Metabolic Efficiency Promotes Protection From Pressure Overload in Hearts Expressing Slow Skeletal Troponin I

Cardiac hypertrophy model

Surgeries to induce chronic pressure-overload via transverse aortic constriction (TAC) were done at 2–3 months of age in male mice expressing either cTnI, the adult isoform of cardiac troponin I (NTG); or ssTnI, the fetal isoform (ssTnI). Mice were bred from heterozygous males and NTG females and the male offspring were randomly selected to undergo TAC or sham surgery. TAC was induced by placement of a titanium metal microclip to constrict the ascending aorta (ID 0.4 mm). The ssTnI model has already been described in detail elsewhere^{16, 17}. Mice were maintained on the CD-1 background. Sham surgeries were performed on both NTG and ssTnI mice to serve as controls. At 10 weeks post-surgery, mice were anaesthetized, and in vivo cardiac function was determined via echocardiography, as described previously¹⁸. At 12–13 weeks post-surgery, the hearts were isolated and perfused to assess cardiac metabolism and isolated heart function. All procedures were approved by the University of Illinois at Chicago Animal Care and Use Committee and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised 2011).

Isolated Heart Perfusion

12–13 weeks after TAC or sham surgery, hearts were isolated and retrogradely perfused with Krebs buffer (in mM: 0.4 palmitate, 10 glucose, 0.5 lactate). The ratio of palmitate:BSA was 3:1. Hearts were perfused in a 14.1 T NMR magnet. The contribution of ¹³C palmitate ([4,6,8,10,12,14,16-¹³C7]palmitate) or ¹³C glucose ([1, 6-¹³C2]glucose) to acetyl CoA was used to determine the relative contribution of exogenous LCFA versus glucose to acetyl CoA production by the TCA cycle. The ¹³C endpoint enrichment of the glutamate pool in acid soluble extracts was quantified via NMR spectroscopy in 5 mm ¹³C probe (Bruker Instruments, Billerica, MA) ^{1, 19}. The glutamate pool is in equilibrium with the TCA cycle intermediate alpha-ketoglutarate and therefore enrichment of the glutamate pool can be used to determine the fractional enrichment of [2-¹³C] acetyl CoA¹⁹. The relative anaplerotic flux was determined from the ¹³C labeling of the glutamate pool as described previously⁴. Left ventricular function (developed pressure, heart rate) was monitored by a fluid-filled balloon inserted into the left ventricle. The energetic status of the mice (PCr:ATP) was determined by dynamic mode ³¹P NMR.

Protein Expression

Protein expression was measured by Western blot in heart tissue using commercially available antibodies (AMP kinase α, 2532S Cell Signaling; β-myosin heavy chain, M8424-.2ML Sigma Aldrich; calsequestrin, PA1-913 Pierce Thermo Scientific; malic enzyme 1, ab97445 Abcam; phospho-AMP kinase α, 2535S Cell Signaling; pyruvate dehydrogenase, ab110330 Abcam; phosphorylated pyruvate dehydrogenase, ab92696 Abcam; pyruvate dehydrogenase kinase 4, ab63157 Abcam; pyruvate carboxylase, ab128952 Abcam). Protein concentrations in whole cell lysates were determined by BCA protein assay. Gels were loaded with 10–60 μg of protein to measure protein expression levels. Calsequestrin was used as a loading control in all gels. Intensity of the bands of interest was normalized to the intensity of the loading control and the relative increase in expression over baseline (NTG) was reported. When the comparison of more than one gel was required, each gel was normalized to the baseline samples within that gel.

Statistical Analysis

Differences in the mean within groups (NTG sham vs. NTG TAC or ssTnI sham vs. ssTnI TAC) were determined by student’s t test. Differences in the mean across groups were determined by one-way ANOVA followed by Tukey-Kramer post hoc test (Prism 4, Graphpad Software Inc.). Means were said to be significantly different when p<0.05. Data is presented as mean±SE.

Cardiac Hypertrophy

Heart weight and heart weight to tibia length, measured in isolated hearts at the end of perfusion, increased 25% in NTG mice 12–13 weeks after transverse aortic constriction (TAC). However, in ssTnI mice, TAC resulted in only a modest increase in both heart weight and heart weight to tibia length, neither one of which were significantly different (Figure 1A&B). Thickening of the left ventricular posterior wall and dilation of the left ventricule during diastole was evident via echocardiography in NTG hearts after TAC, while no significant changes in these parameters where evident in ssTnI hearts after TAC (Figure 1C&D). In agreement with this attenuation of hypertrophic remodeling in ssTnI mice, TAC also failed to upregulate the expression of the fetal isoform of myosin heavy chain, β-myosin heavy chain, which is typically induced in response to chronic pressure-overload²⁰. In contrast, there was a dramatic upregulation of β-myosin heavy chain observed in NTG mice 12–13 weeks after TAC (Figure 1E).

