Improving cardiac conduction with a skeletal muscle sodium channel by gene and cell therapy.

The voltage-gated Na+ channel is a critical determinant of the action potential (AP) upstroke. Increasing Na+ conductance may speed AP propagation. In this study, we propose use of the skeletal muscle Na+ channel SkM1 as a more favorable gene than the cardiac isoform SCN5A to enhance conduction velocity in depolarized cardiac tissue. We used cells that electrically coupled with cardiac myocytes as a delivery platform to introduce the Na+ channels. Human embryonic kidney 293 cells were stably transfected with SkM1 or SCN5A. SkM1 had a more depolarized (18 mV shift) inactivation curve than SCN5A. We also found that SkM1 recovered faster from inactivation than SCN5A. When coupled with SkM1 expressing cells, cultured myocytes showed an increase in the dV/dtmax of the AP. Expression of SCN5A had no such effect. In an in vitro cardiac syncytium, coculture of neonatal cardiac myocytes with SkM1 expressing but not SCN5A expressing cells significantly increased the conduction velocity under both normal and depolarized conditions. In an in vitro reentry model induced by high-frequency stimulation, expression of SkM1 also enhanced angular velocity of the induced reentry. These results suggest that cells carrying a Na+ channel with a more depolarized inactivation curve can improve cardiac excitability and conduction in depolarized tissues.

Two-pore K+ channels, NO and metabolic inhibition.

Ischemic preconditioning is a potent endogenous mechanism protecting many organs from the devastating effects of prolonged ischemia. In the heart, NO is one mediator of this myoprotective response thought to involve activation of the K(ATP) channel. Ischemic preconditioning is known to be induced by metabolic inhibition using sodium cyanide (NaCN) in single cardiomyocytes. In the present study, we show for the first time that the end effector channel activated by NaCN has been incorrectly identified. The channel activated is not K(ATP) but instead belongs to the relatively new family of two-pore domain potassium channels (K2P). Further when activated by metabolic ischemia, the amplitude of K2P current is directly modulated by activators and inhibitors of the NO pathway.

A transgenic mouse model of heart failure using inducible Galpha q.

Receptors coupled to Galpha q play a key role in the development of heart failure. Studies using genetically modified mice suggest that Galpha q mediates a hypertrophic response in cardiac myocytes. Galpha q signaling in these models is modified during early growth and development, whereas most heart failure in humans occurs after cardiac damage sustained during adulthood. To determine the phenotype of animals that express increased Galpha q signaling only as adults, we generated transgenic mice that express a silent Galpha q protein (Galpha qQ209L-hbER) in cardiac myocytes that can be activated by tamoxifen. Following drug treatment to activate Galpha q Q209L-hbER, these mice rapidly develop a dilated cardiomyopathy and heart failure. This phenotype does not appear to involve myocyte hypertrophy but is associated with dephosphorylation of phospholamban (PLB), decreased sarcoplasmic reticulum Ca2+-ATPase activity, and a decrease in L-type Ca2+ current density. Changes in Ca2+ handling and decreased cardiac contractility are apparent 1 week after Galpha qQ209L-hbER activation. In contrast, transgenic mice that express an inducible Galpha q mutant that cannot activate phospholipase Cbeta (PLCbeta) do not develop heart failure or changes in PLB phosphorylation, but do show decreased L-type Ca2+ current density. These results demonstrate that activation of Galpha q in cardiac myocytes of adult mice causes a dilated cardiomyopathy that requires the activation of PLCbeta. However, increased PLCbeta signaling is not required for all of the Galpha q-induced cardiac abnormalities.

Human mesenchymal stem cells as a gene delivery system to create cardiac pacemakers.

We tested the ability of human mesenchymal stem cells (hMSCs) to deliver a biological pacemaker to the heart. hMSCs transfected with a cardiac pacemaker gene, mHCN2, by electroporation expressed high levels of Cs+-sensitive current (31.1+/-3.8 pA/pF at -150 mV) activating in the diastolic potential range with reversal potential of -37.5+/-1.0 mV, confirming the expressed current as I(f)-like. The expressed current responded to isoproterenol with an 11-mV positive shift in activation. Acetylcholine had no direct effect, but in the presence of isoproterenol, shifted activation 15 mV negative. Transfected hMSCs influenced beating rate in vitro when plated onto a localized region of a coverslip and overlaid with neonatal rat ventricular myocytes. The coculture beating rate was 93+/-16 bpm when hMSCs were transfected with control plasmid (expressing only EGFP) and 161+/-4 bpm when hMSCs were expressing both EGFP+mHCN2 (P<0.05). We next injected 10(6) hMSCs transfected with either control plasmid or mHCN2 gene construct subepicardially in the canine left ventricular wall in situ. During sinus arrest, all control (EGFP) hearts had spontaneous rhythms (45+/-1 bpm, 2 of right-sided origin and 2 of left). In the EGFP+mHCN2 group, 5 of 6 animals developed spontaneous rhythms of left-sided origin (rate=61+/-5 bpm; P<0.05). Moreover, immunostaining of the injected regions demonstrated the presence of hMSCs forming gap junctions with adjacent myocytes. These findings demonstrate that genetically modified hMSCs can express functional HCN2 channels in vitro and in vivo, mimicking overexpression of HCN2 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart or other electrical syncytia.

Human mesenchymal stem cells make cardiac connexins and form functional gap junctions.

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (I(j)) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (V(j)) dependence. I(j) records from hMSC-HeLaCx43 and hMSC-HeLaCx40 cell pairs also showed symmetrical and asymmetrical V(j) dependence, while hMSC-HeLaCx45 pairs always produced asymmetrical I(j) with pronounced V(j) gating when the Cx45 side was negative. Symmetrical I(j) suggests that the dominant functional channel is homotypic, while the asymmetrical I(j) suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (gamma(j)) of 30-80 pS. The macroscopic I(j) obtained from hMSC-cardiac myocyte cell pairs exhibited asymmetrical V(j) dependence, while single channel events revealed gamma(j) of the size range 40-100 pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.