We explored the molecular mechanisms that link the genetic defect to the cardiac phenotype in HCM, with a particular focus on myocyte disarray and interstitial fibrosis. We provide several lines of evidence to implicate aldosterone in the pathogenesis of cardiac hypertrophy, fibrosis, and disarray in HCM, including elevated myocardial aldosterone and aldosterone synthase mRNA levels in patients with HCM. Aldosterone provoked expression of cardiac hypertrophic and profibrotic responses in cultured myocytes and fibroblasts through activation of PKD and increased expression of P13K-p110δ
in cardiac myocytes and fibroblasts, respectively. Blockade of PKD and PI3K-p110δ
abrogated the hypertrophic and profibrotic effects of aldosterone. We also performed 2 independent studies and showed that blockade of MR in a genetically engineered mouse model of HCM mutation normalized myocardial collagen content and attenuated myocyte disarray, phenotypes associated with SCD and heart failure in HCM7,27
and improvement in diastolic function. Finally, we provide evidence implicating impaired N-cadherin–mediated myocyte-myocyte attachment, as a consequence of phosphorylation of β
-catenin, in the pathogenesis of myocyte disarray, the hallmark of HCM. Collectively, the results in human hearts, cultured cells, and a genetic animal model of HCM identify aldosterone as a fundamental molecular link between sarcomeric gene mutations and cardiac phenotypes in HCM. These results, if confirmed in additional studies and for other HCM-causing mutations, in conjunction with the results of recent clinical studies showing the beneficial effects of blockade of MR in heart failure13,14
illustrate the need for clinical studies in humans to determine the potential beneficial effects of blockade of MR in HCM.
Aldosterone, discovered 50 years ago,28
is known primarily for its effects on sodium transport in renal tubules, whereas its direct effects on cardiac structure and function are largely unrecognized. The observed increased myocardial aldosterone levels in patients with HCM is in accordance with the results of recent studies showing extraadrenal gland synthesis of aldosterone, including studies in cardiac myocytes and fibroblasts and the myocardium of patients with heart failure.19,27,29,30
In the adrenal glands and cultured cells, expression of CYP11B2
is regulated by a diverse array of factors, including angiotensin II,31
and calmodulin-dependent protein kinases.35
These factors, alone or in combination, are plausible candidates to affect expression of CYP11B2
in HCM hearts. In the present study, myocardial cAMP levels and lipid peroxide levels, an overall index of oxidative stress, were not significantly elevated (data not shown), perhaps because they were less stable in the stored tissue. The molecular mechanisms that regulate expression of CYP11B2
and production of aldosterone in the heart in general and in HCM in particular remain to be established.
Aldosterone imparts a diverse array of biological effects comprising genomic (slow) and nongenomic (rapid) components, including but not limited to changes in the intracellular calcium concentration,36
and activation of extracellular signal-regulated kinases p22/44,25
The present findings implicate PKD and PI3K-p110δ
as the molecular mediators of cardiac hypertrophic and profibrotic effects and provide for novel mechanisms for the actions of aldosterone. Notably, neither PKD nor PI3K-p110δ
has been implicated previously in cardiac hypertrophy and fibrosis. The results, in conjunction with the existing data implicating PKD in cell proliferation, invasiveness, apoptosis, and cellular morphology (reviewed in Rykx et al38
), render PKD as an attractive molecular target in cardiomyopathies. Similarly, identification of PI3K-p110δ
as an effector for aldosterone and its involvement in cardiac fibrosis has broader clinical implications, because fibrosis is a common pathological phenotype of cardiovascular diseases and is considered a predictor of cardiac arrhythmias and SCD.39
Furthermore, because p110δ
is also implicated in the regulation of actin cytoskeleton dynamics and cell migration,40
upregulation of its expression by aldosterone could also contribute to regulation of cardiac myocyte organization, ie, myocyte disarray.
The present data indicate that impaired myocyte-myocyte attachment at AJ because of impaired phospho-β
-catenin and N-cadherin complexing is responsible, at least in part, for myocyte disarray in HCM. The extracellular domains of cadherins form the intercellular bonds between adjacent myocytes, and the cytoplasmic domains attach to cytoskeletal actin through β
-catenin and other effector proteins. The integrity of AJ is essential for the proper alignment of myocytes. Alternative mechanisms for the observed reduction of myocyte disarray could include normalization of interstitial fibrosis and thus restoration of myocardial architecture. It is also possible that increased extracellular matrix proteins in HCM, by activating the integrin/wnt signaling pathways, affect the integrity of the β
-catenin–cadherin–axin complex and thus myocyte morphology and arrangement.41
Accordingly, multiple interdependent mechanisms could be involved in the pathogenesis of myocyte disarray in HCM and its attenuation by spironolactone.
The results of 2 independent, randomized, placebo-controlled studies with spironolactone in cTnT-Q92 mice were concordant and in accordance with the mechanisms delineated for the effects of aldosterone in cell culture studies. Detailed quantification of the extent of myocyte disarray in the primary and replication studies, scored in a total of 11 880 microscopic fields in 23 mice per group, showed a 50% reduction with spironolactone (4.2±1.8 in nontransgenic mice, 19.3±8.0 in the placebo mice, and 9.5±3.0 in the spironolactone group; P
<0.001), which is beyond the 3% to 4% interindividual and intraindividual variabilities. Reversal of interstitial fibrosis with spironolactone is also in agreement with the results of previous observations in other animal models of cardiovascular disorders.19-22,29,42
As is often the case in humans with cTnT mutations,43,44
the cTnT-Q92 mice do not exhibit cardiac hypertrophy.17,45
Therefore, the impact of spironolactone on potential regression of cardiac hypertrophy in vivo could not be assessed. Treatment with spironolactone further reduced the heart weight/body weight ratio (3.5±0.5 versus 4.0±0.7 in the placebo group, P
<0.001). Because myocyte CSA did not change, the reduction in heart weight/body weight ratio in the spironolactone group most likely represents the reduction in interstitial fibrosis. Because human patients with the cTnT-Q92 mutation exhibit extensive myocyte disarray, a potential risk factor for SCD,44
the findings raise the possibility of reducing the risk of SCD in HCM through blockade of MR, a hypothesis that awaits testing in humans.
In summary, the present data implicate aldosterone in the pathogenesis of the cardiac phenotype in HCM and identify novel mechanisms for its hypertrophic and profibrotic effects by implicating PKD and PI3K-p110δ, respectively. Our data also implicate impaired N-cadherin–β-catenin complexing at AJ as a mechanism for myocyte disarray, the pathological hallmark of HCM. Finally, our data establish the reversibility of cardiac phenotypes through blockade of MR. These findings, in the absence of a specific therapy for human HCM, if confirmed in additional studies, emphasize the need for clinical studies to determine potential salutary effects of blockade of MR in human patients with HCM.