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   Heart diseases are a common cause of morbidity and mortality in dogs. In addition to the investigative methods that are considered the gold standard (echocardiography and electrocardiography), we have the option of using blood cardiac biomarker concentrations when they are not available. Cardiac troponin I (cTnI) and N-terminus of pre-brain natriuretic peptide (NT-proBNP) are mainly investigated. Their concentrations can then be used in the differentiation of respiratory problems, detection of occult dilated cardiomyopathy, detection of stage B2 in dogs with myxomatous mitral valve disease, prediction of the first appearance of heart failure symptoms in myxomatous mitral valve disease, and monitoring of heart failure. By early diagnosis and close monitoring of the progression of heart disease, we can select the most appropriate therapy.

   Keywords: dog, cardiac biomarkers, myxomatous mitral valve disease, dilated cardiomyopathy

   Heart disease is an important cause of morbidity and mortality in dogs and is more prevalent in certain breeds. For example, myxomatous mitral valve disease affects 100 % Cavalier King Charles Spaniels over 11 years of age and dilated cardiomyopathy affects about 44 % Dobermans over 6 years of age. The N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a biomarker of myocardial stretch and cardiac troponin I (cTnI) is a biomarker of myocardial damage and their blood concentrations increase due to increasing filling pressure and myocardial remodeling in most cardiac diseases (O'Shaughnessy S., 2021).

Heart disease in dogs
   As already mentioned the two most common heart diseases that occur in dogs are myxomatous mitral valve disease and dilated cardiomyopathy.

   a. Myxomatous mitral valve disease
   The most common canine cardiology patients in veterinarians' offices are dogs with myxomatous mitral valve disease (MMVD). These are usually small to medium sized dogs, where the main symptoms are respiratory signs and a systolic murmur detected on auscultation. In 2019, a consensus of specialists in canine cardiology was published regarding recommendations for the diagnosis and therapy of myxomatous mitral valve disease in dogs. Mitral valve disease and its progression to heart failure has been divided into four stages.

1. Stage A
   Dogs with a higher than average risk of developing heart failure without obvious structural abnormalities at the time of examination. Small breeds of dogs, including breeds with a known predisposition to develop myxomatous mitral valve disease (e.g., cavalier King Charles spaniel, dachshund, miniature and toy poodle).
2. Stage B
   Stage B dogs already have structural abnormalities (e.g. presence of MMVD) but do not have clinical signs of heart failure associated with heart disease.
   1. Stage B1
      Asymptomatic dogs with mitral regurgitation caused by MMVD who do not have pathological findings on radiographs or echocardiography. They have normal left atrial and ventricular size, normal systolic function and normal vertebral heart size (VHS) or vertebral left atrial size (VLAS).
   2. Stage B2
      Asymptomatic MMVD causing mitral regurgitation leading to remodeling heart (enlargement of the left atrium and ventricle. In these dogs we find echocardiographic LA:Ao ratio ≥1.6, left ventricular internal diameter in diastole normalized to body weight LVIDDN≥1.7 and radiological vertebral score VHS˃10.5.
3. Stage C
   Dogs in stage C have clinical signs of heart failurebut are not refractory to standard heart failure treatment.
4. Stage D
   Stage D patients are refractory to standard therapy heart failure and e.g. need a daily dose of furosemide higher than 8 mg/kg.
   In the context of initiating early therapy to prolong the life of the affected dog, early detection of stage B2 is important, where we can start therapy as recommended (Keene B.W., 2019).

   b. Dilated cardiomyopathy
   Dilated cardiomyopathy (DCM) is the most common heart disease in large and giant breed dogs. It is a primary heart muscle disease characterized by ventricular dilatation and systolic dysfunction and slow progression. One of the most commonly affected and also studied breeds is the Doberman.

   There are three stages of dilated cardiomyopathy that an affected individual will go through during the course of the disease. The progression of DCM is from phenotypically normal dogs to clinically affected dogs. Clinically apparent dilated cardiomyopathy is only the "tip of the iceberg". There is a long preclinical phase with progressive reduction in systolic function and ventricular dilation.

1. Stage
   Dogs predisposed to dilated cardiomyopathy are without detectable overt clinical signs and also without morphological or electrocardiographic (ECG) signs. Dogs at this stage can only be detected by a possible genetic test. This stage lasts for months to years.
2. Stage
   This stage is called Occult. However, this is not accurate because morphological changes on the heart and changes on the ECG can already be detected, but clinical signs are absent and the dog appears healthy. A more accurate term is therefore preclinical stage. It lasts 3 to 4 years. About 30 % dogs die at this stage from sudden cardiac death caused by ventricular tachyarrhythmia - ventricular fibrillation.
   Stage 3
   This stage is called Obvious. The owner observes clinical signs of congestive heart failure such as exercise intolerance, inappetence, syncope, cough, respiratory problems or enlarged abdomen. Clinical examination reveals tachypnea, tachycardia and sometimes arrhythmias, and changes are evident on ECG and echocardiography. The average age of development of symptoms of congestive heart failure is 6.7 years.
   In the context of the occurrence of a long preclinical phase, screening is recommended. This screening will identify individuals for whom we can initiate therapy to delay the onset of the overt stage of the disease and thus prolong their lives. It is recommended to perform this examination every year from the age of 2 years (Kvapil R., 2016).

