hearts were paced at a cycle length of 200 ms. Hearts stabilized for 10–20 min and then right ventricular systolic pressure (RVSP) was measured, as described[18]. RV monophasic action potentials were recorded by placing Teflon-coated, 0.25 mm silver electrodes gently on the RV[23]. The signals were recorded using PowerLab and analyzed using Chart V 5.5.6. MAPD was measured as the time to achieve 20% (MAPD20) and 90% (MAPD90) of complete repolarization[23].

Simultaneous measurement of metabolism and function in the RV working heart model

In a separate set of experiments, an isolated working heart model was used to simultaneously measure metabolism and RV work (heart rate × RVSP). The heart was rapidly excised and subsequently mounted on an aortic perfusion cannula for about 10 min. The pulmonary veins, main PA and superior vena cava were isolated and cannulated. The inferior vena cava was isolated and ligated. To initiate flow to the RV and LV, perfusate was delivered to the superior vena cava and left atria cannulae at a constant preload (45 mm Hg and 115 mm Hg, respectively). Hearts worked against a 80 mm Hg aortic afterload, and a 45 mm Hg PA afterload. Cardiac output was measured with Transonic ultrasound flow probes in the afterload lines and cardiac work was calculated as the product of systolic pressure and cardiac output, as described[24]. Glycolysis and glucose oxidation were measured by perfusing hearts with Krebs–Henseleit buffer containing 11 mM [5-³H/U-¹⁴C] glucose, 0.5 mM lactate, 1.2 mM palmitate, 3% albumin, and 100 U/ml insulin, as previously described[25]. The effect of dichloroacetate on glucose oxidation and cardiac function was evaluated by quantitative collection of ³H₂O (glycolysis) and ¹⁴CO₂ (glucose oxidation), at 10 minute intervals after adding 1 mM dichloroacetate to the perfusate.

Hemodynamics, Histology and Cardiac function

Four weeks after monocrotaline, pulmonary hypertension was evident. The PAAT was shortened in Monocrotaline vs Control rats and was improved in the Dichloroacetate+Monocrotaline group (12.4±0.3, 35.0±0.7 vs 20.6±2.7 ms, respectively; n=6–12/group, P<0.001, Fig. 1A). Monocrotaline increased the RV/(LV+Septum) weight ratio versus control and this was reduced in the Dichloroacetate+Monocrotaline group (0.61±0.04, 0.27±0.01 vs 0.39±0.04, respectively n=10–11, P<0.05) (Fig. 1B). Dichloroacetate also reversed monocrotaline-induced hypertrophy of individual RV myocytes on histology (Fig. 1C). Increased RVFW thickness in Monocrotaline vs Control was also reduced in the Dichloroacetate+Monocrotaline group (1.67±0.06, 0.95±0.02 vs 1.37±0.10 mm, respectively; n=6–13, P<0.001, Fig. 1D). RV systolic function was reduced in Monocrotaline versus control rats, suggesting impaired RV contractility, and RVFW systolic shortening was significantly improved by dichloroacetate (85±10%, 19±10% vs 65±14%, respectively, n=3, P<0.05, Fig. 1E). In the Langendorff model, developed RVSP was significantly elevated in Monocrotaline vs Control and was decreased in the Dichloroacetate+Monocrotaline group (63±7, 31±3, 43±3 mm Hg, respectively, n=8–9, P<0.05, Fig 1F). In vivo, thermodilution CO was reduced in Monocrotaline vs Control (58.3±4.9 vs 154.2±16.2 ml/min, n=6, P<0.001, Fig. 1G) and this improved in the Dichloroacetate+Monocrotaline group to 124.6±15.7 ml/min, n=6, P<0.005). Dichloroacetate did not cause hemodynamic or histological changes in control rats (Fig. 1).

Metabolic changes in RVH

In the RV working heart model, cardiac work tended to increase during a 40-minute period of observation. However, the only significant increase in cardiac work occurred in Monocrotaline hearts that received dichloroacetate (1 mM) in the perfusate (n=4–5, p=0.05) (Figure 2A).

Basal rates of glucose oxidation did not differ among the groups (Fig. 2B). However glycolysis rates were increased in Monocrotaline versus Control (4025±611 vs 1872±430 nmol/min*g dry wt, n=5, respectively. P<0.05. Fig. 2C). Addition of 1 mM dichloroacetate to the perfusate increased glucose oxidation in Monocrotaline RVH from 772±323 to 3426±312 nmol/min*g dry weight (n=5, P<0.001, Fig. 2B), with no change in glycolytic rates (Fig. 2C).

