Elevated levels of the cardiac transcription factor Heart- and neural crest derivatives-expressed protein 1 (Hand1) have been reported in several cases of adult cardiac dysrhythmia. Cardiac dysrhythmia is also known as arrhythmia or irregular heartbeat and covers a large heterogeneous group of conditions in which there is abnormal electrical activity in the heart which results in irregular heart rate or rhythm. In many cases cardiac dysrhythmia is not life-threatening, however in some cases it can result in cardiac arrest.
Hand1 is a basic helix-loop-helix transcription factor expressed at high levels in the embryonic heart, and is essential for normal chamber formation during embryonic development. Hand1 expression has been shown to be elevated in human and rat hearts from cardiac disease cases when compared with aged matched control cases.
Ross A. Breckenridge, Robert Orford and colleagues at the MRC National Institute for Medical Research (NIMR) developed a mouse model of cardiac arrhythmia to investigate the role that Hand1 plays in cardiac dysrhythmia. The group developed an inducible transgenic system to over-express Hand1 in adult mouse heart using a binary genetic (Tet-on) approach (TetO Hand1 transgenic mouse model). Over expression of Hand1 in adult mouse heart resulted in mild cardiac hypertrophy with a reduction in life expectancy, no significant fibrosis, myocyte disarray or congestive heart failure was observed, but the mice did have a reduced threshold for induced ventricular tachycardia, indicating a predisposition to cardiac arrhythmia.
Breckenridge RA, … Robert Orford et al. 2009. “Overexpression of the transcription factor Hand1 causes predisposition towards arrhythmia in mice”. Journal of Molecular and Cellular Cardiology, 47 (2009) , Pages 133-141
Robert Orford (NIMR) commissioned Cambridge Research Biochemicals (CRB) to raise a custom sheep polyclonal antibody to the peptide sequence: C-GRESKRKRELPQQP-amide, which corresponded to an N’ terminal region of the mouse Hand1 protein. Crude anti-serum was purified using a thiopropyl Sepharose affinity column derivatised with the peptide antigen.
The affinity purified Hand1 antibodies were then used in Western blotting studies to determine the specificity of the custom generated antibody. The Hand1 antibodies detected a 27kDa band in HL1 atrial cells transfected with the recombinant Flag tagged Hand1 protein. Pre-blocking of the Hand1 antibody with the immunogenic peptide abolished the 27 kDa band. An anti- FLAG antibody was used to confirm expression of the flag tagged Hand1 protein and the banding detected coincided with the molecular weight banding observed with the custom generated Hand1 antibody (data not shown).
The Hand1 antibody was then used to determine the levels of Hand1 in Hand1 unregulated transgenic mouse hearts with and without doxycycline induction. Doxycycline was administered to induce expression of the Hand1 gene in the transgenic mice.
The study findings suggest that the over expression of Hand1 offers a useful mouse model of cardiac arrhythmia and sudden death. Moreover, Hand1 may provide a molecular link between the failing heart and tacharrhythmia. The Hand1 antibody generated will prove to be a useful tool for studying the functional and clinical importance of Hand1 in cardiac dysrhythmia.