We investigated the effects of CPT1b deficiency on the pathological development of cardiac hypertrophy and remodeling under the left ventricular pressure-overload condition in mice. We previously demonstrated cardiac hypertrophy in mice with other FAO enzyme deficiencies, which is most predominant in homozygous long-chain acyl-CoA dehydrogenase deficiency.30
The most important finding based on the genetic mouse model of CPT1b deficiency is that CPT1b deficiency causes lipotoxicity in the heart under the pressure-overload condition and leads to exacerbate cardiac pathology.
CPT1b is the predominant CPT1 isoform expressed in the heart and plays an essential role in myocardial FAO. Repressing myocardial FAO has been proposed as a therapeutic target to improve cardiac efficiency in the failing heart. Inhibitors of CPT1 have already been developed and tested in preclinical animal studies and small clinical trials.18, 19
However, it remains controversial due to the mixed results from animal studies. Schwarzer et al.
showed that etomoxir failed to reverse pressure-overload induced heart failure in vivo
Strikingly, the most studied compound etomoxir has been shown to exert adverse effects that eventually lead to cardiac hypertrophy.31–34
It was proposed that etomoxir treatment may induce cardiac hypertrophy via increased cellular oxidative stress and NF-κB activation.35
Wolkowicz et al.
showed that another CPT1 inhibitor, 2-tetradecylglycidic acid induces myocardial hypertrophy via the AT1 receptor.11
It is clear that the potential adverse effects of CPT1 inhibitors may not be just a nonspecific side effect. It may be associated with the irreversible inhibition of CPT1 activity in the heart. The present study provides evidence to support that partial CPT1b deficiency in a mouse model of CPT1 knockout is detrimental to cardiac structure/function due to pressure-overload induced LV systolic dysfunction. CPT1b+/− mice showed a more pronounced systolic dysfunction, but remained in concentric hypertrophy. It is likely that most CPT1b+/− hearts were still at the stage prior to the transition to dilated cardiomyopathy. Therefore, we could not rule out the possibility that CPT1 inhibition might exert beneficial effects only in dilated cardiomyopathy. Additionally, it is possible that inhibitors and the gene knockout will exhibit distinct functions when the corresponding proteins have non-enzymatic functions as a scaffold.
To our knowledge, the present study is the first study based on a gene targeted mouse model with CPT1b deficiency. While a complete knockout of CPT1b causes embryonic lethality, the heterozygous CPT1b knockout mice are overtly normal. It is noted that in the heterozygous CPT-1b knockout heart, CPT1b expression was blunted in both transcript and protein levels. Since CPT1a is also expressed in the adult heart at low level, a compensatory upregulation of CPT1a is possible. In fact, the inverse response of increased Cpt-1b expression in livers of diet challenged CPT-1a+/− mice was reported.36
However, transcript expression of CPT1a was unchanged in the CPT1b deficient hearts. Since the total CPT1 activity was decreased by about 30% compared to WT littermates and CPT1 activity is upregulated during at least the early stage of cardiac hypertrophy, the depression of CPT1 activity in the CPT1b deficient heart appears to be the key determinant for the detrimental response to pressure-overload induced cardiac pathology. The partial CPT1b deficiency in the heart seems insufficient to affect basal cardiac performance and cardiac metabolism. This result does not support previous observations that partial inhibition of CPT1 activity by CPT1 inhibitors, such as etomoxir, leads to cardiac dysfunction and hypertrophy under physiological conditions.35,11
Therefore, it is likely that the detrimental cardiac effects of etomoxir treatment under normal physiological conditions may be associated with effects unrelated to CPT1 inhibition or more severe cardiac CTP1 inhibition (30% in CPT1b+/−
vs 40–50% with etomoxir treatment37
The most important finding in the present study is that the CPT1b+/−
mice were much more susceptible to pressure-overload induced pathological cardiac hypertrophy, suggesting that partial CPT1b deficiency is detrimental with pathological development under mechanical stress conditions. This result is in sharp contrast to those using CPT1 inhibitors in human studies.18,19
The reasons for this obvious discrepancy may be derived from different degrees of CPT1 inhibition among studies, non-specific effects of CPT1 inhibitors, or the existence of certain levels of CPT1a activity in CPT1b knockout hearts. Importantly, the current finding on the detrimental effects of CPT1b deficiency is against the basic concept of fatty acid oxidation inhibition as a therapeutic approach in treating heart failure patients. A specific site and specific quantity of the inhibition could be crucial. Moreover, our results demonstrated that mitochondrial FAO and CPT1b activity were upregulated in mitochondria samples from hearts with pressure-overload induced hypertrophy. It is likely that an increased CPT1 activity is crucial to maintain mitochondrial FAO among the remaining mitochondria during the development of pathological cardiac hypertrophy. However, the suppressed mitochondrial biogenesis and function in the heart may potentially impair overall myocardial FAO.