RV O₂-consumption was reduced in Monocrotaline vs Control and this was restored in Monocrotaline+Dichloroacetate (148±27, 277±27 vs 230±22 pmol/sec*ml, respectively, n=6–7, P<0.05, Fig. 3A&B). Consistent with the increased glycolysis, FDG uptake was elevated in RV of monocrotaline rats (Fig. 3C). The RV/LV FDG uptake was increased in Monocrotaline vs Control (1.50±0.41 vs 0.46±0.03, n=7–9, P<0.05, Fig. 3D). Dichloroacetate tended to reduce FDG uptake ratio to 1.19±0.34 (n=11, P=NS)

Immnunostaining showed qualitatively greater expression of the cardiac glucose transporter Glut1[26] in monocrotaline-induced RVH, both in the cytosol and at the plasma membrane (where it colocalized with dystrophin). Glut1 expression was decreased toward normal in the Monocrotaline+Dichloroacetate group (Fig. 4A). Consistent with the immunofluorescence, Monocrotaline RVH was accompanied by a 3-fold increase expression of Glut1 mRNA (n=6, respectively, P<0.001, Fig. 4B). Dichloroacetate reduced Glut1 mRNA by 43% (n=6, P<0.05 vs Monocrotaline). Glut1 protein expression was also increased in Monocrotaline group and decreased in the Monocrotaline+Dichloroacetate group (n=4/group; P<0.05, Fig. 4C).

To confirm whether the effect of dichloroacetate was mediated via PDK inhibition, we measure PDH phosphorylation. Phosphorylated PDH increased 2.5-fold in the Monocrotaline group and this decreased in Monocrotaline+Dichloroacetate group (n=7–8, P<0.05, Fig. 4D). Dichloroacetate did not change Glut1 and PDH expression in control rats (Data not shown).

Expression of both Glut1 and phosphorylated PDH did not change in LV in either Monocrotaline or Monocrotaline+Dichloroacetate groups (See Supplemental Fig. 1).

Electrical remodeling in RVH in the Monocrotaline model

The time to achieve 20%and 90% of complete repolarization (MAPD20 and MAPD90) in the RV were significantly increased in Monocrotaline vs Control (Fig. 5A, B&C). Dichloroacetate shortened MAPD20 nearly to the Control levels. To determine whether dichloroacetate acutely changes MAPD, 1 mM dichloroacetate was added to the perfusate. MAPD did not change within 50 min (n=2, P=NS, see Supplemental Fig. 2), suggesting that shortened MAPD in Monocrotaline+Dichloroacetate group is more likely due to the documented re-expression of Kv channels following chronic in vivo dichloroacetate treatment (Fig. 6) than to an acute effect of dichloroacetate on channel activity.

Consistent with the prolongation of MAPD, the QTc was prolonged in Monocrotaline vs Control groups and was normalized in the Monocrotaline+Dichloroacetate group (n=6, P<0.05, Fig. 5D&E). Dichloroacetate had no effect on MAPD or QTc interval in normal rats (Fig. 5).

Expression of Kv1.2, Kv1.5 and Kv4.2 channel mRNA in the RV was downregulated in Monocrotaline vs Control groups (n=6, P<0.01, Fig. 6 A–C) and was partially restored by dichloroacetate. Kv1.4 and Kv2.1 mRNA did not change among the groups (n=4, P>0.05, Supplemental Fig. 3). At the protein level, Kv1.2, Kv1.5 and Kv4.2 expression was also significantly reduced in Monocrotaline vs Control groups (n=3–7, P<0.05, Fig. 6D, E&F). The expression of Kv1.5 protein was significantly increased in the Monocrotaline+Dichloroacetate group (n=7, respectively, P<0.05, Fig. 6E).

