Development of an AAV9-RNAi-mediated silencing strategy to abrogate TRPM4 expression in the adult heart

Abstract

The cation channel transient receptor potential melastatin 4 (TRPM4) is a calcium-activated non-selective cation channel and acts in cardiomyocytes as a negative modulator of the L-type Ca2+ influx. Global deletion of TRPM4 in the mouse led to increased cardiac contractility under β-adrenergic stimulation. Consequently, cardiomyocyte-specific inactivation of the TRPM4 function appears to be a promising strategy to improve cardiac contractility in heart failure patients. The aim of this study was to develop a gene therapy approach in mice that specifically silences the expression of TRPM4 in cardiomyocytes. First, short hairpin RNAmiR30 (shRNAmiR30) sequences against the TRPM4 mRNA were screened in vitro using lentiviral transduction for a stable expression of the shRNA cassettes. Western blot analysis identified three efficient shRNAmiR30 sequences out of six, which reduced the endogenous TRPM4 protein level by up to 90 ± 6%. Subsequently, the most efficient shRNAmiR30 sequences were delivered into cardiomyocytes of adult mice using adeno-associated virus serotype 9 (AAV9)-mediated gene transfer. Initially, the AAV9 vector particles were administered via the lateral tail vein, which resulted in a downregulation of TRPM4 by 46 ± 2%. Next, various optimization steps were carried out to improve knockdown efficiency in vivo. First, the design of the expression cassette was streamlined for integration in a self-complementary AAV vector backbone for a faster expression. Compared to the application via the lateral tail vein, intravenous application via the retro-orbital sinus has the advantage that the vector solution reaches the heart directly and in a high concentration, and eventually a TRPM4 knockdown efficiency of 90 ± 7% in the heart was accomplished by this approach. By optimization of the shRNAmiR30 constructs and expression cassette as well as the route of AAV9 vector application, a 90% reduction of TRPM4 expression was achieved in the adult mouse heart. In the future, AAV9-RNAi-mediated inactivation of TRPM4 could be a promising strategy to increase cardiac contractility in preclinical animal models of acute and chronic forms of cardiac contractile failure.

Ca2+ mishandling in heart failure: Potential targets

“…However, this model has not yet been tested in an experimental model of HF. 218 Similar beneficial effects of TRPMs in HF have beenwere observed in a study in whichwith TRPM7 kinase-deficient mice, that which developed increased cardiac hypertrophy, fibrosis, and cardiac dysfunction after chronic angiotensin II treatment, indicating that TRPM7 might play a protective role against angiotensin II effects. 219 On the other hand, angiotensin II stimulation of rat cardiac fibroblasts increases TRPM7 expression, increasing Ca 2+ influx, NFAT activation, and α-SMA expression.…”

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel—Part 2: TRPM4 in Health and Disease

“…Despite the conflicting data, TRPM4 inhibition can be a way of increasing inotropy. Recently a strategy using in vivo AAV9-RNAi-mediated silencing of cardiac TRPM4 was developed in the hope of increasing the cardiac contractility in animal models of cardiac failure, where a 90% reduction of TRPM4 protein expression was achieved in the adult mouse heart [153].…”

Pharmacological Modulation and (Patho)Physiological Roles of TRPM4 Channel—Part 1: Modulation of TRPM4

“…These models complicate both the pharmacological and genetic approaches, as the compound must be added in a way that provides an effective concentration [12]. Furthermore, KD and/or overexpression might need to be performed in an organ-/tissue-specific manner [13,14]. The in vivo approach, however, can be translated to humans more easily than in vitro methods.…”