Chronic Calmodulin-Kinase II Activation Drives Disease Progression in Mutation-Specific Hypertrophic Cardiomyopathy

Abstract

Background: Although the genetic causes of hypertrophic cardiomyopathy (HCM) are widely recognized, considerable lag in the development of targeted therapeutics has limited interventions to symptom palliation. This is in part attributable to an incomplete understanding of how point mutations trigger pathogenic remodeling. As a further complication, similar mutations within sarcomeric genes can result in differential disease severity, highlighting the need to understand the mechanism of progression at the molecular level. One pathway commonly linked to HCM progression is calcium homeostasis dysregulation, though how specific mutations disrupt calcium homeostasis remains unclear. Methods: To evaluate the effects of early intervention in calcium homeostasis, we used 2 mouse models of sarcomeric HCM (cardiac troponin T R92L and R92W) with differential myocellular calcium dysregulation and disease presentation. Two modes of intervention were tested: inhibition of the autoactivated calcium-dependent kinase (calmodulin kinase II [CaMKII]) via the AC3I peptide and diltiazem, an L-type calcium channel antagonist. Two-dimensional echocardiography was used to determine cardiac function and left ventricular remodeling, and atrial remodeling was monitored via atrial mass. Sarcoplasmic reticulum Ca 2+ ATPase activity was measured as an index of myocellular calcium handling and coupled to its regulation via the phosphorylation status of phospholamban. Results: We measured an increase in phosphorylation of CaMKII in R92W animals by 6 months of age, indicating increased autonomous activity of the kinase in these animals. Inhibition of CaMKII led to recovery of diastolic function and partially blunted atrial remodeling in R92W mice. This improved function was coupled to increased sarcoplasmic reticulum Ca 2+ ATPase activity in the R92W animals despite reduction of CaMKII activation, likely indicating improvement in myocellular calcium handling. In contrast, inhibition of CaMKII in R92L animals led to worsened myocellular calcium handling, remodeling, and function. Diltiazem-HCl arrested diastolic dysfunction progression in R92W animals only, with no improvement in cardiac remodeling in either genotype. Conclusions: We propose a highly specific, mutation-dependent role of activated CaMKII in HCM progression and a precise therapeutic target for clinical management of HCM in selected cohorts. Moreover, the mutation-specific response elicited with diltiazem highlights the necessity to understand mutation-dependent progression at a molecular level to precisely intervene in disease progression.

Effects of MYBPC3 loss-of-function mutations preceding hypertrophic cardiomyopathy

“…In addition, we performed prespecified pathway analysis for calcium-handling genes, NMD genes, and hypertrophy-associated genes. Further supporting an absence of calcium mishandling in the early developmental stage, we found no significantly dysregulated calcium-handling genes in either heterozygous or homozygous models ( Figure 5C), including ATPA2A (Serca2) and CAMK2D, both of which are dysregulated in HCM patient cardiac tissue and in mouse models of HCM (28,30). We also found no significant differential expression of NMD genes in MYBPC3 mutation lines, including in frameshift mutation lines (in which NMD degrades mutant mRNA transcripts) versus promoter deletion or start site mutation lines (in which mRNA loss of function occurs without NMD).…”

Modifications of Sarcoplasmic Reticulum Function Prevent Progression of Sarcomere-Linked Hypertrophic Cardiomyopathy Despite a Persistent Increase in Myofilament Calcium Response

“…Changes in CaMKII signaling have been previously reported in human and mouse models of HCM (Helms et al, 2016;Lehman et al, 2019). We, therefore, also assessed the levels of expression and phosphorylation of CaMKII in our mice.…”

“…We determined cardiac function (using both standard echocardiographic parameters and speckle strain measurements), histology, and myofilament function. We also established levels of Ca 2+ /calmodulindependent protein kinase II (CaMKII) phosphorylation, which has been reported to be a key signaling pathway in human HCM (Helms et al, 2016) and in mouse HCM models (Lehman et al, 2019). We observed that KO of PLN in hearts of cTnT-R92Q mice resulted in decreased CaMKII phosphorylation and a prevention of the development of the HCM phenotype, although the increase in myofilament Ca 2+ sensitivity was preserved.…”

Sarcomeric mutations in cardiac diseases

“…The situation is compounded further when considering that hundreds of different mutations occur in a myriad of proteins, each presumably resulting in distinct molecular and patient phenotypes. As one example, two different mutations that affect the same residue in troponin T (R92L and R92W) lead to divergent phenotypes [10]. However, the converse is also often true and presents an equally challenging paradox: How can different mutations in different proteins ultimately activate similar pathways that lead to the classical descriptions of HCM or DCM?…”

“…Because an increase in Ca 2+ sensitivity of tension can account for slowed relaxation as well as the preserved (or even hyper-contractile) systolic function often seen in HCM patients, the hypothesis is attractive both for its simplicity and because it is suggestive of therapeutic remedies that target Ca 2+ sensitivity [1,2]. Nonetheless such generalizations are not universal [3,7,10] and are likely to be over-simplifications that lack the sensitivity or specificity necessary for predicting individual patient disease burden. Clearly, despite the enormous progress made in identifying the root genetic causes of cardiomyopathies, there is still much more to learn regarding if, when, and how a given mutation will impact patient lives.…”