Targeted Molecular Therapy for Preventing Heart Failure and Sudden Cardiac Death

Overview

Heart failure resulted in more than 650,000 reported sudden cardiac deaths in 2005, making it the leading cause of mortality in the US, according to the National Center for Health Statistics (National Vital Statistics Report (2008), vol.56, issue 10). A lack of mechanistic understanding about heart failure and sudden cardiac death has, however, hampered the development of effective therapies against them. Held on September 28th, 2010, the symposium Targeted Molecular Therapy for Preventing Heart Failure and Sudden Cardiac Death reviewed the biology of and current therapeutic approaches to treating sudden cardiac death. Speakers discussed research efforts to identify useful molecular targets and useful therapeutic approaches to address this unmet medical need.

Walter J. Koch from Thomas Jefferson University introduced a discussion of new approaches to treating chronic heart failure, discussing G protein-coupled receptor- (GPCR-)related targets. He elucidated the role of G protein-coupled receptor kinases (GRKs) in myocardial function as well as their involvement in cardiovascular pathological processes. In particular, Koch spoke of his group's investigation into βARKct (a β-adrenergic receptor kinase peptide), which can inhibit GRK2 activation. Gene therapy with βARKct successfully inhibited GRK2 in mice with chronic heart failure, thereby improving left ventricle function and morphology and rescuing heart failure. Interestingly, neurohormonal feedback, an indirect result of the improved contractile function caused by GRK2 inhibition, decreased sympathetic nervous system (SNS) activity and aldosterone levels.

Comparing GRK2 inhibition and GRK2 knockout studies, Koch revealed that although they show very similar phenotypic results, they might be quite different mechanistically, and their respective mechanisms remain not yet fully understood. His group's work points to a new class of drug therapies—drugs that inhibit GRK2 and more generally, drugs that target other GPCR systems from the "inside out" by focusing on the various components of their intracellular signal transduction pathways.

Koch closed his discussion with a related line of his investigations: his work elucidating a new role, in maladaptive hypertrophy, for another receptor kinase GRK5. Results suggest that GRK5 knock out mice in the adaptive hypertrophy phase (approximately the first 12 weeks after a myocardial infarction, or MI) show less of the typical thickening of ventricular walls and septum. That same class of mice, however, shows more hypertrophy in the wall-thinning, maladaptive phase. Thus, GRK5 activity, like other receptor kinase activity, is an intriguing target for post-MI therapies, which may make heart recovery more effective.

Roger J. Hajjar from Mount Sinai School of Medicine, working from the observation that heart muscle relaxation in failing hearts is defective, described his research group's and others' investigation into sarcoplasmic reticulum (SR) uptake of calcium ions during relaxation, defects in which process have been associated with heart failure. Although his group has a persistent interest in targeting the mechanisms of Ca²⁺ cycling, with and without gene therapy, Hajjar has worked a great deal on the activity of the SR Ca²⁺ ATPase (SERCA2a) in particular. His talk at this discussion group meeting detailed the process of taking that target from the identification stage to the clinical trial stage.

Gene therapy and molecular therapy, though relatively well adapted to target dysfunctional myocytes, bring a range of logistical challenges. One such challenge, the search for an effective vector for SERCA2a gene therapy, led Hajjar and his group to adeno-associated virus (AAV), which has the advantage of being small enough and serotypically varied enough to introduce SERCA2a into the myocytes of failing hearts. Small animal studies of SERCA2a therapy in mice showed improved stroke volume, and Hajjar explained that SERCA2a overexpression as a result of its introduction via an AAV vector also improved the ratio of oxygen consumed by the mouse tissues to the maximum energy output of the animals' hearts; in short, the therapy increased their energy utilization efficiency.

Although there was some concern that increasing the quantity of Ca²⁺ in the SR and possibly inducing a leak would disrupt the ventricular rhythm, further investigation revealed that the total number of ventricular arrhythmias actually decreased, as did the number of sustained cardiovascular arrhythmias (CVAs). The improved survival rate for mice provided the basis for larger animal studies and eventually Phase I and Phase II clinical trials, in which patients underwent cardiac catheterization to achieve maximum perfusion of the treatment.

Though extremely promising, these trials have yielded somewhat mixed results. By Hajjar's evaluation, research still needs to tackle the immune response either from neutralizing antibodies or T-cells, so that patients who have the antibodies do not need to be excluded from the treatment as they were in the initial trials. And, although patients in the high dose group did very well, with much more time elapsed between cardiac events and much improved cardiac function, the study demonstrated, surprisingly, no dose-response relationship. These perplexing issues, Hajjar suggested, are the questions he hopes to answer in future clinical trials.

