Functional characterization of the human α-cardiac actin mutations Y166C and M305L involved in hypertrophic cardiomyopathy.

Inherited cardiomyopathies are caused by point mutations in sarcomeric gene products, including α-cardiac muscle actin (ACTC1). We examined the biochemical and cell biological properties of the α-cardiac actin mutations Y166C and M305L identified in hypertrophic cardiomyopathy (HCM). Untagged wild-type (WT) cardiac actin, and the Y166C and M305L mutants were expressed by the baculovirus/Sf9-cell system and affinity purified by immobilized gelsolin G4-6. Their correct folding was verified by a number of assays. The mutant actins also displayed a disturbed intrinsic ATPase activity and an altered polymerization behavior in the presence of tropomyosin, gelsolin, and Arp2/3 complex. Both mutants stimulated the cardiac ß-myosin ATPase to only 50 % of WT cardiac F-actin. Copolymers of WT and increasing amounts of the mutant actins led to a reduced stimulation of the myosin ATPase. Transfection of established cell lines revealed incorporation of EGFP- and hemagglutinin (HA)-tagged WT and both mutant actins into cytoplasmic stress fibers. Adenoviral vectors of HA-tagged WT and Y166C actin were successfully used to infect adult and neonatal rat cardiomyocytes (NRCs). The expressed HA-tagged actins were incorporated into the minus-ends of NRC thin filaments, demonstrating the ability to form hybrid thin filaments with endogenous actin. In NRCs, the Y166C mutant led after 72 h to a shortening of the sarcomere length when compared to NRCs infected with WT actin. Thus our data demonstrate that a mutant actin can be integrated into cardiomyocyte thin filaments and by its reduced mode of myosin interaction might be the basis for the initiation of HCM.

Autocrine signaling via A(1) adenosine receptors causes downregulation of M(2) receptors in adult rat atrial myocytes in vitro.

G protein-activated K(+) channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G(ßγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R. Adenovirus-driven overexpression of the A(1)-R caused an increase in current induced by adenosine (I(K(Ado))), whereas the M(2)-R-activated current (I(K(ACh))) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M(2)-R resulted in a reduction in I(K(ACh)) without affecting I(K(Ado)), arguing against a competition of the two receptors for common signaling complexes. The negative effect of A(1)-R overexpression on I(K(ACh)) was reduced by the A(1)-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I(K(ACh)) and I(K(Ado)), suggesting common pathways of A(1)-R and M(2)-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I(K(ACh)) and I(K(Ado)). Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A(1)-R in an autocrine fashion. Chronic activation of A(1)-R causes parallel downregulation of both A(1)-R and M(2)-R.

Adenovirus-mediated delivery of short hairpin RNA (shRNA) mediates efficient gene silencing in terminally differentiated cardiac myocytes.

RNA interference (RNAi) represents the most frequently utilized technique to analyze proteins by loss of function assays. Protein synthesis is impaired by sequence-specific degradation of mRNA, which is triggered by short (19-28 nt) silencing RNAs (siRNA). Efficient gene silencing using RNAi has been demonstrated in numerous cell lines and primary cultured cells. Incorporation of siRNA into terminally differentiated mammalian cells, such as adult cardiac myocytes is limited by their resistance to standard transfection protocols. Viral delivery of short-hairpin RNA (shRNA) overcomes these limitations and allows efficient gene silencing in these cells. This chapter describes the generation and characterization of recombinant siRNA-encoding adenoviruses and their application to adult cardiac myocytes, which represent a standard experimental model in research related to cardiac physiology and pathophysiology. Feasibility of this approach is demonstrated by effective ablation (>80%) of both, a transgene encoding for eGFP and the endogenous muscarinic M(2) acetylcholine receptor.

G protein-activated (GIRK) current in rat ventricular myocytes is masked by constitutive inward rectifier current (I(K1)).