Lipotoxicity in addition to the pressure-overload associated systolic dysfunction appears to be the cause of the detrimental effect of CPT1b deficiency under pathological conditions. Patients with inborn errors of FAO typically manifest cardiomyopathy with diminished systolic function.38
Moreover, intramyocardial lipid accumulation is associated with contractile dysfunction in heart tissues from patients with non-ischemic heart failure.39
The reduction of mitochondrial biogenesis should be the consequence of the progression of cardiac pathology and cardiomyocyte apoptosis. The increased sympathetic activity in response to hemodynamic overload might lead to increased lipolysis. While this appears to be the case, it is insufficient to activate PPAR signaling to increase the expression of fatty acid uptake proteins. Instead, the expression of fatty acid uptake proteins was further decreased, possibly due to the exacerbated cardiac pathological development. Therefore, it is plausible that the increased myocardial triglyceride content in CPT1b+/−
hearts is due to the relative reduction of mitochondrial FAO and mitochondrial biogenesis. As a result, myocardial lipid accumulates and feeds the ceramide synthesis. Our observation is in agreement with the previous report that oxfenicine induced myocardial lipid accumulation in rats.40, 41
Myocardial lipid accumulation and the elevation of ceramide content in the heart should be the mechanisms underlying the detrimental effects during the development of pathological cardiac hypertrophy in response to the pressure-overload condition. The cytotoxic effect of ceramide in cardiomyocytes has been well established.42, 43
Ceramide induces apoptosis of cardiomyocyte in vitro
and in vivo
In the PPARγ overexpression heart, myocardial lipid accumulation and increased ceramide content has been observed accompanying cardiomyocyte apoptosis.47
Therefore, the increased ceramide content in the CPT1b deficient heart subjected to TAC is likely to trigger the apoptosis signaling in the heart and aggravates the pathological development.
One limitation of the current study is that the CPT1b deficiency pre-existed, hence we are cautious to avoid over-interpreting this result as CPT-1 inhibitors are used as treatment in heart failure patients. We cannot rule out the possibility that the initial development of the pressure-overload induced cardiac hypertrophy is exceptionally susceptible to CPT1b deficiency. A conditional gene-targeting model of CPT1b could provide more in-depth insights into the effects of CPT1b deficiency in the heart during the various stages of cardiac hypertrophy and heart failure. Despite this limitation, the current finding clearly demonstrates the necessity to further evaluate the use of CPT1 inhibitors as a therapeutic approach to treat patients with cardiac hypertrophy and even heart failure.
The present study demonstrates in a genetic mouse model that partial CPT-1 deficiency is not sufficient to cause cardiac dysfunction under the normal physiological conditions. However, it is detrimental in animals subjected to TAC-induced LV pressure-overload. CPT1b deficiency exacerbates the cardiac pathological development induced by left ventricular pressure-overload due to myocardial lipotoxicity.