CASAAV-mediated Mosaic Gene Silencing Technology To Help Distinguish Heart Failure Cause from Effect

Clinicians understand heart failure (HF) as a syndrome which is characterized by a cluster of symptoms and signs that are the physiological consequence of cardiac dysfunction. Patients with HF commonly experience shortness of breath, cough and wheeze, fatigue, peripheral swelling, and loss of appetite.¹ By the time HF becomes clinically apparent, the changes to myocardium are already profound. A period of subclinical HF progression can occur undetected for years. During the preclinical period, cellular remodeling of cardiomyocytes occurs, but its upstream regulators are poorly understood.

As scientists, we are challenged to distinguish key upstream cardiomyocyte regulators of early cellular remodeling from the massive changes that occur in more advanced failing muscle. When we are able to separate cause from effect in HF progression, a new generation of diagnostic and therapeutic solutions could be available. Clinical practice will improve with a better understanding of the early pathological alterations in cardiac muscle cells which occur within otherwise grossly normal muscle.

The cardiomyocyte structures responsible for regulating intracellular calcium transients and with that both systolic and diastolic function are the t-tubule membrane invaginations and associated cardiac dyads. Scientists have recently made major strides in understanding t-tubule organization and its alteration in failing hearts.² Key proteins involved in maintaining t-tubule structures and their microdomains have been proposed, of which the most well-characterized include junctophilin-2 (JPH2)^{3, 4} and caveolin 3 (CAV3).⁵ Recently, a membrane scaffolding protein bridging integrator I (BIN1, also known as amphiphysin II) has emerged as being responsible for t-tubule membrane microfolds,⁶ calcium channel trafficking⁷ and dyad microdomain regulation in response to stress.⁸ T-tubule remodeling is an early cardiomyocyte change in failing heart muscle.^{3, 9} Is it possible that, in acquired HF, reduction of JPH2, CAV3, and/or cardiac BIN1 (cBIN1) leads to early t-tubule remodeling and progressive functional cardiac decline?^{2, 3, 5, 9–11} The fundamental question remains whether reductions in JPH2, CAV3 or cBIN1 are result of HF progression or a cause.

Fortunately, a new technology introduced by Guo et al¹¹ published in this issue offers the opportunity for scientists to separate changes that are causal of heart failure from those only secondary to progressive disease. The authors developed a genetic mosaic knockout platform to identify t-tubule regulators in postnatal maturation. Combining CRISPR technology together with somatic mutagenesis (CASAAV, CRISPR/Cas9-AAV9-based somatic mutagenesis), the authors used the AAV9 vehicle to deliver tandem guide RNAs targeting a gene of interest together with cardiac troponin T promoter-driven Cre to Rosa^{Cas9GFP/Cas9GFP} neonatal mice. As a result, a gene is knocked down in some but not all cardiomyocytes. The rationale behind the genetic mosaic knockout is that the heart can still function normally even when essential genes are deleted in a small fraction of cardiomyocytes. This permits the distinction of indirect effects secondary to whole organ dysfunction from cell-autonomous direct effects of gene deletion. AAV transduction of cardiomyocytes is monitored through Cre-dependent GFP expression from the Rosa^Cas9GFP allele.

As proof of concept, the authors¹² chose JPH2 as target protein based on its association with dyad microdomain and t-tubule organization.^{3, 4, 13} It was first confirmed that CASAAV system conducts efficient and specific gene silencing in postnatal cardiomyocytes, consistent with an earlier report.¹⁴ The mosaic nature of gene deletion is accomplished through incremental dosage of AAV injection, which allowed a wide range of transduction frequency (from 22% to 75%) in cardiomyocytes. Deletion was efficient in transduced cells. A higher dose of virus affected a larger percentage of cardiomyocytes. Consistent with genomic deletion of Jph2 which is an essential protein, use of middle to high AAV-gRNA (Jph2) dosage was associated with cardiac dysfunction such as hypertrophy, ventricular dilatation and activation of cardiac stress genes. Low dosage of AAV-gRNA (Jph2) that achieved 22% transduction frequency did not affect overall cardiac function and morphology, thereby serving as mosaic deletion platform to allow study possible cell-autonomous effect of Jph2 deletion in the context of normal functioning myocardium.

In well-functioning hearts Jph2 deletion has almost no effect on transduced cardiomyocyte membrane organization and function, indicating a lack of a cell-autonomous role for JPH2. Furthermore, in the presence of organ dysfunction when high dose of AAV-gRNA(Jph2) was injected, non-transduced cells expressing normal levels of JPH2 ironically exhibited more severe t-tubule defects compared to Jph2-deleted cells. This result suggests that reduced JPH2 does not alter t-tubule organization. Like most proteins, JPH2 still impacts heart function. Thus the previously observed effect of reduced JPH2^{3, 4, 10} on T-tubule organization is more likely secondary to overall organ dysfunction and global stress rather than a direct cell-autonomous role on t-tubules. In addition to helping define the role of JPH2, the authors expanded their platform to screen 8 potential targets including transcription factors (NKX2–5, TBX5, MEF2C and TEAD1) and known t-tubule associated proteins (RYR2, CACNA1C, CAV3 and NCX1) in a pursuit of identifying candidates for t-tubule maturation and organization. Of the proteins studied, ryanodine receptor 2 (RYR2) is the only protein demonstrating a cell-autonomous role in t-tubule organization. Despite the well-recognized role of cBIN1 in t-tubule structure and microdomain organization,^6–8 this study did not examine cBIN1. Because RyR2 has been demonstrated to interact with cBIN1 in the heart,⁸ it is very possible that cBIN1 may serve as an early factor in t-tubule remodeling together with RyR2.

Postnatal gene editing, achieved by combining AAV9 delivery to transgenic mice with cardiomyocyte specific Cas9 expression,^{14, 15} bypasses embryonic lethality and allows study of function of essential genes in the maturing and already adult heart. The study by Guo et al¹¹ introduces a novel technique to determine upstream regulators of t-tubule and cardiomyocyte function by exploiting the mosaic nature of gene editing mediated by AAV9. This protocol affords investigators the ability to distinguish an upstream cell-autonomous role in failing hearts from secondary changes brought upon by organ dysfunction.

HF is associated with growing morbidity and mortality. Early diagnosis and thus interventions earlier in the disease process should allow clinicians the ability to reverse disease progression. The technical platform developed by Guo et al.¹² offers a new method for scientists to identify the gene expression changes which are upstream triggers of cardiomyocyte dysfunction. In the present study, JPH2 deletion had minimal impact on t-tubule organization and function, but may exacerbate and manifest the failing phenotype.¹² It is possible that other proteins such as cBIN1 may serve as potential upstream regulators of t-tubules, together with RyR2. The technology introduced by Guo et al.¹² is an important new tool to help advance our understanding of heart failure progression.

Figure 1.

Ventricular cardiomyocytes depend on t-tubules invaginations and their microdomains for normal calcium transients. The cellular remodeling of acquired heart failure includes subcellular t-tubule remodeling which then leads to clinically significant systolic and diastolic dysfunction. CASAAV-mediated Mosaic Gene Silencing Technology provides the opportunity to generate targeted knockouts in a small percentage of cardiomyocytes in vivo, allowing the study of cell-autonomous cellular remodeling without the complication of overall organ dysfunction.