The Role of CaMKII Activation in Hypertrophic Cardiomyopathy

The classic definition of Hypertrophic Cardiomyopathy (HCM) as originally described by Teare is deceptively simple, "left ventricular hypertrophy in the absence of any identifiable cause” ¹. This definition fits the majority of referral patients who are identified either by their symptoms or incidental findings on noninvasive testing. Longitudinal studies, however, including a seminal study performed by Frank and Braunwald in 1964, clearly described the disorder much as we know it today, a complex, progressive and highly variable cardiomyopathy ². Subsequent linkage studies in the early 1990’s established mutations in the components of the cardiac sarcomere as the molecular cause of the familial form of HCM. Since then, the growing accessibility of genetic screening and advent of specialized centers of care have greatly expanded our understanding of what is now considered to be a genetic cardiomyopathy until proven otherwise ³.

Even with this sophisticated understanding of the genetic etiology of the disorder, many clinically relevant questions remain regarding the basic mechanisms whereby mutations in sarcomeric proteins lead to the variable degrees of myocardial remodeling that define HCM. In other words, how does disrupted myofilament function trigger a myocellular response that alters the geometry of the heart leading to arrhythmias, impaired relaxation, hyperdynamics and, in some cases, left ventricular outflow tract obstruction? Recent longitudinal studies and a pilot clinical trial of genotyped HCM cohorts have helped to refine and integrate these important questions ^{4, 5}. In particular, the observation that patients carrying sarcomeric mutations (Sarc -positive HCM) have worse outcomes that may not necessarily be defined by the degree of hypertophy has refocused research efforts on both the preclinical state and the temporal development of the disorder ⁶. Thus, we now know that Sarc-positive patients may exhibit impaired relaxation prior to the onset of symptoms or overt left ventricular hypertrophy, exhibit a non-linear trajectory and focal patterns of ventricular remodeling and finally, that diltiazem can slow the initial development of left ventricular hypertrophy in some cohorts ^{4, 5} , ⁷. This far more nuanced clinical understanding of Sarc-positive HCM establishes a framework for testable hypotheses that will finally allow for proactive management and perhaps even genotype-driven care instead of the current symptom palliation.

Building on these central observations and previous results from transgenic animal models of HCM demonstrating early activation of CaMKIIδ activity, in the current issue of Circulation, Helms et al., have utilized a robust set of myomectomy samples from patients with extensively phenotyped and fully genotyped hypertrophic cardiomyopathy to delineate the potential role of CaMKIIδ activation in disease pathogenesis. The study is specifically focused on identifying pathways known to be involved in the regulation of myocellular calcium homeostasis that may distinguish the natural history of HCM patients with sarcomeric gene mutations (Sarc-positive HCM) from those who do not carry sarcomeric mutations (Sarc-negative HCM). Extensive transcriptional profiling of the known components of the Ca²⁺ regulatory machinery, CaMKII isoforms and the mediators of hypertrophic growth revealed only a decrease in SERCA2A transcripts in both HCM groups as compared to controls. Importantly, the SERCA2A results were validated at the protein level where the SERCA2A/PLN complex was decreased for both HCM cohorts, indicative of extensive myocellular remodeling. Of particular note, CaMKIIδ expression was differentially upregulated only in the Sarc-positive HCM samples. This isolated upregulation suggested that CaMKIIδ may act as a nodal point for the often aggressive cardiac remodeling observed in Sarc-positive HCM patients.

To address this hypothesis, the phosphorylation states of CaMKIIδ and its relevant substrates were measured and revealed a significant level of auto-phosphorylated CaMKIIδ and PLN only in the Sarc-positive HCM samples and a trend towards an increase in HDAC4 phosphorylation in both HCM cohorts (Sarc-positive and Sarc-negative) vs controls. The phosphorylation of HDAC4 by CaMKIIδ is an important component of the signaling cascade that drives pathologic cardiac remodeling, linking myocellular Ca²⁺ dysregulation to hypertrophic growth ⁸. Subsequent probing of the downstream targets of HDAC4, NFAT and MEF2, however, revealed no induction of transcriptional activity in the HCM samples. Similarly, the increase in phosphorylated PLN observed in the Sarc-positive HCM group did not translate into a change in the Ca²⁺ sensitivity of SERCA2A uptake capacity despite a decrease in the activity and total capacity of SERCA2A for Ca²⁺ in all HCM groups. Finally, to determine the potential contribution of the calcium release axis to myocellular Ca²⁺ dysregulation in HCM, RyR2 abundance, phosphorylation state and ryanodine binding capacity were assessed. While a trend towards a decrease in RyR2 abundance and a concomitant small decrease in [³H] ryanodine binding were noted for the Sarc-positive HCM subset, no significant changes were noted in RyR2 phosphorylation potential nor any response to oxidation or nitrosylation was observed for any group.