Biomarkers of heart disease
   The definition of biomarkers is: they are quantitative indicators of biological processes occurring in organs or organ systems. They are an important resource in the assessment of normal, pathological and pharmaceutically influenced processes. They provide information on diagnosis, extent of disease and prognosis. They are biochemical substances that can be measured in assessing disease progression or in evaluating the benefit of therapy. (de Lima G.V., 2017). From a clinical point of view, a reliable biomarker must meet three criteria: 1. it must be easy to measure, 2. it must provide new or valuable information in the diagnosis of the disease or the efficacy of therapy, and 3. it helps the clinician to treat the patient with a more convenient and easy to use test (Baisan R.A., 2016).

   Cardiac biomarkers are a group of proteins that have been intensively studied in human and veterinary medicine in recent decades with regard to their diagnostic and prognostic significance in heart disease. In addition, cardiac biomarkers have been evaluated for their efficacy in assessing the clinical stage of heart disease, risk stratification of patients, selection of therapeutic regimens and prognosis of cases. Cardiac biomarkers are broadly divided into markers of myocardial injury (cardiac troponins, creatine kinase MB isoenzyme), myocyte stress (natriuretic peptides), inflammation (acute phase proteins), cardiac remodeling (matrix metalloproteinases), neurohormonal and endothelial dysfunction (endothelin, aldosterone, renin, noradrenaline) according to their role in the pathophysiology of heart failure. It is unlikely that all of the above biomarkers will eventually be applied in clinical practice. However, some of them (cardiac troponins, natriuretic peptides) are already commercially available and have been established as part of diagnostic investigation in cardiac diseases (Polizopoulou Z.S., 2017). They provide information on myocardial damage not only in cardiac diseases but also in some critical non-cardiac diseases (Table 2).

1. Natriuretic peptides
      Natriuretic peptides include atrial natriuretic peptide (ANP), brain or B-type natriuretic peptide (BNP) C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), ventricular natriuretic peptide (VNP), and urodilatin. In cardiomyocytes, ANP, BNP and VNP are formed. Natriuretic peptides modulate blood volume and blood pressure, antagonise the renin-angiotensin-aldosterone system (RAAS), induce bronchodilation and inhibit smooth muscle cell proliferation. In principle, therefore, they produce the opposite effect to the RAAS and the sympathetic nervous system in an attempt to protect the cardiovascular system from overload.
      Natriuretic peptides are synthesized as pre-prohormones with a carbon C-terminus and a nitrogen N-terminus. ANP is mainly produced by the atria in response to increased tension on the atrial wall and increases intravascular volume and pressure. After secretion from atrial storage granules, proANP is cleaved into a biologically active C-terminus and N-terminus. BNP has the same structural and biological properties. BNP stores are smaller than ANP, so its secretion induces greater synthesis and causes ischemia and stretching of cardiomyocytes. Its production is also induced by angiotensin II, endothelin-1 and adrenergic antagonists. Pre-prohormone is cleaved into a peptide and proBNP. ProBNP is then cleaved into the biologically inert NT-proBNP and the biologically active molecule BNP. Under normal conditions, BNP is produced by cardiomyocytes in the atrium. Due to chronic pressure and volume loading, ventricular cardiomyocytes gradually become the main source of BNP. The half-life of BNP in blood in dogs is 90 seconds and the half-life of NT-proBNP is considerably longer (Ettinger S.J., 2010).
2. Troponins
      Each cardiomyocyte consists of myofibrils arranged in parallel. Myofibrils consist of linearly arranged sarcomeres, which are the functional contractile units of the cell. The sarcomere contains two types of protein fibers. Thin actin filaments and thick myosin filaments. Myosin filaments are composed of serially arranged myosin molecules and each has a helical tail and two globular heads. During muscle contraction, many heads of the myosin filament repeatedly interact with actin to form cross-bridges that attract the thin filament, leading to shortening of the sarcomere. During muscle relaxation, actin and myosin binding sites are blocked by the tropomyosin protein and troponin complex. Troponin has 3 subunits that together act as a molecular switch for cardiomyocyte contraction. Cardiac troponin T (cTnT) attaches the troponin complex to tropomyosin. Other subunits are responsible for inhibition and generation of contraction by calcium and ATP. In the absence of calcium, cardiac troponin I (cTnI), an inhibitory subunit, inhibits ATP hydrolysis necessary for actin-myosin interaction. Calcium initiates contraction by removing the blockade of fiber interaction by binding to the calcium-binding subunit cardiac troponin C (cTnC).
      Cardiac troponin I has less than 50 % homology to the isoform found in skeletal muscle and contains a unique N-terminal peptide. It is not found in skeletal muscle and is therefore unique to the heart. The genetic homology between the canine and feline genes and the human gene for cTnI is 95 and 96 %, respectively. Troponins are purely intracellular proteins and their finding in the blood reflects release from the intracellular compartment of the cardiomyocyte. The majority of troponin forms the so-called structural pool and only a small amount is found freely in the cytosol, the so-called cytosolic pool. Troponins are heart-specific but not disease-specific. Elevated cTnI levels are caused by myocardial damage regardless of the cause. Possible causes of myocardial damage in primary heart disease and in patients without heart disease are listed in Table 2. Troponins cannot replace other cardiac diagnostics (echocardiography, electrocardiography). Myocardial damage occurs in the following cardiac diseases: subaortic stenosis, pulmonic stenosis, MMVD, DCM, arrhythmogenic cardiomyopathy, myocarditis, dirofilariosis, cardiac hemangioma and pericardial effusion. Diseases not directly related to the heart can also cause myocardial damage. These are: Pancreatitis, pyometra, parvovirus enteritis, leptospirosis, leishmaniasis, babesiosis, erhlichiosis, generalized inflammation, SIRS - total inflammatory response syndrome, menigitis-arteritis, IMHA -immune mediated hemolytic anemia, anemia, neoplasia, lymphoma, meningioma, hemangiosarcoma, respiratory disease, brachycephalic syndrome, hypoadrenocorticism, hyperadrenocorticism, heat stroke, and gastric dilatation-volvulus syndrome (Langhorn R., 2016).

