Background

Barth syndrome (BTHS) is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and delayed growth. BTHS is caused by mutations in the tafazzin (Taz) gene. Taz catalyzes the final step in the biosynthesis of cardiolipin, a mitochondrial phospholipid. We previously identified a cardiac-specific defect in mitochondrial calcium (Ca²⁺) uptake in Taz-knockdown (KD) mice, due to structural reorganization of the mitochondrial Ca²⁺ uniporter (MCU). Here, we investigated the consequences of defective cardiolipin remodeling and MCU loss on cardiac mechano-energetic coupling in Taz-KD mice.

Methods and Results

To investigate the mechanisms underlying cardiomyopathy in Taz-KD mice, we analyzed cellular contractility, cytosolic Ca²⁺ handling, and mitochondrial redox state (by NAD(P)H and FAD autofluorescence), in isolated, field-stimulated cardiac myocytes.

Taz-KD cardiac myocytes displayed enhanced contractility and shorter diastolic sarcomere length compared to wild-type (WT) at baseline (0.5 Hz pacing). However, diastolic cytosolic Ca²⁺ levels ([Ca²⁺]_c) and amplitude of Ca²⁺ transients did not differ between genotypes. Although the rate of [Ca²⁺]_c decay was markedly faster than WT, Taz-KD myocytes exhibited prolonged kinetics of sarcomere re-lengthening that progressed between 10 and 50 weeks of age. Combined β-adrenergic stimulation (with isoproterenol) and 5 Hz pacing increased sarcomere shortening in WT and Taz-KD, but this was not accompanied by an increase in Ca²⁺ transient amplitudes in Taz-KD myocytes. Since [Ca²⁺]_c decay kinetics are governed primarily by SR Ca²⁺ ATPase (SERCA) activity, we determined SR Ca²⁺ load by caffeine pulses after pacing at 0.5 Hz and isoproterenol/5Hz. While caffeine-induced SR Ca²⁺ release was similar between genotypes under both conditions, rate of [Ca²⁺]_c decay after caffeine was accelerated in Taz-KD vs. WT myocytes, indicating accelerated Ca²⁺ extrusion via the Na⁺/Ca²⁺ exchanger.

In Taz-KD myocytes, defective mitochondrial Ca²⁺ accumulation hindered the Ca²⁺-dependent regeneration of NAD(P)H and FADH₂ by the Krebs cycle dehydrogenases, inducing oxidation of mitochondrial redox state during combined β-adrenergic and 5 Hz stimulation. To interrogate whether in Taz-KD myocytes, compromised Ca²⁺-dependent adaptation of Krebs cycle activity and thereby, ATP production influence contractile function, we exposed myocytes to pinacidil, which sensitizes sarcolemmal ATP-dependent K⁺ (K_ATP) channels. While in WT myocytes, pinacidil did not affect cellular contractility, in Taz-KD myocytes pinacidil blunted the increase in sarcomere shortening in response to Iso/5Hz, suggesting that drug-primed K_ATP channels become activated by a reduced mitochondrial ATP/ADP ratio under these conditions. Finally, in mechanically prestreched myocytes, the physiological potentiation of force development in response to an increase in pacing frequency was completely absent and even negative in Taz-KD cardiac myocytes.

Conclusions

We identify disruption of the mechano-energetic coupling reserve as a primary defect in BTHS cardiomyopathy. Impaired bioenergetic adaptation due to deficient mitochondrial Ca²⁺ uptake is accompanied by maximal recruitment of cardiac inotropic and lusitropic reserve, which prevents the physiological increase in contractility during elevations of workload. These data provide mechanistic insight into the inability of BTHS patients to increase contractility during physical exercise.