It is illustrative to compare the current results to those of Coppini, et al, who utilized combined Sarc-positive and Sarc-negative HCM myomectomy samples to explore the roles of dysregulated signaling cascades in arrhythmogenicity and impaired relaxation ⁹. They found a marked decrease in Ca²⁺ kinetics (both time to peak and decay) and a consistent increase in intracellular diastolic [Ca²⁺]_i. Similar to the current findings, the SERCA2A/PLN complex was decreased and both the amount and phosphorylation potential of CaMKII (and several downstream targets) was increased in HCM samples, leading to the conclusion that CaMKII likely occupies a central role in disease pathogenesis. In the current study, by expanding their sample size to facilitate a comparison of Sarc-positive versus Sarc-negative HCM and incorporating ⁴⁵Cauptake to directly measure SERCA2A activity, Helms et al both confirmed and significantly extended the previous results with the intriguing observation that the increase in auto-activated CaMKIIδ was specific for Sarc-positive HCM alone. Surprisingly, no additional functional effect on SERCA2A actvity and only minor effects on other downstream CaMKII substrates were observed. While these results, coupled to previous studies in murine models, are strikingly concordant regarding a likely central role of CaMKII in the pathogenesis of HCM, the trajectory of the complex remodeling cascade over time is unclear ¹⁰. This is inherent to the limitations in using explanted tissue to study heart failure. Myomectomy samples (representing established, treated disease) constrain the ability to identify early disease mechanisms, especially those involving signaling cascades. The use of genotyped HCM samples represents a significant advance in that, unlike heart failure tissue, the primary biophysical mechanism is known and as shown in the current study, can both identify potential modulators of discrete disease subsets and provide an important framework for developing novel hypotheses of disease progression.

Perhaps given the near protean complexity of CaMKII signaling in myocardial remodeling, this lack of clarity in the context of a progressive and highly variable genetic cardiomyopathy is to be expected ¹¹. Pathogenic insight into complex cardiomyopathic disorders that develop over decades requires multiple levels of investigation before robust connections can be established. It is illustrative to frame the question of how mutations in the cardiac sarcomere cause HCM in the context of timescales. The timescales involved in disease pathogenesis range from nanosecond dynamics that drive the allosteric interactions between sarcomeric proteins to the milliseconds required for the calcium transient and myofilament activation, to the beat to beat regulation of cardiac function by the autonomic nervous system, and the decade required to remodel the myocardium and disrupt function. Inherent in this system are constants and variables. For example, the presence of the primary mutation and the effect of this mutation on sarcomeric dynamics is a constant that likely triggers a variable myocellular response that can wax and wane dependent on hemodynamic demand and external stressors. Based on recent advances in our understanding of myocellular CaMKII biology, it may be uniquely positioned to play this nodal role in Sarc-positive HCM remodeling ¹¹. It is known that sarcomeric mutations alter Ca²⁺ dissociation kinetics at the myofilament level, a change that could directly alter local nanodomains where CaMKII resides, regulated by tight spatiotemporal controls ^{12, 13}. While the initial molecular response may be compensatory, prolonged or even variable, local mechanical strain may lead to auto-activation of CaMKII and pathogenic remodeling ¹⁴. An important question remaining; how to take into account the lack of downstream activation of hypertrophic signaling, RYR2 Ca²⁺ release and SERCA2A Ca²⁺ uptake activity in the current study? As noted by the authors, this is likely due to the established disease state of the tissue. Moreover, in the context of the oft-described non-linear cardiac remodeling in Sarc-positive HCM, an important avenue to investigate is whether cyclic activation and deactivation of CaMKII responsive pathways over time underlies these periods of accelerated remodeling, an important therapeutic target.

In the main, this benchmark translational study establishes an important role for CaMKII regulation in the pathogenesis of HCM. Of course, many questions remain, again bridging the wide temporal divide that characterizes this unique disorder; sharing some but perhaps not many molecular aspects of heart failure. While these future discoveries will require multiple methodologies to overcome the limitations of myomectomy tissue, the close concordance with previous work from mouse models of HCM and the potential application of engineered heart tissue systems can be leveraged to fill the gaps ¹⁵. The long-term goal for the management of HCM remains to identify the earliest stages of the disorder via genetic screening and to use targeted and eventually even genotype-specific therapies to alter the natural history of disease, the current study brings us closer to that future.