Creatine kinase-mediated improvement of function in failing mouse hearts provides causal evidence the failing heart is energy starved

Abstract

ATP is required for normal cardiac contractile function, and it has long been hypothesized that reduced energy delivery contributes to the contractile dysfunction of heart failure (HF). Despite experimental and clinical HF data showing reduced metabolism through cardiac creatine kinase (CK), the major myocardial energy reserve and temporal ATP buffer, a causal relationship between reduced ATP-CK metabolism and contractile dysfunction in HF has never been demonstrated. Here, we generated mice conditionally overexpressing the myofibrillar isoform of CK (CK-M) to test the hypothesis that augmenting impaired CK-related energy metabolism improves contractile function in HF. CK-M overexpression significantly increased ATP flux through CK ex vivo and in vivo but did not alter contractile function in normal mice. It also led to significantly increased contractile function at baseline and during adrenergic stimulation and increased survival after thoracic aortic constriction (TAC) surgery-induced HF. Withdrawal of CK-M overexpression after TAC resulted in a significant decline in contractile function as compared with animals in which CK-M overexpression was maintained. These observations provide direct evidence that the failing heart is "energy starved" as it relates to CK. In addition, these data identify CK as a promising therapeutic target for preventing and treating HF and possibly diseases involving energy-dependent dysfunction in other organs with temporally varying energy demands.

Figure 1. Effects of CK-M overexpression on protein content and CK activity.

(A) Western blots and summary results, (B) CK-M protein expression, and (C) total CK activity (IU/mg protein) in control mice on a doxycycline (doxy) diet (n = 6), control on regular (reg) diet (n = 10), CK-M/tet on a doxycycline diet (n = 6), and CK-M/tet on regular diet (n = 7) mouse hearts. Results are mean ± SD. ^#P < 0.01, ^‡P < 0.005.

Figure 2. CK expression and activity in TAC hearts.

Effect of TAC and CK-M overexpression on CK-M protein expression (A and B) and CK activity (C) in control sham, control TAC, and CK-M TAC isolated mouse hearts. Results are mean ± SD; n = 6–17 each group. *P < 0.05, ^†P < 0.001.

Figure 3. Perfused heart ³¹P MR spectroscopy.

Representative ³¹P saturation transfer MR spectra are shown for (A and B) sham, control, and CK-M overexpressors, respectively; (C and D) 4-week TAC, control, and CK-M overexpressors; and for (E and F) 9-week TAC, control, and CK-M overexpressors. Spectra were acquired in the presence of saturating irradiation (arrows) either in the control (left spectrum in each pair) or γ-ATP position (right spectrum). The decrease in the height of PCr peak between control and γ-ATP saturation (dotted lines) is directly related to the rate of ATP synthesis through the CK reaction.

Figure 4. Summary of energetic findings in perfused hearts.

ATP concentration (μmol/g of wet weight), PCr/ATP ratio, myocardial T₁′ for PCr(s), pseudo-first-order forward rate constant, k_f (s^–1), and ATP flux through CK (μmol/g/s) in control (white bars) and CK-M overexpressing (black bars) mouse hearts in the sham (A), 4-week TAC (B), and 9-week TAC (C) groups. Results are mean ± SD; n = 5–10 in each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5. Functional and survival effects of CK-M overexpression in TAC hearts.

(A) Time course of experimental protocol and (B) typical transverse in vivo ¹H MR images of the mid-LV at end-systole and end-diastole after 8 weeks of sham or TAC surgery in control and CK-M mice. (C) Summary MRI findings (mean ± SD) at 4 and 8 weeks showing EF, SV, and CO in CK-M sham (open circles, n = 8) and control sham (X, n = 9) mice, as well as in CK-M TAC (filled circles, dashed lines, n = 8) and control TAC mice (filled diamonds, solid lines, n = 13). (D) In vivo cardiac PCr/ATP ratio in control sham (n = 9), control with TAC (n = 13 at 4 weeks, n = 9 at 8 weeks), CK-M overexpressor (Overexp) sham (n = 8), and CK-M with TAC (Overexp TAC; n = 8 at 4 weeks, n = 7 at 8 weeks) mouse hearts. (E) Kaplan-Meier survival curve showing improved survival following TAC in CK-M mice (dotted line n = 18) as compared with control mice (solid line, n = 17). *P < 0.05, **P < 0.001, ***P < 0.0001.

Figure 6. Reversible functional effects of CK-M overexpression in TAC hearts.

(A) Time course of experimental protocol and (B) summary of in vivo cardiac functional parameters (mean ± SD) derived from MRI; EF, SV, and CO in control animals (gray bars), CK-M animals with the CK-M transgene turned “on” during the entire experiment (black bars), and CK-M animals with the CK-M transgene turned “off” after 4 weeks of TAC (cross-hatched bars). n = 5–10 animals in each group. *P < 0.05, ^‡P < 0.01, ^#P = 0.07, **P < 0.001.

Figure 7. Functional response to adrenergic stress.

Effects of dobutamine stress on heart rate (HR; A and B), EF (C and D), and LV ESV (E and F) in sham (A, C, and E) and TAC animals (B, D, and F) in both control (gray bars) and CK-M “on” (black bars) animals (n = 6–10 in each group). The change induced by dobutamine (as percentage of baseline values) is shown in the right plot of each panel. Note that although the contractile response of control and CK-M overexpressing animals was similar under sham conditions (A, C, and E), the dobutamine-induced changes in mean EF and ESV were significantly greater in CK-M TAC hearts than in control TAC hearts (B, D, and F). Results are mean ± SD. ^‡P < 0.005, **P < 0.001.

Figure 8. In vivo determination of ATP synthesis rates through CK in the mouse heart.

(A) Typical transverse ¹H MR image of a mouse at the mid-LV with the nominal location of ³¹P MR cardiac voxel denoted between the white lines. (B) ³¹P MR spectrum with control saturation and TR = 10 seconds and NEX = 16. (C) Spectrum with γ-phosphate of ATP saturated with TR = 6 seconds, NEX = 32. (D) Spectrum with γ-phosphate of ATP saturation with TR = 1.5 seconds, NEX = 96. β-ATP; β-phosphate of ATP. (E–I) Summary of in vivo energetics (mean + SD) for control no-TAC (Control, n = 11), control with TAC (Control TAC, n = 10), CK-M overexpressors no-TAC (Overexp, n = 8), and CK-M overexpressors with TAC (Overexp TAC, n = 7) mice. (E) PCr/ATP ratio. (F) PCr concentration (μmol/g wet weight). (G) ATP concentration (μmol/g wet weight). (H) CK forward pseudo-first-order rate constant (k_f, s^–1). (I) rate of ATP synthesis through CK (CK flux, μmol/g/s). Some of the data in control mice, but not CK-M overexpressers, were previously reported (12). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 9. Isolated myocyte function and calcium transients.

Sarcomere shortening (A), sarcomere shortening time (B), calcium transient amplitude (C), and calcium relaxation time (D) in myocytes isolated from control (n = 3 sham and n = 6 TAC) and CK-M–overexpressing (n = 3 sham and n = 6 TAC) hearts, with 23–51 myocytes in each group. *P < 0.05 versus baseline control; ^#P < 0.05 versus baseline CK-M; ^§P < 0.05 versus TAC control.