Clinical use of cardiac biomarkers
   Examination of the heart to confirm the causative diagnosis includes specialised tests such as electrocardiographic (ECG) and echocardiographic examinations. These examinations are not always readily available. Therefore, efforts are made to use at least more easily accessible examinations such as radiological examinations or cardiac biomarker testing. Cardiac biomarker testing does not reveal a causal diagnosis, but it can differentiate possible causes of clinical symptoms that may be the same in diseases of, for example, the respiratory system. In this context, these are symptoms of respiratory distress or cough. In practice, the detection of NT-proBNP concentration in the IDEXX Laboratories Cardiopet® test is often used, the interpretation criteria of which are shown in Table 1.

   A number of studies have emerged that confirm the feasibility of these examination methods. In heart disease, the timeliness of confirmation of the diagnosis always matters, even in asymptomatic patients of risk groups such as breed affiliation in DCM or breed affiliation or detection of cardiac murmur on routine examination in MMVD. Also, early detection of asymptomatic patients at a certain stage of the disease (stage B2 in MMVD, occult stage in DCM) is currently recommended therapy to delay the onset of overt symptoms of congestive heart failure based on studies. The results of cardiac biomarker testing can help in the following five areas of heart disease diagnosis and therapy (see Table 3):

1. Differentiation of causes (cardiac versus non-cardiac) of respiratory disorders
   Oyama M.A. et all in a 2008 study found that serum NT-proBNP concentration is useful for diagnosing canine heart disease and assessing its severity.
2. Detection of occult dilated cardiomyopathy
   Wess G et all in a 2010 study found that cTnI measurement is a suitable test for detecting cardiomyopathy.
3. Prediction of first occurrence of congestive heart failure and mortality in dogs with MMVD
   Borgarelli M et all in 2021 found that NT-proBNP concentration can be used to identify dogs with preclinical MMVD at higher risk of developing heart failure or death.
4. Monitoring the progress of MMVD
   Willshaw J et all in a 2021 study found that NT-proBNP concentration was associated with a higher likelihood of being in stage B2 MMVD when medication could be initiated.
5. Determining the prognosis of heart disease
   Ljungvall I. et all in a 2010 study found that increasing cTnI concentrations help detect progression of cardiac remodeling in dogs with MMVD. A 2012 study by Hezzel M.J. et all found that serum cTnI concentration has prognostic value in dogs with MMVD. By detecting NT-proBNP and cTnI concentrations together, the determination of prognosis is better than measuring each biomarker separately. Repeated detection of biomarker concentrations helps to refine prognosis.

Conclusion
   Cardiac biomarker testing (cTnI and NT-proBNP) is not a substitute for specialised cardiac examinations such as echocardiography and electrocardiography. It is an alternative when these examinations are not available. Their use ranges from differentiating the cause of respiratory distress to monitoring the progression of heart failure. They can play an important role in detecting stages of the disease that have not yet manifested clinically, but medication can already be started to delay the symptoms of heart failure. This can be used to monitor breeds with a predisposition to certain heart diseases e.g. myxomatous mitral valve degeneration in Cavalier King Charles Spaniels or dilated cardiomyopathy in Dobermans. Repeated examination of their concentration also allows us to adjust and tailor effective therapy for heart failure.

MVDr. Roman Kvapil
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LITERATURE

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