Introduction:
Hyperpolarization-activated cation (HCN) channels encode the pacemaker current If that contributes to generation and regulation of sinus rhythm, with HCN4 representing a key component in the cardiac conduction system. Recent studies have identified an increased ventricular expression of HCN4 in patients with hypertrophic cardiomyopathy and heart failure, however the mechanistic involvement of HCN4 in pathogenesis remains unresolved.
Methods:
We generated transgenic mice that overexpress human HCN4 under the regulation of a cardiac-specific cTNI promoter (HCN4+ mice) to study the contribution of HCN4 overexpression to structural heart disease. Molecular mechanisms were analyzed by quantitative Real-time PCR, western blot and immunohistochemistry.
Results:
HCN4+ mice were viable and exhibited high expression of HCN4 in both atria and ventricles, with HCN4 protein abundantly targeted to the plasma membrane. Real-time PCR analysis showed a 71% reduction in transcript levels of the endogenous murine HCN4 gene. Morphological evaluation at different developmental stages revealed right ventricular dilation and progressive decrease in wall thickness after one month of age, lacking overt myocardial hypertrophy, significant fibrosis or signs of myocardial inflammation. On the cellular level, the changes were characterized by apoptotic cell loss induced by strong transient expression of mu-calpain Ca2+-sensor and tissue transglutaminase (243% and 397% of wild type, respectively), which resulted in a robust activation of caspase 3. Of note, markers of pathological hypertrophy, that is atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and beta-myosin heavy chain (beta-MHC) remained unchanged. At the same time transcription of the physiological cell growth markers glycogen synthase kinase 3-beta (GSK3B) and mammalian target of rapamycin (mTOR) was elevated.
Conclusion:
HCN4 overexpression leads to calpain-mediated apoptosis and is associated with right ventricular dilated cardiomyopathy in a transgenic mouse model.
Real-Time Optical Manipulation of Cardiac Conduction in Intact Hearts