ssTnI Expression Prevented the Decline in Contractile Function Following TAC

Although no changes in in vivo systolic function were apparent from echocardiography (Supplementary Figure 1), diastolic function was significantly affected (Figure 2) following TAC. In NTG mice, there was a clear development of a restrictive filling pattern, indicated by the increase in the E/A ratio (Figure 2A) due to an increase in the E Wave (Figure 2B). A restrictive filling pattern was confirmed by rapid transmitral deceleration of early filling (Figure 2C). This was contrasted by an inverse change in the E/A ratio in ssTnI hearts following TAC, which actually decreased due to a significant increase in the A wave (Figure 2D). An increase in the A wave was necessitated in order to compensate for the prolonged isovolumic relaxation in ssTnI TAC mice (Figure 2E), a potential consequence of the increased calcium sensitivity of the myofilament inherent in the ssTnI mouse²¹. There were no changes in in vivo heart function observed in sham ssTnI mice compared to NTG sham mice.

In isolated perfused hearts, there was a clear reduction in contractile function observed in NTG hearts after TAC (Figure 3A–C) that was absent in ssTnI mice. The decline in rate-pressure product (Figure 3A) and contractility (slower kinetics of contraction (Figure 3B) and relaxation (Figure 3C)) in NTG hearts following the development of hypertrophy was absent in ssTnI hearts.

ssTnI expression induced a greater shift in the metabolic response to TAC

The metabolism of exogenous palmitate and glucose was assessed in isolated hearts at 12–13 weeks post-TAC via ¹³C end-point enrichment analysis of the glutamate pool. Despite the absence of significant contractile changes, hearts from ssTnI mice displayed a robust metabolic remodeling in response to TAC (Figure 4). The relative contribution of palmitate to acetyl CoA formation was significantly decreased (Figure 4A&B), while the relative contribution of glucose increased (Figure 4C). These results suggest that, in ssTnI mice, TAC caused a decrease in palmitate oxidation and an increase in glucose oxidation. In NTG mice, there was a very modest metabolic response to TAC that did not result in significant changes in the relative contributions of the exogenous substrates examined to acetyl CoA formation (Figure 4B&C). The increase in glucose oxidation was at least partially mediated by a reduction in PDK4 expression (Figure 4D). PDK4 controls PDH activity through phosphorylation, which reduces PDH activity. There was also a reduction in the phosphorylation of PDH in ssTnI mice exposed to TAC (Figure 4E), which is in agreement with a reduction in PDK4 expression. No significant changes in total PDH expression was evident in any of the models (Figure 4F).

Attentuated Anaplerotic Flux Response in ssTnI hearts Following TAC Improves Efficiency of Glucose Oxidation

Since the increase in glucose oxidation observed in ssTnI hearts following TAC is indicative of improved coupling of glycolysis and glucose oxidation, we investigated whether the level of anaplerotic flux into the TAC cycle following TAC was altered by ssTnI expression. As expected from previous studies, TAC induced elevations in anaplerotic activity in NTG littermates^1,3,4,8. However, by increasing the relative contribution of glucose to acetyl CoA formation in the ssTnI hearts, the TAC-induced increase in anaplerosis was prevented (Figure 5A). Anaplerotic entry of pyruvate into the TCA cycle is controlled by two enzymes within the heart: malic enzyme 1 (ME1) and pyruvate carboxylase (PC)²². Both ME1 and PC expression were induced by TAC in NTG mice (Figure 5B&C), while PC expression was not induced in ssTnI mice exposed to TAC. Interestingly, however, ME1 expression was higher both at baseline and after TAC in ssTnI hearts (Figure 5B). These results suggest that the downregulation of PDK4 expression plays a key role in reducing anaplerotic flux in the ssTnI hearts exposed to TAC. The downregulation of PDK4 expression reduces PDH phosphorylation to increase PDH activity, thereby allowing PDH to outcompete ME1 for pyruvate.

Energetic Status is Maintained in ssTnI Hearts Following TAC

Hypertrophic remodeling and heart failure are associated with a decline in energetic stores, specifically the PCr:ATP ratio^{1, 2, 9, 23, 24}. As discussed, the increase in anaplerosis in the hypertrophic heart leads to metabolic inefficiencies that may contribute to the decline in energy stores. Since hearts from ssTnI mice showed evidence of improved energetic efficiency of glucose metabolism through a reduction in anaplerosis, the energetic status of the hearts following TAC was investigated. In agreement with the attenuation of anaplerosis in the ssTnI TAC mice, energetic stores were also maintained, as indicated by the PCr:ATP ratio (Figure 6A&B). The lack of a decline in PCr:ATP was confirmed by a failure of TAC to induce an increase in phosphorylation of the energy sensing AMPK in ssTnI TAC mice (Figure 6C&D). In NTG mice, the decline in the PCr:ATP ratio was associated with an increase in AMPK phosphorylation, indicating energy deprivation in these hearts. In contrast, the increased flux from glucose into acetyl-CoA within the TCA cycle in the TAC ssTnI hearts occurred independently of AMPK activation.