Inwardly-rectifying K+ channel subunits are not homogenously expressed in different cardiac tissues. In ventricular myocytes (VM) the background current-voltage relation is dominated by I(K1), carried by channels composed of Kir2.x subunits, which is less important in atrial myocytes (AM). On the other hand in AM a large G protein gated current carried by Kir3.1/3.4 complexes can be activated by stimulation of muscarinic M(2) receptors (I(K(ACh))), which is assumed to be marginal in VM. Recent evidence suggests that total current carried by cardiac inward-rectifiers (I(K(ATP)), I(K(ACh)), I(K1)) in both, AM and VM is limited, due to K+ accumulation/depletion. This lead us to hypothesize that in conventional whole celI recordings I(K(ACh)) in VM is underestimated as a consequence of constitutive I(K1). In that case a reduction in density of I(K1) should be paralleled by an increase in density of I(K(ACh)). Three different experimental strategies have been used to test for this hypothesis: (i) Adenovirus-driven expression of a dominant-negative mutant of Kir2.1, one of the subunits supposed to form I(K1) channels, in VM caused a reduction in I(K1)-density by about 80 %. In those cells I(K(ACh)) was increased about 4 fold. (ii) A comparable increase in I(K(ACh)) was observed upon reduction of I(K1) caused by adenovirus-mediated RNA interference.(iii) Ba2+ in a concentration of 2 microM blocks I(K1) in VM by about 60 % without affecting atrial I(K(ACh)). The reduction in I(K1) by 2 microM Ba2+ is paralleled by a reversible increase in I(K(ACh)) by about 100%. These data demonstrate that the increase in K+ conductance underlying ventricular I(K(ACh)) is largely underestimated, suggesting that it might be of greater physiological relevance than previously thought.

Expression of cTnI-R145G affects shortening properties of adult rat cardiomyocytes.

The R145G amino acid exchange in the inhibitory subunit (cTnI) of cardiac troponin, which regulates muscle contraction, is related to familial hypertrophic cardiomyopathy. Information on its impact on contractility of adult cardiomyocytes is scarce. We studied shortening of adult rat cardiomyocytes before and during ss-adrenergic stimulation using adenovirus-driven expression of human cTnI-wild type (wt) and cTnI-R145G. Baseline sarcomere shortening was significantly decreased by cTnI-R145G expression. Upon ss-adrenergic stimulation using isoproterenol (ISO), nearly identical amplitudes of shortening were obtained with cells expressing cTnI-R145G and control cardiomyocytes (native and cTnI-wt). However, rates of shortening and relengthening were depressed in cTnI-R145G-expressing cells but were comparable to those of control cells upon addition of forskolin or ISO and ICI118,551. This indicates that cTnI-R145G expression influences the response to ss-adrenergic stimulation dependent on the receptor subtype.

Gene silencing in adult rat cardiac myocytes in vitro by adenovirus-mediated RNA interference.

RNA interference (RNAi) by short double stranded RNA (siRNA) represents an efficient and frequently used tool for gene silencing to study gene function. Whereas efficient ablation of genes has been demonstrated in neonatal cardiac myocytes, thus far information on successful application of this technique in adult cardiac myocytes (ACM), a standard experimental model in cardiac physiology and pathophysiology, is sparse. Here we demonstrate efficient ablation of a transgene encoding for enhanced green fluorescent protein (EGFP) and a cell specific endogenous gene encoding for an inward-rectifier channel subunit (Kir2.1) in ACM in vitro using adenovirus driven transcription of siRNA hairpins. EGFP fluorescence and density of background inward rectifier current (IK1) were reduced by > 90% within about 6-8 days after transformation with the corresponding virus. In Kir2.1-silenced myocytes resting membrane potential was significantly reduced. Survival of these cells in culture was compromised, presumably due to Ca2+ -overload caused by the depolarization. The sequence-specific knockdowns of EGFP and Kir2.1 were confirmed on the RNA level using real-time RT-PCR. In Kir2.1-silenced myocytes density of transient outward current, carried predominantly by Kv4.x subunits remained unaffected. This communication for the first time demonstrates proof of principle of efficient RNA interference using adenovirus-based vectors and demonstrates its large potential in phenotyping of ACM.