Evangelia Kranias, from the University of Cincinnati College of Medicine, talked of her particular research program to rescue heart failure and improve heart recovery, much of which resonated with the principles behind Hajjar's work on Ca²⁺ cycling. Kranias ventured from SERCA2a, the calcium ion pump, to the proteins that regulate its activity. Phospholamban (PLN) is a small phosphoprotein that is dephosphorylated in human heart failure, so Kranias and her associates observed that in addition to SERCA2a being down regulated as Hajjar had noted, a greater percentage of the existing pumps are in the inhibited state by PLN. Therefore, the group hypothesized, it might be possible to inhibit PLN to treat heart failure.

But this modulation would require knowledge of PLNs "partners"—the molecules that regulate its activity. Hax-1 was one such molecule, and Kranias's group suggested it as a possible therapeutic target. Hax-1 interacts physically with PLN, though its specific role, either enhancing phosphorylation or decreasing dephosphorylation, remains unclear. Her group also uncovered an extremely promising therapeutic target called Protein Phosphatase Inhibitor-1, or just Inhibitor-1. This inhibitor shuts off PLN's phosphatase when the heart is stimulated and allows the kinases to "do their job unopposed," as Kranias put it. Inhibitor-1 was shown to be extremely beneficial in animal models under three major stress conditions: Transverse aortic constriction (TAC) to induce hypertrophy and heart failure, chronic isoproterenol (ISO) to stimulate β-adrenergic receptors and induce remodeling and hypertrophy, and finally Ischemia reperfusion injury in vivo and ex vivo to elucidate acute effects. In all three cases mice with the increased Inhibitor-1 experienced better cardiac function and less remodeling than their counterparts with untreated heart failure.

PLN is also regulated in part by Heat Shock Protein 20 (Hsp20). Kranias's work shows that Hsp20, because it increases phosphorylation of PLN without seeming to affect other heart phosphoproteins, can increase SR calcium cycling, maintain myocyte functioning, and increase cell survival during such stress conditions as those mentioned above. Overexpression of another small protein Histidine Rich Calcium Binding protein (HRC), unlike of Inhibitor-1, worsens cardiac functioning, especially cardiac relaxation, by interacting with SERCA2a to inhibit Ca²⁺ uptake. Kranias closed her discussion by talking about her group's research into existing human mutations that might affect the expression of all of these important proteins.

Fadi Akar from Mount Sinai School of Medicine moved in his talk away from a discussion of a calcium gradient myocytes to an analysis of the electochemical gradient across the mitochondrial membrane. Disruptions to this gradient, in particular to the membrane potential (ΔΨ_m) can increase the quantity of reactive oxygen species (ROS) and ultimately, Akar contends, lead to arrhythmias.

He argues that one particular receptor, called the inner mitochondrial anion channel (IMAC), is the "principle mediator" of arrhythmias under oxidative stress conditions. ROS buildup, triggered by even a single stressor event, causes oscillations in ΔΨ_m, which eventually disrupts the myocyte's action potential and makes the cell unexcitable. Akar and his group attempted to protect the cells' electrophysiology by blocking the IMAC channel using antagonists to the receptor that gates the channel.

The results of these tests on a cellular level and eventually on an organ level affirmed the group's assertions about the relationship between mitochondrial disruptions and overall arrhythmias following ischemia. The IMAC blocker could delay, in a dose-dependent manner, the action potential shortening that is a hallmark of ischemic injury, and it could also prevent arrhythmias during reperfusion. Post conditioning tests demonstrated that indeed promotion of the mitochondrial membrane potential during reperfusion is cardio-protective—an important find.

Although the cardio-protective efficacy of these blockers does suggest important directions for future research, the available blockers have considerable side effects, namely that they also block Ca²⁺ channels and thus reduce contractility. These side effects make them impracticable as therapies, at least presently. The next step is to examine, as Akar's group did in their proof of concept studies, whether genetically altering the expression of the gating receptor (via the PKBS gene) can be an effective substitute for blocking it pharmacologically. Wherever the particular study goes from here, regulation of the IMAC receptor and stabilization of the mitochondrial membrane potential more generally will remain an intriguing target for preventing post-ischemia arrhythmias.

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This meeting is part of our Translational Medicine Initiative, sponsored by the Josiah Macy Jr. Foundation.

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