RVH induced by pulmonary artery banding

In order to distinguish whether dichloroacetate’s effects on RV function and hypertrophy reflected direct effects on the RV, independent of effects on the pulmonary vasculature, we used the PAB model. Histological study 7 weeks following PAB, showed RV myocyte hypertrophy in the PAB group, and this was partially reversed by concomitant dichloroacetate therapy (Fig 7a). The RVSP in RVH induced by PAB was reduced by dichloroacetate (23±2, 57±5 and 45±4 mm Hg, respectively, n=3–4, P<0.05, Fig 7B). Likewise, the peak pressure gradient between the pulmonary artery and RV pressure was significantly reduced by dichloroacetate (Fig 7B). Dichloroacetate improved RVFW systolic thickening (Control: 74±8%; PAB: 30±3%; PAB+Dichloroacetate: 54±6%, n=3–14, P<0.05, Fig. 7C). The CO, which was reduced in PAB group versus control, was partially restored in the PAB+Dichloroacetate group (177±11, 108±10 and 135±10 ml/min, respectively, n=4–9, P< 0.05, Fig 7D).

Similar to the monocrotaline model, hypertrophied RV myocytes in the PAB model had greater Glut1 expression than control RV myocytes and chronic dichloroacetate therapy normalized both RV myocyte size and Glut1 expression (Fig. 8A).

The reduced RV O₂-consumption in PAB relative to Control was restored in the PAB+Dichloroacetate group (296±23, 208±19 vs 309±33 pmol/sec*ml, n=6–7, P < 0.05, Fig. 8B), consistent with a dichloroacetate-mediated increase in glucose oxidation.

Dichloroacetate improves metabolism in hypertrophied hearts

Our findings suggest that the hypertrophied RV is an example of hibernation, meaning the RV is viable but its function is depressed. Depressed RV function in both models results in part from PDH inhibition, caused by PDK-mediated phosphorylation. The metabolic shift to glycolysis appears to be responsible for much of the observed RV dysfunction and neither the metabolic or functional impairment occur in the LV (Fig. 3C and supplemental Fig. 1).

PDK is a key regulator of glucose oxidation. Activated PDK phosphorylates and inhibits PDH (Fig. 4D), blocking the entry of pyruvate into the mitochondria inhibits formation of acetyl CoA and slows the Krebs’ cycle[30]. In contrast to the normal RV, which can vary its substrate utilization from fatty acids to glucose as needed, RVH is associated with a persistent reliance on glucose metabolism[27]. Dichloroacetate, a prototypic inhibitor of PDK activates PDH, thereby increasing acetyl coenzyme A and enhancing Krebs’ cycle[31]. The ¹⁴C-glucose technique[25] showed that dichloroacetate rapidly increases glucose oxidation and concordantly enhances RV function (Fig. 2), which in light of the constant afterload in this model is consistent with an increase in contractility. Dichloroacetate’s ability to increase O₂ consumption (by enhancing glucose oxidation) was evident in both the monocrotaline and PAB models (Fig. 3A and 8B). The relatively greater increase in glucose oxidation relative to glycolysis caused by dichloroacetate reflects better coupling and would be predicted to reduce lactate and proton production. This has the potential to increase cardiac efficiency, since less energy is used to correct this ionic imbalance.

The beneficial effects of chronic dichloroacetate likely relate both to the enhanced myocardial energetics that occurs when oxidative metabolism is enhanced and the restoration of Kv channel expression. In the working heart model, dichloroacetate rapidly improves RV function (Fig. 2A). This improvement is too rapid to attribute to transcriptional restoration of Kv expression. We also show that dichloroacetate does not acutely shorten MAPD-Supplemental Figure 2). Moreover, expression of the Glut1 transporter was also normalized by dichloroacetate in both models (Fig. 4 A, B&C and Fig. 8A). We believe that the upregulated Glut1 and impaired oxygen consumption in RVH reflect the need to increase glucose import in RVH to compensate for the lower yield of ATP in glycolysis vs oxidative glucose metabolism.

Published evidence for impaired metabolism in RVH is limited. However, a proteomic study of monocrotaline-induced RVH found a pattern of enzyme expression consistent with impaired oxidative metabolism, including down-regulation of PDH (subunit beta E1), isocitrate dehydrogenase, succinyl coenzyme A ligase, NADH dehydrogenase, ubiquinol-cytochrome C reductase, and propionyl coenzyme A carboxylase[32]. We noted a similar reversible mitochondrial metabolic abnormality in the pulmonary vasculature in PAH and in human cancers[1, 19]. In both cases these abnormalities are initiated and maintained, in part, by pathological PDK activation and in both cases several weeks of dichloroacetate therapy is beneficial, regressing vascular obstruction in PAH[1] and stopping tumor growth in a xenotransplantation model of human lung cancer[19]. Similar benefits of PDK inhibition have been reported in experimental LVH[21, 33] and myocardial infarction[22], conditions associated with enhanced glycolysis [21, 33].

