ASK1 has emerged as a kinase of central importance in cardiac myocytes given its dominant role in regulating MAPK signaling and subsequent control of cell death. To gain additional insight into the full range of ASK1 function in the heart we generated inducible transgenic mice to overexpress this protein in the adult heart. While overexpression approaches in the mouse heart can produce non-physiologic effects, numerous kinases and phosphatases have been overexpressed as a means of gaining functional insight. For example, overexpression of activated calcineurin or activated MEK1 in the mouse heart provided important mechanistic data regarding the function of these signaling factors in regulating the cardiac hypertrophic response.29,30
Also to be considered, not all overexpressed signaling proteins in the heart produce a phenotype, as ERK2 overexpression with the αMHC promoter was without effect, nor did it enhance pressure overload hypertrophy.30
In a similar manner, ASK1 transgenic mice had no discernable phenotype up to 12 months of age, despite very high levels of overexpression.
ASK1 overexpression in the heart was likely without baseline effect given the many levels of regulation imposed on this kinase. ASK1 is uniquely activated by phosphorylation of threonine 845, while dephosphorylation of serine 83, serine 967, and serine 1034 results in activation.27
We previously determined that calcineurin enhances ASK1 activation through direct dephosphorylation of serine 967 and an indirect increase in threonine 845 phosphorylation.19
ASK1 is also bound to and inhibited by thioredoxin and glutaredoxin, which upon oxidative stress are released allowing ASK1 activation.31,32
Even though we failed to identify increases in thioredoxin protein in the hearts of ASK1 DTG mice at baseline or after TAC, endogenous levels of thioredoxin may be in excess and fully capable of silencing overexpressed ASK1.
The most prominent biologic effect observed in ASK1 overexpressing mice was a sensitization to cell death following stimulation. ASK1 transgenic mice exhibited greater cardiac TUNEL after MI and following 8 weeks of pressure overload stimulation. ASK1 DTG mice also exhibited a more than 2-fold increase in I-R injury compared with control mice. The I-R model is particularly relevant given ROS generation during reperfusion, which should liberate ASK1 from thioredoxin and glutaredoxin. Previous work showed that Ask1−/−
mice had reduced cardiomyocyte apoptosis and TUNEL in response to angiotensin II infusion,15
or in response to 4 weeks of pressure overload stimulation or 4 weeks after MI.16
Cardiomyocytes generated from Ask1−/−
mice were also resistant to H2
- and calcium overload-induced apoptosis.16,17
Finally, deletion of Ask1
in the mouse rescued cardiomyopathy and the increase in cardiac apoptosis associated with a cardiac-specific deletion of the c-Raf-1
Thus, our results in ASK1 overexpressing TG mice are consistent with data obtained in Ask1−/−
mice, together indicting that ASK1 plays a critical role in regulating cardiomyocyte death, possibly due to increased JNK1/2 activity and upregulated Bax.
In contrast to the cell death observations, ASK1 transgenic mice showed no increase in myocyte or whole organ hypertrophy following TAC or isoproterenol stimulation for 2 weeks compared with controls. Even after 8 weeks of TAC, ASK1 transgenic mice showed no increase in myocyte cross-sectional areas compared with controls. Importantly, pressure overload stimulation is known to potently activate ASK1 in the adult mouse heart,16
so if ASK1 truly functioned as a hypertrophic regulator, the overexpressing mice should have shown enhancement in this process. Ironically, we previously observed increased cardiomyocyte hypertrophy by adenoviral infection with a dominant negative ASK1 mutant, while overexpression of WT ASK1 suppressed hypertrophy due to calcineurin, phenylephrine and FBS stimulation.19
Not surprisingly, results in cultured neonatal myocytes are sometimes at odds with results obtained in genetically modified mouse models, in part because of the variability in the culture model itself. Indeed, other studies in cultured myocytes suggest that overexpression of activated ASK1 actually induced cardiomyocyte hypertrophy in culture, while overexpression of dominant negative ASK1 attenuated hypertrophy.18
Previous results in genetically modified mouse models are similarly unclear in defining the role of ASK1 in regulating cardiac hypertrophy. For example, Ask1−/−
mice showed reduced hypertrophy following angiotensin II infusion, suggesting that ASK1 could positively regulate cardiac growth in vivo
However, TAC stimulation did not result in less cellular hypertrophy in Ask−/−
mice compared with WT mice, suggesting that ASK1 is not required in vivo
for successful pressure overload hypertrophy.16
More recently, Ask1−/−
mice were actually shown to have enhanced physiologic hypertrophy following swimming exercise, suggesting that ASK1 antagonizes the adaptive growth response.21
Our results in ASK1 overexpressing TG mice suggest that ASK1 is not a central regulator of the pathologic hypertrophy response. The discordance in results from the various studies in Ask1−/−
mice and in cultured myocytes may reflect secondary effects associated with increased cell death or greater propensity towards cardiomyopathy. Indeed, 8 weeks of TAC stimulation produced increased heart-weights normalized to body-weight in ASK1 transgenic mice, although more careful inspection of these mice revealed greater ventricular dilation as the causative factor in affecting total heart weights. It is even more complicated when one attempts to invoke an underlying mechanism for an effect on hypertrophy, as ASK1 transgenic mice showed inhibition NFAT activity following TAC stimulation, but hypertrophy was not inhibited. To explain this effect, it is likely that ASK1 regulates other growth effecting pathways that might counteract this anti-hypertrophic effect, such as alterations in MAPK signaling. Second, ASK1 overexpression induces greater cell death with some degree of cardiomyopathy that likely secondarily enhances the cardiac hypertrophic response through greater neuroendocrine dysfunction. Thus, ASK1 is likely a disease modifying kinase that can secondarily impacts cardiac hypertrophy and heart failure through a primary mechanism involving cell death, ventricular remodeling, and other uncharacterized effects.
We previously observed that ASK1 overexpression in neonatal cardiomyocytes induced activation of p38 and JNK, as well as inhibition of calcineurin-NFAT signaling.19
Otsu and colleagues similarly observed that TAC stimulation in Ask1−/−
mice resulted in defective cardiac JNK activition.16
We failed to observe an increase in p38 activation in the heart with ASK1 overexpression after TAC stimulation, although we did observe enhanced activation of MKK4/6 and JNK1/2. It is possible that p38 was not induced in the hearts of ASK1 DTG mice because stimuli other than TAC are needed to induce coupling between ASK1 and the p38 branch of the MAPK cascade when ASK1 is in abundance. Indeed, ASK1 might serve a scaffold function, such that its overexpression affects p38 signaling in a different manner from that observed in Ask1−/−
mice. However, ASK1 overexpression did show coupling to the JNK1/2 signaling branch in the heart after TAC stimulation, an effect known to alter the cell death response.33
The interconnectivity between calcineurin-NFAT and ASK1 signaling circuits is even more intricate, as calcineurin appears to be required for ASK1 activation through dephosphorylation of serine 967 in cultured cardiomyocytes.19
Here we extended this later observation in our transgenic mice. Specifically, we crossed the ASK1 transgene into the CnAβ−/−
background as a way of reducing total calcineurin activity in the heart, as we have previously characterized.26
Consistent with our in vitro
loss-of-function experiments, the CnAβ−/−
background attenuated ASK1’s ability to promote cardiomyopathy upon pressure overload stimulation. Calcineurin is known to play a critical role in altering the decision of death versus survival of cardiomocytes in response to stress stimulation.23
Thus, ASK1 is a highly interconnected signaling effector that holds potential therapeutic relevance, such that inhibitors against this kinase might be cardioprotective in response to diverse disease stimuli that result in cardiomyopathy.