Dichloroacetate and electrical remodeling in RVH

Cellular electrophysiological studies have shown that repolarization abnormalities noted on EKG in animals and humans with ventricular hypertrophy are caused by depression of K⁺ currents[34]. It is an underappreciated fact that patients with RVH have mild prolongation of the QTc interval, particularly on right-sided leads[8]. A decrease in the I_Kr in RVH appears to explain the prolongation of MAPD[28]. In present study, multiple Kv channels including Kv1.2, Kv1.5 and Kv4.2 were downregulated in RVH and this was accompanied with the prolongation of MAPD and the QTc interval. Inhibition of repolarizing K⁺ currents and MAPD prolongation likely adds to the negative inotropic effects of glycolytic metabolism and further reduces RV contractile function [10] (Fig. 8D). Conversely, dichloroacetate’s beneficial electrical effects are, at least in part, due to increased expression of several repolarizing Kv channels, including Kv4.2 and Kv 1.5 (Fig. 6 B, C, E&F). Dichloroacetate’s effect in shortening MAPD20 may relate to its ability to increase expression of both Kv4.2 and Kv1.5 which have the properties of fast activation[6]. Acutely, application of dichloroacetate does not shorten MAPD, consistent with the dichloroacetate’s effects on MAPD relating to re-expression of Kv channels, rather than enhanced channel activation (Supplemental Fig. 2). This suggests that the changes in mitochondrial metabolism induced by PDK contribute significantly to the electrical remodeling in RVH induced by PAH. Consistent with our findings, in a patch clamp of surviving cardiomyocytes from infarcted rat heart, dichloroacetate (1.5 mM) increases the depressed I_to current density; whereas 3-bromopyruvate, a PDH complex inhibitor, reduces the current density[22]. Together with our observations, the literature[22] supports the hypothesis that the downregulation of Kv channels that underlies reduced K⁺ currents in RVH reflects transcriptional effects induced by PDK-mediated metabolic changes.

Limitations

We realize that our data on the metabolic shift and electrical remodeling in RVH is only one step towards a better understanding of the pathophysiology in RVH and RV failure. The initiating stimulus for PDK activation is uncertain. However, since these changes occur only when the experimental RVH is severe we speculate that it reflects a supply-demand mismatch in coronary perfusion of the RV and its increased metabolic demands. Consistent with this, several papers suggest that the hypertrophied RV may be ischemic[2, 35]. Future studies are required to determine the mechanism by which inhibition of PDH downregulates expression of the repolarizing Kv channels. Additional studies are also planed to assess other metabolic therapies in RVH, including agents that alter fatty acid oxidation, such as trimetazidine.

Although dichloroacetate has been safely used in humans to treat lactic acidosis related to mitochondrial diseases [36], the long-term safety of dichloroacetate in patients with RVH remains to be determined. Moreover, the safety of drugs that reduce hypertrophy, traditionally thought to be a beneficial compensatory mechanism in conditions of increased afterload, remains to be established. In the case of dichloroacetate we believe the fact that reduction in RVH relates to positive changes in vascular remodeling and myocardial energetics favors the interpretation that regression of RVH reflects disease regression and is thus “beneficial”.

While we found that alpha actin protein did not increase in RVH, Bakerman et al. noted an increase in alpha-skeletal actin mRNA in the RV of calves exposed to acute hypoxia[37]. While an RVH-associated increase in the reporter protein is a theoretical concern, and could have complicated interpretation of the GLUT immunoblot, no increase in actin was observed. The differences between Bakerman’s study and ours include the species and model of RVH used and the fact we measured protein while they measured mRNA. Changes in mRNA do not always translate into changes in protein. Finally Zhang et al., also using a rodent model, found no change in actin expression in a monocrotaline rodent model[7].

Conclusion

Electrical remodeling and RV dysfunction in RVH due to PAH and PAB relate to a glycolytic shift mediated, in large part, by PDK activation and inhibition of the PDH complex. Dichloroacetate, reverses the electrical remodeling and enhances RV function by enhancing glucose oxidation (Fig. 8D). The relevance of these findings is enhanced by the fact that dichloroacetate has been safely and chronically administered in humans[36], suggesting a trial in RVH associated with PAH or pulmonic stenosis could be safely conducted.